AU760154B2 - Antigens and their detection - Google Patents

Description

WO 99/61458 PCT/AU99/00385 1 Antigens and Their Detection TECHNICAL FIELD The invention relates to novel nucleotide sequences located in a gene which encodes a bacterial flagellin antigen, and the use of those nucleotide-sequences for the detection of bacteria which express particular flagellin antigens, on the basis of that antigen alone, or in conjunction with the 0 antigen expressed by that strain.

BACKGROUND ART The flagellum of many bacteria appears to be made up of a single protein known as flagellin. The serotyping schemes of E. coli and Salmonella enterica are based on highly variable antigenic surface structures which include the lipopolysaccharide which carries the 0 antigen and flagellin which is now known to be the carrier of the classical H antigen. In many strains of S. enterica there are two loci (fliC and fljB) which encode flagellin, and a regulatory system which allows one only to be expressed at any time; and which also provides for expression to rapidly alternate between the two forms first identified as two phases (H1 and H2) for the H antigen of most strains. In E. coli there are 54 forms of H antigen recognised and until recently they were all thought to be encoded at the fliC locus, as has been'shown for E. coli K-12. However in the 1980s Ratiner [Ratiner Y A "Phase variation of the H antigen in Escherichia coli strain Bi327-41, the standard strain for Escherichia coli flagellin antigen H3" FEMS Microbiol. Lett 15 (1982) 33- 36; Ratiner Y A "Presence of two structural genes determining antigenically different phase-specific flagellins in some Escherichia coli strains" FEMS Microbiol. Lett. 19 (1983) 37-41; Ratiner Y A "Two genetic arrangements determining flagellin antigen specificities in two diphasic Escherichia coli strains" FEMS Microbiol.

Lett. 29 ,(1985) 317-323; Ratiner Y A "Different alleles of the flagellin gene hagB in Escherichia coli standard H WO 99/61458 PCT/AU99/00385 -2test strains" FEMS Microbiol Lett. 48 (1987) 97-104.] showed that in some cases there are two loci and that expression can alternate. The matter was further complicated by a recent paper by Ratiner [Ratiner Y A (1998)"New flagellin-specifying genes in some Escherichia coli strains" J. Bacteriol. 180 979-984] showing three loci (flk, fll and flm) for flagellin in addition to fliC although the fljB locus has not been found in E. coli.

However E. coli strains are normally identified by the combination of one 0 antigen and one H antigen [and K antigen when present as a capsule antigen], with no problems reported for the vast majority of cases with alternate phases, while S. enterica strains are normally identified by the combination of O, H1 and H2 antigens. It is still not clear how widespread in E. coli H antigens determined by flagellin genes other than fliC are.

Typing is typically carried out using specific antisera. The incidence of pathogenic E. coli in association with human and animal disease supports the need for suitable and rapid typing techniques.

DESCRIPTION OF THE INVENTION In a first aspect, the present invention provides a novel nucleic acid molecule encoding all or part of an E.

coli flagellin protein.

The present invention provides, for the first time, full length sequence for a flagellin gene for the following E. coli type strains: H6 (SEQ ID NO: H9(SEQ ID NO: 11), H10(SEQ ID NO: 12), H14(SEQ ID NO: H18(SEQ ID NO: 18), H23(SEQ ID NO: 22), H51(SEQ ID NO: H45(SEQ ID NO: 43), H49(SEQ ID NO: 48), H19(SEQ ID NO: 19), H30(SEQ ID NO: 29), H32(SEQ ID NO: 31), H26(SEQ ID NO: 25), H41(SEQ ID NO: 39), H15(SEQ ID NO: 16), ID NO: 20), H28(SEQ ID NO: 27), H46(SEQ ID NO: 44), H31(SEQ ID NO: 30), H34(SEQ ID NO: 33), H43(SEQ ID NO: 41) and H52(SEQ ID NO: 51). Corrected full length sequences have been obtained for H7(SEQ ID NO: 9) and WO 99/61458 PCT/AU99/00385 3 H12(SEQ ID NO: 14) type strains.

Partial flagellin gene sequence, including the central variable region, has been obtained for the following E. coli H type strains: H40(SEQ ID NO: 38), H8(SEQ ID NO: 10), H21(SEQ ID NO: 21), H47(SEQ ID NO: 46), H11(SEQ ID NO: 13), H17(SEQ ID NO: 17), H25(SEQ ID NO: 24), H42(SEQ ID NO: 40), H27(SEQ ID NO: 26), H35(SEQ ID NO: 34), H2(SEQ ID NO: 67), H3(SEQ ID NO: 68), H24(SEQ ID NO: 23), H37(SEQ ID NO: 35), H50(SEQ ID NO: 49), H4(SEQ ID NO: H44(SEQ ID NO: 42), H38(SEQ ID NO: 36), H39(SEQ ID NO: 37), H55(SEQ ID NO: 53), H29(SEQ ID NO: 28), H33(SEQ ID NO: 32), H5(SEQ ID NO: H54(SEQ ID NO: 52) and H56(SEQ ID NO: 54).

Comparison of sequences demonstrates that unique flagellin genes have now been sequenced (partially or completely) for the following E. coli H type strains: H1, H2, H3, H5, H6, H7, H9, H11, H12, H14, H15, H18, H19, H21, H23, H24, H25, H26, H27, H28, H29, H30, H31, H32, H33, H34, H35, H37, H38, H39, H41, H42, H43, H45, H46, H48, H49, H51, H52, H54, and H56 and either H8 or H40, or H50 and H4 or H17.

By comparison of these sequences, the present inventors were able to identify specific sequences for each of the above H serotypes.

The present invention also provides fliC sequences from 10 different H7 strains, in addition to that from the H7 type strain, and two sequences specific to H7 of 0157 and 055 E. coli strains.

The present invention encompasses all or part of the flagellin genes sequenced for H2, H3, H5, H6, H9, H11, H14, H18, H19, H20, H21, H23, H24, H25, H26, H27, H28, H29, H30, H31, H32, H33, H34, H35, H37, H38, H39, H41, H42, H43, H44, H45, H46, H47, H48, H49, H51, H52, H54, H56, H8, H40, H15, H10, or H50, H4 and H17 type strains. Of these flagellin genes sequenced, those from the type strains for H8 and H40 are identical, those from type strains H10 and H50, H1 and H12, H38 and H55, H21 and WO 99/61458 PCT/AU99/00385 -4- 4 H47, and H4, H17 and H44 type strains are highly similar.

The invention also encompasses newly provided sequence for H7 and H12 as well as novel primers for the specific amplification of H1, H7, H12 and H48 as well as for the other above mentioned newly sequenced flagellin genes.

By cloning and expression of these sequenced flagellin genes in a fliC deletion E. coli K-12 strain, and use of anti-H antiserum, we have confirmed the H specificities encoded by 39 falgellin genes. The 39 H specificities are H1, H2, H4, H5, H6, H7, H9, H10, Hll, H12, H14, H15, H16, H18, H19, H20, H21, H23, H24, H26, H27, H28, H29, H30, H31, H32, H33, H34, H38, H39, H41, H42, H43, H45, H46, H49, H51, H52, and H56, encoded by flagellin genes obtained from H type strains for Hl, H2, H4, H5, H6, H7, H9, H10, Hll, H12, H14, H15, H3, H18, H19, H21, H23, H24, H26, H27, H28, H29, H30, H31, H32, H33, H34, H38, H39, H41, H42, H43, H45, H46, H49, H51, H52, and H56 respectively.

The nucleic acid molecules of the invention may be variable in length. In one embodiment they are oligonucleotides of from about 10 to about 20 nucleotides in length. The oligonucleotides of the invention are specific for the flagellin gene from which they are derived and are derived from the central region of the gene. In one embodiment, oligonucleotides in accordance with the present invention, which also include oligonucleotides from the previously sequenced E. coli H1, H7, H12 and H48 genes, are those shown in Table 3.

The 45 sequences (see Table 3) provide a panel to which newly sequenced genes can be compared to select specific oligonucleotides for those newly sequenced genes.

In a second aspect the invention provides a method of detecting the presence of E. coli of a particular H serotype in a sample, the method comprising the step of specifically hybridising at least one nucleic acid molecule derived from a flagellin gene, wherein the at WO 99/61458 PCT/AU99/00385 5 least one nucleic acid molecule is specific for a particular flagellin gene associated with the H serotype, to any E. coli in the sample which contain the gene, and detecting any specifically hybridised nucleic acid molecules, wherein the presence of specifically hybridised nucleic acid molecules identifies the presence of the H serotype in the sample.

In one preferred embodiment the detection method is a Southern blot method. More preferably, the nucleic acid molecule is labelled and hybridisation of the nucleic acid molecule is detected by autoradiography or detection of fluorescence.

Preferred nucleic acid molecules for the detection of particular flagellin genes are listed in Table 3.

In a third aspect the invention provides a method of detecting the presence of E. coli of a particular H serotype in a sample, the method comprising the step of specifically hybridising at least one pair of nucleic acid molecules to any E. coli in the sample which contains the flagellin gene for the particular H serotype, wherein at least one of the nucleic acid molecules is specific for the particular flagellin gene associated with the H serotype, and detecting any specifically hybridised nucleic acid molecules, wherein the presence of specifically hybridised nucleic acid molecules identifies the presence of the H serotype in the sample.

In one preferred embodiment the detection method is a polymerase chain reaction method. More preferably, the nucleic acid molecules are labelled and hybridisation of the nucleic acid molecule is detected by electrophoresis.

It is recognised that there may be instances where spurious hybridisation will arise through the initial selection of a sequence found in many different genes but this is typically recognisable by, for instance, comparison of band sizes against controls in PCR gels, and an alternative sequence can be selected.

WO 99/61458 PCT/AU99/00385 6 In a fourth aspect the invention provides a method for detecting the presence of a particular 0 serotype and H serotype of E. coli in a sample, the method comprising the following steps: specifically hybridising at least one nucleic acid molecule, derived from and specific for a gene encoding a transferase or a gene encoding an enzyme for the transport or processing of a polysaccharide or oligosaccharide unit, the gene being involved in the synthesis of a particular E. coli 0 antigen, to any E.

coli in the sample which contain the gene; specifically hybridising at least one nucleic acid molecule derived from and specific for a particular flagellin gene associated with that H serotype, to any E.

coli in the sample which contain the gene; and detecting any specifically hybridised nucleic acid molecules.

Preferred nucleic acid molecules for the detection of particular flagellin genes are listed in Table 3.

In one preferred embodiment, the sequence of the nucleic acid molecule specific for the 0 antigen is specific to the nucleotide sequence encoding the 0111 antigen. More preferably, the sequence is derived from a gene selected from the group consisting of wbdH (nucleotide position 739 to 1932 of Figure wzx (nucleotide position 8646 to 9911 of Figure wzy (nucleotide position 9901 to 10953 of Figure wbdM (nucleotide position 11821 to 12945 of Figure 5) and fragments of those molecules of at least 10-12 nucleotides in length. Particularly preferred nucleic acid molecules are those set out in Tables 8 and 8A, with respect to the above mentioned genes.

In another preferred embodiment, the sequence of the nucleic acid molecule specific for the 0 antigen is specific to the nucleotide sequence encoding the 0157 antigen. More preferably, the sequence is derived from a gene selected from the group consisting of wbdN WO 99/61458 PCT/AU99/00385 7 (nucleotide position 79 to 861 of Figure wbdO (nucleotide position 2011 to 2757 of Figure wbdP (nucleotide position 5257 to 6471 of Figure wbdR (nucleotide position 13156 to 13821 of Figure wzx (nucleotide position 2744 to 4135 of Figure 6) and wzy (nucleotide position 858 to 2042 of Figure 6) and fragments of those molecules of at least 10-12 nucleotides in length. Particularly preferred nucleic acid molecules are those set out in Tables 9 and 9A, with respect to the above mentioned genes.

In one preferred embodiment the detection method is a Southern blot method. More preferably, the nucleic acid molecule is labelled and hybridisation of the nucleic acid molecule is detected by autoradiography or detection of fluorescence.

In a fifth aspect the invention provides a method for detecting the presence of a particular 0 serotype and H serotype of E. coli in a sample, the method comprising the following steps: specifically hybridising at least one pair of nucleic acid molecules, at least one of which is derived from and specific for a gene encoding a transferase or a gene encoding an enzyme for the transport or processing of a polysaccharide or oligosaccharide unit, the gene being involved in the synthesis of the particular E. coli 0 antigen, to any E. coli in the sample which contain the gene; specifically hybridising at least one pair of nucleic acid molecules, at least one of which is derived from and specific for a particular flagellin gene associated with the particular H serotype, to any E. coli in the sample which contain the gene; and detecting any specifically hybridised nucleic acid molecules.

Preferred nucleic acid molecules for the detection of particular flagellin genes are listed in Table 3.

WO 99/61458 PCT/AU99/00385 8 In one preferred embodiment, the sequence of the nucleic acid molecule specific for the 0 antigen is specific to the nucleotide sequence encoding the 0111 antigen. More preferably, the sequence is derived from a gene selected from the group consisting of wbdH (nucleotide position 739 to 1932 of Figure wzx (nucleotide position 8646 to 9911 of Figure wzy (nucleotide position 9901 to 10953 of Figure wbdM (nucleotide position 11821 to 12945 of Figure 5) and fragments of those molecules of at least 10-12 nucleotides in length. Particularly preferred nucleic acid molecules are those set out in Tables 8and 8A, with respect to the above mentioned genes.

In another preferred embodiment, the sequence of the nucleic acid molecule specific for the 0 antigen is specific to the nucleotide sequence encoding the 0157 antigen. More preferably, the sequence is derived from a gene selected from the group consisting of wbdN(nucleotide position 79 to 861 of Figure wbdO (nucleotide position 2011 to 2757 of Figure wbdP (nucleotide position 5257 to 6471 of Figure wbdR (nucleotide position 13156 to 13821 of Figure wzx (nucleotide position 2744 to 4135 of Figure 6) and wzy (nucleotide position 858 to 2042 of Figure 6) and fragments of those molecules of at least 12 nucleotides in length. Particularly preferred nucleic acid molecules are those set out in Tables 9 and 9A, with respect to the above mentioned genes.

In one preferred embodiment the detection method is a polymerase chain reaction method. More preferably, the nucleic acid molecules are labelled and hybridisation of the nucleic acid molecule is detected by electrophoresis.

The present inventors believe that based on the teachings of the present invention and available information concerning 0 antigen gene clusters, and through use of experimental analysis, comparison of nucleic acid sequences or predicted protein structures, nucleic acid molecules in accordance with the invention WO 99/61458 PCT/AU99/00385 -9 can be readily derived for any particular 0 antigen of interest. Suitable bacterial strains can typically be acquired commercially from depositary institutions.

There are currently 166 defined E. coli 0 antigens.

Samples of the 166 different E. coli 0 antigen serotypes are available from Statens Serum Institut, Copenhagen, Denmark.

The inventors envisage rare circumstances whereby two genetically similar gene clusters encoding serologically different 0 antigens have arisen through recombination of genes or mutation so as to generate polymorphic variants.

In these circumstances multiple pairs of oligonucleotides may be selected to provide hybridisation to the specific combination of genes. The invention thus envisages the use of a panel containing multiple nucleic acid molecules for use in the method of testing for 0 antigen in conjunction with H antigen, wherein the nucleic acid molecules are derived from genes encoding transferases and/or enzymes for the transport or processing of a polysaccharide or oligosaccharide unit including wzx or wzy genes, wherein the panel of nucleic acid molecules is specific to a particular 0 antigen. The panel of nucleic acid molecules can include nucleic acid molecules derived from 0 antigen sugar pathway genes where necessary.

The inventors also found two mutated flagellin genes from H type strains for H35 and H54 which have insertion sequences inserted into normal flagellar genes identical or near identical to that that of the H1 and H21 type strains respectively. Thus, primers for H11 and H21 (listed in Table 3) would also amplify fragments in and H54, which differ in sizes to those in H11 and H21 respectively. The inventors also provide two pairs of primers each for H35 and H54 based on the insertion sequence (see H35 and H54 columns in Table The use of one of them in combination with one of the H1 or H21 primers will generate a PCR band only in H35 or H54 respectively, and this will also differentiate H35 and H54 WO 99/61458 PCT/AU99/00385 10 from H11 and H21 respectively.

The present invention also relates to methods of detecting the presence of particular E. coli H antigens or H antigen and 0 antigen combinations where one or more nucleic acid molecules which generate a particular size fragment indicative of the presence of that H antigen are used or in which the combination of one antigen specific primer for that H antigen with another primer for a related H antigen provides for the detection of the particular H antigen by hybridisation to the relevant gene. Preferably, the H antigen is H11, H21, H35 or H54.

The pairs of nucleic acid molecules where the method of the fifth aspect is used may both hybridise to the relevant H or 0 antigen gene or alternatively only one may hybridise to the relevant gene and the other to another site.

The inventors recognise in applying the methods of the invention for detecting combinations of 0 and H antigens to samples, that the methods do not indicate whether a positive result for a particular 0 and H antigen combination arises because the 0 and H antigen are present on a single E. coli strain present in the sample or are present on different E. coli strains present in the sample. Because the ability to identify the presence of E. coli strains with particular 0 and H antigen combinations is highly desirable (due to the relationship between particular combinations and pathogenicity) the determination that a particular combination is present in a sample can be followed by isolation of single colonies and checking whether the they contain the relevant combination by using the same method again or using antibody labelled magnetic beads to separate cells expressing the particular 0 or H antigen and then testing the isolated cells for the other serotype.

In addition, as mentioned above, the present inventors have established the existence of H7 primers specific to the 0157 and 055 serotypes. Using such WO 99/61458 PCT/AU99/00385 11 primers it is possible to detect particular 0 and H antigen combinations with the use of H specific nucleic acid molecules.

In a sixth aspect the invention provides a method for detecting the presence of a particular 0 serotype and H serotype of E. coli in a sample, the method comprising the following steps: specifically hybridising at least one nucleic acid molecule, derived from and specific for a gene encoding a flagellin associated with a particular E. coli H antigen serotype to any E. coli carrying the gene and present in the sample; and detecting the at least one specifically hybridised nucleic acid molecule, wherein the at least one nucleic acid molecule is specific for the particular combination of 0 and H antigen.

Preferably the combination is 055:H7 or 0157:H7.

The ability to detect the 0157:H7 combination from a particular H7 primer or pair is of particular use given the association of this combination with pathogenic strains.

In a seventh aspect the present invention provides a method for testing a food derived sample for the presence of one or more particular E. coli 0 antigens and H antigens comprising testing the sample by a method of the fourth, fifth or sixth aspect the invention.

In an eighth aspect the present invention provides a method for testing a faecal derived sample for the presence of one or more particular E. coli 0 antigens and H antigens comprising testing the sample by a method of the fourth, fifth or sixth aspect the invention.

In a ninth aspect the present invention provides a method for testing a patient or animal derived sample for the presence of one or more particular E. coli 0 antigens and H antigens comprising testing the sample by a method of the fourth, fifth or sixth aspect the invention.

WO 99/61458 PCT/AU99/00385 12 Preferably, the method of the seventh, eighth or ninth aspect of the invention is a polymerase chain reaction method. More preferably the oligonucleotide molecules for use in the method are labelled. Even more preferably the hybridised nucleic acid molecules are detected by electrophoresis.

In the above described methods it will be understood that where pairs of nucleic acid molecules are used one of the nucleic acid molecules may hybridise to a sequence that is not from the O antigen transferase, wzx or wzy gene or the flagellin gene. Further where both hybridise to these genes the 0 antigen molecules may hybridise to the same or a different one of these genes.

In a tenth aspect the present invention provides a kit for identifying the H serotype of E. coli, the kit comprising: at least one nucleic acid molecule derived from and specific for an E. coli flagellin gene.

In an eleventh aspect the present invention provides a kit for identifying the H and O serotype of E. coli, the kit comprising: at least one nucleic acid molecule derived from and specific for an E. coli flagellin gene; and at least one nucleic acid molecule derived from and specific for a gene encoding a transferase or a gene encoding an enzyme for the transport or processing of a polysaccharide or oligosaccharide unit, the gene being involved in the synthesis of a particular E. coli 0 antigen.

The nucleic acid molecules may be provided in the same or different vials. The kit may also provide in the same or separate vials a second set of specific nucleic acid molecules.

Particularly preferred nucleic acid molecules for inclusion in the kits are those specified in Tables 3, 8, 8A, 9 and 9A as described above.

WO 99/61458 PCT/AU99/00385 13

DEFINITIONS

In this specification, we have used term "flagellin gene" in many cases where previously one would have used "fliC", to allow for the uncertainty as to locus introduced by recent observations. However, uncertainty as to the locus does not alter the fact that most E. coli strains express a single H antigen and that a single flagellin gene sequence per strain is required to give the genetic basis for H antigen variation Any use of the name fliC in this specification where a different locus is later shown to be involved would not affect the validity of conclusions drawn regarding application of information based on the sequence, where the conclusions do not relate to the map position. Thus it is generally the nucleic acid molecule itself which is of importance rather than the name attributed to the gene. When it is known or suspected that the gene encoding the H antigen is not in the fliC locus, we use the term flagellin rather than fliC.

The phrase, "a nucleic acid molecule derived from a gene" means that the nucleic acid molecule has a nucleotide sequence which is either identical or substantially similar to all or part of the identified gene. Thus a nucleic acid molecule derived from a gene can be a molecule which is isolated from the identified gene by physical separation from that gene, or a molecule which is artificially synthesised and has a nucleotide sequence which is either identical to or substantially similar to all or part of the identified gene. While some workers consider only the DNA strand with the same sequence as the mRNA transcribed from the gene, here either strand is intended.

Transferase genes are regions of nucleic acid which have a nucleotide sequence which encodes gene products that transfer monomeric sugar units.

Flippase or wzx genes are regions of nucleic acid which have a nucleotide sequence which encodes a gene WO 99/61458 PCT/AU99/00385 14 product that flips oligosaccharide repeat units generally composed of three to six monomeric sugar units to the external surface of the membrane.

Polymerase or wzy genes are regions of nucleic acid which have a nucleotide sequence which encodes gene products that polymerise repeating oligosaccharide units generally composed of 3-6 monomeric sugar units.

The nucleotide sequences provided in this specification are described as anti-sense sequences. This term is used in the same manner as it is used in Glossary of Biochemistry and Molecular Biology Revised Edition, David M. Glick, 1997 Portland Press Ltd., London on page 11 where the term is described as referring to one of the two strands of double-stranded DNA usually that which has the same sequence as the mRNA. We use it to describe this strand which has the same sequence as the mRNA.

NOMENCLATURE

Synonyms for E. coli 0111 rfb Current names Our names Bastin et al. 1991 wbdH orfl gmd orf2 wbdI orf3 orf3.4* manC orf4 rfbM* manB orf5 rfbK* wbdJ orf6 orf6.7* wbdK orf7 orf7.7* wzx orf8 orf8.9 and rfbX* wzy orf9 wbdL orfl0 wbdM orfll Nomenclature according to Bastin et al. 1991 "Molecular cloning and expression in Escherichia coli K-12 of the rfb gene cluster determining the O antigen of an E. coli 0111 strain". Mol.

Microbiol. 5:9 2223-2231.

Other Synonyms wzy rfc wzx rfbX rmlA rfbA rmlB rfbB rmlC rfbC rmlD rfbD glf orf6* wbbI orf3#, orf8* of E. coli K-12 WO 99/61458 PCT/AU99/00385 15 wbbJ orf2#, orf9* of E. coli K-12 wbbK orfl#, orfl0* of E. coli K-12 wbbL orf5#, orf 11* of E. coli K-12 Nomenclature according to Yao, Z. And M. A. Valvano 1994.

"Genetic analysis of the O-specific lipopolysaccharide biosynthesis region (rfb) of Eschericia coli K-12 W3110: identification of genes the confer groups-specificty to Shigella flexineri serotypes Y and 4a". J. Bacteriol. 176: 4133-4143.

Nomenclature according to Stevenson et al. 1994. "Structure of the O-antigen of E. coli K-12 and the sequence of its rfb gene cluster". J. Bacteriol 176: 4144-4156.

The O antigen genes of many species were given rfb names (rfbA etc) and the 0 antigen gene cluster was often referred to as the rfb cluster. There are now new names for the rfb genes as shown in the table. Both terminologies have been used herein, depending on the source of the information.

In the claims that follow and in the summary of the invention, except where the context requires otherwise due to express language or necessary implication, the word "comprising" is used in the sense of "including", i.e. the features specified may be associated with further features in various embodiments of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows Eco R1 restriction maps of cosmid clones pPR1054, pPR1055, pPR1056, pPR1058, pPR1287 which are subclones of E. coli 0111 0 antigen gene cluster. The thickened line is the region common to all clones.

Broken lines show segments that are non-contiguous on the chromosome. The deduced restriction map for E. coli strain M92 is shown above.

Figure 2 shows a restriction mapping analysis of E.

coli 0111 0 antigen gene cluster within the cosmid clone pPR1058. Restriction enzymes are: BamHl; Bg: BglII, E: EcoRl; H: HindIII; K: KpnI; P: PstI; S: Sall and X: Xhol. Plasmids pPR1230, pPR1231, and pPR1288 are deletion derivatives of pPR1058. Plasmids pPR 1237, pPR1238, pPR1239 and pPR1240 are in pUC19. Plasmids pPR1243, pPR1244, pPR1245, pPR1246 and pPR1248 are in pUC18, and pPR1292 is in pUC19. Plasmid pPR1270 is in WO 99/61458 PCT/AU99/00385 16 pT7T319U. Probes 1, 2 and 3 were isolated as internal fragments of pPR1246, pPR1243 and pPR1237 respectively.

Dotted lines indicate that subclone DNA extends to the left of the map into attached vector.

Figure 3 shows the structure of E. coli 0111 O antigen gene cluster.

Figure 4 shows the structure of E. coli 0157 O antigen gene cluster.

Figure 5 shows the nucleotide sequence (SEQ ID NO: 45) of the E. coli 0111 0 antigen gene cluster. Note: (1) The first and last three bases of a gene are underlined and of italic respectively.; The region which was previously sequenced by Bastin and Reeves 1995 "Sequence and anlysis of the 0 antigen gene (rfb) cluster of Escherichia coli 0111" Gene 164: 17-23 is marked.

