CA2263819A1 - Insecticidal protein toxins from photorhabdus - Google Patents

Abstract

Proteins from the genus Photorhabdus are toxic to insects upon exposure. Photorhabdus luminescens (formerly Xenorhabdus luminescens) have been found in mammalian clinical samples and as a bacterial symbiont of entomopathogenic nematodes of genus Heterorhabditis. These protein toxins can be applied to, or genetically engineered into, insect larvae food and plants for insect control.

Description

CA 022638l9 l999-02-26 W098/08932 PCT~S97/07657 INSECTICIDAL PROTEIN TOXINS FROM PHOTOR~ABDUS
Cross-reference to Related Appllcation Thls patent application is a continuation-in-part of U.S.
Patent Application Serial Number 08/743,699 filed on November 6, 1996, which is a continuation-in-part of U.S. Patent Application Serial Number 08/705,484 filed on August 28, 1996, ~ which is a continuation-in-part of U.S. Patent Application Serial Number 08/608,423 filed February 28, 1996, which is a continuation-in-part of U.S. Patent Application Serial Number 08/395,947 filed February 28, 1995, which was a continuation-in-part of U.S. Patent Application Serial Number 08/063,615 filed May 18, 1993. This application is also a continuation-in-part of provisional U.S.
i5 Patent Application Serial Number 60/007,255 filed November 6, 1995.

Field of the Invention The present invention relates to toxins isolated from bacteria and the use of said toxins as insecticides.

Background of the Invention Many insects are widely regarded as pests to homeowners, to picnickers, to gardeners, and to farmers and others whose investments in agricultural products are often destroyed or diminished as a result of insect damage to field crops.
Part1cularly in areas where the growing season is short, significant insect damage can mean the loss of all profits to growers and a dramatic decrease in crop yield. Scarce supply of particular agricultural products invariably results in higher costs to food processors and, then, to the ultimate consumers of food plants and products derived from those plants.
Preventing insect damage to crops and flowers and eliminating the nuisance of insect pests have typically relied on strong organic pesticides and insecticides with broad toxicities. These synthetic products have come under attack by the general population as being too harsh on the environment and on those exposed to such agents. Similarly in non-agricultural settings, homeowners would GO be satisfied to have insects avoid ~heir homes or outdoor meals without needing to klll the insects.
The extensive use of chemical insecticides has raised environmental and health concerns for farmers, companies that SUBSTITUTE SHEET (RULE 26) CA 022638l9 l999-02-26 W098/08932 PCT~S97/07657 produce the insecticides, government agencies, public interest groups, and the public in general. The development of less intrusive pest management strategies has been spurred along both by societal concern for the environment and by the development of biological tools which exploit mechanisms of insect management.
Biological control agents present a promising alternative to chemical insecticides.
Organisms at every evolutionary development level have devised means to enhance their own success and survival. The use of biological molecules as tools of defense and aggression is known throughout the animal and plant kingdoms. In addition, the relatively new tools of the genetic engineer allow modifications to biological insecticides to accomplish particular solutions to particular problems.
One such agent, Bacillus thuringiensis (Bt), is an effective insecticidal agent, and is widely commercially used as such. In fact, the insecticidal agent of the Bt bacterium is a protein which has such limited toxicity, it can be used on human food crops on the day of harvest. To non-targeted organisms, the Bt toxin is a digestible non-toxic protein.
Another known class of biological insect control agents are certain genera of nematodes known to be vectors of transmission for insect-killing bacterial symbionts. Nematodes containing insecticidal bacteria invade insect larvae. The bacteria then kill the larvae. The nematodes reproduce in the larval cadaver. The nematode progeny then eat the cadaver from within. The bacteria-containing nematode progeny thus produced can then invade additional larvae.
In the past, insecticidal nematodes in the Steinernema and Heterorhabditis genera were used as insect control agents.
Apparently, each genus of nematode hosts a particular species of bacterium. In nematodes of the Heterorhabditis genus, the symbiotic bacterium is Photorhabdus luminescens.
Although these nematodes are effective insect control agents, it is presently difficult, expensive, and inefficient to produce, maintain, and distribute nematodes for insect control.
It has been known in the art that one may isolate an insecticidal toxin from Photorhabdus luminescens that has activity only when injected into Lepidopteran and Coleopteran insect larvae.
This has made it impossible to effectively exploit the insecticidal properties of the nematode or its bacterial symbiont. What would be useful would be a more practical, less labor-intensive wide-area delivery method of an insecticidal toxin which would retain its SU8ST~TlJTE ~}~EET (RULE 2~) CA 022638l9 l999-02-26 WOg8/08932 PCT~S97107657 biological properties after delivery It would be quite desirous to discover toxlns with oral activity produced by the genus Photorhabdus. The isolation and use of these toxins are desirous due to efficacious reasons. Until applicants' discoveries, these toxins had not been isolated or characterized.
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$l~mm~ry of the Invention The native toxins are protein complexes that are produced and secreted by growing bacteria cells of the genus Photorhabdus, of interest are the proteins produced by the species Photorhabdus luminescens. The protein complexes, with a molecular size of approximately 1,000 kDa, can be separated by SDS-PAGE gel analysis into numerous component proteins. The toxins contain no hemolysin, lipase, type C phospholipase, or nuclease activities. The toxins exhibit significant toxicity upon exposure administration to a number of insects.
The present invention provides an easily administered insecticidal protein as well as the expression of toxin in a heterologous system.
The present invention also provides a method for delivering insecticidal toxins that are functional active and effective against many orders of insects.
Objects, advantages, and features of the present invention will become apparent from the following specification.

~rief Description of the Drawings Fig. 1 is an illustration of a match of cloned DNA isolates used as a part of sequence genes for the toxin of the present invention.
Fig. 2 is a map of three plasmids used in the sequencing process.
Fig. 3 is a map illustrating the inter-relationship of several partial DNA fragments.
Fig. 4 is an illustration of a homology analysis between the protein sequences of TcbAii and TcaBii proteins.
Fig. 5 is a phenogram of Photorhabdus strains. Relationship of Photorhabdus Strains was defined by rep-PCR.
~0 The upper axis of Fig. 5 measures the percentage similarity of strains based on scoring of rep-PCR products (i.e., 0.0 [no similarity] to 1.0 [100~ similarity]~. At the right axis, the numbers and letters indicate the various strains tested; 14=W-14, SU85TTTUTE ~HE~ tRULE 26) CA 022638l9 l999-02-26 Hm=Hm, H9=H9, 7=WX-7, 1=WX-1, 2=WX-2, 88=HP88, NC-1=NC-1, 4=WX-4, 9=WX-9, 8=WX-8, 10=WX-10, WIR=WIR, 3=WX-3, 11=WX-11, 5=WX-5, 6=WX-6, 12=WX-12, x14=WX-14, 15=WX-15, Hb=Hb, B2=B2, 48 through 52=ATCC
43948 through ATCC 43952. Vertical lines separatlng horizontal lines indicate the degree of relatedness (as read from the extrapolated intersection of the vertical line with the upper axis) between strains or groups of strains at the base of the horizontal lines (e.g., strain W-14 is approximately 60% similar to strains H9 and Hm).
Fig. 6 is an illustration of the genomic maps of the W-14 Strain.
Fig. 6A is an illustration of the tca and tcb loci and primary gene products.
Fig. 7 is a phenogram of Photorhabdus strains as defined by rep-PCR. The upper axis of Fig. 7 measures the percentage similarity of strains based on scoring of rep-PCR products (i.e., C.0 [no similarity] to 1.0 [100~ similarity]). At the right axis, the numbers and letters indicate the various strains tested.
Vertical lines separating horizontal lines indicate the degree of relatedness (as read from the extrapolated intersection of the vertical line with the upper axis) between strains or groups of strains at the base of the horizontal lines (e.g., strain Indicus is approximately 30~ similar to strains MPl and HB Oswego). Note that the Photorhabdus strains on the phenogram are as follows: 14 = W-14; Hm = Hm; H9 = H9; 7 = WX-7; l = WX-1; 2 = WX-2; 88 = HP88;
NC1 = NC-l; 4 = WX-4; 9 = WX-9; 8 = WX-8; 10 = WX-10; 30 = W30; WIR
= WIR; 3 - WX-3; 11 = WX-ll; 5 = WX-5; 6 = WX-6; 12 = WX-12; 15 =
WX-15; X14 = WX-14; Hb = Hb; B2 = B2; 48 = ATCC 43948; 49 = ATCC
43949; 50 = ATCC 43950; 51 = ATCC 43951- 52 = ATCC 43952.

Detailed Description of the Invention The present inventions are directed to the discovery of a unique class of insecticidal protein toxins from the ~enus Photorhabdus that have oral toxicity against insects. A unique feature of Photorhabdus is its bioluminescence. Photorhabdus may be isolated from a variety of sources. One such source is nematodes, more particularly nematodes of the genus Heterorhabditis. Another such source is from human clinical samples from wounds, see Farmer et al. 1989 J. Clin. Microbiol. 27 SUBSTITI.JTE SHEET (RULE 26) CA 022638l9 l999-02-26 pp. 1594-1600. These saprohytic strains are deposited in the Amerlcan Type Culture Collectlon (Rockville, MD) ATCC #s 43948, 43949, 43950, 43951, and 43952, and are incorporated herein by reference. It is possible that other sources could harbor Photorhabdus bacteria that produce insecticidal toxins. Such ~ sources in the environment could be elther terrestrial or aquatic based.
The genus Photorhabdus ls taxonomlcally defined as a member of the Family Enterobacteriaceae, although it has certain tralts atypical of this family. For example, stralns of this genus are nltrate reduction negatlve, yellow and red pigment produclng and bioluminescent. This latter trait i9 otherwise unknown wlthin the Enterobacteriaceae. Photorhabdus has only recently been described as a genus separate from the Xenorhabdus (Boemare et al., 1993 Int.
15 J. Syst. Bacterlol. 43, 249-255). Thls differentiation ls based on DNA-DNA hybridlzatlon studies, phenotypic differences (e.g., presence ( Photorhabdus) or absence (Xenorhabdus) of catalase and biolumlnescence) and the Family of the nematode host (Xenorhabdus;
Steinernematidae, Photorhabdus; Heterorhabditidae). Comparatlve, cellular fatty-acid analyses (Janse et al. 1990, Lett. Appl.
Mlcroblol 10, 131-135; Suzuki et al. l990, J. Gen. Appl.
Microbiol., 36, 393-401) support the separation of Photorhabdus f rom Xenorhabdus.
In order to establish that the strain collectlon disclosed hereln was comprlsed of Photorhabdus stralns, the strains were characterlzed based on recognized traits which deflne Photorhabdus and dlfferentiate lt from other Enterobacteriaceae and Xenorhabdus species. (Farmer, 1984 Bergey's Manual of Systemic Bacteriology Vol. 1 pp.510-511; Akhurst and Boemare 1988, J. Gen. Microbiol. 134 30 pp. 1835-1845; Boemare et al. 1993 Int. J. Syst. Bacteriol. 43 pp. 249-255, which are incorporated hereln by reference). The traits studied were the following: gram stain negative rods, organism size, colony plgmentation, inclusion bodies, presence of catalase, ablllty to reduce nltrate, bioluminescence, dye uptake, gelatin hydrolysis, growth on selective media, growth temperature, survival under anerobic conditions and motility. Fatty acid ~ analysis was used to confirm that the strains herein all belong to the single genus Photorhabdus.
Currently, the bacterial genus Photorhabdus is comprised of a single defined specles, Photorhabdus luminescens (ATCC Type straln #29999, Polnar et al., 1977, Nematologlca 23, 97-102). A variety of related strains have been described in the literature (e.g., Akhurst et al. 1988 J. Gen. Microbiol., 134, 1835-1845; Boemare SUBSTITIJTE S~IEE~(RULE-26) CA 022638l9 l999-02-26 W098/08932 PCTrUS97/07657 et al. 1993 Int. J. Syst. Bacteriol. 43 pp. 249-255; Putz et al.
1990, Appl. Environ. Microbiol., 56, 181-186t-;- Numerous Photorhabdus strains have been characterized herein. Because there is currently only one species ( luminescens) defined within the genus Photorhabdus, the luminescens species traits were used to characterize the strains herein. As can be seen in Fig. 5, these strains are quite diverse. It is not unforeseen that in the future there may be other Photorhabdus species that will have some of the attributes of the luminescens species as well as some different characteristics that are presently not defined as a trait of Photorhabdus luminescens. However, the scope of the invention herein is to any Photorhabdus species or strains which produce proteins that have functional activity as insect control agents, regardless of~other traits and characteristics.
Furthermore, as is demonstrated herein, the bacteria of the genus Photorhabdus produce proteins that have functional activity as defined herein. Of particular interest are proteins produced by the species Photorhabdus luminescens. The inventions herein should in no way be limited to the strains which are disclosed herein.
These strains illustrate for the first time that proteins produced by diverse isolates of Photorhabdus are toxic upon exposure to insects. Thus, included within the inventions described herein are the strains specified herein and any mutants thereof, as well as any strains or species of the genus Photorhabdus that have the functional activity described herein.
There are several terms that are used herein that have a particular meaning and are as follows:

By "functional activity" it is meant herein that the protein toxin(s) function as insect control agents in that the proteins are orally active, or have a toxic effect, or are able to disrupt or deter feeding, which may or may not cause death of the insect.
When an insect comes into contact with an effective amount of toxin delivered via transgenic plant expression, formulated protein compositions(s), sprayable protein composition(s), a bait matrix or other delivery system, the results are typically death of the insect, or the insects do not feed upon the source which ma~es the toxins available to the insects.

By the use of the term "genetic material" herein, it is meant to include all genes, nucleic acid, DNA and RNA.

SUBSTITUTE S}~EET (RULE 26) W098/08932 PCTrUS97/07657 By "homolog" it is meant an amino acid sequence that is identified as possessing homology to a reference W-14 toxin polypeptide amino acid sequence.

By "homology" it is meant an amino acid sequence that has a ~ similarity index of at least 33~ and/or an identity index of at least 26% to a reference W-14 toxin polypeptide amino acid sequence, as scored by the GAP algorithm using the BlOsum 62 protein scoring matrix (Wisconsin Package Version 9.0, Genetics Computer Group (GCG), Madison, WI).

By "identity" is meant an amino acid sequence that contains an identical residue at a given position, following alignment with a reference W-14 toxin polypeptide amino acid sequence by the GAP
algorithm.

The protein toxins discussed herein are typically referred to as "insecticides". By insecticides it is meant herein that the protein toxins have a "functional activity" as further defined herein and are used as insect control agents.

By the use of the term "oligonucleotides~ it is meant a macromolecule consisting of a short chain of nucleotides of either RNA or DNA. Such length could be at least one nucleotide, but typically are in the range of about 10 to about 12 nucleotides.
The determination of the length of the oligonucleotide is well within the skill of an artisan and should not be a limitation herein. Therefore, oligonucleotides may be less than 10 or greater than 12.
By the use of the term "Photorhabdus toxin" it is meant any protein produced by a Photorhabdus microorganism strain which has functional activity against insects, where the Photorhabdustoxin could be formulated as a sprayable composition, expressed by a transgenic plant, formulated as a bait matrix, delivered via baculovirus, or delivered by any other applicable host or delivery system.

By the use of the term "toxic" or "toxicity" as used herein it is meant that the toxins produced by Photorhabdus have "functional activity" as defined herein.

SUF~,STrrUTE SHEET ~RULE 2~) CA 022638l9 l999-02-26 By "truncated peptide" it is meant herein to include any peptide that is fragment(s) of the peptides observed to have functional activity.

By "substantial sequence homology" is meant either: a DNA fragment having a nucleotide sequence sufficiently similar to another DNA
fragment to produce a protein having similar biochemical properties;
or a polypeptide having an amino acid sequence sufficiently similar to another polypeptide to exhibit similar biochemical properties.
Fermentation broths from selected strains reported in Table 20 were used to determine the following: breadth of insecticidal toxin production by the Photorhabdus genus, the insecticidal spectrum of these toxins, and to provide source material to purify the toxin complexes. The strains characterized herein have been shown to have oral toxicity against a variety of insect orders.
Such insect orders include but are not limited to Coleoptera, Homoptera, Lepidoptera, Diptera, Acarina, Hymenoptera and Dictyoptera.
As with other bacterial toxins, the rate of mutation of the bacteria in a population causes many related toxins slightly different in sequence to exist. Toxins of interest here are those which produce protein complexes toxic to a variety of insects upon exposure, as described herein. Preferably, the toxins are active against Lepidoptera, Coleoptera, Homopotera, Diptera, Hymenoptera, Dictyoptera and Acarina. The inventions herein are intended to capture the protein toxins homologous to protein toxins produced by the strains herein and any derivative strains thereof, as well as any protein toxins produced by Photorhabdus. These homologous proteins may differ in sequence, but do not differ in function from those toxins described herein. Homologous toxins are meant to include protein complexes of between 300 kDa to 2,000 kDa and are comprised of at least two (2) subunits, where a subunit is a peptide which may or may not be the same as the other subunit.
Various protein subunits have been identified and are taught in the Examples herein. Typically, the protein subunits are between about 18 kDa to about 230 kDa; between about 160 kDa to about 230 kDa;
100 kDa to 160 kDa; about 80 kDa to about 100 kDa; and about 50 kDa to about 80 kDa.
As discussed above, some Photorhabdus strains can be isolated from nematodes. Some nematodes, elongated cylindrical parasitic worms of the phylum Nematoda, have evolved an ability to exploit insect larvae as a favored growth environment. The insect larvae SU8ST~TUTE SH EET tRULE--2~) CA 022638l9 l999-02-26 W098/08932 PCT~US97/07657 provide a source of food for growing.nematodes and an environment in which to reproduce. One dramatic effect that follows invasion of larvae by certain nematodes is larval death. Larval death results from the presence of, in certain nematodes, bacteria that produce an insecticidal toxin which arrests larval growth and inhibits feeding activity.
Interestingly, it appears that each genus of insect parasitlc nematode hosts a particular species of bacterium, uniquely adapted for symbiotic growth with that nematode. In the interim since this research was initiated, the name of the bacterial genus Xenorhabdus was reclassified into the Xenorhabdus and the Photorhabdus.
Bacteria of the genus Photorhabdus are characterized as being symbionts of Heterorhabditus nematodes while Xenorhabdus species are symbionts of the Steinernema species. This change in ~5 nomenclature is reflected in this specification, but in no way should a change in nomenclature alter the scope of the in~entions described herein.
The peptides and genes that are disclosed herein are named according to the guidelines recently published in the Journal of Bacteriology "Instructions to Authors" p. i -xii (Jan. 1996), which is incorporated herein by reference. The following peptides and genes were isolated from Photorhabdus strain W-14.

SU8ST~TUTE SHE~ tRUI E 26) CA 022638l9 l999-02-26 Table 1 Peptide/Gene Nomenclatl~re Toxi n Complex l ~ ~ 4 Peptide Peptide Gene Gene Name Sequence ID No.* Name Sequence ID No.*
tca g~n~mic region TcaA 34 ~ tc~A 33 TcaAi pro-peptide tcaA
TcaAii L15] a 34C tcaA
TcaAi i i [4] a 35 C tcaA
TcaAiV [62] a tcaA
TcaB [3] ~ (19~ 20)b, 26C tcaB 25 TcaBi [3]a, (19, 20)b ~8c tcaB 27 TcaBii [5] ~ 30 tcaB 29 TcaC [2], 32 tcaC 31 tcb ~enomic reqion TcbA 12C, [16]a, (21, tcbA
22, 23, 24) TcbAi pro-peptide tcbA
TCbAi i [1 ~ a ~ (21 ~ 22 ~ 23 ~ tcbA 52 TCbAiii [40] a ssc tcbA 54 tcc genomic re~ion TccA [8] a 57C tccA 56 TccB [ 7] ~ 59 tCC~ 58 TccC 61c tccC 60 tcd aenomic region TcdA (17, 18, 37, 38, tcdA, (36)d, 46 39, 42, 43) , 47 TcdAl pro-peptide tcdA
TcdAii [13]a~ (17~ 18~ 37~ tcdA 48 38 ~ 39) , 49c TCdAiii [41]a~ (42~ 43)b, tcdA 50 TcdB [14] a tcdB
aSequence ID No.~s in brackets are peptide N-termini;
bNumbers in parentheses are N-termini of internal peptide tryptic fragments Cdeduced from gene sequence dinternal gene fragment The sequences listed above are grouped by genomic region. More specifically, the Photorhabdus luminesence bacteria (W-14) has at least four distinct genomic regions- tca, tcb, tcc and tcd. As can be seen in Table 1, peptide products are produced from these distinct genomic regions. Furthermore, as illustrated in the Examples, specifically Examples 1~ and 21, individual gene products produced from three genomic regions are associated with insect activity. There is also considerable homology between these four genomic regions.

SU~STITUTE SHEET (RULE 26) W098/08932 PCTrUS97/07657 As is further illustrated in the Examples, the tcbA gene was expressed in E. coli as two possible biological actlve protein fragments (TcbA and TCbAii/iii). The tcdA gene was also expressed in E. coli. As illustrated in Example 16, when the native unprocessed TcbA toxin was treated with the endogeneous metalloproteases or insect gut contents containing proteases, the TcbA protein toxin was processed into smaller subunits that were less than the size of the native peptides and Southern Corn Rootworm activity increased. The smaller toxin peptides remained associated as part of a toxin complex. It may be desirable in some situations to increase activation of the toxin(s) by proteolytic processing or using truncated peptides. Thus, it may be more desirable to use truncated peptide(s) in some applications, i.e., commercial transgenic plant applications.
In addition to the W-14 strain, there are other species within the Photorhabdus genus that have functional activity which is differential (specifically see Tables 20 and 36). Even though there is differential activity, the amino acid sequences in some cases have substantial sequence homology. Moreover, the molecular probes indicate that some genes contained in the strains are homologous to the genes contained in the W-14 strain. In fact all of the strains illustrated herein have one or more homologs of W-14 toxin genes.
The antibody data in Example 26 and the N-terminal sequence data in Example 25 further support the conclusion that there is homology and identity (based on amino acid sequence) between the protein toxin(s) produced by these strains. At the molecular level, the W-14 gene probes indicated that the homologs or the W-14 genes themselves ~Tables 37, 38, and 39) are dispersed throughout the Photorhabdus genus. Further, it is possible that new toxin genes exist in other strains which are not homologous to W-14, but maintain overall protein attributes ~see specifically Examples 14 and 25).
Even though there is homology or identity between toxin genes produced by the Photorhabdus strains, the strains themselves are qulte diverse. Using polymerase chain reaction technology further discussed in Example 22, most of the strains illustrated herein are quite distinguishable. For example as can be seen in Figs. 5, the percentage relative similarity of some of the strains, such as HP88 and NC-l, was about 0.8, which indicates that the strains are similar, while HP88 and Hb was about 0.1, which indicates substantial diversity. Therefore, even though the insect toxin genes or gene products that the strains produce are the same or similar, the strains themselves are diverse.

SUBSTITUTE S~E~T tRULE 26) W098/08932 PCT~US97/07657 In view of the data further disclosed in the Examples and discussions herein, it is clear that a new and unique family of insecticidal protein toxin(s) has been discovered. It has been further lllustrated herein that these toxin(s) widely exist within bacterial strains of the Photor~abdus genus. It may also be the case that these toxin genes widely exist within the family Enterobacteracaea. Antibodies prepared as described in Example 21 or gene probes prepared as described in Example 25 may be used to further screen for bacterial strains within the family Enterobacteracaea that produce the homologous toxin(s) that have functional activity. It may also be the case that specific primer sets exist that could facilitate the identification of new genes within the Photorhabdus genus or family Enterobacteracaea.
As stated above, the antibodies may be used to rapidly screen bacteria of the genus Photorhabdus or the family Enterbacteracaea for homologous toxin products as illustrated in Example 26. Those skilled in the art are quite familiar with the use of antibodies as an analysis or screening tool (see US Patent No. 5,430,137, which is incorporated herein by reference). Moreover, it is generally accepted in the literature that antibodies are elicited against 6 to 20 amino acid residue segments that tend to occupy exposed surface of polypeptides (Current Protocols in Immunology, Coligan et al, National Institutes of Health, John Wiley & Sons, Inc.). Usually the amino acid consist of contiguous amino acid residues, however, in certain cases they may be formed by non-contiguous amino acids that are constrained by specific conformation. The amino acid segments recognized by antibodies are highly specific and commonly referred to epitopes. The amino acid fragment can be generated by chemical and/or enzymatic cleavage of the native protein, by automated,~-solid-phase peptide synthesis, or by production from genetic engineering organisms. Polypeptide fragmellL~ can be isolated by a variety and/or combination of HPLC and FPLC
chromatographic methods known in the art. Selection of polypeptide fragment can be aided by the use of algorithms, for example Kyte and 35 Doolittle, 1982, Journal of Molecular Biology 157: 105-132 and Chou and Fasman, 1974, Biochemistry 13: 222-245, that predict those sequences most likely to exposed on the surface of the protein. For preparation of immunogen containing the polypeptide fragment of interest, in general, polypeptides are covalently coupled using chemical reactions to carrier proteins such as keyhole limpet hemocyanin via free amino (lysine), sulfhydyl (cysteine), phenolic (tyrosine) or carboY~rlic (aspartate or glutamate) groups. Immunogen with an adjuvant is .njected in animals, such as mice or rabbits, or SUBSTITUTE 5HEET ~RULE 26) CA 022638l9 l999-02-26 WO9~08932 PCT~US97/07657 chickens to elicit an immune responsç against the immunogen.
Analysis of antibody titer in antisera of inject animals against polypeptide fragment can be determined by a variety of immunological methods such as ELISA and Western blot. Alternatively, monoclonal antibodies can be prepared using spleen cells of the injected animal for fusion with tumor cells to produce immortalized hybridomas cells producing a single antibody species. Hybridomas cells are screened using immunological methods to select lines that produce a specific antibody to the polypeptide fragment of interest. Purification of antibodies from different sources can be performed by a variety of antigen affinity or antibody affinity columns or other chromatographic HPLC or FPLC methods.
The toxins described herein are quite unique in that the toxins have functional activity, which is key to developing an insect management strategy. In developing an insect management strategy, it is possible to delay or circumvent the protein degradation process by injecting a protein directly into an organism, avoiding its digestive tract. In such cases, the protein administered to the organism will retain its function until it is denatured, non-specifically degraded, or eliminated by the immune system in higher organisms. Injection into insects of an insecticidal toxin has potential application only in the laboratory, and then only on large insects which are easily injected. The observation that the insecticidal protein toxins herein described exhibits their toxic activity after oral ingestion or contact with the toxins permits the development of an insect management plan based solely on the ability to incorporate the protein toxins into the insect diet. Such a plan could result in the production of insect baits.
The Photorhabdus toxins may be administered to insects in a purified form. The toxins may also be delivered in amounts from about 1 to about 100 mg / liter of broth. This may vary upon formulation condition, conditions of the inoculum source, techniques for isolation of the toxin, and the like. The toxins may be administered as an exudate secretion or cellular protein originally expressed in a heterologous prokaryotic or eukaryotic host. Bacteria are typically the hosts in which proteins are expressed. Eukaryotic hosts could include but are not limited to plants, insects and yeast. Alternatively, the toxins may be produced in bacteria or transgenic plants in the field or in the insect by a baculovirus vector. Typically the toxins will be introduced to the insect by incorporating one or more of the toxins into the insects' feed.

SUBSTITUTE SHFET tRULE 2~) CA 022638l9 l999-02-26 W098t08932 PCT~S97/07657 Complete lethality to feeding insects is useful but is not required to achieve useful toxicity. If the insects avoid the toxin or cease feeding, that a~oidance will be useful in some applications, even if the effects are sublethal. For example, if insect resistant transgenic crop plants are desired, a reluctance of insects to feed on the plants is as useful as lethal toxicity to the insects since the ultimate objective is protection of the plants rather than killing the insect.
There are many other ways in which toxins can be incorporated into an insect's diet. As an example, it is possible to adulterate the larval food source with the toxic protein by spraying the food with a protein solution, as disclosed herein. Alternatively, the purified protein could be genetically engineered into an otherwise harmless bacterium, which could then be grown in culture, and either applied to the food source or allowed to reside in the soil in an area in which insect eradication was desirable. Also, the protein could be genetically engineered directly into an insect food source. For instance, the major food source of many insect larvae is plant material.
By incorporating genetic material that encodes the insecticidal properties of the Photorhabdus toxins into the genome of a plant eaten by a particular insect pest, the adult or larvae would die after consuming the food plant. Numerous members of the monocotyledonous and dlctyledenous genera have been transformed.
Transgenic agronmonic crops as well as fruits and vegetables are of commercial interest. Such crops include but are not limited to maize, rice, soybeans, canola, sunflower, alfalfa, sorghum, wheat, cotton, peanuts, tomatoes, potatoes, and the like. Several techniques exist for introducing foreign genetic material into plant cells, and for obtaining plants that stably maintain and express the introduced gene. Such techniques include acceleration of genetic material coated onto microparticles directly into cells (U.S. Patents 4,945,050 to Cornell and 5,141,131 to DowElanco).
Plants may be transformed using Agrobacterium technology, see U.S.
35 Patent 5,177,010 to University of Toledo, 5,104,310 to Texas A&M, European Patent Application 0131624B1, European Patent Applications 120516, 159418B1 and 176,112 to Schilperoot, U.S. Patents 5,149,645, 5,469,976, 5,464,763 and 4,940,838 and 4,693,976 to Schilperoot, European Patent Applications 116718, 290799, 320500 40 all to MaxPlanck, European Patent Applications 604662 and 627752 to Japan Tobacco, European Patent Applications 0267159, and 0292435 and U.S. Patent 5,231,019 all to Ciba Geigy, U.S. Patents 5,463,174 and 4,762,785 both to Calgene, and U.S. Patents 5,004,863 and SUBS 111 LJTE SHEET (RULE 2~) CA 022638l9 l999-02-26 W098/08932 PCT~US97/07657 5,159,135 both to Agracetus. Other transformation technology includes whiskers technology, see U.S. Patents 5,302,523 and 5,464,765 both to Zeneca. Electroporation technology has also been used to transform plants, see WO 87/06614 to Boyce Thompson Institute, 5,472,869 and 5,384,253 both to Dekalb, W09209696 and - WO9321335 both to PGS. All of these transformation patents and publications are incorporated by reference. In addition to numerous technologies for transformlng plants, the type of tissue which is contacted with the foreign genes may vary as well. Such tissue would include but would not be limited to embryogenic tissue, callus tissue type I and II, hypocotyl, meristem, and the like. Almost all plant tissues may be transformed during dedifferentiation using appropriate techniques within the skill of an artisan.
Another variable is the choice of a selectable marker. The preference for a particular marker is at the discretion of the artisan, but any of the following selectable markers may be used along with any other gene not listed herein which could function as a selectable marker. Such selectable markers include but are not limited to aminoglycoside phosphotransferase gene of transposon Tn5 ~Aph II) which encodes resistance to the antibiotics kanamycin, neomycin and G418, as well as those genes which code for resistance or tolerance to glyphosate; hygromycin; methotrexate;
phosphinothricin (bialophos); imidazolinones, sulfonylureas and triazolopyrimidine herbicides, such as chlorosulfuron; bromoxynil, dalapon and the like.
In addition to a selectable marker, it may be desirous to use a reporter gene. In some instances a reporter gene may be used without a selectable marker. Reporter genes are genes which are typically not present or expressed in the recipient organism or tissue. The reporter gene typically encodes for a protein which provides for some phenotypic change or enzymatic property.
Examples of such genes are provided in K. Weising et al. Ann. Rev.
Genetics, 22, 421 (1988), which is incorporated herein by reference. A preferred reporter gene is the glucuronidase (GUS) gene.
Regardless of transformation technique, the gene is preferably incorporated into a gene transfer vector adapted to express the Photorhabdus toxlns in the plant cell by including in the vector a plant promoter. In addition to plant promoters, promoters from a variety of sources can be used efficiently in plant cells to express foreign genes. For example, promoters of bacterial orlgin, such as the octopine synthase promoter, the nopaline synthase SUBSTITUTE S~EET (RULE 2~) ,, .. ~.~.. ~......................... . . . ..

CA 022638l9 l999-02-26 W098/08932 PCT~US97/07657 promoter, the mannopine synthase promoter; promoters of viral origin, such as the cauliflower mosaic virus (35S and l9S), reengineered 35S, known as 35T (see PCT/US96/16582, WO 97/13402 published April 17, 1997, which is incorporated herein by reference) and the like may be used. Plant promoters include, but are not limited to ribulose-1,6-bisphosphate (RUBP) carboxylase small subunit (ssu), beta-conglycinin promoter, phaseolin promoter, ADH promoter, heat-shock promoters and tissue specific promoters.
Promoters may also contain certain ~nh~ncer sequence elements that may improve the transcription efficiency. Typical enhancers include but are not limited to Adh-intron 1 and Adh-intron 6.
Constitutive promoters may be used. Constitutive promoters direct continuous gene expression in all cells types and at all times (e.g., actin, ubiquitin, CaMV 35S). Tissue specific promoters are responsible for gene expression in specific cell or tissue types, such as the leaves or seeds (e.g., zein, oleosin, napin, ACP) and these promoters may also be used. Promoters may also be are active during a certain stage of the plants' development as well as active in plant tissues and organs. Examples of such promoters include but are not limited to pollen-specific, embryo specific, corn silk specific, cotton fiber specific, root specific, seed endosperm specific promoters and the like.
Under certain circumstances it may be desirable to use an inducible promoter. An inducible promoter is responsible for expression of genes in response to a specific signal, such as:
physical stimulus (heat shock genes); light (RUBP carboxylase);
hormone (Em); metabolites; and stress. Other desirable transcription and translation elements that function in plants may be used. Numerous plant-specific gene transfer vectors are known to the art.
In addition, it is known that to obtain high expression of bacterial genes in plants it is preferred to reengineer the bacterial genes so that they are more efficiently expressed in the cytoplasm of plants. Maize is one such plant where it is preferred to reengineer the bacterial gene(s) prior to transformation to increase the expression level of the toxin in the plant. One reason for the reengineering is the very low G+C content of the native bacterial gene(s) (and consequent skewing towards high A+T
content). This results in the generation of sequences mimicking or duplicating plant gene control sequences that are known to be highly A+T rich. The presence of some A+T-rich sequences within the DNA of the gene(s) introduced into plants (e.g., TATA box regions normally found in gene promoters) may result in aberrant SUBSTITUTE SHEET tRULE 26) W098/08932 PCT~US97/07657 transcription of the gene(s). On the other hand, the presence of other regulatory sequences residing in the transcribed mRNA (e.g., polyadenylation signal sequences (AAUAAA), or sequences complementary to small nuclear RNAs involved in pre-mRNA splicing) may lead to RNA instability. Therefore, one goal in the design of reengineered bacterial gene(s), more preferably referred to as plant optimized gene(s), is to generate a DNA sequence having a higher G+C content, and preferably one close to that of plant genes coding for metabolic enzymes. Another goal in the design of the plant optimized gene(s) is to generate a DNA sequence that not only has a higher G+C content, but by modifying the sequence changes, should be made so as to not hinder translation.
An example of a plant that has a high G+C content is maize.
The table below illustrates how high the G+C content is in maize.
As in maize, it is thought that G+C content in other plants is also high.
Table 2 Com~ilation of G+C Contents of Protein Coding Regions of Maize G~nes Protein ClassRange %G+C Mean ~G+C

Metabolic Enzymes (40)44.4-75.3 59.0 (8.0) Storage Proteins Group I (23~ 46.0-51.9 48.1 (1.3~

Group II (13) 60.4-74.3 67.5 (3.2) Group I + II (36)46.0-74.3 55.1 (9.6) Structural Proteins (18) 48.6-70.5 63.6 (6.7) Regulatory Proteins (5)57.2-68.9 62.0 (4.9) Uncharacterized Proteins (9) 41.5-70.3 64.3 (7.2) All Proteins (108) 44.4-75.3 60.8 (5.2) Number of genes in class given in parentheses.
b Standard deviations given in parentheses.
c Combined groups mean ignored in calculation of overall mean.

SU8Sl~TUTE SHEE~ tRULE 26) CA 022638l9 l999-02-26 W098t08932 PCT~S97/07657 For the data in Table 2, coding regions of the genes were extracted from GenBank (Release 71) entries, and base compositions were calculated using the MacVectorTM program (IBI, New Haven, CT).
Intron sequences were ignored in the calculations. Group I and II
storage protein gene sequences were distinguished by their marked difference in base composition.
Due to the plasticity afforded by the re~lln~Ancy of the genetic code (i.e., some amino acids are specified by more than one codon), evolution of the genomes of different organisms or classes or organisms has resulted in differential usage of redundant codons. This "codon biasn is reflected in the mean base composition of protein coding regions. For example, organisms with relatively low G+C contents utilize codons having A or T in the third position of re~lln~nt codons, whereas those having higher G+C contents utilize codons having G or C in the third position. It is thought that the presence of "minor" codons within a gene's mRNA may reduce the absolute translation rate of that mRNA, especially when the relative abundance of the charged tRNA corresponding to the minor codon is low. An extension of this is that the diminution of translation rate by individual minor codons would be at least additive for multiple minor codons. Therefore, mRNAs having high relative contents of minor codons would have correspondingly low translation rates. This rate would be reflected by the synthesis of low levels of the encoded protein.
In order to reengineer the bacterial gene(s), the codon bias of the plant is determined. The codon bias is the statistical codon distribution that the plant uses for coding its proteins.
After determining the bias, the percent frequency of the codons in the gene(s) of interest is determined. The primary codons preferred by the plant should be determined as well as the second and third choice of preferred codons. The amino acid sequence of the protein of interest is reverse translated so that the resulting nucleic acid sequence codes for the same protein as the native bacterial gene, but the resulting nucleic acid sequence corresponds to the first preferred codons of the desired plant. The new sequence is analyzed for restriction enzyme sites that might have been created by the modification. The identified sites are further modified by replacing the codons with second or third choice preferred codons. Other sites in the sequence which could affect the transcription or translation of the gene of interest are the exon:intron 5' or 3' junctions, poly A addition signals, or RNA
polymerase termination signals. The sequence is further analyzed and modified to reduce the frequency of TA or GC doublets. In SUBSTITlJTE SH EET (RULE 26) CA 022638l9 l999-02-26 W098l08932 PCTrUS97tO7657 addition to the doublets, G or C sequence blocks that have more than about four residues that are the same can affect transcription of the sequence. Therefore, these blocks are also modified by replacing the codons of first or second choice, etc. with the next preferred codon of choice. It is preferred that the plant - optimized gene(s) contains about 63% of first choice codons, between about 22% to about 37% second choice codons, and between 15% and 0% third choice codons, wherein the total percentage is 100%. Most preferred the plant optimized gene(s) contain about 63%
of first choice codons, at least about 22% second choice codons, about 7.5% third choice codons, and about 7.5% fourth choice codons, wherein the total percentage is 100~. The method described above enables one skilled in the art to modify gene(s) that are foreign to a particular plant so that the genes are optimally expressed in plants. The method is further illustrated in application PCT/US96/16582, WO 97/13402 published April 17, 1997.
Thus, in order to design plant optimized gene(s) the amino acid sequence of the toxins are reverse translated into a DNA
sequence, utilizing a nonre~1~n~nt genetic code established from a codon bias table compiled for the gene DNA sequence for the particular plant being transformed. The resulting DNA sequence, which is completely homogeneous in codon usage, is further modified to establish a DNA sequence that, besides having a higher degree of codon diversity, also contains strategically placed restriction enzyme recognition sites, desirable base composition, and a lack of sequences that might interfere with transcription of the gene, or translation of the product mRNA.
It is theorized that bacterial genes may be more easily expressed in plants if the bacterial genes are expressed in the plastids. Thus, it may be possible to express bacterial genes in plants, without optimizing the genes for plant expression, and obtain high express of the protein. See U.S. Patent Nos.
4,762,785; 5,451,513 and 5,545,817, which are incorporated herein by reference.
One of the issues regarding commercial exploiting transgenic plants is resistance management. This is of particular concern with Bacillus thuringiensis toxins. There are numerous companies commerically exploiting Bacillus thuringiensis and there has been much concern about Bt toxins becoming resistant. One strataegy for insect resistant management would be to combine the toxins produced by Photorhabdus with toxins such as Bt, vegetative insect proteins (Ciba Geigy) or other toxins. The combinations could be formulated SUBSTlTl.JTE SHEE r tRULE;~) CA 022638l9 l999-02-26 W098/08932 PCT~US97/07657 for a sprayable application or could be molecular combinations.
Plants could be transformed with Photorhabdus'~genes that produce insect toxins and other insect toxin genes such as Bt as with other insect toxin genes such as Bt.
European Patent Application 0400246Al describes transformation of 2 Bt in a plant, which could be any 2 genes. Another way to produce a transgenic plant that contains more than one insect resistant gene would be to produce two plants, with each plant containing an insect resistant gene. These plants would be backcrossed using traditional plant breeding techniques to produce a plant containing more than one insect resistant gene.
In addition to producing a transformed plant containing plant optimized gene(s), there are other delivery systems where it may be desirable to reengineer the bacterial gene(s). Along the same lines, a genetically engineered, easily isolated protein toxin fusing together both a molecule attractive to insects as a food source and the insecticidal activity of the toxin may be engineered and expressed in bacteria or in eukaryotic cells using standard, well-known techniques. After purificatlon in the laboratory such a toxic agent with "built-in" bait could be packaged inside standard insect trap housings.
Another delivery scheme is the incorporation of the genetic material of toxins into a baculovirus vector. Baculoviruses infect particular insect hosts, including those desirably targeted with the Photorhabdus toxins. Infectious baculovirus harboring an expression construct for the Photorhabdus toxins could be introduced into areas of insect infestation to thereby intoxicate or poison infected insects.
Transfer of the insecticidal properties requires nucleic acid sequences encoding the coding the amino acid sequences for the Photorhabdus toxins integrated into a protein expression vector appropriate to the host in which the vector will reside. One way to obtain a nucleic acid sequence encoding a protein with insecticidal properties is to isolate the native genetic material which produces the toxins from Photorhabdus, using information deduced from the toxin's amino acid sequence, large portions of which are set forth below. As described below, methods of purifying the proteins responsible for toxin activity are also disclosed.
Using N-terminal amino acid sequence data, such as set forth below, one can construct oligonucleotides complementary to all, or a section of, the DNA bases that encode the first amino acids of the toxin. These oligonucleotides can be radiolabeled and used as SUBSTITUTE SHEET ~RULE 26) CA 022638l9 l999-02-26 W098/08932 PCTrUS97/07657 molecular probes to isolate the genetic material from a genomic genetic library built from genetic material isolated from strains of Photorhabdus. The genetic library can be cloned in plasmid, cosmid, phage or phagemid vectors. The library could be transformed into Escherichia coli and screened for toxin production - by the transformed cells using antibodies raised against the toxin or direct assays for insect toxicity.
This approach requires the production of a battery of oligonucleotides, since the degenerate genetic code allows an amino acid to be encoded in the DNA by any of several three-nucleotide combinations. For example, the amino acid arginine can be encoded by nucleic acid triplets CGA, CGC, CGG, CGT, AGA, and AGG. Since one cannot predict which triplet is used at those positions in the toxin gene, one must prepare oligonucleotides with each potential triplet represented. More than one DNA molecule corresponding to a protein subunit may be necessary to construct a sufficient number of oligonucleotide probes to recover all of the protein subunits necessary to achieve oral toxicity.
From the amino acid sequence of the purified protein, genetic materials responsible for the production of toxins can readily be isolated and cloned, in whole or in part, into an expression vector using any of several techniques well-known to one skilled in the art of molecular biology. A typical expression vector is a DNA
plasmid, though other transfer means including, but not limited to, cosmids, phagemids and phage are also envisioned. In addition to features required or desired for plasmid replication, such as an origin of replication and antibiotic resistance or other form of a selectable marker such as the bar gene of Streptomyces hygroscopicus or viridochromogenes, protein expression vectors norm~ d~tionally require an expression cassette which incorporates the cis-acting sequences necessary 'f~ transcription and translation of the gene of interest. The cis-acting sequences required for expression in prokaryotes differ from those required in eukaryotes and plants.
A eukaryotic expression cassette requires a transcriptional promoter upstream (5') to the gene of interest, a transcriptional termination region such as a poly-A addition site, and a ribosome binding site upstream of the gene of interest's first codon. In bacterial cells, a useful transcriptional promoter that could be included in the vector is the T7 RNA Polymerase-binding promoter.
Promoters, as previously described herein, are known to efficiently promote transcription of mRNA. Also upstream from the gene of interest the vector may include a nucleotide sequence encoding a SUBSTITUTE St~EET (RULE 26) CA 022638l9 l999-02-26 W098t08932 PCTAUS97/07657 signal sequence known to direct a covalently linked protein to a particular compartment of the host cells such as the cell surface.
Insect viruses, or baculoviruses, are known to infect and adversely affect certain insects. The affect of the viruses on insects is slow, and viruses do not stop the feeding of insects.
Thus viruses are not viewed as being useful as insect pest control agents. Combining the Photorhabdus toxins genes into a baculovirus vector could provide an efficient way of transmitting the toxins while increasing the lethality of the virus. In addition, since different baculoviruses are specific to different insects, it may be possible to use a particular toxin to selectively target particularly damaging lnsect pests. A particularly useful vector for the toxins genes is the nuclear-polyhedrosis virus. Transfer vectors using this virus have been described and are now the vectors of choice for transferring foreign genes into insects. The virus-toxin gene recombinant may be constructed in an orally transmissible form. Baculoviruses normally infect insect victims through the mid-gut intestinal mucosa. The toxin gene inserted behind a strong viral coat protein promoter would be expressed and should rapidly kill the infected insect.
In addition to an insect virus or baculovirus or transgenic plant delivery system for the protein toxins of the present invention, the proteins may be encapsulated using Bacillus thuringiensis encapsulation technology such as but not limlted to 25 U.S. Patent Nos. 4,695,455; 4,695,462; 4,861,595 which are all incorporated herein by reference. Another dellvery system for the protein toxins of the present invention is formulation of the protein into a bait matrix, which could then be used in above and below ground insect bait stations. Examples of such technology 30 include but are not limited to PCT Patent Application WO 93/23998, which i9 incorporated herein by reference.
As is described above, it might become necessary to modify the sequence encoding the protein when expressing it in a non-native host, since the codon preferences of other hosts may differ from that of Photorhabdus. In such a case, translation may be quite inefficient in a new host unless compensating modifications to the coding sequence are made. Additionally, modifications to the amino acid sequence might be desirable to avoid inhibitory cross-reactivity with proteins of the new host, or to refine the insecticidal properties of the protein in the new host. A
genetically modified toxin gene might encode a toxin exhibiting, for example, enhanced or reduced toxicity, altered insect SUBST~TUTE SH EET (RULE 2~) CA 022638l9 l999-02-26 W098/~g32 PCT~S97tO7657 resistance development, altered stability, or modified target species specificity.
In addition to the Photorhabdus genes encoding the toxins, the scope of the present invention is intended to include related nucleic acid sequences which encode amino acid biopolymers - homologous to the toxin proteins and which retain the toxic effect of the Photorhabdus proteins in insect species after oral ingestion.
For instance, the toxins used in the present invention seem to first inhibit larval feeding before death ensues. By manipulating the nucleic acid sequence of Photorhabdus toxins or its controlling sequences, genetic engineers placing the toxin gene into plants could modulate its potency or its mode of action to, for example, keep the eating-inhibitory activity while eliminating the absolute toxicity to the larvae. This change could permit the transformed plant to survive until harvest without having the unnecessarily dramatic effect on the ecosystem of wiping out all target lnsects.
All such modifications of the gene encoding the toxin, or of the protein encoded by the gene, are envisioned to fall within the scope of the present invention.
Other envisioned modifications of the nucleic acid include the addition of targeting sequences to direct the toxin to particular parts of the insect larvae for improving its efficiency.
Strains W-14, ATCC 55397, 43948, 43949, 43950, 43951, 43952 have been deposited in the American Type Culture Collection, 12301 Parklawn Drive, Rockville, MD 20852 USA. Amino acid and nucleotide sequence data for the W-14 native toxin (ATCC 55397) is presented below. Isolation of the genomic DNA for the toxins from the bacterial hosts is also exemplified herein. The other strains identified herein have been deposited with the United States Department of Agriculture, 1815 North Uni~ersity Drive, Peoria, IL
61604.
Standard and molecular biology techniques were followed and taught in the specification herein. Additional information may be found in Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989), Molecular Cloning. A T~horatory M~nual, Cold Spring ~arbor Press;
Cnrrent Protocalsin Molecular Biology, ed. F. M. Ausubel et al., (1997), which are both incorporated herein by reference.
-The following abbreviations are used throughout the Examples: Tris= tris (hydroxymethyl) amino methane; SDS = sodium dodecyl sulfate;
EDTA = ethylenediaminetetraacetic acid, IPTG = isopropylthio-B-galactoside, X-gal = 5-bromo-4-chloro-3-indoyl-B-D-galactoside, SUBSTITUTE SHEET (RULE 26) . ~

CA 022638l9 l999-02-26 W098/08932 PCT~US97/07657 CTAB = cetyltrimethyl~mmQ~ium bromide; kbp = kilobase pairs; dATP, dCTP, dGTP, dTTP, I = 2'-deoxynucleoside 5'-triphosphates of adenine, cytosine, guanine, thymine, and inosine, respectively; ATP
= adenosine 5' triphosphate.

E~ le 1 Purification of Toxin from Photorhabdus luminescens ~n~
Demonstration of Toxlcity after Oral Delivery of Purified Toxi~

The insecticidal protein toxin of the present invention was purified from Photorhabdus luminescens strain W-14, ATCC Accession Number 55397. Stock cultures of Photorhabdus luminescens were maintained on petri dishes cont~in'ng 2% Proteose Peptone No. 3 (i.e., PP3, Difco Laboratories, Detroit MI) in 1.5~ agar, incubated at 25~C and transferred weekly. Colonies of the primary form of the bacteria were inoculated into 200 ml of PP3 broth supplemented with 0.5~ polyoxyethylene sorbitan mono-stearate (Tween 60, Sigma Chemical Company, St. Louis, MO~ in a one liter flask. The broth cultures were grown for 72 hours at 30~C on a rotary shaker. The toxin proteins can be recovered from cultures grown in the presence or absence of Tween; however, the absence of Tween can affect the form of the bacteria grown and the profile of proteins produced by the bacteria. In the absence of Tween, a variant shift occurs insofar as the molecular weight of at least one identified toxin 25 subunit shifts from about 200 ~Da to about 185 kDa.
The 72 hour cultures were centrifuged at 10,000 x g for 30 minutes to remove cells and debris. The supernatant fraction that contained the insecticidal activity was decanted and brought to 50 mM K2HPO4 by adding an appropriate volume of 1.0 M K2HPO4. The pH
was-a~juste~ to 8.6 by adding potassium hydroxide. This supernatant fraction was then mixed with DEAE-Se~ha~1 (Pharmacia LKB Biotechnology) which had been equilibrated with 50 mM K2HPO4.
The toxic activity was adsorbed to the DEAE resin. This mixture was then poured into a 2.6 x 40 cm column and washed with 50 mM
K2HPO4 at room temperature at a flow rate of 30 ml/hr until the effluent reached a steady baseline W absorbance at 280 nm. The column was then washed with 150 mM KCl until the effluent again reached a steady 280 nm baseline. Finally the column was washed with 300 mM KCl and fractions were collected.
Fractions containing the toxin were pooled and filter sterilized using a 0.2 micron pore membrane filter. The toxin was then concentrated and equilibrated to 100 mM KPO4, pH 6.9, using an ultrafiltration membrane with a molecular weight cutoff of 100 kDa SUBSTITUTE SHEE~ (~ULE 26) CA 022638l9 l999-02-26 W098/08932 PCT~US97/07657 at 4~C (Centriprep 100, Amicon Division-W.R. Grace and Company). A

3 ml sample of the toxin concentrate was applied to the top of a 2.6 x 95 cm Sephacryl S-400 HR gel filtration column (Pharmacia LKB
Biotechnology). The eluent buffer was 100 mM KPO4, pH 6.9, which was run at a flow rate of 17 ml/hr, at 4~C. The effluent was ~ monitored at 280 nm.
Fractions were collected and tested for toxic activity.-Toxicity of chromatographic fractions was examined in a biological assay using Manduca sexta larvae. Fractions were either applied directly onto the insect diet (Gypsy moth wheat germ diet, ICN
Biochemicals Division - ICN Biomedicals, Inc.) or administered by intrahemocelic injection of a 5 ~l sample through the first proleg of 4th or 5th instar larva using a 30 gauge needle. The weight of each larva within a treatment group was recorded at 24 hour intervals. Toxicity was presumed if the lnsect ceased feeding and died within several days of consuming treated insect diet or if death occurred within 24 hours after injection of a fraction.
The toxic fractions were pooled and concentrated using the Centriprep-100 and were then analyzed by HP~C using a 7.5 mm x 60 cm TSK-GEL G-4000 SW gel permeation column with lO0 mM potassium phosphate, pH 6.9 eluent buffer running at 0.4 ml/min. This analysis revealed the toxin protein to be contained within a single sharp peak that eluted from the column with a retention time of approximately 33.6 minutes. This retention time corresponded to an estimated molecular weight of 1,000 kDa. Peak fractions were collected for further purification while fractions not containing this protein were discarded. The peak eluted from the HPLC absorbs W light at 218 and 280 nm but did not absorb at 405 nm.
Absorbance at 405 nm was shown to be an attribute of xenorhabdin antibiotic compounds.
Electrophoresis of the pooled peak fractions in a non-denaturing agarose gel (Metaphor Agarose, FMC BioProducts) showed that two protein complexes are present in the peak. The peak material, buffered in 50 mM Tris-HCl, pH 7.0, was separated on a 1.5% agarose stacking gel buffered with 100 mM Tris-HCl at pH 7.0 and 1.9% agarose resolving gel buffered with 200 mM Tris-borate at pH 8.3 under standard buffer conditions (anode buffer lM Tris-HCl, pH 8.3; cathode buffer 0.025 M Tris, 0.192 M glycine). The gels were run at 13 mA constant current at 15~C until the phenol red tracking dye reached the end of the gel. Two protein bands were visualized in the agarose gels using Coomassie brilliant blue staining.

SUBSTITUTE SHFF~ (RULE 26) .... _.. A__ ... .~ _ WO98108932 PCTrUS97/07657 The slower migrating band was referred to as "protein band 1"
and faster migrating band was referred to as "protein band 2." The two protein bands were present in approximately equal amounts. The Coomassie stained agarose gels were used as a guide to precisely excise the two protein bands from unstained portions of the gels.
The excised pieces containing the protein bands were macerated and a small amount of sterile water was added. As a control, a portion of the gel that contained no protein was also excised and treated in the same manner as the gel pieces cont~'ning the protein.
Protein was recovered from the gel pieces by electroelution into lO0 mM Tris-borate pH 8.3, at 100 volts (constant voltage) for two hours. Alternatively, protein was passively eluted from the gel pieces by adding an equal volume of 50 mM Tris-HCl, pH 7.0, to the gel pieces, then incubating at 30~C for 16 hours. This allowed the protein to diffuse from the gel into the buffer, which was then collected.
Results of insect toxicity tests using HPLC-purified toxin (33.6 min. peak) and agarose gel purified toxin demonstrated toxicity of the extracts. Injection of 1.5 ~g of the HPLC purified protein kills within 24 hours. Both protein bands l and 2, recovered from agarose gels by passive elution or electroelution, were lethal upon injection. The protein concentration estimated for these samples was less than 50 ng/larva. A comparison of the weight gain and the mortality between the groups of larvae injected with protein bands 1 or 2 indicate that protein band 1 was more toxic by injection delivery.
When HPLC-purified toxin was applied to larval diet at a concentration of 7.5 ~g/larva, it caused a halt in larval weight gain (24 larvae tested). The larvae begin to feed, but after consuming only a very small portion of the toxin treated diet they began to show pathological symptoms induced by the toxin and the larvae cease feeding. The insect frass became discolored and most larva showed signs of diarrhea. Significant insect mortality resulted when several 5 ~g toxin doses were applied to the diet over a 7-10 day period.
Agarose-separated protein band l significantly inhibited larval weight gain at a dose of 200 ng/larva. Larvae fed similar concentrations of protein band 2 were not inhibited and gained weight at the same rate as the control larvae. Twelve larvae_were fed eluted protein and 45 larvae were fed protein-containing agarose pieces. These two sets of data indicate that protein band 1 was orally toxic to Manduca sexta. In this experiment it appeared that protein band 2 was not toxic to Manduca sexta.

SUBSTITUTE SH EET tRULE 2~) CA 022638l9 l999-02-26 W098/08932 PCTrUS97/07657 Further analysis of protein bands 1 and 2 by SDS-PAGE under denaturlng conditions showed that each band was composed of several smaller protein su~units. Proteins were visualized by Coomassie brilliant blue staining followed by silver stainlng to achieve maximum sensitivity.
- The protein subunits in the two bands were very similar.
Protein band l contains 8 protein subunits of 25.1, 56.2, 60.8, 65.6, 166, 171, 184 and 208 kDa. Protein band 2 had an identical profile except that the 2~.1, 60.8, and 65.6 kDa proteins were not 10 present. The 56.2, 60.8, 65.6, and la4 kDa proteins were present in the complex of protein band 1 at approximately equal concentrations and represent 80% or more of the total protein content of that complex.
The native HPLC-purified toxin was further characterized as follows. The toxin was heat labile in that after being heated to 60~C for 15 minutes it lost its ability to kill or to inhibit weight gain when injected or fed to Manduca sexta larvae. Assays were designed to detect lipase, type C phospholipase, nuclease or red blood cell hemolysis activities and were performed with purified toxin. None of these activities were present. Antibiotic zone inhibition assays were also done and the purified toxin failed to inhibit growth of Gram-negative or -positive bacteria, yeast or filamentous fungi, indicating that the toxic is not a xenorhabdin antibiotic.
The native HP~C-purified toxin was tested for ability to kill insects other than Manduca sexta. Table 3 lists insects killed by the HP~C-purified Photorhabdus luminescens toxin in this study.

Table 3 ~n~ects Killed by Photorh~hdus luminescens Toxin Genus and Route of Common N~me Order species Delivery 35 Tobacco Lepidoptera Manduca sexta Oral and horn worm injected Mealworm Coleoptera Tenebrio moli tor Oral 40 Pharaoh ant Hymenoptera Monomorium pharoanis Oral German Dictyoptera Blattella germanica Oral and cockroach injected 45 Mosquito Diptera Aedes aegypti Oral SUE~STmlTE 5H EET ~RULE 26) .. . .

Fl~rther (~h~racterization of the H;gh Molecul~r Weiqht Toxin Complex In yet further analysis, the toxin protein complex was subjected to further characterization from W-14 growth medium. The culture conditions and initial purification steps through the S-400 5 HR column were identical to those described above. After isolation of the high molecular weight toxin complex from the S-400 HR column fractions, the toxic fractions were equillbrated with 10 ml~ Tris-HCl, pH 8.6, and concentrated in the centriplus 100 (Amicon) concentrators. The protein toxin complex was then applied to a weak 10 anion exchange (WAX) column, Vydac 301VPH575 (Hesparia, CA), at a flow rate of 0.5 ml/min. The proteins were eluted with a linear potassium chloride gradient, 0-250 mM KCl, in 10 mM Tris-HCl pH 8.6 for 50 min. Eight protein peaks were detected by absorbance at 280 nm.
Bioassays using neonate southern corn rootworm (Diabrotica undecimpunctata howardi, SCR) larvae and tobacco horn worm (Manduca sexta, THW) were performed on all fractions eluted from the HPLC
column. THW were grown on Gypsy Moth wheat germ diet (ICN) at 25~C
with a 16 hr light 8 hr dark cycle. SCR were grown on Southern Corn 20 Rootworm Larval Insecta-Diet (BioServ) at 25~C with a 16 hr light /
8 hr dark cycle.
The highest mortality for SCR and THW larvae was observed for peak 6, which eluted with ca. 112 mM to 132mM KCl. SDS-PAGE
analysis of peak 6 showed predominant peptides of 170 kDa, 66 kDa, 25 63 kDa, 59.5 kDa and 31 kDa. Western blot analysis was performed on peak 6 protein fraction with a mixture of polyclonal antibodies made against TcaAii-syn, TcaAiii-syn, TcaBii-syn, TcaC-syn, and TcbAii-syn peptides (described in Example 21) and C5F2, a monoclonal antibody against the TcbAiii peptide. Peak 6 contained immuno-reactive bands 30 of 170 kDa, 90 kDa, 66 kDa, 59.5 kDa and 31 kDa. These are very close to the predicted sizes for the TcaC (166 kDa), TcaAii+ TcaA
(92 kDa), TcaAiii (66 kDa), TcaBii (60 kDa) and TcaAii (25 kDa), respectively. Peak 6 which was further analyzed by native agarose gel electrophoresis, as described herein, migrated as a single band 35 with similar mobility to that of band 1.
The protein concentration of the purified peak 6 toxin protein was determined using the BCA reagents (Pierce). Dilutions of the protein were made in 10 mM Tris, pH 8.6 and applied to the diet bioassays. After 240 hours all neonate larvae on diet bioassays 40 that received 450 ng or greater of the peak 6 protein fraction were dead. The group of larvae that received 90 ng of the same fraction SUBSTITUTE SH EET (RULE 26) W098/08932 PCT~US97/07657 had 40% mortality. After 240 hrs the survivors that received 90 ng and 20 ng of peak 6 protein fraction were ca.~~l0~ and 70~, respectively, of the control weight.

~m~le ~
Insecticide Utility The Photorh~h~r~ luminescens utility and toxicity were further characterized. Photorhabdus luminescens (strain W-14) culture broth was produced as follows. The production medium was 2~ Bacto Proteose Peptone Number 3 (PP3, Difco Laboratories, Detroit, Michigan) in Milli-Q deionized water. Seed culture flasks consisted of 175 ml medium placed in a 500 ml tribaffled flask with a Delong neck, covered with a Kaput and autoclaved for 20 minutes, T=250~F. Production flasks consisted of 500 mls in a 2.8 liter 500 ml tribaffled flask with a Delong neck, covered by a Shin-etsu silicon foam closure. These were autoclaved for 45 minutes, T=250 F. The seed culture was incubated at 28 C at 150 rpm in a gyrotory shaking incubator with a 2 inch throw. After 16 hours of growth, 1% of the seed culture was placed in the production flask which was allowed to grow for 24 hours before harvest. Production of the toxin appears to be during log phase growth. The microbial broth was transferred to a lL centrifuge bottle and the cellular biomass was pelleted l30 minutes at 2500 RPM at 4 C, [R.C.F. = about 1600] HG-4L Rotor RC3 Sorval centrifuge, Dupont, Wilmington, DE~.
The primary broth was chilled at 4~C for 8 - 16 hours and recentrifuged at least 2 hours (conditions above) to further clarify the broth by removal of a putative mucopolysaccharide which precipitated upon standing. (An alternative processing method-combined both steps and involved the use of a 16 hour clarification centrifugation, same conditions as above.~ This broth was then stored at 4 C prior to bioassay or filtration.
Photorhabdus culture broth and protein toxin(s) purified from this broth showed activity (mortality and/or growth inhibition, reduced adult emergence) against a number of insects. More specifically, the activity is seen against corn rootworm (larvae and adult), Colorado potato beetle, and turf grubs, which are members of the insect order Coleoptera. Other members of the - Coleoptera include wireworms, pollen beetles, flea beetles, seed beetles and weevils. Activity has also been observed against aster leafhopper, which is a member of the order, Homoptera. Other members of the Homoptera include planthoppers, pear pyslla, apple SUBSTITUTE S~EET (RULE 26) . -- . ~ .

sucker, scale insects, whiteflies, a~d spittle bugs, as well as numerous host specific aphid species. The broth and purified fractions are also active against beet armyworm, cabbage looper, black cutworm, tobacco budworm, European corn borer, corn earworm, and codling moth, which are members of the order Lepidoptera.
Other typical members of this order are clothes moth, Indian mealmoth, leaf rollers, cabbage worm, cotton bollworm bagworm, Eastern tent cate~pillar, sod webworm, and fall armyworm. Activity is also seen against fruitfly and mosquito larvae, which are members of the order Diptera. Other members of the order Diptera are pea midge, carrot fly, cabbage root fly, turnip root fly, onion fly, crane fly, house fly, and various mosquito species. Activity is seen against carpenter ant and Argentine ant, which are members of the order that also includes fire ants, oderous house ants, and little black ants.
The broth/fraction is useful for reducing populations of insects and were used in a method of inhibiting an insect population. The method may comprise applying to a locus of the insect an effective insect inactivating amount of the active described. Results are reported in Table 4.
Activity against corn rootworm larvae was tested as follows.
Photorhabdus culture broth (filter sterilized, cell-free) or purified HPLC fractions were applied directly to the surface ~about 1.5 cm2) of 0.2~ ml of artificial diet in 30 ~l aliquots following dilution in control medium or l0 mM sodium phosphate buffer, pH
7.0, respectively. The diet plates were allowed to air-dry in a sterile flow-hood and the wells were infested with single, neonate Diabrotica undecimpunctata howardi (Southern corn rootworm, SCR) hatched from sterilized eggs, with second instar SCR grown on artificial diet or with second instar Diabrotica virgifera virgifera (Western corn rootworm, WCR) reared on corn seedlings grown in Metromix . Second instar larvae were weighed prior to addition to the diet. The plates were sealed, placed in a humidified growth chamber and maintained at 27~C for the appropriate period (4 days for neonate and adult SCR, 2-5 days for WCR larvae, 7-14 days for second instar SCR). Mortality and weight determinations were scored as indicated. Generally, 16 insects per treatment were used in all studies. Control mortalities were as follows: neonate larvae, ~5~, adult beetles, 5~.
Activity against Colorado potato beetle was tested as follows Photorhabdus culture broth or control medium was applied to the surface (about 2.0 cm2) of l.5 ml of standard artificial diet held in the wells of a 24-well tissue culture plate. Each well received SUBSTITUTE S}~EET (RULE 26) W098/08932 PCT~US97/07657 50 ~1 of treatment and was allowed to air dry. Individual second instar Colorado potato beetle (Leptinotarsa decemlineata, CPB) larvae were then placed onto the diet and mortality was scored after 4 days. Ten larvae per treatment were used in all studies.
Control mortality was 3.3%.
Activity against Japanese beetle grubs and beetles was tested as follows. Turf grubs (Popillia japonica, 2-3rd instar) were collected from infested lawns and maintained in the laboratory in soil/peat mixture with carrot slices added as additional diet.
Turf beetles were pheromone-trapped locally and maintained in the laboratory in plastic containers with maple leaves as food.
Following application of undiluted Photorhabdus culture broth or control medium to corn rootworm artificial diet (30 ~1/1.54 cm2, beetles) or carrot slices (larvae), both stages were placed singly in a diet well and observed for any mortality and feeding. In both cases there was a clear reduction in the amount of feeding (and feces production) observed.
Activity against mosquito larvae was tested as follows. The assay was conducted in a 96-well microtiter plate. Each well contained 200 ~1 of aqueous solution (Photorhabdus culture broth, control medium or H20) and approximately 20, 1-day old larvae (Aedes aegypti). There were 6 wells per treatment. The results were read at 2 hours after infestation and did not change over the three day observation period. No control mortality was seen.
Activity against fruitflies was tested as follows. Purchased Drosophila melanogaster medium was prepared using 50% dry medium and a 50% liquid of either water, control medium or Photorhabdus culture broth. This was accomplished by placing 8.0 ml of dry medium in each of 3 rearing vials per treatment and adding 8.0 ml of the appropriate liquid. Ten late instar Drosophila melanogaster maggots were then added to each vial. The vials were held on a laboratory bench, at room temperature, under fluorescent ceiling lights. Pupal or adult counts were made after 3, 7 and 10 days of exposure. Incorporation of Photorhabdus culture broth into the diet media for frultfly maggots caused a slight (17%) but significant reduction in day-10 adult emergence as compared to water and control medium (3% reduction).
Activity against aster leafhopper was tested as follows. The ingestion assay for aster leafhopper (Macrosteles severini) is ~0 designed to allow ingestion of the active without other external contact. The reservoir for the active/"food" solution is made by making 2 holes in the center of the bottom portion of a 35 x 10 mm Petri dish. A 2 inch Parafilm M square is placed across the top of SUBST~TUTE S~EET (RULE-26) CA 022638l9 l999-02-26 the dish and secured with an "O" ring. A l oz. plastic cup is then infested with approximately 7 leafhoppers and--the reservoir is placed on top of the cup, Parafilm down. The test solution is then added to the reservoir through the holes. In tests using undiluted Photorhabdus culture broth, the broth and control medium were dialyzed against water to reduce control mortality. Mortality is reported at day 2 where 26.5% control mortality was seen. In the tests using purified fractions (200 mg protein/ml) a final concentration of 5% sucrose was used in all treatments to improve survivability of the aster leafhoppers. The assay was held in an incubator at 28~C, 70% RH with a 16/8 photoperiod. The assay was graded for mortality at 72 hours. Control mortality was 5.5%.
Activity against Argentine ants was tested as follows. A 1.5 ml aliquot of~lO0% Photorhabdus culture broth, control medium or water was pipetted into 2.0 ml clear glass vials. The vials were plugged with a piece of cotton dental wick that was moistened with the appropriate treatment. Each vial was placed into a separate 60xl6mm Petri dish with 8 to 12 adult Argentine ants (Linepithema humile). There were three replicates per treatment. Bioassay plates were held on a laboratory bench, at room temperature under fluorescent ceiling lights. Mortality readings were made after 5 days of exposure. Control mortality was 24%.
Activity against carpenter ant was tested as follows. Black carpenter ant workers ( Camponotus pennsylvanicus) were collected from trees on DowElanco property in Indianapolis, IN. Tests with Photorhabdus culture broth were performed as follows. Each plastic bioassay container (7 1/8" x 3") held fifteen workers, a paper harborage and 10 ml of broth or control media in a plastic shot glass. A cotton wick delivered the treatment to the ants through a hole in the shot glass lid. All treatments contained 5% sucrose.
Bioassays were held in the dark at room temperature and graded at 19 days. Control mortality was 9%. Assays delivering purified fractions utilized artificial ant diet mixed with the treatment (purified fraction or control solution) at a rate of 0.2 ml treatment/2.0 g diet in a plastic test tube. The final protein concentration of the purified fraction was less than 10 ~g/g diet.
Ten ants per treatment, a water source, harborage and the treated diet were placed in sealed plastic containers and maintained in the dark at 27~C in a humidified incubator. Mortality was scored at day 10. No control mortality was seen.
Activity against various lepidopteran larvae was tested as follows. Photorhabdus culture broth or purified fractions were SUBSTrrUTE SHEET tRULE 26) wo g8/08932 rCT/US97tO7657 applied directly to the surface (abo.ut 1.5 cm2) of 0.25 ml of standard artificial diet in 30 ~1 aliquots following dilution in control medium or 10 mM sodium phosphate buffer, pH 1.0, respectively. The diet plates were allowed to air-dry in a sterile flow-hood and the wells were infested with single, neonate larva.
European corn borer (Ostrinia nubilalis) and corn earworm (Helicoverpa zea) eggs were supplied from commercial sources and hatched in-house, whereas beet armyworm (Spodoptera exigua), cabbage looper (Trichoplusia ni), tobacco budworm (~eliothis 0 virescens), codling moth (Laspeyresia pomonella) and black cutworm (Agrotis ipsilon) larvae were supplied internally. Following infestation with larvae, the diet plates were sealed, placed in a humidified growth chamber and maintained in the dark at 27~C for the appropriate period. Mortality and weight determinations were scored at days 5-7 for Photorhabdus culture bro~h and days 4-7 for the purified fraction. Generally, 16 insects per treatment were used in all studies. Control mortality ranged from 4-12.5~ for control medium and was less than 10~ for phosphate buffer.

SUBSTITUTE St~ EET tRULE 26) CA 022638l9 l999-02-26 W09~08932 PCTAUS97~7657 Table.4 ~ffect of Photorhabdus l~l~inescens (Straln W-14) Cultl~re Broth and Purified Toxin Fraction on Mortality and Growth Inhibition of Different Insect Orders/Species lnsect ~rder/~pecles~roth ~url~led ~raCtlOn Mort. ~ Mort.
~:ULI!;O~ ~'h:.KA
Corn Rootworm Southern/neonate larva 100 na 100 na Southern/2nd instar na 38.5 nt nt Southern/adult 45 nt nt nt Western/2nd instar na 35 nt nt Colorado Potato Beetle 93 nt nt nt 2nd instar Turf Grub na a.f. nt nt 3rd instar na a.f. nt nt adult VI~ ~
Fruit Fly (adult emergence) 17 nt nt nt Mosquito larvae 100 na nt nt n~u~KA
Aster Leafhopper 96.5 na 100 na HY__~;NO~ l ~ tA
Argentine Ant 75 na nt na Carpenter Ant 71 na 100 na L h ~ IvO~l~A
Beet Armyworm 12.5 36 18.75 41.4 Black Cutworm nt nt 0 71.2 Cab~age Looper nt nt 21.9 66.8 Codling Moth nt nt 6.25 45.9 Corn Earworm 56.3 94.2 97.9 na European Corn Borer 96.7 98.4 100 na Tobacco Budworm 13.5 52.5 19.4 85.6 Mort. = mortallty, ~.l. = g~owth lnhl~ltlon, na = not applicable, nt = not tested, a.f. = anti-feedant Example 3 Insecticide Utility upon Soil Application Photorhabdus luminescens (strain W-14) culture broth was shown to be actlve against corn rootworm when applied directly to soil or a soil-mix (Metromix ). Activity against neonate SCR and WCR in SU~STITUTE St~ EET (RULEi~6~

CA 022638l9 l999-02-26 W098/08932 PCTrUS97/07657 Metromix was tested as follows (Table 5). The test was run using corn seedlings (United Agriseeds brand CL614) that were germinated in the llght on moist filter paper for 6 days. After roots were approximately 3-6 cm long, a single kernel/seedling was planted in a 591 ml clear plastlc cup with 50 gm of dry Metromix . Twenty - neonate SCR or WCR were then placed directly on the roots of the seedling and covered with Metromix . Upon infestation, the seedlings were then drenched with 50 ml total volume of a diluted broth solution. After drenching, the cups were sealed and left at room temperature in the light for 7 days. Afterwards, the seedlings were washed to remove all Metromix and the roots were excised and weighed- Activity was rated as the percentage of corn root rem~ining relative to the control plants and as leaf damage induced by feeding. Leaf damage was scored visually and rated as either -, +, ++, or +++, with - representing no damage and +++
representing severe damage.
Activity against neonate SCR in soil was tested as follows (Table 6). The test was run using corn seedlings (United Agriseeds brand CL614) that were germinated in the light on moist filter paper for 6 days. After the roots were approximately 3-6 cm long, a slngle kernel/seedling was planted in a 591 ml clear plastic cup with 150 gm of soil from a field in Lebanon, IN planted the previous year with corn. This soil had not been previouslv treated with insecticides. Twenty neonate SCR were then placed directly on the roots of the se~dling and covered with soil. After infestation, the seedlings were drenched with 50 ml total volume of a diluted broth solution. After drenching, the unsealed cups were incubated in a high relative humidity chamber (80%) at 78~F.
Afterwards, the seedlings were washed to remove all soil and the roots ~ere e~'cised and weighed. Activity was rated as the percentage of corn root remaining relative to the co~mC~ol plants and as leaf damage induced by feeding. Leaf damage was scored visually and rated as either -, +, ++, or +++, with - representing no damage and +++ representing severe damage.

SUBSTITUTE SHEE~ (RULE 26) CA 022638l9 l999-02-26 W098/08932 PCTrUS97/07657 T~hle.5 Effect of Photorh~hdus luminescens (Strain W-14) Culture Broth on Rootworm Larvae after Post-Infestation Drenching (Metromix ) Treatment Larvae Leaf Damage Root Weight (g) %
Southern Corn Rootworm Water - - 0.4916 + 0.023 100 Medium (2.0% v/v) - - 0.4416 + 0.029 100 10 Broth (6.25%v/v) - - 0.4641 + 0.081 100 Water + +++ 0.1410 + 0.00628.7 Media (2.0% v/v) ++++ 0.1345 + 0.02830.4 15 Broth (1.56% v/v) + - 0.4830 + 0.031 104 Western Corn Rootworm Water - - 0.4446 + 0.019 100 Broth (2.0% v/v) - - 0.4069 + 0.026 100 Water + - 0.2202 + 0.015 49 Broth (2.0% v/v) + - 0.3879 + 0.013 95 Table 6 Effect of Photorhabdus luminescens (Strain W-14) Culture Broth on Southern ~orn Rootworm Larvae after Post-Infestation Drenching (Soil) 30 Treatment Larvae Leaf Damage Root Weight(g~ %
Water - - 0.214~ + 0.014 100 Broth (50% v/v) - - 0.2260 + 0.016 103 35 Water + +++ 0.0916 + 0.009 43 Broth (50% v/v) + - 0.2428 + 0.032 113 Activity of Photorhabdus luminescens (strain W-14) culture broth against second instar turf grubs in Metromix was observed in tests conducted as follows (Table 7). Approximately 50 gm of dry Metromix was added to a 591 ml clear plastic cup. The Metromix was then drenched with 50 ml total volume of a 50% (v/v) diluted Photorhabdus broth solution. The dilution of crude broth was made with water, with 50% broth being prepared by adding 25 ml of crude 45 broth to 25 ml of water for 50 ml total volume. A 1% (w/v) solution of proteose peptone #3 (PP3), which is a 50% dilution of the normal media concentration, was used as a broth control. After drenching, five second instar turf grubs were placed on the top of the moistened Metromix . ~ealthy turf grub larvae burrowed rapidly into the Metromix . Those larvae that did not burrow within lh were SUBSTITUTE St~EET (RULE 26) CA 022638l9 l999-02-26 removed and replaced with fresh larvae. The cups were sealed and placed in a 28~C incubator, in the dark. After seven days, larvae were removed from the Metromix and scored for mortality. Activity was rated the percentage of mortality relative to control.

Table 7 Effect of Photorh~hdus 1l7~inescens tStrain W-14) Culture ~roth on Turf Grub after Pre-Infestation Drenching (Metromix) lO Treatment Mortality* Mortality %
Water 7/15 47 Control medium 15 (1.0% w/v) 12/19 63 Broth (50% v/v) 17/20 85 *expressed as a ratio of dead/living larvae Example 4 Insecticide Utility upon Leaf Application Activity of Photorhabdus broth against European corn borer was seen when the broth was applied directly to the surface of maize leaves (Table 8~. In these assays Photorhabdus broth was diluted 100-fold with culture medium and applied manually to the surface of excised maize leaves at a rate of about 6.0 ~l/cm2 of leaf surface.
The leaves were air dried and cut into equal slzed strips approximately 2 x 2 inches. The leaves were rolled, secured with paper clips and placed in 1 oz plastic shot glasses with 0.25 inch of 2% agar on the bottom surface to provide moisture. Twelve neonate European corn borers were then placed onto the rolled leaf and the cup was sealed. After incubation for 5 days at 27~C in the dark, the samples were scored for feeding damage and recovered larvae.

SUBSTITUTE Si~FF~ tRULE 26) Table 8 Effect of Photorhabdus luminescens (Strain W-14) Culture Broth on European Corn Borer T,~rvae Following Pre-Infestatlon Application to Excised Maize Leaves Treatment Leaf Damage Larvae Recovered Weight(mg) Water Extensive 55/120 0.42 mg Control Medium Extensive 40/120 0.50 mg Broth (1.0% v/v) Trace 3/120 0.15 mg Activity of the culture broth against neonate tobacco budworm (Heliothis virescens) was demonstrated using a leaf dip methodology. Fresh cotton leaves were excised from the plant and leaf disks were cut with an 18.5 mm cork-borer. The disks were individually emersed in control medium (PP3) or Photorhabdus luminescens (strain W-14) culture broth which had been concentrated approximately 10-fold using an Amicon (Beverly, MA), Proflux M12 tangential filtration system with a 10 kDa filter. Excess liquid was removed and a straightened paper clip was placed through the center of the dlsk. The paper clip was then wedged into a plastic, 1.0 oz shot glass containing approximately 2.0 ml of 1% Agar. This served to suspend the leaf disk above the agar. Following drying of the leaf disk, a single neonate tobacco budworm larva was placed on the disk and the cup was capped. The cups were then sealed in a plastic bag and placed in a darkened, 27~C incubator for 5 days.
At this time the remaining larvae and leaf material were weighed to establish a measure of leaf damage (Table 9).

Table30 Effect of P~otorhabdus luminescens (Strain W-14) Culture Broth on Tobacco Budworm Neonates in a Cotton-T~eaf Dip Assay Final Weights (mg) Treatment Leaf Disk Larvae 35 Control leaves 55.7 + 1.3 na*
Control Medium 34.0 + 2.9 4.3 + 0.91 Photorhabdus broth 54.3 + 1.4 0.0**
* - not applicable, ** - no live larvae found Ex~rle 5 Part A
Characterization of Toxin Peptide Components In a subsequent analysi~ the toxin protein subunits of the bands isolated as in Exampl~ ere resolved on a 7~ SDS

SU8S7~TUTE SHEET ~RULE 26) CA 022638l9 l999-02-26 W098/08932 PCTrUS97/076S7 polyacrylamide electrophoresis gel with a ratio of 30:0.8 (acrylamide:BIS-acrylamide). This gel matrlx facilitates better resolution of the larger proteins. The gel system used to estimate the Band 1 and Band 2 subunit molecular weights in Example 1 was an 18% gel with a ratio of 38:0.18 (acrylamide:BIS-acrylamide), which - allowed for a broader range of size separation, but less resolution of higher molecular weight components.
In this analysis, 10, rather than 8, protein bands were resolved. Table 10 reports the calculated molecular weights of the 10 resolved bands, and directly compares the molecular weights estimated under these conditions to those of the prior example. It is not surprising that additional bands were detected under the different separation conditions used in this example. Variations between the prior and new estimates of molecular weight are also to :5 be expected given the differences in analytical conditions. In the analysis of this example, it is thought that the higher molecular weight estimates are more accurate than in Example l, as a result of improved resolution. However, these are estimates based on SDS
PAGE analysis, which are typically not analytically precise and result in estimates of peptides and which may have been further altered due to post- and co-translational modifications.
Amino acid sequences were determined for the N-terminal portions of five of the 10 resolved peptides. Table 10 + correlates the molecular weight of the proteins and the identified sequences. In SEQ ID NO:2, certain analyses suggest that the prollne at residue 5 may be an asparagine (asn). In SEQ
ID NO:3, certain analyses suggest that the amino acid residues at positions 13 and 14 are both arginine (arg). In SEQ ID NO:4, certain analyses suggest that the amino acid residue at position 6 may be either alanine (ala) or serine (ser). In SEQ ID NO:5, certain analyses suggest that the amino acid residue at position 3 may be aspartic acid (asp).

SUBSTTTUTE SHEFr tRuLF 26) CA 022638l9 l999-02-26 W098/08932 PCT~US97/07657 Table.10 ESTIMATE NEW ESTIMATE* S~O. LISTING
208 200.2 kDa SEQ ID NO:1 184 175.0 kDa SEQ ID NO:2 65.6 68.1 kDa SEQ ID NO:3 60.8 65.1 kDa SEQ ID NO:4 56.2 58.3 kDa SEQ ID NO:5 25.1 23.2 kDa SEQ ID NO:15 *New estimates are based on SDS PAGE and are not based on gene sequences. SDS PAGE is not analytically precise.
le 5 Part B
~h~racterization of Toxin Peptide Com~onents New N-terminal sequence, SEQ ID NO:15, Ala Gln Asp Gly Asn Gln Asp Thr Phe Phe Ser Gly Asn Thr, was obtained by further N-terminal sequencing of peptides isolated from Native HP~C-purified toxin as described in Example 5, Part A, above. This peptide comes from the tcaA gene. The peptide labeled TcaAii, starts at position 254 and goes to position 491, where the TcaAiii peptide starts, SEQ ID
NO:4. The estimated size of the peptide based on the gene sequence is 25,240 Da.

