WO1997017432A1 - Toxines proteiques insecticides provenant de photorhabdus - Google Patents

Toxines proteiques insecticides provenant de photorhabdus Download PDF

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Publication number
WO1997017432A1
WO1997017432A1 PCT/US1996/018003 US9618003W WO9717432A1 WO 1997017432 A1 WO1997017432 A1 WO 1997017432A1 US 9618003 W US9618003 W US 9618003W WO 9717432 A1 WO9717432 A1 WO 9717432A1
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WIPO (PCT)
Prior art keywords
seq
protein
photorhabdus
toxin
insect
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PCT/US1996/018003
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English (en)
Inventor
Jerald C. Ensign
David J. Bowen
James Petell
Raymond Fatig
Sue Schoonover
Richard H. Ffrench-Constant
Thomas A. Rocheleau
Michael B. Blackburn
Timothy D. Hey
Donald J. Merlo
Gregory L. Orr
Jean L. Roberts
James A. Strickland
Lining Guo
Todd A. Ciche
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Wisconsin Alumni Research Foundation
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Priority to UA97084103A priority Critical patent/UA82485C2/uk
Priority to AU10509/97A priority patent/AU729228B2/en
Application filed by Wisconsin Alumni Research Foundation filed Critical Wisconsin Alumni Research Foundation
Priority to RO97-01251A priority patent/RO121280B1/ro
Priority to JP51836997A priority patent/JP3482214B2/ja
Priority to EP96941335A priority patent/EP0797659A4/fr
Priority to CA002209659A priority patent/CA2209659C/fr
Priority to MX9705101A priority patent/MX9705101A/es
Priority to RU97113033/13A priority patent/RU2216174C2/ru
Priority to IL121243A priority patent/IL121243A/en
Priority to PL96321212A priority patent/PL186242B1/pl
Priority to BR9606889A priority patent/BR9606889A/pt
Priority to SK931-97A priority patent/SK93197A3/sk
Priority to KR1019990701698A priority patent/KR20000037116A/ko
Priority to JP10511612A priority patent/JP2000515024A/ja
Priority to TR1999/01126T priority patent/TR199901126T2/xx
Priority to IL12859097A priority patent/IL128590A0/xx
Priority to CA002263819A priority patent/CA2263819A1/fr
Priority to SK246-99A priority patent/SK24699A3/sk
Priority to AU28299/97A priority patent/AU2829997A/en
Priority to PCT/US1997/007657 priority patent/WO1998008932A1/fr
Priority to EP97922696A priority patent/EP0970185A4/fr
Priority to BR9711441-3A priority patent/BR9711441A/pt
Priority to PL97332033A priority patent/PL332033A1/xx
Publication of WO1997017432A1 publication Critical patent/WO1997017432A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • C12N5/12—Fused cells, e.g. hybridomas
    • C12N5/14—Plant cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8279—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance
    • C12N15/8286—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance for insect resistance
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
    • A01G13/00—Protecting plants
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
    • A01N63/50—Isolated enzymes; Isolated proteins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/24—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/52—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
    • Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
    • Y02A40/146—Genetically Modified [GMO] plants, e.g. transgenic plants

Definitions

  • the present invention relates to toxins isolated from bacteria and the use of said toxins as insecticides.
  • 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.
  • 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.
  • the relatively new tools of the genetic engineer allow modifications to biological insecticides to accomplish particular solutions to particular problems.
  • Bacillus thuringiensis is an effective insecticidal agent, and is widely commercially used as such.
  • 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.
  • 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.
  • insecticidal nematodes in the Steinernema and Heterorhabditis genera were used as insect control agents.
  • each genus of nematode hosts a particular species of bacterium.
  • the symbiotic bacterium is Photorhabdus luminescens.
  • 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
  • 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.
  • 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 TcbA ii and TcaB ii proteins.
  • Fig. 5 is a phenogram of Photorhabdus strains. Relationship of Photorhabdus Strains was defined by rep-PCR.
  • 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% similarityl).
  • Fig. 6 is an illustration of the genomic maps of the W-14 Strain.
  • 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
  • Heterorhabdi tis Another such source is from human clinical samples from wounds, see Farmer et al. 1989 J. Clin. Microbiol. 27 pp. 1594-1600. These saprohytic strains are deposited in the American Type Culture Collection (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 either terrestrial or aquatic based.
  • the genus Photorhabdus is taxonomically defined as a member of the Family Enterobacteriaceae, although it has certain traits atypical of this family. For example, strains of this genus are nitrate reduction negative, yellow and red pigment producing and bioluminescent. This latter trait is otherwise unknown within the Enterobacteriaceae.
  • Photorhabdus has only recently been described as a genus separate from the Xenorhabdus (Boemare et al., 1993 Int. J. Syst. Bacteriol. 43, 249-255).
  • This differentiation is based on DNA-DNA hybridization studies, phenotypic differences (e.g., presence (Photorhabdus) or absence (Xenorhabdus) of catalase and bioluminescence) and the Family of the nematode host (Xenorhabdus; Steinernematidae, Photorhabdus; Heterorhabditidae). Comparative, cellular fatty-acid analyses (Janse et al. 1990, Lett. Appl. Microbiol 10, 131-135; Suzuki et al. 1990, J. Gen. Appl. Microbiol., 36, 393-401) support the separation of Photorhabdus from Xenorhabdus.
  • Photorhabdus luminescens ATCC Type strain #29999, Poinar et al., 1977, Nematologica 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;
  • 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.
  • 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.
  • the bacteria of the genus Photorhabdus produce proteins that have functional activity as defined herein.
  • proteins produced by the species Photorhabdus luminescens 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.
  • 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
  • the protein toxins 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.
  • the results are typically death of the insect, or the insects do not feed upon the source which makes the toxins available to the insects.
  • the protein toxins discussed herein are typically referred to as "insecticides”.
  • insecticides it is meant herein that the protein toxins have a "functional activity” as further defined herein and are used as insect control agents.
  • oligonucleotides it is meant a
  • RNA or DNA 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
  • 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.
  • oligonucleotides may be less than 10 or greater than 12.
  • genetic material By the use of the term “genetic material” herein, it is meant to include all genes, nucleic acid, DNA and RNA.