Figure 6 shows the nucleotide sequence (SEQ ID NO: 56) of the E. coli 0157 0 antigen gene cluster. Note: (1) The first and last three bases of a gene (region) are underlined and of italic respectively The region previously sequenced by Bilge et al. 1996 "Role of the Escherichia coli 0157-H7 0 side chain in adherence and analysis of an rfb locus". Inf. and Immun 64:4795-4801 is marked.

Figures 7 to 9 show the nucleotide sequences (SEQ ID NOS: 66 to 68 respectively) obtained for flagellin genes from E. coli type strains for H1 to H3 respectively. The primer positions listed in Table 3 are based on treating the first nucleotide of each of these sequences as No. 1.

Figures 10 to 18 show the nucleotide sequences (SEQ ID NOS: 6 to 14 respectively) obtained for flagellin genes from E. coli type strains for H4 to H12 respectively. The primer positions listed in Table 3 are based on treating the first nucleotide of each of these sequences as No. 1.

Figures 19 and 20 show the nucleotide sequences (SEQ ID NOS: 15 to 16 respectively) obtained for flagellin genes from E. coli type strains for H14 and respectively. The primer positions listed in Table 3 are WO 99/61458 PCT/AU99/00385 17 based on treating the first nucleotide of each of these sequences as No. 1.

Figures 22 and 26 show the nucleotide sequences (SEQ ID NOS: 17 to 21 respectively) obtained for flagellin genes from E. coli type strains for H17 and H21 respectively. The primer positions listed in Table 3 are based on treating the first nucleotide of each of these sequences as No. 1.

Figures 27 to 39 show the nucleotide sequences (SEQ ID NOS: 22 to 34) obtained for flagellin genes from E.

coli type strains for H23 to H35 respectively. The primer positions listed in Table 3 are based on treating the first nucleotide of each of these sequences as No. 1.

Figures 40 to 49 show the nucleotide sequences (SEQ ID NOS: 35 to 44) obtained for flagellin genes from E.

coli type strains for H37 to H46 respectively. The primer positions listed in Table 3 are based on treating the first nucleotide of each of these sequences as No. 1.

Figures 50 to 55 show the nucleotide sequences (SEQ ID NOS: 46 to 51) obtained for flagellin genes from E.

coli type strains for H47 to H52 respectively. The primer positions listed in Table 3 are based on treating the first nucleotide of each of these sequences as No. 1.

Figures 56 to 58 show the nucleotide sequences (SEQ ID NOS: 52 to 54) obtained for flagellin genes from E.

coli type strains for H54 to H56 respectively. The primer positions listed in Table 3 are based on treating the first nucleotide of each of these sequences as No. 1.

Figure 59 shows the nucleotide sequence (SEQ ID NO: 55) obtained for the flagellin gene from E. coli H7 strain M1179. The primer positions listed in Table 3 are based on treating the first nucleotide of each of these sequences as No. 1.

Figures 60 to 68 show the nucleotide sequences (SEQ ID NOS: 57 to 65 respectively) obtained for flagellin genes from E. coli strains M1004, M1211, M1200, M1686, M1328, M917, M527, M973, and M918 respectively. The primer WO 99/61458 PCT/AU99/00385 18 positions listed in Table 3 are based on treating the first nucleotide of each of these sequences as No. 1.

Figure 69 shows the nucleotide sequence (SEQ ID NO: 1) of the fliC gene and DNA flanking the. fliC gene from the H25 type strain.

Figure 70A shows the nucleotide sequence (SEQ ID NO: 2) obtained from the 5' end of the insert of plasmid pPR1989. The insert of plasmid pPR1989 encodes the second flagellin gene of the H55 type strain.

Figure 70B shows the nucleotide sequence (SEQ ID NO: 3) obtained from the 3' end of the insert of plasmid pPR1989. The insert of plasmid pPR1989 encodes the second flagellin gene of the H55 type strain.

Figure 71 shows the nucleotide sequence (SEQ ID NO:4) obtained from the 5' end of the insert of plasmid pPR1993.

The insert of plasmid pPR1993 encodes the second flagellin gene of the H36 strain.

Figure 72 shows the nucleotide sequence (SEQ ID obtained from the 3' end of the insert of plasmid pPR1993.

The insert of plasmid pPR1993 encodes the second flagellin gene of the H36 type strain.

Figure 73 A shows the sequence of polylinker and the SD sequence of plasmid pTrc99A.

Figure 73B shows the sequence of the junction region between the SD sequence and the start of flagellin gene in the plasmids used for the expression of flagellin genes.

BEST METHOD OF CARRYING OUT THE INVENTION In carrying out the methods of the invention with respect to the testing of particular sample types including samples from food, patients, animals and faeces the samples are prepared by routine techniques routinely used in the preparation of such samples for DNA based testing. The steps for testing the samples using particular nucleic acid molecules in assay formats such as Southern blots and PCR are performed under routinely determined conditions appropriate to the sample and the WO 99/61458 PCT/AU99/00385 19 nucleic acid molecules.

H antigen Materials and Methods 1. Bacterial strains and plasmid: There are 54 H types in E. coli [Ewing, Edwards and Ewing's identification of the Enterobacteriaceae., Elsevier Science Publishers, Amsterdam, The Netherlands, 1986]: note H antigens from 1 to 57 were listed and that 13, 22 and 57 are not valid. All the standard H type strains except H16 were obtained from the Institute of Medical and Veterinary Science, Adelaide, Australia. The primary stocks are hold at the Statens Serum Institut, Copenhagen, Denmark.

The additional H7 strains used are listed in Table 1.

We do not have the type strain for H16. It is known that the H3 type strain is biphasic and can also express the H16 flagellin gene [Ratiner, Y. A. (1985) "Two genetic arrangements determining flagellar antigen specificities in two diphasic E. coli strains. FEMS Microbiol Lett 19: 317-323]. We have sequenced and cloned the H16 flagellin gene from the H3 type strain (see below).

E. coli K-12 strain C600 hsm hsr fliC::TnlO [Kuwajiwa, G. (1988) "Flagellin domain that affects H antigenicity of E. coli K-12" J. Bacteriol. 170; 485-488] (laboratory stock no. M2126) was obtained from Dr Benita Westerlund-Wikstrom of the Department of Biosciences, University of Helsinkin, Finland. E. coli K-12 strain EJ2282 (laboratory no. P5560) is a fliC deletion strain, and was obtained from Dr Masatoshi Enomoto of the Department of Biology, Okayama University, Japan [Tominaga, A. M. Mahmound, T. Mokaihara and M.

Enomoto (1994) "Molecular characterization of intact but cryptic, flagellin genes in the genus Shigella.: Mol.

Microbiol. 12: 277-285].

Plasmid pTrc99A was purchased from Pharmacia LKB (Melbourne, VIC, Australia).

WO 99/61458 PCT/AU99/00385 20 2. Antisera Antisera against H1, H3, H8, H14, H15, H17, H23, H24, H26, H29, H30, H31, H32, H33, H35, H36, H37, H38, H39, H43, H44, H46, H47, H48, H49, H52, H53, H54, H55, and H56 were obtained from the Institute of Medical and veterinary Science, Adelaide, Australia. Antisera against H2, H4, H5, H6, H7, H9, H10, H11, H12, H16, H18, H19, H21, H27, H28, H34, H40, H41, H42, H45, and H51 were obtained from Denka Seiken Co., Ltd, Tokyo, Japan.

Antisera to type H50 was not available from any known source.

The antisera available were checked against the appropriate type strains to confirm the specificities of both flagellin H antigen and H antisera: 52 sera (all those except anti-H16 serum listed above) gave a positive reaction with the corresponding type strains for that serum.

3. Agglutination test: Bacteria from 1 ml of an overnight culture grown in Luria broth (Difco Tryptone, 10g/l; Difico yeast extract, NaCI, 0.5 g/l; pH 7.2) at 30oC was centrifuged 4000 rpm/10 min) and the bacteria pellet resuspended in 100 ml of saline. The agglutination test was carried out by mixing equal volumes (5 ml) of both the cells and antiserum on a slide. The slide was rocked for 1 minute and then observed for agglutination. For all agglutination tests, saline containing no antiserum was mixed with cells to be used as a negative control.

For testing the H specificities of strain M2126 or strain P5560 carrying plasmid containing cloned flagellin genes, cells of M2126 or P5560 were used as an additional negative control.

All agglutination tests were first carried out using undiluted antisera (note that the antisera we used have been diluted before reaching our hands), except for anti- WO 99/61458 PCT/AU99/00385 21 H11, anti-H34, anti-H52 and anti-H26 serum for which we used 1:10 dilutions to avoid background agglutination. In cases for which cross-reactions have been reported, we carried out agglutination tests using serial dilutions of sera (see section 10.1) 4. Motility test: The motility of strain M2126 or strain P5560 carrying cloned flagellin genes was examined microscopically. 1 ml of overnight culture grown in Luria broth (Difco Tryptone, Difico yeast extract, 5g/l; NaCI, 0.5 g/l; pH 7.2) at 30oC was inoculated into 10 ml of Luria broth, and the culture was shaken at 100 rpm at 30oC to early log phase (OD 625 A loopful of culture was placed on a slide and examined under a microscope. Motility of individual cells was easily distinguished from Brownian movement and streaming, and presence or absence of motility recorded.

Isolation of chromosomal DNA: Chromosomal DNA from all the 53 H type strains and the strains listed in Table 1 was isolated using the Promega Genomic isolation kit (Madison WI USA). Each chromosomal DNA sample was checked by gel electrophoresis of the DNA and by PCR amplification of the mdh gene using oligonucleotides based on the E. coli K-12 mdh gene [Boyd, Nelson, Wang, Whittam, T.S. and Selander, Molecular genetic basis of allelic polymorphism in malate dehydrogenase (mdh) in natural populations of Escherichia coli and Salmonella enterica. Proc. Natl.

Acad. Sci. USA 91 (1994) 1280-1284].

6. PCR amplification of flagellin gene: Flagellin genes from different strains were first PCR amplified using one of the following four pairs of oligonucleotides: #1285 (5'-atggcacaagtcattaatac) and #1286 WO 99/61458 PCT/AU99/00385 22 #1417 (5'-ctgatcactcaaaataatatcaac) and #1418 #1431 (5'-atggcacaagtcattaatacccaac) and #1432 #1575 (5'-gggtggaaacccaatacg) and #1576(5'-gcgcatcaggcaatttgg) PCR reactions were carried out under the following conditions: denaturing, 94 0 C/30'; annealing, temperature varies (refer to Table extension, 72 0 cycles. The PCR product was purified using the Promega Wizard PCR purification kit (Madison WI USA) before being sequenced.

The H36 and H53 type strains gave two PCR bands using primer pairs #1431/#1432 and #1417/#1418 respectively, and were not sequenced.

7. Enzymes and buffers: Restriction endonucleases and DNA T4 ligase were purchased from Boehringer Mannheim (Castle Hill, NSW, Australia). Restriction enzymes were used in the recommended commercial buffer.

8. Sequencing of the flagellin genes: Each PCR product was first sequenced using the oligonucleotide primers used for the PCR amplification.

Primers based on the obtained sequence were then used to sequence further, and this procedure was repeated until the entire PCR product was sequenced.

The sequencing reactions were performed using the DyeDeoxy Terminator Cycle Sequencing method (Applied Biosystems, CA, USA), and reaction products were analysed using fluorescent dye and an ABI377 automated sequencer (CA, USA).

Sequence data were processed and analysed using Staden programs [Sacchi CT, Zanella R C, Caugant D A, Frasch C E, Hidalgo N T, Milagres L G, Pessoa L L, Ramos S R, Camargo M C C and Melles C E A "Emergence of a new WO 99/61458 PCT/AU99/00385 23 clone of serogroup C Neisseria meningitidis in Sao Paulo, Brazil" J. Clin. Microbiol. 30 (1992) 1282-1286; Staden, Automation of the computer handling of gel reading data produced by the shotgun method of DNA sequencing. Nucl. Acids Res. 10 (1982a) 4731-4751; Staden, An interactive graphics program for comparing and aligning nucleic acid and amino acid sequences. Nucl.

Acids Res. 10 (1982b) 2951-2961; Staden, Computer methods to locate signals in nucleic acid sequences. Nucl. Acids Res. 12 (1984a) 505-519; Staden, Graphic methods to determine the function of nucleic acid sequences. A summary of ANALYSEQ options.

Nucl. Acids Res. 12 (1984b) 521-538; Staden, The current status and portability of our sequence handling software. Nucl. Acids Res. 14 (1986) 217-231].

We were able to PCR amplify flagellin genes from H type strains for H7, 23, 12, 51, 45, 49, 19, 9, 30, 32, 26, 41, 15, 20, 28, 46, 31, 14, 18, 6, 34, 48, 43, 10, 52, and also from H7 strains m1004, m527, m1686, m1211, m1328, m973, m1179, m1200, m917, and m918 using primers #1575 and #1576 which are based on sequences 51-34 bp upstream and 37-54 bp downstream of start and end of the E. coli K-12 fliC gene respectively. Thus, the full sequence of the flagellin gene from these strains was obtained and the use of flanking sequence for primers makes it highly likely that they are at the fliC locus.

For other strains, we were only able to amplify the flagellin gene using one or more of the other three pairs of primers, which are based on sequence within the fliC gene, and thus only partial sequence was obtained. These amplicons may be of the fliC gene or one of the alternative flagellin genes. The flagellin gene sequences from H type strains for H40, 8, 21, 47, 11, 27, 35, 2, 3, 24, 37, 50, 4, 44, 38, 55, 29, 33, 5, and 56 obtained are lacking 18 and 14 codons at 5' and 3' ends respectively.

The flagellin gene sequence of H39 obtained using primers WO 99/61458 PCT/AU99/00385 24 #1285/#1286 lacks 18 and 19 codons at 5' and 3' ends respectively. The flagellin gene sequence of H type strains of H17, 25 and 42 lack 23 and 21 codons at 5' and 3' ends respectively. The flagellin gene sequence of the H type strain for H54 lacks 23 and 12 codons at the 5' and 3' ends respectively. There is very little variation in the sequence at the two ends of flagellin genes and antigenic variation is due to variation in the central region of the gene. The absence of sequence for the ends of some of the flagellin genes is not important for the purpose of the present invention relating to the detection of antigenic variation by DNA sequence based means.

The fliC genes from H type strains of H1, H7 and H12 have been sequenced previously [Schoenhals, G. and Whitfield, Comparative analysis of flagellin sequences from Escherichia coli strains possessing serologically distinct flagellar filaments with a shared complex surface pattern. J. Bacteriol. 175 (1993) 5395-5402] and we did not sequence the gene from the H1 strain.

We have sequenced fliC genes from a set of H7 strains with different 0 antigens, including that of fliC from the H7 type strain as one of the set: we have found four differences from the published H7 sequence (GenBank accession number L07388) which we believe are due to errors in the published sequence.

We have also re-sequenced the fliC gene from the H12 type strain, and have found one difference from the published H12 sequence (GenBank accession number L07389) which we believe is due to an error in the published sequence.

The flagellin genes from type strains H35 and H54 were also amplified using primers #1431/#1432, which are based on sequence within the fliC gene. Sequence data revealed that these two genes would be non-functional due to insertion sequence inserted in the middle of them. We have sequenced them to facilitate selection of primers for the functional flagellin genes.

WO 99/61458 PCT/AU99/00385 25 9. Cloning of flagellin genes DNA was digested for 2 hr at 37 0 C with appropriate restriction enzyme(s). The reaction product was then extracted once with phenol, and twice with ether. DNA was precipitated with 2 vols of ethanol and resuspended in water before the ligation reaction was carried out.

Ligation was carried out O/N at 4 0 C and the ligated DNA was electroporated into one of the E. coli fliC mutant strains.

9.1. Cloning of flagellin genes from type strains for H1, H2, H3, H5, H6, H7, H9, HlO, H11, H12, H14, H15, H18, H19, H21, H24, H26, H27, H28, H29, H31, H34, H38, H39, H41, H42, H43, H45, H46, H49, H51, H52, and H56: The full flagellin gene was PCR amplified using primers #1868 and #1870 (Table 3A). Both these primers are based on the sequences of the H7 flagellin gene of the H7 type strain. #1868 is the 5' primer: there is an NcoI site incorporated into the primer (Table 3B) and the flagellin gene starts at base 3 of the NcoI site. The 3' primer #1870 has a BamHI site incorporated downstream of the stop codon of the flagellin gene (Table 3B). PCR reactions were carried out under the following conditions: denaturing, 94oC/30'; annealing, temperature varies (refer to Table 3A)/30'; extension, 72oC/1'; 30 cycles. The PCR product was purified using the Promega Wizard PCR purification kit (Madison WI USA) before being digested by restriction enzymes NcoI and BamHI and cloned into the NcoI/BamHI sites of plasmid pTrc99A.

Plasmid pTrc99A has a strong trc promoter upstream of the polylinker. Downstream of the promoter, it contains the ribosome binding site (SD sequence, see Fig 73) which is located 8bp upstream of the ATG site within the NcoI site. The polylinker and the SD sequence of pTrc99A are shown in Fig 73.

The plasmids generated were given pPR numbers, and WO 99/61458 PCT/AU99/00385 26 they are listed in Table 3A. In these plasmids, the expression module consists of the trc promoter, the SD sequence, and the full flagellin gene. The sequence of the junction region between the SD sequence and the start of flagellin gene is shown in Fig 73.

For flagellin genes from type strains for H6, H7, H9, H12, H14, H18, H19, H20, H26, H28, H31, H41, H43, H46, H49, H51, and H52, we have the full sequence for each gene and the primer sequences (#1868 and #1870) are conserved among them. The cloned genes therefore have the same sequence as those from the type strains.

For flagellin genes from type strains for Hi, H15 and H34, we also have the full sequence. The previously published sequence of the flagellin gene from the H1 type strain was extracted from GenBank (accession number L07387) and used. Primer #1868 is conserved in all three.

But, primer #1870 has the third base of the fifth last codon in the Hi sequence changed from A to G, and the third base of the second last codon changed from C to T in the H15 and H34 sequences: these changes did not change the amino acid coded, so the cloned genes encode the same gene products as those of the type strains.

For flagellin genes from type strains for H2, H3, H11, H21, H24, H27, H29, H38, H39, H42, and H56, we do not have the full sequences. In the plasmids carrying genes from these type strains, the expression module consists of the trc promoter, the SD sequence, and the full flagellin gene with the first and the last 21 base pairs being determined by the primer sequences which are based on the H7 flagellin gene of the H7 type strain. The sequence of the junction region between the SD sequence and the start of flagellin gene is shown in Fig 73.

9.2. Cloning of the flagellin gene from type strain of H23: The full flagellin gene was PCR amplified using primers #1868 and #1869 (Table 3A). #1868 is the WO 99/61458 PCT/AU99/00385 27 primer: there is an NcoI site incorporated into the primer (Table 3B) and the flagellin gene starts at base 3 of the NcoI site. The 3' primer #1869 has a SalI site incorporated downstream of the stop codon of the flagellin gene (Table 3B). PCR reactions were carried out under the following conditions: denaturing, 94oC/30'; annealing, 55oC/30'; extension, 72oC/1'; 30 cycles. The PCR product was purified using the Promega Wizard PCR purification kit (Madison WI USA) before being digested by restriction enzymes NcoI and SalI and cloned into the NcoI/SalI sites of plasmid pTrc99A to give plasmid pPR1942.

Plasmid pTrc99A has a strong trc promoter upstream of the polylinker. Downstream of the promoter, it contains the ribosome binding site (SD sequence, see Fig 73) which is located 8bp upstream of the ATG site within the NcoI site. The polylinker and the SD sequence of pTrc99A are shown in Fig 73.

The expression module of pPR1942 consists of the trc promoter, the SD sequence, and the full flagellin gene.

The sequence of the junction region between the SD sequence and the start of flagellin gene is shown in Fig 73.

9.3. Cloning of flagellin genes from type strains of H32 and H33: The full flagellin gene was PCR amplified using primers #1868 and #1871 (Table 3A). #1868 is the primer: there is an NcoI site incorporated into the primer (Table 3B) and the flagellin gene starts at base 3 of the NcoI site. The 3' primer #1871 has a PstI site incorporated downstream of the stop codon of the flagellin gene (Table 3B). PCR reactions were carried out under the following conditions: denaturing, 94oC/30'; annealing, temperature varies (refer to Table 3A)/30'; extension, 72oC/1'; 30 cycles. The PCR product was purified using the Promega Wizard PCR purification kit (Madison WI USA) before being digested by restriction enzymes NcoI and PstI WO 99/61458 PCT/AU99/00385 28 and cloned into the NcoI/PstI sites of plasmid pTrc99A.

Plasmid pTrc99A has a strong trc promoter upstream of the polylinker. Downstream of the promoter, it contains the ribosome binding site (SD sequence, see Fig 73) which is located 8bp upstream of the ATG site within the NcoI site. The polylinker and the SD sequence of pTrc99A are shown in Fig 73.

For flagellin genes from type strains for H30 and H32, we have the full sequence. Primer #1868 sequence is conserved in both of them. But, primer #1871 has the third base of the fourth last codon in both sequences changed from G to A to remove a PstI site (see Table 3B): this change did not change the amino acid coded. The expression module consists of the trc promoter, the SD sequence, and the full flagellin gene coding for a gene product which is same as that of the type strain. The sequence of the junction region between the SD sequence and the start of flagellin gene is shown in Fig 73.

We do not have the full sequence for the flagellin gene from the H33 type strain. In the plasmid containing the H33 type strain gene, the expression module consists of the trc promoter, the SD sequence, and the full flagellin gene with the first and the last 21 base pairs been determined by the primer sequences which were used for the cloning of H30 and H32. The sequence of the junction region between the SD and the start of flagellin gene is shown in Fig 73.

9.4. Flagellin genes from type strains for H4 and H17: For the flagellin genes of H4 and H17 type strains the full sequence was not obtained, and the sequenced parts were PCR amplified and cloned into plasmid pPR1951 to give in each case a gene in which the first 26 and the last 31 codons are based on the sequence of the H7 flagellin gene of the H7 type strain.

9.4.1 Construction of expression plasmid vector WO 99/61458 PCT/AU99/00385 29 pPR1951: The first 26 codons of the H7 flagellin gene was first PCR amplified using primers #1868 and #1872 (Table 3B). #1868 is the 5' primer: there is an NcoI site incorporated into the primer (Table 3B) and the flagellin gene starts at base 3 of the NcoI site. Primer #1872 was made to have the last two codons (codons 25 and 26) changed from CTG TCG (Leucine and Serine) to GGA TCC (Glycine and Serine) to generate a BamHI site. This PCR fragment was digested with NcoI and BamHI before being cloned into the NcoI/BamHI sites of pTrc99A to make plasmid pPR1949.

The last 31 codons (including the stop codon) of the H7 flagellin gene was PCR amplified using primers #1884 and #1871 (Table 3A). The 5' primer, #1884, has the first two of the 31 codons changed from TCG AAA (Serine and Lysine) to TCT AGA (Serine and Arginine) to generate a XbaI site (Table 3B). The 3' primer #1871 has a PstI site incorporated downstream of the stop codon (Table 3B). This PCR fragment was digested with XbaI and PstI, and then cloned into the XbaI/PstI sites of pPR1949 to make plasmid pPR1951.

9.4.2 Cloning of flagellin genes from the H4 and H17 type strains: The central regions of flagellin genes from type strains H4 and H17 were PCR amplified using primers #1878 and #1885 (Table 3B), which have a BamHI and a XbaI incorporated at their ends respectively. PCR reactions were carried out under the following conditions: denaturing, 94oC/30'; annealing, 65oC/30'; extension, 72oC/1'; 30 cycles. The PCR product was purified using the Promega Wizard PCR purification kit (Madison WI USA) before being digested by restriction enzymes BamHI and XbaI and cloned into the XbaI/BamHI sites of plasmid pPR1951 to make plasmids pPR1955 (H4) and pPR1957 (H17).

The expression module of plasmids pPR1955 and pPR1957 WO 99/61458 PCT/AU99/00385 30 consists of the trc promoter, the SD sequence, the first 24 codons of the H7 flagellin gene (of the H7 type strain), 2 codons encoding Glycine and Serine, 292 or 293 codons of the central region based on the flagellin gene obtained from the H4 or H17 type strain respectively, 2 codons encoding Serine and Arginine, and then the last 29 codons of the H7 flagellin gene (of the H7 type strain).

Expression of flagellin gene plasmids in E. coli strains lacking the fliC gene, and identification of the H antigens encoded by these plasmids: Plasmids carrying flagellin genes as described in section 9 (see Table 3A for a list) were electroporated into strains M2126 or P5560. Strains M2126 and P5560 do not have functional fliC genes, and are not motile when examined under a microscope. Transformants carrying any of the plasmids listed in Table 3A are motile when examined under a microscope. Thus, the flagellin genes in all of the plasmids are expressed.

The antigenic specificity of the flagellin of each transformant was then determined by slide agglutination.

10.1 Flagellin genes from type strains for H2, H6, H7, H9, H11, H14, H15, H18, H19, H20, H21, H23, H24, H26, H27, H28, H29, H30, H31, H32, H33, H34, H39, H41, H42, H43, H45, H46, H49, H51, H52, and H56: As shown in Table 3A, strains with plasmids carrying these flagellin genes expressed the same H antigen as their respective flagellin parent strain.

For flagellin specificities H2, H5, H6, H7, H9, H14, H18, H19, H20, H23, H24, H26, H27, H28, H29, H31, H33, H39, H51, H52, and H56, there was no cross reaction reported between these flagellins and flagellin antisera for other H antigens [Ewing, W. Edwards and Ewing's identification of the Enterobacteriaceae., Elsevier Science Publishers, Amsterdam, The Netherlands, 1986], and we conclude that we have in each case sequenced the gene WO 99/61458 PCT/AU99/00385 31 encoding the flagellin of the expected specificity from the respective type strain.

It has been observed that cross reactions exist between some type strains and certain antisera at different levels of dilution (of the antisera), being H11 with anti-H21 and anti-H40, H21 with anti-H11, H30 with anti-H32, H32 with anti-H30, H34 with anti-H24 and anti- H31, H41 with anti-H37 and anti-H39, H42 with anti-H6, H43 with anti-H37, H45 with anti-H20, H46 with anti-H17, and H49 with anti-H39 [Ewing, W. Edwards and Ewing's identification of the Enterobacteriaceae., Elsevier Science Publishers, Amsterdam, The Netherlands, 1986]. We have tested strain M2126 or strain P5560 carrying plasmids containing flagellin genes obtained from each of these type strains (H11, H21, H30, H32, H34, H41, H42, H43, H46, and H49) with the appropriate cross-reacting antisera.