Ex~ple 6 Characterizatlon of Toxin Peptide Components In yet another analysis, the toxin protein complex was re-isolated from the Photorhabdus luminescens growth medium (after culture without Tween) by performing a 10% - 80% ~mm~nium sulfate precipitation followed by an ion exchange chromatography step (Mono Q) and two molecular sizing chromatography steps. These conditions were like those used in Example 1. During the first molecular sizing step, a second biologically active peak was found at about 100 + 10 kDa. Based upon protein measurements, this fraction was 20 - 50 fold less active than the larger, or primary, active peak of about 860 + 100 kDa (native). During this isolation experiment, a smaller active peak of about 325 + 50 kDa that retained a considerable portion of the starting biological activity was also resolved. It is thought that the 325 kDa peak is related to or derived from the 860 kDa peak.
A 56 kDa protein was resolved in this analysis. The N-terminal sequence of this protein is presented in SEQ ID NO:6. It SU85Tl rUTE St~EFr tRULE 26) CA 022638l9 l999-02-26 W098~8932 PCTrUS97/07657 is noteworthy that this protein shares significant identity and conservation with SEQ ID NO:5 at the N-terminus, suggesting that the two may be encoded by separate members of a gene family and that the proteins produced by each gene are sufficiently slmilar to both be operable in the insecticidal toxin complex.
- A second, prominent 185 kDa protein was consistently present in amounts comparable to that of protein 3 from Table 10, and may be the same protein or protein fragment. The N-terminal sequence of this 185 kDa protein is shown at SEQ ID NO:7.
Additional N~terminal amino acid sequence data were also obtained from isolated proteins. None of the determined N-terminal sequences appear identical to a protein identified in Table 10.
Other proteins were present in isolated preparation. One such protein has an estimated molecular weight of 108 kDa and an N-terminal sequence as shown in SEQ ID NO:8. A second such protein has an estimated molecular weight of 80 kDa and an N-terminal sequence as shown in SEQ ID NO:9.
When the protein material in the approximately 325 kDa active peak was analyzed by size, bands of approximately 51, 31, 28, and 22 kDa were observed. As in all cases in which a molecular weight was determined by analysis of electrophoretic mobility, these molecular weights were subject to error effects introduced by buffer ionic strength differences, electrophoresis power differences, and the like. One of ordinary skill would understand that definitive molecular weight values cannot be determined using these standard methods and that each was sub~ect to variation. It was hypothesized that proteins of these sizes are degradation products of the larger protein species (of approximately 200 kDa size) that were observed in the larger primary toxin complex.
Finally, several preparations included a protein having the N-terminal sequence shown in SEQ ID NO:10. This sequence was strongly homologous to known chaperonin proteins, accessory proteins known to function in the assembly of large protein complexes. Although the applicants could not ascribe such an assembly function to the protein identified in SEQ ID NO:10, it was consistent with the existence of the described toxin protein complex that such a chaperonin protein could be involved in its assembly. Moreover, although such proteins have not directly been suggested to have toxic activity, this protein may be important to determining the overall structural nature of the protein toxin, and thus, may contribute to the toxic activity or durability of the complex in vivo after oral delivery.

SU8STITUTE S~ EET (RULE 26) CA 022638l9 l999-02-26 W098/08932 PCTrUS97/07657 Subsequent analysis of the stability of the protein toxin complex to proteinase K was undertaken. It was determined that after 24 hour incubation of the complex in the presence of a 10-fold molar excess of proteinase K, activity was virtually eliminated (mortality on oral application dropped to about 5%).
These data confirm the proteinaceous nature of the toxin.
The toxic activity was also retained by a dialysis membrane, again confirming the large size of the native toxin complex.

Example 7 Isolatio~. Characterization and Partial Amino Acid Sequenci ng of Photorhabdus Toxins Isolation and N-Ter~-n~1 Amino Acid Se~uenci~g In a set of experiments conducted in parallel to Examples 5 and 6, ammonium sulfate precipitation of Photorhabdus proteins was performed by adjusting P~otorhabdus broth, typically 2-3 liters, to a final concentration of either 10% or 20~ by the slow addition of ammonium sulfate crystals. After stirring for 1 hour at 4~C, the 20 material was centrifuged at 12,000 x g for 30 minutes. The supernatant was adjusted to 80~ ammonium sulfate, stirred at 4~C
for 1 hour, and centrifuged at 12,000 x g for 60 minutes. The pellet was resuspended in one-tenth the volume of 10 mM Na2 PO4, pH
7.0 and dialyzed against the same phosphate buffer overnight at 25 4~C. The dialyzed material was centrifuged at 12,000 x g for 1 hour prior to ion exchange chromatography.
A HR 16/50 Q Sepharose (Pharmacia) anion exchange column was equilibrated with lO mM Na2 P04, pH 7Ø Centrifuged, dialyzed ammonium sulfate pellet was applied to the Q Sepharose column at a rate of 1.5 ml/min and washed extensively at 3.0 ml/min with equilibration buffer until the optical density (O.D. 280) reached less than 0.100. Next, either a 60 minute NaCl gradient ranging from 0 to 0.5 M at 3 ml/min, or a series of step elutions using 0.1 M, 0.4 M and finally 1.0 NaCl for 60 minutes each was applied to the column. Fractions were pooled and concentrated using a Centriprep 100. Alternatively, proteins could be eluted by a single 0.4 M NaCl wash without prior elution with 0.1 M NaCl.
Two milliliter aliquots of concentrated Q Sepharose samples were loaded at 0.5 ml/min onto a HR 16/~0 Superose 12 (Pharmacia) 40 gel filtration column equilibrated with 10 mM Na2 P04, pH 7Ø The column was washed with the same buffer for 240 min at 0.5 ml/min and 2 min samples were collected. The void volume material was SUBS ~ JTE SHE T tRULE 26) W098/08932 PCT~S97/07657 collected and concentrated using a Centriprep 100. Two milliliter aliquots of concentrated Superose 12 samples were loaded at 0.5 ml/min onto a HR 16/50 Sepharose 4B-CL (Pharmacla) gel filtration column equilibrated with 10 mM Na2 P04, pH 7Ø The column was washed with the same buffer for 240 min at 0.5 ml/min and 2 min samples were collected.
The excluded protein peak was subjected to a second fractionation by application to a gel filtration column that used a Sepharose C~-4B resin, which separates proteins ranging from about 30 kDa to 1000 kDa. This fraction was resolved into two peaks; a minor peak at the void volume (>1000 kDa) and a major peak which eluted at an apparent molecular weight of about 860 kDa. Over a one week period subsequent samples subjected to gel filtration showed the gradual appearance of a third peak (approximately 325 kDa) that seemed to arise from the major peak, perhaps by limited proteolysis. Bioassays performed on the three peaks showed that the void peak had no activity, while the 860 kDa toxin complex fraction was highly active, and the 325 kDa peak was less active, although quite potent. SDS PAGE analysis of Sepharose CL-4B toxin complex peaks from different fermentation productions revealed two distinct peptide patterns, denoted "P" and "S". The two patterns had marked differences in the molecular weights and concentrations of peptide components in their fractions. The "S" pattern, produced most frequently, had 4 high molecular weight peptides (> 150 kDa) while the "P" pattern had 3 high molecular weight peptides. In addition, the "S" peptide fraction was found to have 2-3 fold more activlty against European Corn Borer. This shift may be related to variations in protein expression due to age of inoculum and/or other factors based on growth parameters of aged cultures.
Milligram quantities of peak toxin complex fractions determined to be "P" or "S" peptide patterns were subjected to preparative SDS PAGE, and transblotted with TRIS-glycine (SeprabuffTM to PVDF membranes (ProBlottTM, Applied Biosystems) for 3-4 hours. Blots were sent for amino acid analysis and N-terminal amino acid sequencing at Harvard MicroChem and Cambridge ProChem, respectively. Three peptides in the "S" pattern had unique N-terminal amino acid sequences compared to the sequences identified ~ in the previous example. A 201 kDa (TcdAii) peptide set forth as SEQ ID NO:13 below shared between 33% amino acid identity and 50~
similarity (similarity and identity were calculated by hand) with SEQ ID NO:1 (TcbAii)(in Table 10 vertical lines denote amlno acid SUBSTITUTE SHEE~ (RULE-26) .~

CA 022638l9 l999-02-26 W098/08932 PCTrUS97/07657 identities and colons indicate conservative amino acid substitutions). A second peptide of 197 kDa, -SEQ ID NO:14 (TcdB), had 42% identity and 58~ similarity with SEQ ID NO:2 (TcaC) (similarity and identity were calculated by hand). Yet a third peptide of 205 kDa was denoted TCdAii~ In addition, a limited N-terminal amlno acid sequence, SEQ ID NO:16 (TcbA), of a peptide of at least 235 kDa was identical with the amino acid sequence, SEQ ID
NO:12, deduced from a cloned gene (tcbA), SEQ ID NO:11, containing a deduced amino acid sequence corresponding to SEQ ID NO:1 (TcbAii). This indicates that the larger 235+ kDa peptide was proteolytically processed to the 201 kDa peptide, (TcbAii), (SEQ ID
NO:1) during fermentation, possibly resulting in activation of the molecule. In yet another sequence, the sequence originally reported as SEQ ID NO:5 (TcaBii) reported in Example 5 above, was found to contain an aspartic acid residue (Asp) at the third position rather than glycine (Gly) and two additional amino acids Gly and Asp at the eighth and ninth positions, respectively. In yet two other sequences, SEQ ID NO:2 (TcaC) and SEQ ID NO:3 (TcaBi), additional amino acid sequence was obtained.
Densitometric quantitation was performed using a sample that was identical to the "S" preparation sent for N-terminal analysis.
This analysis showed that the 201 kDa and 197 kDa peptides represent 7.0% and 7.2~, respectively, of the total Coomassie brillant blue stained protein in the "S" pattern and are present in amounts similar to the other abundant peptides. It was speculated that these peptides may represent protein homologs, analogous to the situation found with other bacterial toxins, such as various CryI Bt toxins. These proteins vary from 40-90~ similarity at their N-terminal amino acid sequence, which encompasses the toxic fragment.

Internal Amino Acid Sequencing To facilitate cloning of toxin peptide genes, internal amino acid sequences of selected peptides were obtained as followed.
Milligram quantities of peak 2A fractions determined to be "P" or "S" peptide patterns were subjected to preparative SDS PAGE, and transblotted with TRIS-glycine (SeprabuffTM to PVDF membranes (ProBlottTM, Applied Biosystems) for 3-4 hours. Blots were sent for amino acid analysis and N-terminal amino acid sequencing at Harvard MicroChem and Cambridge ProChem, respectively. Three peptides, referred to as TcbAii (containing SEQ ID NO:1), TcdAii, and TcaB
(containing ,EQ ID NO:3) were subjected to trypsin digestion by SUBSTITUTE SHEET tRULE 26) CA 022638l9 l999-02-26 W098/08932 PCTrUS97/07657 Harvard MicroChem followed by HPLC chromatography to separate individual peptides. N-terminal amino acid analysis was performed on selected tryptic peptide fragments. Two internal peptides were sequenced for the peptide TCdAii (205 kDa peptide) referred to as TcdAii-PT111 (SEQ ID NO:17) and TcdAii-PT79 (SEQ ID NO:18). Two internal peptides were sequenced for the peptide TcaBi (68 kDa peptide) referred to as TcaBi-PT158 ~SEQ ID NO:19) and TcaBi-PT108 (SEQ ID NO:20). Four internal peptides were sequenced for the peptide TCbAii ~201 kDa peptide) referred to as TcbAii-PT103 (SEQ
ID NO:21), TCbAii-pT56 (SEQ ID NO:22), TcbAii-PT81(a) (SEQ ID
NO:23), and TcbAii-PT81(b) (SEQ ID NO:24).

Table 11 N-Terminal Aml n~ Acid Sequences (similarity and identity were calculated by hand) 201 kDa (33% identity & 50~ similarity to SEQ ID NO.l) L I G Y N N Q F S G * A SEQ ID NO:13 F I Q G Y S D L F G N - A SEQ ID NO:1 197 kDa (42% identity & 58% similarity SEQ ID NO.2) M Q N S Q T F S V G E L SEQ ID NO.14 M Q D S P E V S I T T L SEQ ID NO.2 Example 8 Construction of a Cos~id Library of Photorhabdus l~minescens W-14~0 Genomic DNA and its Scree~ing to Isolate Genes Encoding Peptides Comprising the Toxic Protein Preparation As a prerequisite for the production of Photorhabdus lnsect toxic proteins in heterologous hosts, and for other uses, it is necessary to isolate and characterize the genes that encode those peptides. This objective was pursued in parallel. One approach, described later, was based on the use of monoclonal and polyclonal antibodies raised against the purified toxin which were then used to isolate clones from an expression library. The other approach, described in this example, is based on the use of the N-terminal and internal amino acid sequence data to design degenerate oligonucleotides for use in PCR amplication. Either method can be used to identify DNA clones that contain the peptide-encoding genes so as to permit the isolation of the respective genes, and the determination of their DNA base sequence.

SU8SllTUTE Stl EE~ tRULE 26) CA 022638l9 l999-02-26 W098/08932 PCT~US97/07657 Genomic DNA Isolation Photorhabdus luminescens strain W-14 (ATCC accession number S5397) was grown on 2% proteose peptone #3 agar (Difco Laboratories, Detroit, MI) and insecticidal toxin competence was maintained by repeated bioassay after passage, using the method described in Example 1 above. A 50 ml shake culture was produced in a 175 ml baffled flask in 2~ proteose peptone #3 medium, grown at 28~C and 150 rpm for approximately 24 hours. 15 ml of this culture was pelleted and frozen in its medium at -20OC until it was thawed for DNA isolation. The thawed culture was centrifuged, (700 x g, 30 min) and the floating orange mucopolysaccharide material was removed. The rem~'ning cell material was centrifuged ~25,000 x g, 15 min) to pellet the bacterial cells, and the medium was removed and discarded.
Genomic DNA was isolated by an adaptation of the CTAB method described in section 2.4.1 of Current Protocols in Molecular Biology (Ausubel et al. eds, John Wiley & Sons, 1994) [modified to include a salt shock and with all volumes increased 10-fold]. The pelleted bacterial cells were resuspended in TE buffer (10 mM Tris-20 HCl, 1 mM EDTA, pH 8.0) to a final volume of 10 ml, then 12 ml of 5 M NaCl was addedi this mixture was centrifuged 20 min at 15,000 x g. The pellet was resuspended in 5.7 ml TE and 300 ml of 10~ SDS
and 60 ml of 20 mg/ml proteinase K (Gibco BRL Products, Grand Island, NY; in sterile distilled water) were added to the suspension. This mixture was incubated at 37~C for 1 hr; then approximately 10 mg lysozyme (Worthington Biochemical Corp., Freehold, NJ) was added. After an additional 45 min, 1 ml of 5 M
NaCl and 800 ml of CTAB/NaCl solution (10~ w/v CTAB, 0.7 M NaCl) were added. This preparation was incubated 10 min at 65~C, then gently agitated and further incubated and agitated for approximately 20 min to assist clearing of the cellular material.
An equal volume of chloroform/isoamyl alcohol solution (24:1, v/v) was added, mixed gently and centrifuged. After two extractions with an equal volume of PCI (phenol/chloroform/isoamyl alcohol;
35 50:49:1, v/v/v; e~uilibrated with 1 M Tris-HCl, pH 8.0;
Intermountain Scientific Corporation, Kaysville, UT), the DNA was precipitated with 0.6 volume of isopropanol. The DNA precipitate was gently removed with a glass rod, washed twice with 70~ ethanol, dried, and dissolved in 2 ml STE (10 mM Tris-HC1 pH 8.0, 10 mM
NaCl, 1 mM EDTA). This preparation contained 2.5 mg/ml DNA, as determined by optical density at 260 nm (i.e., OD260).

SUBSTITUTE SHEET(RULE-26) CA 022638l9 l999-02-26 W09~08932 PCT~US97/076S7 The molecular size range of the isolated genomic DNA was evaluated for suitability for library construction. CHEF gel analysis was performed in 1.5~ agarose (Seakem LE, FMC BloProducts, Rockland, ME) gels with 0.5 X TBE buffer (44.5 mM Tris-HCl pH 8.0, 44.5 mM H3BO3, 1 mM EDTA) on a BioRad CHEF-DR II apparatus with a Pulsewave 760 Switcher (Bio-Rad Laboratories, Inc., Richmond, CA).
The running parameters were: initial A time, 3 sec; final A time, 12 sec; 200 volts; running temperature, 4-18~C; run time, 16.5 hr.
Ethidium bromide staining and examination of the gel under ultraviolet light indicated the DNA ranged from 30-250 kbp in size.

Construction of Library A partial Sau3A 1 digest was made of this Photorha~dus genomic DNA preparation. The method was based on section 3.1.3 of Ausubel (supra.). Adaptions included running smaller scale reactions under various conditions until nearly optimal results were achieved.
Several scaled-up large reactions with varied conditions were run, the results analyzed on CHEF gels, and only the best large scale preparation was carried forward. In the optimal case, 200 ~g of Photorhabdus genomic DNA was incubated with 1.5 units of Sau3A 1 (New England Biolabs, "NEB", Beverly, MA) for 15 min at 37~C in 2 ml total volume of lX NEB 4 buffer (supplied as lOX by the manufacturer). The reaction was stopped by adding 2 ml of PCI and centrifuging at 8000 x g for 10 min. To the supernatant were added 200 ~l of 5 M NaC1 plus 6 ml of ice-cold ethanol. This preparation was chilled for 30 min at -20~C, then centrifuged at 12,000 x g for 15 min. The supernatant was removed and the precipitate was dried in a vacuum oven at 40~C, then resuspended in 400 ~l STE.
Spectrophotometric assay indicated about 40% recovery of the input DNA. The digested DNA was size fractionated on a~sucrose gradient according to section 5.3.2 of CPMB (op. cit.). A 10% to 40~ (w/v) linear sucrose gradient was prepared with a gradient maker in Ultra-ClearTM tubes (Beckman Instruments, Inc., Palo Alto, CA) and the DNA sample was layered on top. After centrifugation, (26,000 rpm, 17 hr, Beckman SW41 rotor, 20~C), fractions (about 750 ~l) were drawn from the top of the gradient and analyzed by CHEF gel electrophoresis (as described earlier). Fractions containing Sau3A
l fragments in the size range 20-40 kbp were selected and DNA was precipitated by a modification (amounts of all solutions increased approximately 6.3-fold) of the method in section 5.3.3 of Ausubel (supra.~. After overnight precipitation, the DNA was collected by centrifugation (17,000 x g, 15 min), dried, redissolved in TE, -~7-SUBSTITUTE SH~ET (RULE 26) ~ . , CA 022638l9 l999-02-26 W098/08932 PCTrUS97/07657 pooled into a final volume of 80 ~l, and reprecipitated with the addition of 8 ~l 3 M sodium acetate and 220 ~l ethanol. The pellet collected by centrifugation as above was resuspended in 12 ~l TE.
Concentration of the DNA was determined by Hoechst 33258 dye (Polysciences, Inc., Warrington, PA) fluorometry in a Hoefer TKO100 fluorimeter (Hoefer Scientific Instruments, San Francisco, CA).
Approximately 2.5 ~g of the size-fractionated DNA was recovered.
Thirty ~g of cosmid pWE15 DNA (Stratagene, La Jolla, CA) was digested to completion with 100 units of restriction enzyme BamH 1 10 (NEB) in the manufacturer's buffer (final volume of 200 ~1, 37~C, 1 hr). The reaction was extracted with 100 ~1 of PCI and DNA was precipitated from the aqueous phase by addition of 20 ~l 3M sodium acetate and 550 ~1 -20~C absolute ethanol. After 20 min at -70~C, the DNA was collected by centrifugation (17,000 x g, 15 min), dried 15 under vacuum, and dissolved in 180 ~l of 10 mM Tris-HCl, pH 8Ø
To this were added 20 ~1 of lOX CIP buffer (100 mM Tris-HCl, pH
8.3; lO mM ZnC12; 10 mM MgC12), and 1 ~l (0.25 units) of 1:4 diluted calf intestinal alkaline phosphatase (Boehringer Mannheim Corporation, Indianapolis, IN). After 30 min at 37~C, the 20 following additions were made: 2 ~l 0.5 M EDTA, pH 8.0; 10 ~1 10 SDS; 0.5 ~1 of 20 mg/ml proteinase K (as above), followed by incubation at 55~C for 30 min. Following sequential extractions with 100 ~l of PCI and 100 ~1 phenol (Intermountain Scientific Corporation, equilibrated with 1 M Tris-HC1, pH 8.0), the 25 dephosphorylated DNA was precipitated by addition of 72 ~1 of 7.5 M
ammonium acetate and 550 ~1 -20~C ethanol, incubation on ice for 30 min, and centrifugation as above. The pelleted DNA was washed once with 500 ~l -20~C 70~ ethanol, dried under vacuum, and dissolved in 20 ~l of TE buffer.
Ligation of the size-fractionated Sau3A 1 fragments to the BamH 1-digested and phosphatased pWE15 vector was accomplished using T4 ligase (NEB) by a modification (i.e., use of premixed lOX
ligation buffer supplied by the manufacturer) of the protocol in section 3.33 of Ausubel. Ligation was carried out overnight in a 35 total volume of 20 ~l at 1~~C, followed by storage at - 20~C.
Four ~1 of the cosmid DNA ligation reaction, containing about 1 ~g of DNA, was packaged into bacteriophage lambda using a commercial packaging extract (Gigapack III Gold Packaging Extract, Stratagene), following the manufacturer's directions. The packaged preparation was stored at 4~C until use. The packaged cosmid preparation was used to infect Escherichia coli XL1 Blue MR cells SUBSTITUTE SH EE~ (RULE ~6) CA 022638l9 l999-02-26 W098/08932 PCTrUS97/07657 (Stratagene) according to the Gigapack III Gold protocols ("Titering the Cosmid Library"), as follows. XL1 Blue MR cells were grown in LB medium (g/L: Bacto-tryptone, 10; Bacto-yeast extract, 5; Bacto-agar, 15; NaCl, 5; [Difco Laboratories, Detroit, MI]) containing 0.2% (w/v) maltose plus 10 mM MgSO4, at 37~C. After - 5 hr growth, cells were pelleted at 700 x g (15 min) and resuspended in 6 ml of lO mM MgSO4. The culture density was adjusted with 10 mM MgSO4 to OD600= 0.5. The packaged cosmid library was diluted 1:10 or 1:20 with sterile SM medium (0.1 M
NaCl, 10 mM MgSO4 50 mM Tris-HCl pH 7.5, 0.01% w/v gelatin), and 25 ~l of the diluted preparation was mixed with 25 ~l of the diluted XL1 Blue MR cells. The mixture was incubated at 25~C for 30 min (without shaking), then 200 ~l of LB broth was added, and incubation was continued for approximately 1 hr with occasional gentle shaking. Aliquots (20-40 ~1) of this culture were spread on LB agar plates containing 100 mg/l ampicillin (i.e., LB-Ampl0O) and incubated overnight at 37~C. To store the library without amplification, single colonies were picked and inoculated into individual wells of sterile 96-well microwell plates; each well containing 75 ~l of Terrific Broth (TB media: 12 g/l Bacto-tryptone, 24 g/l Bacto-yeast extract, 0.4% v/v glycerol, 17 mM
KHaPO4, 72 mM K,HPO4) plus 100 mg/l ampicillin (i.e., TB-Amp1O0) and incubated (without shaking) overnight at 37~C. After replicating the 96-well plate into a copy plate, 75 ~l/well of filter-sterilized TB:glycerol (1:1, v/v; with, or without, 100 mg/1 ampicillin) was added to the plate, lt was shaken brlefly at 100 rpm, 37~C, and then closed with Parafilm~ (American National Can, Greenwich, CT) and placed in a -70~C freezer for storage. Copy plates were grown and processed identically to the master plates.
A total of 40 such master plates (and their copies) were prepared.

Screening of the Lihrary with Radiolabeled DNA Probes To prepare colony filters for probing with radioactively labeled probes, ten 96-well plates of the library were thawed at 25~C (bench top at room temperature). A replica plating tool with 96 prongs was used to inoculate a fresh 96-well copy plate containing 75 ~l/well of TB-Amp10O. The copy plate was grown overnight (stationary) at 37~C, then shaken about 30 min at 100 rpm at 37~C. A total of 800 colonies was represented in these copy plates, due to nongrowth of some isolates. The replica tool was used to inoculate duplicate impressions of the 96-well arrays onto Magna NT (MSI, Westboro, MA) nylon membranes (0.45 micron, 220 x SUBSTrTUTE S}tEET (RULE 26) .. . . . . .......

CA 022638l9 l999-02-26 W098/08932 PCT~US97/07657 250 mm) which had been placed on solid LB-Amp100 (100 ml/dish) in Bio-assay plastic dishes (Nunc, 243 x 243 x 18 mm; Curtin Mathison Scientific, Inc., Wood Dale, IL). The colonies were grown on the membranes at 37~C for about 3 hr.
A positive control colony (a bacterial clone containing a GZ4 sequence insert, see below) was grown on a separate Magna NT
membrane (Nunc, 0.45 micron, 82 mm circle) on LB medium supplemented with 35 mg/l chloramphenicol (i.e., LB-Cam3s), and processed alongside the library colony membranes. Bacterial colonies on the membranes were lysed, and the DNA was denatured and neutralized according to a protocol taken from the GeniusT~ System User's Guide version 2.0 (Boehringer Mannheim, Indianapolis, IN).
Membranes were placed colony side up on filter paper soaked with 0.5 N NaOH plus 1.5 M NaCl for 15 min to denature, and neutralized on filter paper soaked with 1 M Tris-HC1 pH 8.0, l.S M NaCl for 15 min. After W -crosslinking using a Stratagene W Stratalinker set on auto crosslink, the membranes were stored dry at 25~C until use.
Membranes were trimmed into strips containing the duplicate impressions of a single 96-well plate, then washed extensively by the method of section 6.4.1 in CPMB (op. cit. ): 3 hr at 25~C in 3X
SSC, 0.1% (w/v) SDS, followed by 1 hr at 65~C in the same solution, then rinsed in 2X SSC in preparation for the hybridization step (20X SSC = 3 M NaCl, 0.3 M sodium citrate, pH 7.0).

Amplification of a Specific Genomic Fragment of a ~caC Gene Based on the N-terminal amino acid sequence determined for the purified TcaC peptide fraction [disclosed herein as SEQ ID NO:2], a pool of degenerate oligonucleotides (pool S4Psh) was synthesized by standard ~-cyanoethyl chemistry on an Applied BioSystem ABI394 DNA rRMA Synthesizer (Perkin Elmer, Foster City, CA). The oligonucleotides were deprotected 8 hours at 55~~ ssolved in water, quantitated by spectrophotometric measurement, and diluted for use. This pool corresponds to the determined N-terminal amino acid sequence of the TcaC peptide. The determined amino acid sequence and the corresponding degenerate DNA sequence are given below, where A, C, G, and T are the standard DNA bases, and I
represents inosine:
Amino Met Gln Asp Ser Pro Glu Val Acid S4Psh 5' ATG CA(A/G) GA(T/C) (T/A)(C/G)(T/A) CCI GA(A/G) GT 3' Another set of degenerate oligonucleotides was synthesized ~pool P2.3.5R), representing the complement of the coding strand~5 for the determined amino acid sequence of the SEQ ID NO:17:

5lJ8STll-UTE St~FET tRULE 26) CA 022638l9 l999-02-26 Amino Acid Ala Phe Asn Ile Asp Asp Val Codon~ 5' GCN TT~T/C) AA(T/C) AT(A/T/C) GA(T/C) GA(T/C) GT 3' P2,3.5R 3'CG(A/C/G/T) AA(A/G) TT(A/G) TA(T/A/G~ CT(A/G) CT(A/G) CA 5' These oligonucleotides were used as primers in Polymerase Chain Reactions (PCR , Roche Molecular Systems, Branchburg, NJ) to amplify a specific DNA fragment from genomic DNA prepared from 0 Photorhabdus strain W-14 (see above). A typical reaction (50 ~1) contained 125 pmol of each primer pool P2Psh and P2.3.5R, 253 ng of genomic template DNA, 10 nmol each of dATP, dCTP, dGTP, and dTTP, lX GeneAmp PCR buffer, and 2.5 units of AmpliTaq DNA polymerase (both from Roche Molecular Systems; lOX GeneAmp buffer is 100 mM
5 Tris-HCl pH 8.3, 500 mM KCl, 0.01% w/v gelatin). Amplifications were performed in a Perkin Elmer Cetus DNA Thermal Cycler (Perkin Elmer, Foster City, CA) using 35 cycles of 94~C (1.0 min), 55~C
(2.0 min), 72~C (3.0 min), followed by an extension period of 7.0 min at 72~C. Amplification products were analyzed by electrophoresis through 2% w/v NuSleve 3:1 agarose (FMC
BioProducts) in TEA buffer (40 mM Tris-acetate, 2 mM EDTA, pH 8.0).
A specific product of estimated size 250 bp was observed amongst numerous other amplification products by ethidium bromide (0.5 ~g/ml) staining of the gel and examination under ultraviolet light.
The region of the gel containing an approximately 250 bp product was excised, and a small plug (0.5 mm dia.) was removed and used to supply template for PCR amplification (40 cycles). The reaction (50 ~1) contained the same components as above, minus genomic template DNA. Following amplification, the ends of the fragments were made blunt and were phosphorylated by incubation at 25~C for 20 min with 1 unit of T4 DNA polymerase (NEB), 1 nmol ATP, and 2.15 units of T4 kinase (Pharmacia Biotech Inc., Piscataway, NJ).
DNA fragments were separated from residual primers by electrophoresis through 1% w/v GTG agarose (FMC) in TEA. A gel slice containing fragments of apparent size 250 bp was excised, and the DNA was extracted using a Qiaex kit (Qiagen Inc., Chatsworth, CA).
The extracted DNA fragments were ligated to plasmid vector pBC
~S~+) (Stratagene) that had been digested to completion with restriction enzyme Sma 1 and extracted in a manner similar to that described for pWE15 DNA above. A typical ligation reaction (16.3 ~1) contained 100 ng of digested pBC KS(+) DNA, 70 ng of 250 bp fragment DNA, 1 nmol [Co(NH3)6]Cl3, and 3.9 Weiss units of T4 DNA
ligase (Collaborative Biomedical Products, Bedford, MA), in lX

SVBST~TUTE SHEET (RULE 26) CA 022638l9 l999-02-26 W098t08932 PCTrUS97/07657 ligation buffer (50 mM Tris-HCl, pH.7.4; 10 mM MgCl.; 10 mM
dithiothreitol; 1 mM spermidine, 1 mM ATP, 100 mg/ml bovine serum albumin). Following overnight incubation at 14~C, the ligated products were transformed into frozen, competent Escherichia coli DH5a cells (Gibco BRL) according to the suppliers' recomm~n~tions, and plated on LB-Cam3splates, containing IPTG (119 ~g/ml) and X-gal (50 ~g/ml). Independent white colonies were picked, and plasmid DNA was prepared by a modified alkaline-lysis/PEG precipitation method (PRISM~M Ready Reaction DyeDeoxyTM Terminator Cycle Sequencing Kit Protocols; ABI/Perkin Elmer). The nucleotide sequence of both strands of the insert DNA was determined, using T7 primers [pBC KS(+) bases 601-623: TAAAACGACGGCCAGTGAGCGCG) and LacZ
primers [pBC KS(+) bases 792-816: ATGACCATGATTACGCCAAGCGCGC) and protocols supplied with the PRISMTM sequencing kit (ABI/Perkin Elmer). Nonincorporated dye-terminator dideoxyribonucleotides were removed by passage through Centri-Sep 100 columns ~Princeton Separations, Inc., Adelphia, NJ) according to the manufacturer's instructions. The DNA sequence was obtained by analysis of the samples on an ABI Model 373A DNA Sequencer (ABI/Perkin Elmer). The DNA sequences of two isolates, GZ4 and HB14, were found to be as illustrated in Fig. 1.
This sequence illustrates the following features: 1) bases 1-20 represent one of the 64 possible sequences of the S4Psh degenerate oligonucleotides, ii) the sequence of amino acids 1-3 and 6-12 correspond exactly to that determined for the N-terminus of TcaC (disclosed as SEQ ID NO:2), iii) the fourth amino acid encoded is a cysteine residue rather than serine. This difference is encoded within the degeneracy for the serine codons (see above), iv) the fifth amino acid encoded is prollne, corresponding to the 30 TcaC N-terminal sequence given as SEQ ID NO:2, v) bases 257-276 encode one of the 192 possible sequences designed into the degenerate pool, vi) the TGA termination codon introduced at bases 268-270 is the result of complementarity to the degeneracy built into the oligonucleotide pool at the corresponding position, and does not indicate a shortened reading frame for the corresponding gene.

Labeling of a TcaC Peptide Gene-specific Probe --DNA fragments corresponding to the above 276 bases were 40 amplified (35 cycles) by PCR in a 100 ~l reaction volume, using 100 pmol each of P2Psh and P2.3.5R primers, 10 ng of plasmids GZ4 or HB14 as templates, 20 nmol each of dATP, dCTP, dGTP, and dTTP, 5 SUBSTITUTE SHEET (RULE 26) CA 022638l9 l999-02-26 W098/08932 PCT~US97/07657 units of AmpliTAq DNA polymerase, and lX concentration of GeneAmp buffer, under the same temperature regimes as~described above. The amplification products were extracted from a 1~ GTG agarose gel by Qiaex kit and quantitated by fluorometry.
The extracted amplification products from plasmid HB14 - template (approximately 400 ng) were split into five aliquots and labeled with 32P-dCTP using the High Prime Labeling Mix (Boehringer Mannheim) according to the manufacturer's instructions.
Nonincorporated radioisotope was removed by passage through NucTrap~
Probe Purification Columns (Stratagene), according to the supplier's instructions. The specific activity of the labeled DNA
product was determined by scintillation counting to be 3.11 x 10~
dpm/~g. This labeled DNA was used to probe membranes prepared from 800 members of the genomic library.
Screening with a TcaC-peptide Gene Specific Probe The radiolabeled HB14 probe was boiled approximately 10 min, then added to "minimal hyb" solution. [Note: The "minimal hyb"
method is taken from a CERES protocol; "Restriction Fragment Length Polymorphism Laboratory Manual version 4.0", sections 4-40 and 4-47; CEREStNPI, Salt Lake City, UT. NPI is now defunct, with its successors operating as Linkage Genetics]. "Minimal hyb" solution contains 10~ w/v PEG (polyethylene glycol, M.W. approx. 8000), 7~
w/v SDS; 0.6X SSC, 10 mM sodium phosphate buffer (from a lM stock containing 95 g/l NaH2PO4 lH2O and 84.5 g/l Na2HPO~ 7H2O), 5 mM EDTA, and 100 mg/ml denatured salmon sperm DNA. Membranes were blotted dry briefly then, without prehybridization, 5 strips of membrane were placed in each of 2 plastic boxes containing 75 ml of ~'minimal hyb" and 2.6 ng/ml of radiolabeled HB14 probe. These were incubated overnight with slow shaking (50 rpm) at 60~C. The filters were washed three times for approximately 10 min each at 25~C in "minimal hyb wash solution" (0.25X SSC, 0.2~ SDS), followed by two 30-min washes with slow shaking at 60~C in the same solution. The filters were placed on paper covered with Saran Wrap~
(Dow Brands, Indianapolis, IN) in a light-tight autoradiographic cassette and exposed to X-Omat X-ray film (Kodak, Rochester, NY) with two DuPont Cronex Lightning-Plus C1 enhancers (Sigma Chemical Co., St. Louis, MO), for 4 hr a~ -70~C. Upon development (standard photographic procedures), significant signals were evident in both replicates amongst a high background of weaker, more irregular signals. The filters were again washed for about 4 hr at 68~C in "minimal hyb wash solution" and then placed again in the cassettes SUBSTITUTE S~tEET (RULE 26) W098/08932 PCT~US97/07657 and film was exposed overnight at -7.0~C. Twelve possible positives were identified due to strong signals on both of the duplicate 96-well colony impressions. No signal was seen wlth negative control membranes (colonies of X~l Blue MR cells containing pWE15), and a very strong signal was seen with positive control membranes (DH5~
cells containing the GZ4 isolate of the PCR product) that had been processed concurrently with the experimental samples.
The twelve putative hybridization-positive colonies were retrieved from the frozen 96-well library plates and grown overnight at 37~C on solid LB-Ampl~O medium. They were then patched (3/plate, plus three negative controls: XL1 Blue MR cells containing the pWE15 vector) onto solid LB-Amp1O0. Two sets of membranes (Magna NT nylon, 0.45 micron) were prepared for hybridization. The first set was prepared by placing a filter directly onto the colonies on a patch plate, then removing it with adherent bacterial cells, and processing as below. Filters of the second set were placed on plates containing LB-Ampl00 medium, then inoculated by transferring cells from the patch plates onto the filters. After overnight growth at 37~C, the filters were removed from the plates and processed.
Bacterial cells on the filters were lysed and DNA denatured by placing each filter colony-side-up on a pool (1.0 ml) of 0.5 N NaOH
in a plastic plate for 3 min. The filters were blotted dry on a paper towel, then the process was repeated with fresh 0.5 N NaOH.
After blotting dry, the filters were neutralized by placing each on a 1.0 ml pool of 1 M Tris-HCl, pH 7.5 for 3 min, blotted dry, and reneutralised with fresh buffer. This was followed by two similar soakings (5 min each) on pools of 0.5 M Tris-HCl pH 7.5 plus 1.5 M
NaC1. After blotting dry, the DNA was W crosslinked to the filter (as above), and the filters were washed (2S~C, 100 rpm) in about 100 ml of 3X SSC plus 0.1%(w/v) SDS (4 times, 30 min each with fresh solution for each wash). They were then placed in a minimal volume of prehybridization solution [6X SSC plus 1~ w/v each of Ficoll 400 (Pharmacia), polyvinylpyrrolidone (av. M.W. 360,000;
Sigma ) and bovine serum albumin Fraction V; (Sigma)] for 2 hr at 65~C, 50 rpm. The prehybridization solution was removed, and replaced with the HB14 32P-labeled probe that had been saved from the previous hybridization of the library membranes and which had been denatured at 95~C for 5 min. Hybridization was performed at 40 60~C for 16 hr with shaking at 50 rpm.
Followin~ removal of the labeled probe solution, the membranes were washed 3 times at 25~C (50 rpm, 15 min) in 3X SSC (about 150 ml each wash). They were then washed for 3 hr at 68~C (50 rpm) in SUBST~TUTE SHEET (RULE 26) W098/08932 PCTrUS97/07657 0.25X SSC plus 0.2% SDS (minlm~l hyb wash solution), and exposed to X-ray film as described above for 1.5 hr at 25~C (no ~nh~ncer screens). This exposure revealed very strong hybridization signals to cosmid isolates 22G12, 25A10, 26A5, and 26B10, and a very weak signal with cosmid isolate 8B10. No signal was seen wlth the negative control (pWE15) colonles, and a very strong signal was seen with positive control membranes (DH5a cells containing the GZ4 isolate of the PCR product) that had been processed concurrently with the experimental samples.
Amplification of a Specific Genomic Fragment of a TcaB Gene Based on the N-terminal amino acid sequence determined for the purified TcaBi peptide fraction (disclosed here as SEQ ID NO:3) a pool of degenerate oligonucleotides (pool P8F) was synthesized as described for peptide TcaC. The determined amino acid sequence and the corresponding degenerate DNA sequence are given below, where A, C, G, and T are the standard DNA bases, and I represents inosine:

Amino Acid Leu Phe Thr Gln Thr heu Lys Glu Ala Arg P8F S' TTT ACI CA(A/G) ACI (C/T)TI AAA GAA GCI (A/C)G 3' (C/T)TI

Another set of degenerate oligonucleotides was synthesized (pool P8.108.3R), representing the complement of the coding strand for the determined amino acid sequence of the TcaBi-PT108 internal peptide (disclosed herein as SEQ ID NO:20):
Amino Acid Met Tyr Tyr Ile Gln Ala Gln Gln Codons ATG TA(T/C) TA(~/C) AT(T/C/A) CA(A/G) GC(A/C/G/T) CA(A/G CA(A/G) P8.108.3R 3' AT(A/G) AT(A/G) TA(A/G/T) GT(T/C) CGI GT(T/C) GT 5' TAC

These oligonucleotides were used as primers for PCR using HotStart 50 TubesTM (Molecular Bio-Products, Inc., San Diego, CA) to amplify a speclfic DNA fragment from genomic DNA prepared from Photorhabdus strain W-14 (see above). A typical reaction (50 ~1) contained (bottom layer) 25 pmol of each primer pool P8F and P8.108.3R, with 2 nmol each of dATP, dCTP, dGTP, and dTTP, in lX
GeneAmp PCR buffer, and (top layer) 230 ng of genomic template DNA, 8 nmol each of dATP, dCTP, dGTP, and dTTP, and 2.5 units of 4~ AmpliTaq DNA polymerase, in lX GeneAmp PCR buffer. Amplifications were performed by 35 cycles as described for the TcaC pept1de.
Amplification products were analyzed by electrophoresis through SUBSTITUTE St~ EET (RULE~) ...... ~

CA 022638l9 l999-02-26 W098/08932 PCTrUS97/07657 0.7% w/v SeaKem LE agarose (FMC) in TEA buffer. A specific product of estimated size 1600 bp was observed.
Four such reactions were pooled, and the amplified DNA was extracted from a 1.0% SeaKem LE gel by Qiaex kit as described for the TcaC peptide. The extracted DNA was used directly as the template for sequence determination (PRISM Sequencing Kit) using the P8F and P8.108.3R primer pools. Each reaction contained about 100 ng template DNA and 25 pmol of one primer pool, and was processed according to standard protocols as described for the TcaC
peptide. An analysis of the sequence derived from extension of the P8F primers revealed the short DNA sequence (and encoded amino acid sequence):
GAT GCA TTG NTT GCT
Asp Ala Leu (Val) Ala which corresponds to a portion of the N-terminal peptide sequence disclosed as SEQ ID NO:3 (TcaBi).

Labeling of a TcaBi-peptlde Gene-specific Probe Approximately 50 ng of gel-purified TcaBi DNA fragment was labeled with 32P-dCTP as described above, and nonincorporated radioisotopes were removed by passage through a NICK Column~
(Pharmacia). The specific activity of the labelled DNA was determined to be 6 x 109 dpm/~g. This labeled DNA was used to probe colony membranes prepared from members of the genomic library that had hybridized to the TcaC-peptide specific probe.
The membranes containing the 12 colonies identified in the TcaC-probe library screen (see above) were stripped of radioactive TcaC-specific label by boiling twice for approximately 30 min each time in 1 liter of O.lX SSC plus 0.1 % SDS. Removal of radiolabel was checked with a 6 hr film exposure. The stripped membranes were then incubated with the TcaBi peptide-specific probe prepared above. The labeled DNA was denatured by boiling for 10 min, and then added to the filters that had been incubated for 1 hr in 100 ml of "minimal hyb" solution at 60~C. After overnight hybridization at this temperature, the probe solution was removed, and the filters were washed as follows (all in 0.3X SSC plus 0.1 SDS): once for 5 min at 25~C, once for 1 hr at 60~C in fresh solution, and once for 1 hr at 63~C in fresh solution. After 1.5 hr exposure to X-ray film by standard procedures, 4 strongly-hybridizing colonies were observed. These were, as with the TcaC-specific probe, isolates 22G12, 25A10, 26A5, and 26B10.

SUBSTITUTE SH~ET (RULE 26) CA 022638l9 l999-02-26 W098/08932 PCT~S97/07657 The same TcaBi probe solution .was diluted with an equal volume (about 100 ml) of "minimal hyb" solution, and then used to screen the membranes containing the 800 members of the genomic library.
After hybridization, washing, and exposure to X-ray film as described above, only the four cosmid clones 22G12, 25A10, 26A5, ~ and 26B10, were found to hybridize strongly to this probe.

Isolation of Subclones Containing Genes ~nco~ing TcaC and YcaB
Peptides, and Determi~tion of ~NA Base Sequence Thereof Three hybridization-positive cosmids in strain XLl Blue MR
were grown with shaking overnight (200 rpm) at 30~C in 100 ml TB-Amp1O0. After harvesting the cells by centrifugation, cosmid DNA
was prepared using a commercially available kit (BIGprepTM, 5 Prime 3 Prime, Inc., Boulder, CO), following the manufacturer's protocols. Only one cosmid, 26A5, was successfully isolated by this procedure. When digested with restriction enzyme EcoR 1 (NEB) and analyzed by gel electrophoresis, fragments of approximate sizes 14, 10, 8 (vector), 5, 3.3, 2.9, and 1.5 kbp were detected. A
second attempt to isolate cosmid DNA from the same three strains (8 ml cultures; TB-Ampl00, 30~C) utilized a boiling miniprep method (Evans G. and G. Wahl., 1987, "Cosmid vectors for genomic walking and rapid restriction mapping." in Guide to Molecular Cloning Techniques. Meth. Enzymology, Vol. 152, S. Berger and A. Kimmel, eds., pgs. 604-610). Only one cosmid, 25A10, was successfully isolated by this method. When digested with restriction enzyme EcoR I (NEB) and analyzed by gel electrophoresis, this cosmid showed a fragmentation pattern identical to that previously seen with cosmid 26A5.
A 0.15 ~g sample of 26A5 cosmid DNA was used to transform 50 ml of E. coli DH5a cells (Gibco BRL), by the supplier's protocols.
A single colony isolate of that strain was inoculated into 4 ml of TB-Amp100, and grown for 8 hr at 37~C. Chloramphenicol was added to a final concentration of 225 ~g/ml, incubation was continued for another 24 hr, then cells were harvested by centrifugation and frozen at -20~C. Isolation of the 26A5 cosmid DNA was by a standard alkaline lysis miniprep (Maniatis et al., op . ci t ., p. 382), modified by increasing all volumes by 50~ and with stirring or gentle mixing, rather than vortexing, at every step.
After washing the DNA pellet in 70% ethanol, it was dissolved in TE
containing 25 ~g~ml ribonuclease A (Boehringer Mannheim).

SUBSTITUTE SHFET tRULE 26) CA 022638l9 l999-02-26 W098/08932 PCT~US97/07657 Identification of ~coR I Fragm~nts Hybri~izing to GZ4-derived and I~a~i - Probes Approximately 0.4 ~g of cosmid 25A10 (from XL1 Blue MR cells) and about 0.5 ~g of cosmid 26A5 ~from chloramphenicol-amplified DH5a cells) were each digested with about 15 units of EcoR I (NEB) for 85 min, frozen overnight, then heated at 65~C for five min, and electrophoresed in a 0.7% agarose gel (Seakem LE, lX TEA, 80 volts, 90 min). The DNA was stained with ethidium bromide as described above, and photographed under ultraviolet light. The EcoR I digest of cosmid 25A10 was a complete digestion, but the sample of cosmid 26A5 was only partially digested under these conditions. The agarose gel containing the DNA fragments was subjected to depurination, denaturation and neutralization, followed by Southern blotting onto a Magna NT nylon membrane, using a high salt (20X
SSC) protocol, all as described in section 2.9 of Ausubel et al.
(CPMB, op. cit. ) . The transferred DNA was then W -crosslinked to the nylon membrane as before.
An TcaC-peptide specific DNA fragment corresponding to the insert of plasmid isolate GZ4 was amplified by PCR in a 100 ml reaction volume as described previously above. The amplification products from three such reactions were pooled and were extracted from a 1~ GTG agarose gel by Qiaex kit, as described above, and quantitated by fluorometry. The gel-purified DNA (100 ng) was labeled with 32P-dCTP using the High Prime Labeling Mix (Boehringer Mannheim) as described above, to a specific activity of 6.34 x loB
dpm/~g.
The 32P-labeled GZ4 probe was boiled 10 min, then added to ~minimal hyb" buffer (at 1 ng/ml), and the Southern blot membrane containing the digested cosmid DNA fragments was added, and 30 incubated for 4 hr at 60~C with gentle shaking at 50 rpm. The membrane was then washed 3 times at 25~C for about 5 min each (minimal hyb wash solution), followed by two washes for 30 min each at 60~C. The blot was exposed to film (with enhancer screens) for about 30 min at -70~C. The GZ4 probe hybridized strongly to the 5.0 kbp (apparent size) EcoR I fragment of both these two cosmids, 26A5 and 25A10.
The membrane was stripped of radioactivity by boiling for about 30 min in O.lX SSC plus 0.1 % SDS, and absence of radiolabel was checked by exposure to film. It was then hybridized at 60~C
for 3.5 hours with the (denatured) TcaBi probe in "minimal hyb"
buffer previously used for screening the colony membranes (above~, washed as described previously, and exposed to film for 40 min at -SUBSTITUTE S~EET (RULE 26) CA 022638l9 l999-02-26 W098/08932 PCT~US97/07657 70~C with two enhancer screens. With both cosmids, the TcaBi probe hybridized lightly with the about 5.0 kbp EcoR 1 fragment, and strongly with a fragment of approximately 2.9 kbp.
The sample of cosmid 26A5 DNA previously described, (from DH5a cells) was used as the source of DNA from which to subclone the bands of interest. This DNA (2.5 ~g) was digested with about 3 units of EcoR I (NEB) in a total volume of 30 ~l for 1.5 hr, to give a partial digest, as confirmed by gel electrophoresis. Ten ~g of pBC KS (+) DNA (Stratagene) were digested for 1.5 hr with 20 units of EcoR I in a total volume of 20 ~l, leading to total digestion as confirmed by electrophoresis. Both ~coR I-cut DNA
preparations were diluted to 50 ~l with water, to each an equal volume of PCI was added, the suspension was gently mixed, spun in a microcentrifuge and the aqueous supernatant was collected. DNA was 15 precipitated by 150 ~l ethanol, and the mixture was placed at -20~C
overnight. Following centrifugation and drying, the EcoR I -digested pBC KS (+) was dissolved in 100 ~l TE; the partially digested 26A5 was dissolved in 20 ~l TE. DNA recovery was checked by fluorometry.
In separate reactions, approximately 60 ng of EcoR I -digested pBC KS(+) DNA was ligated with approximately 180 ng or 270 ng of partially digested cosmid 26A5 DNA. Ligations were carried out in a volume of 20 ~l at 15~C for 5 hr, using T4 ligase and buffer from New England BioLabs. The ligation mixture, diluted to 100 ~l with sterile TE, was used to transform frozen, competent DH5a cells (Gibco BRL) accordiny to the supplier's instructions. Varying amounts (25-200 ~l) of the transformed cells were plated on freshly prepared solid LB-Cam3smedium with 1 mM IPTG and 50 mg/l X-gal.
PlaFës were-incubated at 37~C about 20 hr, then chilled in the dark for approximately 3 hr to intensify color for insert-~election.
White colonies were picked onto patch plates of the same composition and incubated overnight at 37~C.
Two colony lifts of each of the selected patch plates were prepared as follows. After picking white colonies to fresh plates, round Magna NT nylon membranes were pressed onto the patch plates, the membrane was lifted off, and subjected to denaturation, neutralization and W crosslinking a~ described above for the library colony membranes. The crosslinked colony lifts were vigorously washed, including gently wiping off the excess cell debris with a tissue. One set was hybridized with the GZ4(TcaC) probe solution described earlier, and the other set was hybridized with the TcaBi probe solution described earlier, accordiny to the SU~ST~TUTE 5H~ET (RULE 26) 'mlnim~l hyb' protocol, followed by washing and film exposure as described for the library colony membranes.
Colonies showing hybridization signals either only with the GZ4 probe, with both GZ4 and TcaBi probes, or only with the TcaBi probe, were selected for further work and cells were streaked for single colony isolation onto LB-Cam3s media with IPTG and X-gal as before. Approximately 35 single colonies, from 16 different isolates, were picked into liquid LB-Cam3s media and grown overnight at 37~C; the cells were collected by centrifugation and plasmid DNA
was isolated by a standard alkaline lysis miniprep according to Maniatis et al. (op. cit. p. 368). DNA pellets were dissolved in TE + 25 ~g/ml ribonuclease A and DNA concentration was determined by fluorometry. The EcoR I digestion pattern was analyzed by gel electrophoresis. The following isolates were picked as useful.
Isolate A17.2 contains religated pBC KS(+) only and was used for a (negative) control. Isolates D38.3 and C44.1 each contain only the 2.9 kbp, TcaBi -hybridizing EcoR I fragment inserted into pBC
KS(+). These plasmids, named pDAB2000 and pDAB2001, respectively, are illustrated in Fig. 2.
Isolate A35.3 contains only the approximately 5 kbp, GZ4)-hybridizing EcoR 1 fragment, inserted into pBC KS(+). This plasmid was named pDAB2002 (also Fig. 2). These isolates provided templates for DNA sequencing.
Plasmids pDAB2000 and pDAB2001 were prepared using the BIGprepTM kit as before. Cultures (30 ml) were grown overnight in TB-Cam3s to an OD600 of 2, then plasmid was isolated according to the manufacturer's directions. DNA pellets were redissolved in 100 ~1 TE each, and sample integrity was checked by EcoR I digestion and gel electrophoretic analysis.
Sequencing reactions were run in duplicate, with one replicate using as template pDAB2000 DNA, and the other replicate using as template pDAB2001 DNA. The reactions were carried out using the dideoxy dye terminator cycle sequencing method, as described above for the sequencing of the GZ4/HB14 DNAs. Initial sequencing runs utilized as primers the LacZ and T7 primers described above, plus primers based on the determined sequence of the TcaBi PCR
amplification product (TH1 = ATTGCAGACTGCCAATCGCTTCGG, TH12 =
GAGAGTATCCAGACCGCGGATGATCTG).
After alignment and editing of each sequencing output, each 40 was truncated to between 250 to 350 bases, depending on the integrity of the chromatographic data as interpreted by the Perkin Elmer Applied Biosystems Division SeqEd 675 software. Subsequent SUBSTITUTE S~t EE~ (RULE 26) CA 022638l9 l999-02-26 W098/08932 PCTrUS97/076~7 sequencing "steps" were made by selecting appropriate sequence for new primers. With a few exceptions, primers (synthesized as described abo~e) were 24 bases in length with a 50% G+C
composition. Sequencing by this method was carried out on both strands of the approximately 2.9 kbp EcoR I fragment.
~ To further serve as template for DNA sequencing, plasmid DNA
from lsolate pDAB2002 was prepared by BIGprepTM kit. Sequencing ~ reactions were performed and analyzed as described above.
Initially, a T3 primer (pBS SK (+) bases 774-796:
CGCGCAATTAACCCTCACTAAAG) and a T7 primer (pBS KS (+) bases 621-643:
GCGCGTAATACGACTCACTATAG) were used to prime the sequencing reactions from the flanking vector sequences, reading into the insert DNA. Another set of primers, (GZ4F:
GTATCGATTACAACGCTGTCACTTCCCi TH13: GGGAAGTGACAGCGTTGTAATCGATAC;
TH14: ATGTTGGGTGCGTCGGCTAATGGACATAAC; and LW1-204:
GGGAAGTGACAGCGTTGTAATCGATAC) was made to prime from internal sequences, which were determined previously by degenerate oligonucleotide-mediated sequencing of subcloned TcaC-peptide PCR
products. From the data generated during the initial rounds of sequencing, new sets of primers were designed and used to walk the entire length of the about 5 kbp fragment. A total of 55 oligo primers was used, enabling the identification of 4832 total bp of contiguous sequence.
When the DNA sequence of the EcoR I fragment insert of pDAB2002 is combined with part of the determined sequence of the pDAB2000/pDAB2001 isolates, a total contiguous sequence of 6005 bp was generated (disclosed herein as SEQ ID NO:25). When long open reading frames were translated into the corresponding amino acids, the sequence clearly shows the TcaBi N-terminal peptide (disclosed as SEQ ID NO:3), encoded by bases 68-124, immediately following a methionine residue (start of translation). Upstream lies a potential ribosome binding site (bases 51-58), and downstream, at bases 215-277 is encoded the TcaBi-PTl58 internal peptide (disclosed herein as SEQ ID NO:19). Further downstream, in the same reading frame, at bases 1787-1822, exists a sequence encoding the TcaBi-PT108 internal peptide (disclosed herein as SEQ ID
NO:20). Also in the same reading frame, at bases 1946-1972, is encoded the TcaBii N-terminal peptide (disclosed herein as SEQ ID
- NO:5), and the reading frame continues uninterrupted to a translation termination codon at nucleotides 3632-3634.
The lack of an in-frame stop codon between the end of the sequence encoding TcaBi -PT108 and the start of the TcaBii encoding SUBSTlTUTE S~EE~ ~RULE 26) CA 022638l9 l999-02-26 W098/08932 PCTrUS97/076S7 region, and the lack of a discernible ribosome binding site immediately upstream of the TcaBii coding region, indicate that peptides TcaBii and TcaBi are encoded by a single open reading frame of 3567 bp beginning at base pair 65 in SEQ ID N0:25), and are most likely derived from a single primary gene product TcaB of 1189 amino acids (131,586 Daltons; disclosed herein as SEQ ID
NO:26) by post-translational cleavage. If the amino acid immediately preceding the TcaBii N-terminal peptide represents the C-terminal amino acid of peptlde TcaBi, then the predicted mass of 10 TcaBii (627 amino acids) is 70,814 Daltons (disclosed herein as SEQ
ID N0:28), somewhat higher than the size observed by SDS-PAGE ~68 kDa). This peptide would be encoded by a contiguous stretch of 1881 base pairs (disclosed herein as SEQ ID N0:27). It is thought that the native C-terminus of TcaBi lies somewhat closer to the C-15 terminus of TcaBi-PT108. The molecular mass of PT108 [3.438 kDa;
determined during N-terminal amino acid sequence analysis of this peptide] predicts a size of 30 amino acids. Using the size of this peptide to designate the C-terminus of the TcaBi coding region ~Glu at position 604 of SEQ ID NO:28], the derived size of TcaBi is 20 determined to be 604 amino acids or 68,463 Daltons, more in agreement with experimental observations.
Translation of the TcaBii peptide coding region of 1686 base pairs (disclosed herein as SEQ ID N0:29) yields a protein of 562 amino acids (disclosed herein as SEQ ID N0:30) with predicted mass 25 of 60,789 Daltons, which corresponds well with the observed 61 kDa.
A potential ribosome binding site (bases 3682-3687) is found 48 bp downstream of the stop codon for the tcaB open reading frame.
At bases 3694-3726 is found a sequence encoding the N-terminus of peptlde TcaC, (disclosed as SEQ ID NO.2). The open reading frame initiated by this N-terminal peptide continues uninterrupted to base 6005 (2361 base pairs, disclosed herein as the first 2361 base pairs of SEQ ID N0.31). A gene ( tcaC) encoding the entire TcaC
peptide, (apparent size about 165 kDa; about 1500 amino acids), would comprise about 4500 bp.
Another isolate containing cloned EcoR I fragments of cosmid 26A5, E20.6, was also identified by its homology to the previously mentioned GZ4 and TcaBi probes. Agarose gel analysis of EcoR I
digests of the DNA of the plasmid harbored by thls strain (pDAB2004, Fig. 2), revealed lnsert fragments of estimated sizes 40 2.9, 5, and 3.3 kbp. DNA sequence analysis initiated from primers designed from the sequence of plasmid pDA~32002 revealed that the SUBSTITU~E S~EET (RULE 26) CA 022638l9 l999-02-26 W098~8932 PCT~US97/07657 3.3 kbp EcoR I fragment of pDAB2004.lies adjacent to the 5 kbp EcoR
I fragment represented in pDAB2002. The 2361 base pair open reading frame discovered ln pDAB2002 continues uninterrupted for another 2094 bases in pD~32004 [disclosed herein as base pairs 2362 to 4458 of SEQ ID NO:31]. DNA sequence analysis using the parent ~ cosmid 26A5 DNA as template confirmed the continuity of the open reading frame. Altogether, the open reading frame (tcaC SEQ ID
NO:31) comprises 4455 base pairs, and encodes a protein (TcaC) of 1485 amino acids [disclosed herein as SEQ ID NO:32]. The calculated molecular size of 166,214 Daltons is consistent with the estimated size of the TcaC peptide (165 kDa), and the derived amino acid sequence matches exactly that disclosed for the TcaC N-terminal sequence [SEQ ID NO:2].
The lack of an amino acid sequence corresponding to SEQ ID
NO:17; used to design the degenerate oligonucleotide primer pool in the discovered sequence indicates that the generation of the PCR~
products found in isolates GZ4 and HB14, which were used as probes in the initial library screen, were fortuitously generated by reverse-strand priming by one of the primers in the degenerate pool. Further, the derived protein sequence does not include the internal fragment disclosed herein as SEQ ID NO:18. These sequences reveal that plasmid pDAB2004 contains the complete coding region for the TcaC peptide.
Further analysis of SEQ ID NO:25 reveals the end of an open reading frame (bases 1-43), which encodes the final 13 amino acids of the TcaAili peptide, disclosed herein as SEQ ID NO:35. Only 24 bases separate the end of the TcaAiii coding region and the start of the TcaBi coding region. Included within the 24 bases are sequences that may serve as a ribosome binding site. Although possible, it is not likely that a Photorhabdus gene promoter is encoded within this short region. We propose that genomic region tca, which includes three long open reading frames [ tcaA tSEQ ID
NO:33), tcaB (SEQ ID NO:25, bases 65-36334), and tcaC (SEQ ID
NO:31),which is separated from the end of tcaB by only 59 bases] is regulated as an operon, with transcription initiating upstream of the start of the tcaA gene (SEQ ID NO:33), and resulting in a polycistronic messenger RNA.

SUBSTITUTE SHEET (RULE 26) CA 022638l9 l999-02-26 W098/08932 PCTrUS97/07657 Scre~ning of the Photorh~hdus G~n~mic Library for G~nes ~ncoding the TcbAi Peptide This example describes a method used to identify DNA clones that contain the TCbAii peptide-encoding genes, the isolation of the gene, and the determination of its partial DNA base sequence.

Primers and PCR Reacti~n~
The TcbAii polypeptide of the insect active preparation is about 206 kDa. The amino acid sequence of the N-terminus of this peptide is disclosed as SEQ ID NO:1. Four pools of degenerate oligonucleotide primers ("Forward primers~: TH-4, TH-5, TH-6, and TH-7) were synthesized to encode a portion of this amino acid sequence, as described in Example 8, and are shown below.

T~hle 12 Amino Acid Phe Ile Gln Gly Tyr Ser Asp Leu Phe TH-4 5'-TT(T/C) ATI CA(A/G) GGI TA(T/C) TCI GA(T/C) CTI TT-3' TH-5 5'-TT(T/C) ATI CA(A/G) GGI TA(T~C) AG(T/C) GA(T/C) CTI TT-3' TH-6 S'-TT(T/C) ATI CA(A/G) GGI TA(T/C) TCI GA(T/C) TT(A/G) TT-3' TH-7 5'-TT(T/C) ATI CA(A/G) GGI TA(T/C) AG(T/C) GA(T/C) TT(A/G) TT-3' In addition, a primary ("a") and a secondary ("b") sequence of an internal peptide preparation (TcbAii-PT81) have been determined and are disclosed herein as SEQ ID NO:23 and SEQ ID NO:24, respectively. Four pools of degenerate oligonucleotides ("Reverse Primers": TH-8, TH-9, TH-10 and TH-11) were similarly designed and synthesized to encode the reverse complement of sequences that encode a portion of the peptide of SEQ ID NO:23, as shown below.

SUBSTITUTE S~ E~ (RULE 26) -al H H H H
V ~) U U

H H H H
U U ~ U

U U U U
~ ~ _ U U U U
~ ~ -- _ ~
C~ U U C- U

E~

H H
~ C~ U U

)~I H H H H

~ H -- H

~ _ _ _ H H H H

~, O

SUBSTrrUTE S~EET tRULE 26) , . .. .

CA 022638l9 l999-02-26 W098/08932 PCTrUS97tO7657 Sets of these primers were used in PCR reactions to amplify TcbAii- encoding gene fragments from the genomic Photorhabdus luminescens W-14 DNA prepared in Example 6. All PCR reactions were run with the "Hot Start" technique using AmpliWaxTM gems and other Perkin Elmer reagents and protocols. Typically, a mixture (total volume 11 ~l) of MgCl2, dNTP's, 10X GeneAmp PCR Buffer II, and the primers were added to tubes containing a single wax bead. [10X
GeneAmp PCR Buffer II is composed of 100 mM Tris-HCl, pH 8.3; and 500 mM KCl.] The tubes were heated to 80~C for 2 minutes and allowed to cool. To the top of the wax seals, a solution containing 10X GeneAmp PCR Buffer II, DNA template, and AmpliTaq DNA polymerase were added. Following melting of the wax seal and mixing of components by thermal cycling, final reaction conditions (volume of 50 ~l) were: 10 mM Tris-HCl, pH 8.3; 50 mM KCl; 2.5 mM
MgCl2; 200 ~M each in dATP, dCTP, dGTP, dTTP; 1.25 mM in a single Forward primer pool; 1.25 ~M in a single Reverse primer pool, 1.25 units of AmpliTaq DNA polymerase, and 170 ng of template DNA.
The reactions were placed in a thermocycler (as in Example 8) and run with the following program:
Table 14 Temperature Time Cycle Repetition 94~C 2 minutes lX

94~C 15 seconds 55-65~C 30 seconds 30X

72~C 1 minute 72~C ~ m1nutes lX

15~C Constant A series of amplifications was run at three different annealing temperatures (55~, 60O, 65OC) using the degenerate primer SUBSTiTt.JTE S~EET (RULE 26) CA 022638l9 l999-02-26 W098/08932 PCTrUS97/07657 pools. Reactions with annealing at 65~C had no amplification products visible following agarose gel electrophoresis. Reactions having a 60~C annealing regime and containing primers TH-5+TH-10 produced an amplification product that had a mobility corresponding to 2.9 kbp. A lesser amount of the 2.9 kbp product was produced - under these conditions with primers TH-7+TH-lO. When reactions were annealed at 55~C, these primer pairs produced more of the 2.9 - k~p product, and this product was also produced by primer pairs TH-5+TH-8 and TH-5+TH-11. Additional very faint 2.9 kbp bands were seen in lanes containing amplification products from primer pairs TH-7 plus TH-8, TH-9, TH-10, or TH-ll.
To obtain sufficient PCR amplification product for cloning and DNA sequence determination, 10 separate PCR reactions were set up using the primers TH-5+TH-10, and were run using the above conditions with a 55~C annealing temperature. All reactions were pooled and the 2.9 kbp product was purified by Qiaex extraction from an agarose gel as described above.
Additional sequences determined for TcbAii internal peptides are disclosed herein as SEQ ID NO:21 and SEQ ID NO:22. As before, degenerate oligonucleotides (Reverse primers TH-17 and TH-18) were made corresponding to the reverse complement of sequences that encode a portion of the amino acid sequence of these peptides.
T~hle 15 From SEO ID NO:21 Amino Acid Met Glu Thr Gln Asn Ile Gln Glu Pro TH-17 3'-TAC CTT/C TGI GTT/C TTA/G TAI GTT/C GTT/C GG-5' T~hle 16 From S~O ID NO:22 Amino Acid AsnPro Ile Asn Ile Asn Thr Gly Ile Asp TH-18 3'-TT(A/G) GGI TAI TT(A/G) TAI TT(A?G) TGI CCI TAI CT(A/G)-5' Degenerate oligonucleotides TH-18 and TH-17 were used in an amplification experiment with Photorhabdus luminescens W-14 DNA as template and primers TH-4, TH-5, TH-6, or TH-7 as the 5'- (Forward) primers. These reactions amplified products of approximately 4 kbp and 4.5 kbp, respectively. These DNAs were transferred from agarose gels to nylon membranes and hybridized with a 32P-labeled probe (as described above~ prepared from the 2.9 kbp product SU8STITUTE SHEFr (RULE 2fi) CA 022638l9 l999-02-26 amplified by the TH-5+TH10 primer p~ir. Both the 4 kbp and the 4.5 kbp amplification products hybridized strongly to the 2.9 kbp probe. These re9ults were used to construct a map ordering the TcbAii internal peptide sequences as shown in Fig. 3. Approximate distances between the primers are shown in nucleotides in Fig. 3.

DNA Se~uence of ~he 2.9 kbp TcbAii-encoding Fragment Approximately 200 ng of the purified 2.9 kbp fragment ~prepared above) was precipitated with ethanol and dissolved in 17 ml of water. One-half of this was used as sequencing template with 25 pmol of the TH-5 pool as primers, the other half was used as template for T~-10 priming. Sequencing reactions were as given in Example 8. No reliable sequence was produced using the TH-10 primer pool; however, reactions with TH-5 primer pool produced the sequence disclosed below:.
1 AATC~l~ll'~ ATCCCTATGC CGNGCCGGGT TCGGTGGAAT CGATGTCCTC ACCGGGGGTT
61 TATTNGAGGG ANTNGTCCCG TGAGGCCAAA AANTGGAATG AAAGAAGTTC AAl"rlNllAC

241 GGAAATNCAC AAGTTGAGGT GAl~illG TNGCNANCTT ~'l'C'~lllAGG TGGGGAGAAA
301 C~ Nl~ANC A~Nll~l~A AACTGTCCGG GAAATCGTCC ATGANCGTGA NCCAGGNTTN

Based on this sequence, a sequencing primer (TH-21, 5'-CCGGGCGACGTTTATCTAGG-3') was designed to reverse complement bases 120-139, and initiate polymerization towards the 5' end (i.e., T~-5 end) of the gel-purified 2.9 kbp TcbAii-encoding PCR fragment. The determined sequence is shown below, and is compared to the biochemically determined N-terminal peptide sequence of TcbAii SEQ
ID NO:l.
TcbAii 2.9 kbp PCR Fraqment Sequence Confirmation [Underlined amino acids = encoded by degenerate oligonucleotides]
SEQ ID NO:1 F I O G Y S D L F G - - A
2.9 kbp seq GC ATG CAG GGG TAT AGT GAC CTG TTT GGT AAT CGT GCT
M Q G Y S D L F G N R A >
From the homology of the derived amino acid sequence to the biochemically determined one, it is clear that the 2.9 kbp PCR
fragment represents the Tc~A coding region. This 2.9 kbp fragment was then used as a hybridization probe to screen the Photorhabdus W-14 genomic library prepared in Example a for cosmids containing the TcbAii-encoding gene.

SUBST~TUTE S~tlEET (RULE 26) CA 022638l9 l999-02-26 W098/08932 PCTrUS97/07657 Scre~nina the Photorh~hdus Cosmid T,i hr~ry The 2.9 kb gel-purified PCR fragment was labeled with 32p using the Boehringer Mannheim High Prime labeling kit as described in Example 8. Filters containing remnants of approximately 800 colonies from the cosmid library were screened as described ~ previously (Example 8), and positive clones were streaked for isolated colonies and rescreened. Three clones (8A11, 25G8, and . 26D1) gave positive results through several screening and characterization steps. No hybridization of the TcbAii-specific probe was ever observed with any of the four cosmids identified in Example 8, and which contain the tcaB and tcaC genes. DNA from cosmids 8A11, 25G8, and 26Dl was digested with restriction enzymes Bgl II, EcoR I or ~ind III (either alone or in combination with one another), and the fragments were separated on an agarose gel and transferred to a nylon membrane as described in Example 8. The membrane was hybridized with l2P-labeled probe prepared from the 4.5 kbp fragment (generated by amplification of Photorhabdus genomic DNA with primers TH-5+TH-17). The patterns generated from cosmid DNAs 8All and 26Dl were identical to those generated with similarly-cut genomic DNA on the same membrane. It is concluded that cosmids 8All and 26Dl are accurate representations of the genomic TcbAii encoding locus. However, cosmid 25G8 has a single Bgl II fragment which is slightly larger than the genomic DNA.
This may result from positioning of the insert within the vector.
DNA ~equence of the tcbA- encodin~ Gene The membrane hybridization analysis of cosmid 26Dl revealed that the 4.5 kbp probe hybridized to a single large EcoR I
fragment (greater than 9 kbp). This fragment was gel purified and ligated into the EcoR I site of pBC KS (+) as described in Example 8, to generate plasmid pBC-S1/R1. The partial DNA sequence of the insert DNA of this plasmid was determined by "primer walking" from the flanking vector sequence, using procedures described in Example 8. Further sequence was generated by extension from new oligonucleotides designed from the previously determined sequence.
When compared to the determined DNA sequence for the ~cbA gene identlfied by other methods (disclosed herein as SEQ ID N0:11 as described in Example 12 below), complete homology was found to nucleotides 1-272, 319-826, 2578-3036, and 3068-3540 (total bases =
1712). It was concluded that both approaches can be used to identify DNA fragments encoding the TcbAii peptide.

SUBSTITUTE St~E~T tRUL~ 26) W098/08932 PCT~US97/07657 Analysis of the Derived Ami n~ Acid Sequence of the tc~A Gene The sequence of the DNA fragment identified as SEQ ID NO:ll encodes a protein whose derived amino acld sequence is disclosed herein as SEQ ID NO:12. Several features verify the identity of the gene as that encoding the TcbAii protein. The TcbAii N-terminal peptide (SEQ ID NO:l; Phe Ile Gln Gly Tyr Ser Asp Leu Phe Gly Asn Arg Ala) is encoded as amino acids 88-100. The TcbAii internal peptide TcbAii-PT81(a) (SEQ ID NO:23) is encoded as amino acids 1065-1077, and TcbAii-PT81(b) (SEQ ID NO:24) is encoded as amino acids 1571-1592. Further, the internal peptide TcbAii-PT56 (SEQ ID NO:22) is encoded as amino acids 1474-1488, and the internal peptide TCbAii-pTlo3 (SEQ ID NO:21) is encoded as amino acids 1614-1639. It is obvious that this gene is an authentic clone encoding the TCbAii peptide as isolated from insecticidal protein preparations of Photorhabdus luminescens strain W-14.
The protein isolated as peptide TcbAii is derived from cleavage of a longer peptide. Evidence for this is provided by the fact that the nucleotides encoding the TcbAii N-terminal peptide SEQ ID NO:1 are preceded by 261 ~ases (encoding 87 N-terminal-proximal amino acids) of a longer open reading frame (SEQ ID
NO:11). This reading frame begins with nucleotides that encode the amino acid sequence Met Gln Asn Ser Leu, which corresponds to the N-terminal sequence of the large peptide TcbA, and is disclosed herein as SEQ ID NO:16. It is thought that TcbA is the precursor protein for TcbAii-Relationship of tc~A, tcaB and tcaC G~nes The tcaB and tcaC genes are closely linked and may betranscribed as a single mRNA (Example 8). The tcbA gene is borne on cosmids that apparently do not overlap the ones harboring the tcaB and tcaC cluster, since the respective genomic library screens identified different cosmids. ~owever, comparison of the amino sequences encoded by the tcaB and tcaC genes with the tcbA gene reveals a substantial degree of homology. The amino acid conservation {Protein Alignment Mode of MacVectorT~ Sequence Analysis Software, scoring matrix pam250, hash value = 2; Oxford Molecular Group, Campbell, CA) is shown in Fig. 4. On the score line of each panel in Fig. 4, up carats (~) indicate homology or conservative amino acid changes,and down carats (v) indicate nonhomology.

SUBSTTTUTE S~ EET (RULE-26~

.. . .

CA 022638l9 l999-02-26 W098/08932 rCT~US97/07657 This analysis shows that the amino acid sequence of the TcbA
peptide from residues 1739 to 1894 is highly homologous to amino acids 441 to 603 of the TcaBi peptide ~162 of the total 627 amino acids of TcaB; SEQ ID NO:28). In addition, the sequence of TcbA
amino acids 1932 to 2459 is highly homologous to amino acids 12 to 531 of peptide TcaBii (520 of the total 562 amino acids; SEQ ID
NO:30). Considering that the TcbA peptide (SEQ ID NO:12) comprises - 2505 amino acids, a total of 684 amino acids (27%) at the C-proximal end of it is homologous to the TcaBi or TcaBii peptides, and the homologies are arranged colinear to the arrangement of the putative TcaB preprotein (SEQ ID NO:26). A sizeable gap in the TcbA homology coincides with the junction between the TcaBi and TcaBii portions of the TcaB preprotein. Clearly the TcbA and TcaB
gene products are evolutionarily related, and it is proposed that they share some common function(s) in Photorhabdus.

~x~ple 10 Characteri~ation of ~inc-metalloproteases ~n Photorhabd~s Broth:
Protease Inhibition. Classification and Purification Protease Inhibition and Classification Assays: Protease assays were performed using FITC-casein dissolved in water as substrate (0.08% final assay concentration). Proteolysis reactions were performed at 25~C for 1 h in the appropriate buffer with 25 ~l of Photorhabdus broth (150 ~1 total reaction volume). Samples were also assayed in the presence and absence of dithiothreitol. After incubation, an equal volume of 12% trichloroacetic acid was added to p-rP-~lplta-te undigested protein. Following precipitation for 0.5 h and subsequent centrifugation, 100 ~l of the supernatant was placed into a 96-well microtiter plate and the pH of the solution was adjusted by addition of an equal volume of 4N NaOH.
Proteolysis was then quantitated using a Fluoroskan II fluorometric plate reader at excitation and emission wavelengths of 485 and 538 nm, respectively. Protease activity was tested over a range from 35 pH 5.0-10.0 in 0.5 units increments. The following buffers were used at 50 mM final concentration: sodium acetate (pH 5.0 - 6.5);
Tris-HCL (pH 7.0 - 8.0); and bis-Tris propane (pH 8.5-10.0). To identify the class of protease(s) observed, crude broth was treated with a variety of protease inhibitors (0.5 ~g/~l final concentration) and then examined for protease activity at pH 8.0 SUBSllTlJTE ~H EET (RULE 26) CA 022638l9 l999-02-26 using the substrate described above. The protease inhibitors used included E-64 (L-trans-expoxysaccinylleucylamido[4-,-guanidino]-butane), 3,4 dichloroisocoumarin, Leupeptin, pepstatin, amastatin, ethylenediaminetetraacetic acid (EDTA) and l,10 phenanthroline.
Protease assays performed over a pH range revealed that indeed protease(s) were present which exhibited m~im~l activity at about pH 8.0 (Table 17). Addition of DTT did not have any effect on protease activity. Crude broth was then treated with a variety of protease inhibitors (Table 18). Treatment of crude broth with the inhibitors described above revealed that 1,10 phenanthroline caused complete inhibition of all protease activity when added at a final concentration of 50 ~g, with the ICso = 5 ~g in 100 ~l of a 2 mg/ml crude broth solution. These data indicate that the most abundant protease(s) found in the Photorhabdus broth are from the zinc-metalloprotease class of enzymes.
T~hle 17 Effect of pH on the Protease Activity Found in a Day 1 Productionof Photorhabdus luminescens ~Strain W-14 pH Flu. Unitsa Percent Activityb 5.0 3013 i 78 17 5.5 7994 i 448 45 6.0 12965 i483 74 30 6.5 14390 +1291 82 7.0 14386 i1287 82 7.5 14135 i198 80 8.0 17582 i831 100 8.5 16183 i953 92 40 9.0 16795 i760 96 9 5 16279 i1022 93 10.0 15225 i210 87 45 a ~lu. unlts = ~luorescence unlts ~Maxlmum = about ~ o~;
background = about 2200).
b Percent activity relative to the maximum at pH 8.0 SUBSTrrUTE S}~ ~ (RULE 26) W098l08932 PCT~US97/07657 T~hle 18 ~ffect Qf Different Protease Inhihitors on the Protease Activity at pH 8 Fol~nd i~ a Day 1 Production of photorh~hdu5 l~minescens (Strain W-14) - Inhibitor Corrected Flu. Unitsa Percent Inhibition~
Control 13053 0 E-64 14259 o 10 1,10 PhenanthrolineC 15 99 3,4 Dichloroisocoumarind 7956 39 Leupeptin 13074 o PepstatinC 13441 o Amastatin 12474 4 15 DMSO Control 12005 8 Methanol Control12125 7 a ~orrecte~ ~lu. un1ts = ~luorescence Unlts - bac~groun~jZ~
flu. units).
b Percent Inhibition relative to protease activity at pH 8Ø
c Inhibitors were dissolved in methanol.
d Inhibitors were dissolved in DMSO.

The isolation of a zinc-metalloprotease was performed by applying dialyzed 10-80~ ammonium sulfate pellet to a Q Sepharose 25 column equilibrated at 50 mM Na2PO4, pH 7.0 as described in Example 5 for Photorhabdus toxin. After extensive washing, a 0 to 0.5 M
NaC1 gradient was used to elute toxin protein. The majority of biological activity and protein was eluted from 0.15 - 0.45 M NaCl.
However, it was observed that the majority of proteolytic activity 30 was present in the 0.25-0.35 M NaCl fraction with some activity in the 0.15-0.25 M NaCl fraction. SDS PAGE analysis of the 0.25-0.35 M NaCl fraction showed a major peptide band of approximately 60 kDa. The 0.15-0.25 M NaC1 fraction contained a similar 60 kDa band but at lower relative protein concentration. Subsequent gel 35 filtration of this fraction using a Superose 12 HR 16/50 column resulted in a major peak migrating at 57.5 kDa that contained a predominant (> 90% of total stained protein) 58.5 kDa band by SDS
PAGE analysis. Additional analysis of this fraction using various protease inhibitors as described above determined that the protease was a zinc-metalloprotease. Nearly all of the protease activity present in Photorhabdus broth at day 1 of fermentation corresponded to the about 58 kDa zinc-metalloprotease.
In yet a second isolation of zinc-metalloprotease(s), W-14 Pho~orhabdus broth grown for three days was taken and protease 4s activity was visualized uslng sodium dodecyl sulfate-polyacrylamide SUBSTrrUTE S~tE~T (RUL~ 26) CA 022638l9 l999-02-26 W098/08932 PCT~US97/07657 gel electrophoresis ~SDS-PAGE) laced with gelatin as described in Schmidt, T.M., Bleakley, B. and Nealson, K.M. 1988. SDS running gels (5.5 x 8 cm) were made with 12.5 % polyacrylamide (40% stock solution of acrylamide/bis-acrylamide; Sigma Chemical Co., St.
Louis, MO) into which 0.1% gelatln final concentration (Biorad EIA
grade reagent; Richmond CA) was incorporated upon dissolving in water. SDS-stacking gels (1.0 x 8 cm) were made with 5~
polyacrylamide, also laced with 0.1~ gelatin. Typically, 2.5 ~g of protein to be tested was diluted in 0.03 ml of SDS-PAGE loading buffer without dithiothreitol (DTT) and loaded onto the gel.
Proteins were electrophoresed in SDS running buffer (Laemmli, U.K.
1970. Nature 227, 680) at 0~ C and at 8 mA. After electrophoresis was complete, the gel was washed for 2 h in 2.5~ (v/v) Triton X-100. Gels were then incubated for 1 h at 37 ~C in 0.1 M glycine (pH 8.0). After incubation, gels were fixed and stained overnight with 0.1% amido black in methanol-acetic acid- water (30:10:60, vol /vol./vol.; Sigma Chemical Co.). Protease activity was visualized as light areas against a dark, amido black stained background due to proteolysis and subsequent diffusion of incorporated gelatin. At least three distinct bands produced by proteolytic activity at 58-, 41-, and 38 kDa were observed.
Activity assays of the different proteases in W-14 day three culture broth were performed using FITC-casein dissolved in water as substrate (0.02~ final assay concentration). Proteolysis 25 experiments were performed at 37~C for 0-0.5 h in O.lM Tris-HCl (pH
8.0) with different protein fractions in a total volume of 0.15 ml.
Reactions were terminated by addition of an equal volume of 12%
trichloroacetic acid (TCA) dissolved in water. After incubation at room temperature for 0.25 h, samples were centrifuged at 10,000 x g 30 for 0.25 h and 0.10 ml aliquots were removed and placed into 96-well microtiter plates. The solution was then neutralized by the addition of an equal volume of 2 N sodium hydroxide, followed by quantitation using a Fluoroskan II fluorometric plate reader with excitation and emission wavelengths of 485 and 538 nm, respectively. Activity measurements were performed using FITC-Casein with different protease concentrations at 37~C for o-lO min.
A unit of activity was arbitrarily defined as the amount of enzyme needed to produce 1000 fluorescent units/min and specific activity was defined as units/mg of protease.