  • Table 18 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 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
  • insect orders include but are not limited to Coleoptera, Homoptera, Lepidoptera,
  • Toxins of interest are those which produce protein complexes toxic to a variety of insects upon exposure, as described herein.
  • 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
  • Photorhabdus toxin any protein produced by a Photorhabdus microorganism strain which has functional activity against insects, where the Photorhabdus toxin could be formulated as a sprayable composition, expressed by a transgenic plant, formulated as a bait matrix, delivered via a Baculovirus, or delivered by any other applicable host or delivery system.
  • 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.
  • 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.
  • 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 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.
  • parasitic nematode hosts a particular species of bacterium, uniquely adapted for symbiotic growth with that nematode.
  • 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 nomenclature is reflected in this specification, but in no way should a change in nomenclature alter the scope of the inventions 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.
  • the sequences listed above are grouped by genomic region.
  • the tcbA gene was expressed in E. coli as two protein fragments TcbA and TcbA iii as illustrated in the Examples. It may be beneficial to have proteolytic clippage of some sequences to obtain the higher activity of the toxins for commercial
  • the toxins described herein are quite unique in that the toxins have functional activity, which is key to 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.
  • 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
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • Plants may be transformed using Agrobacterium technology, see U.S. 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 all to
  • tissue which is contacted with the foreign genes may vary as well.
  • 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.
  • selectable marker 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.
  • selectable markers include but are not limited to aminoglycoside phosphotransferase gene of
  • 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.
  • reporter gene 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.
  • GUS glucuronidase
  • the gene is preferably incorporated into a gene transfer vector adapted to express the Photorhabdus toxins in the plant cell by including in the vector a plant promoter.
  • promoters from a variety of sources can be used efficiently in plant cells to express foreign genes.
  • promoters of bacterial origin such as the octopine synthase promoter, the nopaline synthase promoter, the mannopine synthase promoter;
  • Plant promoters of viral origin such as the cauliflower mosaic virus (35S and 19S)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 enhancer 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.
  • 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.
  • RNA instability may lead to RNA instability.
  • 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
  • AAUAAA polyadenylation signal sequences
  • RNA instability may lead to RNA instability.
  • 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.
  • 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.
  • 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 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.
  • 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 amino acid sequence of the toxins are reverse translated into a DNA sequence, utilizing a nonredundant 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.
  • 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.
  • 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.
  • 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 purification 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.
  • oligonucleotides complementary to all, or a section of, the DNA bases that encode the first amino acids of the toxin can be radiolabeled and used as 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
  • oligonucleotides require 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.
  • 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.
  • expression vector is a DNA plasmid, though other transfer means including, but not limited to, cosmids, phagemids and phage are also envisioned.
  • transfer means including, but not limited to, cosmids, phagemids and phage are also envisioned.
  • cosmids 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
  • 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.
  • a transcriptional promoter upstream 5'
  • a transcriptional termination region such as a poly-A addition site
  • a ribosome binding site upstream of the gene of interest's first codon.
  • T7 RNA Polymerase-binding promoter is the T7 RNA Polymerase-binding promoter. Promoters, as previously described herein, are known to
  • the vector may include a nucleotide sequence encoding a 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
  • Insect viruses 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.
  • 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.
  • different baculoviruses are specific to different insects, it may be possible to use a particular toxin to
  • 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.
  • the proteins may be encapsulated using Baci ll us churingi ensi s encapsulation technology such as but not limited to U.S. Patent Nos. 4,695,455; 4,695,462; 4,861,595 which are all incorporated herein by reference.
  • Baci ll us churingi ensi s encapsulation technology such as but not limited to U.S. Patent Nos. 4,695,455; 4,695,462; 4,861,595 which are all incorporated herein by reference.
  • Another delivery 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 include but are not limited to PCT Patent Application WO
  • a genetically modified toxin gene might encode a toxin exhibiting, for example, enhanced or reduced toxicity, altered insect resistance development, altered stability, or modified target species specificity.
  • 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.
  • the toxins used in the present invention seem to first inhibit larval feeding before death ensues.
  • nucleic acid examples include the addition of targeting sequences to direct the toxin to particular parts of the insect larvae for improving its
  • 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.
  • Tris tris (hydroxymethyl) amino methane
  • SDS sodium dodecyl sulfate
  • EDTA ethylenediaminetetraacetic acid
  • IPTG IPTG
  • insecticidal protein toxin of the present invention was purified from P. luminescens strain W-14, ATCC Accession Number
  • 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 subunit shifts from about 200 kDa 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 K 2 HPO 4 by adding an appropriate volume of 1.0 M K 2 HPO 4 .
  • the pH was adjusted to 8.6 by adding potassium hydroxide.
  • This supernatant fraction was then mixed with DEAE-Sephacel (Pharmacia LKB Biotechnology) which had been equilibrated with 50 mM K 2 HPO 4 .
  • 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 K 2 HPO 4 at room temperature at a flow rate of 30 ml/hr until the effluent reached a steady baseline UV 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 KPO 4 , pH 6.9, using an ultrafiltration membrane with a molecular weight cutoff of 100 kDa 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 ⁇ 95 cm Sephacryl S-400 HR gel filtration column (Pharmacia LKB Biotechnology).
  • the eluent buffer was 100 mM KPO 4 , pH 6.9, which was run at a flow rate of 17 ml/hr, at 4°C.
  • the effluent was monitored at 280 nm.
  • the toxic fractions were pooled and concentrated using the Centriprep-100 and were then analyzed by HPLC using a 7.5 mm x 60 cm TSK-GEL G-4000 SW gel permeation column with 100 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
  • Electrophoresis of the pooled peak fractions in a non-denaturing agarose gel 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 1M 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.
  • 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 containing the protein. Protein was recovered from the gel pieces by electroelution into 100 mM Tris-borate pH 3.3, at 100 volts (constant voltage) for two hours.
  • 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.
  • Agarose-separated protein band 1 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.
  • Protein bands 1 and 2 were composed of several smaller protein subunits. Proteins were visualized by Coomassie brilliant blue staining followed by silver staining to achieve maximum sensitivity. The protein subunits in the two bands were very similar. Protein band 1 contains 8 protein subunits of 25.1, 56.2, 60.3, 65.6, 166, 171, 184 and 208 kDa. Protein band 2 had an identical profile except that the 25.1, 60.8, and 65.6 kDa proteins were not present. The 56.2, 60.8, 65.6, and 184 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 M. 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 HPLC-purified toxin was tested for ability to kill insects other than Manduca sexta.