For strain M2126 or strain P5560 carrying plasmids containing flagellin genes obtained from type strains H11, H34, H41, H42, H43, H45, H46, and H49, no cross reaction was found. We conclude that we have in each case sequenced the gene coding the flagellin of the expected specificity from the respective type strain.

Cross reaction was observed for strain P5560 carrying plasmid pPR1948 (containing the flagellin gene obtained from the H30 type strain) with anti-H32 serum, strain P5560 carrying pPR1940 (containing the flagellin gene obtained from the H32 type strain) with anti-H30 serum and strain M2126 carrying plasmid pPR1995 (containing the flagellin gene obtained from the H21 type strain) with anti-H11 serum.

We note that the reported cross reactions between the type strain and anti-H32, the H32 type strain and and the H21 type strain and anti-H11 happened at a higher level of dilution (of antisera) than for all other type strains with the antisera mentioned above [Ewing, W. Edwards and Ewing's identification of the WO 99/61458 PCT/AU99/00385 32 Enterobacteriaceae., Elsevier Science Publishers, Amsterdam, The Netherlands, 1986]. We conclude that except for these three cases, the antiserum used were supplied at a dilution which did not exhibit cross reactions. For the three strains carrying flagellin genes cloned form type strains for H30, H32, and H21, it was necessary to further dilute the antiserum.

Strain P5560 carrying plasmid pPR1948 (containing the flagellin gene obtained from the H30 type strain) agglutinates with anti-H30 serum when the antiserum is diluted to 1:60, but agglutinates with anti-H32 serum only at a dilution of 1:10 and not at a 1:20 dilution (note that the antisera we used have been diluted before reaching our hands). In contrast, strain P5560 carrying plasmid pPR1940 (containing flagellin gene obtained from the H32 type strain) agglutinates with anti-H32 serum when the antiserum is diluted at 1:100, but agglutinates with serum only at a 1:10 dilution and not at a 1:10 dilution. Thus, we conclude that the flagellin genes we sequenced from type strains for H30 and H32 encode flagellins of H30 and H32 specificities respectively.

Strain M2126 carrying plasmid pPR1995 (containing the flagellin gene obtained from the H21 type strain) agglutinates with anti-H21 serum when the antiserum is diluted to 1:40, but agglutinates only with undiluted anti-Hll serum and not at a 1:10 dilution (note that the antisera we used have been diluted before reaching our hands). In contrast, strain M2126 carrying plasmid pPR1981 (containing flagellin gene obtained from the H11 type strain) did not agglutinate with anti-H21 serum. Thus, we conclude that the flagellin genes we sequenced from type strains for H21 encodes flagellin of H21 specificity.

10.2 Flagellin genes from type strains of HI and H12: These two genes are very similar in sequence, with 8 a.a difference between the gene products. It has been WO 99/61458 PCT/AU99/00385 33 known that some cross-reaction exists between anti-H12 serum and the H1 type strain and between anti-Hi serum and the H12 type strain [Ewing, W. Edwards and Ewing's identification of the Enterobacteriaceae., Elsevier Science Publishers, Amsterdam, The Netherlands, 1986].

Strain M2126 carrying pPR1920 (carrying a flagellin gene from the H1 type strain, Table 3A) agglutinates with anti- H1 serum when the antiserum is diluted to 1:100, but agglutinates only with undiluted anti-H12 serum and not at a 1:10 dilution (please note that the antisera we used have been diluted before reaching our hands). In contrast, strain M2126 carrying plasmid pPR1990 (carrying a flagellin gene from the H12 type strain, Table 3A) agglutinates with anti-H12 serum when the antiserum is diluted at 1:100, but agglutinates only with undiluted anti-Hi serum and not at a 1:10 dilution. Thus, we conclude that the flagellin genes we sequenced from type strains for H1 and H12 encode flagellins of H1 and H12 specificities respectively.

10.3. Flagellin gene coding for H16: Strain P5560 carrying plasmid pPR1969 agglutinated with anti-H16 serum. pPR1969 carries a flagellin gene amplified from the H3 type strain. It has been shown that this H3 type strain is a biphasic strain which can express H3 and H16 specificities [Ratiner, Y. A. (1985) "Two genetic arrangements determining flagellar antigen specificities in two diphasic E. coli strains. FEMS Microbiol Lett 19: 317-323]. Thus, the H3 type strain has two flagellin genes coding for H3 and H16 specificities.

We conclude that we have cloned and sequenced the H16 flagellin gene from this H3 type strain.

10.4 Flagellin gene coding for H4: The flagellin genes obtained from type strains for H4 and H17 are nearly identical, with 4 a.a. difference in the gene products. Plasmid pPR1955 carries a flagellin WO 99/61458 PCT/AU99/00385 34 gene from the H4 type strain, and plasmid pPR1957 carries a flagellin gene from the H17 type strain. Strain P5560 carrying plasmid pPR1955 or plasmid pPR1957 agglutinated with anti-H4 serum, but not with anti-H17 serum. It has been shown that the type strain for H17 is a biphasic strain which can express H17 and H4 [Ratiner, Y. A. (1985) "Two genetic arrangements determining flagellar antigen specificities in two diphasic E. coli strains. FEMS Microbiol Lett 19: 317-323]. The flagellin gene obtained from type strain for H44 is also highly similar to that obtained from the H4 type strain, with 2 a.a. difference in the gene products. It has been shown that the H44 type strain has two complete flagellin genes, being H4 and H44 [Ratiner, Y. A. (1998) "New flagellin specifying genes in some E. coli strains" J. Bacteriol 180: 979-984]. Thus, we conclude that all the three flagellin genes (obtained from type strains for H4, H17 and H44, and sequenced) encode the H4 flagellin, and that the flagellin genes for H17 and H44 specificities have not yet been cloned.

10.5 Flagellin gene coding for The flagellin genes obtained from type strains for and H50 are nearly identical, with 3 a.a. difference in the gene products. Strain P5560 carrying plasmid pPR1923 (which carries a flagellin gene from the H10 type strain) agglutinated with anti-H10 serum. We conclude that the sequence obtained from the H10 type strain encodes the flagellin. It is not clear if the sequence obtained from the H50 type strain encodes H10 or H50 (see below section for 10.6 Flagellin gene coding for H38: The flagellin genes obtained from type strains for H38 and H55 are nearly identical, with only 1 a.a.

difference in the gene products. Strain M2126 carrying plasmid pPR1984 (carrying the flagellin gene from the type strain H38) agglutinated with anti-H38 serum, but not with WO 99/61458 PCT/AU99/00385 35 serum. It also has been shown that the type strain for H55 has two complete flagellin genes coding for and H38 specificities [Ratiner, Y. A. (1998) "New flagellin specifying genes in some E. coli strains" J.

Bacteriol 180: 979-984]. Thus, we conclude that both cloned genes encode the H38 flagellin.

10.7 Summary: Flagellin genes coding for 39 H antigens have been identified, being those for specificities Hi, H2, H4, H6, H7, H9, H10, H11, H12, H14, H15, H16, H18, H19, H21, H23, H24, H26, H27, H28, H29, H30, H31, H32, H33, H34, H38, H39, H41, H42, H43, H45, H46, H49, H51, H52, and H56.

11. Comparison and alignment of the flagellin genes: Programs Pileup [Devereux, Haeberli, P. and Smithies, A comprehensive set of sequence analysis programs for the VAX. Nucl. Acids Res. 12 (1984) 387- 395]and Multicomp [Reeves, Farnell, L. and Lan, R.: MULTICOMP: a program for preparing sequence data for phylogenetic analysis. CABIOS 10 (1994) 281-284] were used.

The previously published sequence of H1 (GenBank accession number L07387) was extracted from GenBank and used. Because we did not sequence H36 and H53 flagellin genes and we did not have the H16 type strain, we only compared 51 flagellin genes of H type strains and the fliC genes from the additional 10 H7 strains.

Among the H7 fliC genes, the percentage of DNA difference ranged from 0.0 to 2.39%. The flagellin genes from type strains for H40 and H8 are identical. Some others are nearly identical: H21 and H47 difference), H12 and H1 difference), H10 and difference), H38 and H55 difference), H4, H44 and H17 are very similar, the pairwise difference ranging from 0.33% to 0.87%.

WO 99/61458 PCT/AU99/00385 36 For the flagellin genes obtained from type strains for H4, H17 and H44, we have shown that all the three genes encode flagellin with the H4 specificity (see above). For the flagellin genes obtained from type strains fro H21 and H47, and H38 and H55, we have confirmed the specificities for one for each pair and have good reason to conclude that both genes of each pair encode the same H specificity (see above section), being that for H21 and H38 specificities respectively.

For the flagellin genes obtained from type strains for H10 and H50, we have confirmed that the one from the type strain encodes H10 specificity. As these two genes are highly similar, we have presumed that they both encode H10 specificity.

In the cases where the flagellin gene from two type strains is near identical, we conclude that both genes code for flagellin of the same H specificity and that one or other strain has an additional locus which carries the functional gene, although the flagellin genes sequenced do not appear to be mutated.

We have shown by cloning and expression that the flagellin genes obtained from the H1 and H12 type strains encode H1 and H12 specificities respectively (see above section). The neucleotide difference between these two genes is higher at 2.6% (see above), but still within the normal range for variation within a gene in E. coli. The two antigens cross react, and this cross reaction must be due to the high level similarity of the flagellins encoded by these two genes.

As discussed above, genes encoding some H antigens have been shown to be located at loci other than fliC. H3, H36, H47, H53 have been shown to be at a locus called flkA, H44 and H55 at fllA, and H54 at flmA [Ratiner Y A (1998)"New flagellin-specifying genes in some Escherichia coli strains" J. Bacteriol. 180 979-984]. However, these strains may carry a fliC in addition to flkA, fllA or flmA [Ratiner Y A (1998)"New flagellin-specifying genes in some WO 99/61458 PCT/AU99/00385 37 Escherichia coli strains" J. Bacteriol. 180 979-984].

The flagellin gene encoding H48 was previously sequenced from E. coli strain K-12 [Kuwajima G, Asaka J, Fujiwara T, Node K and Kondo E "Nucleotide sequence of the hag gene encoding flagellin of Escherichia coli" J Bacteriol. 168 (1986) 1479-1483]. We have sequenced the fliC gene from the H48 type strain, and found that it is identical to that from K-12.

The H54 gene is known to be at flmA [Ratiner Y A (1998)"New flagellin-specifying genes in some Escherichia coli strains" J. Bacteriol. 180 979-984] and the finding of a non-functional presumptive fliC locus in the H54 strain shows that it is present but not expressed. However, we have not amplified and sequenced the functional flmA gene of this strain.

Using the 43 unique sequences (being the 39 identified genes with confirmed specificities and the flagellin genes obtained from the H8 (or H40), H25, H37, and H48 type strains) and the sequences from the two nonfunctional flagellin genes (from H type strains H35 and H54) (see Table 3) we have been able to determine antigen specific primers for each of the H antigen specificities and thereby show that it is practicable to detect E.coli strains carrying specific H antigens without false positives from strains of other H types. There is no reason to expect that the addition of 11 sequences to the 43 unique sequences obtained will affect the general conclusion, as unlike previous reports, our study covers flagellin sequences for a substantial majority of known E.

coli H antigen specificities.

Our study of 11 H7 genes from strains of eight different 0 antigens shows limited variation which was such that the variation within genes for H antigens does not affect the ability to select antigen specific primers.

O:H combinations in general define a strain and as some of the strains thus defined were quite distant from each other in a study by Whittam [Whittam T S, wolfe M L, WO 99/61458 PCT/AU99/00385 38 Wachsmuth I K, Orskov I and Wilson R A "Clonal relationships among Escherichia coli strains that cause hemorrhagic colitis and infantile diarrhea" Infect. Immun.

61 (1993) 1619-1629] the variation we observe is thought to represent that generally present in H7 genes. We also obtained more than one sequences for flagellin genes for H specificities H4, H10, and H38, and again the level of variation within a given specifities is very low.

However, there is a low possibility that primers chosen without knowledge of the variation within genes of each H specificity could fail to give positive results with some isolates due to chance choice of primers which cover a base or bases which contribute to this low level variation. The variation within the H7 genes is in the normal range for variation within a gene in E. coli and if this possibility did occur it would be easy to use an alternate primer pair. For example, if a first primer in a primer pair is unable to hybridise to a target region because of low level variation in that region, a positive result may be achieved by using a second primer in that pair together with a third primer, whether or not the third primer is specific for the flagellin gene. Where the third primer is not specific for the flagellin gene, the specificity of the primer pair derives from the specificity of the second primer. The observation that the overall level of variation within gene for a given H specificity is very low making it extremely unlikely that the regions covered by the two primers specific for H specificity would both have undergone change in the same strain.

There are 54 known H antigens for E. coli and of these there are 11 H antigen specificities for which we do not as yet have sequence. It will be easy to determine these sequences and determine primer pairs specific for these H antigens by comparing these sequences with the obtained sequences (see Table and also modify the primers selected for any H antigen for which we already WO 99/61458 PCT/AU99/00385 39 know the sequence in the unlikely event that there is a possibility of false positives with the primers selected.

The sequences for the remaining H antigens can be obtained in one of the following ways: i. where we have two bands by PCR (H36 and H53 type strains), we purify each and sequence, and also clone each into a strain mutated in its fliC gene and determine the H antigen expressed by use of specific sera. In this way a specific sequence can be related to an H antigen specificity. The other band which represents an H antigen gene for a different specificity is expected to include a mutant gene or a gene similar to one of those for a known H specificity, but if not may represent a new specificity for which primer pairs could be selected. It may be difficult to obtain expression of flagellin genes when cloned from E. coli due to cloning together with regulatory sequences which prevent expression. This is easily avoided by cloning the major segment of the gene into a functioning fliC gene to replace the equivalent segment of that gene, using standard site directed mutagenesis to give suitable restriction sites within the cloned gene and incorporating those restriction sites into primers used to amplify the major segment of the gene to be studied to facilitate the cloning. We have cloned and sequenced the PCR bands from the H36 and the H55 type strains using this method (see section 16).

2. Where two or more strains have the same flagellin gene sequence, the genes are cloned as above and the H antigen specificity represented by this sequence is determined. This identifies the strain in which the expected gene is expressed and also those strains for which we have sequenced a gene which is not being expressed. We then clone the gene for the antigen expressed in these strains by making a bank of plasmid clones using chromosomal DNA and select for a clone which WO 99/61458 PCT/AU99/00385 40 is expressing an H antigen different from the one represented by the known sequence. This can be done by taking advantage of the fact that the H antigen is on flagellin, the protein of the bacterial flagellum used for movement of the bacteria. In the presence of antibodies specific to that flagellum the bacteria cannot swim. For selection the clones are placed in a situation in which motile cells can swim away from the others and be collected. There are many versions of these techniques and any could be used. One version is to place the bacteria on a nutrient agar plate with reduced agar content such that bacteria can swim away from the site of inoculation. This is easily seen as growth on the plate and a sample of the bacteria which are motile can be recovered and cultivated. In this way bacteria carrying cloned H antigen genes can be selected. If the medium in the plate has antibody added to it only bacteria which express an H antigen different to that recognised by the antiserum will be able to swim. Specifically if the antiserum used is specific for the H antigen expressed by the gene for which we have sequence, only clones which express a different H antigen, such as those expressing the H antigen expressed by the H type strains used to make the plasmid, will be selected. Once the clone is obtained, the H antigen gene can be sequenced.

Our work has shown that there are at least 7 cases where the H antigen type strains carry two H antigen genes which appear to be complete and have the potential to function. However, while E. coli does not (in general) have a capacity to express more than one flagellin gene, it is striking that there are several loci for flagellin genes [Ratiner Y A (1998)"New flagellin-specifying genes in some Escherichia coli strains" J. Bacteriol. 180 979- 984]. Several of the pairs of H type strains with identical or near identical sequence do not include any of the H antigen types shown by Ratiner [Ratiner Y A WO 99/61458 PCT/AU99/00385 41 (1998)"New flagellin-specifying genes in some Escherichia coli strains" J. Bacteriol. 180 979-984] to map other than at fliC although these predominate. This suggests that there are additional cases where the expressed gene is not the only flagellin gene present. However the fact that many of the cases where we obtained flagellin genes of identical or near identical sequence and/or two flagellin genes from one strain involve type strains found by Ratiner [Ratiner Y A (1998)"New flagellin-specifying genes in some Escherichia coli strains" J. Bacteriol. 180 979- 984] to map away from fliC are among those near identical to others, indicates that the phenomenon is of limited extent. Nonetheless it remains possible even where only one gene has been obtained by PCR, that it is one of a pair of flagellin genes, the other not being amplified by the primers used, and further that it is the one not amplified which is expressing the H antigen of the strain.

It will therefore be necessary to clone as described above each of the flagellin genes we have sequenced and confirm that it expresses the expected antigen to ensure that the invention give results corresponding to those of the traditional serotyping scheme. In the event that it does not, the gene for the type antigen can be cloned and sequenced by the means described above.

The 11 H7 fliC sequences fell into three groups, one comprising the genes from the 0157:H7 and 055:H7 strains, which were identical, as expected given the proposed relationship between the clones. It has been shown that E.

coli 0157:H7 and 055:H7 clones are closely related [Whittam T S, wolfe M L, Wachsmuth I K, Orskov I and Wilson R A "Clonal relationships among Escherichia coli strains that cause hemorrhagic colitis and infantile diarrhea" Infect. Immun. 61 (1993) 1619-1629] thus it was expected that the H7 fliC genes from 0157 and 055 would be identical. Among the H7 fliC sequences, we can identify primers specific to the H7 fliC gene for each of the three H7 groups. Two of these primers in combination with an H7 WO 99/61458 PCT/AU99/00385 42 specific primer gave two primer pairs specific for the H7 gene of from the 0157:H7 and 055:H7 clones.

13. Specific oligonucleotide primers for each of the 43 flagellin genes Two oligonucleotide primers were chosen based on each of the 43 sequences. None of them had more than identity with any other of 61 flagellin gene sequences.

Thus, these primers are specific for each H type. These primers are listed in Table 3.

The flagellin gene of the H54 type strain is a mutated gene. It has an insertion sequence (IS1222) inserted into a normal flagellin gene of H21. Thus, primers for H21 would amplify a fragment of different size in H54. We also provide 2 primers based on the insertion sequence (see H54 row in Table and the use of one of them in combination with one of the H21 primers will generate a PCR band only in H54, which will also differentiate those strain carrying the mutated H21 gene from those expressing the H21 flagellin gene.

The flic gene of H35 type strain is also a mutated gene. It has an insertion sequence (IS1) inserted into a normal flagellin gene of Hl1. Thus, primers for H11 would amplify a fragment of different size in H35. We also provide 2 primers based on the insertion sequence (see row in Table and the use of one of them in combination with one of the H11 primers will generate a PCR band only in H35, which will also differentiate those strain carrying the mutated H11 gene from those expressing the H11 flagellin gene.

14. Testing of the H7 specific oligonucleotide primers Primer pair #1806/#1809 (see Table 3) was used to carry out PCR on chromosomal DNA samples of all the 54 H type strains and the H7 strains listed in Table 1. PCR reactions were carried out under the following conditions: denaturing, 94 0 C/30'; annealing, 58 0 C/30'; extension, WO 99/61458 PCT/AU99/00385 43 72 0 30 cycles. PCR reaction was carried out in an volume of 50ul for each of the chromosomal sample. After the PCR reaction, 5pl PCR product from each sample was run on an agarose gel to check for amplified DNA.

Primer pairs #1806/#1809 produced a band of predicted size with all the 11 strains expressing H7, but gave no band with other H type strains. Thus, these primers are H7 specific.

15. Testing of oligonucleotide primers specific to H7 of 0157 and 055: Based on a comparison of the fliC sequences of 11 different H7 strains, we have identified two oligonucleotides [#1696 (5'-GGCCTGACTCAGGCGGCC) at positions 178 to 195 in M527 and #1697 GAGTTACCGGCCTGCTGA) positions 1700-1683 in M527] which are unique to H7 of 0157 and 055. Although not identical to any parts of the fliC sequences of any other H7 strains, these two primers are identical or have high level similarity to fliC genes of some other H types. However a combination of one of these primers with one of the H7 specific primers can give specificity for H7 of 0157:H7 and 055:H7 E. coli.

Primer pairs #1696/#1809 and #1697/#1806 were used to carry out PCR on chromosomal DNA samples of all the H type strains and the H7 strains listed in Table 1. PCR reactions were carried out under the following conditions: denaturing, 94 0 C/30'; annealing, 61 0 C/30' (for #1696/#1809) or 60 0 C/30'(for#1697/#1806); extension, 72 0 30 cycles. PCR reaction was carried out in an volume of 50ll for each of the chromosomal samples. After the PCR reaction, 5~i PCR product from each sample was run on an agarose gel to check for amplified DNA.

Both primer pairs produced a band of predicted size with both of the 0157:H7 strains (strains M1004 and M527, see Table and the 055:H7 strain (strain M1686, see Table but gave no band with other strains. Thus, these WO 99/61458 PCT/AU99/00385 44 two pairs of primers are specific to H7 genes of 0157:H7 and 055:H7 E. coli strains.

16. Identification of flagellin genes for the remaining H specificities.

16.1. Sequencing the potential flkA gene coding for the H36 flagellin: Using primers #1431 atg gca caa gtc att aat ace caa c) and #1432 cta ace ctg cag cag aga ca), we have amplified two bands from the H36 type strain. PCR reaction was carried out under the following conditions: denaturing, 94oC/30'; annealing, 57oC/30'; extension, 72oC/1'; 30 cycles. These two PCR fragments were then cloned into the pGEM-T vector using the Promega pGEM-T cloning kit (Madison WI USA) to make plasmids pPR1992 and pPR1993. Inserts from both plasmids were first sequenced using the M13 universal primers (which bind to the pGEM-T DNA flanking the insertion site). For pPR1992, primers based on the sequence obtained were then used to sequence further, and this procedure was repeated until the insert was fully sequenced.

The sequence of the insert of pPR1992 is identical to that of the H12 flagellin gene sequence except perhaps for the first 8 and last 7 codons which are encoded by the PCR primers in plasmid pPR1992. We have only sequenced the two ends of the insert of plasmid pPR1993 (Figures 71 and 72), and the sequences of the two ends of the insert of pPR1993 are very similar to ends of other sequenced flagellin genes. We conclude that the insert of plasmid pPR1993 encodes a flagellin gene. The full sequence of the insert of plasmid pPR1993 can be obtained using the same method as for the sequencing of the insert of plasmid pPR1992. It is known that flkA gene encodes the H36 flagellin [Ratiner, Y. A. (1998) "New flagellin specifying genes in some E. coli strains" J. Bacteriol 180: 979-984], and it is highly likely that plasmid pPR1993 contains the WO 99/61458 PCT/AU99/00385 45 flkA gene of the H36 type strain. H specificities can be confirmed by slide agglutination.

The currently uncharacterised sequence of both ends and of DNA flanking these two sequenced genes can be obtained by PCR walking and sequencing. Methods for PCR walking from a known sequence to an unknown region in chromosomal DNA are available (see [Siebert, P. D. A.

Chenchi, D. E. Kellogg, A. Lukyanov and S. A. Lukyanov (1995) "An improved PCR method for walking in uncloned genomic DNA." Nuc. Acids Res. 23: 1087-1088]).

The sequenced genes then can be PCR amplified and cloned using the method(s) described in section 9.

Flagellins expressed by strain M2126 carrying these plasmids then can be determined by use of specific sera.

The sequences flanking the flkA gene can then be used to PCR amplify other flkA genes (see below).

16.2 The flkA genes coding for H3, H47 and H53: It has been shown that flagellins H3, H47 and H53 are encoded by flkA genes in the type strains [Ratiner, Y. A.

(1998) "New flagellin specifying genes in some E. coli strains" J. Bacteriol 180: 979-984]. These genes can be PCR amplified using primers based on the sequences flanking the flkA gene in the H36 type strain. These PCR fragments can then be sequenced, and the genes expressed in strain M2126 for the identification of these genes.

16.3 The fllA genes coding for H44 and It is known that flagellins H44 and H55 are coded by fllA genes.

16.3.1 The H55 flagellin gene: Using primers #1868 and #1870 (Table 3B), we have amplified two bands from the H55 type strain. PCR reaction was carried out under the following conditions: denaturing, 94oC/30'; annealing, 50oC/30'; extension, 72oC/1'; 30 cycles. These two PCR fragments were then WO 99/61458 PCT/AU99/00385 46 cloned into the pGEM-T vector using the Promega pGEM-T cloning kit (Madison WI USA) to make plasmids pPR1994 and pPR1989. Inserts from both plasmids were first sequenced using the M13 universal primers (which bind to the pGEM-T DNA flanking the insertion site). Primers based on the sequence obtained were then used to sequence further, and this procedure was repeated until both inserts were fully or partly sequenced.

The sequence of the insert of pPR1994 is highly similar to that of the flagellin gene of the H38 type strain, with 1 amino acid difference in the gene products.

We have only sequenced the two ends of the insert of plasmid pPR1989 (figures 70A and 70B), and the sequences of the two ends of the insert of pPR1989 are very similar to ends of other sequenced flagellin genes. We conclude that the insert of plasmid pPR1989 encodes a flagellin gene. The full sequence of the insert of plasmid pPR1989 can be obtained using the same method as for the sequencing of the insert of plasmid pPR1994. It is known that the H55 type strain carries flagellin genes for both H38 and H55, and that the H55 flagellin gene is at the fllA locus [Ratiner, Y. A. (1998) "New flagellin specifying genes in some E. coli strains" J. Bacteriol 180: 979-984]. Thus, it is highly likely that plasmid pPR1989 contains the fllA gene of the H55 type strain.

The currently uncharacterised sequence of both ends and of DNA flanking these two sequenced genes can be obtained by PCR walking and sequencing. Methods for PCR walking from a known sequence to an unknown region in chromosomal DNA are available (see [Siebert, P. D. A.