SU85TI~UTE SH EET (RULE 2~) , W098/08932 PCT~US97/07657 Inhibition studies were perfor~ed using two zinc-metalloprotease inhibitors; 1,10 phenanthroline and N-(a-rhamnopyranosyloxyhydroxyphosphinyl)-Leu-Trp(phosphoramidon) with stock solutions of the inhibitors dissolved in 100% ethanol and S water, respectively. Stoc~ concentrations were typically 10 mg/ml and 5 mg/ml for 1,10 phenanthroline and phosphoramidon, respectively, with final concentrations of inhibitor at 0.5-1.0 ~ mg/ml per reaction. Treatment of three day W-14 crude broth with 1,10 phenanthroline, an inhibitor of all zinc metalloproteases, resulted in complete elimination of all protease activity while treatment with phosphoramidon, an inhibitor of thermolysin-like proteases (Weaver, L.H., Kester, W.R., and Matthews, B.W. 1977. J.
Mol. Biol. 114, 119-132), resulted in about 56% reduction of protease activity. The residual proteolytic activity could not be further reduced with additional phosphoramidon.
The proteases of three day W-14 Photorhabdus broth were purified as follows: 4.0 liters of broth were concentrated using an Amicon spiral ultra filtration cartridge Type SlY100 attached to an Amicon M-12 filtration device. The flow-through material having native proteins less than 100 kDa in size (3.8 L) was concentrated to 0.375 L using an Amicon spiral ultra filtration cartridge Type SlY10 attached to an Amicon M-12 filtration device. The retentate material contained proteins ranging in size from 10-100 kDa. This material was loaded onto a Pharmacia HR16/10 column which had been packed with PerSeptive Biosystem (Framington, MA) Poros~ 50 HQ
strong anion exchange packing that had been equilibrated in 10 mM
sodium phosphate buffer (pH 7.0). Proteins were loaded on the column at a flow rate of 5 ml/min, followed by washing unbound protein with buffer until A280 = 0.00. Afterwards, proteins were eluted using a NaCl gradient of 0-1.0 M NaCl in 40 min at a flow rate of 7.5 ml/min. Fractions were assayed for protease activity, supra., and active fractions were pooled. Proteolytically active fractions were diluted with 50% (v/v) 10 mM sodium phosphate buffer (pH 7.0) and loaded onto a Pharmacia HR 10/10 Mono Q column equilibrated in 10 mM sodium phosphate. After washing the column with buffer until A280 = 0.00, proteins were eluted using a NaCl gradient of 0-0.5 M NaCl for 1 h at a flow rate of 2.0 ml/min.
Fractions were assayed for protease activity. Those fract1ons having the greatest amount of phosphoramidon-sensitive protease SU85TlTlJTE SHEET tRULE 26) CA 022638l9 l999-02-26 W098/08932 PCT~US97/07657 activity, the phosphoramidon sensitive activity being due to the 41/38 kDa protease, infra., were pooled. These fractions were found to elute at a range of 0.15-0.25 M NaC1. Fractions containing a predominance of phosphoramidon-insensitive protease activity, the 58 kDa protease, were also pooled. These fractions were found to elute at a range of 0.25-0.35 M NaCl. The phosphoramidon-sensitive protease fractions were then concentrated to a final volume of 0.7S ml using a Millipore Ultrafree~-15 centrifugal filter device Biomax-5K NMWL membrane. This material 10 was applied at a flow rate of 0.5 ml/min to a Pharmacia HR 10/30 column that had been packed with Pharmacla Sephadex G-50 equilibrated ln 10 mM sodium phosphate buffer (pH 7.0)/ 0.1 M NaCl.
Fractions having the maximal phosphoramidon-sensitive protease activity were then pooled and centrifuged over a Millipore Ultrafree~-15 centrifugal filter device Biomax-50K NMWL membrane.
Proteolytic actlvity analysis, supra., indicated this material to have only phosphoramldon-sensitive protease activity. Pooling of the phosphoramidon-insensitive-protease, the 58 kDa protein, was followed by concentrating in a Millipore Ultrafree~-15 centrifugal filter device Biomax-50~ NMWL membrane and further separation on a Pharmacia Superdex-75 column. Fractions containing the protease were pooled.
Analysis of purified 58- and 41/38 kDa purified proteases revealed that, while both types of protease were completely 25 inhiblted with 1,10 phenanthroline, only the 41/38 kDa protease was inhibited with phosphoramidon. Further analysis of crude broth indicated that protease activity of day 1 W-14 broth has 23% of the total protease activity due to the 41/38 kDa protease, increasing to 44~ in day three W-14 broth.
Standard SDS-PAGE analysis for ex~min'ng protein purity and obtaining amino terminal sequence was performed using 4-20~
gradient MiniPlus SepraGels purchased from Integrated Separation Systems (Natick, MA). Proteins to be amino-terminal sequenced were blotted onto PVDF membrane following purification, infra., (ProBlott~ Mem~ranes; Applied Biosystems, Foster City, CA), visualized with 0.1~ amido black, exclsed, and sent to Cambridge Prochem; Cam~ridge, MA, for sequencing.
Deduced amino terminal sequence of the 58- (SEQ ID NO:45) and 41/38 kDa (SEQ ID NO:44) proteases from three day old W-14 broth SUBSTTTIJTE SH FE7 (RULE 26) CA 022638l9 l999-02-26 WO ~l08932 PCT~US97/07657 were DV-GSEKANEKLK (SEQ ID NO: 45) and DSGDDDKVTNTDIHR (SEQ ID
NO:44), respectively.
Sequencing of the 41/38 kDa protease revealed several amino termini, each one having an additional amino acid removed by proteolysis. Examination of the primary, secondary, tertiary and quartenary sequences for the 38 and 41 kDa polypeptides allowed for deduction of the sequence shown above and revealed that these two ~ proteases are homologous.
~x~le 11. Part A
Screening o~ Photorhabdus Genomic Library Via Use of Antibodies for Genes Encodi~g TcbA Peptide In parallel to the sequencing described above, suitable '5 probing and sequencing was done based on the TcbAii peptide (SEQ ID
NO:1). This sequencing was performed by preparing bacterial culture broths and purifying the toxin as described in Examples l and 2 above.
Genomic DNA was isolated from the Photorhabdus luminescens strain W-14 grown in Grace's insect tissue culture medium. The bacteria were grown in 5 ml of culture medium in a 250 ml Erlenmeyer flask at 28~C and 250 rpm for approximately 24 hours.
Bacterial cells from 100 ml of culture medium were pelleted at 5000 x g for 10 minutes. The supernatant was discarded, and the cell pellets then were used for the genomic DNA isolation.
The genomic DNA was isolated using a modification of the CTAB
method described in Section 2.4.3 of Ausubel ( supra . ) . The section entitled "Large Scale CsCl prep of bacterial genomic DNA" was followed through step 6. At this point, an additional chloroform/isoamyl alcohol ~24:1) extraction was performed followed by a phenol/chloroform/isoamyl (25:24:1) extraction step and a final chloroform/isoamyl/alcohol (24:1) extraction. The DNA was precipitated by the addition of a 0.6 volume of isopropanol. The precipitated DNA was hooked and wound around the end of a bent glass rod, dipped brlefly into 70~ ethanol as a final wash, and dissolved in 3 ml of TE buffer.
The DNA concentration, estimated by optical density at 280/260 nm, was approximately 2 mg/ml.
Using this genomic DNA, a library was prepared. Approximately 50 ~g of genomic DNA was partly digested with Sau3 A1. Then NaCl density gradient centrifugation was used to size fractionate the partially digested DNA fragments. Fractions containing DNA

SUBSTITUTE ~HEET (RULE 26) ~ CA 022638l9 l999-02-26 W098/08932 PCT~S97/07657 fragments with an average size of 1~ kb, or larger, as determined by agarose gel electrophoresis, were ligated into the plasmid BluScript, Stratagene, La Jolla, California, and transformed into an E. coli DH5~ or DHB10 strain.
Separately, purlfied aliquots of the protein were sent to the biotechnology hybridoma center at the University of Wisconsin, Madison for production of monoclonal antibodies to the proteins.
The material that was sent was the HP~C purified fraction containing native bands 1 and 2 which had been denatured at 65~C, and 20 ~g of which was injected into each of four mice. Stable monoclonal antibody-producing hybridoma cell lines were recovered after spleen cells from unimmunized mouse were fused with a stable myeloma cell line. Monoclonal antibodies were recovered from the hybridomas.
Separately, polyclonal antibodies were created by taking natlve agarose gel purified band 1 (see Example 1) protein which was then used to immunize a New Zealand white rabbit. The protein was prepared by excising the band from the native agarose gels, briefly heating the gel pieces to 65~C to melt the agarose, and immediately emulsifying with adjuvant. Freund's complete adjuvant was used for the primary immunizations and Freund's incomplete was used for 3 additional injections at monthly intervals. For each injection, approximately 0.2 ml of emulsified band 1, containing 50 to 100 micrograms of protein, was delivered by multiple subcontaneous injections into the back of the rabbit. Serum was obtained 10 days after the final injection and additional bleeds were performed at weekly intervals for 3 weeks. The serum complement was inactivated by heating to 56~C for 15 minutes and then stored at -20~C.
The monoclonal and polyclonal antibodies were then used to screen the genomic library for the expression of antigens which could be detected by the epitope. Positive clones were detected on nitrocellulose filter colony lifts. An imm1]nohlot analysis of the positive clones was undertaken.
An analysis of the clones as defined by both immunoblot and Southern analysis resulted in the tentative identification of four genomic regions.
In the first region was a gene encoding the peptide designated here as TcbAii. Full DNA sequence of this gene ( tcbA) was obtained. It is set forth as SEQ ID NO:11. Confirmation that the se~uence encodes the internal sequence of SEQ ID NO:1 is d~ ~lstrated by the presence of SEQ ID NO:1 at amino acid number 88 SU85TI~UTE S~E T tRULE 26) W098/08932 rcTrusg7/o7657 from the deduced amino acid sequence created by the open reading frame of SEQ ID NO:ll. This can be confirmed~~by referring to SEQ
ID NO:12, which is the deduced amino acid sequence created by SEQ
ID NO:ll.
The second region of toxin peptides contains the segments referred to above as TcaBi, TcaB1i and TcaC. Following the screening of the library with the polyclonal antisera, this second region of toxin genes was identified by several clones which produced different size proteins, all of which cross-reacted with the polyclonal antibody on an imml~noblot and were also found to share DNA homology on a Southern Blot. Sequence comparison revealed that they belonged to the gene complex designated TcaB and TcaC above.
Two other regions of antibody toxin clones were also isolated in the polyclonal screen. These regions produced proteins that cross-react with a polyclonal antibody and also shared DNA homology with the regions as determined by Southern blotting. Thus, it appears that the Photorhabdus luminescens extracellular protein genes represent a family of genes which are evolutionarily related.
To further pursue the concept that there might be evolutionarily related variations in the toxin peptides contained within this organism, two approaches have been undertaken to examine other strains of Photorha~dus luminescens for the presence of related proteins. This was done both by PCR amplification of genomic DNA and by immunoblot analysis using the polyclonal and monoclonal antibodies.
The results indicate that related proteins are produced by Photorhabdus. luminescens strains WX-2, WX-3, WX-4, WX-5, WX-6, WX-7, WX-8, WX-11, WX-12, WX-15 and W-14.
Ex~mrle 11. Part B
Sequence and Analysis of tcc ~oxin Clones Further DNA sequencing was performed on plasmids isolated from E. coli clones described in Example 11, Part A. The nucleotide sequence from the third region of E. coll clones was shown to be - three closely linked open reading frames at this genomic locus.
This locus was designated tcc with the three open reading frames designated tccA SEQ ID NO:56, tccB SEQ ID NO:58 and tccC SEQ ID
NO:60. The close linkage between these open reading frames is revealed by examination of SEQ ID NO:56, in which 93 bp separate the stop codon of tccA from the start codon of tccb (bases 2992-2994 of SEQ ID NO:56), and by examination of SEQ ID NO:58, in which SUBSTITUTE SHEET (RULE 26) , .. , . ,~ . .. . .. .. ..

CA 022638l9 l999-02-26 W098/08932 PCT~US97/07657 131 bases separate the stop codon of tccB and the tccC (bases 4930-4932 of SEQ ID NO:58). The physical map is presented in Fig. 6B.
The deduced amino acid sequence from the tccA open reading frame indicates that the gene encodes a protein of 105,459 Da.
This protein was designated TccA (SEQ ID NO:57). The first 12 amino acids of this protein match the N-terminal sequence obtained from a 108 kDa protein, SEQ ID NO:8, previously identified as part of the toxin complex.
The deduced amino acid sequence from the tccB open reading 10 frame indicates that this gene encodes a protein of 175,716 Da.
This protein was designated TccB (SEQ ID NO:59). The first 11 amino acids of this protein match the N-terminal sequence obtained from a protein with estimated molecular weight of 185 kDa, SEQ ID
NO:7. Similarity analysis revealed that the TccB protein is related to the proteins identified as TcbA SEQ ID NO:12; 37% similarity and 28% identity, TcdA SEQ ID NO:47; 35~ similarity and 28%identity, and TcaB SEQ ID NO:26; 32~ similarity and 26% identity (using the GAP algorithm Wisconsin Package Version 9.0, Genetics Computer Group (GCG) Madison Wisconsin).
The deduced amino acid sequence of tccC indicated that this open reading frame encodes a protein of 111,694 Da and the protein product was designated TccC (SEQ ID NO:61).

Ex~rle 12 Characterization of ~hotorhabdus Strains In order to establish that the collection described herein was comprised of Photorhabdus strains, the strains herein were assessed in terms of recognized microbiological traits that are characteristic of Photorhabdus and which differentiate it from other Enterobacteriaceae and Xenorhabdus spp. (Farmer, J. J. 1984.
Bergey's Manual of Systemic Bacteriology, Vol 1. pp. 510-511. (ed.
Kreig N. R. and Holt, J. G.). Williams & Wilkins, Baltimore;
Akhurst and Boemare, 1988, Boemare et al., 1993). These characteristic traits are as follows: Gram's stain negative rods, organism size of 0.5-2 ~m in width and 2-10 ~m in length, red/yellow colony pigmentation, presence of crystalline inclusion bodies, presence of catalase, inability to reduce nitrate, presence of bioluminescence, ability to take up dye from growth media, positive for protease production, growth-temperature range below 37~C, survival under anaerobic conditions and positively motile.

SUBST~TUTE S}~EET tRULE 26) CA 022638l9 l999-02-26 W098/08932 PCT~US97/07657 (Table 20). Reference Escherichia coli, Xenorhabdus and Photorh~h~l7s strains were included in all tests for comparison.
The overall results are consistent with all strains being part of the family Enterobacteriaceae and the genus Photorhabdus.
A luminometer was used to establish the bioluminescence of each strain and provide a quantitative and relative measurement of light production. For measurement of relative light emitting units, the broths from each strain (cells and media) were measured at three time intervals after inoculation in liquid culture (6, 12, and 24 hr) and compared to background luminosity (uninoculated media and water). Prior to measuring light emission from the various broths, cell density was established by measuring light absorbance (560 nM) in a Gilford Systems (Oberlin, OH) spectrophotometer using a sipper cell. Appropriate dilutions were then made (to normalize optical density to 1.0 unit) before measuring luminosity. Aliquots of the diluted broths were then placed into cuvettes (300 ~l each) and read in a Bio-Orbit 1251 Luminometer (Bio-Orbit Oy, Twiku, Finland). The integration period for each sample was 45 seconds. The samples were continuously mixed (spun in baffled cuvettes) while being read to provide oxygen availability. A positive test was determined as being 2 5-fold background luminescence (about 5-10 units). In addition, colony luminosity was detected with photographic film overlays and visually, after adaptation in a darkroom. The Gram's staining characteristics of each strain were established with a commercial Gram's stain kit (BBL, Cockeysville, MD) used in conjunction with Gram's stain control slides (~isher Scientific, Pittsburgh, PA).
Microscopic evaluation was then performed using a Zeiss microscope (Carl Zeiss, Germany) 100X oil immersion objective lens (with 10X
ocular and 2X body magnification). Microscopic examination of individual strains for organism size, cellular description and inclusion bodies (the latter after logarithmic growth) was performed using wet mount slides (lOX ocular, 2X body and 40X
objective magnification) with oil immersion and phase contrast microscopy with a micrometer (Akhurst, R.J. and Boemare, N.E. 1990.
Entomopathogenic Nematodes in Biological Control (ed. Gaugler, R.
and Kaya, H.). pp. 75-90. CRC Press, Boca Raton, USA.; Baghdiguian S., Boyer-Giglio M.H., Thaler, J.O., Bonnot G., ~oemare N. 1993.
Biol. Cell 79, 177-185.). Colony pigmentation was observed after SUBSTITUTE SH EE~ (RULE 26) CA 022638l9 l999-02-26 W098/08932 rCTAUS97/07657 lnoculation on Bacto nutrient agar, .(Difco Laboratories, Detroit, MI) prepared as per label instructions. Incubation occurred at 28~C and descriptions were produced after 5-7 days. To test for the presence of the enzyme catalase, a colony of the test organism was removed on a small plug from a nutrient agar plate and placed into the bottom of a glass test tube. One ml of a household hydrogen peroxide solution was gently added down the side of the tube. A positive reaction was recorded when bubbles of gas (presumptive oxygen) appeared immediately or within 5 seconds.
Controls of uninoculated nutrient agar and hydrogen peroxide solution were also examined. To test for nitrate reduction, each culture was inoculated into 10 ml of Bacto Nitrate Broth (Difco Laboratories, Detroit, MI). After 24 hours incubation at 28~C, nitrite production was tested by the addition of two drops of sulfanilic acid reagent and two drops of alpha-naphthylamine reagent (see Difco Manual, 10th edition, Difco Laboratories, Detroit, MI, 1984). The generation of a distinct pink or red color indicates the formation of nitrite from nitrate. The ability of each strain to uptake dye from growth media was tested with Bacto MacConkey agar containing the dye neutral red; Bacto Tergitol-7 agar containing the dye bromothymol blue and Bacto EMB Agar containing the dye eosin-Y (agars from Difco Laboratories, Detroit, MI, all prepared according to label instructions). After inoculation on these media, dye uptake was recorded after incubation at 28~C for 5 days. Growth on these latter media is characteristic for members of the family Enterobacteriaceae.
Motility of each strain was tested using a solution of Bacto Motility Test Medium (Difco Laboratories, Detroit, MI) prepared as per label lnstructions. A butt-stab inoculation was performed with each strain and motility was judged macroscopically by a diffuse zone of growth spreading from the line of inoculum. In many cases, motility was also observed microscopically from liquid culture under wet mount slides. Biochemical nutrient evaluation for each strain was performed using BBL Enterotube II (Benton, Dickinson, Germany). Product instructions were followed with the exception that incubation was carried out at 28~C for 5 days. Results were consistent with previously cited reports for Photorhabdus. The production of protease was tested by observing hydrolysis of gelatin using Bacto gelatin (Difco Laboratories, Detroit, MI) SUBSTITIITE 5~EET (RUL~ 2~) CA 022638l9 l999-02-26 plates made as per label instructions. Cultures were inoculated and the plates were incubated at 28~C for 5 days. To assess growth at different temperatures, agar plates [2% proteose peptone #3 with two percent Bacto-Agar (Difco, Detroit, MI) in deionized water]
were streaked from a common source of inoculum. Plates were sealed with Nesco film and incubated at 20, 28 and 37~C for up to three weeks. Plates showing no growth at 37~C showed no cell viability after transfer to a 28~C incubator for one week. Oxygen requirements for Photorhabdus strains were tested in the following manner. A butt-stab inoculation into fluid thioglycolate broth medium (Difco, Detroit, MI) was made. The tubes were incubated at room temperature for one week and cùltures were then examined for type and extent of growth. The indicator resazur1n demonstrates the level of medium oxidation or the aerobiosis zone (Difco Manual, 10th edition, Difco Laboratories, Detroit, MI). Growth zone resuits obtained for the Photorhabdus strains tested were consistent with those of a facultative anaerobic microorganism.

SUBSTITUTE S~EEl- (RULE 26) . . .

CA 022638l9 l999-02-26 W098/08932 PCTrUS97/07657 T~hle 19 T~xonomic Traits of Photorh~hdus Strains Traits Assessed*
~traln A ~ ~ v ~ l J K ~ M N ~ P ~
-W-14 t + + r~ ~ f _ + + + ~ + + + + + + _ W~ + + r~ ~ + _ + + + ~ + + + + + +
w~-~ _ + + rd ~ + _ + + + ~ + + + + + + _ w~-~ _ + + rd ~ + _ + + + Y l + + + + + + _ W~-4 _ + + rd ~ + _ + + + Yl + + + + + + _ W~-5 _ + + rd ~ + _ + + + L~ + + i + + +
W~-6 _ + + rd ~ + _ + + + LY + + + + + +
W~-7 _ + + r~ ~ + _ + + + ~ + + + + + + _ w~-~ _ + + rd ~ + _ + + + ~ + + + + + + _ W~-~ _ + + rd ~ + _ + + + Yl + + + + + + _ W~-l~ _ + + rd ~ + _ + + + ~o + + + + + +
W~-ll _ + + r S + _ + + + ~o + + + + + + _ W~-l~ _ + + rd ~ + _ + + + ~ + t + + + + _ W~-14 _ + + rd. ~ + _ + + + L~ + + + + + + _ W~-l~ _ + + rd ~ + _ + + + L~ + + + + + +
_ + + ru ~ + _ + + + LY + + + + + + _ ~D _ + + rd ~ + _ + + + yl + + + + + + _ ~m _ + + Ld ~ + _ + + + lY + + + + + + _ ~8~ _ t + rd ~ + _ + + + LY + + + + + + _ NC-l _ + + rd ~ + _ + + + ~ + + + + + + _ w~0 _ + + rd ~ + _ + + + Yl + + + + + + _ Wl~ _ + + rd ~ + _ + + + ~o + + + + + + _ ~z _ + + rd ~ + _ + + + R + + + + + + _ 43~4~ _ + + rd ~ + _ + + + O + + + + + + _ 4~Y~Y _ + + rd ~ + _ + + + O + + + + + + _ 4~Y~ _ + + rd ~ + _ + + + o + + + + + +
4~51 _ + + ra ~ + _ + + + O + + + + + + _ 4~Y~ _ + + rd ~ + _ + + + ~ + + + + + +
* - A = ~ram's ta-n, ~=Cry tallLe _nclus1on bodles, C=Bioluminescence, D=Cell form, E=Motility, F=Nitrate reduction, G=Presence of catalase, H=Gelatin hydrolysis, I=Dye uptake, J=Pigmentation, K=Growth on EMB agar, L=Growth on MacConkey agar, M=Growth on Tergitol-7 agar, N=Facultative anaerobe, O=Growth at 10 20~C, P=Growth at 28~C, Q=Growth at 37~C, t - +~- = posltlve or negative for trait, rd=rod, S=sized within Genus descriptors, RO=red-orange, LR = light red, R= red, O= orange, Y= yellow, T=
tan, LY= light yellow, YT= yellow tan, and LO= light orange.

Cellular fatty acid analysis ls a recognized tool for bacterial characterization at the genus and species level (Tornabene, T. G. 1985. Lipld Analysis and the Relationship to Chemotaxonomy in Methods in Microblology, Vol. 18, 209-234.;
Goodfellow, M. and O'Donnell, A. G. 1993. Roots of Bacterial Syste~atics in ~andbook of New Bacterial Systematics (ed.
Goodfellow, M. & O'Donnell, A. G.) pp. 3-54. London: Academic Press Ltd.), these references are incorporated herein ~y reference, and were used to confirm that our collection was related at the genus level. Cultures were shipr ~d to an external, contract laboratory SU~3ST~TUTE SH EE~ (RULE -26) . .

W09~08932 PCTfUS97/07657 for fatty acid methyl ester analysis- (FAME) using a Microbial ID
(MID~, Newark, DE, USA) Microbial Identification System (MIS). The MIS system conslsts of a Hewlett Packard HP5890A gas chromatograph with a 25mm x 0.2mm 5% methylphenyl silicone fused silica capillary 5 column. Hydrogen is used as the carrier gas and a flame-ionization detector functions in conjunction with an automatic sampler, integrator and computer. The computer compares the sample fatty acid methyl esters to a microbial fatty acid library and against a calibration mix of known fatty acids. As selected by the contract laboratory, strains were grown for 24 hours at 28~C on trypticase soy agar prior to analysis. Extraction of samples was performed by the contract lab as per standard FAME methodology. There was no direct identification of the strains to any luminescent bacterial group other than Photorhabdus. When the cluster analysis was ~5 performed, which compares the fatty acid profiles of a group of isolates, the strain fatty acid profiles were related at the genus level.
The evolutionary diversiey of the Photor~abdus strains in our collection was measured by analysis of PCR (Polymerase Chain Reaction) mediated genomic fingerprinting using genomic DNA from each strain. This technique is based on families of repetitive DNA
sequences present throughout the genome of diverse bacterial species (reviewed by Versalovic, J., Schneider, M., DE Bruijn, F. J. and Lupski, J. R. 1994. Methods Mol. Cell. Biol., 5, 25-40.).
Three of these, repetitive extragenic palindromic sequence (REP), enterobacterial repetitive intergenic consensus (ERIC) and the BOX
element are thought to play an important role in the organization of the bacterial genome. Genomic organization is believed to be shaped by selection and the differential dispersion of these elements within the genome of closely related bacterial strains can be used to discriminate these strains (e.g., Louws, F. J., Fulbright, D. W., Stephens, C. T. and DE Bruijn, F. J. 1994. Appl.
Environ. Micro. 60, 2286-2295). ~ep-PCR utilizes oligonucleotide ~ primers complementary to these repetitive sequences to amplify the variably sized DNA fragments lying between them. The resulting products are separated by electrophoresis to establish the DNA
"fingerprint" for each strain.
To isolate genomic DNA from our strains, cell pellets were resuspended in TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) to a SUBSTITUTE SHEET (RULE 26~

CA 022638l9 l999-02-26 W098/08932 PCTrUS97/07657 final volume of 10 ml and 12 ml of 5. M NaCl was then added. This mixture was centrifuged 20 min. at 15,000 x g. The resulting pellet was resuspended in 5.7 ml of TE and 300 ~l of 10% SDS and 60 ~1 20 mg/ml proteinase K (Gibco BR~ Products, Grand Island, NY) were added. This mixture was incubated at 37 ~C for l hr, approximately 10 mg of lysozyme was then added and the mixture was incubated for an additional 45 min. One milliliter of 5M NaCl and 800 ~l of CTAB/NaCl solution (10% w/v CTAB, 0.7 M NaCl) were then added and the mixture was incubated 10 min. at 65~C, gently agitated, then incubated and agitated for an additional 20 min. to aid in clearing of the cellular material. An equal volume of chloroform/isoamyl alcohol solution t24:1, v/v) was added, mixed gently then centrifuged. Two extractions were then performed with an equal volume of phenol/chloroform/isoamyl alcohol (50:49:1).
Genomic DNA was precipitated with 0.6 volume of isopropanol.
Precipitated DNA was removed with a glass rod, washed twice with 70~ ethanol, dried and dissolved in 2 ml of STE ~10 mM Tris-HCl pH8.0, 10 mM NaCl, 1 mM EDTA). The DNA was then quantitated by optical density at 260 nm. To perform rep-PCR analysis of 20 Photorhabdus genomic DNA the following primers were used, REPlR-I;
5'-IIIICGICGICATCIGGC-3' and REP2-I; 5'-ICGICTTATCIGGCCTAC-3'. PCR
was performed using the following 25~1 reaction: 7.75 ~l H2O, 2.5 ~l 10X LA buffer (PanVera Corp., Madison, WI), 16 ~l dNTP mix (2.5 mM each), 1 ~l of each primer at 50 pM/~ l DMSO, 1.5 ~l 25 genomic DNA (concentrations ranged from 0.075-0.480 ~g/~l) and 0.25 ~l TaKaRa EX Taq (PanVera Corp., Madison, WI). The PCR
amplification was performed in a Perkin Elmer DNA Thermal Cycler (Norwalk, CT) using the following conditions: 95~C/7 min. then 35 cycles of; 94~C/1 min.,44~C/1 min., 65~C/8 min., followed by 15 min.
30 at 65~C. After cycling, the 25 ~l reaction was added to 5 ~l of 6X
gel loading buffer (0.25% bromophenol blue, 40% w/v sucrose in H2O). A 15x20cm 1%-agarose gel was then run in TBE buffer (o.09 M
Tris-borate, 0.002 M EDTA) using 8 ~l of each reaction. The gel was run for approximately 16 hours at 45v. Gels were then stained in 20 ~g/ml ethidium bromide for 1 hour and destained in TBE buffer for approximately 3 hours. Polaroid~ photographs of the gels were then taken under W illumination.
The presence or absence of bands at specific sizes for each strain was scored from the photographs and entered as a similarity SUBSTrTUTE S~E~T (RULE 26) . , . -- .

CA 022638l9 l999-02-26 W098/08932 PCTrUS97tO7657 matrix in the numerical taxonomy sof~ware program, NTSYS-pc (Exeter Software, Setauket, NY). Controls of E. coli strain HB101 and Xanthnm~n~.q oryzae pv. oryzae assayed at the same time produced PCR
"fingerprints" corresponding to published reports (Versalovic, J., Koeuth, T. and Lupski, J. R. 1991. Nucleic Acids Res. 19, 6823-6831; Vera Cruz, C. M., Halda-Alija, L., Louws, F., Skinner, D. Z., George, M. L., Nelson, R. J., DE Brui~n, F. J., Rice, C. and Leach, ~ J. E. 1995. Int. Rice Res. Notes, 20, 23-24.; Vera Cruz, C. M., Ardales, E. Y., Skinner, D. Z., Talag, J., Nelson, R. J., Louws, F. J., Leung, H., Mew, T. W. and Leach, J. E. 1996. Phytopathology (in press, respectively). The data from Pho~orhabdus strains were then analyzed with a series of programs within NTSYS-pc; SIMQUAL
(Similarity for Qualitative data) to generate a matrix of similarity coefficients (using the Jaccard coefficient) and SAHN
'5 (Sequential, Agglomerative, Heirarchical and Nested) clustering [using the UPGMA (Unweighted Pair-Group Method with Arithmetic Averages) method] which groups related strains and can be expressed as a phenogram (Fig. 5). The COPH (cophenetic values) and MXCOMP
(matrix comparison) programs were used to generate a cophenetic value matrix and compare the correlation between this and the original matrix upon which the clustering was based. A resulting normalized Mantel statistic (r) was generated which is a measure of the goodness of fit for a cluster analysis (r=0.8-0.9 represents a very good fit). In our case r = 0.919. Therefore, our collection is comprised of a diverse group of easily distinguishable strains representative of the Photorhabdus genus.

Ex~le 13 In~ecticidal Utility of Toxin(s) Produced by Various Photorh~hdus Strain.q Initial l'seed'' cultures of the various Photorhabdus strains were produced by inoculating 175 ml of 2~ Proteose Peptone #3 (PP3 (Difco Laboratories, Detroit, MI) liquid media with a primary variant subclone in a 500 ml tribaffled flask with a Delong neck, covered with a Kaput. Inoculum for each seed culture was derived from oil-overlay agar slant cultures or plate cultures. After inoculation, these flasks were incubated for 16 hrs at 28~C on a rotary shaker at 150 rpm. These seed cultures were then used as SUBSTITUTE SH EET tRULE 26) ... ...

CA 022638l9 l999-02-26 WO98/08932 PCTrUS97tO7657 uniform inoculum sources for a given fermentation of each strain.
Additionally, overlaying the post-log seed culture with sterile mineral oil, adding a sterile magnetic stir bar for future resuspension and storing the culture in the dark, at room temperature provided long-term preservation of inoculum in a toxin-competent state. The production broths were inoculated by adding 1% of the actively growing seed culture to fresh 2% PP3 media (e.g., 1.75 ml per 175 ml fresh media). Production of broths occurred in either 500 ml tribaffled flasks (see above), or 2800 ml baffled, convex bottom flasks (500 ml volume) covered by a silicon foam closure. Production flasks were incubated for 24-48 hrs under the above mentioned conditions. Following incubation, the broths were dispensed into sterile 1 L polyethylene bottles, spun at 2600 x g for 1 hr at 10~C and decanted from the cell and debris pellet.
The liquid broth was then vacuum filtered through Whatman GF/D (2.7 ~M retention) and GF/B (1.0 ~M retention) glass filters to remove debris. Further broth clarification was achieved with a tangential flow microfiltration device (Pall Filtron, Northborough, MA) using a 0.5 ~M open-channel filter. When necessary, additional clarification could be obtained by chilling the broth (to 4~C) and centrifuging for several hours at 2600 x g. Following these procedures, the broth was filter sterilized using a 0.2 ~M
nitrocellulose membrane filter. Sterile broths were then used directly for ~iological assay, biochemical analysis or concentrated (up to 15-fold) using a lO,OOO MW cut-off, M12 ultra-filtration device (Amicon, Beverly MA) or centrifugal concentrators (Millipore, ~edford, MA and Pall Filtron, Northborough, MA) with a 10,000 MW pore size. In the case of centrifugal concentrators, the broth was spun at 2000 x g for approximately 2 hr. The lO,OOO MW
permeate was added to the corresponding retentate to achieve the desired concentration of components greater than 10,000 MW. Heat inactivation of processed broth samples was acheived by heating the samples at 100~C in a sand-filled heat block for 10 minutes.
The broth(s) and toxin complex(es) from different Pho~orhabdus strains are useful for reducing populations of insects and were used ln a method of inhibiting an insect population which comprises applying to a locus of the insect an effective insect inactivating amount of the active described. A demonstration of the breadth of insecticidal activity observed from broths of a selected group of SUBSTITUTE SH EET (RULE ~6) CA 022638l9 l999-02-26 W098/08932 rCTAUS97/07657 Photorhabdus strains fermented as described above is shown in Table 20. It is possible that additional insecticidal activitles could be detected with these strains through increased concentration of the broth or by employing different fermentation methods.
Consistent with the activity being associated with a protein, the insecticidal activity of all strains tested was heat labile (see above).
~ Culture broth(s) from diverse Photorhabdus strains show differential insecticidal activity (mortality and/or growth inhibition, reduced adult emergence) against a number of ~insects.
More specifically, the activity is seen against corn rootworm larvae and boll weevil larvae which are members of the insect order Coleoptera. Other members of the Coleoptera include wireworms, pollen beetles, flea beetles, seed beetles and Colorado potato beetle. Activity is also observed against aster leafhopper and corn plant hopper, which are members of the order Homoptera. Other members of the Homoptera include planthoppers, pear psylla, apple sucker, scale insects, whiteflies, spittle bugs as well as numerous host specific aphid species. The broths and purified toxin complex(es) are also active against tobacco budworm, tobacco hornworm and European corn borer which are members of the order Lepidoptera. Other typical members of this order are beet armyworm, cabbage looper, black cutworm, corn earworm, codling moth, clothes moth, Indian mealmoth, leaf rollers, cabbage worm, cotton bollworm, bagworm, Eastern tent caterpillar, sod webworm and fall armyworm. Activity is also seen against fruitfly and mosquito larvae which are members of the order Diptera. Other members of the order Diptera are, pea midge, carrot fly, cabbage root fly, turnip root fly, onion fly, crane fly and house fly and various mosquito species. Activity with broth(s) and toxin complex(es) is also seen against two-spotted spider mite which is a member of the order Acarina which includes strawberry spider mites, broad mites, citrus red mite, European red mite, pear rust mite and tomato russet mite.
Activity against corn rootworm larvae was tested as follows.
Photorhabdus culture broth(s) (0-15 fold concentrated, filter sterilized), 2% Proteose Peptone #3, purified toxin complex(es), 10 mM sodium phosphate buffer , pH 7.0 were applied directly to the surface (about 1.5 cm2) of artificial diet (Rose, R. I. and McCabe, SUBST~TUTE S~ E~ tRULE 26) CA 022638l9 l999-02-26 W098t08932 PCTrUS97/076~7 J. M. (1973). J. Econ. Entomol. 66, .(398-400) in 40 ~1 aliquots.
Toxin complex was diluted in 10 mM sodium phosphate buffer, pH 7Ø
The dlet plates were allowed to air-dry in a sterile flow-hood and the wells were infested with single, neonate Diabrotica undecimpunctata howardi (Southern corn rootworm, SCR) hatched from surface sterilized eggs. The plates were sealed, placed in a humidified growth chamber and maintained at 27~C for the appropriate period (3-5 days). Mortality and larval weight determinations were then scored. Generally, 16 insects per treatment were used in all studies. Control mortality was generally less than 5%.
Activity against boll weevil (Anthnmnn~5 grandis) was tested as follows. Concentrated (1-10 fold) Photorhabdus broths, control medium (2% Proteose Peptone #3), purified toxin complex(es) [0.23 15 mg/ml] or 10 mM sodium phosphate buffer, pH 7.0 were applied in 60 ~l aliquots to the surface of 0.35 g of artificial diet (Stoneville Yellow lepidopteran diet) and allowed to dry. A single, 12-24 hr boll weevil larva was placed on the diet, and the wells were sealed and held at 25~C, 50% RH for 5 days. Mortality and larval weights were then assessed. Control mortality ranged between 0-13~.
Activity against mosquito larvae was tested as follows. The assay was conducted in a 96-well microtiter plate. Each well contained 200 ~l of aqueous solution (lO-fold concentrated Photorhabdus culture broth(s), control medium (2% Proteose Peptone 25 #3), 10 mM sodium phosphate buffer, toxin complex(es) @ 0.23 mg/ml or H20) and approximately 20, 1-day old larvae (Aedes aegypti).
There were 6 wells per treatment. The results were read at 3-4 days after infestation. Control mortality was between 0-20%.
Activity against fruitflies was tested as follows. Purchased Drosophila melanogaster medium was prepared using 50% dry medium and a 50% liquid of either water, control medium (2% Proteose Peptone #3), 10-fold concentrated Photor~abdus culture broth(s), purified toxin complex(es) [0.23 mg/ml] or lO mM sodium phosphate buffer , pH 7Ø This was accomplished by placing 4.0 ml of dry medium in each of 3 rearing vials per treatment and adding 4.0 ml of the appropriate liquid. Ten late instar Drosop~ila melanogaster maggots were then added to each 25 ml vial. The vials were held on a laboratory bench, at room temperature, under fluorescent ceiling lights. Pupal or adult counts were made after 15 days of exposure.

SUBSTJTUTE SHEE~ (RULE 26) CA 022638l9 l999-02-26 W098/08932 PCTrUS97tO7657 Adult emergence as compared to water.and control medium (0-16%
reduction). -~-~-~
Activity against aster leafhopper adults (Macrosteles severini) and corn planthopper nymphs ( Peregrinus maidis) was ~ 5 tested with an ingestion assay designed to allow ingestion of the active without other external contact. The reservoir for the active/"foodr' solution is made by making 2 holes in the center of the bottom portion of a 35X10 mm Petri dish. A 2 inch Parafilm M
square is placed across the top of the dish and secured with an "O"
ring. A 1 oz. plastic cup is then infested with approximately 7 hoppers and the reservoir is placed on top of the cup, Parafilm down. The test solution is then added to the reservoir through the holes. In tests using 10-fold concentrated Photorhabdus culture broth(s), the broth and control medium (2% Proteose Peptone #3) were dialyzed against 10 mM sodium phosphate buffer, pH 7.0 and sucrose (to 5%) was added to the resulting solution to reduce control mortality. Purified toxin complex(es) [0.23 mg/ml] or 10 mM sodium phosphate buffer, pH 7.0 was also tested. Mortality is reported at day 3. The assay was held in an incubator at 28~C, 70%
RH with a 16/8 photoperiod. The assays were graded for mortality at 72 hours. Control mortality was less than 6%.
Activity against lepidopteran larvae was tested as follows.
Concentrated (lO-fold) Photorhabdus culture broth(s), control medium (~% Proteose Peptone #3), purified toxin complex(es) [0.23 mg/ml] or 10 mM sodium phosphate buffer, pH 7.0 were applied directly to the surface (about 1.5 cm2) of standard artificial lepidopteran diet (Stoneville Yellow diet) in 40 ~l aliquots. The diet plates were allowed to air-dry in a sterile flow-hood and each well was infested with a single, neonate larva. European corn borer (Ostrinia nubilalis) and tobacco hornworm (Manduca sextaJ eggs were obtained from commercial sources and hatched in-house, whereas tobacco budworm (Heliothis virescens) larvae were supplied internally. Following infestation with larvae, the diet plates were sealed, placed in a humidified growth chamber and maintained in the dark at 27~C for the appropriate period. Mortality and weight determinations were scored at day 5. Generally, 16 insects per treatment were used ln all studies. Control mortality generally ranged from about 4 to about 12.5% for control medium and was less than 10~ for phosphate buffer.

SUBSTITUTE SHEE~ (RULE 26) W O 98/08932 PCT~US97/07657 Activity against two-spotted spider mite ( Tetranychus urticae) was determined as follows. Young squash plants were trimmed to a single cotyledon and sprayed to run-off with 10-fold concentrated broth(s), control medium (2% Proteose Peptone #3), purified toxin complex(es), 10 mM sodium phosphate buffer, pH 7Ø After drying, the plants were infested with a mixed population of spider mites and held at lab temperature and humidity for 72 hr. Live mites were then counted to determine levels of control.

SUBSTITUTE SHEET tRULE 26) , W098/08932 PCTAUS97~7657 T~hle.20 Observed I~ec~icidal S~ectrum of Broths from ~iffer~nt Photorhabdus Strain.q 5 ~hotorha~dus ~traln ~ensltlve* lnsect ~pecles WX-1 3**, 4, 5, 6, 7, 8 WX-2 2, 4 - WX-3 1, 4 WX-4 1, 4 WX-7 3, 4, 5, 6, 7, 8 WX-8 1, 2, 4 WX-9 1, 2, 4 WX-11 1, 2, 4 WX-12 2, 4, 5, 6, 7, 8 WX-14 1, 2, 4 WX-15 1, 2, 4 W30 3, 4, 5, 8 NC-l 1, 2, 3, 4, 5, 6, 7, 8, 9 WIR 2, 3, 5, 6, 7, 8 HP88 1, 3, 4, 5, 7, 8 Hb 3, 4, 5, 7, 8 Hm 1, 2, 3, 4, 5, 7, 8 H9 1, 2, 3, 4, 5, 6, 7, 8 W-14 1, 2, 3, 4, 5, 6, 7, 8, 10 * = 2 25~ mortality and/or growth inhibition vs. control ** _ 1; Tobacco budworm, 2; European corn borer, 3;
Tobacco hornworm, 4; Southern corn rootwor~, 5;
Boll weevil, 6; Mosquito, 7; Fruit Fly, 8;
Aster Leafhopper, 9; Corn planthopper, 10;
Two-spotted spider mlte.

SUBSTITUTE S~tEET (RULE~-26) W098/08932 PCT~US97/07657 E~m~}e. 14 Non W-14 Photorhab~1~s Strains:
Puri~ication~ Ckaracterization and Activity Spectrum Purification The protocol, as follows, is similar to that developed for the purification of W-14 and was established based on purifying those fractions having the most activity against Southern corn root worm (SCR), as determined in bioassays (see Example 13). Typically, 4-20 L of broth that had been filtered, as described in Example 13,were received and concentrated using an Amicon spiral ultra filtration cartridge Type SlY100 attached to an Amicon M-12 filtration device. The retentate contained native proteins consisting of molecular sizes greater than 100 kDa, whereas the flow through material contained native proteins less than 100 kDa in size. The majority of the activity against SCR was contained in the 100 kDa retentate. The retentate was then continually diafiltered with 10 mM sodium phosphate (pH = 7.0) until the filtrate reached an A280 < 0.100. Unless otherwise stated, all procedures from this point were performed in buffer as defined by 10 mM sodium phosphate (pH 7.0). The retentate was then concentrated to a final volume of approximately 0.20 L and filtered using a 0.45 mm NalgeneTM Filterware sterile filtration unit. The filtered material was loaded at 7.5 ml/min onto a Pharmacia HR16/10 column which had been packed with PerSeptive Biosystem Poros~ 50 HQ
strong anion exchange matrix equilibrated in buffer using a PerSeptive Biosystem Sprint~ HPLC system. After loading, the -column was washed with buffer until an A280 < 0.100 was achieved.
Proteins were then eluted from the column at 2.5 ml/min using 30 buffer with 0.4 M NaCl for 20 min for a total volume of 50 ml. The column was then washed using buffer with 1.0 M NaCl at the same flow rate for an additional 20 min (final volume = 50 ml).
Proteins eluted with 0.4 M and 1.0 M NaCl were placed in separate dialysis bags (Spectra/Por~ Membrane MWCO: 2,000) and allowed to dialyze overnight at 4~ C in 12 L buffer. The majority of the activity against SCR was contained in the 0.4 M fraction. The 0.4 M fraction was further purified by application of 20 ml to a Pharmacia XK 26/100 column that had been prepacked with Sepharose CL4B (Pharmacia) using a flow rate of 0.75 ml/min. Fractions were _99_ SUBSTITUTE S}~EET (RULE 26) CA 022638l9 l999-02-26 W098/08932 PCTrUS97/07657 pooled based on A280 peak profile and concentrated to a final volume of 0.75 ml using a Millipore Ultrafree~-15 centrifugal filter device Biomax-50K NMWL membrane. Protein concentrations were determined using a Biorad Protein Assay Kit with bovine gamma globulin as a standard.

~h~racteriz~tion The native molecular weight of the SCR toxin complex was determined using a Pharmacia HR 16/50 that had been prepacked with Sepharose CL4B in buffer. The column was then calibrated using proteins of known molecular size thereby allowing for calculation of the toxin approximate native molecular size. As shown in Table 21, the molecular size of the toxin complex ranged from 777 kDa with strain Hb to 1,900 kDa with strain WX-14. The yield of toxin complex also varied, from strain WX-12 producing 0.8 mg/L to strain Hb, which produced 7.0 mg/L.
Proteins found in the toxin complex were examined for individual polypeptide size using SDS-PAGE analysis. Typically, 20 mg protein of the toxin complex from each strain was loaded onto a 2-15% polyacrylamide gel (Integrated Separation Systems) and electrophoresed at 20 mA in Biorad SDS-PAGE buffer. After completion of electrophoresis, the gels were stained overnight in Biorad Coomassie blue R-250 (0.2~ in methanol: acetic acid: water;
40:10:40 v/v/v). Subsequently, gels were destained in 25 methanol:acetic acid: water; 40:10:40 (v/v/v). The gels were then rinsed with water for 15 min and scanned using a Molecular Dynamics Personal Laser Densitometer~. Lanes were quantitated and molecular sizes were calculated as compared to Biorad high molecular weight standards, which ranged from 200-45 kDa.
Sizes of the individual polypeptides comprising the SCR toxin complex from each strain are listed in Table 22. The sizes of the individual polypeptides ranged from 230 kDa with strain WX-l to a size of 16 kDa, as seen with strain WX-7. Every strain, with the exception of strain Hb, had polypeptides comprising the toxin 35 complex that were in the 160-230 kDa range, the 100-160 kDa range, and the 50-80 kDa range. These data indicate that the toxin complex may vary in peptide composition and components from strain to strain, however, in all cases the toxin attributes appears to consist of a large, oligomeric protein complex.

SlJBSTlTlJTE Stl EET tRULE 26) CA 022638l9 l999-02-26 W098/08932 PCT~US97/07657 T~hle 21 ~racterizatiQn of a Toxin Complex from Non W-14 Photorhabdus Strains ~traln Approx. Yleld Native Active Molecular Wt.a Fraction (mg/L~b ~Y ~-/Z,~OO 1.
Hb Hm 1,400,000 1.1 HP88 813,000 2.5 NCl 1,092,000 3.3 WIR 979,000 1.0 WX-l 973,000 0.8 WX-2 951,000 2.2 WX-7 1,000,000 1.5 WX-12 898,000 0.4 WX-14 1,900,000 l.9 W-14 860,000 7.5 a Natlve molecular welght ~etermlned uslng a Pharmacla 16/50 column packed with Sepharose CL4B
b Amount of toxin complex recovered from culture broth.

Activity Spectrum As shown in Table 23, the toxin complexes purified from strains Hm and H9 were tested for activity against a variety of insects, with the toxin complex from strain W-14 for comparison.
The assays were performed as described in Example 13. The toxin complex from all three strains exhibited activity against tobacco bud worm, European corn borer, Southern corn root worm, and aster leafhopper. Furthermore, the toxin complex from strains Hm and W-14 also exhibited activity against two-spotted spider mite. In addition, the toxin complex from W-14 exhibited activity against mosquito larvae. These data indicate that the toxin complex, while having similarities in activities between certain orders of insects, can also exhibit differential activities against other orders of insects.

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o o o o o a~ r ~ a~ o r N ~D O r c~ ~ r ~D ~ N a~ r~ ~ r r ~ w m m SUBSTITUTE SHEE~ (RULE 26) CA 022638l9 l999-02-26 W098/08932 PCT~US97/07657 T~hle 23 Observed I~Yecticidal Spectr1~m of a Purified Toxin Complex from Photorhabdus Str~in~

~hotorha~dus ~traln ~ensltlve* lnsect ~pecles Hm Toxin Complex 1~*, 2, 3, 5, 6, 7, 8 H9 Toxin Complex 1, 2, 3, 6, 7, 8 ~0 W-14 Toxin Complex 1, 2, 3, 4, 5, 6, 7, 8 ~ = , 2~ mortallty or growth lnhlbltlon * = > 25% mortality or growth inhibition ** = l, Tobacco bud worm; 2, European corn borer; 3, Southern corn root worm; 4, Mosquito; 5, Two-spotted spider mite-6, Aster Leafhopper; 7, Fruit Fly; 8, Boll Weevil Example 15 Sub- Fractionation of Photorhabdus Protein Toxin Complex The Photorhabdus protein toxin complex was isolated as described in Example 14. Next, about 10 mg toxin was applied to a MonoQ 5/5 column equilibrated with 20 mM Tris-HCl, pH 7.0 at a flow rate of lml/min. The column was washed with 20 mM Tris-HCl, pH 7.0 until the optical density at 280 nm returned to baseline absorbance. The proteins bound to the column were eluted with a linear gradient of 0 to 1.0 M NaCl in 20 mM Tris-HCl, pH 7.0 at 1 ml/min for 30 min. One ml fractions were collected and subjected to Southern corn rootworm (SCR) bioassay (see Example 13). Peaks of activity were determined by a series of dilutions of each fraction in SCR bioassays. Two actlvity peaks against SCR were observed and were named A (eluted at about 0.2-0.3 M NaCl) and B
(eluted at 0.3-0.4 M NaCl). Activity peaks A and B were pooled separately and both peaks were further purified using a 3-step procedure described below.
Solid (NH4)2SO4 was added to the above protein fraction to a final concentration of 1.7 M. Proteins were then applied to a phenyl-Superose 5/5 column equilibrated with 1.7 M (NH4)2SO4 in 50 mM potassium phosphate buffer, pH 7 at 1 ml/min. Proteins bound to the column were eluted with a linear gradient of 1.7 M (NH4)2SO4, 0% ethylene glycol, 50 mM potassium phosphate, pH 7.0 to 25~
ethylene glycol, 25 mM potassium phosphate, pH 7.0 (no (NH4)2SO4) at 0.5 ml/min. Fractions were dialyzed overnight against 10 mM
sodium phosphate buffer, pH 7Ø Activities in each fraction against SCR were determined by bioassay.

SUBS~ITUTE SHEET(RU~ E2~) CA 022638l9 l999-02-26 W098/08932 PCTAUSg7/07657 The fractions with the highest.actlvity were pooled and applied to a MonoQ 5/5 column which was equilibrated with 20 mM
Tris-HCl, pH 7.0 at 1 ml/min. The proteins bound to the column were eluted at 1 ml/min by a linear gradient of 0 to lM NaCl in 20 mM Tris-HCl, pH 7Ø
~ For the final step of purification, the most active fractions above ~determined by SCR bioassay) were pooled and subjected to a second phenyl-Superose 5/5/ column. Solid ~NH4)2SO4 was added to a final concentration of 1.7 M. The solution was then loaded onto the column equilibrated with 1.7 M ~NH4)2SO4 in 50 mM potassium phosphate buffer, pH 7 at lml/min. Proteins bound to the column were eluted with a linear gradient of 1.7 M ~NH4)2SO4, 50 mM
potasslum phosphate, pH 7.0 to lO mM potassium phosphate, pH 7.0 at 0.5 ml/min. Fractions were dialyzed overnight against 10 mM
sodium phosphate buffer, pH 7Ø Activities in each fraction against SCR were determined by bioassay.
The final purified protein by the above 3-step procedure from peak A was named toxin A and the final purified protein from peak B
was named toxin B.
~hAracterization and Ami no Acid Sequencing of Toxi n A and Toxin B
In SDS-PAGE, both toxin A and toxin B contained two major ~> 90~ of total Commassie stained protein) peptides: 192 kDa (named A1 and B1, respectively) and 58 kDa ~named A2 and B2, respectively). Both toxin A and toxin B revealed only one major band in native PAGE, indicating A1 and A2 were subunits of one protein complex, and B1 and B2 were subunits of one protein complex. Further, the native molecular weight of both toxin A and toxin B were determined to be 860 kDa by gel filtration chromatography. The relative molar concentrations of A1 to A2 was judged to be a 1 to 1 equivalence as determined by densiometric analysis of SDS-PAGE gels. Similarly, B1 and B2 peptides were present at the same molar concentration.
Toxin A and toxin B were electrophoresed in 10~ SDS-PAGE and transblotted to PVDF membranes. Blots were sent for amino acid ~ analysis and N-terminal amino acid sequencing at Harvard MicroChem and Cambridge ProChem, respectively. The N-terminal amino sequence of B1 was determined to be identical to SEQ ID NO:l, the~ TcbA
- region of the tcbA gene (SEQ ID NO:12, position 87 to 99). A
uni~ue N-terminal sequence was obtained for peptide B2 (SEQ ID
NO:40). The N-terminal amino acid sequence of peptide B2 was identical to the TcbAiii region of the derived amino acid sequence SUBSTITUTE SHEET (RULE 26) for the tcbA gene (SEQ ID NO:12, position 1935 to 1945).
Therefore, the B toxin contained predominantly two peptides, TcbA
and TcbAiii, that were observed to be derived from the same gene product, TcbA.
The N-terminal sequence of A2 (SEQ ID NO:41) was unique in comparison to the TCbAiii peptide and other peptides. The A2 peptide was denoted TCdAiii (see Example 17). SEQ ID NO:6 was determined to be a mixture of amino acid sequences SEQ ID NO:40 and 41.
Peptides A1 and A2 were further subjected to internal amino acid sequencing. For internal amino acid sequencing, 10 ~g of toxin A was electrophoresized in 10% SDS-PAGE and transblotted to PVDF membrane. After the blot was stained with amido black, peptides A1 and A2, denoted TcdAii and TcdAiii, respectively, were excised from the blot and sent to Harvard MicroChem and Cambridge ProChem. Peptides were subjected to trypsin digestion followed by HPLC chromatography to separate individual peptides. N-terminal amino acid analysis was performed on selected tryptic peptide fragments. Two internal amino acid sequences of peptide Al (TcdAii-PK71, SEQ ID NO:38 and TcdAii-PK44, SEQ ID NO:39) were found to have significant homologies with deduced amino acid sequences of the TcbAii region of the tcbA gene (SEQ ID NO:12).
Similarly, the N-terminal sequence (SEQ ID NO:41) and two internal sequences of peptides A2 (TcdAiii-PKS7, SEQ ID NO:42 and TcdAiii-PK20, SEQ ID NO.43) also showed significant homology with deduced amino acid sequences of TcbAiii region of the tcbA gene (SEQ ID
NO:12).
In summary of above results, the toxin complex has at least two active protein toxin complexes against SCR; toxin A and toxin B. Toxin A and toxin B are similar in their natlve and subunits molecular weight, however, their peptide compositions are different. Toxin A contained peptides TcdAii and TcdAiii as the major peptides and the toxin B contains TcbAii and TcbAiii as the major peptides.

Purification and Characterization of Toxin C Tca Peptides The Photorhabdus protein toxin complex was isolated as described above. Next, about 50 mg toxin was applied to a MonoQ
10/10 column equilibrated with 20 mM Tris-HCl, pH 7.0 at a flow rate of 2 ml/min. The column was washed with 20 mM Tris-HCl, pH7.0 - 1 o O -SUBSTiTUTE ~HEET (RULE 26) W098/08932 PCTrUS97/07657 until the optical density at 280 nm.returned to baseline level.
The proteins bound to the column were eluted with a linear gradient of 0 to lM NaCl in 20 mM Tris-HCl, pH 7.0 at 2 ml/min for 60 min.
2 ml fractions were collected and subjected to Western analysis using pAb TcaBii-syn antibody (see Example 21) as the primary antibody. Fractions reacted with pAb TcaBii-syn antibody were combined and solid (NH4)2SO4 was added to a final concentration of 1.7 M. Proteins were then applied to a phenyl-Superose 10/10 column equilibrated with 1.7 M (NH4)2SO4 in 50 mM potassium phosphate buffer, pH 7 at 1~1/min. Proteins bound to the column were eluted with a linear gradient of 1.7 M (NH4)2SO4, 50 mM
potassium phosphate, pH 7.0 to 10 mM potassium phosphate, pH 7.0 at 1 ml/min for 120 min. 2ml Fractions were collected, dialyzed overnight against 10 mM sodium phosphate buffer, pH 7.0, and analyzed by Western blots using pAb TcaBii-syn antibody as the primary antibody.
Fractions cross-reacted with the antibody were pooled and applied to a MonoQ 5/5 column which was equilibrated with 20 mM
Tris-HCl, pH 7.0 at lml/min. The proteins bound to the column were eluted at lml/min by a linear gradient of 0 to lM NaCl in 20 mM
Tris-HCl, pH 7.0 for 30 min.
Fractions above reacted with pAb TcaBii-syn antibody were pooled and subjected to a phenyl-Superose 5/5/ column. Solid (NH4)2SO4 added to a final concentration of 1.7 M. The solution 25 was then applied onto the column equilibrated with 1.7 M (NH4)2SO4 in 50 mM potassium phosphate buffer, pH 7 at lml/min. Pro~eins bound to the column were then eluted with a linear gradient of 1.7 M (NH4)2SO4, 50 mM potassium phosphate, pH 7.0 to 10 mM potassium phosphate, pH 7.0 at 0.5 ml/min for 60 min. Fractions were dialyzed overnight against 10 mM sodium phosphate buffer, pH 7Ø
For the final purification step, fractions reacted with pAb TcaBii-syn antibody above determined by Western analysis were combined and applied to a Mono Q 5/5 column equilibrated with 20 mM
Tris-HCl, pH 7.0 at lml/min. The proteins bound to the column were eluted at lml/min by a linear gradient of 0 to lM NaCl in 20 mM
Tris-HCl, pH 7.0 for 30 min.
The final purified protein fraction contained 6 major peptides examined by SDS-PAGE: 165 kDa, 9o kDa, 64 kDa, 62 kDa, 58 kDa, and 22 kDa. The LD50 of the insecticidal activities of this purified SU8STITUTE S~tEET (RULE 26) fraction were determined to be 100 ~g and 500 ng against SCR and ECB, respectively.
The above peptides were blotted to PVDF membranes and blots were sent for amino acids analysis and 5 amino acid long N-terminal sequencing at Harvard MicroChem and Cambridge ProChem, respectively. The N-terminal amino acid sequence of the 165 kDa peptide was determined to be identical to peptide TcaC (SEQ ID 2, position 1 to 5). The N-terminal amino acid sequence of the 90 kDa peptide was determined to be TcaAii region of the derived amino 10 acid sequence for the tcaA gene (SEQ ID NO 33, position 254 to 258). The N-terminal amino acid sequence of 64 kDa peptide was determined to be identical to peptide TcaBi (SEQ ID 3, position 1 to 5). The N-terminal amino acid sequence of the 62 kDa peptide was determined to be TcaAii region of the derived amino acid 15 sequence for the tcaA gene (SEQ ID NO 33, position 489 to 493).
The N-terminal amino acid sequence of 58 kDa peptide was determined to be identical to peptide TcaBii (SEQ ID 5, position 1 to 5). The N-terminal amino acid sequence of the 22 kDa peptide (SEQ ID NO 62) was determined to be TcaAi region, denoted TcaAiv, of the derived amino acid sequence for the tcaA gene (SEQ ID NO 34, position 98 to 102). It is noted that all tcaA, tcaB, and tcaC genes reside in the same tca operon (Fig. 6A).
Five ~g of purified Tca fraction, purified toxin A, and purified toxin B were analyzed by Western blot using the following antibodies individually as primary antibody: pAb TcaBii-syn antibody, mAb CF52 antibody, pAb TcdAii-syn antibody, and pAb Tcdiii-syn antibody (Example 21). With pAb TcaBii-syn antibody only the purified Tca peptides fraction reacted, but not toxin A or toxin B. With mAb CF52 antibody, only toxin B reacted but not Tca peptides fraction or toxin A. With either pAb TcdAii-syn antibody or pAb Tcdiii-syn antibody only toxin A reacted, but not Tca peptides fraction or toxin B. This indicated that the insecticidal activity observed in the purified Tca peptides fraction is independent of toxin A and toxin B. The purified Tca peptide fraction is a third unique protein toxin, denoted toxin C.

SUBSTITUTE SHEET tRULE 26) W098/08932 PCTrUS97/07657 Example 16 Cleavage and Activation of TcbA Peptide In the toxin B complex, peptide TcbAii and TCbAiii originate from the single gene product TcbA (Example 15). The processing of TcbA peptide to TCbAii and TCbAiii is presumably by the action of Photorhabdus protease(s), and most likely, the metalloproteases described in Example 10. In some cases, it was noted that when Photorhabdus W-14 broth was processed, TcbA peptide was present in toxin B complex as a major component, in addition to peptides TcbAii and TCbAiii. Identical procedures, described for the purification of toxin B complex (Example 15), were used to enrich peptide TcbA from toxin complex fraction of W-14 broth. The final purified material was analyzed in a 4-20~ gradient SDS-PAGE and major peptides were quantified by densitometry. It was determined that TcbA, TCbAii and TCbAiii comprised 58~, 36~, and 6%, respectively, of total protein. The identities of these peptides were confirmed by their respective molecular sizes in SDS-PAGE and Western blot analysis using monospecific antibodies. The native molecular weight of this fraction was determined to be 860 kDa.
The cleava~e of TcbA was evaluated by treating the above purified material with purified 38 kDa and 58 kDa W-14 Photorhabdus metalloproteases (Example 10), and trypsin as a control enzyme (Sigma, MO). The standard reaction consisted 17.5 ~g the above 25 purified fraction, 1.5 unit protease, and 0.1 M Tris buffer, pH 8.0 in a total volume of 100 ~1. For the control reaction, protease was omitted. The reaction mixtures were incubated at 37~C for 90 min. At the end of the reaction, 20 ~1 was taken and boiled with SDS-PAGE sample buffer immediately for electrophoresis analysis in a 4-20% gradient SDS-PAGE. It was determined from SDS-PAGE that in both 38 kDa and 58 kDa protease treatments, the amount of peptides TcbAii and TCbAiii increased about 3-fold while the amount of TcbA
peptide decreased proportionally (Table 24). The relative reduction and augmentation of selected peptides was confirmed by Western blot analyses. Furthermore, gel filtration of the cleaved material revealed that the native molecular size of the complex remained the same. Upon trypsin treatment, peptides TcbA and TcbAii were nonspecifically digested into small peptides. This indicated that 38 kDa and 58 kDa Photorhabdus proteases can SU8STITUTE S~EET tRULE 26) CA 022638l9 l999-02-26 W098/08932 PCT~S97/07657 specifically process peptide TcbA into peptides TcbAii and TcbAiii.
Protease treated and untreated control of the remaining 80 ~l reaction mixture were serial diluted with 10 mM sodium phosphate buffer, pH 7.0 and analyzed by SCR bioassay. By comparing activity in several dilution, it was determined that the 38 kDa protease treatment increased SCR insecticidal activity approximately 3 to 4 fold. The growth inhibition of remaining insects in the protease treatment was also more severe than control (Table 24).

Table 24 ConverSiQn and Activation of Peptide TcbA into Peptides TcbAl and ~çh~iii by Protease Treatment ~ontrol ~8 k~a protease treatment lc~A (~ o~ total proteln) TcbAii(~ of total protein) 36 64 TcbAiii(~ of total protein) 6 18 L~50 (~g proteln) ~ 2 SCR Weight (mg/insect)* 0.2 0.1 *: an 1ndlcat1on o~ growth lnhlbltlon by measurlng the average weight of live insect after 5 days on diet in the assay.

Activation and Procession of Toxin B by SCR Gut Proteases In yet a second demonstration of proteolytic activation, it was examined whether W-14 toxins are processed by insects. Toxin B
purified from Photorhabdus W-14 broth (see Example 15) was comprised of predominantly intact TcbA peptides as judged by SDS-PAGE and Western blot analysis using monoclonal antibody. The LD50 of this fraction against SCR was determined to be around 700 ng.
SCR larva were grown on coleopteran diet until they reached the fourth instar stage (about 100-125 mg total weight each insect). SCR gut content was collected as follows: the guts were removed using dissecting scissors and forceps. After removing the excess fatty material that coats the gut lining, about 40 guts were homogenized in a microcentrifuge tube containing 100 ~1 sterile water. The tube was then centrifuged at 14,000 rpm for 10 minutes and the pellet discarded. The supernatant was stored at a -70~C
freezer until use.
The processing of toxin B by insect gut was evaluated by treating the above purified toxin B with the SCR gut content ~0 collected. The reaction consisted 40 ~g toxin B (1 mg/ml), 50 ~l SU8STlTl.JTE SHEET (RULE 26) CA 022638l9 l999-02-26 W098/08932 PCT~US97/07657 SCR gut content, and O.lM Tris buffer, pH 8.0 in a total volume of lO0 ~l. For the control reaction, SCR gut content was omitted.
The reaction mlxtures were incubated at 37~C for overnight. At the end of reaction, 10 ~l was withdraw and boiled with equal volume 2x SDS-PAGE sample buffer for SDS-PAGE analysis. The remaining 90 ~l reaction mixture was serial diluted with 10 mM sodium phosphate buffer, pH 7.0 and analyzed by SCR bioassay. SDS-PAGE analysis indicated in SCR gut content treatment, peptide TcbA was dlgested completely into smaller peptides. Analysis of the undenatured toxin fraction showed that the native size, about 860 kDa, remained the same even though larger peptides were fragmented. In SCR
bioassays, it was found that the LD50 of SCR gut treated toxin B to be about 70 ng; representing a 10-fold increase. In a separate experiment, protease K treatment completely eliminated toxin activity.

Example 17 Screening of the ~ibrary for a Gene Encodi~g the TcdAl Peptide The cloning and characterization of a gene encoding the TcdA
peptide, described as SEQ ID NO:17 (internal peptide TcdAii-PT111 N-terminal sequence) and SEQ ID NO:18 (internal peptide TcdAii-PT79 N-terminal sequence) was completed. Two pools of degenerate oligonucleotides, designed to encode the amino acid sequences of SEQ ID NO:17 (Table 25) and SEQ ID NO:18 (Table 26), and the reverse complements of those sequences, were synthesized as described in Example 8. The DNA sequence of the oligonucleotides is given below:

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SUBSTlTUTE SHE~T tRULE ~6) , ~

W098/08932 PCTrUS97/07657 Polymerase Chain Reactions (PC~) were performed essentially as described in Example 8, using as forward primers P2.3.6.CB or P2.3.5, and as reverse primers P2.79.R.1 or P2.79R.CB, in all forward/reverse combinations, using Pho~orhabdus W-14 genomic DNA
~ 5 as template. In another set of reactions, primers P2.79.2 or P2.79.3 were used as forward primers, and P2.3.5R, P2.3.5RI, and P2.3R.CB were used as reverse primers in all forward/reverse ~ combinations. Only in the reactions containing P2.3.6.CB as the forward primers combined with P2.79.R.1 or P2.79R.CB as the reverse primers was a non-artifactual amplified product seen, of estimated size (mobility on agarose gels) of 2500 base pairs. The order of the primers used to obtain this amplification product indicates that the peptide fragment TcdAii-PT111 lies amino-proximal to the peptide fragment TcdAii-PT79.
The 2500 bp PCR products were ligated to the plasmid vector pCR'~II (Invitrogen, San Diego, CA) according to the supplier's instructions, and the DNA sequences across the ends of the insert fragments of two isolates (HS24 and HS27) were determined using the supplier's recommended primers and the sequencing methods described previously. The sequence of both isolates was the same. New primers were synthesized based on the determined sequence, and used to prime additional sequencing reactions to obtain a total of 2557 bases of the insert [SEQ ID NO:36]. Translation of the partial peptide encoded by SEQ ID No: 36 yields the 845 amino acid sequence disclosed as SEQ ID NO:37. Protein homology analysis of this portion of the TcdAii peptide fragment reveals substantial amino acid homology ((68~ similarity,and 53~ identity using the Wisconsin Package Version 8.0, Genetics Computer Group (GCG), Madison, WI) to residues 542 to 1390 of protein TcbA [SEQ ID NO:12] or(60%
similarity, and 54% identity using the Wisconsin Package Version 9.0, Genetics Computer Group (GCG), Madison, WI to residues 567 to 1389)). It is therefore apparent that the gene represented in part by SEQ ID NO:36 produces a protein of slmilar, but not identical, amino acid se~uence as the TcbA protein, and which likely has similar, but not identical biological activity as the TcbA protein.
In yet another instance, a gene encoding the peptides TcdAii-PK44 and the TcdAiii 58 kDa N-terminal peptide, described as SEQ ID
NO:39 (internal peptide TcdAii-PK44 sequence), and SEQ ID
NO:41(TcdAiil 58 kDa N-terminal peptide sequence) was isolated.

SUBSTITUTE SHEET (RULE 26) CA 022638l9 l999-02-26 W098/08932 PCT~US97/07657 Two pools of degenerate oligonucleotides, deslgned to encode the amino acid sequences described as SEQ ID NO:39 (Table 28) and SEQ
ID NO:41 (Table 27), and the reverse complements of those sequences, were synthesized as described in Example 8, and their DNA sequences.

SUBSTlTUTE SHEET lRULE-26) ,., ~
H

~I H U ~ U
N~ ~
a U~ U ~ H H

>t H H

N ~ ~ ~D ~ U ~ U

O O ,, ~ ,1 ~ E~
-d H L --1J lJ
~ ~ ~ U U

@ H H H
C~ _ _ _ _ # ~ ~ ~1 SUBSTITUTE SH EET ~RULE 26) CA 022638l9 l999-02-26 Polymerase Chain Reactions (PCR) were performed essentially as described in Example 8, using as forward primers Al.44.1 or A1.44.2, and reverse primers A2.3R or A2.4R, in all forward/reverse combinations, using Photorhabdus W-14 genomic DNA as template. In another set of reactions, primers A2.1 or A2.2 were used as forward primers, and A1.44.lR, and A1.44.2R were used as reverse primers in all forward/reverse combinations. Only in the reactions containing A1.44.1 or A1.44.2 as the forward primers combined with A2.3R as the reverse primer was a non-artifactual amplified product seen, of estimated size (mobility on agarose gels) of 1400 base pairs. The order of the primers used to obtain this amplification product indicates that the peptide fragment-TcdAii-PK44 lies amino-proximal to the 53 kDa peptide fragment of TcdAiii.
The 1400 bp PCR products were ligated to the plasmid vector pCRI~II according to the supplier's instructions. The DNA sequences across the ends of the insert fragments of four isolates were determined using primers similar in sequence to the supplier~s recommended primers and using sequencing methods described previously. The nucleic acid sequence of all isolates differed as expected in the regions corresponding to the degenerate primer sequences, but the amino acid sequences deduced from these data were the same as the actual amino acid sequences for the peptides determined previously, (SEQ ID NOS:41 and 39).
Screening of the W-14 genomic cosmid library as described in Example 8 with a radiolabeled probe comprised of the DNA prepared above (SEQ ID NO:36) identified five hybridizing cosmid isolates, namely 17D9, 20B10, 21D2, 27B10, and 26D1. These cosmids were distinct from those previously identified with probes corresponding to the genes described as SEQ ID NO:11 or SEQ ID NO:25.
Restriction enzyme analysis and DNA blot hybridizations identified three EcoR I fragments, of approximate sizes 3.7, 3.7, and 1.1 kbp, that span the region comprising the DNA of SEQ ID NO:36. Screening of the W-14 genomic cosmid library using as probe the radiolabeled 1.4 kbp DNA fragment prepared in this example identified the same 35 five cosmids (17D9, 20B10, 21D2, 27B10, and 26D1). DNA blot hybridization to EcoR I-digested cosmid DNAs also showed hybridization to the same subset of EcoR I fragments as seen with the 2.5 kbp TcdAii gene probe, indicating that both fragments are encoded on the genomic DNA.

SUBSTITUTE SHEET tRULE 26~

CA 022638l9 l999-02-26 WOg8/08932 PCT~S97/07657 DNA sequence determination of the cloned EcoR I fragments revealed an uninterrupted reading frame of 7551 base pairs (SEQ ID
NO:46), encoding a 282.9 kDa protein of 2516 amino acids (SEQ ID
NO:47). Analysis of the amino acid sequence of this protein ~ 5 revealed all expected internal fragments of peptides TcdAii(SEQ ID
NOS:17, 18, 37, 3B and 39) and the TcdAiii peptide N-terminus (SEQ
ID NO:41~ and all TCdAiii internal peptides (SEQ ID NOS:42 and 43).
- The peptides isolated and identified as TcdAii and TcdAiil are each products of the open reading frame, denoted tcdA, disclosed as SEQ
10 ID NO:46. Further, SEQ ID NO:47 shows, starting at position 89, the sequence disclosed as SEQ ID NO:13, which is the N-terminal sequence of a peptide of size approximately 201 kDa, indicating that the initial protein produced from SEQ ID NO: 46 is processed in a manner similar to that previously disclosed for SEQ ID NO:12.
In addition, the protein is further cleaved to generate a product of size 209.2 kDa, encoded by SEQ ID NO:48 and disclosed as SEQ ID
NO:49 (TcdAii peptide), and a product of size 63.6 kDa, encoded by SEQ ID NO:50 and disclosed as SEQ ID NO:51 (TcdAiii peptide). Thus, it is thought that the insecticidal activity identified as toxin A
(Example 15) derived from the products of SEQ ID NO:46, as exemplified by the full-length protein of 282.9 kDa disclosed as SEQ ID NO:47, is processed to produce the peptides disclosed as SEQ
ID NOS:49 and 51. It is thought that the insecticidal activity identified as toxin B ~Example 15) derives from the products of SEQ
ID NO:ll, as exemplified by the 280.6 kDa protein disclosed as SEQ
ID NO:12. This protein is proteolytically processed to yield the 207.6 kDa peptide disclosed as SEQ ID NO:53, which is encoded by SEQ ID NO:52, and the 62.9 kDa peptide having N-terminal sequence disclosed as SEQ ID NO:40, and further disclosed as SEQ ID NO:55, which is encoded by SEQ ID NO:54.
Amino acid sequence comparisons between the proteins disclosed as SEQ ID NO:12 and SEQ ID NO:47 reveal that they have 69%
similarity and 54% identity using the Wisconsin Package Version 8.0, Genetics Computer Group (GCG), Madison, WI or 60% similarity and 54% identity using version 9.0 of the program. This high degree of evolutionary relationship is not uniform throughout the entire amino acid sequence of these peptides, but is higher towards the carboxy-terminal end of the proteins, since the peptides disclosed as SEQ ID NO:51 (derived from SEQ ID NO:47) and SEQ ID

SUBSTITlJTE St}EET (RULE 26) W098/08g32 PCT~US97/076S7 NO:55 (derived from SEQ ID NO:12) ha-ve 76~ similarity and 64%
identity using the Wisconsin Package Version 8.0, Genetics Computer Group (GCG), Madison, WI or 71~ similarity and 64% identity using version 9.0 of the program.

Example 18 Control of European Cornborer-In~uced Leaf Damage on Maize Plants by Spray Application of Photorhabdus (Strain W-14) Broth The ability of Photorha~dus toxin(s) to reduce plant damage caused by insect larvae was demonstrated by measuring leaf damage caused by European corn borer (Ostrinia nubilalis) infested onto maize plants treated with Photorhabdus broth. Fermentation broth from Photorhabdus strain W-14 was produced and concentrated approximately 10-fold using ultrafiltration (10,000 MW pore-size) as described in Example 13. The resulting concentrated broth was then filter sterilized using 0.2 micron nitrocellulose membrane filters. A similarly prepared sample of uninoculated 2~ proteose peptone #3 was used for control purposes. Maize plants (an inbred line) were grown from seed to vegetative stage 7 or 8 in pots containing a soilless mixture in a greenhouse (27~C day; 22~C
night, about 50%RH, 14 hr day-length, watered/fertilized as needed). The test plants were arranged in a randomized complete block design (3 reps/treatment, 6 plants/treatment) in a greenhouse with temperature about 22~C dayi 18~C night, no artificial light and with partial shading, about 50~RH and watered/fertilized as needeG: Treàtments (uninoculated media and concentrated Photorhabdus broth) were applied with a syringe sprayer, 2.0 mls applied from directly (about 6 inches) over the whorl and 2.0 additional mls applied in a circular motion from approximately one foot above the whorl. In addition, one group of plants received no treatment. After the treatments had dried (approxlmately 30 minutes), twelve neonate European corn borer larvae (eggs obtained from commercial sources and hatched in-house) were applied directly to the whorl. After one week, the plants were scored for damage to the leaves using a modlfied Guthrie Scale (Koziel, M. G., Beland, G. L., Bowman, C., Carozzi, N. B., Crenshaw, R., Crossland, L., Dawson, J., Desai, N., ~ill, M., Kadwell, S., Launis, K., Lewis, SUBSTITUTE St~EET (RULE 26) CA 022638l9 l999-02-26 wo g8/08932 rCT/US97/07657 K., Maddox, D., McPherson, K., Meghj.i, M. Z., Merlin, E., Rhodes, R., Warren, G. W., Wright, M. and Evola, S. V. 1993).
Bio/Technology, 11, 194-195.) and the scores were compared statistically [T-test (LSD) p~0.05 and Tukey's Studentized Range ~HSD) Test p<0.1]. The results are shown in Table 29. For reference, a score of 1 represents no damage, a score of 2 represents fine "window pane" damage on the unfurled leaf with no ~ pinhole penetration and a score of 5 represents leaf penetration with elongated lesions and/or mid rib feeding evident on more than three leaves (lesions < 1 inch). These data indicate that broth or other protein containing fractions may confer protection against specific insect pests when delivered in a sprayable formulation or when the gene or derivative thereof, encoding the protein or part thereof, is delivered via a transgenic plant or microbe.
Table 29 Effect of Photorhabdus Culture Broth on European Corn Borer-Induced ~eaf Damage on Maize Treatment Average Guthrie Score No Treatment 5 02a Uninoculated medium 5 15a Photorha~dus Broth 2.24b Means with different letters are statistically different (p~0.05 or p<0.1).

Example l9 G~netic Engineering of Genes for Expression in E. coli Summary of Con~tructions A series of plasmids were constructed to express the tcbA gene of Photorhabdus W-14 in Escherichia coli. A list of the plasmids is shown in Table 30. A brief description of each construction follows as-well as a summary of the E. coli expression data obtained.

SUBSTITIITE SH EET tRULE 26~

W098/08932 ~CTAUS97/07657 T~hle.30 Expression Plasmids for the tcbA Gene ~lasml~ ~ene Vector/~electlon ~'ompartment p~A~ tc~A p~ hl lntracellular p~A~0~6 tc~A pAc~6/~/Amp ~aculovlrus, secreted p~A~O~/ tc~A ~ 7~/Kan ~erlplasm p~Ah~ tcbA ?~ tc~A lntracellular Ab~revlatlons: han=~anamycln, ~'hl=chloramphenlcol, Amp=amplclllln Co~struction of pDAB2025 In Example 9, a large EcoR I fragment which hybridizes to the TcbAii probe is described. This fragment was subcloned into pBC
(Stratagene, La Jolla CA) to create pDAB2025. Sequence analysis indicates that the fragment is 8816 base pairs. The fragment encodes the tcbA gene with the initiating ATG at position 571 and the terminating TAA at position 8086. The fragment therefore carries 570 base pairs of Photorhabdus DNA upstream of the ATG and 730 base pairs downstream of the TAA.
Cons~ruction of Plasmid pDAB2026 The tcbA gene was PCR amplified from plasmld pDAB2025 using the following primers; 5' prlmer (SlAc51) 5' TTT AAA CCA TGG GAA
ACT CAT TAT CAA GCA CTA TC 3' and 3' primer (SlAc31) 5' TTT AAA GCG
GCC GCT TAA CGG ATG GTA TAA CGA ATA TG 3'. PCR was performed using a TaKaRa LA PCR kit from PanVera (Madison, WI) in the following reaction: 57.5 microliters water, 10 microliters lOX LA buffer, 16 microliters dNTPs ~2.5 mM each stock solution), 20 microliters each primer at 10 pmoles/ microliters, 300 ng of the plasmid pDAB2025 containing the W- 14 tcbA gene and one microliter of TaKaRa LA Taq polymerase. The cycling conditions were 98~C/20 sec, 68~C/5 min, 72~C/10 min for 30 cycles. A PCR product of the expected about 7526 bp was isolated in a 0.8% agarose gel in TBE (100 mM Tris, 90 mM boric acid, 1 mM EDTA) buffer and purified using a Qiaex II kit from Qiagen (Chatsworth, CA). The purified tcbA gene was digested with Nco I and Not I and ligated into the baculovirus transfer vector pAcGP67B (PharMingen (San Diego, CA)) and transformed into DH5a ~. coli. The resulting recomblnant is called pDAB2026. The tcbA gene was then cut from pDAB2026 and transferred to pET27b to SUBSTITI.JTE St~EET tRU~E 26) CA 022638l9 l999-02-26 W098/08932 PCTrUS97/07657 create plasmid pDAB2027. A missense mutation in the tcbA gene was repaired in pDAB2027.
The repaired tc~A gene contains two changes from the sequence shown in Sequence ID NO:11; an A~G at 212 changing an asparagine 71 - 5 to serine 71 and a G~A at 229 changing an alanine 77 to threonine 77. These changes are both upstream of the proposed TcbAii N-terminus.

C~structio~ of ~DAR2028 The tcbA codlng region of pDAB2027 was transferred to vector pET15b. This was accomplished using shotgun ligations, the DNAs were cut with restriction enzymes Nco I and Xho I. The resulting recombinant is called pDAB2028.
1~ Ex~ression of TcbA in E. coli ~rom Plasm1d pDAB2028 Expression of tcbA in E. coli was obtained by modification of the methods previously described by Studier et al. (Studier, F.~., Rosenberg, A., Dunn, J., and Dubendorff, J., (1990) Use of T7 RNA
polymerase to direct expression of cloned genes. Methods Enzymol., 185: 60-89.~. Competent E. coli cells strain BL21(DE3) were transformed with plasmid pDAB2028 and plated on LB agar containing 100 ~g/mL ampicillin and 40 mM glucose. The transformed cells were plated to a density of several hundred isolated colonies/plate.
Following overnight incubation at 37~C the cells were scraped from the plates and suspended in LB broth containing 100 ~g/mL
ampicillin. Typical culture volumes were from 200-500 mL. At time zero, culture densities (OD600) were from 0.05-0.15 depending on the experiment. Cultures were shaken at one of three temperatures (22~C, 30~C or 37~C) until a density of 0.15-0.5 was obtained at which time they were induced with 1 mM isopropylthio-~-galactoside (IPTG). Cultures were incubated at the designated temperature for 4-5 hours and then were transferred to 4~C until processing (12-72 hours).

Purification and Characterization of TcbA Expressed in E. coli from Plasmid pDAB2028 E. coli cultures expressing TcbA peptides were processed as follows. Cells were harvested by centrifugation at 17,000 x G and the media was decanted and saved in a separate container.