  • Table 2 lists insects killed by the HPLC-purified P. luminescens toxin in this study.
  • Photorhabdus 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.
  • 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.
  • 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 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 4oC prior to bioassay or filtration.
  • Photorhabdus culture broth and protein toxin(s) purified from this broth showed activity (mortality and/or growth
  • 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.
  • Homoptera Other members of the Homoptera include planthoppers, pear pyslla, apple sucker, scale insects, whiteflies, and 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 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, 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 3.
  • 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
  • Turf grubs Popillia japonica, 2-3rd instar
  • 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 (Photorhabdus culture broth, control medium or H 2 0) 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.
  • 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
  • the ingestion assay for aster leafhopper (Macrosteles severini) is 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 ⁇ 10 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 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.
  • undiluted Photorhabdus culture broth the broth and control medium were dialyzed against water to reduce control mortality.
  • 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.
  • Photorhabdus l uminescens (strain W-14) culture broth was shown to be active against corn rootworm when applied directly to soil or a soil-mix (Metromix ® ).
  • Activity against neonate SCR and WCR in Metromix ® was tested as follows (Table 4). 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 roots were approximately 3-6 cm long, a single kernel/seedling was planted in a 591 ml clear plastic 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 remaining 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.
  • 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 broth to 25 ml of water for 50 ml total volume.
  • PP3 proteose peptone #3
  • positions 13 and 14 are both arginine (arg).
  • amino acid residue at position 6 may be either alanine (ala) or serine (ser).
  • amino acid residue at position 3 may be aspartic acid (asp).
  • 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 HPLC-purif ied toxin as described in Example 5, Part A, above. This peptide comes from the tcaA gene. The peptide labeled TcaA ii , starts at position 254 and goes to position 491, where the
  • TcaA iii peptide starts, SEQ ID NO: 4.
  • the estimated size of the peptide based on the gene sequence is 25,240 Da.
  • the toxin protein complex was re-isolated from the Photorhabdus luminescens growth medium (after culture without Tween) by performing a 10% - 80% ammonium 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).
  • a second, prominent 185 kDa protein was consistently present in amounts comparable to that of protein 3 from Table 9, 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.
  • 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 9. 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.
  • the pellet was resuspended in one-tenth the volume of 10 mM Na 2 ⁇ PO 4 , pH 7.0 and dialyzed against the same phosphate buffer overnight at 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 10 mM Na 2 ⁇ PO 4 , pH 7.0. 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.
  • the excluded protein peak was subjected to a second
  • 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.
  • TcdA ii A 201 kDa (TcdA ii ) peptide set forth as SEQ ID NO:.13 below shared between 33% amino acid identity and 50% similarity with SEQ ID NO:1 (TcbA ii ) (Table 10, in Table 10 vertical lines denote amino acid identities and colons indicate conservative amino acid substitutions).
  • a second peptide of 197 kDa, SEQ ID NO:14 (TcdB) had 42% identity and 58% homology with SEQ ID NO:2 (TcaC).
  • TcdA ii A 201 kDa (TcdA ii ) peptide set forth as SEQ ID NO:.13 below shared between 33% amino acid identity and 50% similarity with SEQ ID NO:1 (TcbA ii ) (Table 10, in Table 10 vertical lines denote amino acid identities and colons indicate conservative amino acid substitutions).
  • a limited N-terminal amino acid sequence, SEQ ID NO: 16 (TcbA), of a peptide of at least 235 kDa was identical in homology with the amino acid sequence, SEQ ID NO: 12, deduced from a cloned gene (tcbA), SEQ ID NO: 11,
  • 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
  • TcbA ii containing SEQ ID NO:1
  • TcdA ii containing SEQ ID NO: 3
  • TcaB containing SEQ ID NO: 3
  • TcaB i 205 kDa peptide
  • TcaB i -PT111 SEQ ID NO: 17
  • TcaB i -PT79 SEQ ID NO: 18
  • TcaB i -PT158 SEQ ID NO: 19
  • TcaB i -PT108 SEQ ID NO:20
  • TcbA ii 201 kDa peptide
  • TCBAII-PT103 SEQ ID NO.21
  • TcbA ii -PT56 SEQ ID NO:22
  • TcbA ii -PT81(a) SEQ ID NO:23
  • TcbA ii -PT81 (b) SEQ ID NO:24.
  • 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
  • Photorhabdus luminescens strain W-14 (ATCC accession number 55397) 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 -20°C 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 remaining 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-HCl, 1 mM EDTA, pH 8.0) to a final volume of 10 ml, then 12 ml of 5 M NaCl was added; 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
  • preparation contained 2.5 mg/ml DNA, as determined by optical density at 260 nm (i.e., OD 260 ).
  • 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
  • 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 NaCl 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
  • the DNA was collected by centrifugation (17,000 x g, 15 min), dried, redissolved in TE, pooled into a final volume of 80 ⁇ l, and reprecipitated with the addition of 8 ⁇ l 3 M sodium acetate and 220 ⁇ l ethanol.
  • 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.
  • the dephosphorylated DNA was precipitated by addition of 72 ⁇ l of 7.5 M ammonium acetate and 550 ⁇ l -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 10X ligation buffer supplied by the manufacturer) of the protocol in section 3.33 of Ausubel. Ligation was carried out overnight in a total volume of 20 ⁇ l at 15°C, followed by storage at -20°C.
  • the packaged preparation was stored at 4°C until use.
  • the packaged cosmid preparation was used to infect Escherichia col i XLl Blue MR cells (Stratagene) according to the Gigapack ® III Gold protocols ("Titering the Cosmid Library"), as follows.
  • XL1 Elue 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 MgSO 4 , at 37°C.