Chenchi, D. E. Kellogg, A. Lukyanov and S. A. Lukyanov (1995) "An improved PCR method for walking in uncloned genomic DNA." Nuc. Acids Res. 23: 1087-1088]).

The sequenced genes then can be PCR amplified and cloned using the method(s) described in section 9.

Flagellins expressed by strain M2126 carrying these plasmids then can be determined by use of specific sera.

WO 99/61458 PCT/AU99/00385 47 16.3.2 The H44 flagellin gene: The sequence information for DNA flanking the fllA gene in the H55 type strain can then be used to PCR, sequence and identify the fllA gene in the H44 type strain.

16.4 The flmA gene coding for H54: This gene can be cloned by making a bank of plasmid clones in strain M2126 using chromosomal DNA of the H54 type strain and selecting for a transformant which is motile on an agar plate. This is done by taking advantage of the fact that the H antigen is on flagellin, the protein of the bacterial flagellum used for movement of the bacteria. Strain M2126 lacks flagellin. Once the clone(s) is obtained and identified by use of anti-H54 serum, the flagellin gene can be sequenced. It is possible that clones expressing different flagellin specificities can be obtained, and each of them can be identified by using different sera.

16.5 The flagellin genes obtained from the H37 and H48 type strains: We have used primers #1868 and #1869 (both were based on the sequence obtained from the H48 type strain, also see section 9) and primers #1868 and #1870 (both were based on the sequences of the H7 flagellin gene of the H7 type strain, also see section 9) to PCR amplify and clone the sequenced flagellin genes from the H48 and H37 type strains respectively. Strain P5560 carrying the plasmid containing either the cloned gene was not motile and did not react with the appropriate antisera. It is highly likely that mutaions have occured due to PCR errors. This can be resolved by re-amplification and re-cloning of the genes.

16.6 The flagellin gene obtained from the H25 type WO 99/61458 PCT/AU99/00385 48 strain: The flagellin gene sequence we first obtained from the H25 type strain lacks 23 and 21 codons at 5' and 3' ends respectively. We could not amplify the full gene from the H25 type strain using primers based on the H7 flagellin gene of the H7 type strain, and it was necessary to get the full sequence of this flagellin gene by other means.

We have used primers (#2650: 5' cag cga tga aat act tgc cat and #2648: 5' caa tgc ttc gtg acg cac) based on the genes (fliD and fliA respectively) flanking fliC gene in E. coli K-12 [Blattner, F. G. I. Plunkett, C. A.

Bloch, N. T. Perna, V. Burland, M. Riley and et al. (1997) "The complete genome sequence of E. Coli Kil2" Science 277: 1453-1474] and primers (#2658: 5' gcc tga gtc aga cct ttg and 2653 5' aac ctg tct gaa gcg cag) based on the flagellin sequence obtained from the H25 type strain to PCR amplify both ends of the flagellin gene. The PCR product was then sequenced, and we have now obtained the full flagellin gene sequence and sequence for the DNA flanking the flagellin gene from type strain H25 (Figure 69). Now, it is straightforward to PCR amplify, clone and express, and identify this gene using the methods described in sections 9 and 16.7 The flagellin genes obtained from the H8 and H40 type strains: The flagellin gene sequences obtained from both the H8 and H40 type strains lack 18 and 15 codons at 5' and 3' ends respectively. We have used primers based on the H7 flagellin gene of the H7 type strain to PCR amplify and clone the full genes from these two strains. Strain M2126 carrying plasmid made this way was not motile under microscope and did not react with the appropriate antisera. This could be due to PCR errors as mentioned in section 16.5 or perhaps the first and last few amino acids encoded by the primers (based on H7 flagellin gene) are WO 99/61458 PCT/AU99/00385 49 uncompatible in this case.

The full sequence of the full gene can be obtained using method described in section 16.6. The flagellin gene can then be PCR amplified, cloned and expressed, and identified using the methods described in sections 9 and The gene products of the flagellin genes obtained from the H8 and H40 type strains are identical. Thus, one of these two H specificities must be encoded by a unknown gene, and it can be cloned and identified using the method described in the section 16.8.

16.8 Flagellin genes coding for H17, H35, and As mentioned above, the sequenced flagellin genes from the H17 and H50 type strains encode H4 and specificities respectively. The flagellin gene sequence obtained from the H35 strain has a insertion and encodes a non-functional gene (see section Thus, genes coding for these flagellins have not been identified, and their location is unknown. One can use primers based on DNA flanking fliC, fllA, flkA, and flmA to do PCR on the type strain for each of the flagellin antigen. PCR products can then be sequenced, and possible genes can be cloned, expressed and identified then.

If the target gene is not PCR amplified using primers based on sequence of these loci or sequence flanking these loci, it can be cloned by making a bank of plasmid clones in strain M2126 using chromosomal DNA of the type strain and selecting for a transformant which is motile on an agar plate. This is done by taking advantage of the fact that the H antigen is on flagellin, the protein of the bacterial flagellum used for movement of the bacteria.

Strain M2126 lacks flagellin. Once the clone(s) is obtained and identified by use of antisera, the flagellin gene can be sequenced. It is possible that clones expressing different flagellin antigens can be obtained, WO 99/61458 PCT/AU99/00385 50 and each of them can be identified by using different antisera. Antiserum for H50 can be prepared using standard methods [Ewing, W.H.:Edwards and Ewing's identification of the Enterobacteriaceae., Elsevier Science Publishers, Amsterdam, The Netherlands, 1986].

0 antigen Materials and Methods-part 1 The experimental procedures for the isolation and characterisation of the E. coli 0111 0 antigen gene cluster (position 3,021-9,981) are according to Bastin et al. 1991 "Molecular cloning and expression in Escherichia coli K-12 of the rfb gene cluster determining the 0 antigen of an E. coli 0111 strain". Mol. Microbiol.

5:9 2223-2231 and Bastin D.A. and Reeves, P.R. 1995 "Sequence and analysis of the 0 antigen gene(rfb)cluster of Escherichia coli 0111". Gene 164: 17-23.

A. Bacterial strains and growth media Bacteria were grown in Luria broth supplemented as required.

B. Cosmids and phage Cosmids in the host strain x2819 were repackaged in vivo. Cells were grown in 250mL flasks containing 30mL of culture, with moderate shaking at 30 0 C to an optical density of 0.3 at 580 nm. The defective lambda prophage was induced by heating in a water bath at 45 0 C for followed by an incubation at 37 0 C with vigorous shaking for 2hr. Cells were then lysed by the addition of 0.3mL chloroform and shaking for a further 10min. Cell debris were removed from ImL of lysate by a 5min spin in a microcentrifuge, and the supernatant removed to a fresh microfuge tube. One drop of chloroform was added then shaken vigorously through the tube contents.

C. DNA preparation Chromosomal DNA was prepared from bacteria grown overnight at 37 0 C in a volume of 30mL of Luria broth.

After harvesting by centrifugation, cells were washed and WO 99/61458 PCT/AU99/00385 51 resuspended in 10mL of 50mMTris-HC1 pH 8.0. EDTA was added and the mixture incubated for 20min. Then lysozyme was added and incubation continued for a further Proteinase K, SDS, and ribonuclease were then added and the mixture incubated for up to 2hr for lysis to occur.

All incubations were at 370C. The mixture was then heated to 65 0 C and extracted once with 8mL of phenol at the same temperature. The mixture was extracted once with 5mL of phenol/chloroform/iso-amyl alcohol at Residual phenol was removed by two ether extractions.

DNA was precipitated with 2 vols. of ethanol at spooled and washed in 70% ethanol, resuspended in l-2mL of TE and dialysed. Plasmid and cosmid DNA was prepared by a modification of the Birnboim and Doly method [Birnboim, H. C. and Doly, J. (1979) "A rapid alkaline extraction procedure for screening recombinant plasmid DNA" Nucl. Acid Res. 7:1513-1523]. The volume of culture was 10mL and the lysate was extracted with phenol/chloroform/iso-amyl alcohol before precipitation with isopropanol. Plasmid DNA to be used as vector was isolated on a continuous caesium chloride gradient following alkaline lysis of cells grown in 1L of culture.

D. Enzymes and buffers.

Restriction endonucleases and DNA T4 ligase were purchased from Boehringer Mannheim (Castle Hill, NSW, Australia)or Pharmacia LKB (Melbourne, VIC Australia).

Restriction enzymes were used in the recommended commercial buffer.

E. Construction of a gene bank.

Individual aliquots of M92 chromosomal DNA (strain Stoke W, from Statens Serum Institut, 5 Artillerivej, 2300 Copenhagen S, Denmark) were partially digested with 0.2U Sau3Al for l-15mins. Aliquots giving the greatest proportion of fragments in the size range of approximately 40-50kb were selected and ligated to vector pPR691 previously digested with BamHl and PvuII. Ligation mixtures were packaged in vitro with packaging extract.

WO 99/61458 PCT/AU99/00385 52 The host strain for transduction was x2819 and recombinants were selected with kanamycin.

F. Serological procedures.

Colonies were screened for the presence of the 0111 antigen by immunoblotting. Colonies were grown overnight, up to 100 per plate then transferred to nitrocellulose discs and lysed with 0.5N HC1. Tween was added to TBS at 0.05% final concentration for blocking, incubating and washing steps. Primary antibody was E. coli 0 group 111 antiserum, diluted 1:800. The secondary antibody was goat anti-rabbit IgG labelled with horseradish peroxidase diluted 1:5000. The staining substrate was 4-chloro-l-napthol. Slide agglutination was performed according to the standard procedure.

G. Recombinant DNA methods.

Restriction mapping was based on a combination of standard methods including single and double digests and sub-cloning. Deletion derivatives of entire cosmids were produced as follows: aliquots of 1.8mg of cosmid DNA were digested in a volume of 20ml with 0.25U of restriction enzyme for 5-80min. One half of each aliquot was used to check the degree of digestion on an agarose gel. The sample which appeared to give a representative range of fragments was ligated at 40C overnight and transformed by the CaC1 2 method into JM109. Selected plasmids were transformed into sf174 by the same method. P4657 was transformed with pPR1244 by electroporation.

H. DNA hybridisation Probe DNA was extracted from agarose gels by electroelution and was nick-translated using [a-32P]dCTP. Chromosomal or plasmid DNA was electrophoresed in 0.8% agarose and transferred to a nitrocellulose membrane. The hybridisation and pre-hybridisation buffers contained either 30% or 50% formamide for low and high stringency probing respectively. Incubation temperatures were 420C and 37 0 C for pre-hybridisation and hybridisation respectively. Low stringency washing of WO 99/61458 PCT/AU99/00385 53 filters consisted of 3 x 20min washes in 2 x SSC and 0.1% SDS. High-stringency washing consisted of 3 x washes in 2 x SSC and 0.1% SDS at room temperature, a lhr wash in 1 x SSC and 0.1% SDS at 580C and 15min wash in 0.1 x SSC and 0.1% SDS at 58 0

C.

I. Nucleotide sequencing of E. coli 0111 O antigen gene cluster (position 3,021-9,981) Nucleotide sequencing was performed using an ABI 373 automated sequencer (CA, USA). The region between map positions 3.30 and 7.90 was sequenced using uni-directional exonuclease III digestion of deletion families made in PT7T3190 from clones pPR1270 and pPR1272. Gaps were filled largely by cloning of selected fragments into M13mpl8 or M13mpl9. The region from map positions 7.90-10.2 was sequenced from restriction fragments in M13mpl8 or M13mpl9. Remaining gaps in both the regions were filled by priming from synthetic oligonucleotides complementary to determined positions along the sequence, using a single stranded DNA template in M13 or phagemid. The oligonucleotides were designed after analysing the adjacent sequence. All sequencing was performed by the chain termination method. Sequences were aligned using SAP [Staden, 1982 "Automation of the computer handling of gel reading data produced by the shotgun method of DNA sequencing". Nuc. Acid Res. 4731-4751; Staden, 1986 "The current status and portability of our sequence handling software". Nuc. Acid Res. 14: 217-231]. The program NIP [Staden, R. 1982 "An interactive graphics program for comparing and aligning nucleic acid and amino acid sequence". Nuc. Acid Res.

2951-2961] was used to find open reading frames and translate them into proteins.

J. Isolation of clones carrying E. coli 0111 O antigen gene cluster The E. coli 0 antigen gene cluster was isolated according to the method of Bastin et al. [1991 "Molecular cloning and expression in Escherichia coli K- WO 99/61458 PCT/AU99/00385 54 12 of the rfb gene cluster determining the O antigen of an E. coli 0111 strain". Mol. Microbiol. 2223- 2231]. Cosmid gene banks of M92 chromosomal DNA were established in the in vivo packaging strain x2819. From the genomic bank, 3.3 x 103 colonies were screened with E.coli 0111 antiserum using an immuno-blotting procedure: colonies (pPR1054, pPR1055, pPR1056, pPR1058 and pPR1287) were positive. The cosmids from these strains were packaged in vivo into lambda particles and transduced into the E. coli deletion mutant Sf174 which lacks all 0 antigen genes. In this host strain, all plasmids gave positive agglutination with 0111 antiserum.

An Eco R1 restriction map of the 5 independent cosmids showed that they have a region of approximately 11.5 kb in common (Figure Cosmid pPR1058 included sufficient flanking DNA to identify several chromosomal markers linked to 0 antigen gene cluster and was selected for analysis of the 0 antigen gene cluster region.

K. Restriction mapping of cosmid pPR1058 Cosmid pPR1058 was mapped in two stages. A preliminary map was constructed first, and then the region between map positions 0.00 and 23.10 was mapped in detail, since it was shown to be sufficient for 0111 antigen expression. Restriction sites for both stages are shown in Figure 2. The region common to the five cosmid clones was between map positions 1.35 and 12.95 of pPR1058.

To locate the 0 antigen gene cluster within pPR1058, pPR1058 cosmid was probed with DNA probes covering 0 antigen gene cluster flanking regions from S. enterica LT2 and E.coli K-12. Capsular polysaccharide (cps) genes lie upstream of 0 antigen gene cluster while the gluconate dehydrogenase (gnd) gene and the histidine (his) operon are downstream, the latter being further from the 0 antigen gene cluster. The probes used were pPR472 (3.35kb), carrying the gnd gene of LT2, pPR685 (5.3kb) carrying two genes of the cps cluster, cpsB and WO 99/61458 PCT/AU99/00385 55 cpsG of LT2, and K350 (16.5kb) carrying all of the his operon of K-12. Probes hybridised as follows: pPR472 hybridised to 1.55kb and 3.5 kb (including 2.7 kb of vector) fragments of Pstl and HindIII double digests of pPR1246 (a HindIII/EcoRl subclone derived from pPR1058, Figure which could be located at map positions 12.95- 15.1; pPR685 hybridised to a 4.4 kb EcoRl fragment of pPR1058 (including 1.3 kb of vector) located at map position 0.00-3.05; and K350 hybridised with a 32kb EcoRl fragment of pPR1058 (including 4.0kb of vector), located at map position 17.30-45.90. Subclones containing the presumed gnd region complemented a gnd-edd- strain GB23152. On gluconate bromothymol blue plates, pPR1244' and pPR1292 in this host strain gave the green colonies expected of a gnd edd genotype. The his' phenotype was restored by plasmid pPR1058 in the his deletion strain Sf174 on minimal medium plates, showing that the plasmid carries the entire his operon.

It is likely that the 0 antigen gene cluster region lies between gnd and cps, as in other E. coli and S.

enterica strains, and hence between the approximate map positions 3.05 and 12.95. To confirm this, deletion derivatives of pPR1058 were made as follows: first, pPR1058 was partially digested with HindIII and self ligated. Transformants were selected for kanamycin resistance and screened for expression of 0111 antigen.

Two colonies gave a positive reaction. EcoRl digestion showed that the two colonies hosted identical plasmids, one of which was designated pPR1230, with an insert which extended from map positions 0.00 to 23.10. Second pPR1058 was digested with Sail and partially digested with Xhol and the compatible ends were re-ligated.

Transformants were selected with kanamycin and screened for 0111 antigen expression. Plasmid DNA of 8 positively reacting clones was checked using EcoRl and Xhol digestion and appeared to be identical. The cosmid of one was designated pPR1231. The insert of pPR1231 WO 99/61458 PCT/AU99/00385 56 contained the DNA region between map positions 0.00 and 15.10. Third, pPR1231 was partially digested with Xhol, self-ligated, and transformants selected on spectinomycin/ streptomycin plates. Clones were screened for kanamycin sensitivity and of 10 selected, all had the DNA region from the Xhol site in the vector to the Xhol site at position 4.00 deleted. These clones did not express the 0111 antigen, showing that the Xhol site at position 4.00 is within the 0 antigen gene cluster. One clone was selected and named pPR1288. Plasmids pPR1230, pPR1231, and pPR1288 are shown in Figure 2.

L. Analysis of the E. coli 0111 0 antigen gene cluster (position 3,021-9,981) nucleotide sequence data Bastin and Reeves [1995 "Sequence and analysis of the 0 antigen gene(rfb)cluster of Escherichia coli 0111".

Gene 164: 17-23] partially characterised the E.coli 0111 O antigen gene cluster by sequencing a fragment from map position 3,021-9,981. Figure 3 shows the gene organisation of position 3,021-9,981 of E. coli 0111 0 antigen gene cluster. orf3 and orf6 have high level amino acid identity with wcaH and wcaG (46.3% and 37.2% respectively), and are likely to be similar in function to sugar biosynthetic pathway genes in the E. coli K-12 colanic gene cluster. orf4 and orf5 show high levels of amino acid homology to manC and manB genes respectively.

orf7 shows high level homology with rfbH which is an abequose pathway gene. orf8 encodes a protein with 12 transmembrane segments and has similarity in secondary structure to other wzx genes and is likely therefore to be the 0 antigen flippase gene.

Materials and Methods-part 2 A. Nucleotide sequencing of 1 to 3,020 and 9,982 to 14,516 of the E. coli 0111 0 antigen gene cluster The sub clones which contained novel nucleotide sequences, pPR1231 (map position 0 and 1,510), pPR1237 (map position -300 to 2,744), pPR1239 (map position 2,744 WO 99/61458 PCT/AU99/00385 57 to 4,168), pPR1245 (map position 9,736 to 12,007) and pPR1246 (map position 12,007 to 15,300) (Figure were characterised as follows: the distal ends of the inserts of pPR1237, pPR1239 and pPR1245 were sequenced using the M13 forward and reverse primers located in the vector.

PCR walking was carried out to sequence further into each insert using primers based on the sequence data and the primers were tagged with M13 forward or reverse primer sequences for sequencing. This PCR walking procedure was repeated until the entire insert was sequenced. pPR1246 was characterised from position 12,007 to 14,516. The DNA of these sub clones was sequenced in both directions.

The sequencing reactions were performed using the dideoxy termination method and thermocycling and reaction products were analysed using fluorescent dye and an ABI automated sequencer (CA, USA).

B. Analysis of the E. coli 0111 0 antigen gene cluster (positions 1 to 3,020 and 9,982 to 14,516 of Figure nucleotide sequence data The gene organisation of regions of E. coli 0111 0 antigen gene cluster which were not characterised by Bastin and Reeves [1995 "Sequence and analysis of the O antigen gene(rfb)cluster of Escherichia coli 0111." Gene 164: 17-23],(positions 1 to 3,020 and 9,982 to 14,516) is shown in Figure 3. There are two open reading frames in region 1. Four open reading frames are predicted in region 2. The position of each gene is listed in Table 9.

The deduced amino acid sequence of orfl (wbdH) shares about 64% similarity with that of the rfp gene of Shigella dysenteriae. Rfp and WbdH have very similar hydrophobicity plots and both have a very convincing predicted transmembrane segment in a corresponding position. rfp is a galactosyl transferase involved in the synthesis of LPS core, thus wbdH is likely to be a galactosyl transferase gene. orf2 has 85.7% identity at amino acid level to the gmd gene identified in the E.

WO 99/61458 PCT/AU99/00385 58 coli K-12 colanic acid gene cluster and is likely to be a gmd gene. orf9 encodes a protein with 10 predicted transmembrane segments and a large cytoplasmic loop.

This inner membrane topology is a characteristic feature of all known 0 antigen polymerases thus it is likely that orf9 encodes an 0 antigen polymerase gene, wzy. orflO (wbdL) has a deduced amino acid sequence with low homology with Lsi2 of Neisseria gonorrhoeae. Lsi2 is responsible for adding GlcNAc to galactose in the synthesis of lipooligosaccharide. Thus it is likely that wbdL is either a colitose or glucose transferase gene.

orfll (wbdM) shares high level nucleotide and amino acid similarity with TrsE of Yersinia enterocolitica. TrsE is a putative sugar transferase thus it is likely that wbdM encodes the colitose or glucose transferase.

In summary three putative transferase genes and an 0 antigen polymerase gene were identified at map position 1 to 3,020 and 9,982 to 14,516 of E. coli 0111 0 antigen gene cluster. A search of GenBank has shown that there are no genes with significant similarity at the nucleotide sequence level for two of the three putative transferase genes or the polymerase gene. Figure provides the nucleotide sequence of the 0111 antigen gene cluster.

Materials and Methods-part 3 A. PCR amplification of 0157 antigen gene cluster from an E. coli 0157:H7 strain (Strain C664-1992, from Statens Serum Institut, 5 Artillerivej, 2300, Copenhagen S, Denmark) E. coli 0157 0 antigen gene cluster was amplified by using long PCR [Cheng et al. 1994, "Effective amplification of long targets from cloned inserts and human and genomic DNA" P.N.A.S. USA 91: 5695-569] with one primer (primer #412: att ggt age tgt aag cca agg gcg gta gcg t) based on the JumpStart sequence usually found in the promoter region of O antigen gene clusters [Hobbs, WO 99/61458 PCT/AU99/00385 59 et al. 1994 "The JumpStart sequence: a 39 bp element common to several polysaccharide gene clusters" Mol.

Microbiol. 12: 855-856], and another primer #482 (cac tgc cat acc gac gac gcc gat ctg ttg ctt gg) based on the gnd gene usually found downstream of the 0 antigen gene cluster. Long PCR was carried out using the Expand Long Template PCR System from Boehringer Mannheim (Castle Hill NSW Australia), and products, 14 kb in length, from several reactions were combined and purified using the Promega Wizard PCR preps DNA purification System (Madison WI USA). The PCR product was then extracted with phenol and twice with ether, precipitated with 70% ethanol, and resuspended in 40mL of water.

B. Construction of a random DNase I bank: Two aliquots containing about 150ng of DNA each were subjected to DNase I digestion using the Novagen DNase I Shotgun Cleavage (Madison WI USA) with a modified protocol as described. Each aliquot was diluted into of 0.05M Tris -HC1 (pH7.5), 0.05mg/mL BSA and MnC12. 5mL of 1:3000 or 1:4500 dilution of DNaseI (Novagen) (Madison WI USA) in the same buffer was added into each tube respectively and 10ml of stop buffer (100mM EDTA), 30% glycerol, 0.5% Orange G, 0.075% xylene and cyanol (Novagen) (Madison WI USA) was added after incubation at 15 0 C for 5 min. The DNA from the two DNaseI reaction tubes were then combined and fractionated on a 0.8% LMT agarose gel, and the gel segment with DNA of about 1kb in size (about 1.5mL agarose) was excised. DNA was extracted from agarose using Promega Wizard PCR Preps DNA Purification (Madison WI USA) and resuspended in 200 mL water, before being extracted with phenol and twice with ether, and precipitated. The DNA was then resuspended in 17.25 mL water and subjected to T4 DNA polymerase repair and single dA tailing using the Novagen Single dA Tailing Kit (Madison WI USA). The reaction product (85ml containing about 8ng DNA) was then extracted with chloroform:isoamyl alcohol (24:1) once and WO 99/61458 PCT/AU99/00385 60 ligated to 3x 10 3 pmol pGEM-T (Promega) (Madison WI USA) in a total volume of 10OmL. Ligation was carried out overnight at 4 0 C and the ligated DNA was precipitated and resuspended in 20mL water before being electroporated into E. coli strain JM109 and plated out on BCIG-IPTG plates to give a bank.

C. Sequencing DNA templates from clones of the bank were prepared for sequencing using the 96-well format plasmid DNA miniprep kit from Advanced Genetic Technologies Corp (Gaithersburg MD USA) The inserts of these clones were sequenced from one or both ends using the standard M13 sequencing primer sites located in the pGEM-T vector.

Sequencing was carried out on an ABI377 automated sequencer (CA USA) as described above, after carrying out the sequencing reaction on an ABI Catalyst (CA USA).

Sequence gaps and areas of inadequate coverage were PCR amplified directly from 0157 chromosomal DNA using primers based on the already obtained sequencing data and sequenced using the standard M13 sequencing primer sites attached to the PCR primers.

D. Analysis of the E. coli 0157 0 antigen gene cluster nucleotide sequence data Sequence data were processed and analysed using the Staden programs [Staden, 1982 "Automation of the computer handling of gel reading data produced by the shotgun method of DNA sequencing." Nuc. Acid Res. 4731-4751; Staden, 1986 "The current status and portability of our sequence handling software". Nuc. Acid Res. 14: 217-231; Staden, R. 1982 "An interactive graphics program for comparing and aligning nucleic acid and amino acid sequence". Nuc. Acid Res. 10: 2951-2961].

Figure 4 shows the structure of E. coli 0157 0 antigen gene cluster. Twelve open reading frames were predicted from the sequence data, and the nucleotide and amino acid sequences of all these genes were then used to search the GenBank database for indication of possible function and WO 99/61458 PCT/AU99/00385 61 specificity of these genes. The position of each gene is listed in Table 9. The nucleotide sequence is presented in Figure 6.

orfs 10 and 11 showed high level identity to manC and manB and were named manC and manB respectively. orf7 showed 89% identity (at amino acid level) to the gmd gene of the E. coli colanic acid capsule gene cluster (Stevenson K. et al. 1996 "Organisation of the Escherichia coli K-12 gene cluster responsible for production of the extracellular polysaccharide colanic acid".J. Bacteriol. 178:4885-4893) and was named gmd.

orf8 showed 79% and 69% identity (at amino acid level) respectively to wcaG of the E. coli colanic acid capsule gene cluster and to wbcJ (orf14.8) gene of the Yersinia enterocolitica 08 0 antigen gene cluster (Zhang, L. et al. 1997 "Molecular and chemical characterization of the lipopolysaccharide O-antigen and its role in the virulence of Y. enterocolitica serotype 08".Mol.