SUBSTrrUTE St{EET (RULE 26) CA 022638l9 l999-02-26 W098/08932 PCT~US97/076S7 The media was concentrated about 8x using the M12 (Amicon, Beverly MA) filtration system and a lO0 kD molecular mass cut-off filter. The concentrated media was loaded onto an anion exchange column and the bound proteins were eluted with 1.0 M NaCl. The 1.0 M NaCl elution peak was found to cause mortality against Southern corn rootworm (SCR) larvae Table 30). The 1.0 M NaCl fraction was dialyzed against 10 mM sodium phosphate buffer pH 7.0, concentrated, and subjected to gel filtration on Sepharose CL-4B
~Pharmacia, Piscataway, NJ). The region of the CL-4B elution profile correspon~lng to calculated molecular weight (about 900 kDa) as the native W-14 toxin complex was collected, concentrated and bioassayed against larvae. The collected 900 kDa fraction was found to have insecticidal activity (see Table 31 below), with symptomology similar to that caused by native W-14 toxin complex.
This fraction was subjected to Proteinase K and heat treatment, the activity in both cases was either eliminated or reduced, providing evidence that the activity is proteinaceous in nature. In addition, the active fraction tested immunologically positive for the TcbA and TCbAiii peptides in immunoblot analysis when tested with an anti-TcbAiii monoclonal antibody (Table 31).

Table 31 Results of I~mllnohlot and SCR Bio~.ssays ~ractlon ~ Actlvlty lmmuno~lot Natlve Size ~ ~ ~rowth ~eptldes L~L-4~
Mortalit Inhibit. De~ected Estimate y d Size]
~l~c~A Me~la l.~ M +++ +++ 'l~cDA
lon ~xchange 'l~cbA Medla ~-4~ +++ +++ 'l~C~A, about TCbAiii 900 kDa TcbA Me~la C~-4~ ++ +++
+ Proteinase K
~l~c~A Medla ~L-4~ - -+ heat treatment 'l~cbA ~ell ~up ~'~-4~ - +++ N'l' about 900 kD
~K = ~rOtelIlaSe K tr atment ~ h~urs; heat reatment = lJ~ or l~
minutes; ND = None Detected; NT = Not Tested. Scoring system for mortality and growth inhibition as co~pared to control samples; 5-24%="+", 25-4g%="++", 50-100%="+++".

The cell pe~let was resuspended in 10 mM sodium phosphate buffer, pH=7.0, and lysed by passage through a Bio-NebTM cell nebulizer (Glas-Col Inc., Terra Haute, IN). The pellets were S~IBSTI~UTE S~EET ~RULE 26) , CA 022638l9 l999-02-26 treated with DNase to remove DNA and centrifuged at 17,000 x g to separate the cell pellet from the cell supernatant. The supernatant fraction was decanted and filtered through a 0.2 micron fllter to remove large particles and subjected to anion exchange chromatography. Bound proteins were eluted with 1.0 M NaCl, dialyzed and concentrated using Biomax~M (Millipore Corp, Bedford, MA) concentrators with a molecular mass cut-off of 50,000 Daltons.
~ The concentrated fraction was subjected to gel filtration chromatography using Sepharose CL-4B beaded matrix. Bioassay data for material prepared in this way is shown in Table 30 and is denoted as "TcbA Cell Sup".
In yet another method to handle large amounts of material, the cell pellets were re-suspended in 10 mM sodium phosphate buffer, pH
= 7.0 and thoroughly homogenized by using a Kontes Glass Company (Vineland, NJ) 40 ml tissue grinder. The cellular debris was pelleted by centrifugation at 25,000 x g and the cell supernatant was decanted, passed through a 0.2 micron filter and subjected to anion exchange chromatography using a Pharmacia 10/10 column packed wlth Poros HQ 50 beads. The bound proteins were eluted by performing a NaCl gradient of 0.0 to 1.0 M. Fractions containing the TcbA protein were combined and concentrated using a 50 kDa concentrator and subjected to gel filtration chromatography using Pharmacia CL-4B beaded matrix. The fractions containing TcbA
oligomer, molecular mass of approximately 900 kDa, were collected and subjected to anion exchange chromatography using a Pharmacia Mono Q 10/10 column equilibrated with 20 mM Tris buffer pH = 7.3.
A gradient of 0.0 to 1.0 M NaCl was used to elute recombinant TcbA
protein. Recombinant TcbA eluted from the column at a salt concentration of approximately 0.3-0.4 M NaCl, the same molarity at which native TcbA oligomer is eluted from the Mono Q 10/10 column.
The recombinant TcbA fraction was found to cause SCR mortality in bioassay experiments similar to those in Table 31.

A second set of expression constructions were prepared and tested for expression of the TcbA protein toxin.
Construction of pDAR2030: An Expressio~ P~asmid for the tcbA
Coding Region The plasmid pDAB2028 (see herein) contains the tcbA coding region in the commercial vector pET15 (Novagen, Madison, WI), SUBSTITUTE SH EET tRULE-26) W098/08932 PCT~US97/07657 encodes an ampicillin selection marker. The plasmid pD~B2030 was created to express the tcbA coding region from a plasmid which encodes a kanamycin selection marker. This was done by cutting pET27 (Novagen, Madison, WI) a kanamycin selection plasmid, and pDA32028 with Xba I and Xho I. This releases the entire multiple cloning site, including the tcbA coding region from plasmid pDAB2028. The two cut plasmids, were mixed and ligated.
Recombinant plasmids were selected on kanamycin and those containing the pDAB2028 fragment were identified by restriction analysis. The new recombinant plasmid is called pDAB2030.

Con.~truction of Plasmid pDAB2031: Correction of Mutations in tcbA
The two mutations in the N-terminus of the tcbA coding region as described in Example 19 (Sequence ID NO:11; A>G at 212 changing an asparagine 71 to serine 71i G>A at 229 changing an alanine 77 to threonine 77) were corrected as follows: A PCR product was generated using the primers TH50 (5' ACC GTC TTC TTT ACG ATC AGT G
3')and SlAc51(5' TTT AAA CCA TGG GAA ACT CAT TAT CAA GCA CTA TC 3') and pDAB2025 as template to generate a 1778 bp product. This PCR
product was cloned into plasmid pCR2.1 (Invitrogen, San Diego, CA) and a clone was isolated and sequenced. The clone was digested with Nco I and Pin AI and a 1670 bp fragment was purified from a 1 agarose gel. A plasmid containing the mutated tcbA codlng region (pDAB2030) was digested with Nco I and Not I and purified away from the 1670 bp fragment in a 0.8~ agarose with Qiaex II (Qiagen, Chatsworth, CA). The corrected Nco I/Pin AI fragment was then ligated into pDAB2030. The ligated DNA was transformed into DH5a E. coli. A clone was isolated, sequenced and found to be correct.
This plasmid, containing the corrected tcbA coding region, is called pDAB2031.

Construction of DDAB2033 and pDA~2034: Expression Plasmids for t cbA
The expression plasmids pDAB2025 and pDAB2027-2031 all rely on the Bacteriophage T7 expression system. An additional vector system was used for bacterial expression of the tcbA gene and its derivatives. The expression vector Trc99a (Pharmacia Biotech, Piscataway, NJ) contains a strong trc promoter upstream of a multiple cloning site with a 5 ' Nco I site which is compatible with the tcbA coding region from pDAB2030 and 2031. However, the plasmid does not have a compatible 3~ site. Therefore, the Hlnd III site of Trc99a was cut and made blunt by treatment with T4 DNA

SUBSTITUTE SHEET (RULE 26) CA 022638l9 l999-02-26 WOg8/08932 PCT~S97/07657 polymerase (Boehringer Mannheim, Indianapolis, IN). The vector plasmid was then cut by Nco I followed by treatment with alkaline phosphatase. The plasmids pDAB2030 and pDAB2031 were each cut with Xho I ( cuts at the 3' end of the tcbA coding region) followed by treatment with T4 DNA polymerase to blunt the ends. The plasmids were then cut with Nco I, the DNAs were extracted with phenol, ethanol precipitated and resuspended in buffer. The Trc99a and pDAB2030 and pDAB2031 plasmids were mixed separately, ligated and transformed into DH5a cells and plated on LB media containing ampicillin and 50 mM glucose. Recombinant plasmids were identified by restriction digestion. The new plasmids are called pDA}32033 (contains the tcbA coding sequence with the two mutations in tCbAi) and pDAB2034 (contains the corrected version of tcbA from pDAB2031).

Construction of Plasmid pDAB2032: An Expression Plasmid for tCbAiiAiii A plasmid encoding the TcbAiiAiii portion of TcbA was created in a similar way as plasmid pDAB2031. A PCR product was generated using TH42 (5' TAG GTC TCC ATG GCT TTT ATA CAA GGT TAT AGT GAT CTG

3') and TH50 (5' ACC GTC TTC TTT ACG ATC AGT G 3') primers and plasmid pDAB2025 as template. This yielded a product of 1521 bp having an initiation codon at the beginning of the coding sequence of tcbAii. This PCR product was isolated in a 1% agarose gel and purified. The purified product was cloned into pCR2.1 as a~ove and a correct clone was identified by DNA sequence analysis. This clone was digested with Nco I and Pin AI, a 1414 bp fragment was isolated in a 1~ agarose gel and ligated into the Nco I and Pin AI
sites of plasmid pDAB2030 and transformed into DH5a E. coli. This new plasmid, designed to express TcbAiiAiii in E. coli, is called pDA}32032.

~xpression of tcbA and tCbAiiAiii from Plasmids pDAB2030 pDAB2031 ~n~ pDAR2032 . 35 Expression of tcbA in E. coli from plasmids pDA}32030, pDP~32031 ~ and pDA}32032 was as described herein, except expression of tcbAiiAiii was done in E. coli strain HMS174 (DE3) (Novagen, Madison, - WI).

SUBSTlTlJTE 5t~ RULE 2~) WO 98/08932 PCT~US97/07657 Expression of t~h~ from Plasmid pDAB2033 The plasmid pDAB2033 was transformed into BL21 cells (Novagen, Madi-son, WI) and plated on LB contalning 100 micrograms/mL
ampicillin and 50 mM glucose. The plates were spread such that several hundred well separated colonies were present on each plate following incubation at either 30~C or 37~C overnight. The colonies were scraped from the plates and suspended in LB
containing 100 micrograms/mL ampicillin, but no glucose. Typical culture volume was 250 mL in a single 1 L baffle bottom flask. The cultures were induced when the culture reached a density of 0.3-0.6 OD600 nm. Most often this density was achieved immediately after suspension of the cells from the plates and did not require a growth period in liquid media. Two induction methods were used.
Method l: cells were induced with 1 mM IPTG at 37~C. The cultures were shaken at 200 rpm on a platform shaker for 5 hours and harvested. Method 2: The cultures were induced with 25 micromolar IPTG at 30~C and shaken at 200 rpm for 15 hours at either 20~C or 30~C. The cultures were stored at 4~C until used for purification.

Purification of TcbA from E. coli Purification, bioassay and immunoblot analysis of TcbA and TcbAiiAiii was as described herein. Results of several representative E. coli expression experiments are shown in Table 32. All materials shown in Table 32 were purified from the media fraction of the cultures. The predicted native molecular weight is approximately 900 kD as described herein. The purity of the samples, the amount of TcbA relative to contaminating proteins, varied with each preparation.

SUBS~ITUTE SHEET (RULE 26) wo g8/08932 rCT/US97/07657 T~hle 32 Bioassay Activity ~n~ Immllnoblot Analysis of TcbA and Derivatives Produced in E. coli and Purified from the Cultl~re Media Plasmla ~'o~lng ~. Southern-Corn ~eptl~es Mlcrograms Regio~ coll Rootworm Bioassay Detected Protein Strain Activity by Applied to Immunoblot Dlet ~rowth Inhibit. Mortal.
pDA~20~ tc~A ~LZl - +++ ~l~cbA + 1-~
(DE3) TcbA
p~A~2031 tcbA BL~l - +++ 'l~cbA + 1-1 (DE3) TcbAiii pOAB~O~ tcbA BL21 - +++ ~l~cbA + l-Z
TcbAiii pDA~203~ tcbAiiAiii~MS174 +I+ + lcbAiiAiii 13-21 (DE3) + TcbAiii ~corlng ystem ~or nortallty an~ grcwth lnhlLltlon on ~cuthern ~orr Rootworm as compared to control samples; 5-24~="+", 25-49~="++", 50-100~="+++".

Example 20 Characterization of Toxin Peptides with Matrix-~sisted Laser Desorption Ionization Time-of-Flight Mass S~ectroscopy Toxins isolated from W-14 broth were purified as described in Example 15. In some cases, the TcaB protein toxin was pretreated with proteases (Example 16) that had been isolated from W-14 broth as previously described (Example 15). Protein molecular mass was determined using matrix-assisted laser desorption ionization time-of-flight mass spectroscopy, hereinafter MALDI-TOF, on a VOYAGER
BIOSPECTROMETRY workstation with DELAYED EXTRACTION technology (PerSeptive Biosystems, Framingham, MA). Typically, the protein of interes~ (100-500 pmoles in 5 ~1) was mixed with 1 ~1 of acetonitrile and dialyzed for 0.5 to 1 h on a M~ pore VS filter having a pore size of 0.025 ~M (Millipore Corp. Bedford, MA).
Dialysis was performed by floating the filter on water(shinny side up) followed by adding protein-acetonitrile mixture as a droplet to the surface of the filter. After dialysis, the dialyzed protein removed using a pipette and was then mixed with a matrix consisting of sinapinic acid and trifluoroacetic acid according to manufacturers instructions. The protein and matrix were allowed to co-crystallize on a about 3 cm2 gold-plated sample plate (PerSeptive Corp.). Excitation of the crystals and subsequent mass analysis was performed using the following conditions: laser setting of 3050; pressure of 4.55e-07; low mass gate of 1500.0;
negative ions off; accelerating voltage of 25,000; grid voltage of SUE~STITUTE SHEET lRULE 26) CA 022638l9 l999-02-26 W098/08932 PCT~US97/07657 90.0%; guide wire voltage of 0.010%; linear mode; and a pulse delay time of 350 ns.
Protein mass analysis data are shown in Table 33. The data obtained from MALDI-TOF was compared to that hypothesized from gene sequence information and as previously determined by SDS-PA&E.
Table 33 Molecular An~lysis of Peptides by MALDI-TOF, SDS-PAGE and Predicted Determinatio~ Based on G~ne Sequence Peptide Predicted (Gene) SDS PAGEMALDI-TOF _ TcbA 280,634 Da 240,000 Da 281,040 Da TCbAi/ii 217,710 Da not resolved216,812 Da TcbAii 207,698 Da 201,000 Da 206,473 Da TcbAiii 62,943 Da 58,000 Da 63,520 Da __________________________________________________________ TcdAii 209,218 Da188,000 Da 208,186 Da TcdAiii 63,520 Da56,000 Da 63,544 Da ---_____________________ _ TcbAi1 Protease Generated 201,000 Da 216,614 Da 215,123 DaA
210,391 DaA
208,680 DaA
25 TcbAiii Protease Generated 56,000 Da 64,111 Da ~Data normalized TcbA, multiple fragments observed at TcbAi/ii Example 21 Production of Peptide Specific Polyclonal Antibodies Nine peptide components of the W-14 toxin complex, namely, TcaA, TcaAiii, TcaBi, TcaBii, TcaC, TcbAi1, TcbA~ TcdAii, and TcdAiii were selected as targets against which antibodies were produced. Comprehensive DNA and deduced amino acid sequence data for these peptides indicated that the sequence homology between some of these peptides was substantial. If a whole peptide was used as the immunogen to induce antibody production, the resulting antibodies might bind to multiple peptides in the toxin preparation. To avoid this problem antibodies were generated that would bind specifically to a unique region of each peptide of interest. The unique region (subpeptide) of each target peptide was selected based on the analyses described below.
Each entire peptide sequence was analyzed using MacVector Protein Analysis Tool (IBI Sequence Analysis Software, International Biotechnologies, Inc., P. O. Box 9558, New Haven, CT
06535) to determine its antigenicity index. This program was designed to locate possible externally-located amino acid SUBSmUTE SHEET (RULF 26) .. . . . ...

W O 98/08932 PCTrUS97/07657 sequences, i.e., regions that might be antigenic sites. This method combined infor~ation from hydrophilicity, surface probability, and backbone flexibility predictions along with the secondary structure predictions in order to produce a composite prediction of the surface contour of a protein. The scores for - each of the analyses were normalized to a value between -1.0 and +1.0 (MacVector Manual). The antigenicity index value was obtained for the entire sequence of the target peptide. From each peptide, an area covering 19 or more amino acids that showed a high antigenicity index from the original sequence was re-analyzed to determine the antigenicity index of the subpeptide without the flanking residues. This re-analysis was necessary because the antigenicity index of a peptlde could be influenced by the flanking amino acid residues. If the isolated subpeptide sequence did not maintain a high antigenicity index, a new region was chosen and the analysis was repeated.
Each selected subpeptide sequence was aligned and compared to all seven target peptide sequences using MacVector alignment program. If a selected subpeptide sequence showed identity (greater than 20%) to another target peptide, a new 19 or more amino acid region was isolated and re-analyzed. Unique subpeptide sequences covering 19 or more amino acid showing high antigenicity index were selected from all target peptides.
The sequences of seven subpeptides were sent to Genemed Biotechnology Inc. The last amino acid residue on each subpeptide was deleted because it showed no apparent effect on the antigenicity index. A cysteine residue was added to the N-terminal of each subpeptide sequence, except TcaBi-syn which contains an internal cysteine residue. The present of a cysteine residue facilitates conjugation of a carrier protein (KLH). The final peptide products corresponding to the appropriate toxin peptides and SEQ ID NO.s are shown in Table 34.

SU85TITUTE Sl~ EET tRULE 26) CA 022638l9 l999-02-26 W098/08932 PCTrUS97/07657 Table 34 Amino Acid Sequ~nces for Synthetic Peptides _ SEO ID No. Pepide Amino Acid Sequence 63 TcaAii-syn NH2-(C) L R G N S P T N P D X D G I F A Q V A
64 TcaAiii-SYn NH2-(C) Y T P D Q T P S F Y E T A F R S A D G
TcaBi-syn NH2-H G Q S Y N D N N Y C N F T L S I N T
66 TcaBiii-SYn NH2-(C) V D P K T L Q R Q Q A G G D G T G S S
67 TcaC-syn NH2-(C) Y K A P Q R Q E D G D S N A V T Y D K
68 TcbAii-syn NH2-(C) Y N E N P S S E D K K W Y F S S K D D
69 TcbAiii-syn NH2-(C) F D S Y S Q L Y E E N I N A G E Q R A
TcdAii-syn NH2-(C) N P N N S S N K L M F Y P V Y Q Y S G N T
71 TCdAiii-syn NH2-(C) V S Q G S G S A G S G N N N L A F G A G

Each conjugated synthetic peptide was in~ected into two rabbits according to Genemed accelerated program. The pre- and post-immune sera were a~ailable for testing after one month.
The preliminary test of both pre- and post-immune sera from each rabbit was performed by Genemed Biotechnologies Inc. Genemed reported that by using both ELISA and Western blot techniques, they detected the reaction of post-immune sera to the respective synthetic peptides. Subsequently, the sera were tested with the whole target peptides, by Western blot analysis. Two batches of partially purified Photorhabdus strain W-14 toxin complex was used as the antigen. The two samples had shown activity against the Southern corn rootworm. Their peptide patterns on an SDS-PAGE gel were slightly different.
Pre-cast SDS-polyacrylamide gels with 4-20% gradient (Integrated Separation Systems, Natick, MA 01760) were used.
Between 1 to 8 ~g of protein was applied to each gel well.
Electrophoresis was performed and the protein was electroblotted onto Hybond-ECL nitrocellulose membrane (Amersham International).
The membrane was blocked with 10% milk in TBST (25 mM Tris HCl pH
1.4, 136 mM NaCl, 2.7 mM KCl, 0.1% Tween 20) for one hour at room temperature. Each rabbit serum was diluted in 10% milk/TBST to 1:500. Other dilutions between 1:50 to 1:1000 were also used. The serum was added to the membrane and placed on a platform rocker for at least one hour. The membrane was washed thoroughly with the blocking solution or TBST. A 1:2000 dilution of secondary antibodies (goat anti-mouse IgG conjugated to horse radish peroxidase; BioRad Laboratories) in 10% milk/TBST was applied to the membrane placed on a platform rocker for one hour. The membrane was subse~uently washed with excess amount of TBST. The SUBSTITUTE SHEET (RULE 26) CA 022638l9 l999-02-26 W098/08932 PCTrUS97/07657 detection of the protein was performed by using an ECL (Enhanced Chemiluminescence) detection kit (Amersham International).
Western blot analyses were performed to identify binding specificity of each anti-synthetlc peptide antibodies. All synthetic polyclonal antibodies showed specificity toward to processed and, when applicable, unprocessed target peptides from protein fractions derived from Photorhabdus culture broth. Various ~ antibodies were shown to recognize either unprocessed or processed recombinant proteins derived from heterologous expression systems such as bacteria or insect cells, using baculovirus expression constructs. In one case, the anti-TcbAiii-syn antibody showed some cross-reactivity to anti-TcdAiii peptide. In a second case, the anti-TcaC-syn antibody, recognized an unidentified 190 kDa peptide in W-14 toxin complex fractions.
Ex~m~le 22 Char~cterization of Photorhabdus Strains In order to establish that the collection described herein was comprised of Photorhabdus strains, the strains herein were assessed in terms of recognized microbiological traits that are characteristic of the bacterial genus Photorhabdus and which differentiate it from other Enterobacteriaceae and Xenorhabdus spp.
(Farmer, J. J. 1984. Bergey's Manual of Systemic Bacteriology, Vol l. pp. 510-511. (ed. Kreig N. R. and Holt, J. G.). Williams &
Wilkins, Baltimore.; Akhurst and Boemare, 1988, J. Gen. Microbiol.
134, 1835-1845; Forst and Nealson, 1996. Microbiol. Rev. 60, 21-43). These characteristic traits are as follows: Gram stain negative rods, organism size of 0.3-2 ~m in width and 2-10 ~m in length [with occasional filaments (15-50 ~m) and spheroplasts], yellow to orange/red colony pigmentation on nutrient agar, presence of crystalline inclusion bodies, presence of catalase, inability to reduce nitrate, presence of bioluminescence, ability to take up dye from growth media, positive for protease production, growth at temperatures below 37~C, survival under anaerobic conditions and positively motile. (Table 33). Test methods were checked using reference Escherichia coli, Xenorhabdus and Photorhabdus strains.
The overall results are consistent with all strains being part of the family Enterobacteriaceae and the genus Photorhabdus. Note that DEP1, DEP2, and DEP3 refer to Photorhabdus strains obtained SUBSTlllJTE SHEET tRULE 2~) CA 022638l9 l999-02-26 W098/08932 PCTrUS97/07657 from the American Type Culture Collection, 12301 Parklawn Drive, Rockville, MD 20852 USA (#29304, 29999 and 51583, respectively).
A luminometer was used to establish the bioluminescence associated with these Photorhabdus strains. To measure the presence or absence of relative light emitting units, the broths from each strain (cells and media) were measured at three time intervals after inoculation in liquid culture (24, 48, 72 hr) and compared to background luminosity (uninoculated media). Several Xenorhabdus strains were tested as negative controls for luminosity. Prior to measuring light emission from the various broths, cell density was established by measuring light absorbance (560 nM) in a Gilford Systems (Oberlin, OH) spectrophotometer using a sipper cell. The resulting light emitting units could then be normalized to density of cells. Aliquots of the broths were placed into 96-well microtiter plates (lOO ~1 each) and read in a Packard LumicountT~ luminometer (Packard Instrument Co., Meriden, CT). The measurement period for each sample was 0.1 to 1.0 second. The samples were agitated in the luminometer for lO sec prior to taking readings. A positive test was determined as being about 5-fold background luminescence (about 1-15 relative light units). In addition, degree of colony luminosity was confirmed with photographic film overlays and by eye, after visual adaptation in a darkroom. The Gram's staining characteristics of each strain were esta~lished with a commercial Gram's stain kit (BBL, Cockeysville, MD) used in conjunction with Gram's stain control slides (Fisher Scientific, Pittsburgh, PA). Microscopic evaluation was then performed using a Zeiss microscope (Carl Zeiss, Germany) lOOX oil immersion objective lens (with lOX ocular and 2X body magnification). Microscopic ~min~tion of individual strains for organism size, cellular description and inclusion bodies (the latter two observations after logarithmic growth) was performed using wet mount slides (lOX ocular, 2X body and 40X objective magnification) and phase contrast microscopy with a micrometer (Akhurst, R. ~. and Boemare, N. E. l990. Entomopathogenic Nematodes in Biological Co~trol (ed. Gaugler, R. and Kaya, H.). pp. 75-90.
CRC Press, Boca Raton, USA.; Baghdiguian S., Boyer-Giglio M. H., Thaler, J. O., Bonnot G., Boemare N. 1993. Biol. Cell 79, 177-185.). Colony pigmentation was observed after inoculation on Bacto nutrient agar, (Difco Laboratories, Detroit, MI) prepared as per SU8STITUTE ~HEET (RULE 26) CA 022638l9 l999-02-26 W098/08932 PCT~US97/07657 label instructions. Incubation occu~red at 28~C and descriptions were produced after 5 days. To test for the presence of the enzyme catalase, a colony of the test organism was removed on a small plug from a nutrient agar plate and placed into the bottom of a glass test tube. One ml of a household hydrogen peroxide solutlon was gently added down the side of the tube. A positive reaction was recorded when bubbles of gas (presumptive oxygen) appeared lmmediately or within 5 seconds. Controls of uninoculated nutrient agar and hydrogen peroxide solution were also examined. To test for nitrate reduction, each culture was inoculated into 10 ml of Bacto Nitrate Broth (Difco Laboratories, Detroit, MI). After 24 hours incubation with gentle agitatlon at 28~C, nitrite production was tested by the addition of two drops of sulfanilic acid reagent and two drops of alpha-naphthylamine reagent (see Difco Manual, 10th edition, Difco Laboratories, Detroit, MI, 1984). The generation of a distinct pink or red color indicates the formation of nitrite from nitrate whereas the lack of color formation indicates that the strain is nitrate reduction negative. In the latter case, finely powdered zinc was added to further confirm the presence of unreduced nitrate; established by the formation of nitrite and the resultant red color. The ability of each strain to uptake dye from growth media was tested with Bacto MacConkey agar containing the dye neutral red; Bacto Tergitol-7 agar containing the dye bromothymol blue and Bacto EMB Agar containing the dye eosin-Y (formulated agars from Difco Laboratories, Detroit, MI, all prepared according to label instructions). After inoculation on these media, dye uptake was recorded after incubation at 28~C for 5 days. Growth on these latter media is characteristic for members of the family Enterobacte~iaceae. Motility of each strain was tested using a solution of Bacto Motility Test Medium (Difco Laboratories, Detroit, MI) prepared as per label instructions. A
butt-stab inoculation was performed with each strain and motility was judged macroscopically by a diffuse zone of growth spreading from the line of inoculum. The production of protease was tested by observing hydrolysis of gelatin using Bacto gelatin (Difco ~ Laboratories, Detroit, MI) made as per label instructions.
Cultures were inoculated and the tubes or plates were incubated at 28~C for 5 days. Gelatin hydrolysis was then checked at room temperature, i.e. less than 22~C. To assess growth at different SUBSTITUTE St~EET tRULE 26) W098/08932 PCT~US97107657 temperatures, agar plates [2% proteose peptone #3 with two percent Bacto-Agar (Difco, Detroit, MI) in deionized water] were streaked from a common source of inoculum. Plates were incubated at 20, 28 and 37~C for up to three weeks. The incubator temperature levels were checked with an electronic thermocouple and meter to insure valid temperature settings. Oxygen requirements for Photorhabdus strains were tested in the following manner. A butt-stab inoculation into fluid thioglycolate broth medium (Difco, Detroit, MI) was made. The tubes were incubated at room temperature for one week and cultures were then examined for type and extent of growth.
The indicator resazurin demonstrates the presence of medium oxygenation or the aerobiosis zone (Difco Manual, l0th edition, Difco Laboratories, Detroit, MI). Growth zone results obtained for the Photorhabdus strains tested were consistent with those of a facultative anaerobic microorganism. In the case of unclear results, the final agar concentration of fluid thioglycolate broth medium was raised to 0.75% and the growth characteristics rechecked.

~UBSTITUTE SHE~T(Rl~ 26) CA 022638l9 l999-02-26 W098/08932 PCT~S97/07657 Table.~5 Taxon~mi~ Traits of Photorhab~us Strai n~

~traln A~ 1 1 JS K L M N O Y ~.2 + rd ~ + ~ + + +
, zealandica - ~. heplalus - + + r~ ~ + - + + + y + + + + + +
~-Arg - t + r~ ~ + - + + + w + + + + + +
~ swego - t + r~ ~ + - + + + W + + + + + +
~ Lewlston - + + r~ ~ + - + + + l + + ~ + + +
~ K-l~Z - + + ra ~ + - + + + Y + + + + + +
HM~ - + + rd ~ + - + + + Rd + + + + + +
T n~lcus - + + rd ~ + - + + + w + + + + + +
~L - + + r~ ~ + - + ~ + Yl + + + + + +
~W~-~ - + + rd ~ + - + + + y + + + + + +
Megl~ls - ~ + rd ~ + - + ~ + R + + + + + +
~ + + rd ~ + - + + + ~ + + + + + +
A. ~ows .- + + ra ~ + - + + + ~X + + + + + +
M~l - + + r~ ~ + - + + + l + + + + + +
MY~ - + + r~ ~ + - + + + l + + + + + +
M~ - + + r~ ~ + - + + + ~ + + + + + +
M~ - + + r~ ~ + - + + + y + + + + + +
M~ - + + r~ ~ + - + + + ~ + + + + + +
~y~ - + + rd ~ + - + + + W + + + + + +
~Ll~ 1 - + + rd ~ + - + + + w + + + + + +
- + + r~ ~ + - + + + W + + + + + +
- + + r~ ~ + - + + + W + + + + + +
l/ - + + r~ ~ + - + + + y + + + + + +
- + + rc ~ + - + + + o + + + + + +
~l - + + rd ~ + - + + + w + + + + + +
- + + r~ ~ + - + + + ~K + + + + + +
~ - + + rd ~ + - + ~ + ~ + + + + + +
*: A=Gram's stain, B=Crystaline inclusion bodies, C=Bioluminescence, D=Cell form, E=Motility, F=Nitrate reduction, G=Presence of catalase, H=Gelatin hydrolysis, I=Dye uptake, J=Pigmentation on Nutrient Agar (some color shifts after Day 5), K=Growth on EMB agar, L=Growth on MacConkey agar, M=Growth on Tergitol-7 agar, N =Facultative anaerobe, O=Growth at 20~C, P=Growth at 28~C, Q=Growth at 37~C.
t: +=positive for trait, - =negative for trait; rd=rod, S=sized within Genus descriptors.
: W = white, CR = cream, Y =yellow, YT=yellow tan, T=tan PO=pale orange, O=orange, PR=pale red, R=red.
The evolutionary diversity of the Photorhabdus strains in our collection was measured by analysis of PCR (Polymerase Chain Reaction) mediated genomic fingerprinting using genomic DNA from each strain. This technique is based on ~amilies of repetitive DNA
sequences present throughout the genome of diverse bacterial species (reviewed by Versalovic, J., Schneider, M., DE Bruijn, F. J. and Lupski, J. R. 1994. Methods Mol. Cell. Biol., 5, 25-40).
Three of these, repetitive extragenic palindromic sequence (REP), enterobacterial repetitive intergenic consensus (ERIC) and the BOX

SUBSTITUTE S}~EET tRULE 26) CA 022638l9 l999-02-26 W098/08932 PCT~US97/07657 element are thought to play an important role in the organization of the bacterial genome. Genomic organization is believed to be shaped by selection and the differential dispersion of these elements within the genome of closely related bacterial strains can be used to discriminate these strains (e.g., Louws, F. J., Fulbright, D. W., Stephens, C. T. and DE Bruijn, F. J. 1994. Appl.
Environ. Micro. 60, 2286-2295). Rep-PCR utilizes oligonucleotide primers complementary to these repetitive se~uences to amplify the variably sized DNA fragments lying between them. The resulting products are separated by electrophoresis to esta~lish the DNA
"fingerprint" for each strain.
To isolate genomic DNA from our strains, cell pellets were resuspended in TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) to a final volume of 10 ml and 12 ml of 5 M NaCl was then added. This 15 mixture was centrifuged 20 min. at 15,000 x g. The resulting pellet was resuspended in 5.7 ml of TE and 300 ~l of 10~ SDS and 60 ~l 20 mg/ml proteinase K (Gibco BRL Products, Grand Island, NY) were added. This mixture was incubated at 37~C for 1 hr, approximately 10 mg of lysozyme was then added and the mixture was incubated for an additional 45 min. One milliliter of 5M NaCl and 800 ~l of CTAP/NaCl solution (10~ w/v CTA~3, 0.7 M NaCl) were then added and the mixture was incubated 10 min. at 65~C, gently agitated, then incubated and agitated for an additional 20 min. to aid in clearing of the cellular material. An equal volume of chloroform/isoamyl alcohol solution (24:1, v/v) was added, mixed gently then centrifuged. Two extractions were then performed with an equal volume of phenol/chloroform/isoamyl alcohol (50:49:1).
Genomic DNA was precipitated with 0.6 volume of isopropanol.
Precipitated DNA was removed with a glass rod, washed twice with 30 70% ethanol, dried and dissolved in 2 ml of STE (10 mM Tris-HCl pH8.0, 10 mM NaCl, 1 mM EDTA). The DNA was then quantitated by optical density at 260 nm. To perform rep-PCR analysis of Photorhabdus genomic DNA the following primers were used, REPlR-I;
5'-IIIICGICGICATCIGGC-3' and REP2-I; 5'-ICGICTTATCIGGCCTAC-3'. PCR
35 was performed using the following 25~1 reaction: 7.75 ~l H2O, 2.5 ~l 10X LA buffer (PanVera Corp., Madison, WI), 16 ~l dNTP mix (2.5 mM each), 1 ~l of each primer at 50 pM/~ 1 DMSO, 1.5 ~1 genomic DNA (concentrations ranged from 0.075-0.480 ~g/~1) and 0.25 ~l TaKaRa EX Taq (PanVera Corp., Madison, WI). The PCR

SUBSTIll~TE SHEET tRULE 26) W098/08932 PCT~S97~7657 amplification was performed in a Per~in Elmer DNA Thermal Cycler (Norwalk, CT) using the following conditions: 95~C/7 min. then 35 cycles of; 94~C/1 min.,44~C/1 min., 65~C/8 min., followed by 15 min.
at 65~C. After cycling, the 25 ~1 reaction was added to 5 ~1 of 6X
gel loading buffer (0.25% bromophenol blue, 40% w/v sucrose in H2O). A 15x20cm l~-agarose gel was then run in TPE buffer (0.09 M
Tris-borate, 0.002 M EDTA) using 8 ~1 of each reaction. The gel was run for approximately 16 hours at 45v. Gels were then stained in 20 ~g/ml ethidium bromide for 1 hour and destained in TBE buffer for approximately 3 hours. Polaroid photographs of the gels were then taken under W illumination.
The presence or absence of bands at specific sizes for each strain was scored from the photographs and entered as a similarity matrix in the numerical taxonomy software program, NTSYS-pc (Exeter Software, Setau~et, NY). Controls of E. coli strain HB101 and Xanthomonas oryzae pv. oryzae assayed under the same conditions produced PCR fingerprints corresponding to published reports (Versalovic, J., Koeuth, T. and Lupski, J. R. 1991. Nucleic Acids Res. 19, 6823-6831i Vera Cruz, C. M., Halda-Alija, L., Louws, F., Skinner, D. Z., George, M. L., Nelson, R. J., DE Bruijn, F. J., Rice, C. and Leach, J. E. 1995. Int. Rice Res. Notes, 20, 23-24.;
Vera Cruz, C. M., Ardales, E. Y., Skinner, D. ~., Talag, J., Nelson, R. J., Louws, F. J., Leung, H., Mew, T. W. and Leach, J. E.
1996. Phytopathology 86, 1352-1359). The data from Photorhabdus strains were then analyzed with a series of programs within NTSYS-pc; SIMQUAL (Similarity for Qualitative data) to generate a matrix of similarity coefficients (using the Jaccard coefficient) and SAHN
(Sequential, Agglomerative, Heirarchical and Nested) clustering [using the UPGMA (Unweighted Pair-Group Method with Arithmetic Averages) method] which groups related strains and can be expressed as a phenogram (Fig. 7). The COPH (cophenetic values) and MXCOMP
(matrix comparison) programs were used to generate a cophenetic value matrix and compare the correlation between this and the original matrix upon which the clustering was based. A resulting normalized Mantel statistic (r) was generated which is a measure of the goodness of fit for a cluster analysis (r=0.8-0.9 represents a very good fit). In our case r-0.924. Therefore, the collection is comprised of a diverse group of easily distinguishable strains representatlve of the Photorhabdus genus.

SUBSTlTUTE ~I-{EET (RULE2~) .. , .. .,.. ,............... .. .. _ .. .

CA 022638l9 l999-02-26 W098/08932 PCTrUS97/07657 Example 23 Insecticidal Utility of Toxin(s) Produced by Various Photorhabdus Strains Initial "storage" cultures of the various Photorhabdus strains were produced by inoculating 175 ml of 2~ Proteose Peptone #3 (PP3) (Difco Laboratories, Detroit, MI) liquid medium with a primary variant colony in a 500 ml tribaffled flask with a Delong neck, covered with a Kaput closure. After inoculation, the flask was incubated for between 24-72 hrs at 28~C on a rotary shaker at 150 rpm, until stationary phase was reached. The culture was transferred to-a sterile bottle containing a sterile magnetic stir bar and the culture was overlayered with sterile mineral oil, to limit exposure to air. The storage culture was kept in the dark, at room temperature. These cultures were then used as inoculum sources for the fermentatlon of each strain.
"Seed" flasks or cultures were produced by either inoculating 2 mls of an oil overlayered storage culture or by transferring a 20 primary variant colony into 175 ml sterile medium in a 500 ml tribaffled flask covered with a Kaput closure. (The use of other inoculum sources is also possible.) Typically, following 16 hours incubation at 28~C on a rotary shaker at 150 rpm, the seed culture was transferred into production flasks. Production flasks were usually inoculated by adding about 1~ of the actively growing seed culture to sterile 2% PP3 medium (e.g. 2.0 ml per 175 ml sterile medium). Production of broths occurred in 500 ml tribaffled flasks covered with a Kaput. Production flasks were agitated at 28~C on a rotary shaker at 150 rpm. Production fermentations were terminated after 24-72 hrs although successful fermentation is not confined to this time duration. Following appropriate incubation, the broths were dispensed into sterile 1.0 L polyethylene bottles, spun at 2600xg for 1 hr at 10~C and decanted from the cell and debris pellet. Further broth clarification was achieved with a tangential flow microfiltration device (Pall Filtron, Northborough, MA) using a 0.5 ~M open-ch~nnel poly-ether sulfone (PES) membrane filter.
The resulting broths were then concentrated (up to 10-fold) using a 10,000 or 100,000 MW cut-off membrane, M12 ultra-filtration device (Amicon, Beverly MA) or centrifugal concentrators (Millipore, Bedford, MA and Pall Filtron, Northborough, MA) with a lO,OOC or SUBS~lTUTE SHEE~ tRU~E ~6) CA 022638l9 l999-02-26 W098/08932 PCTrUS97/07657 100,000 MW pore size. In the case of centrifugal concentrators, the broth was spun at 2000xg for approximately 2 hr. The membrane permeate was added to the corresponding retentate to achieve the desired concentration of components greater than the pore size used. Following these procedures, the broth was used for biochemical analysis or filter sterilized using a 0.2 ~M cellulose nitrate membrane filter for biological assessment. Heat inactivation of processed broth samples was achieved by heating the samples at 100~C in a sand-filled heat block for 10 minutes.
The broth(s) and toxin complex(es) from different Photorhabdus strains are useful for reducing populations of insects and were used in a method of inhibiting an insect population which comprises applying to a locus of the insect an effective insect inactivating amount of the active described. A demonstration of the breadth of insecticidal activity observed from broths of a selected group of Photorhabdus strains fermented as described above is shown in Table 36. It is possible that improved or additional insecticidal activlties could be detected with these strains through increased concentration of the broth or by employing different fermentation methods. Consistent with the activity being associated with a protein, the insecticidal activity of all strains tested was heat labile.
Culture broth(s) from diverse Photorhabdus strains show differential insecticidal activity (mortality and/or growth inhibition) against a number of insects. More specifically, the activity is seen against corn rootworm which is a member of the insect order Coleoptera. Other members of the Coleoptera include boll weevils, wireworms, pollen beetles, flea beetles, seed beetles and Colorado potato beetle. The broths and purified toxin complex(es) are also active against tobacco budworm, tobacco hornworm and European corn borer which are members of the order ~epidoptera. Other typical members of this order are beet armyworm, cabbage looper, black cutworm, corn earworm, codling moth, clothes moth, Indian mealmoth, leaf rollers, cabbage worm, cotton bollworm, bagworm, Eastern tent caterpillar, sod webworm and fall armyworm. Activity ls also observed against German cockroach which is a member of the order Dictyoptera (or Blattodea). Other members of this order are oriental cockroach and American cockroach.

SUBSTITUTE SltEE~ tRULE 26) ... . ... .

CA 022638l9 l999-02-26 W098/08932 PCTrUS97/076S7 Activity against corn rootworm larvae was tested as follows.
Photorhabdus culture broth~s) ~10 fold concentrated, filter sterilized), 2% Proteose Peptone #3 (10 fold concentrated), purified toxin complex(es), 10 mM sodium phosphate buffer, pH 7.0 were applied directly to the surface (about 1.5 cm2) of artificial diet (Rose, R. I. and McCabe, J. M. 1973. J. Econ. Entomol. 66, 398-400) in 40 ~l aliquots. Toxin complex was diluted in 10 mM
sodium phosphate buffer, pH 7Ø The diet plates were allowed to air-dry in a sterile flow-hood and the wells were infested with single, neonate Diabrotica undecimpunctata howardi (Southern corn rootworm, SCR) hatched from surface sterilized eggs. The plates were sealed, placed in a humidified growth chamber and maintained at 27~C for the appropriate period (3-5 days). Mortality and larval weight determinations were then scored. Generally, 16 insects per treatment were used in all studies. Control mortality was generally less than 5%.
Activity against lepidopteran larvae was tested as follows.
Concentrated (10-fold) Photorhabdus culture broth(s), control medium (2% Proteose Peptone #3), purified toxin complex(es), 10 mM
sodium phosphate buffer, pH 7.0 were applied directly to the surface (about 1.5 cm2) of standard artificial lepidopteran diet (Stoneville Yellow diet) in 40 ~l aliquots. The diet plates were allowed to air-dry in a sterile flow-hood and each well was infested with a single, neonate larva. European corn borer (Ostrinia nubilalis) and tobacco hornworm (Manduca sexta) eggs were obtained from commercial sources and hatched in-house, whereas tobacco budworm (Heliothis virescens) larvae were supplied internally. Following infestation with larvae, the diet plates were sealed, placed in a humidified growth chamber and maintained in the dark at 27~C for the appropriate period. Mortality and weight determinations were scored at day 5. Generally, 16 insects per treatment were used in all studies. Control mortality generally ranged from about 0 to about 12.5~ for control medium and was less than 10% for phosphate buffer.
Activity against cockroach was tested as follows. Concentrated (10-fold) Photor~abdus culture broth(s) and control medium (2~
Proteose Peptone #3) were applied directly to the surface (about 1.5 cm2) of standard artificial lepidopteran diet ~Stoneville Yellow diet) in 40 ~l aliquots. The diet plates were allowed to SUBSTITUTE S~tEET (RULE ~6~

CA 022638l9 l999-02-26 W098/08932 rcTrusg71o76s7 air-dry in a sterile flow-hood and e.ach well was infested with a single, CO2 anesthetized first instar German cockroach (Blatella germ~nica). Following infestation, the diet plates were sealed, placed in a humidified growth chamber and maintained in the dark at 27~C for the appropriate period. Mortality and weight determinations were scored at day 5. Control mortality less than 10~ .

SUBSTITUTE S~E~ (RULE 26) .. ~ . . ....

CA 02263819 l999-02-26 Table 36 Observed Insecticidal Spectrum of Broths ~rom Different photorhabdus Strai~

~hotorhab~us ~traln ~ensltlve* lnsect ~pecles P. zealandica 1**, 2, 4 P. hepialus 1, 2, 4 HB-Arg 1, 2, 4 HB Oswego l, 2, 4 HB Lewiston 1, 2, 4 K-122 l, 4 HMGD 1, 4 Indicus l, 2, 4 GD 2, 4 PWH-5 l, 2, 4 Megidis l, 2, 4 HF-85 1, 2, 4 A. Cows 1, 4 MP1 1, 2, 4 MP2 1, 2, 4 MP4 1, 4 GL98 1, 4 GL101 1, 4, 5 GL138 1, 2, 4 GL155 1, 4 ~L217 1, 2, 4 GL257 1, 4 DEPl 1, 4 DEP2 1, 2, 3, 4 * = 3 25% mortality and/or growth inhibition vs. control ** = 1; Tobacco budworm, 2; European corn borer, 3i Tobacco hornworm, 4; Southern corn rootworm, 5;
German cockroach.

SUBSTITUTE SHEET tRUI E 26) ~A~rle~ 24 Southern Analysis of Non-W-14 Photorhabdus Strains Using W-14 Gene Probes P~otorhabdus strais were grown on 2~ proteose peptone #3 agar (Difco Laboratories, Detroit, MI) and insecticidal toxin competence was maintained by repeated bioassay after passage. A 50 ml shake culture was produced in 175 ml baffled flasks in 2~ proteose peptone #3 medium, grown at 28~ and 150 rpm for approximately 24 10 hours. Fifteen ml of this culture were centrifuged (700 x g, 30 min) and frozen in its medium at -20~ until it was thawed (slowly in ice water) for DNA isolation. The thawed W-14 culture was centrifuged (900 x g, 15 min 4~), and the floating orange mucopolysaccharide material was removed. The remaining cell 15 material was centrifuged (25,000 x g, 4~) to pellet the bacterial cells, and the medium was removed and discarded.
Total DNA was isolated by an adaptation of the CTAB method described in section 2.4.1 of Ausubel et al. (1994). The modifications included a high salt shock, and all volumes were increased ten-fold over the "miniprep" recommended volumes. All centrifugations were at 4~C unless otherwise specified. The pelleted bacterial cells were resuspended in TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8) to a final volume of 10 ml, then 12 ml 5 M
NaCl were addedi this mixture was centrifuged 20 min at 15,000 x g.
25 The pellet was resuspended in 5.7 ml TE, and 300 ~1 of 10% SDS and 60 ~1 of 20 mg/ml proteinase K (in sterile distilled water, Gibco BRL Products, Grand Island, NY) were added to the suspension. The mixture was incubated at 37~C for 1 hr; then approxlmately 10 mg lysozyme (Worthington Biochemical Corp., Freehold, NJ) were added.
30 After an additional 45 min incubation, 1 ml of 5 M NaCl and 800 ~1 of CTAB/NaCl solution (10% w/v CTAB, O.7 M NaCl) were added. This preparation was incubated 10 min at 65~C, then gently agitated and further incubated and agitated for approximately 20 min to assist clearing of the cellular material. An equal volume of chloroform/isoamyl alcohol solution (24:1, v:v) was added, mixed very gently, and the phases separated by centrifugation at 12,000 x g for 15 min. The upper (aqueous) phase was gently removed with a wide-bore pipette and extracted twice as above with an equal volume of PCI (phenol/choloroform/ isoamyl alcohol; 50:49:1, v:v:v;
~0 equilibrated with lM Tris-HCl, pH 8.0; Intermountain Scientific Corporation, Kaysville, UT). The DNA precipitated with 0.6 volume of isopropanol was gently removed on a glass rod, washed twice with 70~ ethanol, dried, and dissolved in 2 ml STE (10 mM Tris-HCl, 10 SUBSTITUTE SHEEJ (RULE 26) W098/08932 PCT~US97/076S7 mM NaCl, 1 mM EDTA, pH 8). This preparation contained 2.5 mg/ml DNA, as determined by optical density at 260nm.

Identification of Bgl II/Hind III Fragments Hybridizing to tc-gene Speclfic Probes Approximately 10 ~g of genomic DNA was digested to completion with about 30 units each of Bgl II and ~ind III (NEB) for 180 min, frozen overnight, then heated at 65~C for five min, and electrophoresed in a 0.8~ agarose gel (Seakem LE, lX TEA, 80 volts, 90 min). The DNA was stained with ethidium bromide (50 ~g/ml) as described earlier, and photographed under ultraviolet light. The DNA fragments in the agarose gel were subjected to depurination (5 min in 0.2 M HCl), denaturation (15 min in 0.5 M
NaOH, 1.5 M NaCl), and neutralization (15 min in 0.5 M Tris HCl pH
15 8.0, 1.5 M NaCl), with 3 rinses of distilled water between each step. The DNA was transferred by Southern blotting from the gel onto a NYTRAN nylon membrane (Amersham, Arlington Heights, IL) using a high salt (20X SSC) protocol, as described in section 2.9 of Ausubel et al. (CPMB, op. cit.). The transferred DNA was then W -crosslinked to the nylon membrane using a Stratagene W
Stratalinker set on auto crosslink. The membranes were stored dry at 25~C until use.
Hybidization was performed using the ECLTMdirect (Amersham, Arlington Heights, IL) labeling and detection system following protocols provided by the manufacturer. In brief, probes were prepared by covalently linking the denatured DNA to the enzyme horseradish peroxidase. Once labeled the probe was used under hybridization conditions which maintain the enzymatic activity.
Unhybridized probe was removed by two gentle washes 20 minutes each 30 at 42~C in 0.5xSSC, 0.4% SDS, and 6M Urea. This was followed by two washes 5 minutes each at room temperature in 2xSSC. As directed by the manufacturer, ECLTM reagents were used to detect the hybridizing DNA bands. There are several factors which influence the ability to detect gene relatedness between various Photorhabdus strains and strain W-14. First, high stringency conditions have not been employed in these hybridizations. It is known in the art that varying the stringency of hybridization and wash conditions will influence the pattern and intensity of hybridizing bands. Second, Southern blots' blot to blot variation will influence the mobility of hybridizing bands and molecular weight estamates. Therefore, W-14 was included as a standard on all Southern blots.

SUBST~TUTE 5~EE r ~RULE 2~) , ~

CA 022638l9 l999-02-26 W098/08932 PCTrUS97/07657 Gene specific probes derived from the W-14 toxin genes were used in these hybridizations. The following lists the specific coordinates within each gene sequence to which the probe corresponds. A probe specific for tcaBi/Bii: 1174 to 3642 of Sequence ID #25, a probe specific for tcaC: 3637 to 6005 of - Sequence ID #25, a probe specific for tcbA: 2097 to 4964 of Sequence ID #11, and a probe specific for tcdA: 1660 to 4191 of ~ sequence ID #46. The following tables summarize Southern Blot analyses of Photorhabdus strains. In the event that hybridization of probes occurred, the hybridized fragment~s) were noted as either identical or different from the pattern observed for the W-14 strain.

SUBSTITUTE SH~ET ~RUI ~ 26) ..... ,.. ~. .. , .. ~ .

CA 022638l9 l999-02-26 Table 37 Southern Analysis of Photorhabdus Strains Stralns tc~A tcbA tcaC~tcaBi/ii W~- :L L) L) L) L~
W~
W~ V L) W2~- 4 L~ ~) NL~ L) W~- ~ L) L) L) L~
Wl~- 6 V L) 1) L) W~!;- / L) L) NL~ L) W2~- Y NL~ L) L) L~
W~- 1 () NL) L) J 1) W~- 11 NL) 1~
WX - 1 ;~ L~ J L? L) W~- 14 L) L) L) W~

~m ~b - 1 L) ;~ L) - L) -N ~: - 1 L) - L) L) W l ~ L L) W~) L) L~ L) L) N~ = Not d termlned; - = no detectable ny~rldlzatlon ?roduct;
I = Identical fragment pattern; D = Different fragment pattern.

SUBSlTrUTE S}tEE~ tRULE 26) ... . . ~.. . . . . ....

W098/08932 PCT~US97/07657 T~hle 38 Southern Analysis of Photorhabdu-s Strains ~tralns tc~A tc~A tca~ tcaBi/ii K~ 3 . 3, ~ . ~ L) - Nli ~'W~ + L~ D
lndlcus ~ Megldls J ~ ~

V J ~ -MY 3 ~ -M~ 1 ~ +
A. ~lows J + ~ -~-Arg ~ N~ L
~M~L~ L) L) ~ -Lewlston O L~ L) -~swego N~ = Not determlned; - no detectable hy~rldlzatlon pr~duct;
I = Identical fragment pattern; D = Different fragment pattern.
+ = Hybrldization fragment pattern not determined.

SUBSTITUTE 5}}EET tRULE 26) TAhle 39 Southern An~lysis of Photorhabdus Stralns Stralns tcoA tc~A tca~ tcaBi/Bii ~;~Y ~ + + L~
101 - +
+

~2l/ +
'/ + + L~
M~4 - +
M~
P heplalus +
zealandla + - l1.

V~2 ~3 N~ = Not det rmlnedi - = no detectable hy~r_dlzatlon product;
I = Identical fragment pattern; D = Different fragment pattern.
+ = Hybridization fragment pattern not determined.
From these analyses it is apparent that homologs of W-14 genes are dispersed throughout these diverse Photorhabdus strains, as evidenced by differences in gene fragment sizes between W-14 and the other strains.

Example 25 N-Terminal Amino Acid Sequences of Toxin Complex Peptides from Different Photorhabdus Strains The relationship of peptides isolated from different Photorhabdus strains, as described in Example l~, were subjected to SU8STITUTE St~E~T tRULE 26) W098/08932 PCT~US97/07657 N-terminal amino acid sequencing. The N-terminal amino acid sequences of toxin peptides in several strains were compared to W-14 toxin peptides. In Table 40, a comparison of toxin peptides compared to date showed that identical or homologous (at least 40%
similarity to W14 gene/peptides) toxin peptides were present in all - of the strains. For example, the N-terminal amino acid sequence of TcaC, SEQ ID NO: 2, was found to be identical to that for 160 kDa - peptide in HP88 but also homologs were present in strains WIR, H9, Hb, WX-1, and Hm. Some W-14 peptides or homologs have not been observed in other strains; however, not all peptides have been sequenced for toxin complexes from other strains due to N-terminal blockage or low abundance. In addition, many other N-terminal amino acid sequences (SEQ ID NOS: 82 to 88) have been obtained for toxin complex peptides from other strains that have no similarity to peptides from W-1~ and in some case were identical to each other. For example, an identical amino acid sequence, SEQ ID NO:
82, was obtained for 64 kDa peptide present in both HP88 and Hb strains and a homologous sequence for a 70 kDa peptide in NC-1 strain (SEQ ID NO: 83).

SU~STITUTE S~EET (RULE 2~) CA 022638l9 l999-02-26 W098/08932 PCT~US97/07657 Table 40 A Comparison of Amino Terminal Sequence Homology Between Droteins Isolated From Non-W-14 Strains W-l4 w-14 W-14 ~ traln l~entlcal ~omology Peptide Gene SEQ ID NO:
TcaAii tcaA 15 TcaAiii tcaA 4 TcaBi tcaB 3 76 H9 _ /4 kDa 76 Hm - 71 kDa TcaBii tcaB 5 H9 61 kDa Hm 61 kDa TcaC tcaA 2 72 Hb - 160 kDa HP88 160 kDa 73 WIR - 170 kDa 74 H9 - 180 kDa Hm - 170 kDa WX-1 - 170 kDa TcbAii tcbA
TcbAiii tcbA 40 TccA tccA 8 77 Hb - ~~ kDa TccB tccB 7 WX-l 170 kDa WX-2 180 kDa WX-14 180 kDa WIR 170 kDa 78 H9 - 170 kDa NC-1 140 kDa 79 Hm - 190 kDa TcdAil tcdA
TcdAiii tcdA 41 Hb 57 kDa 81 H9 - 63 kDa ? ? 9 Hb 86 kDa HP88 86 kDa ~omology re~ers t~ amlno ,cld sequences th~t were at ~easL 4U~
similarity to W14 gene / peptides. Similar residues were identified as being a member in one of the following five groups: (P, A, G, S, T); (Q, N, E, B, D, Z); (H, K, R); ~~, I, V, M); and (F, Y, W).

Example 26 Immunological Analysis of Photorh~hdus Strains Culture broths of Photorhabdus strains were concentrated 10 to 15 times using Centriprep-10 ultrafiltration device (Amicon, Inc.
Beverly, MA 01915). The concentration of the protein ranges from 0.3 to 3.0 mg per ml. Ten to 20 ~g of total protein was loaded in each well of a precast 4-20~ polyacrylamide gel (Integrated Separation Systems, Natick, MA 01760). Gel electrophores1s was performed for 1.25 hours using a constant current set at 25 ma per gel. The gel was electro-hlotted on to Hybond-ECLTM nitrocellulose membrane (Amersham Corporation, Arlington Hts, Il 60005) using a semi-dry electro-blotter (Pharmacia Biotech Inc., Piscataway , NJ

SUBSTITUTE St~ EET (RULE 26) CA 022638l9 l999-02-26 08854). A constant current was applied at 0.75 ma per cm for 2.5 hours. The membrane was blocked with 10% milk in TBST (25 mM Tris HCl pH 7.4, 136 mM NaCl, 2.7 mM KCl, 0.1% Tween 20) for one hour at room temperature. Each primary antibody was diluted in 10%
J milk/TBST to 1:500. Other dilution between 1:50 to 1:1000 was also - used. The membrane was incubated in primary antibody for at least one hour. Then it was washed thoroughly with the blocking solution or TBST. A 1:2000 dilution of secondary antibodies (goat anti-mouse IgG or goat anti rabbit TgG conjugated to horseradish peroxidasei BioRad Laboratories, Hercules, CA 94547) in 10~
milk/TBST was applied to the membrane which was placed on a platform rocker for one hour. The membrane was subsequently washed with excess amount of TBST. The detection of the protein was performed by using an ECL (Enhanced Chemiluminescence) detection kit (Amersham International).
A panel of peptide specific-antibodies generated against W-14 peptides were used to characterize the protein composition of broths from nine non-W-14 Photorhabdus strains using Western blot analysis. In addition, one monoclonal antibody (MAb-C5F2) which recognizes TcbAii1 protein in W-14-derived toxin complex was used.
The results (Table 39) showed cross recognition of the antibodies to some of the proteins in these broths. In some cases, the proteins that were recognized by the antibodies were the same size as the W-14 target peptides. In other cases, the proteins that ~5 were recognized by the antibodies were smaller than the W-14 target peptides. This data indicate that some of the non-W-14 Photorhabdus strains may produce similar protelns to the W-14 strain. The difference could be due to deletion or protein processing or degradation process. Some of the strains did not contain protein(s) that could be recognized by some antibodies, however, it is possible that the concentration is significantly lower than those observed for W-14 peptides. When compared for various toxin peptide homologs these results showed peptide diversity among the Photorha~dus strains.

SUBSTlTUTE SHEET (RULE 26) CA 022638l9 l999-02-26 WOg~8932 PCT~US97107657 Table 41 Cross Recognition by Monoclonal Antibo~ies or Polyclonal Antibodies Generate~ Agalnst W-14 Peptides to Protei~(s) in Broths of Selected Non-W-14 Photorh~hdus ~ho~o- MAb ~Ab PA~ ~Ab ~A~- ~A~ ~Ab BAb BAb rhabdus C5F2 TcdA TcdA TcaC TcaB TcbA TcaB TcaA TcaA
Strain ii~-syn il- iii- i- ii- iii-syn synsyn syn syn syn syn M~l - + + + - + + + +
MY~ + + + + - + + + +
M~ - + + + - N'l' + +
A. (~ows - + + + - ~11 + + +
l~-osw - - ~11 + + Nl + + +
~-Arg - + + + - Nl' + + +
~-leu - + + + - Nl + + +
lnalcus + + + + + Nl + + +
+ + + + + + +
W-14 + + + + + + + + +
+: ~osltlve re.ct1on; -: ~egatlve reactlon; Nl': Not lested Additional non-W-14 Photorhabdus strains were characterized by Western blot analysis using the culture broth and/or partial purified protein fractions as antigen. The panel of antibodies include MAb-C5F2, MAb-DE1 (recognizing TcdAii), PAb-DE2 (recognizing TcaB), PAb-TcbAii-syn, PAb- TcaC-syn, PAb TcaBii-syn, PAb-TcbAiii-syn, PAb-TcaBi-syn. These antibodies showed cross-reactivity with proteins in the broth and in the partial purified fractions of non-W-14 strains.
The data indicate that antibodies could be used to identify proteins in the broth as well as in the partially purified protein fractions.

SUBSTITUTE S}t EET (RULE 26) CA 022638l9 l999-02-26 W098/08932 PCTrUS97/07657 Table 42 Cross Recognition by Monoclonal Antibodies or Plyclonal Antibo~ies Generated A~ainqt W-14 Peptides to Protein(s) in Broth~ and/or p~rtial pllrified Protein Fractio~ of Selected Non-W14 Photoxh~h~

~hoto- Monoclonal ~olyclonal Antl~odles rhabdus Antibodies Strain ~ Ma~ Ma~- ~A~- ~Ab ~A~ ~A~ ~A~- ~Ab-C5F2 DEl DE2 TcbAii TcaC- TcaBii TcbAiii TcaBi -syn syn -syn -syn -syn W~-l + + + + + + + +
W2~ + + ~ t + + N'l' +
WX.-.~ +N'l' + N'l' N'l' N'l' N'l' N'l' W~ +N'l' + N'l' N'l' N'l' ~1'1' N'l' W~- b +N'l' N'l'N'l' ~1'1' N'l' ~'1' N'l' W~- 7 + + + ~ + + N'l' +
W~- ~ +N'l' N'l'N'l' N'l' N'l' N'l' N'l' W~- ~3 +N'l' N'l'N'l' N'l' N'l' N'l' N'l' W~-1~ - N'l' N'l'N'l' N'l' N'l' ~1'1' 1\1'1' W~-12 + + + + + + + +
W2~ + + + + N'l' + N'l' +
W~-1~ +N'l' N'l'N'l' N'l' N'l' N'l' N'l' W~ ~ + + + N'l' N'l' N'l' N'l' N'l' ~1~ - N'l' + N'l' + N'l' - +
~Y - - + Nl + + Nl N'l' ~im - N'l' + + + + N'l' + +
- N'l' + - +
N~-l + - + + + + N'l' +
WlK - N'l' + + + + + +
W-14 + + + + + +
-: Negatlve reactlon; +: ~o ltlve reactlon; N'l: No_ testea Example 27 Bacterial ~xpression of the tc~ Coding Region Engineerinq of the tcdA Gene for Bacterial Expression The 5' and 3' ends of the tcdA coding region (SEQ ID NO:46) were modified to add useful cloning sites for inserting the segment into heterologous expression vectors. The ends were modified using unique primers in Polymerase Chain Reactions (PCR), performed essentially as described in Example 8. Primer sets, as described below, were used in conjunctlon with cosmid 21D2.4 as template, to ~ created products with the appropriately modified ends.
The first primer set was used to modify the 5' end of the gene, to insert a unique Nco I site at the initiator codon using the forward primer AOF1 (5' GAT CGA TCG ATC CAT GGC CAA CGA GTC TGT AAA
AGA GAT ACC TGA TG TAT TAA AAA GCC AGT GTG 3') and to add unique Bgl II, Sal I and Not I sites to facilitate insertion of the remainder of the gene using the reverse primer AORl (5' GAT CGA TCG TAC GCG

SU8STITUTE SHEET (RULE ,26) CA 022638l9 l999-02-26 W098/08932 PCTrUS97/07657 GCC GCT CGA TCG ATC GTC GAC CCA TTG ATT TGA GAT CTG GGC GGC GGG TAT
CCA GAT AAT AAA CGG AGT CAC 3').
Another PCR reaction was designed to modify the 3' end of the gene by adding an additional stop codon and convenient restriction sites for cloning. The forward primer AOF2 (5' ACT GGC TGC GTG GTC
GAC TGG CGG CGA TTT ACT 3') was used to amplify across a unique Sal I site in the gene, later used to clone the modified 3' end. The reverse primer AOR2 (5' CGA TGC ATG CTG CGG CCG CAG GCC TTC CTC GAG
TCA TTA TTT AAT GGT GTA GCG AAT ATG CAA AAT 3') was used to insert a second stop codon (TGA) and cloning sites Xho I, Stu I and Not I.
Bacterlal expression vector pET27b (Novagen, Madison, WI), was modified to delete the ~gl II site at position 446, according to standard molecular biology techniqùes.
The 497 bp PCR product from the first amplification reaction (AOFl+AORl), to modify the 5' end of the gene, was ligated to the modified pET27b vector according to the supplier's instructions.
The DNA sequences of the amplified portion of three isolates were determined using the supplier's recommended primers and the sequencing methods described previously. The sequence of all isolates was the same.
One isolate was then used as a cloning vector to insert the middle portion of the tcdA gene on a 6341 bp Bgl II to Sal I
fragment. The resulting clone was called MC4 and contained all but the 3' most portion of the tcdA coding sequence. Finally, to complete the full-length coding region, the 832 bp PCR product from the second PCR amplification (AOF2+AOR2), to modify the 3' end of the gene, was ligated to isolate MC4 on a Sal I to Not I fragment, according to standard molecular biology techniques. The tcdA coding region was sequenced and found to be complete, the resulting plasmid 30 is called pDA~32035.

Construction of Plasmids pDAB2036, pDAB2037 and pDAB2038 for Bacterial Expression of tcdA
The tcdA coding region was cut from plasmid pDAB2035 with restriction enzymes Nco I and Xho I and gel purified. The fragment was ligated into the Nco I and Xho I sites of the expression vector pET15 to create plasmid pDAB2036. Additionally, pDAB2035 was cut with Nco I and Not I to release the tcdA coding region which was ligated into the Nco I and Not I sites of the expression vector 40 pET28b to create plasmid pD~32037. Finally, plasmid pDAB2035 was cut with Nco I and Stu I to release the tcdA coding region. This fragment was ligated into the expression vector Trc99a which was cut with Hind III followed by treatment with T4 DNA polymerase to blunt SUBSTr- ~TE S}~E T (RULE 26) CA 022638l9 l999-02-26 W09~08932 PCTAUS97/07657 the ends. The vector was then cut with Nco I and ligated with the Nco ~/Stu I cut tcdA fragment. The resulting plasmid is called pDAi32038.

Ex~ression of tcdA from Plasmid pDAB2038 - Plasmid pDAB2038 was transformed into BL21 cells and expressed as described above for plasmid pDAB2033 in Example 19.

-Purification of tcd~ from E. coli The expression culture was centrifuged at 10,300 g for 30 min and the supernatant was collected. It was diluted with two volumes of H2O and applied at a flow rate of 7.5 ml/min to a poros 50 HQ
(Perspective Systems, MA) column (1.6 cm x 10 cm) which was pre-equilibrated with 10 mM sodium phosphate buffer, pH 7.0 (Buffer A).
The column was washed with Buffer A until the optical density at 280 nm returned to baseline level. The proteins bound to the column were then eluted with lM NaCl in Buffer A.
The fraction was loaded in 20 ml aliquots onto a gel filtration column, Sepharose CL-4B (2.6 x 100 cm), which was equilibrated with Buffer A. The protein was eluted in Buffer A at a flow rate of 0.75 mL/min. Fractions with a retention time between 260 minutes and 460 minutes were pooled and applied at 1 mL/min to a Mono Q 5/5 column which was equilibrated with 20 mM Tris-HCl, pH 7.0 (Buffer B). The column was washed with Buffer B until the optical density at 280 nm returned to baseline level. The proteins bound to the column were eluted with a linear gradient of 0 to 1 M NaCl in Buffer B at lmL/min for 30 min. One milliliter fractions were collected, serial diluted, and subjected to SCR bioassay. Fractions eluted out between 0.1 and 0.3 M NaCl were found to have the highest insecticidal activity. Western analysis of the active fractions using pAb TcdAii-syn antibody and pAb Tcdiii-syn antibody indicated the presence of peptides TcdAii and TcdAiii.
-SUBSTITUTE SHEET ~RULE 26) CA 022638l9 l999-02-26 W098/08932 PCT~US97/07657 SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT: Ensign, Jerald C
Bowen, David J
Petell, James Fatig, Raymond Schoonover, Sue ffrench-Constant, Richard Orr, Gregory L
Merlo, Donald J
Roberts, Jean L
Rocheleau, Thomas A
(ii) TITLE OF INVENTION: Insecticidal Protein Toxins from Photorhabdus (iii) NUMBER OF SEQUENCES: 88 (iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: DowElanco (B) STREET: 9330 Zionsville Road (C) CITY: Indianapolis (D) STATE: IN
(E) COUNTRY: US
(F) ZIP: 46268 (v) COMPUTER READABLE FORM:
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(D) SOFTWARE: PatentIn Release #1.0, Version #1.30 (vi) CURRENT APPLICATION DATA:
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(C) CLASSIFICATION:
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: US 08/063,615 (B) FILING DATE: 18-MAY-1993 (vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: US 08/395,497 (B) FILING DATE: 28-FEB-1995 (vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: US 60/007,255 (B) FILING DATE: 06-NOV-1995 (vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: US 08/608,423 (B) FILING DATE: 28-FEB-1996 (vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: US 08/705,484 (B) FILING DATE: 28-AUG-1996 (vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: US 08/743,699 (B) FILING DATE: 06-NOV-1996 SUBSTITUTE S~EET (RULE 26) W098/08932 PCTrUS97/07657 (viii) ATTORNEY/AGENT INFORMATION-(A) NAME: Boruckl, Andrea T.
(B) REGISTRATION NUMBER: 33651 (C) REFERENCE/DOCKET NUMBER: 50301E

(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: 317-337-4846 (B) TELEFAX: 317-337-4847 (2) INFORMATION FOR SEQ ID NO:1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 11 amino acids (B) TYPE: amino acid (C) STRANDEDNESS:
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (v) FRAGMENT TYPE: N-terminal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1 (TcbAii N-terminus):
Phe lle Gln Gly Tyr ser Asp Leu Phe Gly Asn (2) INFORMATION FOR SEQ ID NO:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 12 amino acids (B) TYPE: amino acid (C) STRANDEDNESS:
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (v) FRAGMENT TYPE: N-terminal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2 (TcaC N-terminus):
Met Gln Asp ser Pro Glu Val ser Ile Thr Thr Trp (2) INFORMATION FOR SEQ ID NO:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 19 amino acids (B) TYPE: amino acid (C) STRANDEDNESS:
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (v) FRAGMENT TYPE: N-terminal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3 (TcaBi N-terminus):
Ser Glu Ser Leu Phe Thr Gln Thr Leu Lys Glu Ala Arg Arg Asp Ala Leu Val Ala SUBSlTrUTE SHEET lRULE 26) CA 022638l9 l999-02-26 W098/08932 PCTrUS97/07657 (2) INFORMATION FOR SEQ ID NO:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids (B) TYPE: amino acid (C) STRANDEDNESS:
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (v) FRAGMENT TYPE: N-terminal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4 (TcaAiii N-terminus):
Ala Ser Pro Leu Ser Thr Ser Glu Leu Thr Ser Lys Leu Asn ~5 (2) INFORMATION FOR SEQ ID NO:~:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids (B) TYPE: amino acid (C) STRANDEDNESS:
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (v) FRAGMENT TYPE: N-terminal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5 (TcaBii N-terminus):
Ala Gly Asp Thr Ala Asn Ile Gly Asp (2) INFORMATION FOR SEQ ID NO:6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (v) FRAGMENT TYPE: N-terminal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:
45 Leu Gly Gly Ala Ala Thr Leu Leu Asp Leu Leu Leu Pro Gln Ile (2) INFORMATION FOR SEQ ID NO:7:
(i) SEQUENCE CHARACTERISTICS:
~A) LENGTH: 11 amino acids (B) TYPE: amino acid (C) STRANDEDNESS:
(D) TOPOLOGY: linear ~ii) MOLECULE TYPE: protein (v) FRAGMENT TYPE: N-terminal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7 (TccB N-terminus):

Met Leu Ser Thr Met Glu Lys Gln Leu Asn Glu SVBSllTUTE SHE~T (RULE 26) W09~08932 PCTrUS97/07657 (2) INFORMATION FOR SEQ ID NO:8:
~i) SEQ~N~ CHARACTERISTICS:
(A) LENGTH: 9 amino acids (B) TYPE: amino acid (C) STRANDEDNESS:
(D) TOPOLOGY: linear - (ii) MOLECULE TYPE: protein (v) FRAGMENT TYPE: N-terminal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8 ~TccA N-terminus):
Met Asn Leu Ala Ser Pro Leu Ile Ser (2) INFORMATION FOR SEQ ID NO:9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 16 amino acids (B) TYPE: amino acid (C) STRANDEDNESS:
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (v) FRAGMENT TYPE: N-terminal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:
Met Ile Asn Leu Asp Ile Asn Glu Gln Asn Lys Ile Met Val Val Ser (2) INFORMATION FOR SEQ ID NO:10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 amino acids (B) TYPE: amino acid (C) STRANDEDNESS:
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (v) FRAGMENT TYPE: N-terminal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:
45 Ala Ala Lys Asp Val Lys Phe Gly Ser Asp Ala Arg Val Lys Met Leu Arg Gly Val Asn (2) INFORMATION FOR SEQ ID NO:ll:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 7515 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..7515 SUBSTITUTE SH EET ~RULE 26) CA 022638l9 l999-02-26 WO 98/08932 PCT~US97/07657 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:ll (tcbA gene):

Met Gln Asn Ser Leu Ser Ser Thr Ile Asp Thr Ile Cys Gln Lys Leu 1 5 lO 15 Gln Leu Thr Cys Pro Ala Glu Ile Ala Leu Tyr Pro Phe Asp Thr Phe Arg Glu Lys Thr Arg Gly Met Val Asn Trp Gly Glu Ala Lys Arg Ile Tyr Glu Ile Ala Gln Ala Glu Gln Asp Arg Asn Leu Leu His Glu Lys Arg Ile Phe Ala Tyr Ala Asn Pro Leu Leu Lys Asn Ala Val Arg Leu Gly Thr Arg Gln Met Leu Gly Phe Ile Gln Gly Tyr Ser Asp Leu Phe 85 go 95 Gly Asn Arg Ala Asp Asn Tyr Ala Ala Pro Gly Ser Val Ala Ser Met 100 105 llO

Phe Ser Pro Ala Ala Tyr Leu Thr Glu Leu Tyr Arg Glu Ala Lys Asn Leu His Asp Ser Ser Ser Ile Tyr Tyr Leu Asp Lys Arg Arg Pro Asp Leu Ala Ser Leu Met Leu Ser Gln Lys Asn Met Asp Glu Glu Ile Ser 145 ~50 lS5 160 Thr Leu Ala Leu Ser Asn Glu Leu Cys Leu Ala Gly Ile Glu Thr Lys Thr Gly Lys Ser Gln Asp Glu Val Met Asp Met Leu Ser Thr Tyr Arg Leu Ser Gly Glu Thr Pro Tyr His His Ala Tyr Glu Thr Val Arg Glu Ile Val His Glu Arg Asp Pro Gly Phe Arg His Leu Ser Gln Ala Pro Ile Val Ala Ala Lys Leu Asp Pro Val Thr Leu Leu Gly Ile Ser Ser His Ile Ser Pro Glu Leu Tyr Asn Leu Leu Ile Glu Glu Ile Pro Glu Lys Asp Glu Ala Ala Leu Asp Thr Leu Tyr Lys Thr Asn Phe Gly Asp SUBSTlTUTE S~EET (RULE 26) .