  • the packaged cosmid library was diluted 1:10 or 1:20 with sterile SM medium (0.1 M NaCl, 10 mM MgSO 4 , 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 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 at rays onto Magna NT (MSI, Westboro, MA) nylon membranes (0.45 micron, 220 ⁇ 250 mm) which had been placed on solid LB-Amp 100 (100 ml/dish) in Bio-assay plastic dishes (Nunc, 243 ⁇ 243 ⁇ 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
  • 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-HCl pH 8.0, 1.5 M NaCl for 15 min. After UV-crosslinking using a Stratagene UV 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. ci t .
  • This pool corresponds to the determined N-terminal amino acid sequence of the TcaC peptide.
  • oligonucleotides were used as primers in Polymerase Chain Reactions (PCR ® , Roche Molecular Systems, Branchburg, NJ) to amplify a specific UNA tragment from genomic DNA prepared from Photorhabdus strain W-14 (see above).
  • a typical reaction 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, IX GeneAmp ® PCR buffer, and 2.5 units of AmpliTaq ® DNA polymerase (both from Roche Molecular Systems; 10X GeneAmp ® buffer is 100 mM 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 NuSieve ® 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 ⁇ l contained the same components as above, minus genomic template DNA. Following amplification, the ends of the fragments were made blunt and were phosphorylated by
  • T4 DNA polymerase NEB
  • 1 nmol ATP 1 nmol ATP
  • 2.15 units of T4 kinase 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.
  • FMC 1% w/v GTG ® agarose
  • a gel slice containing fragments of apparent size 250 bp was excised, and the DNA was extracted using a Qiaex kit (Qiagen Inc.,
  • the extracted DNA fragments were ligated to plasmid vector pBC KS(+) (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 ⁇ l) contained 100 ng of digested pBC KS(+) DNA, 70 ng of 250 bp fragment DNA, 1 nmol [Co(NH 3 ) 6 ]Cl 3 , and 3.9 Weiss units of T4 DNA ligase (Collaborative Biomedical Products, Bedford, MA), in 1X ligation buffer (50 mM Tris-HCl, pH 7.4; 10 mM MgCl 2 ; 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
  • 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 proline,
  • bases 257-276 encode one of the 192 possible sequences designed into the degenerate pool
  • 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.
  • TcaC peptide gene-specific probe DNA fragments corresponding to the above 276 bases were 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 units of AmpliTAq ® DNA polymerase, and IX 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 32 P-dCTP using the High Prime Labeling Mix
  • 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 ⁇ 10 8 dpm/ ⁇ g. This labeled DNA was used to probe membranes prepared from 800 members of the genomic library.
  • the twelve putative hybridization-positive colonies were retrieved from the frozen 96-well library plates and grown overnight at 37°C on solid LB-Amp 100 medium. They were then patched (3/plate, plus three negative controls: XL1 Blue MR cells containing the pWE15 vector) onto solid LB-Amp 100 .
  • 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-Amp 100 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.
  • 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 NaCl.
  • the DNA was UV crosslinked to the filter (as above), and the filters were washed (25°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 prehybridizat ion 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 3 2 P-labeled probe that had been saved from the previous
  • Hybridization was performed at 60°C for 16 hr with shaking at 50 rpm.
  • 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 0.25X SSC plus 0.2% SDS (minimal hyb wash solution), and exposed to X-ray film as described above for 1.5 hr at 25°C (no enhancer 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 with the negative control (pWE15) colonies, 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.
  • peptide fraction (disclosed here as SEQ ID NO: 3) a pool of degenerate oligonucleotides (pool P8F) was synthesized as described for peptide TcaC.
  • SEQ ID NO: 3 a pool of degenerate oligonucleotides
  • oligonucleotides were used as primers for PCR ® using
  • HotStart 50 TubesTM (Molecular Bio-Products, Inc., San Diego, CA) to amplify a specific DNA fragment from genomic DNA prepared from
  • Amplifications were performed by 35 cycles as described for the TcaC peptide. Amplification products were analyzed by
  • TcaB i which corresponds to a portion of the N-terminal peptide sequence disclosed as SEQ ID NO:3 (TcaB i ). Labeling of a TcaB i -peptide gene-specific probe.
  • TcaB i 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.
  • 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).
  • the 32 P-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 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 1 fragment of both these two cosmids, 26A5 and 25A10.
  • the membrane was stripped of radioactivity by boiling for about 30 min in 0.1X SSC plus 0.1 % SDS, and absence of
  • radiolabel was checked by exposure to film. It was then
  • TcaB i probe hybridized at 60°C for 3.5 hours with the (denatured! TcaB i 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 -70°C with two enhancer screens. With both cosmids, the TcaB i probe hybridized lightly with the about 5.0 kbp EcoR 1 fragment, and strongly with a fragment of
  • DH5 ⁇ cells DH5 ⁇ cells
  • This DNA 2.5 ⁇ g was digested with about 3 units of EcoR 1 (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 1 in a total volume of 20 ⁇ l, leading to total digestion as confirmed by electrophoresis.
  • 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 UV crosslinking as 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 TcaB i probe solution described earlier, according to the 'minimal hyb' protocol, followed by washing and film exposure as described for the library colony membranes.
  • 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, TcaB i -hybridizing EcoR 1 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-Cam 35 to an OD 600 of 2, then plasmid was isolated according to the manufacturer's directions. DNA pellets were redissolved in 100 ⁇ l TE each, and sample integrity was checked by EcoR 1 digestion and gel electrophoretic analysis.
  • ATTGCAGACTGCCAATCGCTTCGG GAGAGTATCCAGACCGCGGATGATCTG).
  • primers were made by selecting appropriate sequence for new primers. With a few exceptions, primers
  • CGCGCAATTAACCCTCACTAAAG CGCGCAATTAACCCTCACTAAAG
  • T7 primer pBS KS (+) bases 621-643: GCGCGTAATACGACTCACTATAG
  • 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 ⁇ 5 kbp fragment. A total of 55 oligo primers was used, enabling the identification of 4832 total bp or contiguous sequence.
  • TcaB i -PT158 internal peptide (disclosed herein as SEQ ID NO:19). Further downstream, in the same reading frame, at bases 1738-1773, exists a sequence encoding the TcaB i -PT108 internal peptide (disclosed herein as SEQ ID NO: 20). Also in the same reading frame, at bases 1897-1923, is encoded the TcaB ii N-terminal peptide (disclosed herein as SEQ ID NO:5), and the reading frame continues uninterrupted to a translation termination codon at nucleotides 3586-3588.