Microbiol. 23:63-76). Colanic acid and the Yersinia 08 O antigen both contain fucose as does the 0157 0 antigen.

There are two enzymatic steps required for GDP-L-fucose synthesis from GDP-4-keto-6-deoxy-D-mannose, the product of the gmd gene product. However, it has been shown recently (Tonetti, M et al. 1996 Synthesis of GDP-Lfucose by the human FX protein J. Biol. Chem. 271:27274- 27279) that the human FX protein has "significant homology" with the wcaG gene (referred to as Yefb in that paper), and that the FX protein carries out both reactions to convert GDP-4-keto-6-deoxy-D-mannose to GDP- L-fucose. We believe that this makes a very strong case for orf8 carrying out these two steps and propose to name the gene fcl. In support of the one enzyme carrying out both functions is the observation that there are no genes other than manB, manC, gmd and fcl with similar levels of similarity between the three bacterial gene clusters for fucose containing structures.

is very similar to wbeE (rfbE) of Vibrio WO 99/61458 PCT/AU99/00385 62 cholerae 01, which is thought to be the perosamine synthetase, which converts GDP-4-keto-6-deoxy-D-mannose to GDP-perosamine (Stroeher, U.H et al. 1995 "A putative pathway for perosamine biosynthesis is the first function encoded within the rfb region of Vibrio cholerae" 01.

Gene 166: 33-42). V. cholerae 01 and E. coli 0157 0 antigens contain perosamine and N-acetyl-perosamine respectively. The V. cholerae 01 manA, manB, gmd and wbeE genes are the only genes of the V. cholerae 01 gene cluster with significant similarity to genes of the E.

coli 0157 gene cluster and we believe that our observations both confirm the prediction made for the function of wbe of V. cholerae, and show that orf5 of the 0157 gene cluster encodes GDP-perosamine synthetase.

orf5 is therefore named per. orf5 plus about 100bp of the upstream region (postion 4022-5308)was previously sequenced by Bilge, S.S. et al. [1996 "Role of the Escherichia coli 0157-H7 0 side chain in adherence and analysis of an rfb locus".Infect. Immun. 64:4795-4801].

orfl2 shows high level similarity to the conserved region of about 50 amino acids of various members of an acetyltransferase family (Lin, et al. 1994 "Sequence analysis and molecular characterisation of genes required for the biosynthesis of type 1 capsular polysaccharide in Staphylococcus aureus". J. Bateriol. 176: 7005-7016) and we believe it is the N-acetyltransferase to convert GDPperosamine to GDP-perNAc. orfl2 has been named wbdR.

The genes manB, manC, gmd, fcl, per and wbdR account for all of the expected biosynthetic pathway genes of the 0157 gene cluster.

The remaining biosynthetic step(s) required are for synthesis of UDP-GalNAc from UDP-Glc. It has been proposed (Zhang, et al. 1997 "Molecular and chemical characterisation of the lipopolysaccharide O-antigen and its role in the virulence of Yersinia enterocolitica serotype 08".Mol. Microbiol. 23:63-76) that in Yersinia enterocolitica UDP-GalNAc is synthesised from UDP-GlcNAc WO 99/61458 PCT/AU99/00385 63 by a homologue of galactose epimerase (GalE), for which there is a galE like gene in the Yersinia enterocolitica 08 gene cluster. In the case of 0157 there is no galE homologue in the gene cluster and it is not clear how UDP-GalNAc is synthesised. It is possible that the galactose epimerase encoded by the galE gene in the gal operon, can carry out conversion of UDP-GlcNAc to UDP- GalNAc in addition to conversion of UDP-Glc to UDP-Gal.

There do not appear to be any gene(s) responsible for UDP-GalNAc synthesis in the 0157 gene cluster.

orf4 shows similarity to many wzx genes and is named wzx and orf2 which shows similarity of secondary structure in the predicted protein to other wzy genes and is for that reason named wzy.

The orfl, orf3 and orf6 gene products all have characteristics of transferases, and have been named wbdN, wbdO and wbdP respectively. The 0157 0 antigen has 4 sugars and 4 transferases are expected. The first transferase to act would put a sugar phosphate onto undecaprenol phosphate. The two transferases known to perform this function, WbaP (RfbP) and WecA (Rfe) transfer galactose phosphate and N-acetyl-glucosamine phosphate respectively to undecaprenol phosphate.

Neither of these sugars is present in the 0157 structure.

Further, none of the presumptive transferases in the 0157 gene cluster has the transmembrane segments found in WecA and WbaP which transfer a sugar phosphate to undecaprenol phosphate and expected for any protein which transferred a sugar to undecaprenol phosphate which is embedded within the membrane.

The WecA gene which transfers GlcNAc-P to undecaprenol phosphate is located in the Enterobactereal Common Antigen (ECA) gene cluster and it functions in ECA synthesis in most and perhaps all E. coli strains, and also in 0 antigen synthesis for those strains which have GlcNAc as the first sugar in the O unit.

It appears that WecA acts as the transferase for WO 99/61458 PCT/AU99/00385 64 addition of GalNAc-1-P to undecaprenol phosphate for the Yersinia enterocolitica 08 0 antigen [Zhang et al.1997 "Molecular and chemical characterisation of the lipopolysaccharide 0 antigen and its role in the virulence of Yersinia enterocolitica serotype 08" Mol.

Microbiol. 23: 63-76.] and perhaps does so here as the 0157 structure includes GalNAc. WecA has also been reported to add Glucose-l-P phosphate to undecaprenol phosphate in E. coli 08 and 09 strains, and an alternative possibility for transfer of the first sugar to undecaprenol phosphate is WecA mediated transfer of glucose, as there is a glucose residue in the 0157 0 antigen. In either case the requisite number of transferase genes are present if GalNAc or Glc is transferred by WecA and the side chain Glc is transferred by a transferase outside of the 0 antigen gene cluster.

orf9 shows high level similarity (44% identity at amino acid level, same length) with wcaH gene of the E.

coli colanic acid capsule gene cluster. The function of this gene is unknown, and we give orf9 the name wbdQ.

The DNA between manB and wdbR has strong sequence similarity to one of the H-repeat units of E. coli K12.

Both of the inverted repeat sequences flanking this region are still recognisable, each with two of the 11 bases being changed. The H-repeat associated protein encoding gene located within this region has a 267 base deletion and mutations in various positions. It seems that the H-repeat unit has been associated with this gene cluster for a long period of time since it translocated to the gene cluster, perhaps playing a role in assembly of the gene cluster as has been proposed in other cases.

Materials and Methods part 4 To test our hypothesis that 0 antigen genes for transferases and the wzx, wzy genes were more specific than pathway genes for diagnostic PCR, we first carried out PCR using primers for all the E. coli 016 O antigen WO 99/61458 PCT/AU99/00385 65 genes (Table The PCR was then carried out using PCR primers for E.coli 0111 transferase, wzx and wzy genes (Table 8, 8A). PCR was also carried out using PCR primers for the E. coli 0157 transferase, wzx and wzy genes (Table 9, 9A).

Chromosomal DNA from the 166 serotypes of E. coli available from Statens Serum Institut, 5 Artillerivej, 2300 Copenhagen Denmark was isolated using the Promega Genomic (Madison WI USA) isolation kit. Note that 164 of the serogroups are described by Ewing W. Edwards and Ewings "Identification of the Enterobacteriacea" Elsevier, Amsterdam 1986 and that they are numbered 1-171 with numbers 31, 47, 67, 72, 93, 94 and 122 no longer valid. Of the two serogroup 19 strains we used 19ab strain F8188-41. Lior H. 1994 ["Classification of Escherichia coli In Escherichia coli in domestic animals and humans pp 31-72. Edited by C.L. Gyles CAB international] adds two more numbered 172 and 173 to give the 166 serogroups used. Pools containing 5 to 8 samples of DNA per pool were made. Pool numbers 1 to 19 (Table 4) were used in the E. coli 0111 and 0157 assay. Pool numbers 20 to 28 were also used in the 0111 assay, and pool numbers 22 to 24 contained E. coli 0111 DNA and were used as positive controls (Table Pool numbers 29 to 42 were also used in the 0157 assay, and pool numbers 31 to 36 contained E. coli 0157 DNA, and were used as positive controls (Table Pool numbers 2 to 20, 43 and 44 were used in the E. coli 016 assay (Tables 4 to Pool number 44 contained DNA of E. coli K-12 strains C600 and WG1 and was used as a positive control as between them they have all of the E. coli K-12 016 0 antigen genes.

PCR reactions were carried out under the following conditions: denaturing 94 0 C/30"; annealing, temperature varies (refer to Tables)/30"; extension, 72 0 cycles. PCR reaction was carried out in an volume of for each pool. After the PCR reaction, 10mL PCR WO 99/61458 PCT/AU99/00385 66 product from each pool was run on an agarose gel to check for amplified DNA.

Each E. coli chromosomal DNA sample was checked by gel electrophoresis for the presence of chromosomal DNA and by PCR amplification of the E. coli mdh gene using oligonucleotides based on E. coli K-12 [Boyd et al.

(1994) "Molecular genetic basis of allelic polymorphism in malate degydrogenase (mdh) in natural populations of Escherichia coli and Salmonella enterica" Proc. Nat.

Acad. Sci. USA. 91:1280-1284.] Chromosomal DNA samples from other bacteria were only checked by gel electrophoresis of chromosomal DNA.

A. Primers based on E. coli 016 0 antigen gene cluster sequence.

The 0 antigen gene cluster of E. coli 016 was the only typical E. coli 0 antigen gene cluster that had been fully sequenced prior to that of 0111, and we chose it for testing our hypothesis. One pair of primers for each gene was tested against pools 2 to 20, 30 and 43 of E.

coli chromosomal DNA. The primers, annealing temperatures and functional information for each gene are listed in Table 8.

For the five pathway genes, there were 17/21, 13/21, 0/21, 0/21, 0/21 positive pools for rmlB, rmlD, rmlA, rmlC and glf respectively (Table For the wzx, wzy and three transferase genes there were no positives amongst the 21 pools of E. coli chromosomal DNA tested (Table In each case the #44 pool gave a positive result.

B. Primers based on the E. coli 0111 0 antigen gene cluster sequence.

One to four pairs of primers for each of the transferase, wzx and wzy genes of 0111 were tested against the pools 1 to 21 of E. coli chromosomal DNA (Table For wbdH, four pairs of primers, which bind WO 99/61458 PCT/AU99/00385 67 to various regions of this gene, were tested and found to be specific for 0111 as there was no amplified DNA of the correct size in any of those 21 pools of E. coli chromosomal DNA tested. Three pairs of primers for wbdM were tested, and they are all specific although primers #985/#986 produced a band of the wrong size from one pool. Three pairs of primers for wzx were tested and they all were specific. Two pairs of primers were tested for wzy, both are specific although #980/#983 gave a band of the wrong size in all pools. One pair of primers for wbdL was tested and found unspecific and therefore no further test was carried out. Thus, wzx, wzy and two of the three transferase genes are highly specific to 0111.

Bands of the wrong size found in amplified DNA are assumed to be due to chance hybridisation of genes widely present in E. coli. The primers, annealing temperatures and positions for each gene are in Table 8.

The 0111 assay was also performed using pools including DNA from O antigen expressing Yersinia pseudotuberculosis, Shigella boydii and Salmonella enterica strains (Table 8A). None of the oligonucleotides derived from wbdH, wzx, wzy or wbdM gave amplified DNA of the correct size with these pools.

Notably, pool number 25 includes S. enterica Adelaide which has the same 0 antigen as E. coli 0111: this pool did not give a positive PCR result for any primers tested indicating that these genes are highly specific for E.

coli 0111.

Each of the 12 pairs binding to wbdH, wzx, wzy and wbdM produces a band of predicted size with the pools containing 0111 DNA (pools number 22 to 24). As pools 22 to 24 included DNA from all strains present in pool 21 plus 0111 strain DNA (Table we conclude that the 12 pairs of primers all give a positive PCR test with each of three unrelated 0111 strains but not with any other strains tested. Thus these genes are highly specific for E. coli 0111.

WO 99/61458 PCT/AU99/00385 68 C. Primers based on the E. coli 0157 O antigen gene cluster sequence.

Two or three primer pairs for each of the transferase, wzx and wzy genes of 0157 were tested against E. coli chromosomal DNA of pools 1 to 19, 29 and (Table For wbdN, three pairs of primers, which bind to various regions of this gene, were tested and found to be specific for 0157 as there was no amplified DNA in any of those 21 pools of E. coli chromosomal DNA tested. Three pairs of primers for wbdO were tested, and they are all specific although primers 1211/#1212 produced two or three bands of the wrong size from all pools. Three pairs of primers were tested for wbdP and they all were specific. Two pairs of primers were tested for wbdR and they were all specific. For wzy, three pairs of primers were tested and all were specific although primer pair #1203/#1204 produced one or three bands of the wrong size in each pool. For wzx, two pairs of primers were tested and both were specific although primer pair #1217/#1218 produced 2 bands of wrong size in 2 pools, and 1 band of wrong size in 7 pools. Bands of the wrong size found in amplified DNA are assumed to be due to chance hybridisation of genes widely present in E.

coli. The primers, annealing temperatures and function information for each gene are in Table 9.

The 0157 assay was also performed using pools 37 to 42, including DNA from 0 antigen expressing Yersinia pseudotuberculosis, Shigella boydii, Yersinia enterocolitica 09, Brucella abortus and Salmonella enterica strains (Table 9A). None of the oligonucleotides derived from wbdN, wzy, wbdO, wzx, wbdP or wbdR reacted specifically with these pools, except that primer pair #1203/#1204 produced two bands with Y.

enterocolitica 09 and one of the bands is of the same size with that from the positive control. Primer pair #1203/#1204 binds to wzy. The predicted secondary h 69 related to that of E. coli 0157. It is also possible that this band is due to chance hybridization of another gene, as the other two wzy primer pairs (#1205/#1206 and #1207/#1208) did not produce any band with Y.

enterocolitica 09. Notably, pool number 37 includes S.

enterica Landau which has the same O antigen as E. coli 0157, and pool 38 and 39 contain DNA of B. abortus and Y.

enterocolitica 09 which cross react serologically with E.

coli 0157. This result indicates that these genes are highly 0157 specific, although one primer pair may have cross reacted with Y. enterocolitica 09.

Each of the 16 pairs binding to wbdN, wzx, wzy, wbdO, wbdP and wbdR produces a band of predicted size with the pools containing 0157 DNA (pools number 31 to 36). As pool 29 included DNA from all strains present in pools 31 to 36 other than 0157 strain DNA (Table we conclude that the 16 pairs of primers all give a positive PCR test with each of the five unrelated 0157 strains.

Thus PCR using primers based on genes wbdN, wzy, wbdO, wzx, wbdP and wbdR is highly specific for E. coli 0157, giving positive results with each of six unrelated 0157 strains while only one primer pair gave a band of the expected size with one of three strains with 0 antigens known to cross-react serologically with E. coli 0157.

In the claims which follow and in the preceding description of the invention, except where the context requires otherwise due to express language or necessary implication, the word "comprising" is used in the sense of "including", i.e. the features specified may be associated with further features in various embodiments of the invention.

It is to be understood that a reference herein to a prior art document does not constitute an admission that the document forms part of the common general knowledge in the art in Australia.

WO 99/61458 WO 9961458PCT/AU99/00385 70 TABLE 1 H7 strains used in this work in addition to the H antigens type strains Name used Serotype original Source* in this name study M527 0157:H7 C664-1992 a M917 Ol8ac:H7 A57 IMVS M918 Ol8ac:H7 A62 IMVS M973 02:H7 A1107 CDC M1004 0157:H7 EH7 b M1179 Ol8ac:H7 D-M3291/54 IMVS M1200 07:H7 A64 c M1211 Ol9ab:H7 F8188-41 IMVS M1328 053:H7 14097 IMVS M1686 055:H7 TB156 d a.

b.

C.

d.

IMVS,

CDC,

Statens Serum Institut, Copenhagen, Denmark.

Dr R. Brown of Royal Children's Hospital, Melbourne, Australia.

Max-Planck Institut fur molekulare Genetik, Berlin, Germany.

Dr P. Tarr of Children's Hospital and Medical Center, University of Washington, USA.

Institute of Medical and veterinary Science, Adelaide, Australia.

Centers for Disease Control and prevention, Atlanta,

USA.

WO 99/61458 WO 9961458PCT/AU99/00385 71 Table 2 Oligonucleotides used to PCR amplify MlC genes different H type strains for sequencing H Type Strains Annealing Temperature Primers Used 1 55 #1575/#1576 2 55 #1285/#1286 3 55 #1285/#1286 4 50 $1431/#1432 60 #1285/#1286 6 55 #1575/#1576 7 55 #1575/#1576 8 55 #1431/#1432 9 60 #1575/#1576 55 #1575/#1576 11 #1285/#1286 12 60 #1575/#1576 14 60 #1575/#1576 60 #1575/#1576 16 60 #1575/# 1576 17 60 #1417/#1418 18 60 #1575/#1576 19 60 #1575/#1576 60 #1 575/#1 576 21 55 #1285/#1286 23 60 #1 575/#1 576 24 60 #1285/#1286 60 #1417/#1418 26 60 #1575/#1 576 27 50 #1431/#1432 28 60 #1575/#1576 29 60 #1285/#1286 60 #15754#1576 31 60 #1 575/#1 576 32 60 #1575/#1 576 33 60 #1285/1286 55 #1575/#1576 50 #1431/#1432 37 60 #1 285/1M286 38 60 #1285/#1 286 39 55 #1285/#1286 55 #1285/#1286 41 60 #1575/#1576 42 60 #1285/#1286 43 60 #1575/#1576 44 60 #1285/#1286 60 #1575/#1576 46 60 #1575/#1576 47 55 #128,/#1286 48 60 #1575/#1576 49 60 #1575/#1 576 60 #1 285/#1 286 51 60 #1575/#1576 52 60 #1 575/#1 576 54 50 #1431/#1432 60 #1285/#1286 I56 60 #1285/#1286 WO 99/61458 PCT/AU99/00385 72 Table 3 Summary of the flagellin sequences obtained and specific H type oligonucleotide primers H type strain(s) the H specificity H type strain from Positions of Positions of sequenced gene(s) coded by the which the flagellin gene primer 1 primer 2 obtained from gene(s) sequence was used for primer choice 1 1 1 892-909 1172-1189 2 2 2 568-587 1039-1056 4,17,44 4 4 466-483 628-648 5 5 697-714 877-897 6 6 6 565-585 799-816 7 7 7 553-570 1483-1500 (primer #1806) (primer #1809) 9 9 9 616-633 838-855 10 10 559-579 697-717 11 11 11 586-606* 791-810* 12 12 12 892-909 1172-1189 14 14 14 586-606 793-813 15 15 640-660 817-834 3 16 3 649-666 925-942 18 18 18 589-606 802-819 19 19 19 607-624 538-855 20 20 574-591 760-780 21,47 21 21 676-693"* 862-879** 23 23 23 637-654 1336-1353 24 24 24 496-516 772-792 26 26 26 553-570 772-789 27 27 27 685-702 799-819 28 28 28 592-609 778-798 29 29 29 538-555 757-774 30 30 814-831 943-962 31 31 31 571-588 790-807 32 32 32 514-831 1057-1074 33 33 33 553-570 718-735 34 34 34 568-585 796-816 38,55 38 38 553-573 709-729 39 39 39 556-573 718-735 41 41 41 598-615 784-801 42 42 42 547-567 715-735 43 43 43 580-597 844-861 45 45 640-657 943-963 46 46 46 565-582 781-801 49 49 49 589-609 754-771 51 51 51 565-582 1042-1059 52 52 52 598-615 829-846 56 56 56 697-714 877-897 8 and 40 8 562-579 1045-1062 25 529-549 703-723 non-functional H 1 gene 769-789* 1045-1065" 37 37 520-537 715-735 48 48 568-585 835-852 54 1 non-functional H21 gene 988-1008** 1344-1364"* See section 13 for choice of primers for the flagellin gene of Hi 1 See section 13 for choice of primers for the flagellin gene of H21 See text WO 99/61458 PCT/AU99/00385 73 Table 3A Cloning, expression and identification of flagellin genes H type strain Primers used Annealing Plasmid Host strain Anti-serum H antigen from which for POP temperature carrying the H used for which reacts encoded by the H antigen amplification of (00) used for antigen gene expression with an E Coli the cloned gene was the H antigen POP NlC deletion gene amplified gene amplification strain carrying the piasmid Hi #1868 &#1870 55 pPR1920 M2126 Hi Hi H2 #1868 &#1870 55 pPR1977 P5560 H2 H2 H3 #1868 &#1870 55 pPR1969 P5560 H16 H16 H4 #1878 &#1885 65 pPR1955 P5560 H4 H4 #1868 &#1870 60 pPR1967 M2126 H5 H6 #1868 &#1870 55 pPR1921 P5560 H6 H6 H7 #1868 &#1870 55 pPR1919 P5560 H7 H7 Hg #1 868 #1 870 55 pPR1922 P5560 H9 H9 H10 #1868 &#1870 55 pPR1923 P5560 H10 HIO H11 #1868 &#1870 55 PPR1981 M2126 H11 H11 H12 #1868 &#1870 60 PPR1990 M2126 H12 H12 H14 #1868 &#1870 55 pPR1924 P5560 H14 H14 #1868 &#1870 55 pPR1925 P5560 Hi5 H17 #1878 &#1885 65 pPR1957 P5560 H4 H4 H18 #1868 &#1870 55 PPR1986 M2126 H18 H18 H19 #1868 &#1870 55 pPR1927 P5560 H19 H19 #1868 &#1870 55 pPR1963 M2126 H20 H21 #1868 &#1870 55 pPR1995 M2126 H21 H21 H23 #1868 &#1869 55 pPR1942 P5560 H23 H23 H24 #1868 &#1870 55 pPRi971 M2126 H24 H24 H26 #1868 &#1870 65 pPR1928 P5560 H26 H26 H27 #1868 &#1870 55 pPR1970 M2126 H27 H27 H28 #1868 &#1870 60 PR1944 P5560 H28 H28 H29 #1868 &#1870 55 PPR1972 M2126 H29 H29 #1868 &#1871 55 PPR1948 P5560 H30 H31 #1868 &#1870 65 pPR1965 M2126 H31 H31 H32 #1868 &#1871 55 pPR1940 P5560 H32 H32 H33 #1868 &#1871 55 PPR1976 M2126 H33 H33 H34 #1868 &#1870 65 pPR1930 P5560 H34 H34 H38 #1868 &#1870 48 PPR1984 M2126 H38 H38 H39 #1868 &#1870 48 pPR1982 M2126 H39 H39 H41 #1 868 #1 870 65 pPR 1931 P5560 H41 H41 H42 #1868 &#1870 50 pPR1979 M2126 ___H42 H42 H43 #1868 &#1870 65 PPR1968 M2126 H43 H43 #1868 &#1870 60 pPR1943 P5560 ___H45 H46 #1868 &#1870 60 pPR1966 M2126 H46 H46 H49 #1868 &#1870 60 pPR1985 M2126 H49 H49 H51 #1868 &#1870 65 pPR1941 P5560 H51 H51 H52 #1868 #1 870 65 pPR1935 P5560 H52 H52 H56 #1868&#1870 50 pPR1978 M2196 H56 H56 WO 99/61458 WO 9961458PCT/AU99/00385 74 Table 3 B Oligonucleotide primers used for PCR amplification and cloning of H antigen genes #1868 catggc tZ ca caa gtc attaatacc -3' NcoI #1969 ata tgL ca ctt aac cct gca gca gag aca g -3' SaII #1870 5' -atg gt ct taa cctgc agc aga gac ag -3' BamL-l #1 81 5 ac tc at taa ccc tgt agc aga gac ag-3 PstI #182 '-ggt eccgca gac tgg ttc ttg ttg at- 3 #1878 51 cgggsactcacga ccttt 3' BamHl #1884 gc~clasrag cgc aga tca ttc agc aggcc -3' XbaI #1885 5' -gcr cta gac atg ttg gac act tcg gtc gc -3' XbaI WO 99/61458 PCT/AU99/00385 75 TABLE 4 Pool Strains of which chromosonal DNA included in the pool Source* No.

1 E. coli type strains for O serotypes 1, 2, 3, 4, 10, 16, 18 and 39 IMVSa 2 E. coli type strains for O serotypes 40, 41, 48, 49, 71, 73, 88 and 100 IMVS 3 E. coli type strains for O serotypes 102, 109, 119, 120, 121, 125, 126 and IMVS 137 4 E. coli type strains for O serotypes 138, 139, 149, 7, 5, 6, 11 and 12 IMVS E. coli type strains for O serotypes 13, 14, 15, 17, 19ab, 20, 21 and 22 IMVS 6 E. coli type strains for O serotypes 23, 24, 25, 26, 27, 28, 29 and 30 IMVS 7 E. coli type strains for O serotypes 32, 33, 34, 35, 36, 37, 38 and 42 IMVS 8 E. coli type strains for O serotypes 43, 44, 45, 46, 50, 51, 52 and 53 IMVS 9 E. coli type strains for O serotypes 54, 55, 56, 57, 58, 59, 60 and 61 IMVS E. coli type strains for O serotypes 62, 63, 64, 65, 66, 68, 69 and 70 IMVS 11 E. coli type strains for O serotypes 74, 75, 76, 77, 78, 79, 80 and 81 IMVS 12 E. coli type strains for O serotypes 82, 83, 84, 85, 86, 87, 89 and 90 IMVS 13 E. coli type strains for O serotypes 91, 92, 95, 96, 97, 98, 99 and 101 IMVS 14 E. coli type strains for O serotypes 103, 104, 105, 106, 107, 108 and 110 IMVS E. coli type strains for O serotypes 112, 162, 113, 114, 115, 116, 117 and IMVS 118 16 E. coli type strains for O serotypes 123, 165, 166, 167, 168, 169, 170 and See b 171 17 E. coli type strains for O serotypes 172, 173, 127, 128, 129, 130, 131 and See c 132 18 E. coli type strains for O serotypes 133, 134, 135, 136, 140, 141, 142 and IMVS 143 19 E. coli type strains for O serotypes 144, 145, 146, 147, 148, 150, 151 and IMVS 152 a. Institute of Medical and Veterinary Science, Adelaide, Australia b. 123 from IMVS; the rest from Statens Serum Institut, Copenhagen, Denmark c. 172 and 173 from Statens Serum Institut, Copenhagen, Denmark, the rest from

IMVS

WO 99/61458 PCT/AU99/00385 76 TABLE Pool Strains of which chromosonal DNA included in the pool Source* No.