Ile Thr Thr Ala Gln Leu Met Ser Pro Ser Tyr Leu Ala Arg Tyr Tyr 5 Gly Val Ser Pro Glu Asp Ile Ala Tyr Val Thr Thr Ser Leu Ser His Val Gly Tyr Ser Ser Asp Ile Leu Val Ile Pro Leu Val Asp Gly Val Gly Lys Met Glu Val Val Arg Val Thr Arg Thr Pro Ser Asp Asn Tyr Thr Ser Gln Thr Asn Tyr Ile Glu Leu Tyr Pro Gln Gly Gly Asp Asn Tyr Leu Ile Lys Tyr Asn Leu Ser Asn Ser Phe Gly Leu Asp Asp Phe Tyr Leu Gln Tyr Lys Asp Gly Ser Ala Asp Trp Thr Glu Ile Ala His Asn Pro Tyr Pro Asp Met Val Ile Asn Gln Lys Tyr Glu Ser Gln Ala Thr Ile Lys Arg Ser Asp Ser Asp Asn Ile Leu Ser Ile Gly Leu Gln Arg Trp His Ser Gly Ser Tyr Asn Phe Ala Ala Ala Asn Phe Lys Ile Asp Gln Tyr Ser Pro Lys Ala Phe Leu Leu Lys Met Asn Lys Ala Ile Arg Leu Leu Lys Ala Thr Gly Leu Ser Phe Ala Thr Leu Glu Arg Ile Val Asp Ser Val Asn Ser Thr Lys Ser Ile Thr Val Glu Val Leu Asn Lys Val Tyr Arg Val Lys Phe Tyr Ile Asp Arg Tyr Gly Ile Ser Glu Glu Thr Ala Ala Ile Leu Ala Asn Ile Asn Ile Ser Gln Gln Ala Val Gly Asn Gln Leu Ser Gln Phe Glu Gln Leu Phe Asn His Pro Pro Leu Asn Gly Ile Arg Tyr Glu Ile Ser Glu Asp Asn Ser Lys His Leu Pro Asn Pro Asp Leu Asn Leu Lys Pro Asp Ser Thr Gly Asp Asp Gln Arg SUBST~TUTE SHEET tRULE 26) .. ,, .. , . ~. , W 098/08932 PCT~US97/076S7 Lys Ala Val Leu Lys Arg Ala Phe Gln Val Asn Ala Ser Glu Leu Tyr Gln Met Leu Leu Ile Thr Asp Arg Lys Glu Asp Gly Val Ile Lys Asn 0 Asn Leu Glu Asn Leu Ser Asp Leu Tyr Leu Val Ser Leu Leu Ala Gln Ile His Asn Leu Thr Ile Ala Glu Leu Asn Ile Leu Leu Val Ile Cys Gly Tyr Gly Asp Thr Asn Ile Tyr Gln Ile Thr Asp Asp Asn Leu Ala Lys Ile Val Glu.Thr Leu Leu Trp Ile Thr Gln Trp Leu Lys Thr Gln Lys Trp Thr Val Thr Asp Leu Phe Leu Met Thr Thr Ala Thr Tyr Ser Thr Thr Leu Thr Pro Glu Ile Ser Asn Leu Thr Ala Thr Leu Ser Ser Thr Leu His Gly Lys Glu Ser Leu Ile Gly Glu Asp Leu Lys Arg Ala Met Ala Pro Cys Phe Thr Ser Ala Leu His Leu Thr Ser Gln Glu Val Ala Tyr Asp Leu Leu Leu Trp Ile Asp Gln Ile Gln Pro Ala Gln Ile Thr Val Asp Gly Phe Trp Glu Glu Val Gln Thr Thr Pro Thr Ser Leu Lys Val Ile Thr Phe Ala Gln Val Leu Ala Gln Leu Ser Leu Ile Tyr Arg Arg Ile Gly Leu Ser Glu Thr Glu Leu Ser Leu Ile Val Thr Gln Ser Ser Leu Leu Val Ala Gly Lys Ser Ile Leu Asp His Gly Leu Leu Thr Leu Met Ala Leu Glu Gly Phe Hls Thr Trp Val Asn Gly Leu Gly Gln Hls Ala Ser Leu Ile Leu Ala Ala Leu Lys Asp Gly Ala Leu Thr Val Thr Asp Val Ala Gln Ala Met Asn Lys Glu Glu Ser Leu Leu Gln SUBSTITUTE S}tEET (RULE 26) CA 022638l9 l999-02-26 W O 98l08932 PCT~US97~7657 Met Ala Ala Asn Gln Val Glu Lys Asp Leu Thr Lys Leu Thr Ser Trp Thr Gln Ile Asp Ala Ile Leu Gln Trp Leu Gln Met Ser Ser Ala Leu Ala Val Ser Pro Leu Asp Leu Ala Gly Met Met Ala Leu Lys Tyr Gly Ile Asp His Asn Tyr Ala Ala Trp Gln Ala Ala Ala Ala Ala Leu Met Ala Asp Hls Ala Asn Gln Ala Gln Lys Lys Leu Asp Glu Thr Phe Ser Lys Ala Leu Cys Asn Tyr Tyr Ile Asn Ala Val Val Asp Ser Ala Ala Gly Val Arg Asp Arg Asn Gly Leu Tyr Thr Tyr Leu Leu Ile Asp Asn Gln Val Ser Ala Asp Val Ile Thr Ser Arg Ile Ala Glu Ala Ile Ala Gly Ile Gln Leu Tyr Val Asn Arg Ala Leu Asn Arg Asp Glu Gly Gln Leu Ala Ser Asp Val Ser Thr Arg Gln Phe Phe Thr Asp Trp Glu Arg 995 lO00 1005 Tyr Asn Lys Arg Tyr Ser Thr Trp Ala Gly Val Ser Glu Leu Val Tyr Tyr Pro Glu Asn Tyr Val Asp Pro Thr Gln Arg Ile Gly Gln Thr Lys Met Met Asp Ala Leu Leu Gln Ser Ile Asn Gln Ser Gln Leu Asn Ala Asp Thr Val Glu Asp Ala Phe Lys Thr Tyr Leu Thr Ser Phe Glu Gln Val Ala Asn Leu Lys Val Ile Ser Ala Tyr His Asp Asn Val Asn Val Asp Gln Gly Leu Thr Tyr Phe Ile Gly Ile Asp Gln Ala Ala Pro Gly Thr Tyr Tyr Trp Arg Ser Val Asp Hls Ser Lys Cys Glu Asn Gly Lys TTT GCC GCT AAT GCT TGG GGT GAG T&G AAT AAA ATT ACC TGT GCT GTC 3408 Phe Ala Ala Asn Ala Trp Gly Glu Trp Asn Lys Ile Thr Cys Ala Val SUBSTITUTE SH E~T tRULE 26) W O 98/08932 PCTrUS97/07657 Asn Pro Trp Lys Asn Ile Ile Arg Pro Val Val Tyr Met Ser Arg Leu Tyr Leu Leu Trp Leu Glu Gln Gln Ser Lys Lys Ser Asp Asp Gly Lys Thr Thr Ile Tyr Gln Tyr Asn Leu Lys Leu Ala His Ile Arg Tyr Asp 1170 1175 llBo Gly Ser Trp Asn Thr Pro Phe Thr Phe Asp Val Thr Glu Lys Val Lys 1185 llgo 1195 1200 Asn Tyr Thr Ser Ser Thr Asp Ala Ala Glu Ser Leu Gly Leu Tyr Cys Thr Gly Tyr Gln Gly Glu Asp Thr Leu Leu Val Met Phe Tyr Ser Met Gln Ser Ser Tyr Ser Ser Tyr Thr Asp Asn Asn Ala Pro Val Thr Gly Leu Tyr Ile Phe Ala Asp Met Ser Ser Asp Asn Met Thr Asn Ala Gln Ala Thr Asn Tyr Trp Asn Asn Ser Tyr Pro Gln Phe Asp Thr Val Met Ala Asp Pro Asp Ser Asp Asn Lys Lys Val Ile Thr Arg Arg Val Asn Asn Arg Tyr Ala Glu Asp Tyr Glu Ile Pro Ser Ser Val Thr Ser Asn Ser Asn Tyr Ser Trp Gly Asp His Ser Leu Thr Met Leu Tyr Gly Gly Ser Val Pro Asn Ile Thr Phe Glu Ser Ala Ala Glu Asp Leu Arg Leu Ser Thr Asn Met Ala Leu Ser Ile Ile His Asn Gly Tyr Ala Gly Thr Arg Arg Ile Gln Cys Asn Leu Met Lys Gln Tyr Ala Ser Leu Gly Asp Lys Phe Ile Ile Tyr Asp Ser Ser Phe Asp Asp Ala Asn Arg Phe Asn Leu Val Pro Leu Phe Lys Phe Gly Lys Asp Glu Asn Ser Asp Asp Ser SUBSTITUTE SH E~ tRULE26) CA 022638l9 l999-02-26 W O 98l08932 PCTrUS97/07657 Ile Cys Ile Tyr Asn Glu Asn Pro Ser Ser Glu Asp Lys Lys Trp Tyr Phe Ser Ser Lys Asp Asp Asn Lys Thr Ala Asp Tyr Asn Gly Gly Thr Gln Cys Ile Asp Ala Gly Thr Ser Asn Lys Asp Phe Tyr Tyr Asn Leu Gln Glu Ile Glu Val Ile Ser Val Thr Gly Gly Tyr Trp Ser Ser Tyr Lys Ile Ser Asn Pro Ile Asn Ile Asn Thr Gly Ile Asp Ser Ala Lys Val Lys Val Thr Val Lys Ala Gly Gly Asp Asp Gln Ile Phe Thr Ala Asp Asn Ser Thr Tyr Val Pro Gln Gln Pro Ala Pro Ser Phe Glu Glu Met Ile Tyr Gln Phe Asn Asn Leu Thr Ile Asp Cys Lys Asn Leu Asn Phe Ile Asp Asn Gln Ala His Ile Glu Ile Asp Phe Thr Ala Thr Ala Gln Asp Gly Arg Phe Leu Gly Ala Glu Thr Phe Ile Ile Pro Val Thr Lys Lys Val Leu Gly Thr Glu Asn Val Ile Ala Leu Tyr Ser Glu Asn Asn Gly Val Gln Tyr Met Gln Ile Gly Ala Tyr Arg Thr Arg Leu Asn Thr~ Pl.c ~la Gln Gln Leu Val Ser Arg Ala Asn Arg Gly Ile Asp Ala Val Leu Ser Met Glu Thr Gln Asn Ile Gln Glu Pro Gln Leu Gly Ala Gly Thr Tyr Val Gln Leu Val Leu Asp Lys Tyr Asp Glu Ser Ile His Gly Thr Asn Lys Ser Phe Ala Ile Glu Tyr Val Asp Ile Phe Lys Glu Asn Asp Ser Phe Val Ile Tyr Gln Gly Glu Leu Ser Glu Thr Ser 1665 1670 1675 16ao Gln Thr Val Val Lys Val Phe Leu Ser Tyr Phe Ile Glu Ala Thr Gly SU~STlTUTE St~EET tRULE 26) .. ...... --W 098/08932 PCT~US97/07657 AAT AAG AAC CAC TTA TGG GTA CGT GCT AAA.TAC CAA AAG GAA ACG ACT 5136 Asn Lys Asn His Leu Trp Val Arg Ala Lys Tyr Gln Lys Glu Thr Thr Asp Lys Ile Leu Phe Asp Arg Thr Asp Glu Lys Asp Pro His Gly Trp TTT CTC AGC GAC GAT CAC AAG ACC TTT AGT GGT CTC TCT TCC GCA CAG 52~2 0 Phe Leu Ser Asp Asp His Lys Thr Phe Ser Gly Leu Ser Ser Ala Gln Ala Leu Lys Asn Asp Ser Glu Pro Met Asp Phe Ser Gly Ala Asn Ala Leu Tyr Phe Trp Glu Leu Phe Tyr Tyr Thr Pro Met Met Met Ala His Arg Leu Leu Gln Glu Gln Asn Phe Asp Ala Ala Asn His Trp Phe Arg Tyr Val Trp Ser Pro Ser Gly Tyr Ile Val Asp Gly Lys Ile Ala Ile Tyr His Trp Asn Val Arg Pro Leu Glu Glu Asp Thr Ser Trp Asn Ala Gln Gln Leu Asp Ser Thr Asp Pro Asp Ala Val Ala Gln Asp Asp Pro Met His Tyr Lys Val Ala Thr Phe Met Ala Thr Leu Asp Leu Leu Met Ala Arg Gly Asp Ala Ala Tyr Arg Gln Leu Glu Arg Asp Thr Leu Ala ~ 1860 1865 1870 Glu Ala Lys Met Trp Tyr Thr Gln Ala Leu Asn Leu Leu Gly Asp Glu Pro Gln Val Met Leu Ser Thr Thr Trp Ala Asn Pro Thr Leu Gly Asn 1890 1895 lgO0 Ala Ala Ser Lys Thr Thr Gln Gln Val Arg Gln Gln Val Leu Thr Gln Leu Arg Leu Asn Ser Arg Val Lys Thr Pro Leu Leu Gly Thr Ala Asn Ser Leu Thr Ala Leu Phe Leu Pro Gln Glu Asn Ser Lys Leu Lys Gly Tyr Trp Arg Thr Leu Ala Gln Arg Met Phe Asn Leu Arg His Asn Leu Ser Ile Asp Gly Gln Pro Leu Ser Leu Pro Leu Tyr Ala Lys Pro Ala SUBST~TUTE S~EEJ (RULE 2~) CA 022638l9 l999-02-26 Asp Pro Lys Ala Leu Leu Ser Ala Ala Val Ser Ala Ser Gln Gly Gly 1985 l990 1995 2000 Ala Asp Leu Pro Lys Ala Pro Leu Thr Ile His Arg Phe Pro Gln Met Leu Glu Gly Ala Arg Gly Leu Val Asn Gln Leu Ile Gln Phe Gly Ser Ser Leu Leu Gly Tyr Ser Glu Arg Gln Asp Ala Glu Ala Met Ser Gln Leu Leu Gln Thr Gln Ala Ser Glu Leu Ile Leu Thr Ser Ile Arg Met Gln Asp Asn Gln Leu Ala Glu Leu Asp Ser Glu Lys Thr Ala Leu Gln Val Ser Leu Ala Gly Val Gln Gln Arg Phe Asp Ser Tyr Ser Gln Leu Tyr Glu Glu Asn Ile Asn Ala Gly Glu Gln Arg Ala Leu Ala Leu Arg Ser Glu Ser Ala Ile Glu Ser Gln Gly Ala Gln Ile Ser Arg Met Ala Gly Ala Gly Val Asp Met Ala Pro Asn Ile Phe Gly Leu Ala Asp Gly Gly Met His Tyr Gly Ala Ile Ala Tyr Ala Ile Ala Asp Gly Ile Glu Leu Ser Ala Ser Ala Lys Met Val Asp Ala Glu Lys Val Ala Gln Ser Glu Ile Tyr Arg Arg Arg Arg Gln Glu Trp Lys Ile Gln Arg Asp Asn Ala Gln Ala Glu Ile Asn Gln Leu Asn Ala Gln Leu Glu Ser Leu Ser Ile Arg Arg Glu Ala Ala Glu Met Gln Lys Glu Tyr Leu Lys Thr Gln Gln Ala Gln Ala Gln Ala Gln Leu Thr Phe Leu Arg Ser Lys Phe Ser Asn Gln Ala Leu Tyr Ser Trp Leu Arg Gly Arg Leu Ser Gly Ile Tyr Phe Gln Phe Tyr Asp Leu Ala Val Ser Arg Cys Leu Met Ala Glu Gln SUBSTITUTE SHEEl- tRULE 26) CA 022638l9 l999-02-26 W 098/08932 rCT~US97/07657 Ser Tyr Gln Trp Glu Ala Asn Asp Asn Ser Ile Ser Phe Val Lys Pro Gly Ala Trp Gln Gly Thr Tyr Ala Gly Leu Leu Cys Gly Glu Ala Leu Ile Gln Asn Leu Ala Gln Met Glu Glu Ala Tyr Leu Lys Trp Glu Ser Arg Ala Leu Glu Val Glu Arg Thr Val Ser Leu Ala Val Val Tyr Asp Ser Leu Glu Gly Asn Asp Arg Phe Asn Leu Ala Glu Gln Ile Pro Ala Leu Leu Asp Lys Gly Glu Gly Thr Ala Gly Thr Lys Glu Asn Gly Leu Ser Leu Ala Asn Ala Ile Leu Ser Ala Ser Val Lys Leu Ser Asp Leu Lys Leu Gly Thr Asp Tyr Pro Asp Ser Ile Val Gly Ser Asn Lys Val Arg Arg Ile Lys Gln Ile Ser Val Ser Leu Pro Ala Leu Val Gly Pro Tyr Gln Asp Val Gln Ala Met Leu Ser Tyr Gly Gly Ser Thr Gln Leu Pro Lys Gly Cys Ser Ala Leu Ala Val Ser His Gly Thr Asn Asp Ser Gly Gln Phe Gln Leu Asp Phe Asn Asp Gly Lys Tyr Leu Pro Phe Glu ~ 2455 2460 GGT ATT GCT CTT GAT GAT CAG GGT ACA CTG AAT CTT CAA TTT 2~ AAT 7440 Gly Ile Ala Leu Asp Asp Gln Gly Thr Leu Asn Leu Gln Phe Pro Asn Ala Thr Asp Lys Gln Lys Ala Ile Leu Gln Thr Met Ser Asp Ile Ile 60 Leu His Ile Arg Tyr Thr Ile Arg *

SUBS~ITUTE SHEET (RULE 26~

CA 022638l9 l999-02-26 W098/08932 PCTrUS97107657 (2) INFORMATION FOR SEQ ID NO:12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2504 amlno acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12 (TcbA protein):
Met Gln Asn Ser Leu Ser Ser Thr Ile Asp Thr Ile Cys Gln Lys Leu Gln Leu Thr Cys Pro Ala Glu Ile Ala Leu Tyr Pro Phe Asp Thr Phe Arg Glu Lys Thr Arg Gly Met Val Asn Trp Gly Glu Ala Lys Arg Ile 2 0 Tyr Glu Ile Ala Gln Ala Glu Gln Asp Arg Asn Leu Leu His Glu Lys Arg Ile Phe Ala Tyr Ala Asn Pro Leu Leu Lys Asn Ala Val Arg Leu Gly Thr Arg Gln Met Leu Gly Phe Ile Gln Gly Tyr Ser Asp Leu Phe Gly Asn Arg Ala Asp Asn Tyr Ala Ala Pro Gly Ser Val Ala Ser Met Phe Ser Pro Ala Ala Tyr Leu Thr Glu Leu Tyr Arg Glu Ala Lys Asn Leu His Asp Ser Ser Ser Ile Tyr Tyr Leu Asp Lys Arg Arg Pro Asp Leu Ala Ser Leu Met Leu Ser Gln Lys Asn Met Asp Glu Glu Ile Ser Thr Leu Ala Leu Ser Asn Glu Leu Cys Leu Ala Gly Ile Glu Thr I.ys Thr Gly Lys Ser Gln Asp Glu Val Met Asp Met Leu Ser Thr Tyr Arg Leu Ser Gly Glu Thr Pro Tyr His His Ala Tyr Glu Thr Val Arg Glu Ile Val His Glu Arg Asp Pro Gly Phe Arg His Leu Ser Gln Ala Pro Ile Val Ala Ala Lys Leu Asp Pro Val Thr Leu Leu Gly Ile Ser Ser His Ile Ser Pro Glu Leu Tyr Asn Leu Leu Ile Glu Glu Ile Pro Glu Lys Asp Glu Ala Ala Leu Asp Thr Leu Tyr Lys Thr Asn Phe Gly Asp Ile Thr Thr Ala Gln Leu Met Ser Pro Ser Tyr Leu Ala Arg Tyr Tyr Gly val Ser Pro Glu Asp Ile Ala Tyr Val Thr Thr Ser Leu Ser His Val Gly Tyr Ser Ser Asp Ile Leu Val Ile Pro Leu Val Asp Gly Val SUBSTITUTE SHEET (RULE 26) WO 98/08932 PCTrUS97/07657 Gly Lys Met Glu Val Val Arg Val Thr Arg Thr Pro Ser Asp Asn Tyr Thr Ser Gln Thr Asn Tyr Ile Glu Leu Tyr Pro Gln Gly Gly Asp Asn Tyr Leu Ile Lys Tyr Asn Leu Ser Asn Ser Phe Gly Leu Asp Asp Phe 0 Tyr Leu Gln Tyr Lys Asp Gly Ser Ala Asp Trp Thr Glu Ile Ala His Asn Pro Tyr Pro Asp Met Val Ile Asn Gln Lys Tyr Glu Ser Gln Ala Thr Ile Lys Arg Ser Asp Ser Asp Asn Ile Leu Ser Ile Gly Leu Gln Arg Trp His Ser Gly Ser Tyr Asn Phe Ala Ala Ala Asn Phe Lys Ile Asp Gln Tyr Ser Pro Lys Ala Phe Leu Leu Lys Met Asn Lys Ala Ile Arg Leu Leu Lys Ala Thr Gly Leu Ser Phe Ala Thr Leu Glu Arg Ile Val Asp Ser Val Asn Ser Thr Lys Ser Ile Thr Val Glu Val Leu Asn Lys Val Tyr Arg Val Lys Phe Tyr Ile Asp Arg Tyr Gly Ile Ser Glu Glu Thr Ala Ala Ile Leu Ala Asn Ile Asn Ile Ser Gln Gln Ala Val Gly Asn Gln Leu Ser Gln Phe Glu Gln Leu Phe Asn His Pro Pro Leu Asn Gly Ile Arg Tyr Glu Ile Ser Glu Asp Asn Ser Lys His Leu Pro Asn Pro Asp Leu Asn Leu Lys Pro Asp Ser Thr Gly Asp Asp Gln Arg Lys Ala Val Leu Lys Arg Ala Phe Gln Val Asn Ala Ser Glu Leu Tyr Gln Met Leu Leu Ile Thr Asp Arg Lys Glu Asp Gly Val Ile Lys Asn Asn Leu Glu Asn Leu Ser Asp Leu Tyr Leu Val Ser Leu Leu Ala Gln Ile His Asn Leu Thr Ile Ala Glu Leu Asn Ile Leu Leu Val Ile Cys Gly Tyr Gly Asp Thr Asn Ile Tyr Gln Ile Thr Asp Asp Asn Leu Ala Lys Ile Val Glu Thr Leu Leu Trp Ile Thr Gln Trp Leu Lys Thr Gln Lys Trp Thr Val Thr Asp Leu Phe Leu Met Thr Thr Ala Thr Tyr Ser Thr Thr Leu Thr Pro Glu Ile Ser Asn Leu Thr Ala Thr Leu Ser Ser Thr Leu His Gly Lys Glu Ser Leu Ile Gly Glu Asp Leu Lys Arg Ala SUBSTITUTE S~EET (RULE 2~) .. . . . . ..

W 098/08932 PCTrUS97/07657 Met Ala Pro Cys Phe Thr Ser Ala Leu His Leu Thr Ser Gln Glu Val 5 Ala Tyr Asp Leu Leu Leu Trp Ile Asp Gln Ile Gln Pro Ala Gln Ile Thr Val Asp Gly Phe Trp Glu Glu Val Gln Thr Thr Pro Thr Ser Leu ,~ 740 745 750 Lys Val Ile Thr Phe Ala Gln Val Leu Ala Gln Leu Ser Leu Ile Tyr Arg Arg Ile Gly Leu Ser Glu Thr Glu Leu Ser Leu Ile Val Thr Gln Ser Ser Leu Leu Val Ala Gly Lys Ser Ile Leu Asp His Gly Leu Leu Thr Leu Met Ala Leu Glu Gly Phe His Thr Trp Val Asn Gly Leu Gly Gln His Ala Ser Leu Ile Leu Ala Ala heu Lys Asp Gly Ala Leu Thr ~20 825 830 Val Thr Asp Val Ala Gln Ala Met Asn Lys Glu Glu Ser Leu Leu Gln Met Ala Ala Asn Gln Val Glu Lys Asp Leu Thr Lys Leu Thr Ser Trp Thr Gln Ile Asp Ala Ile Leu Gln Trp Leu Gln Met Ser Ser Ala Leu Ala Val Ser Pro Leu Asp Leu Ala Gly Met Met Ala Leu Lys Tyr Gly Ile Asp His Asn Tyr Ala Ala Trp Gln Ala Ala Ala Ala Ala Leu Met Ala Asp His Ala Asn Gln Ala Gln Lys Lys Leu Asp Glu Thr Phe Ser Lys Ala Leu Cys Asn Tyr Tyr Ile Asn Ala Val Val Asp Ser Ala Ala Gly Val Arg Asp Arg Asn Gly Leu Tyr Thr Tyr Leu Leu Ile Asp Asn Gln Val Ser Ala Asp Val Ile Thr Ser Arg Ile Ala Glu Ala Ile Ala Gly Ile Gln Leu Tyr Val Asn Arg Ala Leu Asn Arg Asp Glu Gly Gln Leu Ala Ser Asp Val Ser Thr Arg Gln Phe Phe Thr Asp Trp Glu Arg Tyr Asn Lys Arg Tyr Ser Thr Trp Ala Gly Val Ser Glu Leu Val Tyr Tyr Pro Glu Asn Tyr Val Asp Pro Thr Gln Arg Ile Gly Gln Thr Lys Met Met Asp Ala Leu Leu Gln Ser Ile Asn Gln Ser Gln Leu Asn Ala Asp Thr Val Glu Asp Ala Phe Lys Thr Tyr Leu Thr Ser Phe Glu Gln Val Ala Asn Leu Lys Val Ile Ser Ala Tyr His Asp Asn Val Asn Val SUBSTITUTE S~EET tRULE 26) W O 98/08932 PCTrUS97/07657 Asp Gln Gly Leu Thr Tyr Phe Ile Gly Ile Asp Gln Ala Ala Pro Gly 1090 1095 llOo Thr Tyr Tyr Trp Arg Ser Val Asp His Ser Lys Cys Glu Asn Gly Lys Phe Ala Ala Asn Ala Trp Gly Glu Trp Asn Lys Ile Thr Cys Ala Val Asn Pro Trp Lys Asn Ile Ile Arg Pro Val Val Tyr Met Ser Arg Leu Tyr Leu Leu Trp Leu Glu Gln Gln Ser Lys Lys Ser Asp Asp Gly Lys Thr Thr Ile Tyr Gln Tyr Asn Leu Lys Leu Ala His Ile Arg Tyr Asp Gly Ser Trp Asn Thr Pro Phe Thr Phe Asp Val Thr Glu Lys Val Lys Asn Tyr Thr Ser Ser Thr Asp Ala Ala Glu Ser Leu Gly Leu Tyr Cys Thr Gly Tyr Gln Gly Glu Asp Thr Leu Leu Val Met Phe Tyr Ser Met 3 0 Gln Ser Ser Tyr Ser Ser Tyr Thr Asp Asn Asn Ala Pro Val Thr Gly Leu Tyr Ile Phe Ala Asp Met Ser Ser Asp Asn Met Thr Asn Ala Gln Ala Thr Asn Tyr Trp Asn Asn Ser Tyr Pro Gln Phe Asp Thr Val Met Ala Asp Pro Asp Ser Asp Asn Lys Lys Val Ile Thr Arg Arg Val Asn Asn Arg Tyr Ala Glu Asp Tyr Glu Ile Pro Ser Ser Val Thr Ser Asn Ser Asn Tyr Ser Trp Gly Asp His Ser Leu Thr Met Leu Tyr Gly Gly Ser Val Pro Asn Ile Thr Phe Glu Ser Ala Ala Glu Asp Leu Arg Leu Ser Thr Asn Met Ala Leu Ser Ile Ile His Asn Gly Tyr Ala Gly Thr Arg Arg Ile Gln Cys Asn Leu Met Lys Gln Tyr Ala Ser Leu Gly Asp Lys Phe Ile Ile Tyr Asp Ser Ser Phe Asp Asp Ala Asn Arg Phe Asn Leu Val Pro Leu Phe Lys Phe Gly Lys Asp Glu Asn Ser Asp Asp Ser Ile Cys Ile Tyr Asn Glu Asn Pro Ser Ser Glu Asp Lys Lys Trp Tyr Phe Ser Ser Lys Asp Asp Asn Lys Thr Ala Asp Tyr Asn Gly Gly Thr Gln Cys Ile Asp Ala Gly Thr Ser Asn Lys Asp Phe Tyr Tyr Asn Leu SUBSTITUTE SHEET tRULE 26) CA 022638l9 l999-02-26 WO 98/08932 PCT~US97/07657 Gln Glu Ile Glu Val Ile Ser Val Thr Gly Gly Tyr Trp Ser Ser Tyr hys Ile Ser Asn Pro Ile Asn Ile Asn Thr Gly Ile Asp Ser Ala Lys Val Lys Val Thr Val Lys Ala Gly Gly Asp Asp Gln Ile Phe Thr Ala 0 Asp Asn Ser Thr Tyr Val Pro Gln Gln Pro Ala Pro Ser Phe Glu Glu Met Ile Tyr Gln Phe Asn Asn Leu Thr Ile Asp Cys Lys Asn Leu Asn Phe Ile Asp Asn Gln Ala His Ile Glu Ile Asp Phe Thr Ala Thr Ala Gln Asp Gly Arg Phe Leu Gly Ala Glu Thr Phe Ile Ile Pro Val Thr Lys Lys Val Leu Gly Thr Glu Asn Val Ile Ala Leu Tyr Ser Glu Asn Asn Gly Val Gln Tyr Met Gln Ile Gly Ala Tyr Arg Thr Arg Leu Asn Thr Leu Phe Ala Gln Gln Leu Val Ser Arg Ala Asn Arg Gly Ile Asp Ala Val Leu Ser Met Glu Thr Gln Asn Ile Gln Glu Pro Gln Leu Gly Ala Gly Thr Tyr Val Gln Leu Val Leu Asp Lys Tyr Asp Glu Ser Ile His Gly Thr Asn Lys Ser Phe Ala Ile Glu Tyr Val Asp Ile Phe Lys Glu Asn Asp Ser Phe Val Ile Tyr Gln Gly Glu Leu Ser Glu Thr Ser Gln Thr Val Val Lys Val Phe Leu Ser Tyr Phe Ile Glu Ala Thr Gly Asn Lys Asn His Leu Trp Val Arg Ala Lys Tyr Gln Lys Glu Thr Thr Asp Lys Ile Leu Phe Asp Arg Thr Asp Glu Lys Asp Pro His Gly Trp Phe Leu Ser Asp Asp His Lys Thr Phe Ser Gly Leu Ser Ser Ala Gln Ala Leu Lys Asn Asp Ser Glu Pro Met Asp Phe Ser Gly Ala Asn Ala Leu Tyr Phe Trp Glu Leu Phe Tyr Tyr Thr Pro Met Met Met Ala His Arg Leu Leu Gln Glu Gln Asn Phe Asp Ala Ala Asn His Trp Phe Arg Tyr Val Trp Ser Pro Ser Gly Tyr Ile Val Asp Gly Lys Ile Ala Ile Tyr His Trp Asn Val Arg Pro Leu Glu Glu Asp Thr Ser Trp Asn Ala Gln Gln Leu Asp Ser Thr Asp Pro Asp Ala Val Ala Gln Asp Asp Pro SUBSTITUTE SHEE~ tRULE 26~

W 098/08932 rCT~US97/07657 Met His Tyr Lys Val Ala Thr Phe Met Ala Thr Leu Asp Leu Leu Met Ala Arg Gly Asp Ala Ala Tyr Arg Gln Leu Glu Arg Asp Thr Leu Ala Glu Ala Lys Met Trp Tyr Thr Gln Ala Leu Asn Leu Leu Gly Asp Glu Pro Gln Val Met Leu Ser Thr Thr Trp Ala Asn Pro Thr Leu Gly Asn Ala Ala Ser Lys Thr Thr Gln Gln Val Arg Gln Gln Val Leu Thr Gln Leu Arg Leu Asn Ser Arg Val Lys Thr Pro Leu Leu Gly Thr Ala Asn ~0 Ser Leu Thr Ala Leu Phe Leu Pro Gln Glu Asn Ser Lys Leu Lys Gly Tyr Trp Arg Thr Leu Ala Gln Arg Met Phe Asn Leu Arg His Asn Leu Ser Ile Asp Gly Gln Pro Leu Ser Leu Pro Leu Tyr Ala Lys Pro Ala Asp Pro Lys Ala Leu Leu Ser Ala Ala Val Ser Ala Ser Gln Gly Gly Ala Asp Leu Pro Lys Ala Pro Leu Thr Ile His Arg Phe Pro Gln Met Leu Glu Gly Ala Arg Gly Leu Val Asn Gln Leu Ile Gln Phe Gly Ser Ser Leu Leu Gly Tyr Ser Glu Arg Gln Asp Ala Glu Ala Met Ser Gln Leu Leu Gln Thr Gln Ala Ser Glu Leu Ile Leu Thr Ser Ile Arg Met Gln Asp Asn Gln Leu Ala Glu Leu Asp Ser Glu Lys Thr Ala Leu Gln Val Ser Leu Ala Gly Val Gln Gln Arg Phe Asp Ser Tyr Ser Gln Leu Tyr Glu Glu Asn Ile Asn Ala Gly Glu Gln Arg Ala Leu Ala Leu Arg Ser Glu Ser Ala Ile Glu Ser Gln Gly Ala Gln Ile Ser Arg Met Ala Gly Ala Gly Val Asp Met Ala Pro Asn Ile Phe Gly Leu Ala Asp Gly Gly Met His Tyr Gly Ala Ile Ala Tyr Ala Ile Ala Asp Gly Ile Glu Leu Ser Ala Ser Ala Lys Met Val Asp Ala Glu Lys Val Ala Gln Ser ~5 Glu Ile Tyr Arg Arg Arg Arg Gln Glu Trp Lys Ile Gln Arg Asp Asn Ala Gln Ala Glu Ile Asn Gln Leu Asn Ala Gln Leu Glu Ser Leu Ser Ile Arg Arg Glu Ala Ala Glu Met Gln Lys Glu Tyr Leu Lys Thr Gln SUBSTlTUTE Sl~ EET (RU ~ E 26) WO 98/08932 rCT~US97/07657 2210 2215 , 2220 Gln Ala Gln Ala Gln Ala Gln Leu Thr Phe Leu Arg Ser Lys Phe Ser Asn Gln Ala Leu Tyr Ser Trp Leu Arg Gly Arg Leu Ser Gly Ile Tyr Phe Gln Phe Tyr Asp Leu Ala Val Ser Arg Cys Leu Met Ala Glu Gln Ser Tyr Gln Trp Glu Ala Asn Asp Asn Ser Ile Ser Phe Val Lys Pro Gly Ala Trp Gln Gly Thr Tyr Ala Gly Leu Leu Cys Gly Glu Ala Leu Ile Gln Asn Leu Ala Gln Met Glu Glu Ala Tyr Leu Lys Trp Glu Ser Arg Ala Leu Glu Val Glu Arg Thr Val Ser Leu Ala Val Val Tyr Asp Ser Leu Glu Gly Asn Asp Arg Phe Asn Leu Ala Glu Gln Ile Pro Ala Leu Leu Asp Lys Gly Glu Gly Thr Ala Gly Thr Lys Glu Asn Gly Leu Ser Leu Ala Asn Ala Ile Leu Ser Ala Ser Val Lys Leu Ser Asp Leu Lys Leu Gly Thr Asp Tyr Pro Asp Ser Ile Val Gly Ser Asn Lys Val Arg Arg Ile Lys Gln Ile Ser Val Ser Leu Pro Ala Leu Val Gly Pro Tyr Gln Asp Val Gln Ala Met Leu Ser Tyr Gly Gly Ser Thr Gln Leu Pro Lys Gly Cys Ser Ala Leu Ala Val Ser His Gly Thr Asn Asp Ser Gly Gln Phe Gln Leu Asp Phe Asn Asp Gly Lys Tyr Leu Pro Phe Glu Gly Ile Ala Leu Asp Asp Gln Gly Thr Leu Asn Leu Gln Phe Pro Asn Ala Thr Asp Lys Gln Lys Ala Ile Leu Gln Thr Met Ser Asp Ile Ile Leu His Ile Arg Tyr Thr Ile Arg *

(2) INFORMATION FOR SEQ ID NO:13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 12 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13 (TcdAii N-termlnus):
Leu Ile Gly Tyr Asn Asn Gln Phe Ser Gly Xaa Aia SUE~S-rlTUTE S~EET tRULE 26) WO98~8g32 PCTrUS97/07657 (2) INFORMATION FOR SEQ ID NO:14:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 12 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14 (TcdB N-terminus):
Met Gln Asn Ser Gln Thr Phe Ser Val Gly Glu ~eu (2) INFORMATION FOR SEQ ID NO:15:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single ( D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15 (TcaAii N-terminus~:
Ala Gln Asp Gly Asn Gln Asp Thr Phe Phe Ser Gly Asn Thr (2) INFORMATION FOR SEQ ID NO:16:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 5 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii~ MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16 (TcbA N-terminus~:
4 5 Met Gln Asn Ser Leu (2) INFORMATION FOR SEQ ID NO:17:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17 (TcdAii-PTlll internal peptide):

Ala Phe Asn Ile Asp Asp Val Ser Leu Phe SUBSTITUTE St~EET (RULE--26) CA 022638l9 l999-02-26 W098/08932 PCTrUS97/07657 (2~ INFORMATION FOR SEQ ID NO:18:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 16 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: l inear - (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18 (TcdAii- PT79 internal peptide):
Phe Ile Val Tyr Thr Ser Leu Gly Val Asn Pro Asn Asn Ser Ser Asn (2) INFORMATION FOR SEQ ID NO:l9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: l inear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:l9 (TcaBi- PT158 internal peptide):
Ile Ser Asp Leu Val Thr Thr Ser Pro Leu Ser Glu Ala Ile Gly Ser Leu Gln Leu Phe Ile (2) INFORMATION FOR SEQ ID NO:20:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 12 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: l inear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20 (Tca~i- PT 108 internal peptide):
Met Tyr Tyr Ile Gln Ala Gln Gln Leu Leu Gly Pro (2) INFORMATION FOR SEQ ID NO:21:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: l inear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21 (TcbA i- PT103 internal peptide):

SUBST~TUTE SHEET tRULE 26) CA 022638l9 l999-02-26 W O 98l08932 PCT~US97tO7657 Gly Ile Asp Ala Val Leu Ser Met Glu Thr Gln Asn Ile Gln Glu Pro Gln Leu Gly Ala Gly Thr Tyr Val Gln Leu (2) INFORMATION FOR SEQ ID NO:22:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi~ SEQUENCE DESCRIPTION: SEQ ID NO:22 (TcbAii- PT56 internal peptide):
Ile Ser Asn Pro Ile Asn Ile Asn Thr Gly Ile Asp Ser Ala Lys (2) INFORMATION FOR SEQ ID NO:23:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 13 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23 (TcbA- PT81 (a) internal peptide):
Thr Tyr Leu Thr Ser Phe Glu Gln Val Ala Asn Leu ~ys (2) INFORMATION FOR SEQ ID NO:24:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii~ MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24 (TcbAii- PT81 ~b) internal peptide):
Val Leu Gly Thr Glu Asn Val Ile Ala Leu Tyr Ser Glu Asn Asn Gly l 5 10 15 Val Gln Tyr Met Gln Ile SUBSTITUTE SHE~T tRULE 26) CA 022638l9 l999-02-26 W098/08932 PCTrUS97/076S7 (2) INFORMATION FOR SEQ ID NO:25:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6054 ba8e palrs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: l inear ~ (ii) MOLECULE TYPE: DNA ( genomlc) (ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..43 - (D) OTHER INFORMATION: /product= "end Of TcaA
(ix) FEATURE:
(A) NAME/K~Y: RBS
(B) LOCATION: 51 58 (ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 65 3634 (D) OT~ER INFORMATION: /product= "TcaBi"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:

Val Ala Gln Asn Leu Ser Ala Ala Ile Ser Asn Arg Gln ~--Met Ser Glu Ser Leu Phe Thr Gln Thr Leu Lys Glu Ala Arg Arg Asp Ala Leu Val Ala His Tyr Ile Ala Thr Gln Val Pro Ala Asp heu Lys Glu Ser Ile Gln Thr Ala Asp Asp Leu Tyr Glu Tyr 4 0 Leu Leu Leu Asp Thr Lys Ile Ser Asp Leu Val Thr Thr Ser Pro Leu Ser Glu Ala Ile Gly Ser Leu Gln Leu Phe Ile His Arg Ala Ile Glu Gly Tyr Asp Gly Thr Leu Ala Asp Ser Ala Lys Pro Tyr Phe Ala Asp Glu Gln Phe Leu Tyr Asn Trp Asp Ser Phe Asn His Arg Tyr Ser Thr Trp Ala Gly Lys Glu Arg Leu Lys Phe Tyr Ala Gly Asp Tyr Ile Asp 60 Pro Thr Leu Arg Leu Asn Lys Thr Glu Ile Phe Thr Ala Phe Glu Gln Gly Ile Ser Gln Gly Lys Leu Lys Ser Glu Leu Val Glu Ser Lys Leu 6~ 145 150 155 SUBST~TUTE SHEET (RULE 26) ... . ..

CA 022638l9 l999-02-26 Arg Asp Tyr Leu Ile Ser Tyr Asp Thr Leu, Ala Thr Leu Asp Tyr Ile Thr Ala Cys Gln Gly Lys Asp Asn Lys Thr Ile Phe Phe Ile Gly Arg Thr Gln Asn Ala Pro Tyr Ala Phe Tyr Trp Arg Lys Leu Thr Leu Val Thr Asp Gly Gly Lys Leu Lys Pro Asp Gln Trp Ser Glu Trp Arg Ala Ile Asn Ala Gly Ile Ser Glu Ala Tyr Ser Gly His Val Glu Pro Phe Trp Glu Asn Asn Lys Leu His Ile Arg Trp Phe Thr Ile Ser Lys Glu Asp Lys Ile Asp Phe Val Tyr Lys Asn Ile Trp Val Met Ser Ser Asp Tyr Ser Trp Ala Ser Lys Lys Lys Ile Leu Glu Leu Ser Phe Thr Asp Tyr Asn Arg Val Gly Ala Thr Gly Ser Ser Ser Pro Thr Glu Val Ala Ser Gln Tyr Gly Ser Asp Ala Gln Met Asn Ile Ser Asp Asp Gly Thr Val Leu Ile Phe Gln Asn Ala Gly Gly Ala Thr Pro Ser Thr Gly Val Thr Leu Cys Tyr Asp Ser Gly Asn Val Ile Lys Asn Leu Ser Ser Thr Gly-~e~ Ala ~sn Leu Ser Ser Lys Asp Tyr Ala Thr Thr Lys Leu Arg Met Cys His Gly Gln Ser Tyr Asn Asp Asn Asn Tyr Cys Asn Phe Thr Leu Ser Ile Asn Thr Ile Glu Phe Thr Ser Tyr Gly Thr Phe Ser Ser Asp Gly Lys Gln Phe Thr Pro Pro Ser Gly Ser Ala Ile Asp Leu His Leu Pro Asn Tyr Val Asp Leu Asn Ala Leu Leu Asp Ile Ser Leu Asp Ser Leu Leu Asn Tyr Asp Val Gln Gly Gln Phe Gly Gly Ser Asn Pro -17~-SUBSTITUTE S~tEET (RULE 26) ., ~, W O 98/08932 PCTrUS97/07657 Val Asp Asn Phe Ser Gly Pro Tyr Gly Ile Tyr Leu Trp Glu Ile Phe Phe His Ile Pro Phe Leu Val Thr Val Arg Met Gln Thr Glu Gln Arg 0 Tyr Glu Asp Ala Asp Thr Trp Tyr Lys Tyr Ile Phe Arg Ser Ala Gly Tyr Arg Asp Ala Asn Gly Gln Leu Ile Met Asp Gly Ser Lys Pro Arg Tyr Trp Asn Val Met Pro Leu Gln Leu Asp Thr Ala Trp Asp Thr Thr Gln Pro Ala Thr Thr Asp Pro Asp Val Ile Ala Met Ala Asp Pro Met His Tyr Lys Leu Ala Ile Phe Leu His Thr Leu Asp Leu Leu Ile Ala Arg Gly Asp Ser Ala Tyr Arg Gln Leu Glu Arg Asp Thr Leu Val Glu Ala Lys Met Tyr Tyr Ile Gln Ala Gln Gln Leu Leu Gly Pro Arg Pro Asp Ile His Thr Thr Asn Thr Trp Pro Asn Pro Thr Leu Ser Lys Glu Ala Gly Ala Ile Ala Thr Pro Thr Phe Leu Ser Ser Pro Glu Val Met Thr Phe Ala Ala Trp Leu Ser Ala Gly Asp Thr Ala Asn Ile Gly Asp Gly Asp Phe Leu Pro Pro Tyr Asn Asp Val Leu Leu Gly Tyr Trp Asp Lys Leu Glu Leu Arg Leu Tyr Asn Leu Arg His Asn Leu Ser Leu Asp Gly Gln Pro Leu Asn Leu Pro Leu Tyr Ala Thr Pro Val Asp Pro Lys Thr Leu Gln Arg Gln Gln Ala Gly Gly Asp Gly Thr Gly Ser Ser Pro Ala Gly Gly Gln Gly Ser Val Gln Gly Trp Arg Tyr Pro Leu Leu Val Glu Arg Ala Arg Ser Ala Val Ser Leu Leu Thr Gln Phe Gly Asn Ser -17~-SUBSTITI.JTE St~EET (RULE 26) .. . . .

W O 98/08932 PCT~US97/076S7 Leu Gln Thr Thr Leu Glu Hls Gln Asp Asn Glu Lys Met Thr Ile Leu Leu Gln Thr Gln Gln Glu Ala Ile Leu Lys Hls Gln His Asp Ile Gln Gln Asn Asn Leu Lys Gly Leu Gln His Ser Leu Thr Ala Leu Gln Ala Ser Arg Asp Gly Asp Thr Leu Arg Gln Lys Hls Tyr Ser Asp Leu Ile Asn Gly Gly Leu Ser Ala Ala Glu Ile Ala Gly Leu Thr Leu Arg Ser Thr Ala Met Ile Thr Asn Gly Val Ala Thr Gly Leu Leu Ile Ala Gly Gly Ile Ala Asn Ala Val Pro Asn Val Phe Gly Leu Ala Asn Gly Gly Ser Glu Trp Gly Ala Pro Leu Ile Gly Ser Gly Gln Ala Thr Gln Val GGC GCC GGC ATC CAG GAT CAG AGC GCG GGC ATT TCA GAA GTG ACA GCA 2~92 Gly Ala Gly Ile Gln Asp Gln Ser Ala Gly Ile Ser Glu Val Thr Ala Gly Tyr Gln Arg Arg Gln Glu Glu Trp Ala Leu Gln Arg Asp Ile Ala Asp Asn Glu Ile Thr Gln Leu Asp Ala Gln Ile Gln Ser Leu Gln Glu Gln Ile Thr Met Ala Gln Lys Gln Ile Thr Leu Ser Glu Thr Glu Gln Ala Asn Ala Gln Ala Ile Tyr Asp Leu Gln Thr Thr Arg Phe Thr Gly Gln Ala Leu Tyr Asn Trp Met Ala Gly Arg Leu Ser Ala Leu Tyr Tyr Gln Met Tyr Asp Ser Thr Leu Pro Ile Cys Leu Gln Pro Lys Ala Ala Leu Val Gln Glu Leu Gly Glu Lys Glu Ser Asp Ser Leu Phe Gln Val Pro Val Trp Asn Asp Leu Trp Gln Gly ~eu Leu Ala Gly Glu Gly Leu Ser Ser Glu Leu Gln Lys Leu Asp Ala Ile Trp Leu Ala Arg Gly Gly SUBSTITUTE SHEE~ ~RULE 26) .

CA 022638l9 l999-02-26 W O 98/08932 PCT~US97/07657 1005 1010 .1015 1020 Ile Gly Leu Glu Ala Ile Arg Thr Val Ser Leu Asp Thr Leu Phe Gly Thr Gly Thr Leu Ser Glu Asn Ile Asn Lys Val Leu Asn Gly Glu Thr Val Ser Pro Ser Gly Gly Val Thr Leu Ala Leu Thr Gly Asp Ile Phe Gln Ala Thr Leu Asp Leu Ser Gln Leu Gly Leu Asp Asn Ser Tyr Asn Leu Gly Asn Glu Lys Lys Arg Arg Ile Lys Arg Ile Ala Val Thr Leu lOB5 1090 1095 1100 Pro Thr Leu Leu Gly Pro Tyr Gln Asp Leu Glu Ala Thr Leu Val Met Gly Ala Glu Ile Ala Ala Leu Ser His Gly Val Asn Asp Gly Gly Arg Phe Val Thr Asp Phe Asn Asp Ser Arg Phe Leu Pro Phe Glu Gly Arg Asp Ala Thr Thr Gly Thr Leu Glu Leu Asn Ile Phe His Ala Gly Lys Glu Gly Thr Gln His Glu Leu Val Ala Asn Leu Ser Asp Ile Ile Val 1165 1170 1175 llB0 CAT CTG AAT TAC ATC ATT CGA GAC GCG TAA AlllCllllC lll~lCGATT 3654 His Leu Asn Tyr Ile Ile Arg Asp Ala *

SUBST~TUTE SHE~ (RULE 26) ACAAGCCAGC ~'l'~'l''l'C~'l'AC TGGATAACGC ACCTCCCGCA CCGGAAGAGT GG~ lCA 4494 T~lG~l~lll GACCACGGTG AGCGCGTACC TCACTTCATA CCGTGCCAAC ATGGGATGCA 4554 TTCAGTGATA TG~lCG~llC CGGTCAACAA CATCTGGTGG AAATCAAGGG TAATCGCGTC 5454 GGCACCACCG ACCTTATCTA TGCGCAATCC GG~ lGC TCATTTATCT CAACCAAAGT 5634 GTGCCACATA TCGCGCCACA TCACTGGCGT TGTGACCTGT CACTGACCAA ACCCTGGTTG 5~14 CAATTCTGGT TGGATGAAAA ATTACAGCTC ACCAAAGCAG GCAAATCTCC GG~ll~llAT 5934 (2) INFORMATION FOR SEQ ID NO:26:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1189 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: proteln (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26 (TcaB protein):
Met Ser Glu Ser Leu Phe Thr Gln Thr Leu Lys Glu Ala Arg Arg Asp SUBSTITUTE SHEET (RULE 2~) W 098/08932 PCTrUS97/07657 Ala Leu Val Ala His Tyr Ile Ala Thr Gln Val Pro Ala Asp Leu Lys 20 25 . 30 Glu Ser Ile Gln Thr Ala Asp Asp Leu Tyr Glu Tyr Leu Leu Leu Asp Thr Lys Ile Ser Asp Leu Val Thr Thr Ser Pro Leu Ser Glu Ala Ile 0 Gly Ser Leu Gln Leu Phe Ile His Arg Ala Ile Glu Gly Tyr Asp Gly ~ Thr Leu Ala Asp Ser Ala Lys Pro Tyr Phe Ala Asp Glu Gln Phe Leu ~ 85 90 95 Tyr Asn Trp Asp Ser Phe Asn His Arg Tyr Ser Thr Trp Ala Gly Lys Glu Arg Leu Lys Phe Tyr Ala Gly Asp Tyr Ile Asp Pro Thr Leu Arg Leu Asn Lys Thr.Glu Ile Phe Thr Ala Phe Glu Gln Gly Ile Ser Gln ~5 Gly Lys Leu Lys Ser Glu ~eu Val Glu Ser Lys Leu Arg Asp Tyr Leu Ile Ser Tyr Asp Thr Leu Ala Thr Leu Asp Tyr Ile Thr Ala Cys Gln Gly Lys Asp Asn Lys Thr Ile Phe Phe Ile Gly Arg Thr Gln Asn Ala Pro Tyr Ala Phe Tyr Trp Arg Lys Leu Thr Leu Val Thr Asp Gly Gly Lys Leu Lys Pro Asp Gln Trp Ser Glu Trp Arg Ala Ile Asn Ala Gly Ile Ser Glu Ala Tyr Ser Gly His Val Glu Pro Phe Trp Glu Asn Asn Lys Leu His Ile Arg Trp Phe Thr Ile Ser Lys Glu Asp Lys Ile Asp Phe Val Tyr Lys Asn Ile Trp Val Met Ser Ser Asp Tyr Ser Trp Ala Ser Lys Lys Lys Ile Leu Glu Leu Ser Phe Thr Asp Tyr Asn Arg Val Gly Ala Thr Gly Ser Ser Ser Pro Thr Glu Val Ala Ser Gln Tyr Gly Ser Asp Ala &ln Met Asn Ile Ser Asp Asp Gly Thr Val Leu Ile Phe Gln Asn Ala Gly Gly Ala Thr Pro Ser Thr Gly Val Thr Leu Cys Tyr Asp Ser Gly Asn Val Ile Lys Asn Leu Ser Ser Thr Gly Ser Ala Asn Leu Ser Ser Lys Asp Tyr Ala Thr Thr Lys Leu Arg Met Cys His Gly Gln Ser Tyr Asn Asp Asn Asn Tyr Cys Asn Phe Thr Leu Ser Ile Asn Thr Ile Glu Phe Thr Ser Tyr Gly Thr Phe Ser Ser Asp Gly Lys Gln SUE~STITUTE SHEE7 (RULE 26) .....

CA 022638l9 l999-02-26 W O 98/08932 PCTrUS97/07657 Phe Thr Pro Pro Ser Gly Ser Ala Ile Asp Leu His Leu Pro Asn Tyr Val Asp Leu Asn Ala Leu Leu Asp Ile Ser Leu Asp Ser Leu Leu Asn Tyr Asp Val Gln Gly Gln Phe Gly Gly Ser Asn Pro Val Asp Asn Phe Ser Gly Pro Tyr Gly Ile Tyr Leu Trp Glu Ile Phe Phe His Ile Pro Phe Leu Val Thr Val Arg Met Gln Thr Glu Gln Arg Tyr Glu Asp Ala Asp Thr Trp Tyr Lys Tyr Ile Phe Arg Ser Ala Gly Tyr Arg Asp Ala Asn Gly Gln Leu Ile Met Asp Gly Ser Lys Pro Arg Tyr Trp Asn Val Met Pro Leu Gln Leu Asp Thr Ala Trp Asp Thr Thr Gln Pro Ala Thr Thr Asp Pro Asp Val Ile Ala Met Ala Asp Pro Met His Tyr Lys Leu Ala Ile Phe Leu His Thr Leu Asp Leu Leu Ile Ala Arg Gly Asp Ser Ala Tyr Arg Gln Leu Glu Arg Asp Thr Leu Val Glu Ala Lys Met Tyr 35 Tyr Ile Gln Ala Gln Gln Leu Leu Gly Pro Arg Pro Asp Ile His Thr Thr Asn Thr Trp Pro Asn Pro Thr Leu Ser Lys Glu Ala Gly Ala Ile Ala Thr Pro Thr Phe Leu Ser Ser Pro Glu Val Met Thr Phe Ala Ala Trp Leu Ser Ala Gly Asp Thr Ala Asn Ile Gly Asp Gly Asp Phe Leu Pro Pro Tyr Asn Asp Val Leu Leu Gly Tyr Trp Asp Lys Leu Glu Leu 5 o Arg Leu Tyr Asn Leu Arg His Asn Leu Ser Leu Asp Gly Gln Pro Leu Asn Leu Pro Leu Tyr Ala Thr Pro Val Asp Pro Lys Thr Leu Gln Arg Gln Gln Ala Gly Gly Asp Gly Thr Gly Ser Ser Pro Ala Gly Gly Gln Gly Ser Val Gln Gly Trp Arg Tyr Pro Leu Leu Val Glu Arg Ala Arg Ser Ala Val Ser Leu Leu Thr Gln Phe Gly Asn Ser Leu Gln Thr Thr ~5 Leu Glu His Gln Asp Asn Glu Lys Met Thr Ile Leu Leu Gln Thr Gln Gln Glu Ala Ile Leu Lys His Gln His Asp Ile Gln Gln Asn Asn Leu Lys Gly Leu Gln His Ser Leu Thr Ala Leu Gln Ala Ser Arg Asp Gly SUBSTlT lTE SHEET (RULE 2~) ,~.

CA 022638l9 l999-02-26 WO 98/08932 PCTrUS97/07657 770 775 . 780 Asp Thr Leu Arg Gln Lys His Tyr Ser Asp Leu Ile Asn Gly Gly Leu Ser Ala Ala Glu Ile Ala Gly Leu Thr Leu Arg Ser Thr Ala Met Ile - Thr Asn Gly Val Ala Thr Gly Leu heu Ile Ala Gly Gly Ile Ala Asn Ala Val Pro Asn Val Phe Gly Leu Ala Asn Gly Gly Ser Glu Trp Gly Ala Pro heu Ile Gly Ser Gly Gln Ala Thr Gln Val Gly Ala Gly Ile Gln Asp Gln Ser Ala Gly Ile Ser Glu Val Thr Ala Gly Tyr Gln Arg Arg Gln Glu Glu Trp Ala Leu Gln Arg Asp Ile Ala Asp Asn Glu Ile Thr Gln Leu Asp Ala Gln Ile Gln Ser Leu Gln Glu Gln Ile Thr Met Ala Gln Lys Gln Ile Thr Leu Ser Glu Thr Glu Gln Ala Asn Ala Gln Ala Ile Tyr Asp Leu Gln Thr Thr Arg Phe Thr Gly Gln Ala Leu Tyr Asn Trp Met Ala Gly Arg Leu Ser Ala Leu Tyr Tyr Gln Met Tyr Asp Ser Thr Leu Pro Ile Cys Leu Gln Pro Lys Ala Ala Leu Val Gln Glu Leu Gly Glu hys Glu Ser Asp Ser Leu Phe Gln Val Pro Val Trp Asn Asp Leu Trp Gln Gly heu Leu Ala Gly Glu Gly Leu Ser Ser Glu Leu Gln Lys Leu Asp Ala Ile Trp Leu Ala Arg Gly Gly Ile Gly Leu Glu Ala Ile Arg Thr Val Ser Leu Asp Thr Leu Phe Gly Thr Gly Thr Leu Ser Glu Asn Ile Asn Lys Val Leu Asn Gly Glu Thr Val Ser Pro Ser Gly Gly Val Thr Leu Ala Leu Thr Gly Asp Ile Phe Gln Ala Thr Leu Asp Leu Ser Gln Leu Gly Leu Asp Asn Ser Tyr Asn Leu Gly Asn Glu Lys Lys Arg Arg Ile Lys Arg Ile Ala Val Thr Leu Pro Thr Leu Leu Gly Pro Tyr Gln Asp Leu Glu Ala Thr heu Val Met Gly Ala Glu Ile Ala Ala Leu Ser H1s Gly Val Asn Asp Gly Gly Arg Phe Val Thr Asp Phe Asn Asp Ser Arg Phe Leu Pro Phe Glu Gly Arg Asp Ala Thr Thr SUBS rlTUTE SHEET (RULE 26) CA 022638l9 l999-02-26 W 098t08932 PC~US97/07657 Gly Thr Leu Glu Leu Asn Ile Phe His Ala Gly Lys Glu Gly Thr Gln His Glu Leu Val Ala Asn Leu Ser Asp Ile Ile Val His Leu Asn Tyr Ile Ile Arg Asp Ala *

(2) INFORMATION FOR SEQ ID NO:27:
~i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1881 base pairs (B) TYPE: nucleic acld (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genom1c) (ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..1881 (D) OTHER INFORMATION: tcaBi (xl) SEQUENCE DESCRIPTION: SEQ ID NO:27 ( tcaBi coding reglon):

Met Ser Glu Ser Leu Phe Thr Gln Thr Leu Lys Glu Ala Arg Arg Asp 1 5 lO 15 Ala Leu Val Ala His Tyr Ile Ala Thr Gln Val Pro Ala Asp Leu Lys Glu Ser Ile Gln Thr Ala Asp Asp Leu Tyr Glu Tyr Leu Leu Leu Asp Thr Lys Ile Ser Asp Leu Val Thr Thr Ser Pro Leu Ser Glu Ala Ile Gly Ser Leu Gln Leu Phe Ile His Arg Ala Ile Glu Gly Tyr Asp Gly Thr heu Ala Asp Ser Ala Lys Pro Tyr Phe Ala Asp Glu Gln Phe Leu Tyr Asn Trp Asp Ser Phe Asn His Arg Tyr Ser Thr Trp Ala Gly Lys lO0 105 110 Glu Arg Leu Lys Phe Tyr Ala Gly Asp Tyr Ile Asp Pro Thr Leu Arg Leu Asn Lys Thr Glu Ile Phe Thr Ala Phe Glu Gln Gly Ile Ser Gln Gly Lys Leu Lys Ser Glu Leu Val Glu Ser Lys Leu Arg Asp Tyr ~eu Ile Ser Tyr Asp Thr Leu Ala Thr Leu Asp Tyr Ile Thr Ala Cys Gln -lB2-SUBSTITUTE SHEET (RULE 2~) , CA 022638l9 l999-02-26 W 098/08932 PCTrUS97107657 Gly Lys Asp Asn Lys Thr Ile Phe Phe Ile Gly Arg Thr Gln Asn Ala CCC TAT GCA TTT TAT TGG CGA AAA TTA ACT.TTA GTC ACT GAT GGC GGT 624 Pro Tyr Ala Phe Tyr Trp Arg Lys Leu Thr Leu Val Thr Asp Gly Gly Lys Leu Lys Pro Asp Gln Trp Ser Glu Trp Arg Ala Ile Asn Ala Gly Ile Ser Glu Ala Tyr Ser Gly His Val Glu Pro Phe Trp Glu Asn Asn Lys Leu His Ile Arg Trp Phe Thr Ile Ser Lys Glu Asp Lys Ile Asp Phe Val Tyr Lys Asn Ile Trp Val Met Ser Ser Asp Tyr Ser Trp Ala 260 26~ 270 Ser Lys Lys Lys Ile Leu Glu Leu Ser Phe Thr Asp Tyr Asn Arg Val Gly Ala Thr Gly Ser Ser Ser Pro Thr Glu Val Ala Ser Gln Tyr Gly Ser Asp Ala Gln Met Asn Ile Ser Asp Asp Gly Thr Val Leu Ile Phe Gln Asn Ala Gly Gly Ala Thr Pro Ser Thr Gly Val Thr Leu Cys Tyr Asp Ser Gly Asn Val Ile Lys Asn Leu Ser Ser Thr Gly Ser Ala Asn Leu Ser Ser Lys Asp Tyr Ala Thr Thr Lys Leu Arg Met Cys His Gly Gln Ser Tyr Asn Asp Asn Asn Tyr Cys Asn Phe Thr Leu Ser~Il-e Asn Thr Ile Glu Phe Thr Ser Tyr Gly Thr Phe Ser Ser Asp Gly Lys Gln Phe Thr Pro Pro Ser Gly Ser Ala Ile Asp Leu His Leu Pro Asn Tyr Val Asp Leu Asn Ala Leu Leu Asp Ile Ser Leu Asp Ser Leu Leu Asn Tyr Asp Val Gln Gly Gln Phe Gly Gly Ser Asn Pro Val Asp Asn Phe Ser Gly Pro Tyr Gly Ile Tyr Leu Trp Glu Ile Phe Phe Hls Ile Pro SUBSTITUTE S~EE~ ~RULE 26) , . .... .

CA 022638l9 l999-02-26 WO 98t08932 PCT~US97/07657 Phe Leu Val Thr Val Arg Met Gln Thr Glu Gln Arg Tyr Glu Asp Ala Asp Thr Trp Tyr Lys Tyr Ile Phe Arg Ser Ala Gly Tyr Arg Asp Ala Asn Gly Gln Leu Ile Met Asp Gly Ser Lys Pro Arg Tyr Trp Asn Val Met Pro Leu Gln Leu Asp Thr Ala Trp Asp Thr Thr Gln Pro Ala Thr Thr Asp Pro Asp Val Ile Ala Met Ala Asp Pro Met His Tyr Lys Leu 530 535 ' 540 Ala Ile Phe Leu His Thr Leu Asp Leu Leu Ile Ala Arg Gly Asp Ser Ala Tyr Arg Gln Leu Glu Arg Asp Thr Leu Val Glu Ala Lys Met Tyr Tyr Ile Gln Ala Gln Gln Leu Leu Gly Pro Arg Pro Asp Ile His Thr Thr Asn Thr Trp Pro Asn Pro Thr Leu Ser Lys Glu Ala Gly Ala Ile GCC ACA CCG ACA TTC CTC AGT TCA CCG GAG GTG ATG ACG TTC GCT GCC lB72 Ala Thr Pro Thr Phe Leu Ser Ser Pro Glu Val Met Thr Phe Ala Ala TGG ~TA AGC 1881 Trp Leu Ser (2) INFORMATION FOR SEQ ID NO:28:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 627 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28 (TcaBi protein):
Met Ser Glu Ser Leu Phe Thr Gln Thr Leu Lys Glu Ala Arg Arg Asp Ala Leu Val Ala His Tyr Ile Ala Thr Gln Val Pro Ala Asp Leu Lys Glu Ser Ile Gln Thr Ala Asp Asp Leu Tyr Glu Tyr Leu Leu Leu Asp Thr Lys Ile Ser Asp Leu Val Thr Thr Ser Pro Leu Ser Glu Ala Ile SU8SlTI LITE SHEET (RULE 2~) CA 022638l9 l999-02-26 W 098/08932 PCT~US97/07657 Gly Ser Leu Gln Leu Phe Ile His Arg Ala,Ile Glu Gly Tyr Asp Gly Thr Leu Ala Asp Ser Ala Lys Pro Tyr Phe Ala Asp Glu Gln Phe Leu Tyr Asn Trp Asp Ser Phe Asn His Arg Tyr Ser Thr Trp Ala Gly Lys 0 Glu Arg Leu Lys Phe Tyr Ala Gly Asp Tyr Ile Asp Pro Thr Leu Arg Leu Asn Lys Thr Glu Ile Phe Thr Ala Phe Glu Gln Gly Ile Ser Gln Gly Lys Leu Lys Ser Glu Leu Val Glu Ser Lys Leu Arg Asp Tyr Leu Ile Ser Tyr Asp Thr Leu Ala Thr Leu Asp Tyr Ile Thr Ala Cys Gln Gly Lys Asp Asn Lys Thr Ile Phe Phe Ile Gly Arg Thr Gln Asn Ala Pro Tyr Ala Phe Tyr Trp Arg Lys Leu Thr Leu Val Thr Asp Gly Gly Lys Leu Lys Pro Asp Gln Trp Ser Glu Trp Arg Ala Ile Asn Ala Gly Ile Ser Glu Ala Tyr Ser Gly His Val Glu Pro Phe Trp Glu Asn Asn 225 230 ' 235 240 Lys Leu His Ile Arg Trp Phe Thr Ile Ser Lys Glu Asp Lys Ile Asp Phe Val Tyr Lys Asn Ile Trp Val Met Ser Ser Asp Tyr Ser Trp Ala Ser Lys Lys Lys Ile Leu Glu Leu Ser Phe Thr Asp Tyr Asn Arg Val Gly Ala Thr Gly Ser Ser Ser Pro Thr Glu Val Ala Ser Gln Tyr Gly Ser Asp Ala Gln Met Asn Ile Ser Asp Asp Gly Thr Val Leu Ile Phe Gln Asn Ala Gly Gly Ala Thr Pro Ser Thr Gly Val Thr Leu Cys Tyr Asp Ser Gly Asn Val Ile Lys Asn Leu Ser Ser Thr Gly Ser Ala Asn Leu Ser Ser Lys Asp Tyr Ala Thr Thr Lys Leu Arg Met Cys His Gly Gln Ser Tyr Asn Asp Asn Asn Tyr Cys Asn Phe Thr Leu Ser Ile Asn Thr Ile Glu Phe Thr Ser Tyr Gly Thr Phe Ser Ser Asp Gly Lys Gln Phe Thr Pro Pro Ser Gly Ser Ala Ile Asp Leu His Leu Pro Asn Tyr Val Asp Leu Asn Ala Leu Leu Asp Ile Ser Leu Asp Ser Leu Leu Asn Tyr Asp Val Gln Gly Gln Phe Gly Gly Ser Asn Pro Val Asp Asn Phe SUBSTiTUTE St~EET (RULE 26) .. ~ . ...... , .~ ...

CA 022638l9 l999-02-26 Ser Gly Pro Tyr Gly Ile Tyr Leu Trp Glu Ile Phe Phe His Ile Pro Phe Leu Val Thr Val Arg Met Gln Thr Glu Gln Arg Tyr Glu Asp Ala Asp Thr Trp Tyr Lys Tyr Ile Phe Arg Ser Ala Gly Tyr Arg Asp Ala Asn Gly Gln Leu Ile Met Asp Gly Ser Lys Pro Arg Tyr Trp Asn Val Met Pro Leu Gln Leu Asp Thr Ala Trp Asp Thr Thr Gln Pro Ala Thr Thr Asp Pro Asp Val Ile Ala Met Ala Asp Pro Met His Tyr Lys Leu Ala Ile Phe Leu His Thr Leu Asp Leu Leu Ile Ala Arg Gly Asp Ser Ala Tyr Arg Gln Leu Glu Arg Asp Thr Leu Val Glu Ala Lys Met Tyr Tyr Ile Gln Ala Gln Gln Leu Leu Gly Pro Arg Pro Asp Ile His Thr Thr Asn Thr Trp Pro Asn Pro Thr Leu Ser Lys Glu Ala Gly Ala Ile Ala Thr Pro Thr Phe Leu Ser Ser Pro Glu Val Met Thr Phe Ala Ala Trp Leu Ser (2) INFORMATION FOR SEQ ID NO:29:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1689 base pairs (B) TYPE: nucleic acid (C) STRANDEDWESS: double (D) TOPOLOGY: linear (ii) MOLECULE T~PE: DNA (genomic) (ix) FEATURE:
- - (A-3 NAME/KEY: CDS
(B) LOCATION: 1..1689 (D) OTHER INFORMATION: tcaBii (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29 (tcaBii coding regaion):

Ala Gly Asp Thr Ala Asn Ile Gly Asp Gly Asp Phe Leu Pro Pro Tyr l 5 10 15 Asn Asp Val Leu Leu Gly Tyr Trp Asp Lys Leu Glu Leu Arg Leu Tyr Asn Leu Arg His Asn Leu Ser Leu Asp Gly Gln Pro Leu Asn Leu Pro Leu Tyr Ala Thr Pro Val Asp Pro Lys Thr Leu Gln Arg Gln Gln Ala SVBSTITUTE SHEET (RULE 26) .

CA 022638l9 l999-02-26 WO 98/08932 PC~AUS97/076S7 Gly Gly Asp Gly Thr Gly Ser Ser Pro Ala Gly Gly Gln Gly Ser Val Gln Gly Trp Arg Tyr Pro Leu Leu Val Glu Arg Ala Arg Ser Ala Val Ser Leu Leu Thr Gln Phe Gly Asn Ser Leu Gln Thr Thr Leu Glu His -CAG GAT AAT GAA AAA ATG ACG ATA CTG TTG CAG ACT CAA CAG GAA GCC 3845 Gln Asp Asn Glu Lys Met Thr Ile Leu Leu Gln Thr Gln Gln Glu Ala Ile Leu Lys His Gln His Asp Ile Gln Gln Asn Asn Leu Lys Gly Leu Gln Hls Ser Leu Thr Ala Leu Gln Ala Ser Arg Asp Gly Asp Thr Leu Arg Gln Lys ~is Tyr Ser Asp Leu Ile Asn Gly Gly Leu Ser Ala Ala Glu Ile Ala Gly Leu Thr Leu Arg Ser Thr Ala Met Ile Thr Asn Gly Val Ala Thr Gly Leu Leu Ile Ala Gly Gly Ile Ala Asn Ala Val Pro Asn Val Phe Gly Leu Ala Asn Gly Gly Ser Glu Trp Gly Ala Pro Leu Ile Gly Ser Gly Gln Ala Thr Gln Val Gly Ala Gly Ile Gln Asp Gln Ser Ala Gly Ile Ser Glu Val Thr Ala Gly Tyr Gln Arg Arg Gln Glu Glu Trp Ala Leu Gln Arg Asp Ile Ala Asp Asn Glu Ile Thr Gln Leu Asp Ala Gln Ile Gln Ser Leu Gln Glu Gln Ile Thr Met Ala Gln Lys Gln Ile Thr Leu Ser Glu Thr Glu Gln Ala Asn Ala Gln Ala Ile Tyr Asp Leu Gln Thr Thr Arg Phe Thr Gly Gln Ala ~eu Tyr Asn Trp Met Ala Gly Arg Leu Ser Ala Leu Tyr Tyr Gln Met Tyr Asp Ser Thr Leu Pro Ile Cys Leu Gln Pro Lys Ala Ala Leu Val Gln Glu Leu Gly Glu SU8ST~TUTE SHEET tRULE 26) CA 022638l9 l999-02-26 W 098108932 PCTrUS97/07657 340 345 . 350 Lys Glu Ser Asp Ser ~eu Phe Gln Val Pro Val Trp Asn Asp Leu Trp 35~ 360 365 Gln Gly Leu Leu Ala Gly Glu Gly Leu Ser Ser Glu Leu Gln Lys Leu Asp Ala Ile Trp Leu Ala Arg Gly Gly Ile Gly Leu Glu Ala Ile Arg 3~5 390 395 400 Thr Val Ser Leu Asp Thr Leu Phe Gly Thr Gly Thr Leu Ser Glu Asn Ile Asn Lys Val Leu Asn Gly Glu Thr Val Ser Pro Ser Gly Gly Val Thr Leu Ala Leu Thr Gly Asp Ile Phe Gln Ala Thr Leu Asp Leu Ser Gln Leu Gly Leu Asp Asn Ser Tyr Asn Leu Gly Asn Glu Lys Lys Arg Arg Ile Lys Arg Ile Ala Val Thr Leu Pro Thr Leu Leu Gly Pro Tyr Gln Asp Leu Glu Ala Thr Leu Val Met Gly Ala Glu Ile Ala Ala Leu Ser His Gly Val Asn Asp Gly Gly Arg Phe Val Thr Asp Phe Asn Asp Ser Arg Phe Leu Pro Phe Glu Gly Arg Asp Ala Thr Thr Gly Thr Leu Glu Leu Asn Ile Phe His Ala Gly Lys Glu Gly Thr Gln Hls Glu Leu Val Ala Asn Leu Ser Asp Ile Ile Val Hls Leu Asn Tyr Ile Ile Arg Asp AIa *

60 ~2) INFORMATION FOR SEQ ID NO:30:

(i) SEQUENCE CHARACTERISTICS:
~A) LENGTH: 562 amino acids ~B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein SUBSTITUTE SHEET ~RULE 26) W O98/08932 PCT~US97/07657 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:30 (TcaBii protein):
Ala Gly Asp Thr Ala Asn Ile Gly Asp Gly Asp Phe Leu Pro Pro Tyr 1 5 lO 15 Asn Asp Val Leu Leu Gly Tyr Trp Asp Lys Leu Glu Leu Arg Leu Tyr Asn Leu Arg His Asn Leu Ser Leu Asp Gly Gln Pro Leu Asn Leu Pro Leu Tyr Ala Thr Pro Val Asp Pro Lys Thr Leu Gln Arg Gln Gln Ala Gly Gly Asp Gly Thr Gly Ser Ser Pro Ala Gly Gly Gln Gly Ser Val Gln Gly Trp Arg Tyr Pro Leu Leu Val Glu Arg Ala Arg Ser Ala Val Ser Leu Leu Thr Gln Phe Gly Asn Ser Leu Gln Thr Thr Leu Glu His Gln Asp Asn Glu Lys Met Thr Ile Leu Leu Gln Thr Gln Gln Glu Ala Ile Leu Lys His Gln His Asp Ile Gln Gln Asn Asn Leu Lys Gly Leu Gln His Ser Leu Thr Ala Leu Gln Ala Ser Arg Asp Gly Asp Thr Leu Arg Gln Lys His Tyr Ser Asp Leu Ile Asn Gly Gly Leu Ser Ala Ala Glu Ile Ala Gly Leu Thr Leu Arg Ser Thr Ala Met Ile Thr Asn Gly Val Ala Thr Gly Leu Leu Ile Ala Gly Gly Ile Ala Asn Ala Val Pro Asn Val Phe Gly Leu Ala Asn Gly Gly Ser Glu Trp Gly Ala Pro Leu Ile Gly Ser Gly Gln Ala Thr Gln Val Gly Ala Gly Ile Gln Asp Gln Ser Ala Gly Ile Ser Glu Val Thr Ala Gly Tyr Gln Arg Arg Gln Glu Glu Trp Ala Leu Gln Arg Asp Ile Ala Asp Asn Glu Ile Thr Gln Leu Asp Ala Gln Ile Gln Ser Leu Gln Glu Gln Ile Thr Met Ala Gln Lys Gln Ile Thr Leu Ser Glu Thr Glu Gln Ala Asn Ala Gln Ala Ile Tyr Asp Leu Gln Thr Thr Arg Phe Thr Gly Gln Ala Leu Tyr Asn Trp Met Ala Gly Arg Leu Ser Ala Leu Tyr Tyr Gln Met Tyr Asp Ser Thr Leu ~ 65 Pro Ile Cys Leu Gln Pro Lys Ala Ala Leu Val Gln Glu Leu Gly Glu Lys Glu Ser Asp Ser Leu Phe Gln Val Pro Val Trp Asn Asp Leu Trp SUBSTITUTE SHEET (RULE 26) . ~ .. ~. .

W098/08932 PCTrUS97/07657 Gln Gly Leu Leu Ala Gly Glu Gly Leu Ser Ser Glu Leu Gln Lys Leu 370 375 3ao Asp Ala Ile Trp Leu Ala Arg Gly Gly Ile Gly Leu Glu Ala Ile Arg Thr Val Ser Leu Asp Thr Leu Phe Gly Thr Gly Thr Leu Ser Glu Asn Ile Asn Lys Val Leu Asn Gly Glu Thr Val Ser Pro Ser Gly Gly Val Thr Leu Ala Leu Thr Gly Asp Ile Phe Gln Ala Thr Leu Asp Leu Ser Gln Leu Gly Leu Asp Asn Ser Tyr Asn Leu Gly Asn Glu Lys Lys Arg Arg Ile Lys Arg Ile Ala Val Thr Leu Pro Thr Leu Leu Gly Pro Tyr Gln Asp Leu Glu Ala Thr Leu Val Met Gly Ala Glu Ile Ala Ala Leu Ser His Gly Val Asn Asp Gly Gly Arg Phe Val Thr Asp Phe Asn Asp Ser Arg Phe Leu Pro Phe Glu Gly Arg Asp Ala Thr Thr Gly Thr Leu Glu Leu Asn Ile Phe His Ala Gly Lys Glu Gly Thr Gln His Glu Leu Val Ala Asn Leu Ser Asp Ile Ile Val His Leu Asn Tyr Ile Ile Arg Asp Ala *

(2) INFORMATION FOR SEQ ID NO:31:
(i) SEQUENCE CHARACTERISTICS:
~5 (A) LENGTH: 4458 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iX) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..4458 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31 (tcac gene):

Met Gln Asp Ser Pro Glu Val Ser Ile Thr Thr Leu Ser Leu Pro Lys Gly Gly Gly Ala Ile Asn Gly Met Gly Glu Ala Leu Asn Ala Ala Gly Pro Asp Gly Met Ala Ser Leu Ser Leu Pro Leu Pro Leu Ser Thr Gly Arg Gly Thr Ala Pro Gly Leu Ser Leu Ile Tyr Ser Asn Ser Ala Gly SUBSTITUTE SHEET tRULE 26) CA 022638l9 l999-02-26 W 098/08932 rCT~US97/07657 50 55 . 60 Asn Gly Pro Phe Gly Ile Gly Trp Gln Cys Gly Val Met Ser Ile Ser Arg Arg Thr Gln His Gly Ile Pro Gln Tyr Gly Asn Asp Asp Thr Phe Leu Ser Pro Gln Gly Glu Val Met Asn Ile Ala Leu Asn Asp Gln Gly Gln Pro Asp Ile Arg Gln Asp Val Lys Thr Leu Gln Gly Val Thr Leu Pro Ile Ser Tyr Thr Val Thr Arg Tyr Gln Ala Arg Gln Ile Leu Asp . , , Phe Ser Lys Ile Glu Tyr Trp Gln Pro Ala Ser Gly Gln Glu Gly Arg Ala Phe Trp Leu Ile Ser Thr Pro Asp Gly His Leu His Ile Leu Gly Lys Thr Ala Gln Ala Cys Leu Ala Asn Pro Gln Asn Asp Gln Gln Ile Ala Gln Trp Leu Leu Glu Glu Thr Val Thr Pro Ala Gly Glu His Val Ser Tyr Gln Tyr Arg Ala Glu Asp Glu Ala His Cys Asp Asp Asn Glu Lys Thr Ala His Pro Asn Val Thr Ala Gln Arg Tyr Leu Val Gln Val Asn Tyr Gly Asn Ile Lys Pro Gln Ala Ser Leu Phe Val Leu Asp Asn Ala Pro Pro Ala Pro Glu Glu Trp Leu Phe His Leu Val Phe Asp His Gly Glu Arg Asp Thr Ser Leu His Thr Val Pro Thr Trp Asp Ala Gly Thr Ala Gln Trp Ser Val Arg Pro Asp Ile Phe Ser Arg Tyr Glu Tyr Gly Phe Glu Val Arg Thr Arg Arg Leu Cys Gln Gln Val Leu Met Phe CAC CGC ACC GCG CTC ATG GCC GGA GAA GCC AGT ACC AAT GAC GCC CCG lO08 His Arg Thr Ala Leu Met Ala Gly Glu Ala Ser Thr Asn Asp Ala Pro SUBSTrrUTE SH EET tRULE 26) .. ~., ., . . , ~

Glu Leu Val Gly Arg Leu Ile Leu Glu Tyr Asp Lys Asn Ala Ser Val Thr Thr Leu Ile Thr Ile Arg Gln Leu Ser His Glu Ser Asp Gly Arg Pro Val Thr Gln Pro Pro Leu Glu Leu Ala Trp Gln Arg Phe Asp Leu Glu Lys Ile Pro Thr Trp Gln Arg Phe Asp Ala Leu Asp Asn Phe Asn Ser Gln Gln Arg Tyr Gln Leu Val Asp Leu Arg Gly Glu Gly Leu Pro Gly Met Leu Tyr Gln Asp Arg Gly Ala Trp Trp Tyr Lys Ala Pro Gln 2 5 Arg Gln Glu Asp Gly Asp Ser Asn Ala Val Thr Tyr Asp Lys Ile Ala Pro Leu Pro Thr Leu Pro Asn Leu Gln Asp Asn Ala Ser Leu Met Asp Ile Asn Gly Asp Gly Gln Leu Asp Trp Val Val Thr Ala Ser Gly Ile Arg Gly Tyr His Ser Gln Gln Pro Asp Gly Lys Trp Thr His Phe Thr Pro Ile Asn Ala Leu Pro Val Glu Tyr Phe His Pro Ser Ile Gln Phe 4 5 Ala Asp Leu Thr Gly Ala Gly Leu Ser Asp Leu Val Leu Ile Gly Pro Lys Ser Val Arg Leu Tyr Ala Asn Gln Arg Asn Gly Trp Arg Lys Gly Glu Asp Val Pro Gln Ser Thr Gly Ile Thr Leu Pro Val Thr Gly Thr Asp Ala Arg Lys Leu Val Ala Phe Ser Asp Met Leu Gly Ser Gly Gln Gln His Leu Val Glu Ile Lys Gly Asn Arg Val Thr Cys Trp Pro Asn Leu Gly His Gly Arg Phe Gly Gln Pro Leu Thr Leu Ser Gly Phe Ser Gln Pro Glu Asn Ser Phe Asn Pro Glu Arg Leu Phe Leu Ala Asp Ile SUE~STITUTE SH EET tRULE 26~

CA 022638l9 l999-02-26 W 098/08932 PCTrUS97/07657 Asp Gly Ser Gly Thr Thr Asp Leu Ile Tyr Ala Gln Ser Gly Ser Leu Leu Ile Tyr Leu Asn Gln Ser Gly Asn Gln Phe Asp Ala Pro Leu Thr 0 Leu Ala Leu Pro Glu Gly Val Gln Phe Asp Asn Thr Cys Gln Leu Gln Val Ala Asp Ile Gln Gly Leu Gly Ile Ala Ser Leu Ile Leu Thr Val Pro His Ile Ala Pro His His Trp Arg Cys Asp Leu Ser Leu Thr Lys Pro Trp Leu Leu Asn Val Met Asn Asn Asn Arg Gly Ala His His Thr Leu His Tyr Arg Ser Ser Ala Gln Phe Trp Leu Asp Glu Lys Leu Gln Leu Thr Lys Ala Gly Lys Ser Pro Ala Cys Tyr Leu Pro Phe Pro Met His Leu Leu Trp Tyr Thr Glu Ile Gln Asp Glu Ile Ser Gly Asn Arg Leu Thr Ser Glu Val Asn Tyr Ser His Gly Val Trp Asp Gly Lys Glu Arg Glu Phe Arg Gly Phe Gly Cys Ile Lys Gln Thr Asp Thr Thr Thr Phe Ser His Gly Thr Ala Pro Glu Gln Ala Ala Pro Ser Leu Ser Ile Ser Trp Phe Ala Thr Gly Met Asp Glu Val Asp Ser Gln Leu Ala Thr Glu Tyr Trp Gln Ala Asp Thr Gln Ala Tyr Ser Gly Phe Glu Thr Arg Tyr Thr Val Trp Asp His Thr Asn Gln Thr Asp Gln Ala Phe Thr Pro Asn Glu Thr Gln Arg Asn Trp Leu Thr Arg Ala Leu Lys Gly Gln Leu Leu Arg Thr Glu Leu Tyr Gly Leu Asp Gly Thr Asp Lys Gln Thr Val Pro Tyr Thr Val Ser Glu Ser Arg Tyr Gln Val Arg Ser Ile Pro Val SUBSTlTl.JTE SHEET tRULE 26) ... ~ ,.. .