  • TcaB ii N-terminal peptide represents the C-terminal amino acid of peptide TcaB i
  • the predicted mass of TcaB ii (627 amino acids) is 70,814 Daltons (disclosed herein as SEQ ID NO:28), somewhat higher than the size observed by SDS-PAGE (68 kDa).
  • SEQ ID NO:28 the predicted mass of TcaB ii (627 amino acids) is 70,814 Daltons
  • SEQ ID NO:28 the predicted mass of TcaB ii (627 amino acids) is 70,814 Daltons (disclosed herein as SEQ ID NO:28), somewhat higher than the size observed by SDS-PAGE (68 kDa).
  • SEQ ID NO:27 the predicted mass of TcaB ii lies somewhat closer to the C-terminus of TcaB i -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.
  • the size of this peptide to designate the C-terminus of the TcaB i coding region [Glu at position 604 of SEQ ID NO:28]
  • the derived size of TcaB i is determined to be 604 amino acids or 68,463 Daltons, more in agreement with
  • TcaB ii peptide coding region of 1686 base pairs yields a protein of 562 amino acids (disclosed herein as SEQ ID NO: 30) with predicted mass of 60,789 Daltons, which corresponds well with the observed 61 kDa.
  • a potential ribosome binding site (bases 3633-3638) is found 48 bp downstream of the stop codon for the tcaB open reading frame.
  • At bases 3645-3677 is found a sequence encoding the N-terminus of peptide TcaC, (disclosed as SEQ ID NO.2).
  • 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 NO.31).
  • 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.
  • This example describes a method used to identify DNA clones that contain the TcbA ii peptide-encoding genes, the isolation of the gene, and the determination of its partial DNA base sequence Primers and PCR reactions
  • the TcbA ii polypeptide of the insect active preparation is ⁇ 206 kDa.
  • the amino acid sequence of the N-terminus of this peptide is disclosed as SEQ ID NO:1.
  • 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.
  • a primary (“a”) and a secondary (“b") sequence of an internal peptide preparation have been determined and are disclosed herein as SEQ ID No:23 and SEQ ID No:24, respectively.
  • b a secondary sequence of an internal peptide preparation
  • 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.
  • 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.
  • a solution containing 10X GeneAmp ® PCR Buffer II, DNA template, and AmpliTaq ® DNA polymerase were added.
  • final reaction conditions were: 10 mM Tris-HCl, pH 8.3; 50 mM KCl; 2.5 mM MgCl 2 ; 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.
  • thermocycler as in
  • TcbA ii internal peptides are disclosed herein as SEQ ID NO: 21 and SEQ ID NO: 22.
  • 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.
  • oligonucleotides TH-18 and TH-17 were used in an amplification experiment with Photorhabdus l uminescens 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 32 P-labeled probe (as described above) prepared from the 2.9 kbp product amplified by the TH-5+TH10 primer pair. Both the 4 kbp and the 4.5 kbp amplification products hybridized strongly to the 2.9 kbp probe. These results were used to construct a map ordering the TcbA ii internal peptide sequences as shown in
  • 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., TH-5 end) of the gel-purified 2.9 kbp TcbA ii -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:1.
  • the sequence of the DNA fragment identified as SEQ ID NO: 11 encodes a protein whose derived amino acid sequence is disclosed herein as SEQ ID NO: 12.
  • SEQ ID NO: 12 The sequence of the DNA fragment identified as SEQ ID NO: 11 encodes a protein whose derived amino acid sequence is disclosed herein as SEQ ID NO: 12.
  • the TcbA ii N-terminal peptide (SEQ ID NO:1; Phe He Gln Gly Tyr Ser Asp Leu Phe Gly Asn Arg Ala) is encoded as amino acids 88-100.
  • TcbA ii internal peptide TcbA ii - PT81(a) (SEQ ID NO:23) is encoded as amino acids 1065-1077
  • TcbA ii -PT81(b) (SEQ ID NO:24) is encoded as amino acids 1571-1592
  • the internal peptide TcbA ii -PT56 (SEQ ID NO:22) is encoded as amino acids 1474-1488
  • the internal peptide TcbA ii -PT103 (SEQ ID NO:24) is encoded as amino acids 1614-1639. It is obvious that this gene is an authentic clone encoding the TcbA ii peptide as isolated from insecticidal protein preparations of Photorhabdus l uminescens strain W-14.
  • TcbA ii The protein isolated as peptide TcbA ii is derived from cleavage of a longer peptide. Evidence for this is provided by the fact that the nucleotides encoding the TcbA ii N-terminal peptide SEQ ID NO: 1 are preceded by 261 bases (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 correspond to the N-terminal sequence of the large peptide TcbA, and is disclosed herein as SEQ ID 11O:16. It is thought that TcbA is the precursor protein for TcbA ii Relationship of tcbA , tcaB and tcaC genes.
  • tcaB and ccaC genes are closely linked and may be
  • tcbA gene transcribed as a single mRNA (Example 8).
  • the tcbA gene is borne on cosmids that apparently do not overlap the ones harboring the t caB and t caC cluster, since the respective genomic library
  • up carats ( ⁇ ) indicate homology or conservative amino acid changes
  • down carats (v) indicate nonhomology.
  • Protease Inhibition, Classification, and Purification Protease Inhibition and Classification 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 ⁇ l 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 precipitate undigested protein.
  • protease inhibitors included E-64 (L-trans-expoxysaccinylleucylamido[4-, -guaindino]-butane), 3,4 dichloroisocoumarin, Leupeptin, pepstatin, amastatin,
  • EDTA ethylenediaminetetraacetic acid
  • 1,10 phenanthroline 1,10 phenanthroline
  • the isolation of a zinc-metalloprotease was performed by applying dialyzed 10-80% ammonium sulfate pellet to a Q Sepharose column equilibrated at 50 mM Na 2 PO 4 , pH 7.0 as described in Example 5 for Photorhabdus toxin. After extensive washing, a 0 to 0.5 M NaCl 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 was present in the 0.25-0.35 M NaCl fraction with some activity in the 0.15-0.25 M NaCl fraction.
  • SDS-stacking gels (1.0 ⁇ 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).
  • proteolytic 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.