E. coli type strains for O serotypes 153, 154, 155, 156, 157, 158, 159 and IMVS 160 21 E. coli type strains for O serotypes 161, 163, 164, 8, 9 and 124 IMVS 22 As pool #21, plus E. coli 0111 type strain Stoke W. IMVS 23 As pool #21, plus E. coli 0111:H2 strain C1250-1991 See d 24 As pool #21, plus E. coli 0111:H12 strain C156-1989 See e As pool #21, plus S. enterica serovar Adelaide See f 26 Y. pseudotuberculosis strains of O groups IA, IIA, IIB, IIC, III, IVA, IVB, See g VA, VB, VI and VII 27 S. boydii strains of serogroups 1, 3, 4, 5, 6, 8, 9, 10, 11, 12, 14 and 15 See h 28 S. enterica strains of serovars (each representing a different O group) IMVS Typhi, Montevideo, Ferruch, Jangwani, Raus, Hvittingfoss, Waycross, Dan, Dugbe, Basel, 65,:i:e,n,z,15 and 52:d:e,n,x,z15 d. C1250-1991 from Statens Serum nstitut, Copenhagen, Denmark d. C1250-1991 from Statens Serum Institut, Copenhagen, Denmark e. C156-1989 from Statens Serum Institut, Copenhagen, Denmark f. S. enterica serovar Adelaide from IMVS g. Dr S Aleksic of Institute of Hygiene, Germany h. Dr J Lefebvre of Bacterial Identification Section, Laboratoroie de Sante Publique du Quebec, Canada WO 99/61458 PCT/AU99/00385 77 TABLE 6 Pool Strains of which chromosonal DNA included in the pool Source* No.

29 E. coli type strains for O serotypes 153, 154, 155, 156, 158, 159 and 160 IMVS E. coli type strains for O serotypes 161, 163, 164, 8, 9, 111 and 124 IMVS 31 As pool #29, plus E. coli 0157 type strain A2 (0157:H19) IMVS 32 As pool #29, plus E. coli 0157:H16 strain C475-89 See d 33 As pool #29, plus E. coli 0157:H45 strain C727-89 See d 34 As pool #29, plus E. coli 0157:H2 strain C252-94 See d As pool #29, plus E. coli 0157:H39 strain C258-94 See d 36 As pool #29, plus E. coli 0157:H26 See e 37 As pool #29, plus S. enterica serovar Landau See f 38 As pool #29, plus Brucella abortus See g See h 39 As pool #29, plus Y. enterocolitica 09 Y. pseudotuberculosis strains of 0 groups IA, IIA, IIB, IIC, III, IVA, IVB, VA, See i VB, VI and VII 41 S. boydii strains of serogroups 1, 3, 4, 5, 6, 8, 9, 10, 11, 12, 14 and 15 See j 42 S. enterica strains of serovars (each representing a different O group) Typhi, IMVS Montevideo, Ferruch, Jangwani, Raus, Hvittingfoss, Waycross, Dan, Dugbe, Basel, 65:i:e,n,z15 and 52:d:e,n,x,z15 43 E. coli type strains for 0 serotypes 1,2,3,4,10,18 and 29 IMVS 44 As pool #43, plus E. coli K-12 strains C600 and WG1 IVMS See k d. 0157 strains from Statens Serum Institut, Copenhagen, Denmark e. 0157:H26 from Dr R Brown of Royal Children's Hospital, Melbourne, Victoria f. S. enterica serovar Landau from Dr M Poppoff of Institut Pasteur, Paris, France g. B. Abortus from the culture collection of The University of Sydney, Sydney, Australia h. Y. enterocolitica 09 from Dr. K. Bettelheim of Victorian Infectious Diseases Reference Laboratory Victoria, Australia.

i. Dr S Aleksic of Institute of Hygiene, Germany J. Dr J Lefebvre of Bacterial Identification Section, Laboratoroie de Sant6 Publique du Quebec, Canada k. Strains C600 and WG1 from Dr. B.J. Backmann of Department of Biology, Yale University, USA.

TABLE 7 PCR assay result using primers based on the E. coli serotype 016 (strain K-12) 0 antigen gene cluster sequence Gene Function Base Forward primer Reverse primer Length of Number of pools Annealing positions of (base positions) (base positions) the PCR (out of 21) giving temperature the gene fragment band of correct of the PCR size rifl* TDP-rhamnose pathway 90-1175 #1064(91-109) #1065(1175-1157) 1085bp 17 60 0

C

rniiD* TDP-rhamnose pathway 1175-2074 #1066(1175-1 193) #1067 (2075-2058) 9Olbp 13 rinIA* TDP-rhamnose pathway 2132-3013 #1068(2131-2148) #1069(3013-2995) 883bp 0 60 0

C

rinIC* TDP-rhamnose pathway 3013-3570 #1070(3012-3029) #1071(3570-3551) 559bp 0 gtf Galactofuranose pathway 4822-5925 #1074(4822-4840) #1075(5925-5908) llO4bp 0 55 0

C

WZX Flippase 3567-4814 #1072(3567-3586) #1073(4814-4797) 1248bp 0 55 0

C

w~zy 0 polymerase 5925-7091 #1076(5925-5944) #1077(7091-7074) 1167bp 0 60 0

C

7vbbl* Galactofuranosyl 7094-8086 #1078 (7094-7111) #1079(8086-8069) 993bp 0 50 0

C

transferase w(bbI* Acetyltransferase 8067-8654 #1080(8067-8084) #1081(8654-8632) 588bp 0 60 0

C

7wbbK** Glucosyl transferase 5770-6888 #1082(5770-5787) #1083(6888-6871) 11ll9bp 0 55 0

C

7vbbL*** Rhamanosyltransferase 679-1437 #1084(679-697) #1085(1473-1456) 795bp 55 0

C

~,~~Base positions based on GenBank entry U09876, U03041 and L19537 respectively 19 pools giving a band of wrong size TABLE 8 PCR assay data using 0111 primers Gene Base positions of Forward primer Reverse primer Length of the Number of poois Annealing the gene according (base positions) (base positions) PCR fragment (out of 21) giving temperature to SEQ ID NO: 1 band of correct of the PCR size w(bdH 739-1932 #866 (739-757) #867(1941-1924) 1203bp 0 60 0

C

#976(925-942) #978(1731-1714) 807bp 0 60 0

C

#976(925-942) #979(1347-1330) 423bp 0 60 0

C

#977(1165-1182) #978(1731-1714) 567bp 0 60 0

C

7VZX 8646-9911 #969(8646-8663) #970(9908-9891) 1263bp 0 50 0

C

#1060(8906-8923) #1062(9468-9451) 563bp 0 60 0

C

#1061(9150-9167) #1063 (9754-9737) 605bp 0 50 0

C

WZY 9901-10953 #900(9976-9996) #901(10827-10807) 852bp 0 60 0

C

#980(10113-10130) #983(10484-10467) 372bp 0* 61 0

C

zvbdL 10931-11824 #870(10931-10949) #871(11824-11796) 894bp 7 60 0

C

ZwbdM 11821-12945 #868(11821-11844) #869(12945-12924) 1125bp 0 60 0

C

#984(12042-12059) #987(12447-12430) 406bp 0 60 0

C

#985(12258-12275) #986(12698-12681) 441bp 65 0

C

Giving a band of wrong size in all pools One pool giving a band of wrong size TABLEM TABLE 8APCR specificity test data using 0111 primers Gene Base positions Forward primer Reverse primer Length of the Number of pools (pools Annealing of the gene (base positions) (base positions) PCR fragment no. 25-28) giving band of temperature according to SEQ correct size of the PCR ID NO: 1 7vbdH 739-1932 #866 (739-757) #867(1941-1924) 1203bp 0* 60 0

C

#976(925-942) #978(1731-1714) 807bp 0 60 0

C

#976(925-942) #979(1347-1330) 423bp 0 60 0

C

#977(1165-1182) #978(1731-1714) 567bp 0 60 0

C

wVzx 8646-9911- #969(8646-8663) #970(9908-9891) 1263bp 0 55 0

C

#1060(8906-8923) #1062(9468-9451) 563bp 0 60 0

C

#1061(9150-9167) #1063 (9754-9737) 605bp 0* 50 0

C

WZY 9901-10953 #900(9976-9996) #901(10827-10807) 852bp 0 60 0

C

#980(10113-10130) #983(10484-10467) 372bp 60 0

C

7tbdL 10931-11824 #870(10931-10949) #871(11824-11796) 894bp 0 60 0

C

z.'I'dM 11821-12945 #868(11821-11844) #869(12945-12924) 1125bp 0 60 0

C

#984(12042-12059) #987(12447-12430) 406bp 0 60 0

C

#985(12258-12275) #986(12698-12681) 441bp 0* 65 0

C

I pool giving a band of wrong size 2 pools giving 3 bands of wrong sizes, 1 pool giving 2 bands of wrong sizes TABLE 9 PCR results using primers based on the E. coli 0157 sequence Gene Function Base position Forward primer Reverse primer Length of Number of Annealing of the gene (base positions) (base positions) the PCR pools (out of 21) temperature according to fragment giving band of of the PCR ID NO: 2 correct size IwbdN Sugar transferase 79-861 #1197(79-96) #1198 (861-844) 783 0 55 0

C

#1199(184-201) #1200(531-514) 348 0 55 0

C

#1201(310-327) #1202(768-751) 459 0 55 0

C

zvzy 0 antigen 858-2042 #1203(858-875) #1204(2042-2025) 1185 0* 50 0

C

#1205(1053-1070) #1206(1619-1602) 567 0 63 0

C

#1207(1278-1295) #1208(1913-1896) 636 0 60 0

C

zbdO Sugar transferase 2011-2757 #1209(2011-2028) #1210(2757-2740) 747 0 50 0

C

#1211(2110-2127) #1212(2493-2476) 384 0* 62 0

C

#1213(2305-2322) #1214(2682-2665) 378 0 60 0

C

WZX 0 antigen flippase 2744-4135 #1215(2744-2761) #1216(4135-4118) 1392 0 50 0

C

#1217(2942-2959) #1218(3628-3611) 687 63 0

C

7cbdP Sugar transferase 5257-6471 #1221(5257-5274) #1222(6471-6454) 1215 0 #1223(5440-5457) #1224(5973-5956) 534 0 55 0

C

#1225(5707-5724) #1226(6231-6214) 525 0 WbdR N-acetyl 13156-13821 #1229(13261-13278) #1230(13629-13612) 369 0 55 0

C

1#1231(13384-13401) #1232(13731-13714) 348 0 60 0

C

3 bands of wrong size in one pool, 1 band of wrong size in all other pools 3 bands of wrong sizes in 9 pools, 2 bands of wrong size in all other poois 2 bands of wrong sizes in 2 pools, 1 band of wrong size in 7 pools TABLE 9A PCR results using primers based on the E. coli 0157 sequence Gene Function Base position Forward primer Reverse primer Length Number of pools Annealing of the gene (base positions) (base positions) of the (pools no. 37-42) temperatur according to PCR giving band of e of the SEQ ID NO: 2 fragmen correct size PCR t ItbdN Sugar transf erase 79-861 #1197(79-96) #1198 (861-844) 783 0* 55 0

C

#1199(184-201) #1200(531-514) 348 0* 55 0

C

#1201(310-327) #1202(768-751) 459 0 61 0

C

WZY 0 antigen 858-2042 #1203(858-875) #1204(2042-2025) 1185 50 0

C

#1205(1053-1070) #1206(1619-1602) 567 60 0

C

#1207(1278-1295) #1208(1913-1896) 636 0 60 0

C

7vbdO Sugar transferase 2011-2757 #1209(2011-2028) #1210(2757-2740) 747 0 50 0

C

#1211(2110-2127) #1212(2493-2476) 384 61 0

C

#1213(2305-2322) #1214(2682-2665) 378 0 60 0

C

7vzx 0 antigen flippase 2744-4135 #1215(2744-2761) #1216(4135-4118) 1392 0 5 0 c #1217(2942-2959) #1218(3628-3611) 687 0 63 0

C

tvbdP Sugar transferase 5257-6471 #1221(5257-5274) #1222(6471-6454) 1215 0 55 0

C

#1223(5440-5457) #1224(5973-5956) 534 0* 60 0

C

#1225(5707-5724) #1226(6231-6214) 525 0 55 0

C

7vbdR N-acetyl transferase 13156-13821 #1229(13261-13278) #1230(13629-13612) 369 0 50 0

C

#1231(13384-13401) #1232(13731-13714) 348 0 60 0

C

1 band of wrong size in one pool pool #39 giving two bands, one band of correct size, the other band of wrong size in another pool.

2 bands of wrong sizes in one pool S 3 bands of wrong sizes in 2 pools, 2 bands of wrong sizes in 2 other pools EDITORIAL NOTE NO 40235/99 Sequence listing pages 1-56 is part of the description.

Claim pages are to follow.