W 098/08932 PCT~US97/07657 Asn Lys Glu Thr Glu Leu Ser Ala Trp Val Thr Ala Ilë Glu Asn Arg Ser Tyr His Tyr Glu Ar~ Ile Ile Thr Asp Pro Gln Phe Ser Gln Ser Ile Lys Leu Gln His Asp Ile Phe Gly Gln Ser Leu Gln Ser Val Asp Ile Ala Trp Pro Arg Arg Glu Lys Pro Ala Val Asn Pro Tyr Pro Pro Thr Leu Pro Glu Thr Leu Phe Asp Ser Ser Tyr Asp Asp Gln Gln Gln 980 985 ggo Leu Leu Arg Leu Val Arg Gln Lys Asn Ser Trp His His Leu Thr Asp Gly Glu Asn Trp Arg Leu Gly Leu Pro Asn Ala Gln Arg Arg Asp Val Tyr Thr Tyr Asp Arg Ser Lys Ile Pro Thr Glu Gly Ile Ser Leu Glu Ile Leu Leu Lys Asp Asp Gly Leu Leu Ala Asp Glu Lys Ala Ala Val Tyr Leu Gly Gln Gln Gln Thr Phe Tyr Thr Ala Gly Gln Ala Glu Val Thr Leu Glu Lys Pro Thr Leu Gln Ala Leu Val Ala Phe Gln Glu Thr Ala Met Met Asp Asp Thr Ser Leu Gln Ala Tyr Glu Gly Val Ile Glu Glu Gln Glu Leu Asn Thr Ala Leu Thr Gln Ala Gly Tyr Gln Gln Val Ala Arg Leu Phe Asn Thr Arg Ser Glu Ser Pro Val Trp Ala Ala Arg Gln Gly Tyr Thr Asp Tyr Gly Asp Ala Ala Gln Phe Trp Arg Pro Gln Ala Gln Arg Asn Ser Leu Leu Thr Gly Lys Thr Thr Leu Thr Trp Asp ~5 Thr His His Cys Val Ile Ile Gln Thr Gln Asp Ala Ala Gly Leu Thr ACG CAA GCC CAT T.AC GAT TAT CGT TTC CTT ACA CCG GTA CAA CTG ACA 3600 Thr Gln Ala His Tyr Asp Tyr Arg Phe Leu Thr Pro Val Gln Leu Thr SUBSTITUTE S~tEET (RULE 26) W 0 98/08932 PCT~US97/07657 1185 1190 . 1195 1200 Asp Ile Asn Asp Asn Gln His Ile Val Thr Leu Asp Ala Leu Gly Arg Val Thr Thr Ser Arg Phe Trp Gly Thr Glu Ala Gly Gln Ala Ala Gly Tyr Ser Asn Gln Pro Phe Thr Pro Pro Asp Ser Val Asp Lys Ala Leu Ala Leu Thr Gly Ala Leu Pro Val Ala Gln Cys Leu Val Tyr Ala Val Asp Ser Trp Met Pro Ser Leu Ser Leu Ser Gln Leu Ser Gln Ser Gln GAA GAG GCA GAA GCG CTA TGG GCG CAA CTG CGT GCC GCT CAT ATG ATT 3 a 88 Glu Glu Ala Glu Ala Leu Trp Ala Gln Leu Arg Ala Ala His Met Ile Thr Glu Asp Gly Lys Val Cys Ala Leu Ser Gly Lys Arg Gly Thr Ser His Gln Asn Leu Thr Ile Gln Leu Ile Ser Leu Leu Ala Ser Ile Pro Arg Leu Pro Pro His Val Leu Gly Ile Thr Thr Asp Arg Tyr Asp Ser Asp Pro Gln Gln Gln His Gln Gln Thr Val Ser Phe Ser Asp Gly Phe Gly Arg Leu Leu Gln Ser Ser Ala Arg His Glu Ser Gly Asp Ala Trp Gln Arg Lys Glu Asp Gly Gly Leu Val Val Asp Ala Asn Gly Val Leu Val Ser Ala Pro Thr Asp Thr Arg Trp Ala Val Ser Gly Arg Thr Glu Tyr Asp Asp Lys Gly Gln Pro Val Arg Thr Tyr Gln Pro Tyr Phe Leu Asn Asp Trp Arg Tyr Val Ser Asp Asp Ser Ala Arg Asp Asp Leu Phe Ala Asp Thr His Leu Tyr Asp Pro Leu Gly Arg Glu Tyr Lys Val Ile Thr Ala Lys Lys Tyr Leu Arg Glu Lys Leu Tyr Thr Pro Trp Phe Ile SUBSllTUTE SH EET (RULE 26) CA 022638l9 l999-02-26 Val Ser Glu Asp Glu Asn Asp Thr Ala Ser Arg Thr Pro *

(2) INFORMATION FOR SEQ ID NO:32:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1485 amlno acids (B) TYPE: amino acid (D) TOPOLOGY: linear (li) MOLEC~LE TYPE: proteln (xi) SEQUENCE DESCRIPTION: SEQ ID NO:32 (TcaC protein):
Met Gln Asp Ser Pro Glu Val Ser Ile Thr Thr Leu Ser Leu Pro Lys Gly Gly Gly Ala Ile Asn Gly Met Gly Glu Ala Leu Asn Ala Ala Gly Pro Asp Gly Met Ala Ser Leu Ser Leu Pro Leu Pro Leu Ser Thr Gly Arg Gly Thr Ala Pro Gly Leu Ser Leu Ile Tyr Ser Asn Ser Ala Gly Asn Gly Pro Phe Gly Ile Gly Trp Gln Cys Gly Val Met Ser Ile Ser Arg Arg Thr Gln His Gly Ile Pro Gln Tyr Gly Asn Asp Asp Thr Phe Leu Ser Pro Gln Gly Glu Val Met Asn Ile Ala Leu Asn Asp Gln Gly Gln Pro Asp Ile Arg Gln Asp Val Lys Thr Leu Gln Gly Val Thr Leu Pro Ile Ser Tyr Thr Val Thr Arg Tyr Gln Ala Arg Gln Ile Leu Asp Phe Ser Lys Ile Glu Tyr Trp Gln Pro Ala Ser Gly Gln Glu Gly Arg 4 5 Ala Phe Trp Leu Ile Ser Thr Pro Asp Gly His Leu His Ile Leu Gly Lys Thr Ala Gln Ala Cys Leu Ala Asn Pro Gln Asn Asp Gln Gln Ile Ala Gln Trp Leu Leu Glu Glu Thr Val Thr Pro Ala Gly Glu His Va Ser Tyr Gln Tyr Arg Ala Glu Asp Glu Ala His Cys Asp Asp Asn Glu Lys Thr Ala His Pro Asn Val Thr Ala Gln Arg Tyr Leu Val Gln Val ~0 Asn Tyr Gly Asn Ile Lys Pro Gln Ala Ser Leu Phe Val Leu Asp Asn Ala Pro Pro Ala Pro Glu Glu Trp Leu Phe His Leu Val Phe Asp His Gly Glu Arg Asp Thr Ser Leu His Thr Val Pro Thr Trp Asp Ala Gly Thr Ala Gln Trp Ser Val Arg Pro Asp Ile Phe Ser Arg Tyr Glu Tyr SUBST~TUTE SH E T tRULE-26) W O 98/08932 PCTrUS97/07657 Gly Phe Glu Val Arg Thr Arg Arg Leu Cys Gln Gln Val Leu Met Phe His Arg Thr Ala Leu Met Ala Gly Glu Ala Ser Thr Asn Asp Ala Pro Glu Leu Val Gly Arg Leu Ile Leu Glu Tyr Asp Lys Asn Ala Ser Val Thr Thr Leu Ile Thr Ile Arg Gln Leu Ser His Glu Ser Asp Gly Arg ~ 355 360 365 Pro Val Thr Gln Pro Pro Leu Glu Leu Ala Trp Gln Arg Phe Asp Leu Glu Lys Ile Pro Thr Trp Gln Arg Phe Asp Ala Leu Asp Asn Phe Asn Ser Gln Gln Arg Tyr Gln Leu Val Asp Leu Arg Gly Glu Gly Leu Pro Gly Met Leu Tyr Gln Asp Arg Gly Ala Trp Trp Tyr Lys Ala Pro Gln Arg Gln Glu Asp Gly Asp Ser Asn Ala Val Thr Tyr Asp Lys Ile Ala Pro Leu Pro Thr Leu Pro Asn Leu Gln Asp Asn Ala Ser Leu Met Asp Ile Asn Gly Asp Gly Gln Leu Asp Trp Val Val Thr Ala Ser Gly Ile Arg Gly Tyr His Ser Gln Gln Pro Asp Gly Lys Trp Thr His Phe Thr Pro Ile Asn Ala Leu Pro Val Glu Tyr Phe His Pro Ser Ile Gln Phe Ala Asp Leu Thr Gly Ala Gly Leu Ser Asp Leu Val Leu Ile Gly Pro Lys Ser Val Arg Leu Tyr Ala Asn Gln Arg Asn Gly Trp Arg Lys Gly Glu Asp Val Pro Gln Ser Thr Gly Ile Thr Leu Pro Val Thr Gly Thr Asp Ala Arg Lys Leu Val Ala Phe Ser Asp Met Leu Gly Ser Gly Gln Gln His Leu Val Glu Ile Lys Gly Asn Arg Val Thr Cys Trp Pro Asn Leu Gly His Gly Arg Phe Gly Gln Pro Leu Thr Leu Ser Gly Phe Ser Gln Pro Glu Asn Ser Phe Asn Pro Glu Arg Leu Phe Leu Ala Asp Ile Asp Gly Ser Gly Thr Thr Asp Leu Ile Tyr Ala Gln Ser Gly Ser Leu Leu Ile Tyr Leu Asn Gln Ser Gly Asn Gln Phe Asp Ala Pro Leu Thr Leu Ala Leu Pro Glu Gly Val Gln Phe Asp Asn Thr Cys Gln Leu Gln SU~SllTUTE SH~ET tRULE 26) CA 022638l9 l999-02-26 W O 98/08932 PCTrUS97/07657 Val Ala Asp Ile Gln Gly Leu Gly Ile Ala Ser Leu Ile Leu Thr Val Pro His Ile Ala Pro His His Trp Arg Cys Asp Leu Ser Leu Thr Lys Pro Trp Leu Leu Asn Val Met Asn Asn Asn Arg Gly Ala His His Thr 0 Leu His Tyr Arg Ser Ser Ala Gln Phe Trp heu Asp Glu Lys Leu Gln Leu Thr Lys Ala Gly Lys Ser Pro Ala Cys Tyr Leu Pro Phe Pro Met His Leu Leu Trp Tyr Thr Glu Ile Gln Asp Glu Ile Ser Gly Asn Arg Leu Thr Ser Glu Val Asn Tyr Ser His Gly Val Trp Asp Gly Lys Glu Arg Glu Phe Arg Gly Phe Gly Cys Ile Lys Gln Thr Asp Thr Thr Thr Phe Ser His Gly Thr Ala Pro Glu Gln Ala Ala Pro Ser Leu Ser Ile Ser Trp Phe Ala Thr Gly Met Asp Glu Val Asp Ser Gln Leu Ala Thr Glu Tyr Trp Gln Ala Asp Thr Gln Ala Tyr Ser Gly Phe Glu Thr Arg Tyr Thr Val Trp Asp His Thr Asn Gln Thr Asp Gln Ala Phe Thr Pro Asn Glu Thr Gln Arg Asn Trp Leu Thr Arg Ala Leu Lys Gly Gln Leu Leu Arg Thr Glu Leu Tyr Gly Leu Asp Gly Thr Asp Lys Gln Thr Val Pro Tyr Thr Val Ser Glu Ser Arg Tyr Gln Val Arg Ser Ile Pro Val Asn Lys Glu Thr Glu Leu Ser Ala Trp Val Thr Ala Ile Glu Asn Arg Ser T~ IIiG Iyr Glu Arg Ile Ile Thr Asp Pro Gln Phe Ser Gln Ser Ile Lys Leu Gln His Asp Ile Phe Gly Gln Ser Leu Gln Ser Val Asp Ile Ala Trp Pro Arg Arg Glu Lys Pro Ala Val Asn Pro Tyr Pro Pro Thr Leu Pro Glu Thr Leu Phe Asp Ser Ser Tyr Asp Asp Gln Gln Gln Leu Leu Arg Leu Val Arg Gln Lys Asn Ser Trp His His Leu Thr Asp 995 lO00 1005 Gly Glu Asn Trp Arg Leu Gly Leu Pro Asn Ala Gln Arg Arg Asp Val Tyr Thr Tyr Asp Arg Ser Lys Ile Pro Thr Glu Gly Ile Ser Leu Glu 1025 ~030 1035 1040 ~0 Ile Leu Leu Lys Asp Asp Gly Leu Leu Ala Asp Glu Lys Ala Ala Val SUBSTI~UTE ~HEET tRULE 26) Tyr Leu Gly Gln Gln Gln Thr Phe Tyr Thr Ala Gly Gln Ala Glu Val Thr Leu Glu Lys Pro Thr Leu Gln Ala Leu Val Ala Phe Gln Glu Thr Ala Met Met Asp Asp Thr Ser Leu Gln Ala Tyr Glu Gly Val Ile Glu - 1090 1095 llO0 Glu Gln Glu Leu Asn Thr Ala Leu Thr Gln Ala Gly Tyr Gln Gln Val Ala Arg Leu Phe Asn Thr Arg Ser Glu Ser Pro Val Trp Ala Ala Arg i5 1125 1130 1135 Gln Gly Tyr Thr Asp Tyr Gly Asp Ala Ala Gln Phe Trp Arg Pro Gln Ala Gln Arg Asn Ser Leu Leu Thr Gly Lys Thr Thr Leu Thr Trp Asp Thr His His Cys Val Ile Ile Gln Thr Gln Asp Ala Ala Gly Leu Thr Thr Gln Ala His Tyr Asp Tyr Arg Phe Leu Thr Pro Val Gln Leu Thr Asp Ile Asn Asp Asn Gln His Ile Val Thr Leu Asp Ala Leu Gly Arg Val Thr Thr Ser Arg Phe Trp Gly Thr Glu Ala Gly Gln Ala Ala Gly Tyr Ser Asn Gln Pro Phe Thr Pro Pro Asp Ser Val Asp Lys Ala Leu Ala Leu Thr Gly Ala Leu Pro Val Ala Gln Cys Leu Val Tyr Ala Val Asp Ser Trp Met Pro Ser Leu Ser Leu Ser Gln Leu Ser Gln Ser Gln Glu Glu Ala Glu Ala Leu Trp Ala Gln Leu Arg Ala Ala His Met Ile Thr Glu Asp Gly Lys Val Cys Ala Leu Ser Gly Lys Arg Gly Thr Ser His Gln Asn Leu Thr Ile Gln Leu Ile Ser Leu Leu Ala Ser Ile Pro Arg Leu Pro Pro His Val Leu Gly Ile Thr Thr Asp Arg Tyr Asp Ser Asp Pro Gln Gln Gln His Gln Gln Thr Val Ser Phe Ser Asp Gly Phe Gly Arg Leu Leu Gln Ser Ser Ala Arg His Glu Ser Gly Asp Ala Trp Gln Arg Lys Glu Asp Gly Gly Leu Val Val Asp Ala Asn Gly Val Leu Val Ser Ala Pro Thr Asp Thr Arg Trp Ala Val Ser Gly Arg Thr Glu Tyr Asp Asp Lys Gly Gln Pro Val Arg Thr Tyr Gln Pro Tyr Phe Leu Asn Asp Trp Arg Tyr Val Ser Asp Asp Ser Ala Arg Asp Asp Leu Phe SUBSl 11 lJTE SHEET tRULE 2~) WO 98/08932 PCTrUS97/07657 1425 1430 . 1435 1440 Ala Asp Thr His Leu Tyr Afip Pro Leu Gly Arg Glu Tyr Lys Val Ile Thr Ala Lys Lys Tyr Leu Arg Glu Lys Leu Tyr Thr Pro Trp Phe Ile Val Ser Glu Asp Glu Asn Asp Thr Ala Ser Arg Thr Pro *
0 1475 i480 1485 (2) INFORMATION FOR SEQ ID NO:33:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 3288 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA ~genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:33 (tcaA gene):

Met Val Thr Val Met Gln Asn Lys Ile Ser Phe Leu Ser Gly Thr Ser Glu Gln Pro Leu Leu Asp Ala Gly Tyr Gln Asn Val Phe Asp Ile Ala Ser Ile Ser Arg Ala Thr Phe Val Gln Ser Val Pro Thr Leu Pro Val Lys Glu Ala His Thr Val Tyr Arg Gln Ala Arg Gln Arg Ala Glu Asn Leu Lys Ser Leu Tyr Arg Ala Trp Gln Leu Arg Gln Glu Pro Val Ile Lys Gly Leu Ala Lys Leu Asn Leu Gln Ser Asn Val Ser Val Leu Gln Asp Ala Leu Val Glu Asn Ile Gly Gly Asp Gly Asp Phe Ser Asp Leu Met Asn Arg Ala Ser Gln Tyr Ala Asp Ala Ala Ser Ile Gln Ser Leu Phe Ser Pro Gly Arg Tyr Ala Ser Ala Leu Tyr Arg Val Ala Lys Asp Leu His Lys Ser Asp Ser Ser Leu His Ile Asp Asn Arg Arg Ala Asp Leu Lys Asp Leu Ile Leu Ser Glu Thr Thr Met Asn Lys Glu Val Thr Ser Leu Asp Ile Leu Leu Asp Val Leu Gln Lys Gly Gly Lys Asp Ile SUBSllTUTE S~EET (RULE 26) , W O 98/08932 PCTrUS97/07657 Thr Glu Leu Ser Gly Ala Phe Phe Pro Met Thr Leu Pro Tyr Asp Asp His Leu Ser Gln Ile Asp Ser Ala Leu Ser Ala Gln Ala Arg Thr Leu : 210 215 220 Asn Gly Val Trp Asn Thr Leu Thr Asp Thr Thr Ala Gln Ala Val Ser ~ 225 230 235 240 Glu Gln Thr Ser Asn Thr Asn Thr Arg Lys Leu Phe Ala Ala Gln Asp Gly Asn Gln Asp Thr Phe Phe Ser Gly Asn Thr Phe Tyr Phe Lys Ala Val Gly Phe Ser Gly Gln Pro Met Val Tyr Leu Ser Gln Tyr Thr Ser Gly Asn Gly Ile Val Gly Ala Gln Leu Ile Ala Gly Asn Pro Asp Gln Ala Ala Ala Ala Ile Val Ala Pro Leu Lys Leu Thr Trp Ser Met Ala Lys Gln Cys Tyr Tyr Leu Val Ala Pro Asp Gly Thr Thr Met Gly Asp Gly Asn Val Leu Thr Gly Cys Phe Leu Arg Gly Asn Ser Pro Thr Asn Pro Asp Lys Asp Gly Ile Phe Ala Gln Val Ala Asn Lys Ser Gly Ser Thr Gln Pro Leu Pro Ser Phe His Leu Pro Val Thr Leu Glu His Ser Glu Asn Lys Asp Gln Tyr Tyr Leu Lys Thr Glu Gln Gly Tyr Ile Thr Val Asp Ser Ser Gly Gln Ser Asn Trp Lys Asn Ala Leu Val Ile Asn Gly Thr Lys Asp Lys Gly Leu Leu Leu Thr Phe Cys Ser Asp Ser Ser Gly Thr Pro Thr Asn Pro Asp Asp Val Ile Pro Pro Ala Ile Asn Asp Ile Pro Ser Pro Pro Ala Arg Glu Thr Leu Ser Leu Thr Pro Val Ser SUBSTITUTE S~{EET tRULE 26) CA 022638l9 l999-02-26 WO 98/08932 PCTr~S97/07657 Tyr Gln Leu Met Thr Asn Pro Ala Pro Thr Glu Asp Asp Ile Thr Asn His Tyr Gly Phe Asn Gly Ala Ser Leu Arg Ala Ser Pro Leu Ser Thr Ser Glu Leu Thr Ser Lys Leu Asn Ser Ile Asp Thr Phe Cys Glu Lys Thr Arg Leu Ser Phe Asn Gln Leu Met Asp ~eu Thr Ala Gln Gln Ser Tyr Ser Gln Ser Ser Ile Asp Ala hys Ala Ala Ser Arg Tyr Val Arg Phe Gly Glu Thr Thr Pro Thr Arg Val Asn Val Tyr Gly Ala Ala Tyr Leu Asn Ser Thr Leu Ala Asp Ala Ala Asp Gly Gln Tyr Leu Trp Ile Gln Thr Asp Gly Lys Ser Leu Asn Phe Thr Asp Asp Thr Val Val Ala Leu Ala Gly Arg Ala Glu Lys Leu Val Arg Leu Ser Ser Gln Thr Gly sgs 600 605 Leu Ser Phe Glu Glu Leu Asp Trp Leu Ile Ala Asn Ala Ser Arg Ser Val Pro Asp His His Asp Lys Ile Val Leu Asp Lys Pro Val Leu Glu Ala Leu Ala Glu Tyr Val Ser Leu Lys Gln Arg Tyr Gly Leu Asp Ala Asn Thr Phe Ala Thr Phe Ile Ser Ala Val Asn Pro Tyr Thr Pro Asp Gln Thr Pro Ser Phe Tyr Glu Thr Ala Phe Arg Ser Ala Asp Gly Asn CAT GTC ATT GCG CTA GGT ACA GAG GTG A~A TAT GCA GAA AAT GAG CAG 2112 Hls Val Ile Ala Leu Gly Thr Glu Val Lys Tyr Ala Glu Asn Glu Gln Asp Glu Leu Ala Ala Ile Cys Cys Lys Ala Leu Gly Val Thr Ser Asp Glu Leu Leu Arg Ile Gly Arg Tyr Cys Phe Gly Asn Ala Gly Ser Phe Thr Leu Asp Glu Tyr Thr Ala Ser Gln Leu Tyr Arg Phe Gly Ala Ile SU~STITUTE SHEET (RULE 26) W 098/08932 PCTrUS97/07657 CCC CGT TTG TTT GGG CTG ACA TTT GCC CAA,GCC GAA ATT TTA TGG CGT 2304 Pro Arg Leu Phe Gly Leu Thr Phe Ala Gln Ala Glu Ile Leu Trp Arg Leu Met Glu Gly Gly Lys Asp Ile Leu Leu Gln Gln Leu Gly Gln Ala : AAA TCC CTG CAA CCA CTG GCT ATT TTA CGC CGT ACC GAG CAG GTG CTG 2400 Lys Ser Leu Gln Pro Leu Ala Ile Leu Arg Arg Thr Glu Gln Val Leu Asp Trp Met Ser Ser Val Asn Leu Ser Leu Thr Tyr Leu Gln Gly Met Val Ser Thr Gln Trp Ser Gly Thr Ala Thr Ala Glu Met Phe Asn Phe Leu Glu Asn Val Cys Asp Ser Val Asn Ser Gln Ala Ala Thr Lys Glu Thr Met Asp Ser Ala Leu Gln Gln Lys Val Leu Arg Ala Leu Ser Ala Gly Phe Gly Ile Lys Ser Asn Val Met Gly Ile Val Thr Phe Trp Leu Glu Lys Ile Thr Ile Gly Ser Asp Asn Pro Phe Thr Leu Ala Asn Tyr Trp His Asp Ile Gln Thr Leu Phe Ser His Asp Asn Ala Thr Leu Glu Ser Leu Gln Thr Asp Thr Ser Leu Val Ile Ala Thr Gln Gln Leu Ser ~5 CAG CTA GTG TTA ATT GTG AAA TGG CTG AGC CTG ACC GAG CAG GAT CTG 2832 Gln Leu Val Leu Ile Val Lys Trp Leu Ser Leu Thr Glu Gln Asp Leu Gln Leu Leu Thr Thr Tyr Pro Glu Arg Leu Ile Asn Gly Ile Thr Asn Val Pro Val Pro Asn Pro Glu Leu Leu Leu Thr Leu Ser Arg Phe Lys Gln Trp Glu Thr Gln Val Thr Val Ser Arg Asp Glu Ala Met Arg Cys Phe Asp Gln Leu Asn Ala Asn Asp Met Thr Thr Glu Asn Ala Gly Ser Leu Ile Ala Thr Leu Tyr Glu Met Asp Lys Gly Thr Gly Ala Gln Val Asn Thr Leu Leu Leu Gly Glu Asn Asn Trp Pro Lys Ser Phe Thr Ser SUBSTITUTE Sl~ EET tRULE 26) CA 022638l9 l999-02-26 W O 98/08932 PCT~US97/07657 Leu Trp Gln Leu Leu Thr Trp Leu Arg Val Gly Gln Arg Leu Asn Val Gly Ser Thr Thr Leu Gly Asn Leu Leu Ser Met Met Gln Ala Asp Pro Ala Ala Glu Ser Ser Ala Leu Leu Ala Ser Val Ala Gln Asn Leu Ser Ala Ala Ile Ser Asn Arg Gln ~--(2) INFORMATION FOR SEQ ID NO:34:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1095 amino acids (B) TYP~: amino acids (C) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:34 (TcaA protein):
Features From To Descrlption 254 267 SEQ ID NO:15 254 492 TcaAii peptide Met Val Thr Val Met Gln Asn Lys Ile Ser Phe Leu Ser Gly Thr Ser Glu Gln Pro Leu Leu Asp Ala Gly Tyr Gln Asn Val Phe Asp Ile Ala Ser Ile Ser Arg Ala Thr Phe Val Gln Ser Val Pro Thr Leu Pro Val Lys Glu Ala His Thr Val Tyr Arg Gln Ala Arg Gln Arg Ala Glu Asn Leu Lys Ser Leu Tyr Arg Ala Trp Gln Leu Arg Gln Glu Pro Val Ile Lys Gly Leu Ala Lys Leu Asn Leu Gln Ser Asn Val Ser Val Leu Gln Asp Ala Leu Val Glu Asn Ile Gly Gly Asp Gly Asp Phe Ser Asp Leu Met Asn Arg Ala Ser Gln Tyr Ala Asp Ala Ala Ser Ile Gln Ser Leu Phe Ser Pro Gly Arg Tyr Ala Ser Ala Leu Tyr Arg Val Ala Lys Asp Leu His Lys Ser Asp Ser Ser Leu His Ile Asp Asn Arg Arg Ala Asp Leu Lys Asp Leu Ile Leu Ser Glu Thr Thr Met Asn Lys Glu Val Thr Ser Leu Asp Ile Leu Leu Asp Val Leu Gln Lys Gly Gly Lys Asp Ile Thr Glu Leu Ser Gly Ala Phe Phe Pro Met Thr Leu Pro Tyr Asp Asp SUBSTITUTE S~EEET t~ULE 26) CA 022638l9 l999-02-26 W O 98/08932 PCTnUS97/076~7 His Leu Ser Gln Ile Asp Ser Ala Leu Ser Ala Gln Ala Arg Thr Leu Asn Gly Val Trp Asn Thr Leu Thr Asp Thr Thr Ala Gln Ala Val Ser Glu Gln Thr Ser Asn Thr Asn Thr Arg Lys Leu Phe Ala Ala Gln Asp Gly Asn Gln Asp Thr Phe Phe Ser Gly Asn Thr Phe Tyr Phe Lys Ala ~ 260 265 270 Val Gly Phe Ser Gly Gln Pro Met Val Tyr Leu Ser Gln Tyr Thr Ser Gly Asn Gly Ile Val Gly Ala Gln Leu Ile Ala Gly Asn Pro Asp Gln Ala Ala Ala Ala Ile Val Ala Pro Leu Lys Leu Thr Trp Ser Met Ala Lys Gln Cys Tyr Tyr Leu Val Ala Pro Asp Gly Thr Thr Met Gly Asp Gly Asn Val Leu Thr Gly Cys Phe Leu Arg Gly Asn Ser Pro Thr Asn Pro Asp Lys Asp Gly Ile Phe Ala Gln Val Ala Asn Lys Ser Gly Ser Thr Gln Pro Leu Pro Ser Phe His Leu Pro Val Thr Leu Glu His Ser Glu Asn Lys Asp Gln Tyr Tyr Leu Lys Thr Glu Gln Gly Tyr Ile Thr Val Asp Ser Ser Gly Gln Ser Asn Trp Lys Asn Ala Leu Val Ile Asn Gly Thr Lys Asp Lys Gly Leu Leu Leu Thr Phe Cys Ser Asp Ser Ser Gly Thr Pro Thr Asn Pro Asp Asp Val Ile Pro Pro Ala Ile Asn Asp Ile Pro Ser Pro Pro Ala Arg Glu Thr Leu Ser Leu Thr Pro Val Ser Tyr Gln Leu Met Thr Asn Pro Ala Pro Thr Glu Asp Asp Ile Thr Asn His Tyr Gly Phe Asn Gly Ala Ser Leu Arg Ala Ser Pro Leu Ser Thr 485 490 W4 ~ 495 Ser Glu Leu Thr Ser Lys Leu Asn Ser Ile Asp Thr Phe Cys Glu Lys Thr Arg Leu Ser Phe Asn Gln Leu Met Asp Leu Thr Ala Gln Gln Ser Tyr Ser Gln Ser Ser Ile Asp Ala Lys Ala Ala Ser Arg Tyr Val Arg Phe Gly Glu Thr Thr Pro Thr Arg Val Asn Val Tyr Gly Ala Ala Tyr Leu Asn Ser Thr Leu Ala Asp Ala Ala Asp Gly Gln Tyr Leu Trp Ile SUBSTITUTE SH E~T (RULE 26) ... .~._.. . .

CA 022638l9 l999-02-26 W O 98l~8932 PCTrUS97/07657 Gln Thr Asp Gly Lys Ser Leu Asn Phe Thr Asp Asp Thr Val Val Ala Leu Ala Gly Arg Ala Glu Lys Leu Val Arg Leu Ser Ser Gln Thr Gly Leu Ser Phe Glu Glu Leu Asp Trp Leu Ile Ala Asn Ala Ser Arg Ser 0 Val Pro Asp His His Asp Lys Ile Val Leu Asp Lys Pro Val Leu Glu Ala Leu Ala Glu Tyr Val Ser Leu Lys Gln Arg Tyr Gly Leu Asp Ala Asn Thr Phe Ala Thr Phe Ile Ser Ala Val Asn Pro Tyr Thr Pro Asp Gln Thr Pro Ser Phe Tyr Glu Thr Ala Phe Arg Ser Ala Asp Gly Asn His Val Ile Ala.Leu Gly Thr Glu Val Lys Tyr Ala Glu Asn Glu Gln Asp Glu Leu Ala Ala Ile Cys Cys Lys Ala Leu Gly Val Thr Ser Asp Glu Leu Leu Arg Ile Gly Arg Tyr Cys Phe Gly Asn Ala Gly Ser Phe Thr Leu Asp Glu Tyr Thr Ala Ser Gln Leu Tyr Arg Phe Gly Ala Ile Pro Arg Leu Phe Gly Leu Thr Phe Ala Gln Ala Glu Ile Leu Trp Arg Leu Met Glu Gly Gly Lys Asp Ile Leu Leu Gln Gln Leu Gly Gln Ala Lys Ser Leu Gln Pro Leu Ala Ile Leu Arg Arg Thr Glu Gln Val Leu Asp Trp Met Ser Ser Val Asn Leu Ser Leu Thr Tyr Leu Gln Gly Met Val Ser Thr Gln Trp Ser Gly Thr Ala Thr Ala Glu Met Phe Asn Phe Leu Glu Asn Val Cys Asp Ser Val Asn Ser Gln Ala Ala Thr Lys Glu Thr Met Asp Ser Ala Leu Gln Gln Lys Val Leu Arg Ala Leu Ser Ala Gly Phe Gly Ile Lys Ser Asn Val Met Gly Ile Val Thr Phe Trp Leu Glu Lys Ile Thr Ile Gly Ser Asp Asn Pro Phe Thr Leu Ala Asn Tyr Trp His Asp Ile Gln Thr Leu Phe Ser His Asp Asn Ala Thr Leu Glu Ser Leu Gln Thr Asp Thr Ser Leu Val Ile Ala Thr Gln Gln Leu Ser Gln Leu Val Leu Ile Val Lys Trp Leu Ser Leu Thr Glu Gln Asp Leu Gln Leu Leu Thr Thr Tyr Pro Glu Arg Leu Ile Asn Gly Ile Thr Asn SUBSTlTUTE S~EET (RULE 26) Val Pro Val Pro Asn Pro Glu Leu Leu Leu Thr Leu Ser Arg Phe Lys 5Gln Trp Glu Thr Gln Val Thr Val Ser Arg Asp Glu Ala Met Arg Cys Phe Asp Gln Leu Asn Ala Asn Asp Met Thr Thr Glu Asn Ala Gly Ser Leu Ile Ala Thr Leu Tyr Glu Met Asp Lys Gly Thr Gly Ala Gln Val Asn Thr Leu Leu Leu Gly Glu Asn Asn Trp Pro Lys Ser Phe Thr Ser Leu Trp Gln Leu Leu Thr Trp Leu Arg Val Gly Gln Arg Leu Asn Val ~ 0 Gly Ser Thr Thr Leu Gly Asn Leu Leu Ser Met Met Gln Ala Asp Pro Ala Ala Glu Ser Ser Ala Leu Leu Ala Ser Val Ala Gln Asn Leu Ser 1075 1080 lOBS
Ala Ala Ile Ser Asn Arg Gln ~--(2) INFORMATION FOR SEQ ID NO:35 ti) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 603 amino acids (B) TYPE: amino acid 3 5 ( C ) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:35 (TcaAiii protein):
4 0Pro Leu Ser Thr Ser Glu Leu Thr Ser Lys Leu Asn Ser Ile Asp Thr Phe Cys Glu Lys Thr Arg Leu Ser Phe Asn Gln Leu Met Asp Leu Thr Ala Gln Gln Ser Tyr Ser Gln Ser Ser Ile Asp Ala Lys Ala Ala Ser Arg Tyr Val Arg Phe Gly Glu Thr Thr Pro Thr Arg Val Asn Val Tyr Gly Ala Ala Tyr Leu Asn Ser Thr Leu Ala Asp Ala Ala Asp Gly Gln 55Tyr Leu Trp Ile Gln Thr Asp Gly Lys Ser Leu Asn Phe Thr Asp Asp Thr Val Val Ala Leu Ala Gly Arg Ala Glu Lys Leu Val Arg Leu Ser Ser Gln Thr Gly Leu Ser Phe Glu Glu Leu Asp Trp Leu Ile Ala Asn Ala Ser Arg Ser Val Pro Asp His Hls Asp Lys Ile Val Leu Asp Lys Pro Val Leu Glu Ala Leu Ala Glu Tyr Val Ser Leu Lys Gln Arg Tyr SUBSTITIJTE SHEE~ tRULE 26) W O 98/08932 rCTAUS97107657 Gly Leu Asp Ala Asn Thr Phe Ala Thr Phe, Ile Ser Ala Val Asn Pro Tyr Thr Pro Asp Gln Thr Pro Ser Phe Tyr Glu Thr Ala Phe Arg Ser Ala Asp Gly Asn His Val Ile Ala Leu Gly Thr Glu Val Lys Tyr Ala 0 Glu Asn Glu Gln Asp Glu Leu Ala Ala Ile Cys Cys Lys Ala Leu Gly Val Thr Ser Asp Glu Leu Leu Arg Ile Gly Arg Tyr Cys Phe Gly Asn Ala Gly Arg Phe Thr Leu Asp Glu Tyr Thr Ala Ser Gln Leu Tyr Arg Phe Gly Ala Ile Pro Arg Leu Phe Gly Leu Thr Phe Ala Gln Ala Glu Ile Leu Trp Arg Leu Met Glu Gly Gly Lys Asp Ile Leu Leu Gln Gln ; Xx~c Gly Gln Ala Lys Ser Leu Gln Pro Leu Ala Ile Leu Arg Arg Thr Glu Gln Val Leu Asp Trp Met Ser Pro Val Asn Leu Ser Leu Thr Tyr Leu Gln Gly Met Val Ser Thr Gln Trp Ser Gly Thr Ala Thr Ala Glu Met Phe Asn Phe Leu Glu Asn Val Cys Asp Ser Val Asn Ser Gln Ala Xxx Thr Lys Glu Thr Met Asp Ser Ala Leu Gln Gln Lys Val Leu Arg 4 0 Ala Leu Ser Ala Gly Phe Gly Ile Lys Ser Asn Val Met Gly Ile Val Thr Phe Trp Leu Glu Lys Ile Thr Ile Gly Arg Asp Asn Pro Phe Thr Leu Ala Asn Tyr Trp His Asp Ile Gln Thr Leu Phe Ser His Asp Asn Ala Thr Leu Glu Ser Leu Gln Thr Asp Thr Ser Leu Val Ile Ala Thr Gln Gln Leu Ser Gln Leu Val Leu Ile Val Lys Trp Val Ser Leu Thr 5 5 Glu Gln Asp Leu Gln Leu Leu Thr Thr Tyr Pro Glu Arg Leu Ile Asn Gly Ile Thr Asn Val Pro Val Pro Asn Pro Glu Leu Leu Leu Thr Leu Ser Arg Phe Lys Gln Trp Glu Thr Gln Val Thr Val Ser Arg Asp Glu Ala Met Arg Cys Phe Asp Gln Leu Asn Ala Asn Asp Met Thr Thr Glu Asn Ala Gly Ser Leu Ile Ala Thr Leu Tyr Glu Met Asp Lys Gly Thr Gly Ala Gln Val Asn Thr Leu Leu Leu Gly Glu Asn Asn Trp Pro Lys SU85TTTUTE SHFET (RULE 26) .... ... . .

CA 022638l9 l999-02-26 W O 98/08932 PCT~US97/07657 Ser Phe Thr Ser Leu Trp Gln Leu Leu Thr Trp Leu Arg Val Gly Gln Arg Leu Asn Val Gly Ser Thr Thr Leu Gly Asn Leu Leu Ser Met Met Gln Ala Asp Pro Ala Ala Glu Ser Ser Ala ~eu Leu Ala Ser Val Ala Gln Asn Leu Ser Ala Ala Ile Ser Asn Arg Gln *

(2) INFORMATION FOR SEQ ID NO:36:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2557 base pairs (B) TYPE: nucleic acid (C) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:36 (tcdA internal fragment):

ACCATGATAA TAAAGATGGA AAAATTAAAA ATAACCTAAA GAA~ lCC AATTTATATA 120 TCTGGGACTG GTTGAATACT AAGTATACGC CGG~llCATC GGAAGCCGTA GAAACGCAGG 600 qO TAACGGCAGA ACAACTGGCT GATGCCATGA ATCTTGATGC TAATTTGCTG TTGCAAGCCA 900 GTATTCAAGC ACAAAATCAT CAACATCTTC CCCCAGTAAC TCCAGAAAAT GC~~ CCT 960 GCCCCACAGG GC~lllCCGC TTTGGTCGGG CTGGATTATA TTCAATCAAT GAAAGAGACA 1080 ACAGGCTAAT ACATTACAAC G~llll~lGG ATGAATCTCG CAGTGCCGCA TTAAGCACCT 1200 AATACTTACT GATTGATAAT CAG~~ lG CGGCAATAAA AACCACCCGG ATCGCCGAAG 1320 ~ CCATTGCCAG TATTCAACTG TACGTCAACC GGGCATTGGA AAATGTGGAA GAAAATGCCA 1380 ATTCGGGGGT TATCAGCCGC CAAll~lllA TCGACTGGGA CAAATACAAT AAACGCTACA 1440 GCACTTGGGC GG~l~lll~l CAATTAGTTT ACTACCCGGA AAACTATATT GATCCGACCA 1500 TAAACGCCGA TACCGTCGAA GATGCCTTTA l~l~llATCT GACATCGTTT GAACAAGTGG 1620 SUBSTITUTE St~E~T tRULE 2~) .. . . ... .

CA 022638l9 l999-02-26 WO 9~08932 PCT~US97/07657 r~ GGTT GGAACAAAAG GAGATCACCA AACAGACAGG AAATAGTAAA GATGGCTATC 1920 l~rllGCl~A TATGGCATCC AAAGATATGA CCCCAGAACA GAGCAATGTT TATCGGGATA 2220 ATTATGAGAT TC~ llCG GTAAGTAGCC GTAAAGACTA TGGTTGGGGA GATTATTACC 2340 GCAATCAATG CAATTTGATG AATAAATATG GCAAACTAGG TGATAAATTT Ail~l~lATA 2520 (2) INFORMATION FOR SEQ ID NO:37:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 845 amino acids (B) TYPE: amino acids (C) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (partial) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:37 (TcdA internal peptide):
Ala Phe Asn Ile Asp Asp Val Ser Leu Phe Arg Leu Leu Lys Ile Thr Asp His Asp Asn Lys Asp Gly Lys Ile Lys Asn Asn Leu Lys Asn Leu Ser Asn Leu Tyr Ile Gly Lys Leu Leu Ala Asp Ile His Gln Leu Thr Ile Asp Glu Leu Asp Leu Leu Leu Ile Ala Val Gly Glu Gly Lys Thr Asn Leu Ser Ala Ile Ser Asp Lys Gln Leu Ala Thr Leu Ile Arg Lys Leu Asn Thr Ile Thr Ser Trp Leu His Thr Gln Lys Trp Ser Val Phe Gln Leu Phe Ile Met Thr Ser Thr Ser Tyr Asn Lys Thr Leu Thr Pro Glu Ile Lys Asn Leu Leu Asp Thr Val Tyr His Gly Leu Gln Gly Phe Asp Lys Asp Lys Ala Asp Leu Leu His Val Met Ala Pro Tyr Ile Ala Ala Thr Leu Gln Leu Ser Ser Glu Asn Val Ala His Ser Val Leu Leu Trp Ala Asp Lys Leu Gln Pro Gly Asp Gly Ala Met Thr Ala Glu Gly Phe Trp Asp Trp Leu Asn Thr Lys Tyr Thr Pro Gly Ser Ser Glu Ala Val Glu Thr Gln Glu His Ile Val Gln Tyr Cys Gln Ala Leu Ala Gln SUBSTITUTE St~EE~ tRU~ E 2~) WO 98/08932 PCT~US97/07657 Leu Glu Met Val Tyr His Ser Thr Gly Ile Asn Glu Asn Ala Phe Arg Leu Phe Val Thr Lys Pro Glu Met Phe Gly Ala Ala Thr Gly Ala Ala Pro Ala His Asp Ala Leu Ser Leu Ile Met Leu Thr Arg Phe Ala Asp Trp Val Asn Ala Leu Gly Glu Lys Ala Ser Ser Val Leu Ala Ala Phe Glu Ala Asn Ser Leu Thr Ala Glu Gln Leu Ala Asp Ala Met Asn Leu Asp Ala Asn Leu Leu Leu Gln Ala Ser Ile Gln Ala Gln Asn His Gln His Leu Pro Pro Val Thr Pro Glu Asn Ala Phe Ser Cys Trp Thr Ser Ile Asn Thr Ile Leu Gln Trp Val Asn Val Ala Gln Gln Leu Lys Cys Arg Pro Thr Gly Arg Phe Arg Phe Gly Arg Ala Gly Leu Tyr Ser Ile 3 0 Asn Glu Arg Asp Thr Asp Leu Cys Pro Val Gly Lys Arg Gly Arg Arg Ile Asn Arg Arg Val Glu Phe Asn Asn Arg Leu Ile His Tyr Asn Ala Phe Leu Asp Glu Ser Arg Ser Ala Ala Leu Ser Thr Tyr Tyr Ile Arg Gln Val Ala Lys Ala Ala Ala Ala Ile Lys Ser Arg Asp Asp Leu Tyr Gln Tyr Leu Leu Ile Asp Asn Gln Val Ser Ala Ala Ile Lys Thr Thr Arg Ile Ala Glu Ala Ile Ala Ser Ile Gln Leu Tyr Val Asn Arg Ala Leu Glu Asn Val Glu Glu Asn Ala Asn Ser Gly Val Ile Ser Arg Gln Phe Phe Ile Asp Trp Asp Lys Tyr Asn Lys Arg Tyr Ser Thr Trp Ala Gly Val Ser Gln Leu Val Tyr Tyr Pro Glu Asn Tyr Ile Asp Pro Thr Met Arg Ile Gly Gln Thr Lys Met Met Asp Ala Leu Leu Gln Ser Val Ser Gln Ser Gln Leu Asn Ala Asp Thr Val Glu Asp Ala Phe Met Ser Tyr Leu Thr Ser Phe Glu Gln Val Ala Asn Leu Lys Val Ile Ser Ala Tyr His Asp Asn Ile Asn Asn Asp Gln Gly Leu Thr Tyr Phe Ile Gly Leu Ser Glu Thr Asp Ala Gly Glu Tyr Tyr Trp Arg Ser val Asp ~Iis SU8STITUTE SHEET (RULE 26) CA 022638l9 l999-02-26 W098/08932 PCT~S97107G57 Ser Lys Phe Asn Asp Gly Lys Phe Ala Ala Asn Ala Trp Ser Glu Trp His Lys Ile Asp Cys Pro Ile Asn Pro Tyr Lys Ser Thr Ile Arg Pro Val Ile Tyr Lys Ser Arg Leu Tyr Leu Leu Trp Leu Glu Gln Lys Glu 0 Ile Thr Lys Gln Thr Gly Asn Ser Lys Asp Gly Tyr Gln Thr Glu Thr Asp Tyr Arg Tyr Glu Leu Lys Leu Ala His Ile Arg Tyr Asp Gly Thr Trp Asn Thr Pro Ile Thr Phe Asp Val Asn Lys Lys Ile Ser Glu Leu ~ys Leu Glu Lys Asn Arg Ala Pro Gly Leu Tyr Cys Ala Gly Tyr Gln Gly Glu Asp Thr Leu Leu Val Met Phe Tyr Asn Gln Gln Asp Thr Leu 2 5 Asp Ser Tyr Lys Asn Ala Ser Met Gln Gly Leu Tyr Ile Phe Ala Asp Met Ala Ser Lys Asp Met Thr Pro Glu Gln Ser Asn Val Tyr Arg Asp Asn Ser Tyr Gln Gln Phe Asp Thr Asn Asn Val Arg Arg Val Asn Asn Arg Tyr Ala Glu Asp Tyr Glu Ile Pro Ser Ser Val Ser Ser Arg Lys Asp Tyr Gly Trp Gly Asp Tyr Tyr Leu Ser Met Val Tyr Asn Gly Asp 4 0 Ile Pro Thr Ile Asn Tyr Lys Ala Ala Ser Ser Asp Leu Lys Ile Tyr Ile Ser Pro Lys Leu Arg Ile Ile His Asn Gly Tyr Glu Gly Gln Lys ~5 Arg Asn Gln Cys Asn Leu Met Asn Lys Tyr Gly Lys Leu Gly Asp Lys Phe Il~ Val Tyr Thr Ser Leu Gly Val Asn Pro Asn Asn (2) INFORMATION FOR SEQ ID NO:38:
5 5 ( i ~ SEQUENCE CHARACTERISTICS:
~A) LENGTH: 16 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MO~ECULAR TYPE: protein (v) FRAGMENT TYPE: N-terminal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:38 (TcdAii- pk71 internal peptide):
Arg Tyr Tyr Asn Leu Ser Asp Glu Glu Leu Ser Gln Phe Ile Gly SUBSTITUTE SHE T (RULE 26) W098/08932 PCT~US97/07657 Lys (2) INFORMATION FOR SEQ ID NO:39:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: protein (v) FRAGMENT TYPE: N-terminal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:39 (TcdAii- pK44 internal peptide):
Gly Thr Ala Thr Asp Val Ser Gly Pro Val Glu Ile Asn Thr Ala Ile Ser Pro Ala Lys (2) INFORMATION FOR SEQ ID NO:40:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 11 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: protein (v) FRAGMENT TYPE: N-terminal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:40 (TcbAiii N-terminus):
Ala Asn Ser Leu Thr Ala Leu Phe Leu Pro Gln (2) INFORMATION FOR SEQ ID NO:41:
(1) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: protein (v) FRAGMENT TYPE: N-terminal 50 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:41 (TcdAiii N-terminus):
Leu Arg Ser Ala Asn Thr Leu Thr Asp Leu Phe Leu Pro Gln 1 5 lO

SUBSTITUTE SHEET (RULE 26) CA 022638l9 l999-02-26 W098/08932 PCT~US97/07657 (2) INFORMATION FOR SEQ ID NO:42:
(i) SEQUENCE CHARACTERISTICS:
~A) LENGTH: l9 amino acids (B) TYPE: amino acid tC) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: protein (v) FRAGMENT TYPE: N-terminal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:42 (TcdA-pk57 internal peptide):
Arg Ala Leu Glu Val Glu Arg Thr Val Ser Leu Ala Glu Val Tyr Ala Gly Leu Glu (2) INFORMATION FOR SEQ ID NO:43:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 11 amino acids ~B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: protein (v) FRAGMENT TYPE: N-terminal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:43 (TcdA~ pK20 internal peptide):

Ile Arg Glu Asp Tyr Pro Ala Ser Leu Gly Lys (2) INFORMATION FOR SEQ ID NO:44:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 16 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: protein (v) FRAGMENT TYPE: N-terminal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:44:
Asp Asp Ser Gly Asp Asp Asp Lys Val Thr Asn Thr Asp Ile ~is Arg (2) INFORMATION FOR SEQ ID NO:45:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 13 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: protein (v) FRAGMENT TYPE: N-terminal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:45:

SUBSTITUTE SH EE~ (RULE 2~) CA 022638l9 l999-02-26 W098~8932 PCT~US97/07657 Asp Val Xaa Gly Ser G1u Lys Ala Asn Glu Lys Leu Lys 5 (2) INE'ORMATION FOR SEQ ID NO:96:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 7551 base pa1rs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: llnear (li) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:46 (tcdA):

Met Asn Glu Ser Val Lys Glu Ile Pro Asp Val Leu Lys Ser Gln Cys Gly Phe Asn Cys Leu Thr Asp Ile Ser His Ser Ser Phe Asn Glu Phe CGC CAG CAA GTA TCT GAG CAC CTC TCC TGG TCC GAA ACA CAC GAC TT.''. 144 Arg Gln Gln Val Ser Glu His Leu Ser Trp Ser Glu Thr His Asp Leu TAT Cr.T GAT GCA CAA CAG GCA CAA AAG GAT AAT CGC CTG TAT GAA GCG- 192 Tyr .ii s Asp Ala Gln Gln Ala Gln Lys Asp Asn Arg Leu Tyr Glu Ala Arg ~ie Leu Lys Arg Ala Asn Pro Gln Leu Gln Asn Ala Val His Leu Ala Ile Leu Ala Pro Asn Ala Glu Leu Ile Gly Tyr Asn Asn Gln Phe Ser Gly Arg Ala Ser Gln Tyr Val Ala Pro Gly Thr Val Ser Ser Met TTC TC'' CCC GCC GCT TAT TTG ACT GAA CTT TAT CGT GAA GCA CGC AAT 384 4 5 Phe Ser Pro Ala Ala Tyr Leu Thr Glu Leu Tyr Arg Glu Ala Arg Asn Leu His Ala Ser Asp Ser Val Tyr Tyr Leu Asp Thr Arg Arg Pro Asp Leu Lvs Ser Met Ala Leu Ser Gln Gln Asn Met Asp Ile Glu Leu Ser Thr Leu Ser Leu Ser Asn Glu Leu Leu Leu Glu Ser Ile Lys Thr Glu Ser Lys Leu Glu Asn Tyr Thr Lys Val Met Glu Met Leu Ser Thr Phe Arg Pro Ser Gly Ala Thr Pro Tyr ~ls Asp Ala Tyr Glu Asn Val Arc GAA GTT ATC CAG CTA CAA GAT CCT GGA CTT GAG CAA CTC AAT GCA TC.''. 672 Glu Val Ile Gln Leu Gln Asp Pro Gly Leu Glu Gln Leu Asn Ala Ser SU~STITUTE S~ EET (RULE 26) CA 022638l9 l999-02-26 W Og8/08932 P ~ ~US97/07657 Pro Ala Ile Ala GLy Leu Met His Gln Ala Ser Leu Leu Gly Ile Asn Ala Ser Ile Ser Pro Glu Leu Phe Asn Ile Leu Thr Glu Glu Ile Thr 2gS 250 255 Glu Gly Asn Ala Glu Glu Leu Tyr Lys Lys Asn Phe Gly Asn Ile Glu Pro Ala Ser Leu Ala Met Pro Glu Tyr Leu Lys Arg Tyr Tyr Asn Leu Ser Asp Glu Glu Leu Ser Gln Phe Ile Gly Lys Ala Ser Asn Phe Gly Gln Gln Glu Tyr Ser Asn Asn Gln Leu Ile Thr Pro Val Val Asn Ser Ser Asp Gly Thr Val Lys Val Tyr Arg Ile Thr Arg Glu Tyr Thr Thr AAT GCT TAT CAA ATG GAT GTG GAG CTA TTT CCC TTC GGT GGT GAG AP.T 1056 Asn Ala Tyr Gln Met Asp Val Glu Leu Phe Pro Phe Gly Gly Glu Asn Tyr Arg Leu Asp Tyr Lys Phe Lys Asn Phe Tyr Asn Ala Ser Tyr Leu Ser Ile Lys Leu Asn Asp Lys Arg Glu Leu Val Arg Thr Glu Gly Ala Pro Gln Val Asn Ile Glu Tyr Ser Ala Asn Ile Thr Leu Asn Thr Ala g5 385 390 395 900 Asp Ile Ser Gln Pro Phe Glu Ile Gly Leu Thr Arg Val Leu Pro Ser Gly Ser Trp Ala Tyr Ala Ala Ala Lys Phe Thr Val Glu Glu Tyr Asn Gln Tyr Ser Phe Leu Leu Lys Leu Asn Lys Ala Ile Arg Leu Ser Arg Ala Thr Glu Leu Ser Pro Thr Ile Leu Glu Gly Ile Val Arg Ser Val Asn Leu Gln Leu Asp Ile Asn Thr Asp Val Leu Gly Lys Val Phe Leu Thr Lys Tyr Tyr Met Gln Arg Tyr Ala Ile His Ala Glu Thr Ala Leu SUBSTITUTE SHEE~ tRULE 2~) CA 022638l9 l999-02-26 W O 98/08932 PCTrusg7/07657 Ile Leu Cys Asn Ala Pro Ile Ser Gln Arg Ser Tyr Asp Asn Gln Pro 500 505 ' 510 Ser Gln Phe Asp Arg Leu Phe Asn Thr Pro Leu Leu Asn Gly Gln Tyr Phe Ser Thr Gly Asp Glu Glu Ile Asp Leu Asn Ser Gly Ser Thr Gly Asp Trp Ar~ Lys Thr Ile Leu Lys Arg Ala Phe Asn Ile Asp Asp Val Ser Leu Phe Arg Leu Leu Lys Ile Thr Asp His Asp Asn Lys Asp Gly Lys Ile Lys Asn Asn Leu Lys Asn Leu Ser Asn Leu Tyr Ile Gly Lys Leu Leu Ala Asp Ile His Gln Leu Thr Ile Asp Glu Leu Asp Leu Leu Leu Ile Ala Val Gly Glu Gly Lys Thr Asn Leu Ser Ala Ile Ser Asp Lys Gln Leu Ala Thr Leu Ile Arg Lys Leu Asn Thr Ile Thr Ser Trp Leu His Thr Gln Lys Trp Ser Val Phe Gln Leu Phe Ile Met Thr Ser Thr Ser Tyr Asn Lys Thr Leu Thr Pro Glu Ile Lys Asn Leu Leu Asp Thr Val Tyr His Gly Leu Gln Gly Phe Asp Lys Asp Lys Ala Asp Leu Leu His Val Met Ala Pro Tyr Ile Ala Ala Thr Leu Gln Leu Ser Ser Glu Asn Val Ala His Ser Val Leu Leu Trp Ala Asp Lys Leu Gln Pro Gly Asp Gly Ala Met Thr Ala Glu Lys Phe Trp Asp Trp Leu Asn Thr Lys Tyr Thr Pro Gly Ser Ser Glu Ala Val Glu Thr Gln Glu His Ile ~ 65 Val Gln Tyr Cys Gln Ala Leu Ala Gln Leu Glu Met Val Tyr His Ser Thr Gly Ile Asn Glu Asn Ala Phe Arg Leu Phe Val Thr Lys Pro Glu SUBSTITUTE SH ~ RULE 26) .. . .

CA 022638l9 l999-02-26 W 098/08932 PCT~US97/07657 Met Phe Gly Ala Ala Thr Gly Ala Ala Pro Ala His Asp~ Ala Leu Ser Leu Ile Met Leu Thr Arg Phe Ala Asp Trp Val Asn Ala Leu Gly Glu 0 Lys Ala Ser Ser Val Leu Ala Ala Phe Glu Ala Asn Ser Leu Thr Ala Glu Gln Leu Ala Asp Ala Met Asn Leu Asp Ala Asn Leu Leu Leu Gln Ala Ser Ile Gln Ala Gln Asn His Gln His Leu Pro Pro Val Thr Pro GAA AAT GCG TTC TCC TGT TGG ACA TCT ATC AAT ACT ATC CTG CAA TGG 26qO
Glu Asn Ala Phe Ser Cys Trp Thr Ser Ile Asn Thr Ile Leu Gln Trp GTT P ~T GTC GCA CAA CAA TTG AAT GTC GCC CCA CAG GGC GTT TCC GCT 2688 Val Asn Val Ala Gln Gln Leu Asn Val Ala Pro Gln Gly Val Ser Ala Leu Val Gly Leu Asp Tyr Ile Gln Ser Met Lys Glu Thr Pro Thr Tyr Ala Gln Trp Glu Asn Ala Ala Gly Val Leu Thr Ala Gly Leu Asn Ser Gln Gln Ala Asn Thr Leu His Ala Phe Leu Asp Glu Ser Arg Ser Ala Ala Leu Ser Thr Tyr Tyr Ile Arg Gln Val Ala Lys Ala Ala Ala Ala 945 950 955 96~
ATT ~AA AGC CGT GAT GAC TTG TAT CAA TAC TTA CTG ATT GAT AAT CAG 2928 Ile Lvs Ser Arg Asp Asp Leu Tyr Gln Tyr Leu Leu Ile Asp Asn Gln Val Ser Ala Ala Ile Lys Thr Thr Arg Ile Ala Glu Ala Ile Ala Ser Ile Gln Leu Tyr Val Asn Arg Ala Leu Glu Asn Val Glu Glu Asn Ala Asn Ser Gly Val Ile Ser Arg Gln Phe Phe Ile Asp Trp Asp Lys Tyr Asn Lys Arg Tyr Ser Thr Trp Ala Gly Val Ser Gln Leu Val Tyr Tyr 1025 1030 1035 lOgO

Pro Glu Asn Tyr Ile Asp Pro Thr Met Arg Ile Gly Gln Thr Lys Met Met Asp Ala Leu 1eu Gln Ser Val Ser Gln Ser Gln Leu Asn Ala Asp SUBSTITUTE SHEET tRULE 26) .. .. .. . . .

CA 022638l9 l999-02-26 Thr Val Glu Asp Ala Phe Met Ser Tyr Leu Thr Ser Phe Glu Gln Val Ala Asn Leu Lys Val Ile Ser Ala Tyr ~ls Asp Asn Ile Asn Asn Asp Gln Gly Leu Thr Tyr Phe Ile Gly Leu Ser Glu Thr Asp Ala Gly Glu Tyr Tyr Trp Arg Ser Val Asp His Ser Lys Phe Asn Asp Gly Lys Phe Ala Ala Asn Ala Trp Ser Glu Trp His Lys Ile Asp Cys Pro Ile Asn CCT TAT AAA AGC ACT ATC CGT CCA GTG ATA TAT AAA TCC CGC CTG TAT 350g Pro Tyr Lys Ser Thr Ile Arg Pro Val Ile Tyr Lys Ser Arg Leu Tyr Leu Leu Trp Leu Glu Gln Lys Glu Ile Thr Lys Gln Thr Gly Asn Ser Lys Asp Gly Tyr Gln Thr Glu Thr Asp Tyr Arg Tyr Glu Leu Lys Leu Ala His Ile Arg Tyr Asp Gly Thr Trp Asn Thr Pro Ile Thr Phe Asp Val Asn Lys Lys Ile Ser Glu Leu Lys Leu Glu Lys Asn Arg Ala Pro Gly Leu Tyr Cys Ala Gly Tyr Gln Gly Glu Asp Thr Leu Leu Val Met Phe Tyr Asn Gln Gln Asp Thr Leu Asp Ser Tyr Lys Asn Ala Ser Met Gln Gly Leu Tyr Ile Phe Ala Asp Met Ala Ser Lys Asp Met Thr Pro Glu Gln Ser Asn Val Tyr Arg Asp Asn Ser Tyr Gln Gln Phe Asp Thr Asn Asn Val Arg Arg Val Asn Asn Arg Tyr Ala Glu Asp Tyr Glu Ile Pro Ser Ser Val Ser Ser Arg Lys Asp Tyr Gly Trp Gly Asp Tyr Tyr Leu Ser Met Val Tyr Asn Gly Asp Ile Pro Thr Ile Asn Tyr Lys Ala 1330 1335 13~0 Ala Ser Ser Asp Leu Lys Ile Tyr Ile Ser ~ro Lys Leu Arg Ile Ile SU~STITUTE SHEET tRULE 26) W098/08932 PCT~US97/07657 CAT AAT GGA TAm GAA GGA CAG AAG CGC AAT CAA TGC AAT CTG ATG AAT 4128 His Asn Gly Tyr Glu Gly Gln Lys Arg Asn Gln Cys Asn Leu Met Asn Lys Tyr Gly Lys Leu Gly ASD Lys Phe Ile Val Tyr Thr Ser Leu Gly Val Asn Pro Asn Asn Ser Ser Asn Lys Leu Met Phe Tyr Pro Val Tyr Gln Tyr Ser Gly Asn Thr Ser Gly Leu Asn Gln Gly Arg Leu Leu Phe His Arg Asp Thr Thr Tyr Pro Ser Lys Val Glu Ala Trp Ile Pro Gly 1425 1430 1435 14qO

Ala Lys Arg Ser Leu Thr Asn Gln Asn Ala Ala Ile Gly Asp Asp Tyr Ala Thr Asp Ser Leu Asn Lys Pro Asp Asp Leu Lys Gln Tyr Ile Phe Met .hr Asp Ser Lys Gly Thr Ala Thr Asp Val Ser Gly Pro Val Glu Ile Asn Thr Ala Ile Ser Pro Ala Lys Val Gln Ile Ile Val Lys Ala GGT GGC AAG GAG CAA ACT ~TT ACC GCA GAT AAA GAT GTC TCC ATT CAG 4 560 Gly Gly Lys Glu Gln Thr Phe Thr Ala Asp Lys Asp Val Ser Ile Gln Pro Ser Pro Ser Phe Asp Glu Met Asn Tyr Gln Phe Asn Ala Leu Glu Ile Asp Gly Ser Gly Leu Asn Phe Ile Asn Asn Ser Ala Ser Ile Asp Val Thr Phe Thr Ala Phe Ala Glu Asp Gly Arg Lys Leu Gly Tyr Glu Ser Phe Ser Ile Pro Val Thr Leu Lys Val Ser Thr Asp Asn Ala Leu Thr Leu His H's Asn Glu Asn Gly Ala Gln Tyr Met Gln Trp Gln Ser Tyr Arg Thr Arg Leu Asn Thr Leu Phe Ala Arg Gln Leu Val Ala Arg Ala Thr Thr C-l~ Ile Asp Thr Ile Leu Ser Met Glu Thr Gln Asn Ile CAG GAA CCG CAG TTA GGC AAA GGT TTC TAT GCT ACG TTC GTG ATA CCm 4944 SUBSTITUTE SHEET tRULE 2~) . .

Gln Glu Pro Gln Leu Gly Lys Gly Phe Tyr Ala Thr Phe Val Ile Prc Pro Tyr Asn Leu Ser Thr His Gly Asp Glu Arg Trp Phe Lys Leu Tyr Ile Lys His Val Val Asp Asn Asn Ser His Ile Ile Tyr Ser Gly Gln Leu Thr Asp Thr Asn Ile Asn Ile Thr Leu Phe Ile Pro Leu Asp Asp Val Pro Leu Asn Gln Asp Tyr His Ala Lys Val Tyr Met Thr Phe Lys Lys Ser Pro Ser Asp Gly Thr Trp Trp Gly Pro His Phe Val Arg Asp Asp Lys Gly Ile Val Thr Ile Asn Pro Lys Ser Ile Leu Thr His Phe 1730 1735 17~0 Glu Ser Val Asn Val Leu Asn Asn Ile Ser Ser Glu Pro Met Asp Phe Ser Gly Ala Asn Ser Leu Tyr Phe Trp Glu Leu Phe Tyr Tyr Thr Pro Met Leu Val Ala Gln Arg Leu Leu His Glu Gln Asn Phe Asp Glu Ala Asn Arg Trp Leu Lys Tyr Val Trp Ser Pro Ser Gly Tyr Ile Val His Gly Gln Ile Gln Asn Tyr Gln Trp Asn Val Arg Pro Leu Leu Glu Asp Thr ~ Trp Asn Ser Asp Pro Leu Asp Ser Val Asp Pro Asp Ala Val Ala Gln His Asp Pro Met His Tyr Lys Val Ser Thr Phe Met Arg Thr Leu Asp Leu Leu Ile Ala Arg Gly Asp His Ala Tyr Arg Gln Leu Glu Arg Asp Thr Leu Asn Glu Ala Lys Met Trp Tyr Met Gln Ala Leu His Leu Leu Gly Asp Lys Pro Tyr Leu Pro Leu Ser Thr Thr Trp Ser Asp Pro Arg Leu ASD Arg Ala Ala Asp Ile Th- Thr Gln Asn Ala His As~

SUBSlll~JTE SHE~T (RULE 26) . .

CA 022638l9 l999-02-26 AGC GCA ATA GTC GCT CTG CGG CAG AAT ATA,CCT ACA CCG GCA CCT TTA 5808 Ser Ala Ile Val Ala Leu Arg Gln Asn Ile Pro Thr Pro Ala Pro Leu Ser Leu Arg Ser Ala Asn Thr Leu Thr Asp Leu Phe Leu Pro Gln Ile 0 Asn Glu Val Met Met Asn Tyr Trp Gln Thr Leu Ala Gln Arg Val Tyr Asn Leu Arg Hls Asn Leu Ser Ile Asp Gly Gln Pro Leu Tyr Leu Pro Ile Tyr Ala Thr Pro Ala Asp Pro Lys Ala Leu Leu Ser Ala Ala Val Ala Thr Ser Gln Gly Gly Gly Lys Leu Pro Glu Ser Phe Met Ser Leu Trp Arg Phe Pro His Met Leu Glu Asn Ala Arg Gly Met Val Ser Gln Leu Thr Gln Phe Gly Ser Thr Leu Gln Asn Ile Ile Glu Arg Gln Asp Ala Glu Ala Leu Asn Ala Leu Leu Gln Asn Gln Ala Ala Glu Leu Ile Leu Thr Asn Leu Ser Ile Gln Asp Lys Thr Ile Glu Glu Leu Asp Ala Glu Lys Thr Val Leu Glu Lys Ser Lys Ala Gly Ala Gln Ser Arg Phe ~5 GAT AGC TAC GGC AAA CTG TAC GAT GAG AAT ATC AAC GCC GGT GAA AAC 6336 Asp Ser Tyr Gly Lys Leu Tyr Asp Glu Asn Ile Asn Ala Gly Glu Asn Gln Ala Met Thr Leu Arg Ala Ser Ala Ala Gly Leu Thr Thr Ala Val Gln Ala Ser Arg Leu Ala Gly Ala Ala Ala Asp Leu Val Pro Asn Ile Phe Gly Phe Ala Gly Gly Gly Ser Arg Trp Gly Ala Ile Ala Glu Ala Thr Gly Tyr Val Met Glu Phe Ser Ala Asn Val Met Asn Thr Glu Ala Asp Lys Ile Ser Gln Ser Glu Thr Tyr Arg Arg Arg Arg Gln Glu Trp Glu Ile Gln Arg Asn Asn Ala Glu Ala Glu Leu Lys Gln Ile Asp Ala SUBSTlTlJTE SHEET (RULE 26) ... .

CA 022638l9 l999-02-26 WO 98l08932 PCT~US97/07657 CAG CTC AAA TCA CTC GCT GTA CGC CGC GAA GCC GCC GTA TTG CAG AAA ~672 Gln Leu Lys Ser Leu Ala Val Arg Arg Glu ALa Ala Val Leu Gln Lys Thr Ser Leu Lys Thr Gln Gln Glu Gln Thr Gln Ser Gln Leu Ala Phe Leu Gln Arg Lys Phe Ser Asn Gln Ala Leu Tyr Asn Trp Leu Arg Gly Arg Leu Ala Ala Ile Tyr Phe Gln Phe Tyr Asp Leu Ala Val Ala Arg Cys Leu Met Ala Glu Gln Ala Tyr Arg Trp Glu Leu Asn Asp Asp Ser Ala Arg Phe Ile Lys Pro Gly Ala Trp Gln Gly Thr Tyr Ala Gly Leu Leu Ala Gly Glu Thr Leu Met Leu Ser Leu Ala Gln Met Glu Asp Ala His Leu Lys Arg Asp Lys Arg Ala Leu Glu Val Glu Arg Thr Val Ser ~325 2330 2335 Leu Ala Glu Val Tyr Ala Gly Leu Pro Lys Asp Asn Gly Pro Phe Ser Leu Ala Gln Glu Ile Asp Lys Leu Val Ser Gln Gly Ser Gly Ser Ala ~0 2355 2360 2365 Gly Ser Gly Asn Asn Asn Leu Ala Phe Gly Ala Gly Thr Asp Thr Lys Thr Ser Leu Gln Ala Ser Val Ser Phe Ala Asp Leu Lys Ile Arg Glu Asp Tyr Pro Aia Ser Leu Gly Lys Ile Arg Arg Ile Lys Gln Ile Ser Val Thr Leu Pro Ala Leu Leu Gly Pro Tyr Gln Asp Val Gln Ala Ile Leu Ser Tyr Gly Asp Lys Ala Gly Leu Ala Asn Gly Cys Glu Ala Leu Ala Val Ser His Gly Met Asn Asp Ser Gly Gln Phe Gln Leu Asp Phe AAC GAT GGC AAA TTC CTG CCA TTC GAA GGC ATC GCC ATT GAT CAA GGC 7g40 Asn Asp Gly Lys Phe Leu ero Phe Glu G]y Ile Ala Ile Asp Gln Gly ACG CTG ACA CT î AGC TTC CCA AAT GCA TCT ATG CCG GAG AAA GGT AAA 7488 Thr Leu Thr Leu Ser Phe Pro Asn Ala Ser Met Pro Glu Lys Gly Lys SUBSTITUTE SHEE E (RULE 26) CA 022638l9 l999-02-26 W098/08932 PCT~US97/07657 CAA GCC ACT ATG TTA AAA ACC CTG AAC GAT ATC ATT TTG CAT ATT CGC ? 536 Gln Ala Thr Met Leu Lys Thr Leu Asn Asp Ile Ile Leu His Ile Arg Tyr Thr Ile Lys ~--_O
(2) INFORMATION FOR SEQ ID NO:47:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2516 amino acids (B) TYPE: amino aclds (C) STRANDEDNESS: slngle (D) TOPOLOGY: linear (il) MOLECULE TYPE: protein (xl) SEQUENCE DESCRIPTION: SEQ ID NO:47 (TcdA):
Features From To Descriptlon Peptide 1 2516 TcdA proteins Peptide 89 1937 TcdAii pep~ide Fragment 89 100 TcdAii N-termlnus (SEQ ID NO:13) Fragment 284 299 (SEQ ID NO:38) Fragment 554 563 (SEQ ID NO:17) Fragment 1080 1092 (SEQ ID NO:23; 12/13) Fragment 1385 1400 (SEQ ID NO:18) Fragment 1478 lq97 (SEQ ID NO:39) Fragment 1620 1642 (SEQ ID NO:21; 19/23) Fragment 1938 1948 (SEQ ID NO:41) Peptide 1938 2516 TCdAiii peptide Fragment 2327 2345 (SEQ ID NO:42) Fragment 2398 2408 (SEQ ID NO:43) Met Asn Glu Ser Val Lys Glu Ile Pro Asp Val Leu Lys Ser Gln Cys 4 O Gly Phe Asn Cys Leu Thr Asp Ile Ser His Ser Ser Phe Asn Glu Phe Arg Gln Gln Val Ser Glu His Leu Ser Trp Ser Glu Thr His Asp Leu Tyr~::s Asp Pila Gln Gln Ala Gln Lys Asp Asn Arg Leu Tyr Glu Ala Arg Ile Leu Lys Arg Ala Asn Pro Gln Leu Gln Asn Ala Val His Leu Ala Ile Leu Ala Pro Asn Ala Glu Leu Ile Gly Tyr Asn Asn Gln Phe Ser Gly Arg Ala Ser Gln Tyr Val Ala Pro Gly Thr Val Ser Ser Met Phe Ser Pro Ala Ala Tyr Leu Thr Glu Leu Tyr Arg Glu Ala Arg Asn Leu His Ala Ser Asp Ser Val Tyr Tyr Leu Asp Thr Arg Arg Pro Asp Leu Lys Ser Met Ala Leu Ser Gln Gln Asn Met Asp Ile Glu Leu Ser Thr Leu Ser Leu Ser Asn Glu Leu Leu Leu Glu Ser Ile Lys Thr Glu SU8STITUTE SHEET (RULE 26) ... . . . . ..

CA 022638l9 l999-02-26 W O 98/08932 PCTruS97/07657 Ser Lys Leu Glu Asn Tyr Thr Lys Val Met Glu Met Leu Ser Thr Phe Arg Pro Ser Gly Ala Thr Pro Tyr His Asp Ala Tyr Glu Asn Val Arg : Glu Val Ile Gln Leu Gln Asp Pro Gly Leu Glu Gln Leu Asn Ala Ser Pro Ala Ile Ala Gly Leu Met His Gln Ala Ser Leu Leu Gly Ile Asn ~ 225 230 235 ZqO
Ala Ser Ile Ser Pro Glu Leu Phe Asn Ile Leu Thr Glu Glu Ile Thr 2g5 250 255 Glu Gly Asn Ala Glu Glu Leu Tyr Lys Lys Asn Phe Gly Asn Ile Glu Pro Ala Ser Leu Ala Met Pro Glu Tyr Leu Lys Arg Tyr Tyr Asn Leu Ser Asp Glu Glu Leu Ser Gln Phe Ile Gly Lys Ala Ser Asn Phe Gly Gln Gln Glu Tyr Ser Asn Asn Gln Leu Ile Thr Pro Val Val Asn Ser Ser Asp Gly Thr Val Lys Val Tyr Arg Ile Thr Arg Glu Tyr Thr Thr Asn Ala Tyr Gln Met Asp Val Glu Leu Phe Pro Phe Gly Gly Glu Asn Tyr Arg Leu Asp Tyr Lys Phe Lys Asn Phe Tyr Asn Ala Ser Tyr Leu Ser Ile Lys Leu Asn Asp Lys Arg Glu Leu Val Arg Thr Glu Gly Ala Pro Gln V~l Asn Ile Glu Tyr Ser Ala Asn Ile Thr Leu Asn Thr Ala Asp Ile Ser Gln Pro Phe Glu Ile Gly Leu Thr Arg Val Leu Pro Ser Gly Ser Trp Ala Tyr Ala Ala Ala Lys Phe Thr Val Glu Glu Tyr Asn Gln Tyr Ser Phe Leu Leu Lys Leu Asn Lys Ala Ile Arg Leu Ser Arg 435 440 q45 Ala Thr Glu Leu Ser Pro Thr Ile Leu Glu Gly Ile Val Arg Ser Val Asn Leu Gln Leu Asp Ile Asn Thr Asp Val Leu Gly Lys Val Phe Leu Thr Lys Tyr Tyr Met Gln Arg Tyr Ala Ile His Ala Glu Thr Ala Leu Ile Leu Cys Asn Ala Pro Ile Ser Gln Arg Ser Tyr Asp Asn Gln Pro Ser Gln Phe Asp Arg Leu Phe Asn Thr Pro Leu Leu Asn Gly Gln Tyr Phe Ser Thr Gly Asp Glu Glu Ile Asp Leu Asn Ser Gly Ser Thr Gly SUBSTITUTE SHE~T ~RULE 2~) W 098J08932 PCT~US97/07657 Asp Trp Arg Lys Thr Ile Leu Lys Arg Ala Phe Asn Ile Asp Asp Val Ser Leu Phe Arg Leu Leu Lys Ile Thr Asp His Asp Asn Lys Asp Gly Lys Ile Lys Asn Asn Leu Lys Asn Leu Ser Asn Leu Tyr Ile Gly Lys 1 0 Leu Leu Ala Asp Ile His Gln Leu Thr Ile Asp Glu Leu Asp Leu Leu Leu Ile Ala Val Gly Glu Gly Lys Thr Asn Leu Ser Ala, Ile Ser Asp Lys G' n Leu Ala Thr Leu Ile Arg Lys Leu Asn Thr Ile Thr Ser Trp Leu His Thr Gln Lys Trp Ser Val Phe Gln Leu Phe Ile Met Thr Ser Thr Ser Tyr Asn Lys Thr Leu Thr Pro Glu Ile Lys Asn Leu Leu Asp 2 5 Thr Val Tyr His Gly Leu Gln Gly Phe Asp Lys Asp Lys Ala Asp Leu Leu Hls Val Met Ala Pro Tyr Ile Ala Ala Thr Leu Gln Leu Ser Ser Glu Asn Val Ala His Ser Val Leu Leu Trp Ala Asp Lys Leu Gln Pro Gly Asp Gly Ala Met Thr Ala Glu Lys Phe Trp Asp Trp Leu Asn Thr Lys Tyr Thr Pro Gly Ser Ser Glu Ala Val Glu Thr Gln Glu His Ile Val Gln Tyr Cys Gln Ala Leu Ala Gln Leu Glu Met Val Tyr Hls Ser Thr Gly Ile Asn Glu Asn Ala Phe Arg Leu Phe Val Thr Lys Pro Glu Met Phe Gly Ala Ala Thr Gly Ala Ala Pro Ala His Asp Ala Leu Ser Leu I 1 e Met Leu Thr Arg Phe Ala Asp Trp Val Asn Ala Leu Gly Glu Lys Ala Ser Ser Val Leu Ala Ala Phe Glu Ala Asn Ser Leu Thr Ala 5 5 Glu Gln Leu Ala Asp Ala Met Asn Leu Asp Ala Asn Leu Leu Leu Gln Ala Ser Ile Gln Ala Gln Asn His Gln His Leu Pro Pro Val Thr Pro Glu Asn Ala Phe Ser Cys Trp Thr Ser Ile Asn Thr Ile Leu Gln Trp Val Asn Val Ala Gln Gln Leu Asn Val Ala Pro Gln Gly Val Ser Ala Leu Val Gly Leu Asp Tyr Ile Gln Ser Met Lys Glu Thr Pro Thr Tyr ~ 0 Ala Gln Trp Glu Asn Ala Ala Gly Val Leu Thr Ala Gly Leu Asn Ser SUBS 111 lJTE SHEET tRULE 26) .. ..

CA 022638l9 l999-02-26 W 098/08932 PCTrUS97/07657 Gln Gln Ala Asn Thr Leu His Ala Phe Leu Asp Glu Ser Arg Ser Ala Ala Leu Ser Thr Tyr Tyr Ile Arg Gln Val Ala Lys Ala Ala Ala Ala Ile Lys Ser Arg Asp Asp Leu Tyr Gln Tyr Leu Leu Ile Asp Asn Gln Val Ser Ala Ala Ile Lys Thr Thr Arg Ile Ala Glu Ala Ile Ala Ser Ile Gln Leu Tyr Val Asn Arg Ala Leu Glu Asn Val Glu Glu Asn Ala Asn Ser Gly Val Ile Ser Arg Gln Phe Phe Ile Asp Trp Asp Lys Tyr lO10 1015 1020 Asn Lys Arg Tyr Ser Thr Trp Ala Gly Val Ser Gln Leu Val Tyr Tyr Pro Glu Asn Tyr Ile Asp Pro Thr Met Arg Ile Gly Gln Thr Lys Met Met Asp Ala Leu Leu Gln Ser Val Ser Gln Ser Gln Leu Asn Ala Asp Thr Val Glu Asp Ala Phe Met Ser Tyr Leu Thr Ser Phe Glu Gln Val Ala Asn Leu Lys Val Ile Ser Ala Tyr His Asp Asn Ile Asn Asn Asp Gln Gly Leu Thr Tyr Phe Ile Gly Leu Ser Glu Thr Asp Ala Gly Glu Tyr Tyr Trp Arg Ser Val Asp His Ser Lys Phe Asn Asp Gly Lys Phe Ala Ala Asn Ala Trp Ser Glu Trp Hls Lys Ile Asp Cys Pro Ile Asn Pro Tyr Lys Ser Thr Ile Arg Pro Val Ile Tyr Lys Ser Arg Leu Tyr ~5 1155 1160 1165 Leu Leu Trp Leu Glu Gln Lys Glu Ile Thr Lys Gln Thr Gly Asn Ser Lys Asp Gly Tyr Gln Thr Glu Thr Asp Tyr Arg Tyr Glu Leu Lys Leu Ala His Ile Arg Tyr Asp Gly Thr Trp Asn Thr Pro Ile Thr Phe Asp Val Asn Lys Lys Ile Ser Glu Leu Lys Leu Glu Lys Asn Arg Ala Pro Gly Leu Tyr Cys Ala Gly Tyr Gln Gly Glu Asp Thr Leu Leu Val Met Phe Tyr Asn Gln Gln Asp Thr Leu Asp Ser Tyr Lys Asn Ala Ser Met Gln Gly Leu Tyr Ile Phe Ala Asp Met Ala Ser Lys Asp Met Thr Pro Glu Gln Ser Asn Val Tyr Arg Asp Asn Ser Tyr Gln Gln Phe Asp Thr Asn Asn Val Arg Arg Val Asn Asn Arg Tyr Ala Glu Asp Tyr Glu Ile SUBSTITUTE SHEET tRU~ E 26) .. . .