  • FITC-Casein with different protease concentrations at 37° C for 0-10 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.
  • Inhibition studies were performed 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 water, respectively.
  • Stock 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.
  • 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 S1Y100 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 S1Y10 attached to an Amicon M-12 filtration device.
  • the retentate material contained proteins ranging in size from 10-100 kDa.
  • Fractions were assayed for protease activity. Those fractions having the greatest amount of phosphoramidon-sensitive protease 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 NaCl. 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 0.75 ml using a Millipore Ultrafree ® -15 centrifugal filter device Biomax-5K NMWL membrane. This material was applied at a flow rate of 0.5 ml/min to a Pharmacia HR 10/30 column that had been packed with Pharmacia Sephadex G-50 equilibrated in 10 mM sodium phosphate buffer (pH 7.0)/ 0.1 M NaCl.
  • Standard SDS-PAGE analysis for examining 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 . , (ProBlottTM Membranes; Applied Biosystems, Foster City, CA), visualized with 0.1% amido black, excised, and sent to Cambridge Prochem; Cambridge, MA, for sequencing.
  • 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.
  • Genomic DNA was isolated from the Photorhabdus l uminescens 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
  • the genomic DNA was isolated using a modification of the
  • the precipitated DNA was hooked and wound around the end of a bent glass rod, dipped briefly into 70% ethanol as a final wash, and dissolved in 3 ml of TE buffer.
  • the DNA concentration estimated by optical density at 230/260 nm, was approximately 2 mg/ml.
  • polyclonal antibodies were created by taking native 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
  • 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 immunoblot 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 five classes of clones.
  • TcbA ii Full DNA sequence of this gene ( TcbA) was obtained. It is set forth as SEQ ID NO: 11. Confirmation that the sequence encodes the internal sequence of SEQ ID NO:1 is demonstrated by the presence of SEQ ID NO:1 at amino acid number 38 from the deduced amino acid sequence created by the open reading frame of SEQ ID NO: 11. This can be confirmed by
  • SEQ ID NO: 12 is the deduced amino acid sequence created by SEQ ID NO: 11.
  • the second class of toxin peptides contains the segments referred to above as TcaB i , TcaB ii and TcaC.
  • this second class of toxin genes was identified by several clones which produced different size proteins, all of which cross-reacted with the polyclonal antibody on an immunoblot 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.
  • the deduced amino acid from the tccA open reading frame indicates the gene encodes a protein of 105,459 Da. This protein was designated TccA.
  • the first 12 amino acids of this protein match the N-terminal sequence obtained from a 108 kDa protein, SEQ ID NO: 7, previously identified as part of the toxin complex.
  • the deduced amino acid from the tccB open reading frame indicates this gene encodes a protein of 175,716 Da. This protein was designated TccB.
  • 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: 8.
  • TccC 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.
  • 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. (Table 18). Reference Escherichia col i , Xenorhabdus and Photorhabdus strains were included in all tests for comparison. The overall results are consistent with all strains being part ot 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.
  • the broths from each strain 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).
  • cell density was established by measuring light absorbance (560 nM) in a Gilford Systems
  • MIS Microbial Identification System
  • the MIS system consists of a Hewlett Packard HP5890A gas chromatograph with a 25mm ⁇ 0.2mm 5% methylphenyl silicone fused silica capillary 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
  • Genomic organization is believed to be shaped by selection and the differential
  • Rep-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
  • 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 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.
  • 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
  • Photorhabdus genomic DNA the following primers were used, REP1R-I; 5'-IIIICGICGICATCIGGC-3' and REP2-I; 5'-ICGICTTATCIGGCCTAC-3'. PCR was performed using the following 25 ⁇ l reaction: 7.75 ⁇ l H 2 O,
  • 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 uM retention) glass filters to remove debris. Further broth clarification was achieved with a tangential flow microfiltration device (Pall Filtron,
  • processed broth samples was acheived by heating the samples at 100°C in a sand-filled heat block for 10 minutes.
  • 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 19. It is possible that additional insecticidal activities 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 .
  • 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, so ⁇ 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 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) [0.23 mg/ml] or 10 mM sodium phosphate buffer , pH 7.0 were applied directly to the surface (about 1.5 cm 2 ) 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 7.0.
  • 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
  • the assay was conducted in a 96-well microtiter plate. Each well contained 200 ⁇ l of aqueous solution (10-fold concentrated
  • Purchased Drosophi l a melanogaster medium was prepared using 50% dry medium and a 50% liquid of either water, control medium (2% Proteose Peptone #3), 10-fold concentrated Photorhabdus culture broth(s), purified toxin complex(es) [0.23 mg/ml] or 10 mM sodium phosphate buffer , pH 7.0. 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
  • Drosophi la 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.
  • Control mortality was less than 6%.
  • 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) [0.23 mg/ml] or 10 mM sodium phosphate buffer, pH 7.0 were applied directly to the surface ( ⁇ 1.5 cm 2 ) 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.
  • Control mortality generally ranged from 4-12.5% for control medium and was less than 10% for phosphate buffer.
  • Activity against two-spotted spider mite 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) [0.23 mg/ml] or 10 mM sodium phosphate buffer, pH 7.0. 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.
  • the protocol 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).
  • SCR Southern corn root worm
  • 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 S1Y100 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 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.
  • the majority of the activity against SCR was contained in the 0 4 M fraction.
  • the 0.4 M fraction was further purified by
  • 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 20, 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:
  • Sizes of the individual polypeptides comprising the SCR toxin complex from each strain are listed in Table 21.
  • the sizes of the individual polypeptides ranged from 230 kDa with strain WX-1 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 complex that were in the 160-230 kDa range, the 100-160 kDa range, and the 50-80 kDa range.
  • 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.
  • the toxin complex from strains Hm and W-14 also exhibited activity against two-spotted spider mite.
  • the toxin complex from W-14 exhibited activity against mosquito larvae.
  • 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 1ml /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).