WO 99/61458 SEQUENCE LISTING PART <110> THE UNIVERSITY OF SYDNEY <120> ANTIGENS AND THEIR DETECTION <130> REEVES <140> <141> <160> 68 <170> Patentln Ver. PCT/AU99/00385 <210> 1 <211> 1773 <212> DNA <213> Escherichia coli <400> 1 atgcgacgta attttttggc aaaattctaa ggtggaagcc tcattaatac cgctgtcgac acgc tgcggg ccgcacgtaa aaatcaacaa actctgattc accgcgtatc tgaaaattca attcttcaac atgctatcac atactgatga cgctgcacaa gtaaagact t ttgatgttac tgatcaaagt aaaactattc taac tgatct ctgagcaatt catcagtcga tcaccaacct ccgactatgc actccgtgct aatggcctta tagaacgaat ttaatgacac aggttgtgag caatacttaa caacagcctc ttctatcgag ccaagcgatt cgccaacgac caacttgcag tgacctgtct cggtcagact ggttggtgcg tttggggctg atctatcccg gtctgcagca catccttgat ctatgctgct ttatgatgat ttcagcagat tgctggtgat gaatttaagt tgaaggtgct caaattccgg gaacaacacc gaccgaagtg gtctaaagcg acctgcctga accggggtta tcggcgtaag gaacagcaac tgaccagacg accgtagact tcgctgatca cgcctctctt gctaaccgct ggtatttctc cgtgttcgtg tcaa tacagg cagttcaacg aatgatggtc aatggcttct caagccgcta atcgcagcca aaagatggta tctgttaatg tatgcgaacg aaagacggcg gcggcagaca gatgttactg tcaactgagg tcttctctag accaccaacc t cc aac at gt aaccaggtac ccccgccacc gaggaagggg ataacagggt tgaaaacagg ctcaaaataa ctggtctgcg tcacttctaa tggcgcagac aactgaccgt acgaaatcaa gcgttaatgt aaactatctc cagtttctaa gcaatgaacc aattgggggt aggcaacagc ccgcttcagg gtgttgacga cagctcaagg ttcttaagaa atactaatgg atccgctggc gtgccgtgca tgtc tgaagc cgaaagcgca cgcagcaagt ggcggggttt cggggcaaag agtatttcga tgacggcgac aaaatgaatc tatcaacaag cattaacagc catcaaaggt cac tgaaggc tcaggccact atcccgtctc tctttccaaa catcgatctg aaactctctt tgttgatgtt ttccgatacg tgattatgtt taaagtaacc tgccaagcaa ttttgtcaca tggagcaaca taaggtaacc gcttctggat gaaccgtctc gcagtcccgt gatcatccag tctgtcactg tttctgtccg tttacgattt ccgctagaaa gaagccgaag atggcacaag aac cagt ctg gc taaagatg ctgactcagg gcactgtctg accggt ac ta gatgaaattg gatggttcaa aagaaaattg aatgtcagca aacttcggtg tcaagcctgt gttcagtcag ttaaacacca acaggtcagc cttcaaggca gctcttaagt acaactgcga aaagctattg gattccgcta attcaggacg caggcaggta ttacaaggct caatttaccg 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 SUBSTITUTE SHEET (Rule 26) (ROIAU) WO 99/61458 2 WO 996145 -2-PCT/AU99/00385 ataaccccca aataacccCt catttcaccc actaatcgtc cgattaaaaa ccctgcagaa 1680 acggataatc atgccgataa ctcatataac gcagggctgt ttatcgtgaa ttcactctat 1740 accgctgaag gtgtaatgga taaacactcg ctg 1773 <210> 2 <211> 500 <212> DNA <213> Escherichia coli <400> 2 aacagcctct gccattgagc caggcgattg gccaacgacg aac ctgcagc gatctgaact ggtcagaccc gttggcgcga ttaaacctga cgctgatcac gtctgtcttc ccaaccgctt gtatctccgt gtatccgtga caatccagga agttcaacgg acgatggtga ctgggtttaa tcagaacaac cggtctgcgt cacctctaac tgcacagacc gctgactgtt cgaaattaaa cgtgaacgtg aaccatcacc atcaacaaaa atcaacagcg atcaaaggtc actgaaggcg cagtcttcta tcccgtctgg ctggcaaaag atcgacctga accagtcttc caaaagatga tgac tcaggc cactgtctga cgggtac taa acgaaattga acggctccat aaaaaattga aatgtctact cgctgctggc agctcgtaac aatcaacaac ctctgaatcc ccgcgtatcc gaaaattcag ctcttctact 120 180 240 300 360 420 480 500 <210> 3 <211> 500 <212> DNA <213> Escherichia coli <400> 3 ctcagtatgc ccgcgactgg caaacaaaac cagc tat tta ctaccgctaa Cttctctggg ccaccaacct caaatatgtc accaggttcc tgtcaccggc tgatattgtt tgtaaaatat cgcaactgaa cccactgaaa tgctgtacag gtctgaagcg taaagcgcag tcagcaggtt agtacaggtg tctgcttatg tatgcccaca gcgggcaaat gccctggacg aaccgtctgg cagtcccgta atcatccagc ccgtaactta ttgatgatgc ctaatggtag tgac tactga atgcaatcag attctgcggt ttcaggacgc aggccggtaa cgatccagat aggtacattg cgtcacgaac agcgtctaca ccagatcgac aaccaacctg cgactatgcg ctccgtgttg acagatcctg acaactgatg gacagtggtt gctgctgaaa aaattccgtt aacaacacca accgaagtgt gctaaagc ta 120 180 240 300 360 420 480 500 <210> 4 <211> 399 <212> DNA <213> Escherichia coli <400> 4 agcctgtcgc attgagcgtc gcgattgcta aatgatggta ctgcagcgtg ctttcttcta tgttgaccca tctcttctgg accgttttac tttctgttgc tacgtgaact tccaggctga gaataacctg cctgcgtatt agcaaatatt gcagaccact gactgttcag aattactcaa aacaaatctc aacagtgcta aaaggtctga gaaggcgcgc gcaactaacg cgtc tggaag agtcttctct aagatgacgc Ctcaggcttc tgaatgaaat gtactaactc aaat tgaccg gagctccgcc agcaggtcag ccgtaacgcg taacaacaac tgacagcgat tgtatctgag 120 180 240 300 360 SUBSTITUT SHEET (Rule 26) (RO/AU) WO99/61458 3 caaactcagt ttaacggcgt gaaagtcctt gctgaaaat <210> <211> 417 <212> DNA <213> Escherichia coli PCT/AU99/00385 <400> gcacgttagt agactgcttc ttctggtaaa tcgacaaagc gtttcgactc gccgtatcga tgcaacaagc tgttaacggt tggtaacaat cgaagatgca attggctaaa tgctatcacc agatgctgac gggtacctct gcaacttacg aaagtcatgt gcaaaatcgt gttgacaatc aacc ttggca tacgcgaccg gttctggcgc atgttagtgc atctgagcaa tgcaatctac tgcgttctga acaccgtaaa aagtgtctaa aggctaacca agatggtaaa atcagaaggt caccaacccg cctcggtgca caacctgtct catgtctcgt gaccacgcag acgataacgg ggtagcccga ctcgaaacta gtacaaaacc tctgcccgta gcgcagatcc aacgtac 120 180 240 300 360 417 <210> 6 <211> 950 <212> DNA <213> Escherichia coli <400> 6 aacaaaaacc aacagcgcta aaaggtctga gaaggcgcgc gcctctaccg cgtcttgatg tcgaaaaacg ggcttgcaac gcggatttca gtcaaggatg agtac taaat at taccggta gccgatccgt ctcggtgccg aacctgtctg atgtcgaaag agtctgcgct aagatgacgc ctlcaggccgc tgtcagagat gcacgaactc aaattgaccg attcgatgaa aaatcgacag gcgcggcgaa ctgggggtaa atgcggtcgt cggcggcggc taaaagcact ttcaaaaccg aagcgcagtc cgcagat tat gtcgacttct atcgagcgcc tctcttctgg cgcgggccag acgtaacgcc taacaacaac tgattccgac tgtatctggt gattcagatt taccactttg actgacggct acaagtcaat tgattctgca ggtaactgtt ggatgccgca tctggattct ccgtat tcag ccagcaggcg gcgattgcta aacgacggta ttgcagcgta ctgtcttcta cagacccagt ggtgccaatg aatctgaaag gctgatggta ttactgtctt accggtaaat ggtgcagcgg atcgctaaag gcggtcacca gacgccgact ggcaactccg accgctttac tttctctggc ttcgtgaact ttcaggacga tcaacggtgt ataaccagac gatttaccgt cagcaattgc acactgacac acatggaagc aagtggcggg tcgacaaatt acctgaacaa atgcgaccga tgctgtctaa tctgcgtatt ttctaacatc gcagacggct gaccgttcag aatcaaatcc gaacgtgctg gatcagcatt gtccggcatg tgctgcggat cgcgtctaac cactgtagtc agccgctaca ccgctcctcc caccaccacc agtgtccaac 120 180 240 300 360 420 480 540 600 660 720 780 840 900 950 <210> 7 <211> 1212 <212> DNA <213> Escherichia coli <400> 7 aacaaaaacc aacagcgc ta aaaggtctga agtctgcgct gtcgacttct atcgagcgcc tctcttctgg tctgcgtatt aagatgacgc cgcgggccag gcgattgcta accgcttcac ttctaacatc 120 ctcaggccgc acgtaacgcc aacgacggta tctctctggc gcagaccact 180 SUBSTITUTE SHEET (Rule 26) (RO/AIJ) WO 99/61458 -4 WO 9961458PCT/AU99/00385 gaaggcgcgc gcgacgaccg cgtctggatg gcgaaagatg gatttgcaga tcacttaacg ggtgttgatt gtttctagcc gtcgt tcaat acgttgaata actcagaaag gtaacattcc gcggcaaaag cagttctcag gttgatactt aacctgaaca tatgctacag gtgctgtcca tgtctgaaat ggactaactc aaattgatcg gttcgatgaa agattgactc ttagtgattc tcactgctgt tgacgttaca ccggcaatga aagccgatgt atcaactgat aaggtaaaaa caacagataa gggcttcttc tccgctcctc acaccaccac aagtgtccaa aa caacaacaac tgattctgac cgtttccggt gattcaggtt ttctacatta cattactcaa tgcgaaagat caacactctg tttctactcc cgaatacaca taaagtigcc ctacgctaca taaagttgtt tgctgatcca cctcggtgcg caacctgtct catgtcgaaa ttgcagcgtg ctgtcttcta cagacccagt ggcgcgaatg ggac tgaacg attaccggtg ctgactactg gatgcgaaag gcgtcgatta gacaccgata gctgactctg acggtttcaa gttgaattat ctggcacttt gtgcaaaacc gaagcgcagt gcgcagatca tgcgtgagt t ttcaggacga tcaacggcgt atgggcagac gtttctccgt ccgccgggac cgacaggtaa gggctgctac atcatacaga atggactaac acggctcggc cggcacttga caacagcaaa tagacaaagc gtctggattc cccgtattca tccagcaggc gaccgttcag aatcaaatcc gaatgtgctg tattagcatt ttcgggtcag aaaacctgtt aacagtcgat atcacagttc cggcaaagtc gactgcggct tgcgggatat tgataatac t accgactgca tattgcacag cgcagtaacc ggacgccgac aggtaactcg 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1212 <210> 8 <211> 1647 <212> DNA <213> Escherichia coli <400> 8 atggc acaag aaccagtctg gcgaaggatg ctgac tcagg gcgctgtccg accgggac ta gacgaaattg gacggttcga aagaaaattg acggctaata gggggaac tg attctaaatg tttggtacac gccgcttctg gatacgacta tccggtaatt ttaactaaaa actggtgcta gcgtttgatg gacacttata gcagacaccg caagctgatg ggttccgatg tcattaatac cgctgtcgag acgccgcggg ctgcacgtaa aaattaacaa actctgattc accgcgtatc tgaaaattca actctgatac cggcagcaac ttggtgtaac ctgttgctgg cagccgctgc atgatatttc aagc tacagt taactgctgc ataatgc tgg aagccgcgac gtgcatcaat ctgccactgt cttatatgag gaagtatcac gtaagttaac caacagcctc ttctatcgag tcaggcgatt cgccaacgac caacttacag ggatctggac cggtcagacc ggttggtgcg gctggggctg cctgaaagat tcaatatact cgcagatgga tgtzaacctat cagcgctaac tacaattggt tgatgatggc tggtgataca catccaaact gtccattgat aggtgctaag caatggggtt aac tac tgag aacggatacg tcgctgatca cgtctgtctt gctaaccgtt ggtatttctg cgtattcgtg tccattcagg cagttcaacg aatgacggcc aatgggttta atgtctggat gacaaatcgg aataaagtta acctacaata ctggctgctt ggcaaagatc gcagtacttt caagctactt gataaaggaa accaatacat acttatagcg ctcagtgata gatgcggctg actagcaaag ctcaaaataa ctggcttgcg ttacttctaa ttgcgcagac aactgacggt acgaaatcaa gcgtgaacgt agactatcac atgtgaacgg tcacagctgc ctgtagcaag caactagcgc aagacactaa tcc tc aatcc aagatgtaaa atatggatgc tggctaaac t cattcaccag ttgcaaatgc taacaacagg ctccgccaac ccggtaaact cagaatcaac tatcaacaag tattaacagc cattaaaggc caccgaaggc tcaggcttct atcccgtctc actggcaaaa tattgatctg caaaggggaa ggcggcacca tagcgtagat cgatgttggt ttcatattcc tcaggccgga catcgataaa taccggtaac tgctactgct tgacggtaca agtaaaaaat ttctgctgct ttactatgca ggtctacaaa atcagatccg 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 SUBSTITUTE SHEET (Rule 26) (ROIAU) Axin no 11K I AZQ TV SJZ~~JO- 5 I'Ll/A ctggcagctc ttgacgacgc tatcagccag atcgacaaat tccgctcctc cctgggtgcg gtgcaaaacc gtctggattc cgcagtgacc aacctgaaca acaccactac caacctgtct gaagcgcagt cccgtat tca ggacgccgac tatgcgaccg aagtgtccaa catgtcgaaa gcgcagatta tccagcaggc cggtaactcc gtgctggcaa aagctaacca ggttccgcag caggttctgt ctctgctgca gggttaa <210> 9 <211> 1758 <212> DNA <213> Escherichia coli U99/00385 1440 1500 1560 1620 1647 <400> 9 atggcacaag aaccagtctg gcgaaggatg ctgactcagg gcgctgtctg accggaacta gatgaaattg gacggttcga aagaaaatcg attaccaaca acgacaggtc gataaattag aaatctggtg caggctactg ggtaaatctg tcagcaggta acgactaaca agcgaaggta gcaactcctg gctacagtga attaccttta gatgctgcga gtctcttaca actgatacca ggtaaaatca ctggacgacg cgtctggatt tcccgtattc atccagcagg tctctgctgc tcattaatac cgctgtcgag acgccgcggg ctgcacgtaa aaatcaacaa actctgattc accgcgtatc tgaaaattca attctgatac aagctgcaac tttatgatct ggaatggcga attttactac attcagctaa ttaatggttc atatcaccat acgctggtag gtgacggtgc cgacaacctc gtaaagatgt attccggtgt agtcttatgt gcgttaacaa ataaagatta ctactgagac ctatcagctc ccgcagtcac aggacgccga ccggtaactc agggttaa caacagcctc ttctatcgag tcaggcgatt cgccaacgac caacttacag ggatctggac cggccagacc ggttggtgcg tctgggtctg ggtaagtgat gaaaaccgaa taaagtcacc caccaaatct aaaacgtgat ttacaccaca cggtggaagc cgcagctaaa ttctctgaca tccagtagct agtattgagc actgagcaaa ggatgataaa ggataacggc tgctccagca taccagtgct catcgacaaa caacc tgaac ctatgcgacc cgtgctggca tcgctgatca cgtctgtctt gctaaccgtt ggtatttctg cgtatccgtg tccattcagg cagttcaacg aatgacggtg aatggtttta ttaacttctg aataccttgt gttggcggcg actgctggta gcgttagctg aaagatggta caggcatacg gctgatatga ttcaatggca ccgttaatcc gaaaccaaag actattgggt ggtggtatta tctgtgactg attggtactg ggttctgcaa ttccgttctt aacaccacta gaagtgtcca aaagccaacc Ctcaaaataa Ctggcttgcg t tact tc taa ttgcacagac agc tgacggt acgaaatcaa gcgtgaacgt aaactatcac acgtaaatgg ctggcgcgaa taactaccga tagattatac cgggtgtaga ccacccttca ctgtttcttt tagacgatgc aagcgctgct ctgaatatac ctggtgggat cggctgccgc ttaccgcggg ctaacgttgc ttgccgggta ctgtaaatgt cgaccaaccc cctgggtgc ccaacctgtc acatgtcgaa aggtaccgca tatcaacaag tattaacagc cattaaaggc caccgaaggc tcaggcttct atcccgtctt actggcaaaa tatcgacctg taaaggtact gttaaacacc tgctgcattc ttacaacgct cgccgcggcg tgctgatgtg cgaaacggat aggcaacttg taaagccgcg tatcgcaaaa tact tatcag gaca tc ttca tgaatccagt cgactataca tgcttcagcg gaactccgcg gcttgctgcc tat ccagaac tgaagcgcag agcgcagatt gcaggttc tg 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1758 <210> <211> 1383 <212> DNA <213> Escherichia coli SUBSTITUTE SHEET (Rule 26) (RO/AU) WO 99/61458 6 WO 996 145 -6-PCTIAU99/00385 <400> aacaaatctc aacagcgcaa aaaggtctga gaaggtgcgc gcaac taacg cgtctggaag gctgaaaata aatctggcaa cagaaagcaa gatgttggcg aataaagatg gtatccggtg aaaggctcca gctaatggta gacagtaatg aacagtgcgg ctggatctta aatgtcagcg tatctgagca ttgcaatcta ctgcgttctg aacaccgtaa gaagtgtcta cag agtcttctct aagacgatgc cccaggcttc tgaatgaaat gtactaactc aaattgaccg atgaaatgaa aaattgatgc caggcagtga gtaaaactta tttttgtaag aaagtat tga ttgaatacaa aaggtgtttt cacccacggg gccagttcac atgcagccaa cagatggtaa aatcagaagg ccaccaaccc acctcggtgc acaacctgtc acatgtctcg tagctctgct agcaggtcag ccgtaacgca taacaacaac tgacagcgat tgtatctgag aat tcaggtt gaaaac tctc cctgatttct taccgtgaat cgcagctgat tgcaacagaa gggcattaca gaccgcaaat tgccggcgca cactacaaaa gaaaacaggt aacggtaact tggtagcccg gctcgaaact agtacaaaac ttctgcccgt tgcgcagatc attgagcgtc gcgattgcta aatgatggta ctgcagcgta ctttcttcta caaactcagt ggtgctaatg ggcc tggacg aaatttaaag gtggagagcg ggatcgctga ctagcgaaac tttactaaca attgatggtc acaataacta gtggaaaata agcactttag gatactactc attc tggtaa atcgacaagg cgtttcgact agccgtatcg ctgcaacaag tgtcttctgg accgttttac tttctgttgc ttcgtgaact tccaggctga ttaacggcgt atggtgaaac gttttaatat cgacaggtac gcgcggttaa cgaccagtag t tgcaataaa acac tggcgc aagatgttca cagacacago aagccgcaac ttgtaaatgg ctggtgcccc acgaagatgc cattggctaa ctgccatcac aagatgctga cgggtacctc tctgcgtatt ggcaaatatt gcagaccact ttctgttcag aattactcaa gaaagtcctt catcactatc cgatggcgcg tgataattat gaatgatgct tgatactaaa attagctgac agagcttgat atttactatt tgtttacaaa actctctgat cgccacc tac taaagtgatg agcaaaatcg agttgacaat caaccttggc ctacgcgacc tgttctggcg 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1383 <210> 11 <211> 2013 <212> DNA <213> Escherichia coli <400> 11 atggcacaag aaccagtctg gcgaaggatg ctgactcagg gcgctgtccg accgggacta gacgaaattg gatggctcga aagaaaattg gtagcgaata acagatacca tccgatctgt tttggcgtgg gctgcagatg tcgtcgcagg ggaaaaat ta tcattaatac cgctgtcgag acgccgcggg ctgcacgtaa aaattaacaa actccgattc accgcgtatc tgaaaattca actctgatac cagctgcgac atggcgtgac tagctaatat ctgcaaagaa gtgccgattc cgacagtgac c tgcggcaga caacagcctc ttctatcgag tcaggcgatt cgccaacgac caacttacag ggatctggac cggccagacc ggtcggcgcg gctgaatctg aagcgacgat cgcgtataca caccgatgga tggttacacc agcgaagacg tattggtggt tgataatgcg tcgctgatca cgtctgtctt gctaaccgtt ggtatttccg cgtattcgtg tccattcagg cagttcaacg aacgatggcg gctggtttta ttaaaacttrg aacacaatta tcagtgatca tatgatgcag ttaagcatca aaagagcaga acgctgtatt ctcaaaataa ctggcttgcg ttacttctaa ttgcacagac aactgacggt acgaaatcaa gcgtgaacgt aaacgattac acgttaacgg ctggtttcac gtaatgacaa ctgggggagg caagtaaatc ttaatccaaa aagttaatat tagataaaca tatcaacaag tattaacagc cattaaaggc cac tgaaggc tcaggcttct atcccgtctg gctgtccaaa tattgatctg taaaggttct taagggcacc agccaaagc t ggcaaacgct ttatagtttt caccggtgat ttcccaggat gggaaac ttg 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 SUBSTITUTE SHEET (Rule 26) (ROIAU) WO 99/61458 7 WO 996145 -7-PCT/AU99/00385 acaaaaacga gattctaccg gcttccggaa attcttgcag attttggtag gcgactactg ttaattgcta gtt tatagca aatgtcacca accattgata gctgctgcaa gatgtaacct tatgaaacag gatcccctga ggtgcggtgc ctgtccgaag tcgaaagcgc ccgcagcagg atgcaggtaa gtgcggttcc tgtctgttca gtggtgcatt caagtaatgg gtggattcac acagtaagga caacaggaag acgcacatgt tgggcggtac atgctgatgt actttgaaca ctgatggtaa aagctctgga aaaaccgtct cgcagtcccg agatcatcca ttctgtctct cgataccgca agttggggtt gtccgcagga tgcggctaag aaacataaca tacaacggct tgctacctta tggcgctcag cagtgccgaa aggtacagta ttatgtcgaa aaaaaatggg gttaacaaca cgaagccatc ggattccgcg tattcaggac gcaggccggt gctgcagggt gcgacttggg gcaactacaa gcaggaattc gtaagtattg gcggctgatg ggaggaaata accgtaactt ttcaccagtt ggtatggcaa acttacacag gatggtgcac gctattacca gaagctacta agctccatcg gtcaccaacc gccgac tatg aactccgtgc taa atggtttaat ttacaattac agacctcaac agggaggcgc gtagtgcact cagc tgc ttc caggtaccgg tagcaaaagt atctgacaaa tttccaatgg tttcagccaa acagcaccgg ctgcatccag acaaattccg tgaacaacac cgaccgaagt tggcaaaagc ttccaacagc ttctggtaca aaattctcag tgctacagac ttatcttgat gttagataat ccagaacact agacacagtc aagcaatttt ggatgtgaaa tgc tacaaaa tggtaccatc ttccaccgcc ctcctccctc cactaccaac gtccaacatg taaccaggta 1.020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 1860 1920 1980 2013 <210> 12 <211> 1263 <212> DNA <213> Escherichia coli <400> 12 atggcacaag aaccagtctg gcgaaggatg ctgactcagg gcgctgtccg accggtacta gaagagat tg gatgggaaaa aaaaagatcg agtgttaaag gataaagctt gtaaatgaca attaacttca actgttggcc gtgtataaag aatcaatcaa tctacaaccg gcatctgttg attgccaacc gctgactatg aactccgtgc taa tcattaatac cgctgtcgag acgccgcggg ctgcacgtaa aaat taacaa actctgagtc atcgtgtttc tgaacattca attcatctac atggggccac caggatcgtt ctaaaagtag actctacaaa gcgatgtaaa ataaaagcgg ctttcaatgc cgccaagcaa ataaattccg tgaacaacac cgaccgaagt tggcaaaagc caacagcctc ttctatcgag tcaggcgatt cgccaacgac caacttacag tgacctgtct aagtcagact ggt tggggca actaaacctc catcaataag aggtacccta taagtactac tgaaagtgga attggatgct caatgatgct cgctaatatc tttaacagct CtCttctctc cactaccaac gtccaacatg caaccaggta tcgctgatca cgtctgtctt gctaaccgtt ggtatttctg cgtgtgcgtg tctatccagg caatttaacg aatgatggac tccagttttg caagtggcag aaattagttg gatgccgaag actactccta tctgcactta tatatcattc agtgatgctg aacccgctta ggtgccgttc ctgtctgaag tcgaaagcgc ccgcagcagg ctcaaaataa ctggcttgcg ttacttctaa ttgcgcagac agctgactgt acgaaatcaa gcgtgaatgt agactatcac atgc tacaaa taggtgctgg agaaagacgg tagatac tag c tgcagcgac aagccaacca agaccaaaga gtgttttatc aggctcttga agaaccgtct cgcagtcccg agattatcca ttctgtctct tatcaacaag tattaacagc cattaaaggc caccgaaggc tcaggcgacc atctcgcctg tttggctaaa tattgatctg cttgggcacc cgactttaaa taagtactat taagggtaaa ggaagtaac t atcgcttgtc tgtaacaact tat tggtgca tgatgcaatt ggattctgcc tattcaggac gcaggccggt gc tgcagggt 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1263 SUBSTITUTE SHEET (Rule 26) (RO/AU) WO 99/61458 WO 9961458PCT/AU99/00385 -8 <210> 13 <211> 1368 <212> DNA <213> Escherichia coli <400> 13 aacaaatctc aacagtgcta aaaggtctga gaaggtgcgc gcaac taacg cgtctggaag gctgaaaata aatc tggcaa cagaaagcaa gatgttggcg gggaatgata atagctggta aatgataaat gggaatggtg gctgacaaaa tatacgacaa gc taagaaaa ggtaaaacga gaaggtggta aacccgctcg ggtgcagtac Ctgtcttctg tctcgtgcgc agtcttctct aagatgacgc ctcaggcttc tgaatgaaat gtactaactc aaattgaccg atgaaatgaa aaattgatgc ctggcagtga gtgatgctta tttttgttag cagggattga tcacgtttaa tatattctgc aagcttcttt ccaaagttga caggaagcac taacggagac gcccgattct aaactatcga aaaaccgttt cccgtagccg aga tc ctgc a gagctccgcc agcaggtcag ccgtaacgcg taacaacaac tgacagcgat tgtatctgag aattcaggtt gaaaactctc cctgatttct tactgttaac tgcagcagat tgc tacagca tggagttgaa agaaattgat gattacgagt taacaaggct gttagttgtt tgcttctggt ggtaaacgaa caaagcattg cgactctgct tatcgaagat acaagcgggt at tgaacgtc gcgattgcta aatgatggta ctgcagcgtg Ctttcttcta caaactcagt ggtgctaatg ggcctggacg aaatttaaag gtagatagcg ggttcactga ctcgcagcag ttcacaacaa ggtaagtcag gagacagttt gccacacttt aacggtgcaa aacaataaag gatgcagcaa gctaaagttg atcaccaacc gctgactacg acctctgttc tctcttctgg accgttttac tttctgttgc tacgtgaact tccaggctga ttaacggcgt atggtgaaac gttttaatat cgacaggtac gagctgttaa caactaaatc cggctaagaa caac tgcagc tgacatttac acaaaaatag ccgatcttga c ttacgatgt tcatgtatct aatcgttgca acaatctgcg ttggcaacac cgaccgaagt tggcgcag cctgcgtatt agcaaatatt gcagaccact gactgttcag aattactcaa gaaagtcctt catcactatc cgatggcgcg tgataactat agatactaca tgacacaaac taaagcacag ggatggcaat tgtgac agat cgctggcctt tctcaatgca tagtgcagat gagcaaatca atctaccacc ttctgacctc cgtaaacaac gtctaacatg 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1368 <210> 14 <211> 1788 <212> DNA <213> Escherichia coli <400> 14 atggcac aag aaccagtctg gcgaaggatg ctgactcagg gcgctgtccg accgggacta gacgaaattg gacggttcaa aagaaaattg atagccaata aatactataa gatggtgaca tcattaatac cgctgtcgag acgcagcggg cggcccgtaa aaatcaacaa actccgattc accgcgtatc tgaaaattca actcagatac aagcggc gac ctacaacaaa ctgttactat caacagcctc ttctatcgag tcaggcgatt cgccaacgac caacttacag ggatctggac tggccagacc ggttggtgcg gctggggctg cat tagcgac taatgcgctg caaagcagat tcgctgatca Cgtctgtctt gctaaccgtt ggtatctccg cgtatccgtg tccattcagg cagttcaacg aatgacggcc aatggtttta c tgacagcag actgcatcaa gctgctcaaa ctcaaaataa ctggcttgcg tcacctctaa ttgcgcagac aac tgacggt acgaaatcaa gcgtgaacgt agac tatcac acgtgaatgg cgaaaatgga aggcgcttga ctgccacggt tatcaacaag tattaacagc cattaaaggc caccgaaggc tcaggcttct atcccgtctg actggcgaaa gattgatctg ttccggtacg tgctgcaact tcaactgaaa ttatacatac 120 180 240 300 360 420 480 540 600 660 720 SUBSTITUTE SHEET (Rule 26) (RO/AU) WO 99/61458 9- WO 9961458PCT/AU99/00385 aatgcatcag ggtgatgtag gcagcaagcg cagaaagcat gcggctacgc aagactgcat gatgctgcaa ttaaataaag gcaacaatta ggtactgcta actgatgctg gctaacaacg gtaggggctg ggcacagtaa ttccgttctt aacaccacta gaagtgtcca aaagccaacc ctggtaactt cage tagcc t gtgaagtgaa atttaactag ttgatggttt cagtcacgat ctgctaacgc tggctacagc cctataaatc gcgcaaaata atggtgaaat gcaaggtaac aagtatatgt caaaagatcc ccc tgggtgc ccaacctgtc acatgtcgaa aggtaccgca ctcattcagt tctcccgccg ctttgatgtt tgatggtaac at tcaagaaa ggggggaaca aggggtatcg taaacaaggc tggcgttcag tgccgataaa gactacaatt tgttgattct tagtgctaat ac tgaaagct tatccagaac cgaagcgcag agcgcagatc gcaggttctg aatgtatcga gctgggcaaa gatgcgaatg ttaactacaa gctggtgatg acttataact ttcactgata aaagcagttg acgtatcagg gctgacgttt ggttcataca ggaactggta ggtactttaa ctggatgaag cgtctggatt tcccgtattc at tcagcagg tctctgctgc ataatacttc ctgctagtgg gtaaaatcac acgatgctgg gtcaatcaat t taaaacggg cagctagcaa cagctgacgg ctgtatttgc ctaatgcaac ccacgaagta cgggtaaata caacagatgc ctatcagctc ccgcagtcac aggacgccga ccggtaactc agggttaa agcaaaagca tgtttataaa aatcggagga tggtgcgact cgggtttaag tgctgatgct agaaaccgt t tgatacatcc cgcaggtgac agcaacatac t tcaatcgat tgcgccgaaa aactagcgaa cat cgacaaa caacctgaac ctatgcgacc cgtgctggca 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1788 <210> <211> 1653 <212> DNA <213> Escherichia coli <400> atggcacaag aaccagtctg gcgaaggatg ctgactcagg gcgctgtccg accgggac ta gacgaaattg gacggttcga aagaaaattg gttgctaaca aacaaatata gttagtgatg gcaacgaat t tcagc tgccg attactatcg agcaatggag gcttctttga attgacattg acttattcag tctggaaacg gtaacaaaag actgtagatt tataaagatg tcattaatac cgc tgtcgag acgccgcagg c tgcacgtaa aaatcaacaa actccgattc accgcgtatc tgaaaattca actcagatac ctgctgcatc ctgtgagtgc gtgatactgt acaagtatga atgttcagaa atggttctgc ataaacttta ctgaggctag gcggtacc tc taacaggtgc atgttgattt gtgttgctcc tttatctaca ctgacggtaa caacagcctc ttctatcgag tcaggcgatt cgccaacgac caacttacag tgacctggac cggtcagacc ggttggtgcg gctggggctg taaagctgac gggttacgat tcaggcaacc cagtgcaagt atatttgacc acaggatgtt cattgataca tctgtccaca tatctccttt aaaagttgat cactaccgca ggtttatatt ggatgatggt attgacgaca tcgctgatca cgtctgtctt