CA 022638l9 l999-02-26 Pro Ser Ser Val Ser Ser Arg Lys Asp Tyr Gly Trp Gly Asp Tyr Tyr Leu Ser Met Val Tvr Asn Gly Asp Ile Pro Thr Ile Asn Tyr Lvs Ala Ala Ser Ser Asp Leu Lys Ile Tyr Ile Ser Pro Lys Leu Arg Ile Ile His Asn Gly Tyr Glu Gly Gln Lys Arg Asn Gln Cys Asn Leu Met Asn Lys Tyr Gly Lys Leu Gly Asp Lys Phe Ile Val Tyr Thr Ser Leu Gly Val Asn Pro Asn Asn Ser Ser Asn Lys Leu Met Phe Tyr Pro Val Tyr Gln Tyr Ser Gly Asn Thr Ser Gly Leu Asn Gln Gly Arg Leu Leu Phe His Arg Asp Thr Thr Tyr Pro Ser Lys Val Glu Ala Trp Ile Pro Gly 1425 lfi30 1435 1440 Ala Lys Arg Ser Leu Thr Asn Gln Asn Ala Ala Ile Gly Asp Asp Tyr Ala Thr Asp Ser Leu Asn Lys Pro Asp Asp Leu Lys Gln Tyr Ile Phe Met Thr Asp Ser Lys Gly Thr Ala Thr Asp Val Ser Gly Pro Val Glu Ile Asn Thr Ala Ile Ser Pro Ala Lys Val Gln Ile Ile Val Lys Ala Gly Gly Lys Glu Gln Thr Phe Thr Ala Asp Lys Asp Val Ser Ile Gln Pro Ser Pro Ser Phe Asp Glu Met Asn Tyr Gln Phe Asn Ala Leu Glu Ile ASD Gly Ser 51y Leu Asn Phe Ile Asn Asn Ser Ala Ser Ile ASD

Val Thr Phe Thr Ala Phe Ala Glu Asp Gly Arg Lys Leu Gly Tyr Glu Ser Phe Ser Ile Pro Val Thr Leu Lys Val Ser Thr Asp Asn Ala Leu Thr Leu His His Asn Glu Asn Gly Ala Gln Tyr Met Gln Trp Gln Ser Tyr Arg Thr Arg Leu Asn Thr Leu Phe Ala Arg Gln Leu Val Ala Arg Ala Thr Thr Gly Ile Asp Thr Ile Leu Ser Met Glu Thr Gln Asn Ile Gln Glu Pro Gln Leu Gly Lys Gly Phe Tyr Ala Thr Phe Val Ile Pro Pro Tyr Asn Leu Ser Thr His Gly Asp Glu Arg Trp Phe Lys Leu Tyr Ile Lys His Val Val Asp Asn Asn Ser His Ile Ile Tyr Ser Gly Gln SUBSTITUTE SH EE~ (RULE 26~

CA 022638l9 l999-02-26 W 098/08932 PCTrUS97/07657 Leu Thr Asp Thr Asn Ile Asn Ile Thr Leu Phe Ile Pro Leu Asp As~

Val Pro Leu Asn Gln Asp Tyr His Ala Lys Val Tyr Met Thr Phe Lys Lys Ser Pro Ser Asp Gly Thr Trp Trp Gly Pro His Phe Val Arg Asp 0 Asp Lys Gly Ile Val Thr Ile Asn Pro Lys Ser Ile Leu Thr His Phe Glu Ser Val Asn Val Leu Asn Asn Ile Ser Ser Glu Pro Met Asp Phe Ser Gly Ala Asn Ser Leu Tyr Phe Trp Glu Leu Phe Tyr Tyr Thr Pro Met Leu Val Ala Gln Arg Leu Leu His Glu Gln Asn Phe Asp Glu Ala Asn Arg Trp Leu Lys Tyr Val Trp Ser Pro Ser Gly Tyr Ile Val His Gly Gln Ile Gln Asn Tyr Gln Trp Asn Val Arg Pro Leu Leu Glu Asp Thr Ser Trp Asn Ser Asp Pro Leu Asp Ser Val Asp Pro Asp Ala Val Ala Gln His Asp Pro Met His Tyr Lys Val Ser Thr Phe Met Arg Thr Leu Asp Leu Leu Ile Ala Arg Gly Asp His Ala Tyr Arg Gln Leu Glu Arg Asp Thr Leu Asn Glu Ala Lys Met Trp Tyr Met Gln Ala Leu His Leu Leu Gly Asp Lys Pro Tyr Leu Pro Leu Ser Thr Thr Trp Ser Asp Pro Arg Leu Asp Arg Ala Ala Asp Ile Thr Thr Gln Asn Ala His Asp ~5 Ser Ala Ile Val Ala Leu Arg Gln Asn Ile Pro Thr Pro Ala Pro Leu Ser Leu Arg Ser Ala Asn Thr Leu Thr Asp Leu Phe Leu Pro Gln Ile Asn Glu Val Met Met Asn Tyr Trp Gln Thr Leu Ala Gln Arg Val Tyr Asn Leu Arg His Asn Leu Ser Ile Asp Gly Gln Pro Leu Tyr Leu Pro Ile Tyr Ala Thr Pro Ala Asp Pro Lys Ala Leu Leu Ser Ala Ala Val Ala Thr Ser Gln Gly Gly Gly Lys Leu Pro Glu Ser Phe Met Ser Leu Trp Arg Phe Pro His Met Leu Glu Asn Ala Arg Gly Met Val Ser Gln ~ 65 2020 2025 2030 Leu Thr Gln Phe Gly Ser Thr Leu Gln Asn Ile Ile Glu Arg Gln Asp Ala Glu Ala Leu Asn Ala Leu Leu Gln Asn Gln Ala Ala Glu Leu Ile SUBSTITllTE S~tEET (RULE;~63 CA 022638l9 l999-02-26 WO 98/08932 PCTrUS97/07657 Leu Thr Asn Leu Ser Ile Gln Asp Lys Thr Ile Glu 51u Leu Asp Ala Glu Lys Thr Val Leu Glu Lys Ser Lys Ala Gly Ala Gln Ser Arg Phe Asp Ser Tyr Gly Lys Leu Tyr Asp Glu Asn Ile Asn Ala Gly Glu Asn Gln Ala Met Thr Leu Arg Ala Ser Ala Ala Gly Leu Thr Thr Ala Val Gln Ala Ser Arg Leu Ala Gly Ala Ala Ala Asp Leu Val Pro Asn Ile Phe Gly Phe Ala Gly Gly Gly Ser Arg Trp Gly Ala Ile Ala Glu Ala Thr Gly Tyr Val Met Glu Phe Ser Ala Asn Val Met Asn Thr Glu Ala Asp Lys Ile Ser Gln Ser Glu Thr Tyr Arg Arg Arg Arg Gln Glu Trp Glu Ile Gln Arg Asn Asn Ala Glu Ala Glu Leu Lys Gln Ile Asp Ala Gln Leu Lys Ser Leu Ala Val Arg Arg Glu Ala Ala Val Leu Gln Lys Thr Ser Leu Lys Thr Gln Gln Glu Gln Thr Gln Ser Gln Leu Ala Phe Leu Gln Arg Lys Phe Ser Asn Gln Ala Leu Tyr Asn Trp Leu Arg Gly Arg Leu Ala Ala Ile Tyr Phe Gln Phe Tyr Asp Leu Ala Val Ala Arg Cys Leu Met Ala Glu Gln Ala Tyr Arg Trp Glu Leu Asn Asp Asp Ser Ala Arg Phe Ile Lys Pro Gly Ala Trp Gln Gly Thr Tyr Ala Gly Leu Leu Ala Gly Glu Thr Leu Met Leu Ser Leu Ala Gln Met Glu Asp Ala His Leu Lys Arg Asp Lys Arg Ala Leu Glu Val Glu Arg Thr Val Ser Leu Ala Glu Val Tyr Ala Gly Leu Pro Lys Asp Asn Gly Pro Phe Ser Leu Ala Gln Glu Ile Asp Lys Leu Val Ser Gln Gly Ser Gly Ser Ala Gly Ser Gly Asn Asn Asn Leu Ala Phe Gly Ala Gly Thr Asp Thr Lys Thr Ser Leu Gln Ala Ser Val Ser Phe Ala Asp Leu Lys Ile Arg Glu Asp Tyr Pro Ala Ser Leu Gly Lys Ile Arg Arg Ile Lys Gln Ile Ser Val Thr Leu Pro Ala Leu Leu Gly Pro Tyr Gln Asp Val Gln Ala Ile Leu Ser Tyr Gly ASD Lys Ala Gly Leu Ala Asn Gly Cys Glu Ala Leu SUBSlTrUTE S~EE r tRULE 26) CA 022638l9 l999-02-26 W O 98/08932 PCT~US97/07657 2435 2440 . 2445 Ala Val Ser His Gly Met Asn Asp Ser Gly Gln Phe Gln Leu Asp Phe Asn Asp Gly Lys Phe Leu Pro Phe Glu Gly Ile Ala Ile Asp Gln Gly 2465 2470 24~5 2480 Thr Leu Thr Leu Ser Phe Pro Asn Ala Ser Met Pro Glu Lys Gly Lys Gln Ala Thr Met Leu Lys Thr Leu Asn Asp Ile Ile Leu His Ile Arg Tyr Thr Ile Lys (2) INFORMATION FOR SEQ ID NO:48:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 5547 base pairs (B) TY~E: nucleic acld (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii~ MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 48 (tcdAil coding reglon):

Leu Ile Gly Tyr Asn Asn Gln Phe Ser Gly Arg Ala Ser Gln Tyr Val Ala Pro Gly Thr Val Ser Ser Met Phe Ser ero Ala Ala Tyr Leu Thr G1u Leu Tyr Arg Glu Ala Arq Asn Leu His Ala Ser Asp Ser Val Tyr Tyr Leu Asp Thr Arg Arg Pro Asp Leu Lys Ser Met Ala Leu Ser Gln Gln Asn Met Asp Ile Glu Leu Ser Thr Leu Ser Leu Ser Asn Glu Leu Leu Leu Glu Ser Ile Lys Thr Glu Ser Lys Leu Glu Asn Tyr Thr Lys Val Met Glu Met Leu Ser Thr Phe Arg Pro Ser Gly Ala Thr Pro Tyr CAT GAT GCT TAT GAA AAT GTG CGT GAA GTT ATC CAG CTA CAA GAT CCT 38g His Asp Ala Tyr Glu Asn Val Arg Glu Val Ile Gln Leu Gln Asp Pro Gly Leu Glu Gln Leu Asn Ala Ser Pro Ala Ile Ala Gly Leu Met His Gln Ala Ser Leu Leu Gly Ile Asn Ala Ser Ile Ser Pro Glu Leu Phe SUBSTITUTE SHEET (RULE 26) CA 022638l9 l999-02-26 W 09~08932 PCT~US97/07657 Asn Ile Leu Thr Glu Glu Ile Thr Glu Gly Asn Ala Glu Glu Leu Tyr Lys Lys Asn Phe Gly Asn Ile Glu Pro Ala Ser Leu Ala Met Pro Glu 180 185 l90 Tyr Leu Lys Arg Tyr Tyr Asn Leu Ser Asp Glu Glu Leu Ser Gln Phe Ile Gly Lys Ala Ser Asn Phe Gly Gln Gln Glu Tyr Ser Asn Asn Gln Leu Ile Thr Pro Val Val Asn Ser Ser Asp Gly Thr Val Lys Val Tyr Arg Ile Thr Arg Glu Tyr Thr Thr Asn Ala Tyr Gln Met Asp Val Glu Leu Phe Pro Phe Gly Gly Glu Asn Tyr Arg Leu Asp Tyr Lys Phe Lys AAT TTT TAT AAT GCC TCT TAT TTA TCC ATC AAG TTA AAT GAT AA~ AGA 86g Asn Phe Tyr Asn Ala Ser Tyr Leu Ser Ile Lys Leu Asn Asp Lys Arg Glu Leu Val Arg Thr Glu Gly Ala Pro Gln Val Asn Ile Glu Tyr Ser GCA AAT ATC ACA TTA AAT ACC GCT GAT ATC AGT CAA CCT TTT GAA ATT g60 Ala Asn Ile Thr Leu Asn Thr Ala Asp Ile Ser Gln Pro Phe Glu Ile Gly Leu Thr Arg Val Leu Pro Ser Gly Ser Trp Ala Tyr Ala Ala Ala Lys Phe Thr Val Glu Glu Tyr Asn Gln Tyr Ser Phe Leu Leu Lys Leu Asn Lys Ala Ile Arg Leu Ser Arg Ala Thr Glu Leu Ser Pro Thr Ile Leu Glu Gly Ile Val Arg Ser Val Asn Leu Gln Leu Asp Ile Asn Thr Asp Val Leu Gly Lys Val Phe Leu Thr Lys Tyr Tyr Met Gln Arg Tyr Ala Ile His Ala Glu Thr Ala Leu Ile Leu Cys Asn Ala Pro Ile Ser Gln Arg Ser Tyr Asp Asn Gln Pro Ser Gln Phe Asp Arg Leu Phe Asn ACG CCA TTA CTG AAC GGA CAA TAT TTT TCT ACC GGC GAT GAG GAG AT~ 1344 Thr Pro Leu Leu Asn Gly Gln Tyr Phe Ser Thr Gly Asp Glu Glu Ile SUBSTITUTE 51~ FET (RULE 2~) CA 022638l9 l999-02-26 W O 9~08932 rCTAUS97/07657 435 940 . 445 Asp Leu Asn Ser Gly Ser Thr Gly Asp Trp Arg Lys Thr Ile Leu Lys Arg Ala Phe Asn Ile Asp Asp Val Ser Leu Phe Arg Leu Leu Lys Ile ~ 465 470 475 480 Thr Asp His Asp Asn Lys Asp Gly Lys Ile Lys Asn Asn Leu Lys Asn ~ 485 490 495 Leu Ser Asn Leu Tyr Ile Gly Lys Leu Leu Ala Asp Ile His Gln Leu Thr Ile Asp Glu Leu Asp Leu Leu Leu Ile Ala Val Gly Glu Gly Lys Thr Asn Leu Ser Ala Ile Ser Asp Lys Gln Leu Ala Thr Leu Ile Arg Lys Leu Asn Thr Ile Thr Ser Trp Leu His Thr Gln Lys Trp Ser Val Phe Gln Leu Phe Ile Met Thr Ser Thr Ser Tyr Asn Lys Thr Leu Thr Pro Glu Ile Lys Asn Leu Leu Asp Thr Val Tyr His Gly Leu Gln Gly Phe Asp Lys Asp Lys Ala Asp Leu Leu His Val Met Ala Pro Tyr Ile Ala Ala Thr Leu Gln Leu Ser Ser Glu Asn Val Ala His Ser Val Leu Leu Trp Ala Asp Lys Leu Gln Pro Gly Asp Gly Ala Met Thr Ala Glu AAA TTC TGG GAC TGG TTG AAT ACT AAG TAT ACG CCG GGT T2~ GAA 1968 Lys Phe Trp Asp Trp Leu Asn Thr Lys Tyr Thr Pro Gly Ser Ser Glu Ala Val Glu Thr Gln Glu His Ile Val Gln Tyr Cys Gln Ala Leu Ala Gln Leu Glu Met Val Tyr Hls Ser Thr Gly Ile Asn Glu Asn Ala Phe Arg Leu Phe Val Thr Lys Pro Glu Met Phe Gly Ala Ala Thr Gly Ala Ala Pro Ala His Asp Ala Leu Ser Leu Ile Met Leu Thr Arg Phe Ala SU8STITUTE St~EET tRULE 26) .... . . . ,.. --.. ,.~

Asp Trp Val Asn Ala Leu Gly Glu Lys Ala.Ser Ser Val Leu Ala Ala Phe Glu Ala Asn Ser Leu Thr Ala Glu Gln Leu Ala Asp Ala Met Asn Leu Asp Ala Asn Leu Leu Leu Gln Ala Ser Ile Gln Ala Gln Asn His Gln His Leu Pro Pro Val Thr Pro Glu Asn Ala Phe Ser Cys Trp Thr Ser Ile Asn Thr Ile Leu Gln Trp Val Asn Val Ala Gln Gln Leu Asn Val Ala Pro Gln Gly Val Ser Ala Leu Val Gly Leu Asp Tyr Ile Gln Ser Met Lys Glu Thr Pro Thr Tyr Ala Gln Trp Glu Asn Ala Ala Gly Val Leu Thr Ala Gly Leu Asn Ser Gln Gln Ala Asn Thr Leu Hls Ala Phe Leu Asp Glu Ser Arg Ser Ala Ala Leu Ser Thr Tyr Tyr Ile Arg Gln Val Ala Lys Ala Ala Ala Ala Ile Lys Ser Arg Asp Asp Leu Tyr Gln Tyr Leu Leu Ile Asp Asn Gln Val Ser Ala Ala Ile Lys Thr Thr ~5 Arg Ile Ala Glu Ala Ile Ala Ser Ile Gln Leu Tyr Val Asn Arg Ala Leu Giu Asn Val Glu Glu Asn Ala Asn Ser Gly Val Ile Ser Arg Gln TTC TTT ATC GAC TGG GAC AAA TAC AAT A~A CGC TAC AGC ACT TGG GCG 2832 Phe Phe Ile Asp Trp Asp Lys Tyr Asn Lys Arg Tyr Ser Thr Trp Ala Gly Val Ser Gln Leu Val Tyr Tyr Pro Glu Asn Tyr Ile Asp Pro Thr Met Arg Ile Gly Gln Thr Lys Met Met Asp Ala Leu Leu Gln Ser Val Ser Gln Ser Gln Leu Asn Ala Asp Thr Val Gl~ Asp Ala Phe Met Ser Tyr Leu Thr Ser Phe Glu Gln Val Ala Asn Leu Lys Val Ile Ser Ala SUBSTlTlJTE SH EEl- ~RULE 26) CA 022638l9 l999-02-26 WO 98/08932 PCTrUS97/07657 TAT CAC GAT AAT AmT AAT AAC GAT CAA GGG ,CTG ACC TAT TTT ATC GGA 3072 Tyr His Asp Asn 'e Asn Asn Asp Gln Gly Leu Thr Tyr Phe Ile Gly Leu Ser Glu Thr As~ Ala Gly Glu Tyr Tyr Trp Arg Ser Val Asp His 0 Ser Lys Phe Asn Asp Gly Lys Phe Ala Ala Asn Ala Trp Ser Glu Trp lOq5 1050 1055 His Lys Ile Asp Cys Pro Ile Asn Pro Tyr Lys Ser Thr Ile Arg Pro Val Ile Tyr Lys Ser Arg Leu Tyr Leu Leu Trp Leu Glu Gln Lys Glu Ile Thr Lys Gln Thr Gly Asn Ser Lys Asp Gly Tyr Gln Thr Glu Thr Asp Tyr Arg Tyr Glu Leu Lys Leu Ala His Ile Arg Tyr Asp Gly Thr Trp Asn Thr Pro Ile Thr Phe Asp Val Asn Lys Lys Ile Ser Glu Leu Lys Leu Glu Lys Asn Arg Ala Pro Gly Leu Tyr Cys Ala Gly Tyr Gln llqO 1145 1150 Gly Glu Asp Thr Leu Leu Val Met Phe Tyr Asn Gln Gln Asp Thr Leu GAT AGT TAT A~A AAC GCT TCA ATG CAA GGA CTA TAT ATC TTT GCT GAT 3552 Asp Ser Tyr Lys Asn Ala Ser Met Gln Gly Leu Tyr Ile Phe Ala Asp ~5 ATG GCA TCC AAA GAT ATG ACC CCA GAA CAG AGC AAT GTT TAT CGG GAT _600 Met Ala Ser Lys Asp Met Thr Pro Glu Gln Ser Asn Val Tyr Arg Asp Asn Ser Tyr Gln Gln Phe Asp Thr Asn Asn Val Arg Arg Val Asn Asn Arg Tyr Ala Glu Asp Tyr Glu Ile Pro Ser Ser Val Ser Ser Arg Lys GAC TAT GGT TGG GGA GAT TAT TAC CTC AGC ATG GTA TAT AAC GGA GAT 37q4 Asp Tyr Gly Trp Gly Asp Tyr Tyr Leu Ser Met Val Tyr Asn Gly Asp Ile ~ro Thr I~e Asn Tyr Lys Ala Ala Ser Ser Asp Leu Lys Ile Tyr Ile Ser Pro Lys Leu Arg Ile Ile His Asn Gly Tyr Glu Gly Gln Lys CGC rAT CAA TGC AAT CTG ATG AAT AAA TAT GGC AAA CTA GGT GAT AAA 3888 Arg Asn Gln Cys Asn Leu Met Asn Lys Tyr Gly Lys Leu Gly Asp Lys SUBSTITUTE SHEET tRULE 26) ,.. . .... ~ ~_.

CA 022638l9 l999-02-26 W O 98/08932 PCT~US97tO7657 Phe Ile Val Tyr Thr Ser Leu Gly Val Asn Pro Asn Asn Ser Ser Asn Lys Leu Met Phe Tyr Pro Val Tyr Gln Tyr Ser Gly Asn Thr Ser Gly 0 CTC AAT CAA GGG AGA CTA CTA TTC CAC CGT GAC ACC ACT TP.T CCA TCT 4032 Leu Asn Gln Gly Arg Leu Leu Phe Hls Arg Asp Thr Thr Tyr Pro Ser Lys Val Glu Ala Trp Ile Pro Gly Ala Lys Arg Ser Leu Thr Asn Gln Asn Ala Ala Ile Gly Asp Asp Tyr Ala Thr Asp Ser Leu Asn Lys Pro 1365 ~370 1375 Asp Asp Leu Lys Gln Tyr Ile Phe Met Thr Asp Ser Lys Gly Thr Ala ACT GAT GTC TCA GGC CCA GTA GAG ATT AAT ACT GCA ATT TC, CCA GCA 9224 Thr Asp Val Ser Gly Pro Val Glu Ile Asn Thr Ala Ile Ser Pro Ala Lys Val Gln Ile Ile Val Lys Ala Gly Gly Lys Glu Gln Thr Phe Thr Ala Asp Lys Asp Val Ser Ile Gln Pro Ser Pro Ser Phe Asp Glu Met Asn Tyr Gln Phe Asn Ala Leu Glu Ile Asp Gly Ser Gly Leu Asn Phe ATT AAC AAC TCA GCC AGT ATT GAT GTT ACT TTT ACC GCA TTm GCG GAG 4416 Ile Asn Asn Ser Ala Ser Ile Asp Val Thr Phe Thr Ala Phe Ala Glu Asp Gly Arg Lys Leu Gly Tyr Glu Ser Phe Ser Ile Pro Val Thr Leu Lys Val Ser Thr Asp Asn Ala Leu Thr Leu Hls His Asn G~ru ~i Gly Ala Gln Tyr Met Gln Trp Gln Ser Tyr Arg Thr Arg Leu Asn Thr Leu Phe Ala Arg Gln Leu Val Ala Arg Ala Thr Thr Gly Ile Asp Thr Ile Leu Ser Met Glu Thr Gln Asn Ile Gln Glu Pro Gln Leu Gly Lys Gly Phe Tyr Ala Thr Phe Val Ile Pro Pro Tyr Asn Leu Ser Thr His Gly Asp Glu Arg Trp Phe Lys Leu Tyr Ile Lys His Val Val Asp Asn Asn SUBSTlllJTE St~EET (RULE 26) W 098/08932 PCT~US97/07657 1570 1575 . 1580 Ser His Ile Ile Tvr Ser Gly Gln Leu Thr Asp Thr Asn Ile Asn Ile Thr Leu Phe Ile Pro Leu Asp Asp Val Pro Leu Asn Gln Asp Tyr His Ala Lys Val Tyr Met Thr Phe Lys Lys Ser Pro Ser Asp Gly Thr Trp Trp Gly Pro Hls Phe Val Arg Asp Asp Lys Gly Ile Val Thr Ile Asn Pro Lys Ser Ile Leu Thr His Phe Glu Ser Val Asn Val Leu Asn Asn Ile Ser Ser Glu Pro Met Asp Phe Ser Gly Ala Asn Ser Leu Tyr Phe Trp Glu Leu Phe Tyr Tyr Thr Pro Met Leu Val Ala Gln Arg Leu Leu His Glu Gln Asn Phe Asp Glu Ala Asn Arg Trp Leu Lys Tyr Val Trp Ser Pro Ser Gly Tyr Ile Val His Gly Gln Ile Gln Asn Tyr Gln Trp Asn Val Arg Pro Leu Leu Glu Asp Thr Ser Trp Asn Ser Asp Pro Leu Asp Ser Val Asp Pro Asp Ala Val Ala Gln His Asp Pro Met His Tyr Lys Val Ser Thr Phe Met Arg Thr Leu Asp Leu Leu Ile Ala Arg Gly Asp His Ala Tyr Arg Gln Leu Glu Arg Asp Thr Leu Asn Glu Ala Lys Met Trp Tyr Met Gln Ala Leu His Leu Leu Gly Asp Lys Pro Tyr Leu Pro Leu Ser Thr Thr Trp Ser Asp Pro Arg Leu Asp Arg Ala Ala Asp Ile Thr Thr Gln Asn Ala His Asp Ser Ala Ile Val Ala Leu Arg Gln Asn Ile Pro Thr Pro Ala Pro Leu Ser SUBSTlTUTE SHE~T (RULE 26) CA 022638l9 l999-02-26 WOg8/08932 PCT~S97/076S7 (2) INFORMATION FOR SEQ ID NO:49:
(i) SEQ~ENCE CHARACTERISTICS:
(A) LENGTH: 1849 amino acids (B) TYPE: amino acids (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xl) SEQUENCE DESCRIPTION: SEQ ID NO:49 (TcdAii):
Features From To De~crlption Peptide 1 1849 TcdAii peptide Fragment 1 12 TcdAii N-terminus (SEQ ID NO:13) Fragment 196 211 (SEQ ID NO:38) ~l5 Fragment 466 475 (SEQ ID NO:17) Fragment 993 1004 (SEQ ID NO:23; 12/13) Fragment 1297 1312 (SEQ ID NO:18) Fragment 1390 1409 (SEQ ID NO:39) Fragment 1532 1554 (SEQ ID NO:21; 19/23) Leu Ile Gly Tyr Asn Asn Gln Phe Ser Gly Arg Ala Ser Gln Tyr Val Ala Pr~ Gly Thr Val Ser Ser Met Phe Ser Pro Ala Ala Tyr Leu Thr Glu Le~ Tyr Arg Glu Ala Arg Asn Leu His Ala Ser Asp Ser Val Tyr Tyr Leu Asp Thr Arg Arg Pro Asp Leu Lys Ser Met Ala Leu Ser Gln Gln Asr Met Asp Ile Glu Leu Ser Thr Leu Ser Leu Ser Asn Glu Leu Leu Le' Glu Ser Ile Lys Thr Glu Ser Lys Leu Glu Asn Tyr Thr Lys Val Me~ Glu Met Leu Ser Thr Phe Arg Pro Ser Gly Ala Thr Pro Tyr His Asp Ala Tyr Glu Asn Val Arg Glu Val Ile Gln Leu Gln Asp Pro Gly Leu Glu Gln Leu Asn Ala Ser Pro Ala Ile Ala Gly Leu Met His Gln Ala Ser Leu Leu Gly Ile Asn Ala Ser Ile Ser Pro Glu Leu Phe Asn Ile Leu Thr Glu Glu Ile Thr Glu Gly Asn Ala Glu Glu Leu Tyr Lys Lys Asn Phe Gly Asn Ile Glu Pro Ala Ser Leu Ala Met Pro Glu Tyr Leu Lys Arg Tyr Tyr Asn Leu Ser Asp Glu Glu Leu Ser Gln Phe Ile Gly T yS Ala Ser Asn Phe Gly Gln Gln Glu Tyr Ser Asn Asn Gln Leu Il_ Thr Pro Val Val Asn Ser Ser Asp Gly Thr Val Lys Val Tyr Arg Il_ Thr Ara Glu Tyr Thr Thr Asn Ala Tyr Gln Met Asp Val Glu SUBSTITUTE SHEET tRULE 26) CA 022638l9 l999-02-26 Leu Phe Pro Phe Gly Gly Glu Asn Tyr Arg,Leu Asp Tyr Lys Phe Lys Asn Phe Tyr Asn Ala Ser Tyr Leu Ser Ile Lys Leu Asn Asp Lys Arg Glu Leu Val Arg Thr Glu Gly Ala Pro Gln Val Asn Ile Glu Tyr Ser 0 Ala Asn Ile Thr Leu Asn Thr Ala Asp Ile Ser Gln Pro Phe Glu Ile Gly Leu Thr Arg Val Leu Pro Ser Gly Ser Trp Ala Tyr Ala Ala Ala Lys Phe Thr Val Glu Glu Tyr Asn Gln Tyr Ser Phe Leu Leu Lys Leu Asn Lys Ala Ile Arg Leu Ser Arg Ala Thr Glu Leu Ser Pro Thr Ile Leu Glu Gly Ile Val Arg Ser Val Asn Leu Gln Leu Asp Ile Asn Thr Asp Val Leu Gly Lys Val Phe Leu Thr Lys Tyr Tyr Met Gln Arg Tyr 385 390 395 gO0 Ala Ile His Ala Glu Thr Ala Leu Ile Leu Cys Asn Ala Pro Ile Ser Gln Arg Ser Tyr Asp Asn Gln Pro Ser Gln Phe Asp Arg Leu Phe Asn Thr Pro Leu Leu Asn Gly Gln Tyr Phe Ser Thr Gly Asp Glu Glu Ile 435 4qO 445 Asp Leu Asn Ser Gly Ser Thr Gly Asp Trp Arg Lys Thr Ile Leu Lys Arg Ala Phe Asn Ile Asp Asp Val Ser Leu Phe Arg Leu Leu Lys Ile Thr Asp His Asp Asn Lys Asp Gly Lys Ile Lys Asn Asn Leu Lys Asn Leu Ser Asn Leu Tyr Ile Gly Lys Leu Leu Ala Asp Ile His Gln Leu Thr Ile Asp Glu Leu Asp Leu Leu Leu Ile Ala Val Gly Glu Gly Lys Thr Asn Leu Ser Ala Ile Ser Asp Lys Gln Leu Ala Thr Leu Ile Arg Lys Leu Asn Thr Ile Thr Ser Trp Leu His Thr Gln Lys Trp Ser Val Phe Gln Leu Phe Ile Met Thr Ser Thr Ser Tyr Asn Lys Thr Leu Thr Pro Glu Ile Lys Asn Leu Leu Asp Thr Val Tyr His Gly Leu Gln Gly Phe Asp Lys Asp Lys Ala Asp Leu Leu His Val Met Ala Pro Tyr Ile Ala Ala Thr Leu Gln Leu Ser Ser Glu Asn Val Ala His Ser Val Leu Leu Trp Ala Asp Lys Leu Gln Pro Gly Asp Gly Ala Met Thr Ala Glu SUBSTITIJTE SHEET (RULE 26) ... . ~., Lys Phe Trp Asp Trp Leu Asn Thr Lys Tyr Thr Pro Gly Ser Ser Glu Ala Val Glu Thr Gln Glu His Ile Val Gln Tyr Cys Gln Ala Leu Ala Gln Leu Glu Met Val Tyr His Ser Thr Gly Ile Asn Glu Asn Ala Phe Arg Leu Phe Val Thr Lys Pro Glu Met Phe Gly Ala Ala Thr Gly Ala Ala Pro Ala His Asp Ala Leu Ser Leu Ile Met Leu Thr Arg Phe Ala Asp T~ Val Asn Ala Leu Gly Glu Lys Ala Ser Ser Val Leu Ala Ala Phe Glu Ala Asn Ser Leu Thr Ala Glu Gln Leu Ala Asp Ala Met Asn 7gO 745 750 Leu Asp Ala Asn Leu Leu Leu Gln Ala Ser Ile Gln Ala Gln Asn His Gln ~.is Leu Pro Pro Val Thr Pro Glu Asn Ala Phe Ser Cys Trp Thr Ser I e Asn Thr Ile Leu Gln Trp Val Asn Val Ala Gln Gln Leu Asn Val Ala Pro Gln Gly Val Ser Ala Leu Val Gly Leu Asp Tyr Ile Gln Ser Met Lys Glu Thr Pro Thr Tyr Ala Gln Trp Glu Asn Ala Ala Gly Val Leu Thr Ala Gly Leu Asn Ser Gln Gln Ala Asn Thr Leu His Ala Phe Leu Asp Glu Ser Arg Ser Ala Ala Leu Ser Thr Tyr Tyr Ile Arg Gln Val Ala Lys Ala Ala Ala Ala Ile Lys Ser Arg Asp Asp Leu Ty-Gln Tyr Leu Leu Ile Asp Asn Gln Val Ser Ala Ala Ile Lys Thr Thr Arg Ile Ala Glu Ala Ile Ala Ser Ile Gln Leu Tyr Val Asn Arg Ala Leu Glu Asn Val Glu Glu Asn Ala Asn Ser Gly Val Ile Ser Arg Gln Phe Phe Ile Asp Trp Asp Lys Tyr Asn Lys Arg Tyr Ser Thr Trp Ala Gly Val Ser-Gln Leu Val Tyr Tyr Pro Glu Asn Tyr Ile Asp Pro Thr Met Arg Ile Gly Gln Thr Lys Met Met Asp Ala Leu Leu Gln Ser Val Ser Gln Ser Gln Leu Asn Ala Asp Thr Val Glu Asp Ala Phe Met Ser Tyr Leu Thr Ser Phe Glu Gln Val Ala Asn Leu Lys Val Ile Ser Ala Tyr H s Asp Asn Ile Asn Asn Asp Gln Gly Leu Thr Tyr Phe Ile Gly SU85TITUTE SH EET tRULE 2~) W O 98/08932 PCTrUS97/07657 Leu Ser Glu Thr Asp Ala Gly Glu Tyr Tyr Trp Arg Ser Val Asp His Ser Lys Phe Asn Asp Gly Lys Phe Ala Ala Asn Ala Trp Ser Glu Trp Hls Lys Ile Asp Cys Pro Ile Asn Pro Tyr Lys Ser Thr Ile Arg Pro Val Ile Tyr Lys Ser Arg Leu Tyr Leu Leu Trp Leu Glu Gln Lys Glu Ile Thr Lys Gln Thr Gly Asn Ser Lys Asp Gly Tyr Gln Thr Glu Thr Asp Tyr Arg Tyr Glu Leu Lys Leu Ala His Ile Arg Tyr Asp Gly Thr 20 Trp Asn Thr Pro Ile Thr Phe Asp Val Asn Lys Lys Ile Ser Glu Leu Lys Leu Glu Lys Asn Arg Ala Pro Gly Leu Tyr Cys Ala Gly Tyr Gln Gly Glu Asp Thr Leu Leu Val Met Phe Tyr Asn Gln Gln Asp Thr Leu Asp Ser Tyr Lys Asn Ala Ser Met Gln Gly Leu Tyr Ile Phe Ala Asp Met Ala Ser Lys Asp Met Thr Pro Glu Gln Ser Asn Val Tyr Arg Asp 3 5 Asn Ser Tyr Gln Gln ehe Asp Thr Asn Asn Val Arg Arg Val Asn Asn Arg Tyr Ala Glu Asp Tyr Glu Ile Pro Ser Ser Val Ser Ser Arg Lys Asp Tyr Gly Trp Gly Asp Tyr Tyr Leu Ser Met Val Tyr Asn Gly Asp 1~35 12~0 1245 Ile Pro Thr Ile Asn Tyr Lys Ala Ala Ser Ser Asp Leu Lys Ile Tyr Ile Ser Pro Lys Leu Arg Ile Ile His Asn Gly Tyr Glu Gly Gln Lys Arg Asn Gln Cys Asn Leu Met Asn Lys Tyr Gly Lys Leu Gly Asp Lys Phe Ile Val Tyr Thr Ser Leu Gly Val Asn Pro Asn Asn Ser Ser Asn Lys Leu Met Phe Tyr Pro Val Tyr Gln Tyr Ser Gly Asn Thr Ser Gly Leu Asn Gln Gly Arg Leu Leu Phe His Arg Asp Thr Thr Tyr Pro Ser Lys Val Glu Ala Trp Ile Pro Gly Ala Lys Arg Ser Leu Thr Asn Gln 65 Asn Ala Ala Ile Gly Asp Asp Tyr Ala Thr Asp Ser Leu Asn Lys Pro Asp Asp Leu Lys Gln Tyr Ile Phe Met Thr Asp Ser Lys Gly Thr Ala Thr Asp Val Ser Gly Pro Val Glu Ile Asn Thr Ala Ile Ser Pro Ala SUBSTITUTE SHEET tRULE 26) .. ......... . . . ...... .... ..

CA 022638l9 l999-02-26 W 098/08932 PCTrUS97~7657 1395 1400 . 1905 Lys Val Gln Ile Ile Val Lys Ala Gly Gly Lys Glu Gln Thr Phe Thr Ala Asp Lys Asp Val Ser Ile Gln Pro Ser Pro Ser Phe Asp Glu Met Asn Tyr Gln Phe Asn Ala Leu Glu Ile Asp Gly Ser Gly Leu Asn Phe Ile Asn Asn Ser Ala Ser Ile Asp Val Thr Phe Thr Ala Phe Ala Glu Asp Gly Arg Lys Leu Gly Tyr Glu Ser ehe Ser Ile Pro Val Thr Leu 1475 1480 14~5 Lys Val Ser Thr Asp Asn Ala Leu Thr Leu His His Asn Glu Asn Gly Ala Gln Tyr Met Gln Trp Gln Ser Tyr Arg Thr Arg Leu Asn Thr Leu Phe Ala Arg Gln Leu Val Ala Arg Ala Thr Thr Gly Ile Asp Thr Ile Leu Ser Met Glu Thr Gln Asn Ile Gln Glu Pro Gln Leu Gly Lys Gly Phe Tyr Ala Thr Phe Val Ile Pro Pro Tyr Asn Leu Ser Thr His Gly Asp Glu Arg Trp Phe Lys Leu Tyr Ile Lys His Val Val Asp Asn Asn Ser His Ile Ile Tyr Ser Gly Gln Leu Thr Asp Thr Asn Ile Asn Ile Thr Leu Phe Ile Pro Leu Asp Asp Val Pro Leu Asn Gln Asp Tyr His Ala Lys Val Tyr Met Thr Phe Lys Lys Ser Pro Ser Asp Gly Thr Trp Trp Gly Pro His Phe Val Arg Asp Asp Lys Gly Ile Val Thr Ile Asn Pro Lys Ser Ile Leu Thr His Phe Glu Ser Val Asn Val Leu Asn Asn Ile Ser Ser Glu Pro Met Asp Phe Ser Gly Ala Asn Ser Leu Tyr Phe Trp Glu Leu Phe Tyr Tyr Thr Pro Met Leu Val Ala Gln Arg Leu Leu His Glu Gln Asn Phe Asp Glu Ala Asn Arg Trp Leu Lys Tyr Val Trp Ser Pro Ser Gly Tyr Ile Val His Gly Gln Ile Gln Asn Tyr Gln Trp Asn Val Arg Pro Leu Leu Glu Asp Thr Ser Trp Asn Ser Asp Pro Leu Asp Ser Val Asp Pro Asp Ala Val Ala Gln His Asp Pro Met His Tyr Lys Val Ser Thr Phe Met Arq Thr Leu As~ Leu Leu Ile Ala Arg Glv 1765 17,0 1775 SUBS~ITUTE S~tEET tRULE 26) CA 022638l9 1999-02-26 WO 98/08g32 PCT/US97/07657 Asp His Ala Tvr Arg Gln Leu Glu Arg Asp Thr Leu Asn Glu Ala Lys Met Trp Tyr Met Gln Ala Leu His Leu Leu Gly Asp Lys Pro Tyr Leu Pro Leu Ser Thr Thr Trp Ser Asp Pro Arg Leu Asp Arg Ala Ala Asp 0 Ile Thr Thr Gln Asn Ala His Asp Ser Ala Ile Val Ala Leu Arg Gln Asn Ile 2ro Thr Pro Ala Pro Leu Ser (2) INFORMATION FOR SEQ ID NO:50:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1740 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomlc) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:~0 (tCdAiil coding region):

Leu Arg Ser Ala Asn Thr Leu Thr Asp Leu Phe Leu Pro Gln Ile Asn Glu Val Met Met Asn Tyr Trp Gln Thr Leu Ala Gln Arg Val Tyr Asn Leu Arg His Asn Leu Ser Ile Asp Gly Gln Pro Leu Tyr Leu Pro Ile gO 45 Tyr Ala Thr Pro Ala Asp Pro Lys Ala Leu Leu Ser Ala Ala Val Ala Thr Ser Gln Gly Gly Gly Lys Leu Pro Glu Ser Phe Met Ser Leu Trp Arg Phe Pro His Met Leu Glu Asn Ala Arg Gly Met Val Ser Gln Leu Thr Gln Phe Gly Ser Thr Leu Gln Asn Ile Ile Glu Arg Gln Asp Ala Glu Ala Leu Asn Ala Leu Leu Gln Asn Gln Ala Ala Glu Leu Ile Leu Thr Asn Leu Ser Ile Gln Asp Lys Thr Ile Glu Glu Leu Asp Ala Glu Lys Thr Val Leu Glu Lys Ser Lys Ala Gly Ala Gln Ser Arg Phe Asp SUBSTlTl.JTE S}tE~T (RULE 26) W O 98/08932 PCTrUS97/07657 Ser Tyr Gly Lys Leu Tyr Asp Glu Asn Ile ~sn Ala Gly Glu Asn Gln Ala Met Thr Leu Arg Ala Ser Ala Ala Gly Leu Thr Thr Ala Val Gln Ala Ser Arg Leu Ala Gly Ala Ala Ala Asp Leu Val Pro Asn Ile Phe Gly Phe Ala Gly Gly Gly Ser Arg Trp Gly Ala Ile Ala Glu Ala Thr Gly Tyr Val Met Glu Phe Ser Ala Asn Val Met Asn Thr Glu Ala Asp Lys Ile Ser Gln Ser Glu Thr Tyr Arg Arg Arg Arg Gln Glu Trp Glu Ile Gln Arg Asn Asn Ala Glu Ala Glu Leu Lys Gln Ile Asp Ala Gln Leu Lys Ser Leu Ala Val Arg Arg Glu Ala Ala Val Leu Gln Lys Thr Ser Leu Lys Thr Gln Gln Glu Gln Thr Gln Ser Gln Le~ Ala Phe Leu Gln Arg Lys Phe Ser Asn Gln Ala Leu Tyr Asn Trp Leu Arg Gly Arg Leu Ala Ala Ile Tyr Phe Gln Phe Tyr Asp Leu Ala Val Ala Arg Cys Leu Met Ala Glu Gln Ala Tyr Arg Trp Glu Leu Asn Asp Asp Ser Ala Arg Phe Ile Lys Pro Gly Ala Trp Gln Gly Thr Tyr Ala Gly Leu Leu Ala Gly Glu Thr Leu Met Leu Ser Leu Ala Gln Met Glu Asp Ala His Leu Lys Arg Asp Lys Arg Ala Leu Glu Val Glu Arg Thr Val Ser Leu Ala Glu Val Tyr Ala Gly Leu Pro Lys Asp Asn Gly Pro Phe Ser Leu Ala Gln Glu Ile Asp Lys Leu Val Ser Gln Gly Ser Gly Ser Ala Gly Ser Gly Asn Asn Asn Leu Ala Phe Gly Ala Gly Thr Asp Thr Lys Thr SUBSTITUTE SHEET tRUL~ 26) CA 022638l9 l999-02-26 W O 98/08932 PCT~US97tO7657 TCT T.G CAG GCA TCA GTT TCA TTC GCT GAT T.TG AAA ATT CGT GAA GAT 1392 Ser Leu Gln Ala Ser Val Ser ehe Ala Asp Leu Lys Ile Arg Glu Asp TAC CGG GCA TCG CTT GGC AAA ATT CGA CGT ATC AAA CAG ATC AGC GTC 1~90 Tyr Pro Ala Ser Leu Gly Lys Ile Arg Arg Ile Lys Gln Ile Ser Val 465 g70 475 480 Thr Leu Pro Ala Leu Leu Gly Pro Tyr Gln Asp Val Gln Ala Ile Leu Ser Tyr Gly Asp Lys Ala Gly Leu Ala Asn Gly Cys Glu Ala Leu Ala Val Ser His Gly Met Asn Asp Ser Gly Gln Phe Gln Leu Asp Phe Asn Asp Gly Lys Phe Leu Pro Phe Glu Gly Ile Ala Ile Asp Gln Gly Thr Leu Tn- Leu Ser Phe Pro Asn Ala Ser Met Pro Glu Lys Gly Lys Gln Ala Thr Met Leu Lys Thr Leu Asn Asp Ile Ile Leu His Ile Arg Tyr ACC AT~ AAA TAA 1740 Thr Ile Lys ~--(2) INFORMATION FOR SEQ ID NO:51:
(i) SEQUENCE CHARACTERISTICS:
(A) ~ENGTH: 579 amino acids (B) TYPE: amino acids (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULF TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:51 (TcdA~
Leu Ar~ Ser Ala Asn Thr Leu Thr Asp Leu Phe Leu Pro Gl~ sn Glu Val Met Met Asn Tyr Trp Gln Thr Leu Ala Gln Arg Val Tyr Asn Leu Arg Hls Asn Leu Ser Ile Asp Gly Gln Pro Leu Tyr Leu Pro lle Tyr A'a Thr Pro Ala Asp Pro Lys Ala Leu Leu Ser Ala Ala Val Ala ~ 50 55 60 Thr Ser Gln Gly Gly Gly Lys Leu Pro Glu Ser Phe Met Ser Leu Trp Arg Phe Pro His Met Leu Glu Asn Ala Arg Gly Met Val Ser Gln Leu Thr G-_n Phe Gly Ser Thr Leu Gln Asn Ile Ile Glu Arg Gln Asp Ala SUBST~TI.JTE SHE T ~RULE 26) CA 022638l9 l999-02-26 W 098/08932 PCTrUS97/07657 Glu Ala Leu Asn Ala Leu Leu Gln Asn Gln-Ala Ala Glu Leu Ile Leu Thr Asn Leu Ser Ile Gln Asp Lys Thr Ile Glu Glu Leu ASD Ala Glu Lys Thr Val Leu Glu Lys Ser Lys Ala Gly Ala Gln Ser Arg Phe As~
145 lS0 155 160 0 Ser Tyr Gly Lys Leu Tyr Asp Glu Asn Ile Asn Ala Gly Glu Asn Gln Ala Met Thr Leu Arg Ala Ser Ala Ala Gly Leu Thr Thr Ala Val Gln Ala Ser Arg Leu Ala Gly Ala Ala Ala Asp Leu Val Pro Asn Ile Phe Gly Phe Ala Gly Gly Gly Ser Arg Trp Gly Ala Ile Ala Glu Ala Thr Gly Tyr Val Met Glu Phe Ser Ala Asn Val Met Asn Thr Glu Ala Asp Lys Ile Ser Gln Ser Glu Thr Tyr Arg Arg Arg Arg Gln Glu Trp Gl~

Ile Gln Arg Asn Asn Ala Glu Ala Glu Leu Lys Gln Ile Asp Ala Glr.

Leu Lys Ser Leu Ala Val Arg Arg Glu Ala Ala Val Leu Gln Lys Thr Ser Leu Lys Thr Gln Gln Glu Gln Thr Gln Ser Gln Leu Ala Phe Leu Gln Arg Lys Phe Ser Asn Gln Ala Leu Tyr Asn Trp Leu Arg Gly Arg Leu Ala Ala Ile Tyr Phe Gln Phe Tyr Asp Leu Ala Val Ala Arg Cys Leu ~et Ala Glu Gln Ala Tyr Arg Trp Glu Leu Asn Asp Asp Ser Ala dS
Arg Phe Ile Lys Pro Gly Ala Trp Gln Gly Thr Tyr Ala Gly Leu Leu Ala Gly Glu Thr Leu Met Leu Ser Leu Ala Gln Met Glu Asp Ala Hls Leu Lys Arg Asp Lys Arg Ala Leu Glu Val Glu Arg Thr Val Ser Leu Ala Glu Val Tyr Ala Gly Leu Pro Lys Asp Asn Gly Pro Phe Ser Leu Ala Gln Glu Ile Asp Lys Leu Val Ser Gln Gly Ser Gly Ser Ala Gly Ser Gly Asn Asn Asn Leu Ala Phe Gly Ala Gly Thr Asp Thr Lys Thr Ser Leu Gln Ala Ser Val Ser Phe Ala Asp Leu Lys Ile Arg Glu Asp Tyr Pro Ala Ser Leu Gly Lys Ile Arg Arg Ile Lys Gln Ile Ser Val 0 Thr Leu Pro Ala Leu Leu Gly Pro Tyr Gln Asp Val Gln Ala Ile Leu SUBSTITUTE S~IEET ~RU~ E 26) CA 022638l9 l999-02-26 W O 98/08932 PCT~US97/07657 Ser Tyr Gly Asp Lys Ala Gly Leu Ala Asn Gly Cys Glu Ala Leu Ala Val Ser His Gly Met Asn Asp Ser Gly Gln Phe Gln Leu Asp Phe Asn Asp Gly Lys Phe Leu Pro Phe Glu Gly Ile Ala Ile Asp Gln Gly Thr Leu Thr Leu Ser ?he Pro Asn Ala Ser Met Pro Glu Lys Gly Lys Gln Ala Thr Met Leu Lys Thr Leu Asn Asp Ile Ile Leu His Ile Arg Tyr Thr Ile Lys ~--(2) INFORMATION FOR SEQ ID NO:52:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 5532 base palrs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: llnear (1l) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:52 (tcbAii coding region):

Phe Ile Gln Gly Tyr Ser Asp Leu Phe Gly Asn Arg Ala Asp Asn Tyr Ala Ala Pro Gly Ser Val Ala Ser Met Phe Ser Pro Ala Ala Tyr Leu Thr Glu Leu Tyr Arg Glu Ala Lys Asn Leu His Asp Ser Ser Ser Ile Tyr Tyr Leu Asp Lys Arg Arg Pro Asp Leu Ala Ser Leu Met Leu Ser Gln Lys Asn Met Asp Glu Glu Ile Ser Thr Leu Ala Leu Ser Asn Glu Leu Cys Leu Ala Gly Ile Glu Thr Lys Thr Gly Lys Ser Gln Asp Gl~

Val Met Asp Met Leu Ser Thr Tyr Arg Leu Ser Gly Glu Thr Pro Ty-~ 60CAT CAC GCT TAT GAA ACT GTT CGT GAA ATC GTT CAT GAA CGT GAT CCA 384 His His Ala Tyr Glu Thr Val Arg Gl~ Ile Val His Glu Ar~ Asp Pro GGA TT~ CGT CAT TTG TCA CAG GCA CCC ATT GTT GCT GCT AAG CTC GAT 932 Gly Phe Arg His Leu Ser Gln Ala Pro Ile Val Ala Ala Lys Leu Asp 130 135 l40 SU85TITUTE SHE~T (RULE 26) CA 022638l9 l999-02-26 WO 98t08932 PCTrUS97/07657 Pro Val Thr Leu Leu Gly Ile Ser Ser His Ile Ser Pro Glu Leu Tyr AAC TTv CTG ATT GAG GAG ATC CCG GAA AAA GAT GAA GCC GCG CTT GAT 528 Asn Leu Leu Ile Glu Glu Ile Pro Glu Lys Asp Glu Ala Ala Leu Asp ACG rTT TAT AAA ACA AAC TTT GGC GAT ATT ACT ACT GCT CAG TTA ATG -76 Thr Leu Tyr Lys Thr Asn Phe Gly Asp Ile Thr Thr Ala Gln Leu Met Ser P~o Ser Tyr Leu Ala Arg Tyr Tyr Gly Val Ser Pro Glu Asp Ile Ala Tyr Val Thr Thr Ser Leu Ser His Val Gly Tyr Ser Ser Asp Ile Leu Val Ile Pro Leu Val Asp Gly Val Gly Lys Met Glu Val Val Arg GTT ArC CGA ACA CCA TCG GAT AAT TAT ACC AGT CAG ACG AAT TAT ATT 768 Val Thr Arg Thr Pro Ser Asp Asn Tyr Thr Ser Gln Thr Asn Tyr Ile Glu Leu Tyr Pro Gln Gly Gly Asp Asn Tyr Leu Ile Lys Tyr Asn Leu Ser Asn Ser Phe Gly Leu Asp Asp Phe Tyr Leu Gln Tyr Lys Asp Gly Ser Ala Asp Trp Thr Glu Ile Ala His Asn Pro Tyr Pro Asp Met Val Ile Asn Gln Lys Tyr Glu Ser Gln Ala Thr Ile Lys Arg Ser Asp Ser GAC ~-.T ATA CTC AGT ATA GGG TTA CAA AGA TGG CAT AGC GGT AGT TAT 1008 Asp Asn Ile Leu Ser Ile Gly Leu Gln Arg Trp His Ser Gly Ser Tyr Asn ~ A'a Ala Ala Asn Phe Lys Ile Asp Gln Tyr Ser Pro Lys Ala TTC CTG CTT AAA ATG AAT AAG GCT ATT CGG TTG CTC AAA GCT ACC GGC llOq Phe Leu Leu Lys Met Asn Lys Ala Ile Arg Leu Leu Lys Ala Thr Gly CTC ~CT TTT GCT ACG TTG GAG CGT ATT GTT GAT AGT GTT AAT AGC ACC 1152 Leu Ser Phe Ala Thr Leu Glu Arg Ile Val Asp Ser Val Asn Ser Thr AAA TrC ATC ACG GTT GAG GTA TTA AAC AAG GTT TAT CGG GTA AAA TTC '200 Lys Ser Ile Thr Val Glu Val Leu Asn Lys Val Tyr Arg Val Lys Phe TAT ATT GAT CGT TAT GGC ATC AGT GAA GAG ACA GCC GCT ATT TTG GCT 12g8 Tyr I'e Asp Arg Tyr Gly Ile Ser Glu Glu Thr Ala Ala Ile Leu Ala AAT ATT AAT ATC TCT CAG CAA GCT GTT GGC AAT CAG CTT AGC CAG TTT '296 Asn _ e Asn Ile Ser Gln Gln Ala Val Gly Asn Gln Leu Ser Gln Phe SUBSTlllJTE S~EET (RULE 26) CA 022638l9 l999-02-26 W O 98l08932 PCTAUS97tO7657 Glu Gln Leu Phe Asn His Pro Pro Leu Asn Gly Ile Arg Tyr Glu Ile Ser Glu Asp Asn Ser Lys His Leu Pro Asn Pro Asp Leu Asn Leu Lys CCA GAC AGT ACC GGT GAT GAT CAA CGC AAG GCG GTT TTA AAA CGC GCG lq40 Pro Asp Ser Thr Gly Asp Asp Gln Arg Lys Ala Val Leu Lys Arg Ala Phe Gln Val Asn Ala Ser Glu Leu Tyr Gln Met Leu Leu Ile Thr Asp Arg Lys Glu Asp Gly Val Ile Lys Asn Asn Leu Glu Asn Leu Ser Asp Leu Tyr Leu Val Ser Leu Leu Ala Gln Ile llis Asn Leu Thr Ile Ala Glu Leu Asn Ile Leu Leu Val Ile Cys Gly Tyr Gly Asp Thr Asn Ile 530 535 5qO

Tyr Gln Ile Thr Asp Asp Asn Leu Ala Lys Ile Val Glu Thr Leu Leu Trp Ile Thr Gln Trp Leu Lys Thr Gln Lys Trp Thr Val Thr Asp Leu Phe Leu Met Thr Thr Ala Thr Tyr Ser Thr Thr Leu Thr Pro Glu Ile Ser Asn Leu Thr Ala Thr Leu Ser Ser Thr Leu His Gly Lys Glu Ser Leu Ile Gly Glu Asp Leu Lys Arg Ala Met Ala Pro Cys Phe Thr Ser Ala Leu His Leu Thr Ser Gln Glu Val Ala Tyr Asp Leu Leu Leu Trp Ile Asp Gln Ile Gln Pro Ala Gln Ile Thr Val Asp Gly Phe Trp Glu Glu Val Gln-Thr Thr Pro Thr Ser Leu Lys Val Ile Thr Phe Ala Gln Val Leu Ala Gln Leu Ser Leu Ile l~yr Arg Arg Ile Gly Leu Ser Glu Thr Glu Leu Ser Leu Ile Val Thr Gln Ser Ser Leu Leu Val Ala Gly Lys Ser Ile Leu Asp His Gly Leu Leu Thr Leu Met Ala Leu Glu Gly SUBSTITUTE S}~EET (RULE 26) .. .. .. ....

W 098/08932 PCTrUS97/07657 Phe His Thr Trp Val Asn Gly Leu Gly Gln Hls Ala Ser Leu Ile Leu Ala Ala Leu Lys Asp Gly Ala Leu Thr Val Thr Asp Val Ala Gln Ala Met Asn Lys Glu Glu Ser Leu Leu Gln Met Ala Ala Asn Gln Val Glu Lys Asp Leu Thr Lys Leu Thr Ser Trp Thr Gln Ile Asp Ala Ile Leu Gln Trp Leu Gln Met Ser Ser Ala Leu Ala Val Ser Pro Leu Asp Leu Ala Gly Met Met Ala Leu Lys Tyr Gly Ile Asp His Asn Tyr Ala Ala Trp Gln Ala Ala Ala Ala Ala Leu Met Ala Asp His Ala Asn Gln Ala CAG A~A AAA CTG GAT GAG ACG TTC AGT AAG GCA TTA TGT AAC TAT TAT 25q4 Gln Lys Lys Leu Asp Glu Thr Phe Ser Lys Ala Leu Cys Asn Tyr Tyr Ile Asn Ala Val Val Asp Ser Ala Ala Gly Val Arg Asp Arg Asn Gly Leu Tyr Thr Tyr Leu Leu Ile Asp Asn Gln Val Ser Ala Asp Val Ile Thr Ser Arg Ile Ala Glu Ala Ile Ala Gly Ile Gln Leu Tyr Val Asn Arg Ala Leu Asn Arg Asp Glu Gly Gln Leu Ala Ser Asp Val Ser Thr Arg Gln Phe Phe Thr Asp Trp Glu Arg Tyr Asn Lys Arg Tyr Ser Thr Trp Ala Gly Val Ser Glu Leu Val Tyr Tyr Pro Glu Asn Tyr Val Asp Pro Thr Gln Arg Ile Gly Gln Thr Lys Met Met Asp Ala Leu Leu Gln Ser Ile Asn Gln Ser Gln Leu Asn Ala Asp Thr Val Glu Asp Ala Phe Lys Thr Tyr Leu Thr Ser Phe Glu Gln Val Ala Asn Leu Lys Val Ile Ser Ala Tyr ~ls Asp Asn Val Asn Val Asp Gln Gly Leu Thr Tyr Phe SUBSTrrUTE SH EET (RULE 26) CA 022638l9 l999-02-26 Ile Gly Ile Asp Gln Ala Ala Pro Gly Thr Tyr Tyr Trp Arg Ser Val Asp His Ser Lys Cys Glu Asn Gly Lys Phe Ala Ala Asn Ala Trp Gly 1025 1030 1035 lOqO

Glu Trp Asn Lys Ile Thr Cys Ala Val Asn Pro Trp Lys Asn Ile Ile Arg Pro Val Val Tyr Met Ser Arg Leu Tyr Leu Leu Trp Leu Glu Gln CAA TCA AAG AAA AGT GAT GAT GGT AAA ACC ACG ATT TAT CAA TAT AAC 326g Gln Ser Lys Lys Ser Asp Asp Gly Lys Thr Thr Ile Tyr Gln Tyr Asn Leu Lys Leu Ala His Ile Arg Tyr Asp Gly Ser Trp Asn Thr Pro Phe Thr Phe Asp Val Thr Glu Lys Val Lys Asn Tyr Thr Ser Ser Thr Asp Ala Ala Glu Ser Leu Gly Leu Tyr Cys Thr Gly Tyr Gln Gly Glu Asp Thr Leu Leu Val Met Phe Tyr Ser Met Gln Ser Ser Tyr Ser Ser Tyr Thr Asp Asn Asn Ala Pro Val Thr Gly Leu Tyr Ile Phe Ala Asp Met Ser Ser Asp Asn Met Thr Asn Ala Gln Ala Thr Asn Tyr Tr~ Asn Asn Ser Tyr Pro Gln Phe Asp Thr Val Met Ala Asp Pro Asp Ser Asp Asn Lys Lys Val Ile Thr Arg Arg Val Asn Asn Arg Tyr Ala Glu Asp Tyr Glu Ile Pro Ser Ser Val Thr Ser Asn Ser Asn Tyr Ser Trp Gly Asp Hls Ser Leu Thr Met Leu Tyr Gly Gly Ser Val Pro Asn Ile Thr Phe Glu Ser Ala Ala Glu Asp Leu Arg Leu Ser Thr Asn Met Ala Leu Ser Ile Ile Hls Asn Gly Tyr Ala Gly Thr Arg Arg Ile Gln Cys Asn Leu SUBST~TUTE S~EE~ (RULE 26) Met Lys Gln Tyr Ala Ser Leu Gly Asp Lys Phe Ile Ile Tyr Asp Ser Ser Phe Asp Asp Ala Asn Arg Phe Asn Leu Val Pro Leu Phe Lys Phe GGA AAA GAC GAG AAC TCA GAT GAT AGT ATT TGT ATA TAT AAT GAA AAC 3~84 Gly Lys Asp Glu Asn Ser Asp Asp Ser Ile Cys Ile Tyr Asn Glu Asn Pro Ser Ser Glu Asp Lys Lys Trp Tyr Phe Ser Ser Lys Asp Asp Asn Lys Thr Ala Asp Tyr Asn Gly Gly Thr Gln Cys Ile Asp Ala Gly Thr Ser Asn Lys Asp Phe Tyr Tyr Asn Leu Gln Glu Ile Glu Val Ile Ser Val Thr Gly Gly Tyr Trp Ser Ser Tyr Lys Ile Ser Asn Pro Ile Asn 1380 1385 13~0 Ile Asn Thr Gly Ile Asp Ser Ala Lys Val Lys Val Thr Val Lys Ala Gly Gly Asp Asp Gln Ile Phe Thr Ala Asp Asn Ser Thr Tyr Val Pro Gln Gln Pro Ala Pro Ser Phe Glu Glu Met Ile Tyr Gln Phe Asn Asn Leu Thr Ile Asp Cys Lys Asn Leu Asn Phe Ile Asp Asn Gln Ala His Ile Glu Ile Asp Phe Thr Ala Thr Ala Gln Asp Gly Arg Phe Leu Gly Ala Glu Thr Phe Ile Ile Pro Val Thr Lys Lys Val Leu Gly Thr Glu Asn Val Ile Ala Leu Tyr Ser Glu Asn Asn Gly Val Gln Tyr Met Gln Ile Gly Ala Tyr Arg Thr Arg Leu Asn Thr Leu Phe Ala Gln Gln Leu Val Ser Arg Ala Asn Arg Gly Ile Asp Ala Val Leu Ser Met Glu Thr Gln Asn Ile Gln Glu Pro Gln Leu Gly Ala Gly Thr Tyr Val Gln Leu Val Leu Asp Lys Tyr Asp Glu Ser Ile ~ls Gly Thr Asn Lys Ser Phe SUBST~TUTE SHEET tRULE 26) CA 022638l9 l999-02-26 WO 98/08932 PCT~USg7/07657 Ala Ile Glu Tyr Val Asp Ile Phe Lys Glu Asn Asp Ser Phe Val Ile Tyr Gln Gly Glu Leu Ser Glu Thr Ser Gln Thr Val Val Lys Val Phe 0 Leu Ser Tyr Phe Ile Glu Ala Thr Gly Asn Lys Asn His Leu Trp Val Arg Ala Lys Tyr Gln Lys Glu Thr Thr Asp Lys Ile Leu Phe Asp Arg Thr Asp Glu Lys Asp Pro His Gly Trp Phe Leu Ser Asp Asp His Lys Thr Phe Ser Gly Leu Ser Ser Ala Gln Ala Leu Lys Asn Asp Ser Glu Pro Met Asp Phe Ser Gly Ala Asn Ala Leu Tyr Phe Trp Glu Leu Phe Tyr Tyr Thr Pro Met Met Met Ala His Arg Leu Leu Gln Glu Gln Asn Phe Asp Ala Ala Asn His Trp Phe Arg Tyr Val Trp Ser Pro Ser Gly Tyr Ile Val Asp Gly Lys Ile Ala Ile Tyr His Trp Asn Val Arg Pro Leu Glu Glu Asp Thr Ser Trp Asn Ala Gln Gln Leu Asp Ser Thr Asp Pro Asp Ala Val Ala Gln Asp Asp Pro Met His Tyr Lys Val Ala Thr Phe Met Ala Thr Leu Asp Leu Leu Met Ala Arg Gly Asp Ala Ala Tyr Arg Gln Leu Glu Arg Asp Thr Leu Ala Glu Ala Lys Met Trp Tyr Thr Gln Ala Leu Asn Leu Leu Gly Asp Glu Pro Gln Val Met Leu Ser Thr Thr Trp Ala Asn Pro Thr Leu Gly Asn Ala Ala Ser Lys Thr Thr Gln Gln Val Arg Gln Gln Val Leu Thr Gln Leu Arg Leu Asn Ser Arg Val Lys Thr Pro Leu SUBSTITUTE SHEET tRULE 26) .... ... ~.~

CA 022638l9 l999-02-26 W098l08932 PCT~US97tO7657 (2) INFORMATION FOR SEQ ID NO:53:
(i) SEQUENCE CH~RACTERISTICS:
(A) LENGTH: 1844 amlno acids (B) TYPE: amlno acids (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:53 (TcbAll):
Features From To Description Peptide 1 1844 TcbAil peptide Fragment 1 11 (SEQ ID NO:1) Fragment 978 990 (SEQ ID NO:23) Fragment 1387 1401 (SEQ ID No:22) Fragment 1484 1505 (SEQ ID NO:24) Fragment 1527 1552 (SEQ ID NO:21) Phe Ile Gln Gly Tyr Ser Asp Leu Phe Gly Asn Arg Ala Asp Asn Tyr Ala Ala Pro Gly Ser Val Ala Ser Met Phe Ser Pro Ala Ala Tyr Leu Thr Glu Leu Tyr Arg Glu Ala Lys Asn Leu His Asp Ser Ser Ser Ile 3 0 Tyr Tyr Leu Asp Lys Arg Arg Pro Asp Leu Ala Ser Leu Met Leu Ser Gln Lys Asn Met Asp Glu Glu Ile Ser Thr Leu Ala Leu Ser Asn Glu Leu Cys Leu Ala Gly Ile Glu Thr Lys Thr Gly Lys Ser Gln Asp Glu Val Met Asp Met Leu Ser Thr Tyr Arg Leu Ser Gly Glu Thr Pro Tyr 9 0 100 105 llO
His His Ala Tyr Glu Thr Val Arg Glu Ile Val His Glu Arg Asp Pro 4 5 Gly Phe Arg His Leu Ser Gln Ala Pro Ile Val Ala Ala Lys Leu Asp Pro Val Thr Leu Leu Gly Ile Ser Ser His Ile Ser Pro Glu Leu Tyr Asn Leu Leu Ile Glu Glu Ile Pro Glu Lys Asp Glu Ala Ala Leu Asp Thr Leu Tyr Lys Thr Asn Phe Gly Asp Ile Thr Thr Ala Gln Leu Met Ser Pro Ser Tyr Leu Ala Arg Tyr Tyr Gly Val Ser Pro Glu Asp Ile 60 Ala Tyr Val Thr Thr Ser Leu Ser His Val Gly Tyr Ser Ser Asp Ile Leu Val Ile Pro Leu Val Asp Gly Val Gly Lys Met Glu Val Val Arg Val Thr Arg Thr Pro Ser Asp Asn Tyr Thr Ser Gln Thr Asn Tyr Ile SUBSTlTUTE SHEE~ (RULE 2~) CA 022638l9 l999-02-26 W 09~08932 PCT~US97tO76S7 Glu Leu Tyr Pro Gln Gly Gly Asp Asn Tyr.Leu Ile Lys Tyr Asn Leu Ser Asn Ser Phe Gly Leu Asp Asp Phe Tyr Leu Gln Tyr Lys Asp Gly Ser Ala Asp Trp Thr Glu Ile Ala His Asn Pro Tyr Pro Asp Met Val 0 Ile Asn Gln Lys Tyr Glu Ser Gln Ala Thr Ile Lys Arg Ser Asp Ser Asp Asn Ile Leu Ser Ile Gly Leu Gln Arg Trp His Ser Gly Ser Tyr Asn Phe Ala Ala Ala Asn Phe Lys Ile Asp Gln Tyr Ser Pro Lys Ala Phe Leu Leu Lys Met Asn Lys Ala Ile Arg Leu Leu Lys Ala Thr Gly Leu Ser Phe Ala Thr Leu Glu Arg Ile Val Asp Ser Val Asn Ser Thr Lys Ser Ile Thr Val Glu Val Leu Asn Lys Val Tyr Arg Val Lys Phe Tyr Ile Asp Arg Tyr Gly Ile Ser Glu Glu Thr Ala Aia Ile Leu Ala Asn Ile Asn Ile Ser Gln Gln Ala Val Gly Asn Gln Leu Ser Gln Phe Glu Gln Leu Phe Asn His Pro Pro Leu Asn Gly Ile Arg Tyr Glu Ile Ser Glu Asp Asn Ser Lys His Leu Pro Asn Pro Asp Leu Asn Leu Lys Pro Asp Ser Thr Gly Asp Asp Gln Arg Lys Ala Val Leu Lys Arg Ala Phe Gln Val Asn Ala Ser Glu Leu Tyr Gln Met Leu Leu Ile Thr Asp Arg Lys Glu Asp Gly Val Ile Lys Asn Asn Leu Glu Asn Leu Ser Asp Leu Tyr Leu Val Ser Leu Leu Ala Gln Ile His Asn Leu Thr Ile Ala Glu Leu Asn Ile Leu Leu Val Ile Cys Gly Tyr Gly Asp Thr Asn Ile Tyr Gln Ile Thr Asp Asp Asn Leu Ala Lys Ile Val Glu Thr Leu Leu Trp Ile Thr Gln Trp Leu Lys Thr Gln Lys Trp Thr Val Thr Asp Leu Phe Leu Met Thr Thr Ala Thr Tyr Ser Thr Thr Leu Thr Pro Glu Ile Ser Asn Leu Thr Ala Thr Leu Ser Ser Thr Leu His Gly Lys Glu Ser Leu Ile Gly Glu Asp Leu Lys Arg Ala Met Ala Pro Cys Phe Thr Ser Ala Leu His Leu Thr Ser Gln Glu Val Ala Tyr Asp Leu Leu Leu Trp SUBSTITUTE St~EFT ~RULE 26) . . .. . . . .

W 098/08932 rcTrusg7/o7657 Ile Asp Gln Ile Gln Pro Ala Gln Ile Thr Val Asp ~--y Phe Trp Glu Glu Val Gln Thr Thr Pro Thr Ser Leu Lys Val Ile Thr Phe Ala Gln Val Leu Ala Gln Leu Ser Leu Ile Tyr Arg Arg Ile Gly Leu Ser Glu Thr Glu Leu Ser Leu Ile Val Thr Gln Ser Ser Leu Leu Val Ala Gly Lys Ser Ile Leu Asp His Gly Leu Leu Thr Leu Met Ala Leu Glu Gly Phe His Thr TrD Val Asn Gly Leu Gly Gln His Ala Ser Leu Ile Leu Ala Ala Leu Lys Asp Gly Ala Leu Thr Val Thr Asp Val Ala Gln Ala -Met Asn Lys Glu Glu Ser Leu Leu Gln Met Ala Ala Asn Gln Val Glu Lys Asp Leu Thr Lys Leu Thr Ser Trp Thr Gln Ile Asp Ala Ile Leu Gln Trp Leu Gln Met Ser Ser Ala Leu Ala Val Ser Pro Leu Asp Leu Ala Gly Met Met Ala Leu Lys Tyr Gly Ile Asp His Asn Tyr Ala Ala Trp Gln Ala Ala Ala Ala Ala Leu Met Ala Asp His Ala Asn Gln Ala Gln Lys Lys Leu Asp Glu Thr Phe Ser Lys Ala Leu Cys Asn Tyr Tyr 835 840 8g5 Ile Asn Ala Val Val Asp Ser Ala Ala Gly Val Arg Asp Arg Asn Gly Leu Tyr Thr Ty- Leu Leu Ile Asp Asn Gln Val Ser Ala Asp Val Ile Thr Ser Arg Ile Ala Glu Ala Ile Ala Gly Ile Gln Leu Tyr Val Asn Arg Ala Leu Asn Arg Asp Glu Gly Gln Leu Ala Ser Asp Val Ser Thr 9oO 905 910 Arg Gln Phe Phe Thr Asp Trp Glu Arg Tyr Asn Lys Arg Tyr Ser Thr Trp Ala Gly Val Ser Glu Leu Val Tyr Tyr Pro Glu Asn Tyr Val Asp Pro Thr Gln Arg Ile Gly Gln Thr Lys Met Met Asp Ala Leu Leu Glr.

Ser Ile Asn Gln Ser Gln Leu Asn Ala Asp Thr Val Glu Asp Ala Phe ~5 Lys Thr Tyr Leu Thr Ser Phe Glu Gln Val Ala Asn Leu Lys Val Ile Ser Ala Tyr His Asp Asn Val Asn Val Asp Gln Gly Leu Thr Tyr Phe Ile Gly Ile ASD Gln Ala Ala Pro Gly Thr Tyr Tyr Trp Arg Ser Val SUBST~TUTE SHE~T (RULE 26) CA 022638l9 l999-02-26 WO 98/08932 PCT~US97/07657 1010 1015 . 1020 Asp His Ser Lys Cys Glu Asn Gly Lys Phe Ala Ala Asn Ala Trp Gly Glu Trp Asn Lys Ile Thr Cys Ala Val Asn Pro Trp Lys Asn Ile Ile Arg Pro Val Val Tyr Met Ser Arg Leu Tyr Leu Leu Trp Leu Glu Gln Gln Ser Lys Lys Ser Asp Asp Gly Lys Thr Thr Ile Tyr Gln Tyr Asn 5 Leu Lys Leu Ala His Ile Arg Tyr Asp Gly Ser Trp Asn Thr Pro Phe Thr Phe Asp Val Thr Glu Lys Val Lys Asn Tyr Thr Ser Ser Thr Asp Ala Ala Glu Ser Leu Gly Leu Tyr Cys Thr Gly Tyr Gln Gly Glu Asp Thr Leu Leu Val Met Phe Tyr Ser Met Gln Ser Ser Tyr Ser Ser Tyr Thr Asp Asn Asn Ala Pro Val Thr Gly Leu Tyr Ile Phe Ala Asp Met Ser Ser Asp Asn Met Thr Asn Ala Gln Ala Thr Asn Tyr Trp Asn Asn Ser Tyr Pro Gln Phe Asp Thr Val Met Ala Asp Pro Asp Ser Asp Asn Lys Lys Val Ile Thr Arg Arg Val Asn Asn Arg Tyr Ala Glu Asp Tyr Glu Ile Pro Ser Ser Val Thr Ser Asn Ser Asn Tyr Ser Trp Gly Asp His Ser Leu Thr Met Leu Tyr Gly Gly Ser Val Pro Asn Ile Thr Phe 4 5 Glu Ser Ala Ala Glu Asp Leu Arg Leu Ser Thr Asn Met Ala Leu Ser Ile Ile His Asn Gly Tyr Ala Gly Thr Arg Arg Ile Gln Cys Asn Leu Met Lys Gln Tyr Ala Ser Leu Gly Asp Lys Phe Ile Ile Tyr Asp Ser 1285 lZ90 1295 Ser Phe Asp Asp Ala Asn Arg Phe Asn Leu Val Pro Leu Phe Lys Phe Gly Lys Asp Glu Asn Ser Asp Asp Ser Ile Cys Ile Tyr Asn Glu Asn Pro Ser Ser Glu Asp Lys Lys Trp Tyr Phe Ser Ser Lys ASD Asp Asn Lys Thr Ala Asp Tyr Asn Gly Gly Thr Gln Cys Ile Asp Ala Gly Thr Ser Asn Lys Asp Phe Tyr Tyr Asn Leu Gln Glu Ile Glu Val Ile Ser Val Thr Gly Gly Tyr Trp Ser Ser Tyr Lys Ile Ser Asn Pro Ile Asn SUBST~TUTE SHEET (RULE 26) .... ~ .. ~.. ..

WO 98/08932 PCTrUS97/07657 Ile Asn Thr Gly Ile Asp Ser Ala Lys Val Lys Val Thr Val Lys Ala Gly Gly Asp Asp Gln Ile Phe Thr Ala Asp Asn Ser Thr Tyr Val Pro 1~10 1415 1420 Gln Gln Pro Ala Pro Ser Phe Glu Glu Met Ile Tyr Gln Phe Asn Asn 0 Leu Thr Ile Asp Cys Lys Asn Leu Asn Phe Ile Asp Asn Gln Ala His Ile Giu Ile Asp Phe Thr Ala Thr Ala Gln Asp Gly Arg Phe Leu Gly Ala Glu Thr Phe Ile Ile Pro Val Thr Lys Lys Val Leu Gly Thr Glu Asn Val Ile Ala Leu Tyr Ser Glu Asn Asn Gly Val Gln Tyr Met Gln Ile Gly Ala Tyr Arg Thr Arg Leu Asn Thr Leu Phe Ala Gln Gln Leu 25 Val Ser Arg Ala Asn Arg Gly Ile Asp Ala Val Leu Ser Met Glu Thr Gln Asn Ile Gln Glu Pro Gln Leu Gly Ala Gly Thr Tyr Val Gln Leu Val Leu Asp Lys Tyr Asp Glu Ser Ile His Gly Thr Asn Lys Ser Phe Ala Ile Glu Tyr Val Asp Ile Phe Lys Glu Asn Asp Ser Phe Val Ile Tyr Gln Gly Glu Leu Ser Glu Thr Ser Gln Thr Val Val Lys Val Phe 40 Leu Ser Tyr Phe Ile Glu Ala Thr Gly Asn Lys Asn His Leu Trp Val Arg Ala Lys Tyr Gln Lys Glu Thr Thr Asp Lys Ile Leu Phe Asp Arg Thr Asp Glu Lys Asp Pro His Gly Trp Phe Leu Ser Asp Asp His Lys Thr Phe Ser Gly Leu Ser Ser Ala Gln Ala Leu Lys Asn Asp Ser Glu Pro Met Asp Phe Ser Gly Ala Asn Ala Leu Tyr Phe Trp Glu Leu Phe 55 Tyr Tyr Thr Pro Met Met Met Ala His Arg Leu Leu Gln Glu Gln Asn Phe Asp Ala Ala Asn His Trp Phe Arq Tyr Val Trp Ser Pro Ser Glv Tyr Ile Val Asp Gly Lys Ile Ala Ile Tyr His Trp Asn Val Arg Pro Leu Glu Glu Asp Thr Ser Trp Asn Ala Gln Gln Leu Asp Ser Thr Asp 1730 1735 1~40 Pro Asp Ala Val Ala Gln Asp Asp Pro Met His Tyr Lys Val Ala Thr 7 0 Phe Met Ala Thr Leu Asp Leu Leu Met Ala Arg Gly Asp Ala Ala Tyr SU~STTTUTE SH E~ ~RULE-26) , CA 022638l9 l999-02-26 W098/08932 PCT~US97/07657 Arg Gln Leu Glu Arg Asp Thr Leu Ala Glu Ala Lys Met Trp Tyr Thr 5 Gln Ala Leu Asn Leu Leu Gly Asp Glu Pro Gln Val Met Leu Ser Thr Thr Trp Ala Asn Pro Thr Leu Gly Asn Ala Ala Ser Lys Thr Thr Gln Gln Val Arg Gln Gln Val Leu Thr Gln Leu Arg Leu Asn Ser Arg Val Lys Thr Pro Leu 184g (2) INFORMATION FOR SEQ ID NO:54:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1722 base palrs (B) TYP~: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: llnear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:54 (tCbAiii coding region):

Leu Gly Thr Ala Asn Ser Leu Thr Ala Leu Phe Leu Pro Gln Glu Asn Ser Lys Leu Lys Gly Tyr Trp Arg Thr Leu Ala Gln Arg Met Phe Asn TTA CGT CAT AAT CTG TCG ATT GAC GGC CAG CCG CTC TCC TTG CCG CTG lg4 Leu Arg His Asn Leu Ser Ile Asp Gly Gln Pro Leu Ser Leu Pro Leu Tyr Ala Lys Pro Ala Asp Pro Lys Ala Leu Leu Ser Ala Ala Val Ser Ala Ser Gln Gly Gly Ala Asp Leu Pro Lys Ala Pro Leu Thr Ile His - ~' 70 75 80 Arg Phe Pro Gln Met Leu Glu Gly Ala Arg Gly Leu Val Asn Gln Leu Ile Gln Phe Gly Ser Ser Leu Leu Gly Tyr Ser Glu Arg Gln Asp Ala Glu Ala Met Ser Gln Leu Leu Gln Thr Gln Ala Ser Glu Leu Ile Leu Thr Ser Ile Arg Met Gln Asp Asn Gln Leu Ala Glu Leu Asp Ser Glu Lys Thr Ala Leu Gln Val Ser Leu Ala Gly Val Gln Gln Arg Phe Asp SUBSTITUTE St~EET tRULE 26) ~. . . ~ .

CA 022638l9 l999-02-26 W O 98/08932 PCT~US97/076S7 Ser Tyr Ser Gln Leu Tyr Glu Glu Asn Ile Asn Ala Gly Glu Gln Arg Ala Leu Ala Leu Arg Ser Glu Ser Ala Ile Glu Ser Gln Gly Ala Gln 0 Ile Ser Arg Met Ala Gly Ala Gly Val Asp Met Ala Pro Asn Ile Phe Gly Leu Ala Asp Gly Gly Met His Tyr Gly Ala Ile Ala Tyr Ala Ile Ala Asp Gly Ile Glu Leu Ser Ala Ser Ala Lys Met Val Asp Ala Glu Lys Val Ala Gln Ser Glu Ile Tyr Arg Arg Arg Arg Gln Glu Trp Lys Ile Gln Arg Asp Asn Ala Gln Ala Glu Ile Asn Gln Leu Asn Ala Gln Leu Glu Ser Leu Ser Ile Arg Arg Glu Ala Ala Glu Met Gln Lys Glu Tyr Leu Lys Thr Gln Gln Ala Gln Ala Gln Ala Gln Leu Thr Phe Leu Arg Ser Lys Phe Ser Asn Gln Ala Leu Tyr Ser Trp Leu Arg Gly Arg Leu Ser Gly Ile Tyr Phe Gln Phe Tyr Asp Leu Ala Val Ser Arg Cys Leu Met Ala Glu Gln Ser Tyr Gln Trp Glu Ala Asn Asp Asn Ser Ile AGC TTT GTC A~A CCG GGT GCA TGG CAA GGA ACT TAC GCC GGC TTA TTG 1104 Ser Phe Val Lys Pro Gly Ala Trp Gln Gly Thr Tyr Ala Gly Leu Leu Cys Gly Glu Ala Leu Ile Gln Asn Leu Ala Gln Met Glu Glu Ala Tyr Leu Lys Trp Glu Ser Arg Ala Leu Glu Val Glu Arg Thr Val Ser Leu Ala Val Val Tyr Asp Ser Leu Glu Gly Asn Asp Arg Phe Asn Leu Ala Glu Gln Ile Pro Ala Leu Leu Asp Lys Gly Glu Gly Thr Ala Gly Thr Lys Glu Asn Gly Leu Ser Leu Ala Asn Ala Ile Leu Ser Ala Ser Val SUBSTITUTE SHEET tRULE 26) CA 022638l9 l999-02-26 WO 98/08932 PCTrUS97/07657 Lys Leu Ser Asp Leu Lys Leu Gly Thr Asp Tyr Pro Asp Ser Ile Val 450 q55 460 Gly Ser Asn Lys Val Arg Arg Ile Lys Gln Ile Ser Val Ser Leu Pro Ala Leu Val Gly Pro Tyr Gln Asp Val Gln Ala Met Leu Ser Tyr Gly Gly Ser Thr Gln Leu Pro Lys Gly Cys Ser Ala Leu Ala Val Ser His Gly Thr Asn Asp Ser Gly Gln Phe Gln Leu Asp Phe Asn Asp Gly Lys Tyr Leu Pro Phe Glu Gly Ile Ala Leu Asp Asp Gln Gly Thr Leu Asn Leu Gln Phe Pro Asn Ala Thr Asp Lys Gln Lys Ala Ile Leu Gln Thr Met Ser Asp Ile Ile Leu His Ile Arg Tyr Thr Ile Arg ~--(2) INFORMATION FOR SEQ ID NO:55:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 573 a~ino acids ~B) TYPE: amino acids (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:55 (TcbAiii):

Leu Gly Thr Ala Asn Ser Leu Thr Ala Leu Phe Leu Pro Gln Glu Asn Ser Lys Leu Lys Gly Tyr Trp Arg Thr Leu Ala Gln Arg Met Phe Asn Leu Arg His Asn Leu Ser Ile Asp Gly Gln Pro Leu Ser Leu Pro Leu Tyr Ala Lys Pro Ala Asp Pro Lys Ala Leu Leu Ser Ala Ala Val Ser Ala Ser Gln Gly Gly Ala Asp Leu Pro Lys Ala Pro Leu Thr Ile His ~ 65 70 75 80 Arg Phe Pro Gln Met Leu Glu Gly Ala Arg Gly Leu Val Asn Gln Leu Ile Gln Phe Gly Ser Ser Leu Leu Gly Tyr Ser Glu Arg Gln Asp Ala Glu Ala Met Ser Gln Leu Leu Gln Thr Gln Ala Ser Glu Leu Ile Leu SUBSTITUTE S~EET (RULE 26) W098/08932 PCTrUS97/076S7 Thr Ser Ile Arg Met Gln Asp Asn Gln Leu Ala Glu Leu Asp Ser Glu 130 135 ' 140 Lys Thr Ala Leu Gln Val Ser Leu Ala Gly Val Gln Gln Arg Phe Asp Ser Tyr Ser Gln Leu Tyr Glu Glu Asn Ile Asn Ala Gly Glu Gln Arg 0 Ala Leu Ala Leu Arg Ser Glu Ser Ala Ile Glu Ser Gln Gly Ala Gln Ile Ser Arg Met Ala Gly Ala Gly Val Asp Met Ala Pro Asn Ile Phe Gly Leu Ala Asp Gly Gly Met His Tyr Gly Ala Ile Ala Tyr Ala Ile Ala Asp Gly Ile Glu Leu Ser Ala Ser Ala Lys Met Val Asp Ala Glu Lys Val Ala Gln Ser Glu Ile Tyr Arg Arg Arg Arg Gln Glu Trp Lys Ile Gln Arg Asp Asn Ala Gln Ala Glu Ile Asn Gln Leu Asn Ala Gln Leu Glu Ser Leu Ser Ile Arg Arg Glu Ala Ala Glu Met Gln Lys Glu Tyr Leu Lys Thr Gln Gln Ala Gln Ala Gln Ala Gln Leu Thr Phe Leu Arg Ser Lys Phe Ser Asn Gln Ala Leu Tyr Ser Trp Leu Arg Gly Arg Leu Ser Gly Ile Tyr Phe Gln Phe Tyr Asp Leu Ala Val Ser Arg Cys Leu Met Ala Glu Gln Ser Tyr Gln Trp Glu Ala Asn Asp Asn Ser Ile Ser Phe Val Lys Pro Gly Ala Trp Gln Gly Thr Tyr Ala Gly Leu Leu Cys Gly Glu Ala Leu Ile Gln Asn Leu Ala Gln Met Glu Glu Ala Tyr Leu Lys Trp Glu Ser Arg Ala Leu Glu Val Glu Arg Thr Val Ser Leu Ala Val Val Tyr Asp Ser Leu Glu Gly Asn Asp Arg Phe Asn Leu Ala Glu Gln Ile Pro Ala Leu Leu Asp Lys Gly Glu Gly Thr Ala Gly Thr g20 425 430 Lys Glu Asn Gly Leu Ser Leu Ala Asn Ala Ile Leu Ser Ala Ser Val Lys Leu Ser Asp Leu Lys Leu Gly Thr Asp Tyr Pro Asp Ser Ile Val Gly Ser Asn Lys Val Arg Arg Ile Lys Gln Ile Ser Val Ser Leu Pro Ala Leu Val Gly Pro Tyr Gln Asp Val Gln Ala Met Leu Ser Tyr Gly ~0 Gly Ser Thr Gln Leu Pro Lys Gly Cys Ser Ala Leu Ala Val Ser His SUBSTITUTE St~EET tRULE 26) .