  • SCR Southern corn rootworm
  • Peaks of activity were determined by a series of dilutions of each fraction in SCR bioassays. Two activity 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 (NH 4 ) 2 SO 4 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 (NH 4 ) 2 SO 4 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 (NH 4 ) 2 SO 4 , 0% ethylene glycol, 50 mM potassium phosphate, pH 7 0 to 25% ethylene glycol, 25 mM potassium phosphate, pH 7.0 (no (NH 4 ) 2 SO 4 ) at 0.5 ml/min. Fractions were dialyzed overnight against 10 mM sodium phosphate buffer, pH 7.0 Activities in each fraction against SCR were determined by bioassay.
  • Proteins bound to the column were eluted with a linear gradient of 1.7 M (NH 4 ) 2 SO 4 , 50 mM potassium phosphate, pH 7.0 to 10 mM potassium phosphate, pH 7.0 at 0.5 ml/min. Fractions were dialyzed overnight against 10 mM sodium phosphate buffer, pH 7 0.
  • 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.
  • 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,
  • 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
  • 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
  • the N-terminal amino sequence of B1 was determined to be identical to SEQ ID NO:1, the TcbA ii region of the tcbA gene (SEQ ID NO: 12, position 37 to 99).
  • a unique 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 TcbA iii region of the derived amino acid sequence for the tcbA gene (SEQ ID NO:12, position 1935 to 1945). Therefore, the B toxin contained predominantly two peptides, TcbA ii and TcbA iii , 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 TcbA iii peptide and other peptides.
  • the A2 peptide was denoted TcdA iii (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.
  • 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 TcdA ii and TcdA iii , respectively, were excised from the blot and sent to Harvard MicroChem and
  • 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 native and subunits molecular weight, however, their peptide compositions are different.
  • Toxin A contained peptides TcdA ii and TcdA iii as the major peptides and the toxin B contains TcbA ii and TcbA iii as the major peptides.
  • TcbA ii and TcbA iii originate from the single gene product TcbA (Example 15).
  • TcbA peptide to TcbA ii and TcbA iii is presumably by the action of
  • Photorhabdus W-14 broth was processed, TcbA peptide was present in toxin B complex as a major component, in addition to peptides TcbA ii and TcbA iii .
  • 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, TcbA ii and TcbA iii 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.
  • TcbA The cleavage 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 purified fraction, 1.5 unit protease, and 0.1 M Tris buffer, pH 8.0 in a total volume of 100 ⁇ l.
  • protease was omitted.
  • the reaction mixtures were incubated at 37 °C for 90 min. At the end of the reaction, 20 ⁇ l was taken and boiled with SDS-PAGE sample buffer immediately for electrophoresis analysis in a 4-20% gradient SDS-PAGE.
  • Protease treated and untreated control of t he remaining 80 ul react ion mixture were seria l di lut ed 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 23).
  • PCR Polymerase Chain Reactions
  • 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 TcdA ii -PT111 lies amino-proximal to the peptide fragment TcdA ii -PT79.
  • the 2500 bp PCR products were ligated to the plasmid vector pCRTMII (Invitrogen, San Diego, CA) according to the supplier's instructions, and tne 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].
  • TcdA ii -PK44 and the TcdA iii 58 kDa N-terminal peptide, described as SEQ ID NO:9 (internal peptide TcdA ii -PK44 sequence), and SEQ ID NO:41(TcdA iii 58 kDa N-terminal peptide sequence) was isolated.
  • PCR Polymerase Chain Reactions
  • primers A2.1 or A2.2 were used as forward primers, and A1.44.1R, and A1.44.2R were used as reverse primers in all forward/reverse combinations.
  • the 1400 bp PCR products were ligated to the plasmid vector pCRTMII 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
  • 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 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 TcdA ii gene probe, indicating that both fragments are encoded on the genomic DNA.
  • DNA sequence determination of the cloned EcoR I fragments revealed an uninterrupted reading frame of 7551 base pairs (SEQ ID NO: 46), encoding a 232.9 kDa protein of 2516 amino acids (SEQ ID NO: 47). Analysis of the amino acid sequence of this protein revealed all expected internal fragments of peptides TcdA ii (SEQ ID NOS:17, 18, 37, 38 and 39) and the TcdA iii peptide N-terminus (SEQ ID NO: 41) and all TcdA iii internal peptides (SEQ ID NOS:42 and 43).
  • TcdA ii and TcdA iii are each products of the open reading frame, denoted tcdA, disclosed as SEQ 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
  • insecticidal activity identified as toxin B derives from the products of SEQ ID NO: 11, 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: 12.
  • 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.
  • Example 9 a large EcoR I fragment which hybridizes to the TcbA ii probe is described. This fragment was subcloned into pBC (Stratagene, La Jolla CA). Sequence analysis indicates that this 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.
  • the tcbA gene was PCR amplified using the following primers; 5' primer (S1Ac51) 5' TTT AAA CCA TGG GAA ACT CAT TAT CAA GCA CTA TC 3' and 3' primer (S1Ac31) 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, Wisconsin) in the following reaction: 57.5 ml water, 10 ml 10X LA buffer, 16 ml dNTPs (2.5 mM each stock solution), 20 ml each primer at 10 pmoles/ml, 300 ng of the plasmid pDAB634 containing the W-14 tcbA gene and one ml 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 7526bp 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, California).
  • the purified tcbA gene was digested with Nco I and Not I and ligated into the baculovirus transfer vector pAcGP67B (PharMingen (San Diego, California)) and transformed into DH5 ⁇ E. col i .
  • the tcbA gene was then cut from pAcGP67B and transferred to pET27b to create plasmid pDAB635.
  • a missense mutation in the tcbA gene was repaired in pDAB635.
  • the repaired tcbA gene contains two changes from the sequence shown in Sequence ID NO: 11; an A>G at 212 changing an asparagine 71 to serine 71 and a G>A at 229 changing an alanine 77 to threonine 77. These changes are both upstream of the proposed TcbA ii N-terminus.
  • tcbA coding region of pDAB635 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 pET15-tcbA .
  • Competent E. col i cells strain BL21(DE3) were transformed with plasmid pET15-tcbA 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).
  • IPTG isopropylthio- ⁇ -galactoside
  • 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.
  • the media was concentrated about 8x using the M12 (Amicon,
  • the region of the CL- 4B elution profile corresponding 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 30 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.
  • the active fraction tested immunologically positive for the TcbA and TcbA iii peptides in immunoblot analysis when tested with an anti-TcbA iii monoclonal antibody Table 30.