W098/08932 PCT~US97/07657 Gly Thr Asn Asp Ser Gly Gln Phe Gln Leu'Asp Phe Asn As~ Gly Lys Tyr Leu Pro Phe Glu Gly Ile Ala Leu Asp Asp Gln Gly Thr Leu Asn 530 535 5qO
Leu Gln Phe Pro Asn Ala Thr Asp Lys Gln Lys Ala Ile Leu Gln Thr Met Ser Asp Ile Ile Leu Hls Ile Arg Tyr Thr Ile Arg ~--(2) INFORMATION FOR SEQ ID NO:56 (1) SEQUENCE CHARACTERISTICS:
(A~ L~NGTH: 2994 base pa1rs (B) TYPE: nucleic acld (C) STRANDEDNESS: double (D) TOPOLOGY: linear (li~ MOLECULE TYPE: DNA (genomic) (xl) SEQUENCE DESCRIPTION: SEQ ID NO:56 (tccA) 1 Met Asn Gln Leu Ala Ser Pro Leu Ile Ser Arg Thr Glu Glu Ile His 16 17 Asn Leu Pro Gly Lys Leu Thr Asp Leu Gly Tyr Thr Ser Val Phe Asp 32 33 Val Val Arg Met Pro Arg Glu Arg Phe Ile Arg Glu His Arg Ala Asp 48 145 CTC GGG CGC AGT GCT GAA AAA ATG TAT GAC CTG GCA GTG GGC TAT GCT 19249 Leu Gly Arg Ser Ala Glu Lys Met Tyr Asp Leu Ala Val Gly Tyr Ala 64 His Gln Val Leu His His Phe Arg Arg Asn Ser Leu Ser Glu Ala Val 80 81 Gln Phe Gly Leu Arg Ser Pro Phe Ser Val Ser Gly Pro Asp Tyr Ala 96 97 Asn Gln Phe Leu Asp Ala Asn Thr Gly Trp Lys Asp Lys Ala Pro Ser 112 55113 Gly Ser Pro Glu Ala Asn Asp Ala Pro Val Ala Tyr Leu Thr ~is Ile 128 129 Tyr Gln Leu Ala Leu Glu Gln Glu Lys Asn Gly Ala Thr Thr Ile Met 144 145 Asn Thr Leu Ala Glu Arg Arg Pro Asp Leu Gly Ala Leu Leu Ile Asn 160 161 Asp Lys Ala Ile Asn Glu Val Ile Pro Gln Leu Gln Leu Val Asn Glu 176 SUBS 111 ~JTE SHEET tRULE 26~

CA 022638l9 l999-02-26 177 Ile Leu Ser Lys Ala Ile Gln Lys Ly's Leu Ser Leu Thr Asp Leu Glu 192 193 Ala Val Asn Ala Arg Leu Ser Thr Thr Arg Tyr Pro Asn Asn Leu Pro 208 0 209 Tyr His Tyr Gly His Gln Gln Ile Gln Thr Ala Gln Ser Val Leu Gly 224 225 Thr Thr Leu Gln Asp Ile Thr Leu Pro Gln Thr Leu Asp Leu Pro Gln 240 241 Asn Phe Trp Ala Thr Ala Lys Gly Lys Leu Ser Asp Thr Thr Ala Ser 256 257 Ala Leu Thr Arg Leu Gln Ile Met Ala Ser Gln Phe Ser Pro Glu Gln 272 273 Gln Lys Ile Ile Thr Glu Thr Val Gly Gln Asp Phe Tyr Gln Leu Asn 288 289 Tyr Gly Asp Ser Ser Leu Thr Val Asn Ser Phe Ser Asp Met Thr Ile 304 305 Met Thr Asp Arg Thr Ser Leu Thr Val Pro Gln Val Glu Leu Met Leu 320 321 Cys Ser Thr Val Gly Gly Ser Thr Val Val Lys Ser Asp Asn Val Ser 336 337 Ser Gly Asp Thr Thr Ala Thr Pro Phe Ala Tyr Gly Ala Arg Phe Ile 352 353 His Ala Gly Lys Pro Glu Ala Ile Thr Leu Ser Arg Ser Gly Ala Glu 368 369 Ala His Phe Ala Leu Thr Val Asn Asn Leu Thr Asp Asp Lys Leu Asp 384 385 Arg Ile Asn Arg Thr Val Arg Leu Gln Lys Trp Leu Asn Leu Pro Tyr 400 401 Glu Asp Ile Asp Leu Leu Val Thr Ser Ala Met Asp Ala Glu Thr Gly 416 417 Asn Thr Ala Leu Ser Met Asn Asp Asn Thr Leu Arg Met Leu Gly Val 432 SUBSTITUTE SHEET tRULE 26) CA 022638l9 l999-02-26 W O 98/08932 rCT~US97/07657 433 Phe Lys His Tyr Gln Ala Lys Tyr Gly Val Ser Ala Lys Gln Phe Ala 448 449 Gly Trp Leu Arg Val Val Ala Pro Phe Ala Ile Thr Pro Ala Thr Pro 464 - 465 Phe Leu Asp Gln Val Phe Asn Ser Val Gly Thr Phe Asp Thr Pro Phe 480 481 Val Ile Asp Asn Gln Asp Phe Val Tyr Thr Leu Thr Thr Gly Gly Asp 496 497 Gly Ala Arg Val Lys His Ile Ser Thr Ala Leu Gly Leu Asn His Arg 512 513 Gln Phe Leu Leu Leu Ala Asp Asn Ile Ala Arg Gln Gln Gly Asn Val 528 529 Thr Gln Ser Thr Leu Asn Cys Asn Leu Phe Val Val Ser Ala Phe Tyr 544 545 Arg Leu Ala Asn Leu Ala Arg Thr Leu Gly Ile Asn Pro Glu Ser Phe 560 561 Cys Ala Leu Val Asp Arg Leu Asp Ala Gly Thr Gly Ile Val Trp Gln 576 577 Gln Leu Ala Gly Lys Pro Thr Ile Thr Val Pro Gln Lys Asp Ser Pro -592 593 Leu Ala Ala Asp Ile Leu Ser Leu Leu Gln Ala Leu Ser Ala Ile Ala 608 609 Gln Trp Gln Gln Gln His Asp Leu Glu Phe Ser Ala Leu Leu Leu Leu 624 625 Leu Ser Asp Asn Pro Ile Ser Thr Ser Gln Gly Thr Asp Asp Gln Leu 640 641 Asn Phe Ile Arg Gln Val Trp Gln Asn Leu Gly Ser Thr Phe Val Gly 656 SUBSTmlTE SHEET ~RULE ~6) CA 022638l9 l999-02-26 W098/08932 PCT~US97/07657 657 Ala Thr Leu Leu Ser Arg Ser Gly Ala Pro Leu Val Asp Thr Asn Gly 672 673 His Ala Ile Asp Trp Phe Ala Leu Leu Ser Ala Gly Asn Ser Pro Leu 688 689 Ile Asp Lys Val Gly Leu Val Thr Asp Ala Gly Ile Gln Ser Val Ile 704 705 Ala Thr Val Val Asn Thr Gln Ser Leu Ser Asp Glu Asp Lys Lys Leu 720 721 Ala Ile Thr Thr Leu Thr Asn Thr Leu Asn Gln Val Gln Lys Thr Gln 736 737 Gln Gly Val Ala Val Ser Leu Leu Ala Gln Thr Leu Asn Val Ser Gln 752 753 Ser Leu Pro Ala Leu Leu ~eu Arg Trp Ser Gly Gln Thr Thr Tyr Gln 768 769 Trp heu Ser Ala Thr Trp Ala Leu Lys Asp Ala Val Lys Thr Ala Ala 784 785 Asp Ile Pro Ala Asp Tyr ~eu Arg Gln Leu Arg Glu Val Val Arg Arg 800 801 Ser Leu Leu Thr Gln Gln Phe Thr Leu Ser Pro Ala Met Val Gln Thr 816 817 Leu Leu Asp Tyr Pro Ala Tyr Phe Gly Ala Ser Ala Glu Thr Val Thr 832 833 Asp Ile Ser Leu Trp Met Leu Tyr Thr Leu Ser Cys Tyr Ser Asp Leu 848 849 Leu Leu Gln Met Gly Glu Ala Gly Gly Thr Glu Asp Asp Val Leu Ala 864 865 Tyr Leu Arg Thr Ala Asn Ala Thr Thr Pro Leu Ser Gln Ser Asp Ala 880 SUBSTITUTE SHEET (RULE 26) W 098~932 PCT~US97/076S7 881 Ala Gln Thr Leu Ala Thr Leu Leu Gly Trp Glu Val Asn Glu Leu Gln 896 897 Ala Ala Trp Ser Val Leu Gly Gly Ile Ala Lys Thr Thr Pro Gln Leu 912 913 Asp Ala Leu Leu Arg Leu Gln Gln Ala Gln Asn Gln Thr Gly Leu Gly 928 929 Val Thr Gln Gln Gln Gln Gly Tyr Leu Leu Ser Arg Asp Ser Asp Tyr 944 945 Thr Leu Trp Gln Ser Thr Gly Gln Ala Leu Val Ala Gly Val Ser His 960 961 Val Lys Gly Ser Asn End 965 (2) INFO~MATION FOR SEQ ID NO:57 (i) SEQUENCE CHU~RA~TERISTICS:
(A) LENGTH: 965 amino aclds (B) TYPE: amino acid (C) TOPOLOGY: linear (ii) MOLECULE TYPE: proteln (xi) SEQUENCE DESCRIPTION: SEQ ID NO:57 (TCcA peptide) Features From To Description 1 10 SEQ ID NO:8 1 Met Asn Gln Leu Ala Ser Pro Leu Ile Ser Arg Thr Glu Glu Ile His 16 17 Asn Leu Pro Gly Lys Leu Thr Asp Leu Gly Tyr Thr Ser Val Phe Asp 32 33 Val Val Arg Met Pro Arg Glu Arg Phe Ile Arg Glu His Arg Ala Asp 48 49 Leu Gly Arg Ser Ala Glu Lys Met Tyr Asp Leu Ala Val Gly Tyr Ala 64 His Gln Val Leu His His Phe Arg Arg Asn Ser Leu Ser Glu Ala Val 80 81 Gln Phe Gly Leu Arg Ser Pro Phe Ser Val Ser Gly Pro Asp Tyr Ala 96 97 Asn Gln Phe Leu Asp Ala Asn Thr Gly Trp Lys Asp Lys Ala Pro Ser 112 113 Gly Ser Pro Glu Ala Asn Asp Ala Pro Val Ala Tyr Leu Thr His Ile 128 129 Tyr Gln Leu Ala Leu Glu Gln Glu Lys Asn Gly Ala Thr Thr Ile Met 144 145 Asn Thr Leu Ala Glu Arg Arg Pro Asp Leu Gly Ala Leu Leu Ile Asn 160 161 Asp Lys Ala Ile Asn Glu Val Ile Pro Gln Leu Gln Leu Val Asn Glu 176 177 Ile Leu Ser Lys Ala Ile Gln Lys Lys Leu Ser Leu Thr Asp Leu Glu 192 SUBSTITUTE S~EET ~RUJ_F 26) CA 022638l9 l999-02-26 193 Ala Val Asn Ala Arg Leu Ser Thr Thr Arg Tyr Pro Asn Asn Leu Pro 208 209 Tyr His Tyr Gly His Gln Gln Ile Gln Thr Ala Gln Ser Val Leu Gly 224 225 Thr Thr Leu Gln Asp Ile Thr Leu Pro Gln Thr Leu Asp Leu Pro Gln 240 241 Asn Phe Trp Ala Thr Ala Lys Gly Lys Leu Ser Asp Thr Thr Ala Ser 256 0 257 Ala Leu Thr Arg Leu Gln Ile Met Ala Ser Gln Phe Ser Pro Glu Gln 272 273 Gln Lys Ile Ile Thr Glu Thr Val Gly Gln Asp Phe Tyr Gln heu Asn 288 289 Tyr Gly Asp Ser Ser Leu Thr Val Asn Ser Phe Ser Asp Met Thr Ile 304 305 Met Thr Asp Arg Thr Ser Leu Thr Val Pro Gln Val Glu Leu Met Leu 320 321 Cys Ser Thr Val Gly Gly Ser Thr Val Val Lys Ser Asp Asn Val Ser 336 337 Ser Gly Asp Thr Thr Ala Thr Pro Phe Ala Tyr Gly Ala Arg Phe Ile 352 353 His Ala Gly Lys Pro Glu Ala Ile Thr Leu Ser Arg Ser Gly Ala Glu 368 369 Ala His Phe Ala Leu Thr Val Asn Asn Leu Thr Asp Asp Lys Leu Asp 384 385 Arg Ile Asn Arg Thr Val Arg Leu Gln Lys Trp Leu Asn Leu Pro Tyr 400 401 Glu Asp Ile Asp Leu Leu Val Thr Ser Ala Met Asp Ala Glu Thr Gly 416 417 Asn Thr Ala Leu Ser Met Asn Asp Asn Thr Leu Arg Met Leu Gly Val 432 433 Phe Lys ~is Tyr Gln Ala Lys Tyr Gly Val Ser Ala Lys Gln Phe Ala 448 449 Gly Trp Leu Arg Val Val Ala Pro Phe Ala Ile Thr Pro Ala Thr Pro 464 465 Phe Leu Asp Gln Val Phe Asn Ser Val Gly Thr Phe Asp Thr Pro Phe 480 481 Val Ile Asp Asn Gln Asp Phe Val Tyr Thr Leu Thr Thr Gly Gly Asp 496 497 Gly Ala Arg Val Lys His Ile Ser Thr Ala Leu Gly Leu Asn His Arg 512 513 Gln Phe Leu Leu Leu Ala Asp Asn Ile Ala Arg Gln Gln Gly Asn Val 528 529 Thr Gln Ser Thr Leu Asn Cys Asn Leu Phe Val Val Ser Ala Phe Tyr 544 545 Arg Leu Ala Asn Leu Ala Arg Thr Leu Gly Ile Asn Pro Glu Ser Phe 560 561 Cys Ala Leu Val Asp Arg Leu Asp Ala Gly Thr Gly Ile Val Trp Gln 576 577 Gln Leu Ala Gly Lys Pro Thr Ile Thr Val Pro Gln Lys Asp Ser Pro 592 593 Leu Ala Ala Asp Ile Leu Ser Leu Leu Gln Ala Leu Ser Ala Ile Ala 608 609 Gln Trp Gln Gln Gln His Asp Leu Glu Phe Ser Ala Leu Leu Leu Leu 624 625 Leu Ser Asp Asn Pro Ile Ser Thr Ser Gln Gly Thr Asp Asp Gln Leu 640 641 Asn Phe ~le Arg Gln Val Trp Gln Asn Leu Gly Ser Thr Phe Val Gly 656 657 Ala Thr Leu Leu Ser Arg Ser Gly Ala Pro Leu Val Asp Thr Asn Gly 672 673 His Ala Ile Asp Trp Phe Ala Leu Leu Ser Ala Gly Asn Ser Pro Leu 688 689 Ile Asp Lys Val Gly Leu Val Thr Asp Ala Gly Ile Gln Ser Val Ile 704 705 Ala Thr Val Val Asn Thr Gln Ser Leu Ser Asp Glu Asp Lys Lys Leu 720 721 Ala Ile Thr Thr Leu Thr Asn Thr Leu Asn Gln Val Gln Lys Thr Gln 736 737 Gln Gly Val Ala Val Ser Leu Leu Ala Gln Thr Leu Asn Val Ser Gln 752 SUBSTITUTE SHEET tRULE 26) CA 022638l9 l999-02-26 W O 98/08932 PCT~US97/07657 753 Ser Leu Pro Ala Leu Leu Leu Arg Trp Ser Gly Gln Thr Thr Tyr Gln 768 769 Trp Leu Ser Ala Thr Trp Ala Leu Lys Asp Ala Val Lys Thr Ala Ala 784 785 Asp Ile Pro Ala Asp Tyr Leu Arg Gln Leu Arg Glu Val Val Arg Arg 800 801 Ser Leu Leu Thr Gln Gln Phe Thr Leu Ser Pro Ala Met Val Gln Thr 816 817 Leu Leu Asp Tyr Pro Ala Tyr Phe Gly Ala Ser Ala Glu Thr Val Thr 832 833 Asp Ile Ser Leu Trp Met Leu Tyr Thr Leu Ser Cys Tyr Ser Asp Leu 848 849 Leu Leu Gln Met Gly Glu Ala Gly Gly Thr Glu Asp Asp Val Leu Ala 864 865 Tyr Leu Arg Thr Ala Asn Ala Thr Thr Pro Leu Ser Gln Ser Asp Ala 880 881 Ala Gln Thr Leu Ala Thr Leu Leu Gly Trp Glu Val Asn Glu Leu Gln 896 897 Ala Ala Trp Ser Val Leu Gly Gly Ile Ala Lys Thr Thr Pro Gln Leu 912 913 Asp Ala Leu Leu Arg Leu Gln Gln Ala Gln Asn Gln Thr Gly Leu Gly 928 929 Val Thr Gln Gln Gln Gln Gly Tyr Leu Leu Ser Arg Asp Ser Asp Tyr 944 945 Thr Leu Trp Gln Ser Thr Gly Gln Ala Leu Val Ala Gly Val Ser His 960 961 Val Lys Gly Ser Asn 965 (2) INFORMATION FOR SEQ ID NO:58 (i) SEQUENCE CHARACTERISTICS:
~A) LENGTH: 4932 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 58 ( ~CCB) l ~Met Leu Ser Thr Met Glu Lys Gln Leu Asn Glu Ser Gln Arg Asp Ala 16 17 Leu Val Thr Gly Tyr Met Asn Phe Val Ala Pro Thr Leu Lys Gly Val 32 33 Ser Gly Gln Pro Val Thr Val Glu Asp Leu Tyr Glu Tyr Leu Leu Ile 4B

49 Asp Pro Glu Val Ala Asp Glu Val Glu Thr Ser Arg Val Ala Gln Ala 64 Ile Ala Ser Ile Gln Gln Tyr Met Thr Arg Leu Val Asn Gly Ser Glu 80 81 Pro Gly Arg Gln Ala Met Glu Pro Ser Thr Ala Asn Glu Trp Arg Asp 96 97 Asn Asp Asn Gln Tyr Ala Ile Trp Ala Ala Gly Ala Glu Val Arg Asn 112 113 Tyr Ala Glu Asn Tyr Ile Ser Pro Ile Thr Arg Gln Glu Lys Ser His 128 -26g-SUBSTITUTE SH~ET tRULE 2~) CA 022638l9 l999-02-26 129 Tyr Phe Ser Glu Leu Glu Thr Thr Leu Asn Gln Asn Arg Leu Asp Pro 144 145 Asp Arg Val Gln Asp Ala Val Leu Ala Tyr Leu Asn Glu Phe Glu Ala 160 0 161 Val Ser Asn Leu Tyr Val Leu Ser Gly Tyr Ile Asn Gln Asp hys Phe 176 177 Asp Gln Ala Ile Tyr Tyr Phe Ile Gly Arg Thr Thr Thr Lys Pro Tyr 192 193 Arg Tyr Tyr Trp Arg Gln Met Asp Leu Ser Lys Asn Arg Gln Asp Pro 208 209 Ala Gly Asn Pro Val Thr Pro Asn Cys Trp Asn Asp Trp Gln Glu Ile 224 225 Thr Leu Pr~ Leu Ser Gly Asp Thr Val Leu Glu His Thr Val Arg Pro 240 241 Val Phe Tyr Asn Asp Arg Leu Tyr Val Ala Trp Val Glu Arg Asp Pro 256 769 GCA GTA CAG AAG GAT GCT GAC GGT AAA AAC ATC GGT A~A ACC CAT GCC 816 257 Ala Val Gln Lys Asp Ala Asp Gly Lys Asn Ile Gly Lys Thr His Ala 272 273 Tyr Asn Ile Lys Phe Gly Tyr Lys Arg Tyr Asp Asp Thr Trp Thr Ala 288 289 Pro Asn Thr Thr Thr Leu Met Thr Gln Gln Ala Gly Glu Ser Ser Glu 304 305 Thr Gln Arg Ser Ser Leu Leu Ile Asp Glu Ser Ser Thr Thr Leu Arg 320 321 Gln Val Asn Leu Leu Ala Thr Thr Asp Phe Ser Ile Asp Pro Thr Glu 336 337 Glu Thr Asp Ser Asn Pro Tyr Gly Arg Leu Met Leu Gly Val Phe Val 352 353 Arg Gln Phe Glu Gly Asp Gly Ala Asn Arg Lys Asn Lys Pro Val Val 368 369 Tyr Gly Tyr Leu Tyr Cys Asp Ser Ala Phe Asn Arg His Val Leu Arg 384 385 Pro Leu Ser Lys Asn Phe Leu Phe Ser Thr Tyr Arg Asp Glu Thr Asp 400 SUBSTITUTE SHEE7 (RULE 26) CA 022638l9 l999-02-26 W O 98~8932 PCTAUS97/07657 1201 GGT CAA AAC AGC TTG CAA TTT GC& GTA TAC GAT AAA AAG TAT GTA ATT

5401 Gly Gln Asn Ser Leu Gln Phe Ala Val Tyr Asp Lys Lys Tyr Val Ile 416 0417 Thr Lys Val Val Thr Gly Ala Thr Glu Asp Pro Glu Asn Thr Gly Trp 432 5433 Val Ser Lys Val Asp Asp Leu Lys Gln Gly Thr Thr Gly Ala Tyr Val 448 20449 Tyr Ile Asp Gln Asp Gly Leu Thr Leu His Ile Gln Thr Thr Thr Asn 464 25465 Gly Asp Phe Ile Asn Arg His Thr Phe Gly Tyr Asn Asp Leu Val Tyr 480 30481 Asp Ser Lys Ser Gly Tyr Gly Phe Thr Trp Ser Gly Asn Glu Gly Phe 496 35497 Tyr Leu Asp Tyr His Asp Gly Asn Tyr Tyr Thr Phe His Asn Ala Ile 512 40513 Ile Asn Tyr Tyr Pro Ser Gly Tyr Gly Gly Gly Ser Val Pro Asn Gly 528 45529 Thr Trp Ala Leu Glu Gln Arg Ile Asn Glu Gly Trp Ala Ile Ala Pro 544 168 Q ~
50545 Leu Leu Asp Thr Leu His Thr Val Thr Val Lys Gly Ser Tyr Ile Ala 560 _ _.

55561 Trp Glu Gly Glu Thr Pro Thr Gly Tyr Asn Leu Tyr Ile Pro Asp Gly 576 60577 Thr Val Leu Leu Asp Trp Phe Asp Lys Ile Asn Phe Ala Ile G~y Leu 592 65593 Asn Lys Leu Glu Ser Val Phe Thr Ser Pro Asp Trp Pro Thr Leu Thr 608 1825 ACT ATC AAA AAT TTC AGT A~A ATC GCC GAT AAC CGC AAA TTC TAT CAG

70609 Thr Ile Lys Asn Phe Ser Lys Ile Ala Asp Asn Arg Lys Phe Tvr Gln 624 SUBSTlTUTE SHEET (RULE 26) CA 022638l9 l999-02-26 W 098/08932 rcTrusg7/o7657 625 Glu Ile Asn Ala Glu Thr Ala Asp Gly Arg Asn Leu Phe Lys Arg Tyr 640 641 Ser Thr Gln Thr Phe Gly Leu Thr Ser Gly Ala Thr Tyr Ser Thr Thr 656 5657 Tyr Thr Leu Ser Glu Ala Asp Phe Ser Thr Asp Pro Asp Lys Asn Tyr 672 673 Leu Gln Val Cys Leu Asn Val Val Trp Asp His Tyr Asp Arg Pro Ser 688 25689 Gly Lys Lys Gly Ala Tyr Ser Trp Val Ser Lys Trp Phe Asn Val Tyr 704 705 Val Ala Leu Gln Asp Ser Lys Ala Pro Asp Ala Ile Pro Arg Leu Val 720 721 Ser Arg Tyr Asp Ser Lys Arg Gly Leu Val Gln Tyr Leu Asp Phe Trp 736 737 Thr Ser Ser Leu Pro Ala Lys Thr Arg Leu Asn Thr Thr Phe Val Arg 752 753 Thr Leu Ile Glu Lys Ala Asn Leu Gly Leu Asp Ser Leu Leu Asp Tyr 768 769 Thr Leu Gln Ala Asp Pro Ser Leu Glu Ala Asp Leu Val Thr Asp Gly 784 785 Lys Ser Glu Pro Met Asp Phe Asn Gly Ser Asn Gly Leu Tyr Phe Trp 800 801 Glu Leu Phe Phe His Leu Pro Phe Leu Val Ala Thr Arg Phe Ala Asn 816 65817 Glu Gln Gln Phe Ser Pro Ala Gln Lys Ser Leu His Tyr Ile Phe Asp 832 833 Pro Ala Met Lys Asn Lys Pro His Asn Ala Pro Ala Tyr Trp Asn Val 848 SUBSTITUTE SHEET ~RULE 26) 849 Arg Pro Leu Val Glu Gly Asn Ser Asp Leu Ser Arg His Leu ASD Asp 864 865 Ser Ile Asp Pro Asp Thr Gln Ala Tyr Ala His Pro Val Ile Tyr Gln 880 881 Lys Ala Val Phe Ile Ala Tyr Val Ser Asn Leu Ile Ala Gln Gly Asp 896 897 Met Trp Tyr Arg Gln Leu Thr Arg Asp Gly Leu Thr Gln Ala Arg Val 912 913 Tyr Tyr Asn Leu Ala Ala Glu Leu Leu Gly Pro Arg Pro Asp Val Ser 928 929 Leu Ser Ser Ile Trp Thr Pro Gln Thr Leu Asp Thr Leu Ala Ala Gly 944 945 Gln Lys Ala Val Leu Arg Asp Phe Glu His Gln Leu Ala Asn Ser Asp 960 961 Thr Ala Leu Pro Ala Leu Pro Gly Arg Asn Val Ser Tyr Leu Lys Leu 976 977 Ala Asp Asn Gly Tyr Phe Asn Glu Pro Leu Asn Val Leu Met Leu Ser 992 993 His Trp Asp Thr Leu Asp Ala Arg Leu Tyr Asn Leu Arg His Asn Leu 1009 Thr Val Asp Gly Lys Pro ~eu Ser Leu Pro Leu Tyr Ala Ala Pro Val 1025 Asp Pro Val Ala Leu Leu Ala Gln Arg Ala Gln Ser Gly Thr Leu Thr 1041 Asn Gly Val Ser Gly Ala Met Leu Thr Val Pro Pro Tyr Arg Phe Ser SUBSTITUTE S~l EET (RU~ E 26) CA 022638l9 l999-02-26 W098/08932 PCT~US97/07657 1057 Ala Met Leu Pro Arg Ala Tyr Ser Ala Val Gly Thr Leu Thr Ser Phe 1073 Gly Gln Asn Leu Leu Ser Leu Leu Glu Arg Ser Glu Arg Ala Cys Gln 1089 Glu Glu Leu Ala Gln Gln Gln Leu Leu Asp Met Ser Ser Tyr Ala Ile 1105 Thr Leu Gln Gln Gln Ala Leu Asp Gly Leu Ala Ala Asp Arg Leu Ala 1121 Leu Leu Ala Ser Gln Ala Thr Ala Gln Gln Arg His Asp H1s Tyr Tyr 1137 Thr Leu Tyr Gln Asn Asn Ile Ser Ser Ala Glu Gln Leu Val Met Asp 1153 Thr Gln Thr Ser Ala Gln Ser Leu Ile Ser Ser Ser Thr Gly Val Gln 1169 Thr Ala Ser Gly Ala Leu Lys Val Ile Pro Asn Ile Phe Gly Leu Ala 118~ p ~ly Gly Ser Arg Tyr Glu Gly Val Thr Glu Ala Ile Ala Ile Gly 1201 Leu Met Ala Ala Gly Gln Ala Thr Ser Val Val Ala Glu Arg Leu Ala 1217 Thr Thr Glu Asn Tyr Arg Arg Arg Arg Glu Glu Trp Gln Ile Gln Tyr 1233 Gln Gln Ala Gln Ser Glu Val Asp Ala Leu Gln Lys Gln Leu Asp Ala SU8STITUTE SH~T (RULE 26) W O 9~08932 PCTrUS97/07657 lZ49 Leu Ala Val Arg Glu Lys Ala Ala Gln Thr Ser Leu Gln Gln Ala Lys 1265 Ala Gln Gln Val Gln Ile Arg Thr Met Leu Thr Tyr Leu Thr Thr Arg 1281 Phe Thr Gln Ala Thr Leu Tyr Gln Trp Leu Ser Gly Gln Leu Ser Ala 1297 Leu Tyr Tyr Gln Ala Tyr Asp Ala Val Val Ala Leu Cys Leu Ser Ala 1313 Gln Ala Cys Trp Gln Tyr Glu Leu Gly Asp Tyr Ala Thr Thr Phe Ile 1329 Gln Thr Gly Thr Trp Asn Asp His Tyr Arg Gly Leu Gln Val Gly Glu 1345 Thr Leu Gln Leu Asn Leu His Gln Met Glu Ala Ala Tyr Leu Val Arg 1361 His Glu Arg Arg Leu Asn Val Ile Arg Thr Val Ser Leu Lys Ser Leu 1377 Leu Gly Asp Asp Gly Phe Gly Lys Leu Lys Thr Glu Gly Lys Val Asp 1393 Phe Pro Leu Ser Glu Lys Leu Phe Asp Asn Asp Tyr Pro Gly His Tyr 1409 Leu Arg Gln Ile Lys Thr Val Ser Val Thr Leu Pro Thr Leu Val Gly 1425 Pro Tyr Gln Asn Val Lys Ala Thr Leu Thr Gln Thr Ser Ser Ser Ile SUBSTITUTE SHEET (RULE 26) CA 022638l9 l999-02-26 W 098~8932 PCT~US97/07657 1441 Leu Leu Ala Ala Asp Ile Asn Gly Val Lys Arg Leu Asn Asp Pro Thr 1457 Gly Lys Glu Gly Asp Ala Thr His Ile Val Thr Asn Leu Arg Ala Ser 1473 Gln Gln Val Ala Leu Ser Ser Gly Ile Asn Asp Ala Gly Ser Phe Glu 1489 Leu Arg Leu Glu Asp Glu Arg Tyr Leu Ser Phe Glu Gly Thr Gly Ala 4513 GTT TCC AAA TGG ACT CTT AAC TTC CCG CGT TCT GTG GAT GAG CA~ ATT

1505 Val Ser Lys Trp Thr Leu Asn Phe Pro Arg Ser Val Asp Glu His Ile 1521 Asp Asp Lys Thr Leu Lys Ala Asp Glu Met Gln Ala Ala Leu Leu Ala 1537 Asn Met Asp Asp Val Leu Val Gln Val His Tyr Thr Ala Cys Asp Gly 1553 Gly Ala Ser Phe Ala Asn Gln Val Lys Lys Thr Leu Ser End 1565 4709 TAACTAATCC C~CCCACTCT GTTCGCCAGA GTGGGAGAAG ~lll~lc~TA TCTAAAATCA 4768 4770 ATCTTGCGAT ~ ClC~AT TTCATTGGAA GGGAAGCTGT AAAACAAATA AGGAATATGA 4828 (2) INFORMATION FOR SEQ ID NO:59 (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1565 amino acids (B) TYPE: amino acld (C) TOPOLOGY: linear (ii) MOLECULE TYPE: protein 65(xi) SEQUENCE DESCRIPTION: SEQ ID NO:59 (TCcB peptide) Features From To Description 1 Met Leu Ser Thr Met Glu Lys Gln Leu Asn Glu Ser Gln Arg Asp Ala SUBSTITUTE S}t E T (RULE 26) CA 022638l9 l999-02-26 W O 98/08932 PCTrUS97/07657 17 Leu Val Thr Gly Tyr Met Asn Phe Val Ala Pro Thr Leu Lys Gly Val 33 Ser Gly Gln Pro Val Thr Val Glu Asp Leu Tyr Glu Tyr Leu Leu Ile 4a 49 Asp Pro Glu Val Ala Asp Glu Val Glu Thr Ser Arg Val Ala Gln Ala Ile Ala Ser Ile Gln Gln Tyr Met Thr Arg Leu Val Asn Gly Ser Glu 81 Pro Gly Arg Gln Ala Met Glu Pro Ser Thr Ala Asn Glu Trp Arg Asp 97 Asn Asp Asn Gln Tyr Ala Ile Trp Ala Ala Gly Ala Glu Val Arg Asn 113 Tyr Ala Glu Asn Tyr Ile Ser Pro Ile Thr Arg Gln Glu Lys Ser His .
129 Tyr Phe Ser Glu Leu Glu Thr Thr Leu Asn Gln Asn Arg Leu Asp Pro 145 Asp Arg Val Gln Asp Ala Val Leu Ala Tyr Leu Asn Glu Phe Glu Ala 161 Val Ser Asn Leu Tyr Val Leu Ser Gly Tyr Ile Asn Gln Asp Lys Phe 177 Asp Gln Ala Ile Tyr Tyr Phe Ile Gly Arg Thr Thr Thr Lys Pro Tyr 193 Arg Tyr Tyr Trp Arg Gln Met Asp Leu Ser Lys Asn Arg Gln Asp Pro 209 Ala Gly Asn Pro Val Thr Pro Asn Cys Trp Asn Asp Trp Gln Glu Ile 225 Thr Leu Pro Leu Ser Gly Asp Thr Val Leu Glu His Thr Val Arg Pro 241 Val Phe Tyr Asn Asp Arg Leu Tyr Val Ala Trp Val Glu Arg Asp Pro 257 Ala Val Gln Lys Asp Ala Asp Gly Lys Asn Ile Gly Lys Thr His Ala 273 Tyr Asn Ile Lys Phe Gly Tyr Lys Arg Tyr Asp Asp Thr Trp Thr Ala 289 Pro Asn Thr Thr Thr Leu Met Thr Gln Gln Ala Gly Glu Ser Ser Glu 305 Thr Gln Arg Ser Ser Leu Leu Ile Asp Glu Ser Ser Thr Thr Leu Arg 321 Gln Val Asn Leu Leu Ala Thr Thr Asp Phe Ser Ile Asp Pro Thr Glu 337 Glu Thr Asp Ser Asn Pro Tyr Gly Arg Leu Met Leu Gly Val Phe Val 353 Arg Gln Phe Glu Gly Asp Gly Ala Asn Arg Lys Asn Lys Pro Val Val 369 Tyr Gly Tyr Leu Tyr Cys Asp Ser Ala Phe Asn Arg His Val Leu Arg SU8STITUTE S~EET (RUI E 26) W 098/08932 PCTrUS97/07657 385 Pro Leu Ser Lys Asn Phe Leu Phe Ser Thr Tyr Arg Asp Glu Thr Asp 401 Gly Gln Asn Ser Leu Gln Phe Ala Val Tyr Asp Lys Lys Tyr Val Ile 417 Thr Lys Val Val Thr Gly Ala Thr Glu Asp Pro Glu Asn Thr Gly Trp 0 433 Val Ser Lys Val Asp Asp Leu Lys Gln Gly Thr Thr Gly Ala Tyr Val 449 Tyr Ile Asp Gln Asp Gly Leu Thr Leu His Ile Gln Thr Thr Thr Asn 465 Gly Asp Phe Ile Asn Arg His Thr Phe Gly Tyr Asn Asp Leu Val Tyr 481 Asp Ser Lys Ser Gly Tyr Gly Phe Thr Trp Ser Gly Asn Glu Gly Phe 497 Tyr Leu Asp Tyr His Asp Gly Asn Tyr Tyr Thr Phe His Asn Ala Ile 513 Ile Asn Tyr Tyr Pro Ser Gly Tyr Gly Gly Gly Ser Val Pro Asn Gly 529 Thr Trp Ala Leu Glu Gln Arg Ile Asn Glu Gly Trp Ala Ile Ala Pro 545 Leu Leu Asp Thr Leu His Thr Val Thr Val Lys Gly Ser Tyr Ile Ala 561 Trp Glu Gly Glu Thr Pro Thr Gly Tyr Asn Leu Tyr Ile Pro Asp Gly 577 Thr Val Leu Leu Asp Trp Phe Asp Lys Ile Asn Phe Ala Ile Gly Leu 593 Asn Lys Leu Glu Ser Val Phe Thr Ser Pro Asp Trp Pro Thr Leu Thr 609 Thr Ile Lys Asn Phe Ser Lys Ile Ala Asp Asn Arg Lys Phe Tyr Gln 625 Glu Ile Asn Ala Glu Thr Ala Asp Gly Arg Asn Leu Phe Lys Arg Tyr 641 Ser Thr Gln Thr Phe Gly Leu Thr Ser Gly Ala Thr Tyr Ser Thr Thr 657 Tyr Thr Leu Ser Glu Ala Asp Phe Ser Thr Asp Pro Asp Lys Asn Tyr 673 Leu Gln Val Cys Leu Asn Val Val Trp Asp His Tyr Asp Arg Pro Ser 689 Gly Lys Lys Gly Ala Tyr Ser Trp Val Ser Lys Trp Phe Asn Val Tyr 705 Val Ala Leu Gln Asp Ser Lys Ala Pro Asp Ala Ile Pro Arg Leu Val 721 Ser Arg Tyr Asp Ser Lys Arg Gly Leu Val Gln Tyr Leu Asp Phe Trp 737 Thr Ser Ser Leu Pro Ala Lys Thr Arg Leu Asn Thr Thr Phe Val Arg 753 Thr Leu Ile Glu Lys Ala Asn Leu Gly Leu Asp Ser Leu Leu Asp Tyr 76~

SUBSTITUTE SHEET tRULE 26) CA 022638l9 l999-02-26 769 Thr Leu Gln Ala Asp Pro Ser Leu Glu Ala Asp Leu Val Thr Asp Gly 5785 Lys Ser Glu Pro Met Asp Phe Asn Gly Ser Asn Gly Leu Tyr Phe Trp 801 Glu Leu Phe Phe His Leu Pro Phe Leu Val Ala Thr Arg Phe Ala Asn 817 Glu Gln Gln Phe Ser Pro Ala Gln Lys Ser Leu His Tyr Ile Phe Asp 833 Pro Ala Met Lys Asn Lys Pro His Asn Ala Pro Ala Tyr Trp Asn Val 849 Arg Pro Leu Val Glu Gly Asn Ser Asp Leu Ser Arg His Leu Asp Asp 20865 Ser Ile Asp Pro Asp Thr Gln Ala Tyr Ala His Pro Val Ile Tyr Gln 881 Lys Ala Val Phe Ile Ala Tyr val Ser Asn Leu Ile Ala Gln Gly Asp 897 Met Trp Tyr Arg Gln Leu Thr Arg Asp Gly Leu Thr Gln Ala Arg Val 913 Tyr Tyr Asn Leu Ala Ala Glu Leu Leu Gly Pro Arg Pro Asp Val Ser 929 Leu Ser Ser Ile Trp Thr Pro Gln Thr Leu Asp Thr Leu Ala Ala Gly 35945 Gln Lys Ala Val heu Arg Asp Phe Glu His Gln Leu Ala Asn Ser Asp 961 Thr Ala Leu Pro Ala Leu Pro Gly Arg Asn Val Ser Tyr Leu Lys Leu 977 Ala Asp Asn Gly Tyr Phe Asn Glu Pro Leu Asn Val Leu Met Leu Ser 993 His Trp Asp Thr Leu Asp Ala Arg Leu Tyr Asn Leu Arg His Asn Leu 1009 Thr Val Asp Gly Lys Pro Leu Ser Leu Pro Leu Tyr Ala Ala Pro Val 1025 Asp Pro Val Ala Leu Leu Ala Gln Arg Ala Gln Ser Gly Thr Leu Thr 1041 Asn Gly Val Ser Gly Ala Met Leu Thr Val Pro Pro Tyr Arg Phe Ser 1057 Ala Met Leu Pro Arg Ala Tyr Ser Ala Val Gly Thr Leu Thr Ser Phe 1073 Gly Gln Asn Leu Leu Ser Leu Leu Glu Arg Ser Glu Arg Ala Cys Gln 1089 Glu Glu Leu Ala Gln Gln Gln Leu Leu Asp Met Ser Ser Tyr Ala Ile 1105 Thr Leu Gln Gln Gln Ala Leu Asp Gly Leu Ala Ala Asp Arg Leu Ala 1121 Leu Leu Ala Ser Gln Ala Thr Ala Gln Gln Arg His Asp His Tyr Tyr SUE~STITUTE SHE~ ~RULE 26) W 098/08932 PCT~US97/07657 1137 Thr Leu Tyr Gln Asn Asn Ile Ser S~r Ala Glu Gln Leu Val Met Asp 1152 .
1153 Thr Gln Thr Ser Ala Gln Ser Leu Ile Ser Ser Ser Thr Gly Val Gln 1169 Thr Ala Ser Gly Ala Leu Lys Val Ile Pro Asn Ile Phe Gly Leu Ala 0 1185 Asp Gly Gly Ser Arg Tyr Glu Gly Val Thr Glu Ala Ile Ala Ile Gly 1201 Leu Met Ala Ala Gly Gln Ala Thr Ser Val Val Ala Glu Arg Leu Ala 1217 Thr Thr Glu Asn Tyr Arg Arg Arg Arg Glu Glu Trp Gln Ile Gln Tyr 1233 Gln Gln Ala Gln Ser Glu Val Asp Ala Leu Gln Lys Gln Leu Asp Ala 1249 Leu Ala Val Arg Glu Lys Ala Ala Gln Thr Ser Leu Gln Gln Ala Lys 1265 Ala Gln Gln Val Gln Ile Arg Thr Met Leu Thr Tyr Leu Thr Thr Arg 1281 Phe Thr Gln Ala Thr Leu Tyr Gln Trp Leu Ser Gly Gln Leu Ser Ala 1297 Leu Tyr Tyr Gln Ala Tyr Asp Ala Val Val Ala Leu Cys Leu Ser Ala 1313 Gln Ala Cys Trp Gln Tyr Glu Leu Gly Asp Tyr Ala Thr Thr Phe Ile 132e 1329 Gln Thr Gly Thr Trp Asn Asp His Tyr Arg Gly Leu Gln Val Gly Glu 1345 Thr Leu Gln Leu Asn Leu His Gln Met Glu Ala Ala Tyr Leu Val Arg 1361 His Glu Arg Arg Leu Asn Val Ile Arg Thr Val Ser Leu Lys Ser Leu 1377 Leu Gly Asp Asp Gly Phe Gly Lys Leu Lys Thr Glu Gly Lys Val Asp 1393 Phe Pro Leu Ser Glu Lys Leu Phe Asp Asn Asp Tyr Pro Gly His Tyr 1409 Leu Arg Gln Ile Lys Thr Val Ser Val Thr Leu Pro Thr Leu Val Gly 1425 Pro Tyr Gln Asn Val Lys Ala Thr Leu Thr Gln Thr Ser Ser Ser Ile 1441 Leu Leu Ala Ala Asp Ile Asn Gly Val Lys Arg Leu Asn Asp Pro Thr 1457 Gly Lys GlU Gly Asp Ala Thr His Ile Val Thr Asn Leu Arg Ala Ser 1473 Gln Gln Val Ala Leu Ser Ser Gly Ile Asn Asp Ala Gly Ser Phe Glu 1489 Leu Arg Leu Glu Asp Glu Arg Tyr Leu Ser Phe Glu Gly Thr Gly Ala 1505 Val Ser Lys Trp Thr Leu Asn Phe Pro Arg Ser Val Asp Glu His Ile SUBSTITUTE SHEET (RULE ~6) CA 022638l9 l999-02-26 W O 98/08932 PCT~US97/07657 1521 Asp Asp Lys Thr Leu Lys Ala Asp Glu Met Gln Ala Ala Leu Leu Ala 1537 Asn Met Asp Asp Val Leu Val Gln Val His Tyr Thr Ala Cys Asp Gly 1553 Gly Ala Ser Phe Ala Asn Gln Val Lys Lys Thr Leu Ser 1565 (2) INFORMATION FOR SEQ ID NO:60 (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 3132 base pairs (B) TYPE: nucleic acld (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:60 (tccC) 1 Met Ser Pro Ser Glu Thr Thr Leu Tyr Thr Gln Thr Pro Thr Val Ser 16 17 Val Leu Asp Asn Arg Gly Leu Ser Ile Arg Asp Ile Gly Phe His Arg 32 33 Ile Val Ile Gly Gly Asp Tnr Asp Thr Arg Val Thr Arg His Gln Tyr 48 49 Asp Ala Arg Gly His Leu Asn Tyr Ser Ile Asp Pro Arg Leu Tyr Asp 64 65 ~Ala Lys Gln Ala Asp Asn Ser Val Lys Pro Asn Phe Val Trp Gln His 80 81 Asp Leu Ala Gly His Ala Leu Arg Thr Glu Ser Val Asp Ala Gly Arg 96 97 Thr Val Ala Leu Asn Asp Ile Glu Gly Arg Ser Val Met Thr Met Asn 113 Ala Thr Gly Val Arg Gln Thr Arg Arg Tyr Glu Gly Asn Thr ~eu Pro 129 Gly Arg ~eu Leu Ser Val Ser Glu Gln Val Phe Asn Gln Glu Ser Ala SVBST~UTE SHEET (RULE 26) . .

CA 022638l9 l999-02-26 W O 98/08g32 PCTrUS97/07657 145 Lys Val Thr Glu Arg Phe Ile Trp Ala Gly Asn Thr Thr Ser Glu Lys 161 Glu Tyr Asn Leu Ser Gly Leu Cys Ile Arg His Tyr Asp Thr Ala Gly 177 Val Thr Arg Leu Met Ser Gln Ser Leu Ala Gly Ala Met Leu Ser Gln 193 Ser His Gln Leu Leu Ala Glu Gly Gln Glu Ala Asn Trp Ser Gly Asp 25209 Asp Glu Thr Val Trp Gln Gly Met Leu Ala Ser Glu Val Tyr Thr Thr 225 Gln Ser Thr Thr Asn Ala Ile Gly Ala Leu Leu Thr Gln Thr Asp Ala 241 Lys Gly Asn Ile Gln Arg Leu Ala Tyr Asp Ile Ala Gly Gln Leu Lys 257 Gly Ser Trp Leu Thr Val Lys Gly Gln Ser Glu Gln Val Ile Val Lys 273 Ser Leu Ser Trp Ser Ala Ala Gly His Lys Leu Arg Glu Glu His Gly 55289 Asn Gly Val Val Thr Glu Tyr Ser Tyr Glu Pro Glu Thr Gln Arg Leu 305 Ile Gly Ile Thr Thr Arg Arg Ala Glu Gly Ser Gln Ser Gly Ala Arg 321 Val Leu Gln Asp Leu Arg Tyr Lys Tyr Asp Pro Val Gly Asn Val Ile 336 lO09 AGT ATC CAT AAT GAT GCC GAA GCT ACC CGC TTT TGG CGT AAT CAG AAA

SUBSTITUTE ~EEr (RULE26) CA 022638l9 l999-02-26 W 098/08932 PCT~US97/07657 337 Ser Ile Hls Asn Asp Ala Glu Ala Thr Arg Phe Trp Arg Asn Gln Lys 352 353 Val Glu Pro Glu Asn Arg Tyr Val Tyr Asp Ser Leu Tyr Gln Leu Met 368 369 Ser Ala Thr Gly Arg Glu Met Ala Asn Ile Gly Gln Gln Ser Asn Gln 385 Leu Pro Ser Pro Val Ile Pro Val Pro Thr Asp Asp Ser Thr Tyr Thr 400 401 Asn Tyr Leu Arg Thr Tyr Thr Tyr Asp Arg Gly Gly Asn Leu Val Gln 416 417 Ile Arg His Ser Ser Pro Ala Thr Gln Asn Ser Tyr Thr Thr Asp Ile 432 433 Thr Val Ser Ser Arg Ser Asn Arg Ala Val Leu Ser Thr Leu Thr Thr 448 449 Asp Pro Thr Arg Val Asp Ala Leu Phe Asp Ser Gly Gly His Gln Lys 464 465 Met Leu Ile Pro Gly Gln Asn Leu Asp Trp Asn Ile Arg Gly Glu Leu 480 481 Gln Arg Val Thr Pro Val Ser Arg Glu Asn Ser Ser Asp Ser Glu Trp 496 1536 - ~
497 Tyr Arg Tyr Ser Ser Asp Gly Met Arg Leu Leu Lys Val Ser Glu Gln 512 513 Gln Thr Gly Asn Ser Thr Gln Val Gln Arg Val Thr Tyr Leu Pro Gly 528 529 Leu Glu Leu Arg Thr Thr Gly Val Ala Asp Lys Thr Thr Glu Asp Leu 544 545 Gln Val Ile Thr Val Gly Glu Ala Gly Arg Ala Gln Val Arg Val Leu 560 SUBSTITUTE SH EE-r ~RULE 2~) CA 022638l9 l999-02-26 W O 98l08932 PCTAUS97/07657 561 His Trp Glu Ser Gly Lys Pro Thr Asp Ile Asp Asn Asn Gln Val Arg 576 577 Tyr Ser Tyr Asp Asn Leu Leu Gly Ser Ser Gln Leu Glu Leu Asp Ser 592 593 Glu Gly Gln Ile Leu Ser Gln Glu Glu Tyr Tyr Pro Tyr Gly Gly Thr 608 609 Ala Ile Trp Ala Ala Arg Asn Gln Thr Glu Ala Ser Tyr Lys Phe Ile 624 625 Arg Tyr Ser Gly Lys Glu Arg Asp AIa Thr Gly Leu Tyr Tyr Tyr Gly 640 641 Tyr Arg Tyr Tyr Gln Pro Trp Val Gly Arg Trp Leu Ser Ala Asp Pro 656 657 Ala Gly Thr Val Asp Gly Leu Asn Leu Tyr Arg Met Val Arg Asn Asn 672 673 Pro Ile Thr Leu Thr Asp His Asp Gly Leu Ala Pro Ser Pro Asn Arg 688 689 Asn Arg Asn Thr Phe Trp Phe Ala Ser Phe Leu Phe Arg Lys Pro Asp 704 705 Glu Gly Met Ser Ala Ser Met Arg Arg Gly Gln Lys Ile Gly Arg Ala 720 721 Ile Ala Gly Gly Ile Ala Ile Gly Gly Leu Ala Ala Thr Ile Ala Ala 736 737 Thr Ala Gly Ala Ala Ile Pro Val Ile Leu Gly Val Ala Ala Val Gly 752 753 Ala Gly Ile Gly Ala Leu Met Gly Tyr Asn Val Gly Ser Leu Leu Glu 768 769 Lys Gly Gly Ala Leu Leu Ala Arg Leu Val Gln Gly Lys Ser Thr Leu 784 785 Val Gln Ser Ala Ala Gly Ala Ala Ala Gly Ala Ser Ser Ala Ala Ala 800 -28~-SUBSTITUTE SHF~ (RULE 26) CA 022638l9 l999-02-26 5801 Tyr Gly Ala Arg Ala Gln Gly Val Gly Val Ala Ser Ala Ala Gly Ala 816 0817 Val Thr Gly Ala Val Gly Ser Trp Ile As~ Asn Ala Asp Arg Gly Ile 832 15833 Gly Gly Ala Ile Gly Ala Gly Ser Ala Val Gly Thr Ile Asp Thr Met 848 20849 Leu Gly Thr Ala Ser Thr Leu Thr His Glu Val Gly Ala Ala Ala Gly 864 25865 Gly Ala Ala Gly Gly Met Ile Thr Gly Thr Gln Gly Ser Thr Arg Ala 880 30881 Gly Ile His Ala Gly Ile Gly Thr Tyr Tyr Gly Ser Trp Ile Gly Phe 896 35897 Gly Leu Asp Val Ala Ser Asn Pro Ala Gly His Leu Ala Asn Tyr Ala 912 40913 Val Gly Tyr Ala Ala Gly Leu Gly Ala Glu Met Ala Val Asn Arg Ile 928 45929 Met Gly Gly Gly Phe Leu Ser Arg Leu Leu Gly Arg Val Val Ser Pro 944 50945 Tyr Ala Ala Gly Leu Ala Arg Gln Leu Val HiS Phe Ser Val Ala Arg 960 55961 Pro Val Phe Glu Pro Ile Phe Ser Val Leu Gly Gly Leu Val Gly Gly 976 60977 Ile Gly Thr Gly Leu His Arg Val Met Gly Arg Glu Ser Trp Ile Ser 992 65993 Arg Ala Leu Ser Ala Ala Gly Ser Gly Ile Asp His Val Ala Gly Met SUBSTITIJTE SHEET (RULE 26) , . ... ....... ...

CA 022638l9 l999-02-26 WO 98/08932 PCTrUS97/07657 1009 Ile Gly Asn Gln Ile Arg Gly Arg Val Leu Thr Thr Thr Gly Ile Ala 1025 Asn Ala Ile Asp Tyr Gly Thr Ser Ala Val Gly Ala Ala Arg Arg Val 1041 Phe Ser Leu End 1043 (2) INFORMATION FOR SEQ ID NO:61 (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1043 amino acids (B) TYPE: amino acid (C) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:61 (TccC peptide) 1 Met Ser Pro Ser Glu Thr Thr Leu Tyr Thr Gln Thr Pro Thr Val Ser 16 17 Val Leu Asp Asn Arg Gly Leu Ser Ile Arg Asp Ile Gly Phe His Arg 32 33 Ile Val Ile Gly Gly Asp Thr Asp Thr Arg Val Thr Arg His Gln Tyr 48 49 Asp Ala Arg Gly His Leu Asn Tyr Ser Ile Asp Pro Arg Leu Tyr Asp 64 Ala Lys Gln Ala Asp Asn Ser Val Lys Pro Asn Phe Val Trp Gln His 80 81 Asp Leu Ala Gly His Ala Leu Arg Thr Glu Ser Val Asp Ala Gly Arg 96 97 Thr Val Ala Leu Asn Asp Ile Glu Gly Arg Ser Val Met Thr Met Asn 112 113 Ala Thr Gly Val Arg Gln Thr Arg Arg Tyr Glu Gly Asn Thr Leu Pro 128 129 Gly Arg Leu Leu Ser Val Ser Glu Gln Val Phe Asn Gln Glu Ser Ala 144 145 Lys Val Thr Glu Arg Phe Ile Trp Ala Gly Asn Thr Thr Ser Glu Lys 160 161 Glu Tyr Asn Leu Ser Gly Leu Cys Ile Arg His Tyr Asp Thr Ala Gly 176 177 V~al Thr Arg Leu Met Ser Gln Ser Leu Ala Gly Ala Met Leu Ser Gln 192 193 Ser His Gln Leu Leu Ala Glu Gly Gln Glu Ala Asn Trp Ser Gly Asp 208 209 Asp Glu Thr Val Trp Gln Gly Met Leu Ala Ser Glu Val Tyr Thr Thr 224 225 Gln Ser Thr Thr Asn Ala Ile Gly Ala Leu Leu Thr Gln Thr Asp Ala 240 241 Lys Gly Asn Ile Gln Arg Leu Ala Tyr Asp Ile Ala Gly Gln Leu Lys 256 257 Gly Ser Trp Leu Thr Val Lys Gly Gln Ser Glu Gln Val Ile Val Lys 272 273 Ser Leu Ser Trp Ser Ala Ala Gly His Lys Leu Arg Glu Glu His Gly 288 289 Asn Gly Val Val Thr Glu Tyr Ser Tyr Glu Pro Glu Thr Gln Arg Leu 304 305 Ile Gly Ile Thr Thr Arg Arg Ala Glu Gly Ser Gln Ser Gly Ala Arg 320 321 Val Leu Gln Asp Leu Arg Tyr Lys Tyr Asp Pro Val Gly Asn Val Ile 336 337 Ser Ile His Asn Asp Ala Glu Ala Thr Arg Phe Trp Arg Asn Gln Lys 352 SUBSTrrUTE SHE~ (RULE 26) 353 Val Glu Pro Glu Asn Arg Tyr Val Tyr Asp Ser Leu Tyr Gln Leu Met 368 369 Ser Ala Thr Gly Arg Glu Met Ala Asn Ile Gly Gln Gln Ser Asn Gln 384 385 Leu Pro Ser Pro Val Ile Pro Val Pro Thr Asp Asp Ser Thr Tyr Thr 400 401 Asn Tyr Leu Arg Thr Tyr Thr Tyr Asp Arg Gly Gly Asn Leu Val Gln 416 417 Ile Arg His Ser Ser Pro Ala Thr Gln Asn Ser Tyr Thr Thr Asp Ile 432 433 Thr Val Ser Ser Arg Ser Asn Arg Ala Val Leu Ser Thr Leu Thr Thr 448 449 Asp Pro Thr Arg Val Asp Ala Leu Phe Asp Ser Gly Gly His Gln Lys 464 465 Met Leu Ile Pro Gly Gln Asn Leu Asp Trp Asn Ile Arg Gly Glu Leu 480 481 Gln Arg Val Thr Pro Val Ser Arg Glu Asn Ser Ser Asp Ser Glu Trp 496 497 Tyr Arg Tyr Ser Ser Asp Gly Met Arg Leu Leu ~ys Val Ser Glu Gln 512 513 Gln Thr Gly Asn Ser Thr Gln Val Gln Arg Val Thr Tyr Leu Pro Gly 528 529 Leu Glu Leu Arg Thr Thr Gly Val Ala Asp Lys Thr Thr Glu Asp Leu 544 545 Gln Val Ile Thr Val Gly Glu Ala Gly Arg Ala Gln Val Arg Val Leu 560 561 His Trp Glu Ser Gly Lys Pro Thr Asp Ile Asp Asn Asn Gln Val Arg 576 577 Tyr Ser Tyr Asp Asn Leu Leu Gly Ser Ser Gln Leu Glu Leu Asp Ser 592 593 Glu Gly Gln Ile Leu Ser Gln Glu Glu Tyr Tyr Pro Tyr Gly Gly Thr 608 609 Ala Ile Trp Ala Ala Arg Asn Gln Thr Glu Ala Ser Tyr Lys Phe Ile 624 625 Arg Tyr Ser Gly Lys Glu Arg Asp Ala Thr Gly Leu Tyr Tyr Tyr Gly 640 641 Tyr Arg Tyr Tyr Gln Pro Trp Val Gly Arg Trp Leu Ser Ala Asp Pro 656 657 Ala Gly Thr Val Asp Gly Leu Asn Leu Tyr Arg Met Val Arg Asn Asn 672 673 Pro Ile Thr Leu Thr Asp His Asp Gly Leu Ala Pro Ser Pro Asn Arg 688 689 Asn Arg Asn Thr Phe Trp Phe Ala Ser Phe Leu Phe Arg Lys Pro Asp 704 705 Glu Gly Met Ser Ala Ser Met Arg Arg Gly Gln Lys Ile Gly Arg Ala 720 721 Ile Ala Gly Gly Ile Ala Ile Gly Gly Leu Ala Ala Thr Ile Ala Ala 736 737 Thr Ala Gly Ala Ala Ile Pro Val Ile Leu Gly Val Ala Ala Val Gly 752 753 Ala Gly Ile Gly Ala Leu Met Gly Tyr Asn Val Gly Ser Leu Leu Glu 768 769 Lys Gly Gly Ala Leu Leu Ala Arg Leu Val Gln Gly Lys Ser Thr Leu 784 785 Val Gln Ser Ala Ala Gly Ala Ala Ala Gly Ala Ser Ser Ala Ala Ala 800 801 Tyr Gly Ala Arg Ala Gln Gly Val Gly Val Ala Ser Ala Ala Gly Ala 816 817 Val Thr Gly Ala Val Gly Ser Trp Ile Asn Asn Ala Asp Arg Gly Ile 832 833 Gly Gly Ala Ile Gly Ala Gly Ser Ala Val Gly Thr Ile Asp Thr Met 848 849 Leu Gly Thr Ala Ser Thr Leu Thr His Glu Val Gly Ala Ala Ala G~y 864 865 Gly Ala Ala Gly Gly Met Ile Thr Gly Thr Gln Gly Ser Thr Arg Ala 880 881 Gly Ile His Ala Gly Ile Gly Thr Tyr Tyr Gly Ser Trp Ile Gly Phe 896 897 Gly Leu Asp Val Ala Ser Asn Pro Ala Gly His Leu Ala Asn Tyr Ala 912 913 Val Gly Tyr Ala Ala Gly Leu Gly Ala Glu Met Ala Val Asn Arg Ile 928 SUBSTITlJTE St~EET (RULE 26) CA 022638l9 l999-02-26 WO 98~8932 PCTAUS97/07657 929 Met Gly Gly Gly Phe Leu Ser Arg Leu Leu Gly Arg Val Val Ser Pro 944 945 Tyr Ala Ala Gly Leu Ala Arg Gln Leu Val His Phe Ser Val Ala Arg 960 961 Pro Val Phe Glu Pro Ile Phe Ser Val Leu Gly Gly Leu Val Gly Gly 976 977 Ile Gly Thr Gly Leu His Arg Val Met Gly Arg Glu Ser Trp Ile Ser 992 0 993 Arg Ala Leu Ser Ala Ala Gly Ser Gly Ile Asp His Val Ala Glv Met 1008 1009 Ile Gly Asn Gln Ile Arg Gly Arg Val Leu Thr Thr Thr Gly Ile Ala 1024 1025 Asn Ala Ile Asp Tyr Gly Thr Ser Ala Val Gly Ala Ala Arg Arg Val 1040 1041 Phe Ser Leu 1043 (2) INFORMATION FOR SEQ ID NO:62: TcaAiv (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 5 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: protein (v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:62: TcaAiv Asn Ile Gly Gly Asp (2) INFORMATION FOR SEQ ID NO:63: TcaAii-syn (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: protein (v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:63: TcaAii-syn Cys Leu Arg Gly Asn Ser Pro Thr Asn Pro Asp Lys Asp Gly Ile l 5 10 15 Phe Ala Gln Val Ala (2) INFORMATION FOR SEQ ID NO:64: TcaA~ syn (i) SEQUENCE CHARACTERISTICS;
(A) LENGTH: 20 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: protein (v) FRAGMENT TYPE: Internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:64: TcaAiii-syn Cys Tyr Thr Pro Asp Gln Thr Pro Ser Phe Tyr Glu Thr Ala Phe SUBSTITUTE SHEET (RULE 26) , .

Arg Ser Ala Asp Gly (2) INFORMATION FOR SEQ ID NO:65: TcaB -syn (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 19 amino acids (B) TYPE: amlno acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: protein (v) FRAGMENT TYPE: Internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 65: TcaBi-syn His Gly Gln Ser Tyr Asn Asp Asn Asn Tyr Cys Asn Phe Thr Leu Ser Ile Asn Thr (2) INFORMATION FOR SEQ ID NO:66: TcaBii-syn (i) SEQUENCE CHARACTERISTICS:
2 5 (A) LENGTH: 20 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: protein (v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:66: TcaBii-syn Cys Val Asp Pro Lys Thr Leu Gln Arg Gln Gln Ala Gly Gly Asp Gly Thr Gly Ser Ser (2) INFORMATION FOR SEQ ID NO:67: TcaC-syn (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: protein (v) FRAGMENT TYPE: internal (xi~ SEQUENCE DESCRIPTION: SEQ ID NO:67: TcaC-syn 5~
Cys Tyr Lys Ala Pro Gln Arg Gln Glu Asp Gly Asp Ser Asn Ala Val Thr Tyr Asp Lys SUBSTITUTE SltEET tRULE 26) CA 022638l9 l999-02-26 W098/08932 PCTrUS97/0765 (2) INFORMATION FOR SEQ ID NO:68: TcbAii-syn (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: protein (v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:68: TcbAii-syn Cys Tyr Asn Glu Asn Pro Ser Ser Glu Asp Lys ~ys Trp Tyr Phe Ser Ser Lys Asp Asp (2) INFORMATION FOR SEQ ID NO:69: TcbAiii-syn (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: protein (v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:69: TcbA~ syn Cys Phe Asp Ser Tyr Ser Gln Leu Tyr Glu Glu Asn Ile Asn Al~
1 5 lO 15 Gly Glu Gln Arg Ala (2) INFORMATION FOR SEQ ID NO:70: TcdAii-syn (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: protein (v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:70: TcdAii-syn Cys Asn Pro Asn Asn Ser Ser Asn Lys ~eu Met Phe Tyr Pro Val Tyr Gln Tyr Ser Gly Asn Thr (2) INFORMATION FOR SEQ ID NO:71: TcdAiii-syn (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: protein (v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:71: TcdAiii-syn SUBSTlTUT~ SH EET (RULE 26) W098/08932 PCT~S97/07657 Val Ser Gln Gly Ser Gly Ser Ala Gly Ser Gly ~sn Asn Asn Leu Ala Phe Gly Ala Gly (2) INFORMATION FOR SEQ ID NO:72:
SEQUENCE CHARACTERISTICS:
(A) LENGTH: 12 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: protein (v) FRAGMENT TYPE: N-terminal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:72: 160 kDa - Hb Met Gln Asp Ser Pro Glu Val Ala Ile Thr Thr Leu (2) INFORMATION FOR SEQ ID NO:73:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 8 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: protein (v) FRAGMENT TYPE: N-terminal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:73: 170 kDa - WIR
Met Gln Arg Ser Ser Glu Val Ser (2) INFORMATION FOR SEQ ID NO:74:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 12 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: protein . .
(v) FRAGMENT TYPE: N-terminal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:74: 180 kDa - H9 Met Gln Asp Ile Pro Glu Val Gln Leu asn (xi) SEQUENCE DESCRIPTION: SEQ ID NO:75: 170 kDa - Hm(2) INFORMATION FOR SEQ ID NO:75:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 12 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: protein (v) FRAGMENT TYPE: N-terminal Met Gln Asp Ser Pro Glu Val Ser Val Thr Gln Asn SUBSTITUTE SHEET ~RULE 2~) CA 022638l9 l999-02-26 W098/08932 PCTrUS97/07657 (2) INFORMATION FOR SEQ ID NO:76:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii~ MOLECULAR TYPE: protein (v) FRAGMENT TYPE: N-terminal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:76: 74 kDa - H9 Ser Glu Ser Leu Phe Thr Gln Ser Leu Lys Glu Ala Arg Arg Asp l 5 10 15 (2) INFORMATION FOR SEQ ID NO:77:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: protein (v) FRAGMENT TYPE: N-terminal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:77: 71 kDa - Hb Met Asn Leu Ile Glu Ala Lys Leu Gln Glu Asn Arg Asp Ala (2) INFORMATION FOR SEQ ID NO:78:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: protein (v) FRAGMENT TYPE: N-terminal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:78: 170 kDa - H9 Met Leu Ser Thr Met Glu Lys Gln Leu Asn Glu Ser Gln Arg Asp (2) INFORMATION FOR SEQ ID NO:79:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids ~B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: protein (v) FRAGMENT TYPE: N-terminal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:79: 109 kDa - Hm Met ~eu Asp Ile Met Glu Lys Gln Leu Asn Glu Ser Glu Arg Asp SVBSTITU~E S~{ EET tRULE 2~) W098/08932 PCTrUS97/07657 (2) INFORMATION FOR SEQ ID NO:80:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 8 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: slngle (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: protein (v) FRAGMENT TYPE: N-terminal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:80: 170 kDa - WX-1 Met Gln Asp Ser Arg Glu Val Ser (2) INFORMATION FOR SEQ ID NO:81:
(i~ SEQUENCE CHARACTERISTICS:
(A) LENGTH: 12 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: protein (v) FRAGMENT TYPE: N-terminal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:81: 69 kDa - H9 Leu Arg Ser Ala Xxx Ser Ala Leu Thr Thr Leu Leu (2) INFORMATION FOR SEQ ID NO:82:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids (B) TYPE: amino acid ~ (C) STRANDEDNESS: single ~D) TOPOLOGY: linear (ii) MOLECULAR TYPE: protein (v) FRAGMENT TYPE: N-terminal 45 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:82: 64 kDa - HP88 Leu Lys Leu Ala Asp Asn Gly Tyr Phe Asn Glu Pro Leu Asn Val (2) INFORMATION FOR SEQ ID NO:83:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: protein (v) FRAGMENT TYPE: N-terminal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:83: 70 kDa - NC-l Leu Lys Leu Ala Asp Asn Ser Tyr Phe Asn Glu Pro Leu Asn l 5 10 15 SUBSTITUTE SHE~T tRULE 26) CA 022638l9 l999-02-26 W098l08932 PCTAUS97/07657 (2~ INFORMATION FOR SEQ ID NO:84:
(i) SEQ~N~ CHARACTERISTICS:
(A) LENGTH: 15 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: protein (v) FRAGMENT TYPE: N-terminal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:84: 60 kDa - WIR
Ser Lys Asp Glu Ser Lys Ala Asp Ser Gln Leu Val Tyr His Thr (2) INFORMATION FOR SEQ ID NO:85:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids (B) TYPE: amino acid ~C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: protein (v) FRAGMENT TYPE: N-terminal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:85: 58 kDa - NC-l Met Lys Lys Arg Gly Leu Thr Thr Asn Ala Gly Ala Pro Val (2) INFORMATION FOR SEQ ID NO:86:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single ( D) TOPOLOGY: linear (ii) MOLECULAR TYPE: protein (v) FRAGMENT TYPE: N-terminal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:86: 60 kDa - WX-12 Met Leu Asn Pro Ile Val Arg Lys Phe Glu Tyr Gly Glu His Thr (2) INFORMATION FOR SEQ ID NO:87:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: protein (v) FRAGMENT TYPE: N-terminal 60 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:87: 60 kDa - Hm Ala Glu Ile Tyr Asn Lys Asp Gly Asn Lys Leu Asp Leu Tyr Gly SU8ST~TUTE SHEE~ (RULE 2~) CA 022638l9 l999-02-26 W098/08932 PCT~US97/07657 (2) INFORMATION FOR SEQ ID NO:88:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: protein (v) FRAGMENT TYPE: N-terminal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:88: 140 kDa - Hm Asn Leu Ile Glu Ala Thr Leu Glu Gln Asn Leu Arg Asp Ala ~ .
_ SUBSTITUTE SHEET (RULE 26) , .. ,: ...... .. .

Claims (99)

We claim:

1. A composition, comprising an effective amount of a Photorhabdus protein toxin that has functional activity against an insect.

2. The composition of Claim 1, wherein the Photorhabdus toxin is produced by a purified culture of Photorhabdus, a transgenic plant, baculovirus, or heterologous microbial host.

3. The composition of Claim 2, wherein the Photorhabdus toxin produced by a purified culture of Photorhabdus luminescens.

4. The composition of Claim 2, wherein the toxin is produced from a purified culture of Photorhabdus luminescens strain designated ATCC 55397.

5. The composition of Claim 2, wherein the toxin is produced by a purified culture of Photorhabdus luminescens strain designated W-14.

6. The composition of Claim 1, wherein the toxin is produced by a purified culture of Photorhabdus strain designated WX-1, WX-2, WX-3, WX-4, WX-5, WX6, WX-7, WX-8, WX-9, WX-10, WX-11, WX-12, WX-14, WX-15, H9, Hb, Hm, HP88, NC-1, W30, WIR, B2, ATCC# 43948, ATCC# 43949, ATCC# 43950, ATCC#
43951, ATCC# 43952, DEP1, DEP2, DEP3, P. zealandrica, P.
hepialus, HB-Arg, HB Oswego, HB Oswego, HB Lewiston, K-122, HMGD, Indicus, GD, PWH-5, Megidis, HF-85, A. Cows, MP1, MP2, MP3, MP4, MP5, GL98, GL101, GL138, GL55, GL217, or GL257.

7. The composition of Claim 2, wherein the toxin is produced from a purified culture of Photorhabdus luminescens strain designated WX-1, WX-2, WX-3, WX-4, WX-5, WX-6, WX-7, WX-8, WX-9, WX-10, WX-11, WX-12, WX-14, WX-15, H9, Hb, Hm, HP88, NC-1, W30, WIR, B2, ATCC# 43948, ATCC# 43949, ATCC#
43950, ATCC# 43951, ATCC# 43952, DEP1, DEP2, DEP3, P.
zealandrica, P. hepialus, HB-Arg, HB Oswego, HB Oswego, HB
Lewiston, K-122, HMGD, Indicus, GD, PWH-5, Megidis, HF-85, A.
Cows, MP1, MP2, MP3, MP4, MP5, GL98, GL101, GL138, GL55, GL217, or GL257.

8. The composition of Claim 1, wherein the toxin is represented by amino acid sequence is SEQ ID NO:12.

9. The composition of Claim 6, wherein the composition is a mixture of one or more toxins produced from purified cultures of Photorhabdus.

10. The composition of Claim 1 or 6, wherein the insect is of the order Lepidoptera, Coleoptera, Hymenoptera, Diptera, Dictyoptera, Acarina or Homoptera.

11. The composition of Claim 1 or 6, wherein the insect species is from order Coleoptera and is Southern Corn Rootworm, Western Corn Rootworm, Colorado Potato Beetle, Mealworm, Boll Weevil or Turf Grub.

12. The composition of Claim 1 or 6, wherein the insect species is from order Lepidoptera and is Beet Armyworm, Black Cutworm, Cabbage Looper, Codling Moth, Corn Earworm, European Corn Borer, Tobacco Hornworm, or Tobacco Budworm.

13. The composition of Claim 1 or 6, wherein the toxin is formulated as a sprayable insecticide.

14. The composition of Claim 1 or Claim 6, wherein the toxin is formulated as a bait matrix and delivered in an above ground or below ground bait station.

15. A method of controlling an insect, comprising orally delivering to an insect an effective amount of a protein toxin that has functional activity against an insect, wherein the protein is produced by a purified bacterial culture of the genus Photorhabdus.

16. The method of Claim 15, wherein the bacterium is a purified culture of Photorhabdus luminescens.

17. The method of Claim 15, wherein the toxin is produced from a purified culture of Photorhabdus luminescens strain designated ATCC 55397.

18. The method of Claim 16, wherein the toxin is produced from a purified culture of Photorhabdus luminescens strain designated W-14.

19. The method of Claim 15, wherein the toxin is produced from a purified culture of Photorhabdus strains designated WX-1, WX-2, WX-3, WX-4, WX-5, WX-6, WX-7, WX-8, WX-9, WX-10, WX-11, WX-12, WX-14, WX-15, H9, Hb, Hm, HP88, NC-1, W30, WIR, B2, ATCC# 43948, ATCC# 43949, ATCC# 43950, ATCC#
43951, ATCC# 43952, DEP1, DEP2, DEP3, P. zealandrica, P.
hepialus, HB-Arg, HB Oswego, HB Oswego, HB Lewiston, K-122, HMGD, Indicus, GD, PWH-5, Megidis, HF-85, A. Cows, MP1, MP2, MP3, MP4, MP5, GL98, GL101, GL138, GL155, GL217, or GL257.

20. The method of Claim 15, wherein the toxin is produced from a purified culture of Photorhabdus luminescens strains designated WX-1, WX-2, WX-3, WX-4, WX-5, WX-6, WX-7, WX-8, WX-9, WX-10, WX-11, WX-12, WX-14, WX-15, H9, Hb, Hm, HP88, NC-1, W30, WIR, B2, ATCC# 43948, ATCC# 43949, ATCC#
43950, ATCC# 43951, ATCC# 43952, DEP1, DEP2, DEP3, P.
zealandrica, P. hepialus, HB-Arg, HB Oswego, HB Oswego, HB
Lewiston, K-122, HMGD, Indicus, GD, PWH-5, Megidis, HF-85, A.
Cows, MP1, MP2, MP3, MP4, MP5, GL98, GL101, GL138, GL155, GL217, or GL257.

21. The method of Claim 19, wherein a mixture of one or more toxins is produced from a purified culture of Photorhabdus and said toxins are orally delivered to an insect.

22. The method of Claim 15, wherein the toxin is produced by a prokaryotic host transformed with a gene encoding the toxin.

23. The method of Claim 15, wherein the toxin is produced by a eukaryotic host transformed with a gene encoding the toxin.

24. The method of Claim 23, wherein the eukaryotic host is baculovirus.

25. The method of Claim 15 or 19, wherein the insect is of the order Lepidoptera, Coleoptera, Hymenoptera, Diptera, Dictyoptera, Acarina or Homoptera.

26. The method of Claim 15 or 19, wherein the insect species is from order Coleoptera and is Southern Corn Rootworm, Western Corn Rootworm, Colorado Potato Beetle, Mealworm, Boll Weevil or Turf Grub.

27. The method of Claim 15 or 19, wherein the insect species is from order Lepidoptera and is Beet Armyworm, Black Cutworm, Cabbage Looper, Codling Moth, Corn Earworm, European Corn Borer, Tobacco Hornworm, or Tobacco Budworm.

28. The method of Claim 15 or 19, wherein the toxin is formulated as a sprayable insecticide.

29. The method of Claim 15 or Claim 19, wherein the toxin is formulated as a bait matrix and delivered in an above ground or below ground bait station.

30. A method of isolating a gene coding for a protein subunit, comprising the steps of: constructing at least one RNA or DNA oligonucleotide molecule that corresponds to at least a part of a DNA coding region of an amino acid sequence selected from a group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID
NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO: 13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID
NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:36, SEQ ID NO:37, SEQ ID
NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:62, SEQ ID NO:72, SEQ ID NO:73, SEQ ID
NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:82, SEQ ID
NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87, and SEQ ID NO:88, wherein the nucleotide molecule is used to isolate genetic material from Photorhabdus or Photorhabdus luminescens.

31. A method for expressing a protein produced by a purified bacterial culture of the genus Photorhabdus in a prokaryotic or eukaryotic host in an effective amount so that the protein has functional activity against an insect, wherein the method comprises: constructing a chimeric DNA construct having 5' to 3' a promoter, a DNA sequence encoding a protein, a transcription terminator, and then transferring the chimeric DNA construct into the host.

32. The method of Claim 31, wherein the protein has functional activity against insects selected from a group consisting of Coleoptera, Lepidoptera, Diptera, Homoptera, Hymenoptera, Dictyoptera, and Acarina.

33. The method of Claim 31, wherein the protein encoded by the DNA sequence has an N-terminal amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID
NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ
ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:
13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID
NO:22, SEQ ID NO:23, SEQ ID NO-24, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID
NO:42, SEQ ID NO:43, SEQ ID NO:62, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID
NO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID
NO:87, and SEQ ID NO:88.

34. The method of Claim 31, wherein the protein encoded by the DNA sequence includes the amino acid sequence selected from the group consisting of SEQ ID NO:12, SEQ ID NO:26, SEQ
ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID
NO:35, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, and SEQ ID NO:61.

35. A chimeric DNA construct, adapted for expression in a prokaryotic or eukaryotic host comprising, 5' to 3' a transcriptional promoter active in the host; a DNA sequence encoding a Photorhabdus protein that has functional activity against an insect; and a transcriptional terminator.

36. A chimeric DNA construct of Claim 35, wherein the protein encoded by the DNA sequence has an N-terminal amino acid sequence selected from the group consisting of SEQ ID
NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO: 5, SEQ

ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO: 13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ
ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID
NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID
NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:62, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID
NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID
NO:86, SEQ ID NO:87, and SEQ ID NO:88.

37. The chimeric DNA construct of Claim 35, wherein the protein encoded by the DNA sequence has an amino acid sequence selected from the group consisting of SEQ ID NO:12, SEQ ID
NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51, SEQ ID
NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, and SEQ ID
NO:61.

38. The chimeric DNA construct of Claim 35, wherein the DNA sequence encoding the Photorhabdus luminescens protein is selected from the group comprising SEQ ID NO:11, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID
NO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO: 58, and SEQ ID NO:60.

39. The chimeric DNA construct of Claim 35, wherein the host is baculovirus or a plant cell.

40. An isolated and substantially purified preparation comprising, a DNA molecule capable of encoding an effective amount of a protein that is produced by a bacterium of the genus Photorhabdus and that has functional activity against an insect.

41. The preparation of Claim 40, wherein the bacterium is Photorhabdus luminescens.

42. A purified preparation comprising, a protein produced by Photorhabdus or Photorhabdus luminescens having an N-terminal amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID
NO:4, SEQ ID NO:5, SEQ ID NO.6, SEQ ID NO:7, SEQ ID NO:8, SEQ

ID NO:9, SEQ ID NO:10, SEQ ID NO: 13, SEQ ID NO:14, SEQ ID
NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID
NO:24, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID
NO:62, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID
NO:80, SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87, and SEQ ID NO:88.

43. A purified protein preparation comprising, a protein that has an N-terminal amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID
NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ
ID NO:9, and SEQ ID NO:10, SEQ ID NO: 13, SEQ ID NO:14, SEQ ID
NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID
NO:24, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:62, SEQ ID NO:72, SEQ ID
NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81, SEQ ID
NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87, and SEQ ID NO:88.

44. A purified protein preparation comprising, a protein selected from the group of SEQ ID NO:12, SEQ ID NO:26, SEQ ID
NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID
NO:55, SEQ ID NO:57, SEQ ID NO:59, and SEQ ID NO:61.

45. A purified DNA preparation comprising, a DNA
sequence selected from the group consisting of SEQ ID NO:11, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID
NO:33, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58 and SEQ ID NO:60, wherein the DNA sequence is isolated from its native host.

46. A purified protein preparation comprising, a Photorhabdus luminescens protein with at least one subunit having an approximate molecular weight between 18 kDa to about 230 kDa; between about 160 kDa to about 230 kDa; 100 kDa to 160 kDa; about 80 kDa to about 100 kDa; or about 50 kDa to about 80 kDa.

47. A purified protein preparation comprising, a Photorhabdus luminescens protein with at least one subunit having an approximate molecular weight of about 280 kDa.

48. A substantially pure microorganism culture comprising, ATCC 55397.

49. The culture of Claim 48, wherein the culture is a derivative of ATCC 55397 that produces a protein toxin that has functional activity against an insect.

50. A transgenic plant comprising in its genome, a chimeric artificial gene construction imbuing the plant with an ability to express an effective amount of a Photorhabdus protein that has functional activity against an insect.

51. The transgenic plant of Claim 50, wherein the plant is transformed using acceleration of genetic material coated onto microparticles directly into cells, Agrobacteria, whiskers, or electroporation techniques

52. The transgenic plant of Claim 50, wherein the selectable marker is selected from the group consisting of kanamycin, neomycin, glyphosate, hygromycin, methotrexate, phosphinothricin (bialophos), chlorosulfuron, bromoxynil, dalapon and the like.

53. The transgenic plant of Claim 50, wherein the promoter is selected from the group consisting of octopine synthase, nopaline synthase, mannopine synthase, 35S, 19S, 35T, ribulose-1,6-bisphosphate (RUBP) carboxylase small subunit (ssu), beta-conglycinin, phaseolin, alcohol dehydrogenase (ADH), heat-shock, ubiquitin, zein, oleosin, napin, or acyl carier protein (ACP).

54. The transgenic plant of Claim 50, wherein embryogenic tissue, callus tissue type I or II, hypocotyl, meristem, or plant tissue during dedifferentiation is used in preparing the transgenic plant.

55. The transgenic plant of Claim 50, wherein the chimeric gene is a DNA sequence which encodes a Photorhabdus protein that has functional activity against an insect and at least one codon of the gene has been modified so that the codon is a plant preferred codon.

56. A method of controlling an insect comprising orally delivering to an insect an effective amount of a protein toxin, wherein the protein is produced by a transgenic plant, which said insect feeds.

57. A composition of matter, comprising a purified DNA
sequence from a purified bacterial culture from the genus Photorhabdus .

58. A substantially pure microorganism culture comprising, H9.

59. A substantially pure microorganism culture comprising, Hb.

60. A substantially pure microorganism culture comprising, Hm.

61. A substantially pure microorganism culture comprising, HP88.

62. A substantially pure microorganism culture comprising, NC-l.

63. A substantially pure microorganism culture comprising, W30.

64. A substantially pure microorganism culture comprising, WIR.

65. A substantially pure microorganism culture comprising, B2.

66. A substantially pure microorganism culture comprising, P. zealandrica.

67. A substantially pure microorganism culture comprising, P. hepialus.

68. A substantially pure microorganism culture comprising, HB-Arg.

69. A substantially pure microorganism culture comprising, HB Oswego.

70. A substantially pure microorganism culture comprising, HB Lewiston.

71. A substantially pure microorganism culture comprising, K-122.

72. A substantially pure microorganism culture comprising, HMGD.

73. A substantially pure microorganism culture comprising, Indicus.

74. A substantially pure microorganism culture comprising, GD.

75. A substantially pure microorganism culture comprising, PWH-5.

76. A substantially pure microorganism culture comprising, Megidis.

77. A substantially pure microorganism culture comprising, HF-85.

78. A substantially pure microorganism culture comprising, A. Cows.

79. A substantially pure microorganism culture comprising, MP1.

80. A substantially pure microorganism culture comprising, MP2.

81. A substantially pure microorganism culture comprising, MP3.

82. A substantially pure microorganism culture comprising, MP4.

83. A substantially pure microorganism culture comprising, MP5.

84. A substantially pure microorganism culture comprising, GL98.

85. A substantially pure microorganism culture comprising, GL155.

86. A substantially pure microorganism culture comprising, GL101.

87. A substantially pure microorganism culture comprising, GL138. - -

88. A substantially pure microorganism culture comprising, GL217.

89. A substantially pure microorganism culture comprising, GL257.

90. A method of making an antibody against a protein fragment that is part of a protein having functional activity, where the protein is produced by bacteria of the Enterobacteracaea family, wherein the method comprises:

a) isolating a fragment of the protein, where the protein fragment is at least six amino acids b) immunizing a mammalian species with the protein fragment; and c) harvesting serum containing antibody or antibody from the spleen of the mammalian species, where the antibody harvested is antibody to the protein fragment having functional activity.

91. The method of Claim 1, wherein the protein fragment is selected from the group consisting of SEQ ID NO:63, SEQ ID
NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, and SEQ ID NO:71.

92. The method of Claim 90, wherein the bacteria is from the genus Photorhabdus.

93. The method of Claim 90, wherein the bacteria is from the genus Photorhabdus luminescens.

94. A method of selecting a DNA fragment which encodes a portion of a protein that has functional activity, where the protein is produced from a bacteria of the Enterobacteracaea family, wherein the method comprises:

a) isolating a fragment of the DNA sequence having at least 30 nucleotides;

b) tagging the DNA fragment with a radioactive or chemical agent;

c) hybridizing the DNA fragment to a DNA library, where the DNA library is an Enterobacteracaea cDNA or Enterobacteracaea genomic library; and.

d) selecting the fragment that is hybridized to the DNA
in the library that encodes for the protein that has functional activity.

95. The method of Claim 94, wherein the bacteria is from the genus Photorhabdus.

96. The method of Claim 95, wherein the bacteria is from the genus Photorhabdus luminescens.

97. A method of selecting a DNA fragment which encodes a portion of a protein that has functional activity, where the protein is produced from a bacteria of the Enterobacteracaea family, wherein the method comprises:

a) isolating at least two primers, where a primer is a fragment of DNA having at least twelve nucleotides;

b) using the primers from step a), amplifying a DNA
fragment from Enterobacteracaea by using primers with polymerase chain reaction technology and purifying the DNA
fragment;

c) tagging the purified DNA fragment with a radioactive or chemical agent;

d) hybridizing the purified DNA fragment to a DNA
library, where the DNA library is an Enterobacteracaea cDNA or Enterobacteracaea genomic library; and e) selecting a DNA fragment that is equal or larger in size to the purified DNA fragment from the library, where the selected DNA fragment or portion thereof encodes for a protein that has functional activity.

98. The method of Claim 97, wherein the bacteria is from the genus Photorhabdus.

99. The method of Claim 98, wherein the bacteria is from the genus Photorhabdus luminescens.