  • 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 with Poros HQ 50 beads.
  • the bound proteins were eluted by performing a NaCl gradient of 0.0 to 1.0 M.
  • TcbA 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 30.

Abstract

L'invention porte sur des protéines issues de l'espèce bactérienne Photorhabdus et toxiques pour les insectes exposés à celles-ci. Photorhabdus luminescens (ex-Xenorhabdus luminescens), qui a été découvert dans des échantillons cliniques de mammifères, s'est révélé comme étant un symbiote bactérien de nématodes entomopathogènes de l'espèce Heterorhabditis. Il est possible d'appliquer ces toxines protéiques sur de la nourriture pour larves d'insecte ou des plantes leur convenant ou bien de les produire par génie génétique dans ces aliments ou ces plantes et ce, aux fins de la lutte contre les insectes.
PCT/US1996/018003 1995-11-06 1996-11-06 Toxines proteiques insecticides provenant de photorhabdus WO1997017432A1 (fr)

Priority Applications (23)

Application Number Priority Date Filing Date Title
UA97084103A UA82485C2 (uk) 1995-11-06 1996-06-11 Інсектицидні білкові токсини з photorhabdus
SK931-97A SK93197A3 (en) 1995-11-06 1996-11-06 Insecticidal protein toxins from photorhabdus
RO97-01251A RO121280B1 (ro) 1995-11-06 1996-11-06 Toxine proteice insecticide din photorhabdus
JP51836997A JP3482214B2 (ja) 1995-11-06 1996-11-06 ホトルハブダス由来殺虫性タンパク毒素
EP96941335A EP0797659A4 (fr) 1995-11-06 1996-11-06 Toxines proteiques insecticides provenant de photorhabdus
CA002209659A CA2209659C (fr) 1995-11-06 1996-11-06 Toxines proteiques insecticides provenant de photorhabdus
MX9705101A MX9705101A (es) 1995-11-06 1996-11-06 Toxinas proteinicas insecticidas obtenidas a partir de photorhabdus luminescens.
RU97113033/13A RU2216174C2 (ru) 1996-11-06 1996-11-06 Способ борьбы с насекомыми и штамм бактерий photorhabdus luminescens (варианты)
IL121243A IL121243A (en) 1995-11-06 1996-11-06 Pecombinant insecticidal protein toxin from photorhabdus, polynucleotide encoding said protein and method of controlling pests using said protein
PL96321212A PL186242B1 (pl) 1995-11-06 1996-11-06 Polinukleotyd kodujący zrekombinowane białko i zrekombinowane białko o aktywności toksycznej przeciwko szkodliwym owadom
BR9606889A BR9606889A (pt) 1995-11-06 1996-11-06 Toxinas proteicas inseticidas de photorhabdus
AU10509/97A AU729228B2 (en) 1995-11-06 1996-11-06 Insecticidal protein toxins from photorhabdus
TR1999/01126T TR199901126T2 (xx) 1996-08-29 1997-05-05 Photarhabdus' dan elde edilen insektisid i�levli protein toksinleri.
JP10511612A JP2000515024A (ja) 1996-08-29 1997-05-05 ホトラブダス由来殺虫性タンパク質毒素
BR9711441-3A BR9711441A (pt) 1996-08-29 1997-05-05 Toxinas de proteìnas inseticidas provenientes de photorhabdus
IL12859097A IL128590A0 (en) 1996-08-29 1997-05-05 Insecticidal protein toxins from photorhabdus
CA002263819A CA2263819A1 (fr) 1996-08-29 1997-05-05 Toxines proteiniques insecticides isolees a partir de photorhabdus
SK246-99A SK24699A3 (en) 1996-08-29 1997-05-05 Insecticidal protein toxins from photorhabdus
AU28299/97A AU2829997A (en) 1996-08-29 1997-05-05 Insecticidal protein toxins from (photorhabdus)
PCT/US1997/007657 WO1998008932A1 (fr) 1996-08-29 1997-05-05 TOXINES PROTEINIQUES INSECTICIDES ISOLEES A PARTIR DE $i(PHOTORHABDUS)
EP97922696A EP0970185A4 (fr) 1996-08-29 1997-05-05 Toxines proteiniques insecticides isolees a partir de photorhabdus
KR1019990701698A KR20000037116A (ko) 1996-08-29 1997-05-05 포토랍두스 유래의 살충 단백질 독소
PL97332033A PL332033A1 (en) 1995-11-06 1997-05-05 Insecticidal proteinous toxins obtained from photorhabdus

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WO1998008388A1 (fr) * 1996-08-29 1998-03-05 The Minister Of Agriculture Fisheries & Food In Her Britannic Majesty's Government Of The United Kingdom Of Great Britain & Northern Ireland Agents antiparasitaires
WO1998050427A1 (fr) * 1997-05-05 1998-11-12 Dow Agrosciences Llc TOXINES PROTEIQUES INSECTICIDES ISSUES DE $i(XENORHABDUS)
WO1998059067A1 (fr) * 1997-06-20 1998-12-30 Mycogen Corporation Methode pour identifier des microbes pesticides
WO1999042589A2 (fr) * 1998-02-20 1999-08-26 Novartis Pharma Ag. Toxines insecticides de photorhabdus
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JP2004089189A (ja) 2004-03-25
RO121280B1 (ro) 2007-02-28
PL186242B1 (pl) 2003-12-31
KR19980701244A (ko) 1998-05-15
MX9705101A (es) 1997-10-31
HUP9900768A3 (en) 2002-10-28
IL121243A0 (en) 1998-01-04
BR9606889A (pt) 1997-10-28
SK93197A3 (en) 1998-05-06
JP3657593B2 (ja) 2005-06-08
IL121243A (en) 2010-05-31
CA2209659C (fr) 2008-01-15
JP2002509424A (ja) 2002-03-26
EP0797659A1 (fr) 1997-10-01
AU729228B2 (en) 2001-01-25
JP3482214B2 (ja) 2003-12-22
AU1050997A (en) 1997-05-29
PL321212A1 (en) 1997-11-24
KR100354530B1 (ko) 2003-01-06
CA2209659A1 (fr) 1997-05-15
HUP9900768A2 (hu) 1999-06-28

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