gctaaccgtt ggtatttccg cgtattcgtg tccatccagg cagttcaacg aatgacggcc agtgggttta ttggtagctg gctgctaaag attaataacg aagtcttact ccgggcgttg cagatcagca actgggcgct Cttgcagcca accggtaata caggcagctt ggttatagcg gataacaacg tcagtgacta gatgc tgaaa ctcaaaataa ctggcttgcg ttacttctaa ttgcgcagac agctgacggt acgaaatcaa gcgtgaacgt agactatcac atgtgaatgg ctaatgcaac cgtctgattt gcttcggaac cttttgatac gtgataccgc gtgatggtaa taacgaaaaa ataataccaa gtactacgcc tcgataaagc tcgacggcgc gggcgt tgac acggcagcgg ctaaagctgc tatcaacaag tattaacagc cattaaaggc cactgaaggt tcaggcttc t gtctcgtctg gctggcgaaa gattgatctg tggcggggct tgtggtaggc gctggctgga ggcggctagt cacaacggct taagggcac t aattacgtca cggctttagt agcgacaacc gaacactatt tgtatcaacc aactggcgct cacatctgat taaggcagt t aaccaccgcc 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1~02 0 1080 1140 1200 1260 1320 1380 SUBSTITUTE SHEET (Rule 26) (RO/AU) WO 99/61458PC/U /085-1 10 PCT/AU99/00385 gatcccctga aagctctgga cgaagccatc ggtgcggtgc agaaccgtct ggattccgcg ctgtctgaag cgcagtcccg tattcaggac tcgaaagcgc agatcatcca gcaggccggt ccacagcagg ttctgtctct gctgcagggt agctccatcg acaaattccg ctcctccctc gtcaccaacc tgaacaacac cactaccaac gctgactatg cgaccgaagt atccaacatg aactccgtgc tggcaaaagc taaccaggta taa 1440 1500 1560 1620 1653 <210> 16 <211> 1689 <212> DNA <213> Escherichia coli <400> 16 atggcacaag aaccagtctg gcgaaggatg ctgactcagg gcgctgtccg accggtacca gacgaaattg gacggttcca aagaagattg gtggcgaata gcagcagacg gctgcagatg accattaata ccagcaaaag gttcagtcct acgacatcgg gcattatata aaactggcta gatggcacta agtgctgatt acaggtctga tacaccagca gatggctctg actaccgacg gcaatcagcc tccgcggtca caggacgccg gccggtaact cagggctaa tcattaatac cgc tgtcgag acgccgcagg ctgcacgtaa aaatcaacaa actcccagtc atcgcgtatc tgaaaattca actcttctac ctgcagcaac caaatggtac ttattgctag atggcttcgg gcgacttcac tcctgacgcc ttgatgtcgt tcgacagtac ctctgactgg atattgatat ctctgcagtc ccgtaggtac aagatggttc taaccaacgg ctgcaaccaa agatcgataa ccaacc tgaa ac tatgcgac Ccgtgctggc caacagcctc ttctatcgag tcaggcgatt cgccaacgac caacttacag tgacctggac cggtcagacc ggttggcgcg cttgaacctg taaagctgat agttacttat catcaaagac tgattccagt ttacgacgta gaaagcaggt tctggccagt aggtaacctg ccttcagggc tgctgctaac agcgac taaa tgatggtaaa cctgactact ttcaggtaaa agccgcaacc gttccgttca caacaccact cgaagtgtcc aaaagccaac tcgctgatca cgtctgtctt gctaaccgtt ggtatttctg cgtgtgcgtg tctatccagg cagttcaacg aacgatggcc acaggtttta ttaaccgctg actgtcagtg ggcagtgctc gcgctgactt gcttcaagcg gataccgcaa gatggtaaga actcagaaca tctggtgttg ggtaatattg tctacgggct gtgactatcg gataacacca gcggtctatg accaccgatc tccctgggtg accaacctgt aacatgtcga caggtaccgc ctcaaaataa ctggcttgcg ttacttctaa ttgcacagac aactgaccgt acgaaat taa gcgtgaacgt agaccatcac acgttaacgg ctcaactctc ctggttataa cgacttctgc ccaatgacta ccaataatac atctgaaagt ttacagcaaa gtgctggctt cttcaaccat gtctgaccgg ttactgttgg gcgggactac ctaaactgta tagaagcgga cgc tgaaagc ctatccagaa ctgaagcgca aagcgcagat aacaggttct tatcaacaag tattaacagc cattaaaggc cactgaaggc tcaggcaacc atcccgtctg gctggcaaaa tatcgacctg ttctggttct tgcaccgggt agaatccact aattactgca tacttatgac tgctgcccag aaccgttggt agatggttct gacctctgct cactactgaa tgttcgtatc tactggcgct tgctcagtcc tctgcagaaa tggtgatttc cctggatgag ccgtctggat gtcccgtatt cattcagcag gtctctgctg 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1689 <210> 17 <211> 915 <212> DNA <213> Escherichia coli <400> 17 SUBSTITUTE SHEET (Rule 26) (RO/AU) WO 99/61458 11 WO 9961458PCT/AU99/00385 gcgctgtcga gacgctgcgg gccgcacgta gagattaaca aactctgatt gaccgtgtat atgaagattc gacagtacca gcgaaac tga ggtaaacaag gtcgttgatt gcggcggtaa gcactggatg aaccgtctgg cagtcccgta attatccagc cttctatcga gccaggcgat acgccaacga acaacttgca ccgacctgtc ctggtcagac agattggtgc ctttgaatct cggctgctga tcaatttact ctgcaaccgg ctgttggtgc ccgcaatcgc attctgcggt t tcaggacgc aggcg gcgcctctct tgctaaccgc cggtatttct gcgtattcgt ttctattcag ccagttcaac caatgataac gaaaggattt tggtacagca gtcttacact taaatacatg aacggaagtg taaagtcgac caccaacctg cgactatgcg tctggtctgc ttcacttcta ctggcgcaga gaactgaccg gacgaaatca ggtgtgaacg cagacgatca accgtgtccg attgctgctg gacaccgcgt gcagccactg gcgggagccg aaattccgct aacaacacca accgaagtgt gtattaacag acatcaaagg cggc tgaagg ttcaggcctc aatcccgtct tgc tgtcgaa gcat tggct t gcatggcgga cggatgtcaa ctaacagtac tagtcattac ctacagccga cctccctcgg ccaccaacct ccaacatgtc cgctaaagat tctgactcag cgcgctgtca taccggcacg tgatgaaatt aaacgattcg gcaacaaatc tttcagcgcg ggatgc tggg taaatatgcg cagtacggcg accgt taaaa tgccgttcaa gtctgaagcg gaaagcgcag <210> 18 <211> 1665 <212> DNA <213> Escherichia coli <400> 18 atggcacaag aaccagtctg gcgaaggatg ctgactcagg gcgctgtccg acagggacta gacgaaattg gatggttcaa aagaaaat tg acagccaata gtcactacag attttgaatg tttggtgcag tatacggctt ggtggtacca gtcattattg gatgatactg gacttaatgg actgc~taata aacgccgtta acagtcaata cctgccgagt agtaaagc ta gaagctacta cgttcttccc tcattaatac cgctgtcgag acgccgcagg c tgcacgtaa aaatcaacaa actccgattc accgcgtatc tgaaaattca actctgatac ctgctgcaac ctggagttaa cg~tagctgg ctgcccctgg Ctgcttcagt ctgctgcaac ctaaagatgg gtaacttaag caaacaatgc cgacaaagtt aaaatgagac atggcactgc attttgctca tctatgtaag ccaacccgct tgggtgc tat caacagcctc ttctatcgag tcaggcgatt cgccaatgac caacttacag tgacctggac cggtcagacc ggtcggcgca gctgaatctg acttaaagat tagatatatt tgttgatggc tacgccagtg tgatgcgact agtaagtatt ttctttaact taaaactaac taatgccaaa tgatggggta ttacactgca tgcatcagcg agc tgatggc tgccaatggt ggcagcat tg ccagaaccgt tcgc tgatca cgtctgtctt gctaaccgtt ggtatttctg cgtattcgtg tccatccagg cagttcaacg aatgatggtg gctggtttta atggttggtt gctgacaaag agtaaagttt gaatatactt caactggcgg ggcaacggta gctgctgatg gcaggcactg acagtcatta ga tat ttc tg actgttggtg tatttagtcg actattacta aacttaacga gatgacgcta ctggattccg ctcaaaataa ctggcttgcg ttacttctaa t tgcacagac aactgacggt acgaaatcaa gcgtgaacgt aaaccatcac acgtgaatgg taaaactcga ccgtcgcaag ccacggaggc atcataaaga cattcctgaa caacagctca acggtgccgc atactcaagc caacagataa ttgatgcttc taactttacc atggaaaagt gtggtgaaaa ctaatacaac tcgcgtctat cagtcaccaa tatcaacaag tattaacagc tattaaaggc cac tgaaggc tcaggccac t atctcgtctg gctgtccaaa gattgatctg cgaaggtgaa taatacgggg tagcacgga t agatgttggt tactaacaca tcctgaagcg agagcaaaaa tctctatctt taaactgtct aggtacattt aacgtttgct tgcgacatat gagcaaaac t tgcggctacc tagtgaatct cgacaaat tc cctgaacaac 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 SUBSTITUTE SHEET (Rule 26) (RO/AU) Illd-t anh< I ACQ ~Xf llil C l~IAr~l~~VO V~ 7Ut~- 12 J~Ituu accactacca acctgtctga agcgcagtcc cgtattcagg acgccgacta tgcgaccgaa 1560 gtgtccaaca tgtcgaaagc gcagatcatt cagcaggccg gtaactccgt gctggcaaaa 1620 gccaaccagg taccgcagca ggttctgtct ctgctgcagg gttaa 1665 <210> 19 <211> 1842 <212> DNA <213> Escherichia coli Jo0 <400> 19 atggcacaag aaccagtctg gcgaaggatg c tgac tcagg gcgctgtccg accggaacta gacgaaat tg gatggctcga aagaaaattg gtagcgaata aaagctgctg agcaatgtac acaggtatag tttagcgcaa gatacattta gatggtacga acccaaacag gatactacag gaagcaaaag gcttataatg gcgggagccg gataatacca cttcaggata gctgtcacta accagtaaag tccactattt accaccgccg tcctccctcg accaccaacc tccaacatgt aaccaggtac tcattaatac cgc tgtcgag acgccgcagg ctgcacgtaa aaattaacaa actccacctc accgcgtatc tgaaaattca actctgatac ccgctgcgac atggcaccgt tggctgctgc ctgcaactgg cggctgcatc ccgctaaagt ttacatccag gtagtggcac gcaaagatgc gaatgaccat ccgcatatgc ctgcaactgc cggcagatgt aaga tt tcac t caa tggc aa ttgtcaatta atgcgacaga atcccctgaa gtgcggtgca tgtctgaagc cgaaagcgca cacagcaggt caacagcc tc ttctatcgag tcaggcgatt cgccaacgac caacttacag tgacctggac tggtcagacc ggtcggcgcg gctgaatctg tacagataat aac ttatagc taaagatggc ggggacttat taaagattct gactattggt cgatggtaag aaccaaagct ctatggtaac cacttctgct gacctcaatt gggtaatgcc tgccgatatc cttaagtgat agcagcaaac tttcgctcat agatggtagc agctctggac aaaccgtc tg gcagtcccgt gattatocag tctgtctctg tcgctgatca cgtctgtctt gctaaccgtt ggtatttctg cgtattcgtg tccatccagg cagttcaacg aacgatggcg gctggtttta ctgacattgg aaaaatgtcc gacgaaatta acttatcata ctgttgagca tctaaatcgc gcgctgtatt gcaacctggg tctgcggcag ggtggtaatg actactggta gcggtgggag tctatctcag ggtagtgata attgatgaca actaacggta ctgaccaccg gaagccatca gattccgcgg attcaggacg caggccggta ctgcagggtt ctcaaaataa ctggcttgcg t tact tc taa t tgcgcagac aactgacggt acgaaatcaa gcgtgaacgt aaacgattac acgttaacgg ctggttttac agtttgccgc cgttcgctgg aggactctaa cactggcacc aagaagttaa tagatgagaa ataacctgat cagctgttgg ctcaggtgtt ctccgggtga cgctgggcgc cttcgcaaat cttacaacgt gcggcgcaat gcgtgactaa atgcagcaac gctccatcga tcaccaacct ccgactatgc actccgtgct aa tatcaacaag tattaacagc cattaaaggc cactgaaggc tcaggcgacg atcccgtctt gctgtctaaa tattgatctg taaaggttct agcgggtact cgcgactgca taataacggc o tcatacagc aaacgctggc cgttagcaaa gggcaacctg ggccaataca gacagtaatc aaaagacgcg tgcgggagcc aacggcagtt ggcgagcatc gaccagcaat cacagaccaa cgatacaggc caaagccgaa caaattccgc gaacaacacc gaccgaagtg ggcaaaagct 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 1842 <210> <211> 1731 <212> DNA <213> Escherichia coli <400> SUBSTITUTE SHEET (Rule 26) (RCY/AU) WO 99/61458 WO 9961458PCT/AU99/00385 13 atggcacaag aaccagtctg gcgaaggatg c tgactcagg gcgctgtccg accggtacca gacgaaat tg gacggttcca aagaagattg gtggcgaata gcagcagatt gccgcagatg aatggctttg tttacttatg ggcgacactg agtgatggca t taac tcaga ttagctctga acttcgattg attgctgtta ggtttcactg gttcaggcat acactgacta ggttcaggta aaagccgcaa aagttccgtt aacaacacca accgaagtgt gcaaaagcca tcattaatac cgctgtcgag acgccgcagg cggcccgtaa aaattaacaa actcccagtc accgcgtatc tgaaaattca actcttctac ctgcggcgac ctacaggtgc tactgtctag c Lgcaggagc acacgactgc caacattcag aactcactgc atggtggtaa acaaaaatgg tac tgaatgg caggcgctgt ttggtacagt atgatgctgc ctgataacac aagcggtc ta ccaccaccga catccttggg ccaccaacct ccaatatgtc accaggtacc caacagcctc ttctatcgag tcaggcgatt cgccaacgac caacttacag tgatctggac cggtcagacc ggt tggcgcg gttgaaactg taaagcggat cattgcttac cctcgctgat cacttccaat tacgacagct tgttgaaatt taaggatggc taacggtgt t tttaacggct ttcaagcgat aattagttca agacacagct gacttctggc cactaaactg tgtagaagcg tccgctggcc tgctatccag gtctgaagcg gaaagcgcag gcagcaggtt tcgctgatca cgtctgtctt gctaaccgtt ggtatttctg cgtgtgcgtg tctatccagg cagt tcaacg aatgatggcc actggtttta ttggctgctg acagtaagtg ggtacgacta gcctataaac gagctgcagt ggtggtacta tctaagcttt ggaacactcg gttaaatcca ggtactggta gctgctctgc ggttatatct aacaaagctt tatctgcaga gatggtgatt gctctggatg aaccgtctgg cagtcccgta atcatccagc ctgtctctgc ctcaaaataa ctggcttgcg ttacttctaa t tgcgcagac agctgactgt acgaaatcaa gcgtgaacgt agaccatcac acgtgaatgg ctgcaattgg ctgggctgac t tacagccac ttaacaaaga cttacctgac cacaagacgt acattgatac cggaagcgac caattactac atgctggtac aatctgcaag ctgtaggtac cttacaccaa aagatggctc tcactaccga acgcaatcag attctgcagt ttcaggacgc aggccggtaa tgcagggtta tatcaacaag tattaacagc cattaaaggc caccgaaggc tcaggcgacc atcccgtctg gc tggcaaaa tatcgacctg ttctggttct tacccctggg taaaactaca aggcgtgaaa taataataca tccgaaagcg cgtgctgtcc aactggtaat tctgagtggt agctgataac tgaaggtacg caaaacgac t tgatgggagt cactgacggt tgtaaccaac cgc tgcaacc ccagatcgac caccaacctg cgactatgcg ctccgtgctg a 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1731 <210> 21 <211> 1380 <212> DNA <213> Escherichia coli <400> 21 aacaaatctc aacagtgcta aaaggtctga gaaggtgcgc gcaactaacg cgtctggaag gctgaaaata aatctggcaa cagaaagcaa caaattaacg gatggcaaac caattcaaga agtcttctct aagatgacgc ctcaggcttc tgaatgaaat gtactaactc aaattgaccg atgaaatgaa aaattgatgc ccggcagtga gtac tgataa aagtttatgt ttgatgcaac gagctccgcc agcaggtcag ccgtaacgcg taacaacaac tgacagcgat tgtatctgag aattcaggtt gaaaactctc cc tgat ttc t ctatactgtt gagtactgcg taagcttgca attgaacgtc gcgattgcta aatgatggta c tgcagcgta ctttcttcta caaac tcagt ggtgc taatg ggcctggacg aaatttaaag aatgtagata gatggttcac gtggctgcta tctcttctgg accgttttac tttctgttgc ttcgtgaact tccaggctga ttaacggcgt atggtgaaac gttttaatat cgacaggtac gtggcgtagt ttacgaccag aagatttagc cctgcgtatt agcaaatatt gcagaccact ttctgttcag aattactcaa gaaagtcctt catcactatc cgatggcgcg tgataattat acaggataaa cagtgatact tcaagggaat SUBSTITUTE SHEET (Rule 26) (RO/AU) WO 99/61458PC/U /085-1 14 PCT/AU99/00385 aagattgtct ggtaatggta agtactgata agtgcagggc gatctgaacg gttagtgcag ctgagcaaat caatctacca cgttctgacc accgtaaaca gtgtctaaca acgaaggtat aattaaccgc catcaggtac aattgactgc ctgccaagaa atggtaaaac cagaaggtgg ccaacccgct tcggtgcagt acctgtcttc tgtctcgtgc cgaatttaca caatgttgat tagtgcaacc aacaaaagtt aacaggaagc gataacggag tagcccgatt cgaaactatc acaaaaccgt tgcccgtagc gcagatcctg aataccggca ggtaaggctg gttgccccta gaaaataaag acgttagttg actgcttctg ctggtaaacg gacaaagcat ttcgactctg cgtatcgaag caacaagcgg ctgtcgctat t tgaattcac cgacagccct cagcgacac t ttaacggtgc gtaacaataa aagatgcagc tggc taaagt Ccatcaccaa atgctgacta gtacctctgt agatgccaaa tatttcgggg atacaaaaat atctgatctt aacttacgat agtcatgtat aaaatcgttg tgacaatctg ccttggcaac cgcgaccgaa tctggcacag 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 <210> 22 <211> 1767 <212> DNA <213> Escherichia coli <400> 22 atggcacaag aaccagtctg gcgaaggatg ctgactcagg gcgctgtctg accggtacta gatgaaattg gacggttcca aaaaaaatcg attaccaaca ggtgcctatg ttgaatgatg gctgctacag gctactgcta aaaactgtta gatggtaaga actaacgcag tctgctactg ggtacggctg tctaaatcag acagttgatt actgatacat tatgctgtaa gccgataagg aattctgcag ctggctgccc atc cagaac c gaagcgcagt gcgcagat ta caggttctgt tcattaatac cgctgtcgag acgcagcggg cggcacgtaa aaatcaacaa actccgactc accgcgtatc tgaaaattca actctgatac aagcagcaac atataaaaac gtgatgttgt gtgggtttac ataaaactgc atggttcttg tttctattgg caggtatgac gtaagggtgg gggttgatcc taagcaagga tcaactccgg tcaaagatgc ataaagatac Ctgttgatga gtaaattgac tggacgctgc gtctggattc cccgtattca tccagcaggc ctctgctaca caacagcctc ttctatcgag tcaggcgatt cgccaacgac caacttacag cgacctggct tggtcagact ggtaggtgct tctgggcctg tgtcagtgat cactaacaca tac tatcaat gacggatgtc ccgtgatgca gactacgaat tggtgttgct gac tcaagca atccctgacc tgatgacgct tgttgttctt tatcatgact agatggtgct tggtgaagtt tactaadtat cactgatacc tatcagctcc cgcagtcacc ggacgccgac cggtaac tcc gggttaa tcgctgatca cgtctgtctt gctaaccgtt ggtatctctc cgtgtacgtg tctattcagg cagttcaacg aacgacggcc aatggtttta gttactcgcg gcgctgacta aatggtaagg tccatctccg Ct tgcggcgt gatggtacgg gcttatgtag acaactaccg tttggtgaca tcagatgatg gctgatacta tcaaaggtta atcaccaaaa accgttgctg aaaccgacta accagtgcag atcgacaaat aacc tgaaca tatgcgaccg gtgc tggcaa ctcaaaataa ctggcttgcg ttacttctaa tggcgcagac aactgaccgt acgaaatcaa gcgtgaacgt agac tatc ac acgtgaatgg caggcggtac caactgatgc atactgccta gggatcc tac ctttacatgc taaaatttga atgcagcagg atttggttac cgacgta taa tactgggcac aagcaac tgg gtttcgatgc ctaaagaata attatgctgc tcggcgcgac gcacagcaac tccgttcatc acaccactac aagtgtccaa aagccaacca tatcaacaag tattaacagc cattaaaggc caccgaaggt tcaggcaacc atcccgtctg gctggcaaaa tattgacctg ttctgggacg attggtgaat cttcgcgaaa taaatataat cgc tgc tgac tgagccgggt taccgatgcc caacctgacc tgctgctgca aattggtcag catttcttac taacacgaca aggtacatca caccacttct ggtagatagc agttaacctg caaagatcct cctgggtgct caacctgtcc catgtcgaaa ggtaccgcag 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1767 SUBSTITUTE SHEET (Rule 26) (RO/AU) WO 99/61458 WO 9961458PCT/AU99/00385 15 <210> 23 <211> 1383 <212> DNA <213> Escherichia coli <400> 23 aacaaaaacc aacagcgc ta aaaggtctga gaaggcgcgc gcgacgaccg cgtttaagcg gctaagaatg gacctgcagc gatgcagtga gtcgacttcg gaaattgcta actgctgatg gatggtaacg gacgctggtc gcttctctcg gcacaagatg aaaaccgcca ggtggtgaca gctggtgcac aacggtaaaa ttccgctcct aacaccacta gaagtatcca aaa agtctgcgct aagatgacgc ctcaggccgc tgtctgagat ggactaactc aaat tgac cg acaccctgtc aaatcgattc aaaccagtgc caacaaccag aagacgataa gttactatgc tagatacacc aaaccgtttc ttaaacttca gcagcctgta gctatactga atggcacaac tgcaaaacgt ccacgtctcc ccctcggtgc ccaacctgtc acatgtcgaa gtcgacttct atcgagcgcc tttcttctgg tctgcgtatt tgcgggccag acgtaacgcc taacaacaac tgattctgac tgtatctggt tat tcaggta tcatacactg tgccgtgaat t ttgac tgc t tggtgattac tgtcgatatc gacaggtacg ctttggcact ggatgagaaa tgccgccaac tgctgacggc cgaaattgtt tgatctctcc gctggctgcc ggtgcagaac tgaagcgcag agcgcagatc gcgattgcta aacgacggta ttgcagcgtg ctgtcttcta cagactcagt ggtgcaaatg ggtctggatg actcttgggg atcactggtc tacgcgcatg gacaaggcta ccaacgacga gatgctgcaa ggcaatgata gttgatgagg aaagcagtga gtcgatgctg agtgcaacca cttgacgacg cgtctggatt tcccgtattc atccagcagg accgcttcac tttctctggc tgcgtgagtt tccaggatga ttaacggcgt acggtcagac gtttcagcgt ggggggcagg tcggtagcgg tcacagggac ccggtgaggt caagcacata cagccggtat ctgctactta ctaccggtaa ccgatgccgc cgtcaggtaa acacggtaac caatcagcca ccgcggtcac aggacgctga caggtaactc ttctaacatc gcagaccact gactgtacag aatcaaatcc gaacgtac tg tatcaatatt taaaaataat ttctgttgct tgctatcagc tacgggtaat cgctctgaaa tgacttcaca cagcactggt tgcaatcaaa agtcactgtc tgtaaaactg aacttacgat cgcaatcccg gatcgacaaa caacctgaac ctatgcgacc cgtgctgtcc 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1383 <210> 24 <211> 1197 <212> DNA <213> Esoherichia coli <400> 24 gcgctgtcga gacgctgcgg gccgcacgta gaaatcaaca aactctgatt gaccgcgtat atgaaaattc gattcttcaa aatgctatca gatactgatg cttctatcga gccaagcgat acgccaacga acaacttgca ctgacctgtc ccggtcagac aggttggtgc Ctttggggct catctatccc agtctgcagc gcgcctctct tgctaaccgc cggtatttct gcgtgttcgt ttcaatacag tcagttcaac gaatgatggt gaatggcttc gcaagccgct aatcgcagcc tctggtctgc ttcacttcta ctggcgcaga gaactgaccg gacgaaatca ggcgttaatg caaactatct tcagtttcta agcaatgaac aaat tggggg gcattaacag acatcaaagg ccactgaagg ttcaggccac aatcccgtct ttctttccaa ccatcgatct aaaactctct ctgttgatgt tttccgatac cgctaaagat tctgactcag cgcactgtct taccggtact cgatgaaatt agatggttca gaagaaaatt taatgtcagc taacttcggt gtcaagcctg 120 180 240 300 360 420 480 540 600 SUBSTITUTE SHEET (Rule 26) (RO/AIJ) WO 99/61458 WO 9961458PCT/AU99/00385 16 tcgctgcaca ggtaaagact attgatgtta ctgatcaaag aaaaactatt ttaactgatc actgagcaat gcatcagtcg atcaccaacc gccgactatg acatccttga tctatgctgc cttatgatga tttcagcaga ctgctggtga tgaatttaag ttgaaggtgc acaaattccg tgaacaacac cgaccgaagt taaagatggt ttctgttaat ttatgcgaac taaagacggc tgcggcagac tgatgttact ttcaactgag gtcttctcta caccaccaac gtccaacatg aaggcaacag gccgcttcag ggtgttgacg gcagctcaag attcttaaga gatactaatg gatccgctgg ggtgccgtgc ctgtctgaag tcgaaagcgc ctgattatgt gtaaagtaac atgccaagca gttttgtcac atggagcaac gtaaggtaac cgcttctgga agaaccgtct cgcagtcccg agatcatcca tgttcagtca cttaaacacc aacaggtcag acttcaaggc agctcttaag cacaactgcg taaagctatt cgattccgct tattcaggac gcaggca 660 720 780 840 900 960 1020 1080 1140 1197 <210> <211> 1674 <212> DNA <213> Escherichia coli <400> atggcacaag aaccagtctg gcgaaggatg ctgac tcagg gcgc tgtccg acagggacta gacgaaattg gatggctcga aagaaaattg gttgataatg gctgatgcta acagcggctg gatactggct tcttacactt tacttaggcg attgccaaag ggcaacttaa ctgtttgctg gatcaaactg tcaaccttaa gctggcgcag actgaagatg gacggtgaag accaagtctg gacaaattcc ctgaacaaca gcgaccgaag c tggcaaaag <210> 26 <211> 1365 tcattaatac cgctgtcgag acgccgcagg ctgcacgtaa aaatcaacaa actccgattc accgcgtatc tgaaaattca actctgatac ccaaggcgac atggcaaaat atgtattggg taggagtcgc ttgatgctac catctaacac atggctccat ccaaaaatac gtgctcagga ctggtaccgt ataatggttc ttcagacagg acgaaaccgt gttctactgt aagcaaccac gctcctccct ccactaccaa tgtccaacat ccaaccaggt caacagcctc ttctatcgag tcaggcgatt cgccaacgac caacttacag tgacctggac tggtcagacc ggtcggcgcg gc taaatctg tggcaaagat cacttatacc Caaagcggct tgctgatgcc tggtgttgcc cggtaaaatt caccgatacc cgcgaatttg tgcaacgatc tgatttcaaa ctatacagcc tggcgcagat taccgcgacc ctataaagct tgaccctctg cggtgccgtt cctgtctgaa gtcgaaagcg accgcagcag tcgctgatca cgtctgtctt gctaaccgtt ggtatttctg cgtattcgtg tccatccagg cagttcaacg aacgatggcg gctggtttta cttactgatg aaagacaccg gctggcgata tcgacttaca aaggcggatg aatatcggtg aatggcgatg ggggctgctg accttcgata ggcgcgtcta aacgtaggtg gtgtataaag tactacggtt gctgatggtt aaagcccttg caaaaccgtc gcgcagtccc cagatcattc gttctgtctc ctcaaaataa ctggcttgcg ttacttctaa t tgcacagac aactgacggt acgaaatcaa gcgtgaacgt aaacgattac acgtgaatgg ctggttttac ttactaaatt gcattaccta cctacaatgc C tggaacggc gtaccgagca cgctgtatct ataaagcaac gcggcatgac tttctgctga gtaaggctta ataccactgg ttgctgatgg ccatcactaa acgacgcaat tggattccgc gtattcagga agcaggccgg tgc tgcaggg tatcaacaag tattaacagc cattaaaggc cactgaaggc tcaggccact atctcgtctg gctgtctaaa tattgatctg tgctggctct ggcaagcgca cgacaaagcg tgcgggcact agccaataag actgaaaggg agaagttaac cgatagtacc tgtagataaa agctaaattc tgcaatggca tgccgtaacc cgcactgacg taaagtttct agatgcgact cagccagatc cgtcaccaac cgccgactat taactccgtg ttaa 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1674 SUBSTITUTE SHEET (Rule 26) (RO/AU) WO 99/61458 WO 9961458PCT/AU99/00385 17 <212> DNA <213> Escherichia coi <400> 26 aacaaatc tc aacagtgc ta aaaggtctga gaaggtgcgc gcaactaacg cgtc tggaag gccgaaaata aatctggcaa cagaaagcaa caaattaacg gatggtgacg aaagtcggtg aaagatgcct gatggcagtg attgatgcga gcaggtcagt ttaaataacg agcgctgatg ggtggtagcc ccgc tcgaaa gcagtacaaa tcttctgccc cgtgcgcaga agtcttctct aagatgacgc ctcaggcttc tgaatgaaat gtactaactc aaattgaccg atgaaatgaa aaattgatgc ctggcagtga gtactgataa caatttttgt gtacaggtat caattaaata gtaacggcac cagggaagga tcac tacaac ctaaaaaagt gtaagacagt cgattctggt ccatcgacaa accgtttcga gtagccgtat tcctgcaaca tagctctgct agcaggtcag ccgtaacgcg taacaacaac tgacagcgat tgtatctgag aattcaggtt gaaaactctc cc tga t ttc t ctatactgtt tagcgctacc tgatgcgact ccaaggtatt tctaaccgct cgcaacatta taaggttgaa gggtagttct aactgggctt aaaagaagat ggcattggct ctctgctatc cgaagatgct agcgggtacc attgagcgtc gcgattgcta aatgatggta c tgcagcgtg ctttcttcta caaactcagt ggtgctaatg ggcc tggacg aaatttaaag aatgtagata gatggttctc gggcttgcaa actttcacca aatattgatg aaaacgtctg aacaaagccg ttagttgtaa ggcaaaacta gcagcaaaat aaagttgaca accaaccttg gactacgcga tctgttctgg tctcttctgg accgttttac tttctgttgc tacgtgaac t ttcaggcaga ttaacggcgt atggggaaac gctttaatat cgacaggtac gtggagcagt tgactactaa aagccgcagt acaaaggcac gcaaagatgt atcctgttta ctacagcatc atggcgctga tgtatctgag cgttgcaatc atctgcgttc gcaacaccgt ccgaagtgtc cgcag cctgcgtatt ggcaaatatt gcagactact gactgttcag aattactcaa gaaagtcctt catcactatc cgatggcgcg tgataattat tcaaaatgag gagtgataca ttctttagct tgatgcattt aacctttact caaaaatagt ggatctggac ttatgaagtt caaatcagaa tactaccaac tgacctcggt aaacaacctg taacatgtc t 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1365 <210> 27 <211> 1740 <212> DNA <213> Escherichia coli <400> 27 atggcacaag aaccagtc tg gcgaaggatg ctgactcagg gcgctgtccg accgggacta gacgaaattg gacggttcaa aagaaaattg gtggctaata actgctgatg gctgcagatg ggttttggat tatagtgcaa tcattaatac cgctgtcgag acgccgcagg ctgcacgtaa aaatcaacaa actccgattc accgcgtatc tgaaaattca actctgatac c tgcagcgac ctaatggtac taattgcgag cgccaacagc ctattgcagc caacagcctc ttctatcgag tcaggcgatt cgccaacgat caacttacag ggatc tggac tggccagacc ggttggtgcg gctggggctg taaatctgat agttacctat tttggctaat tacagattat tggtacaaat tcgctgatca cgtctgtctt gctaaccgtt ggtatttctg cgtatccgtg tccattcagg cagttcaacg aatgacggcc agtgggttta ttggcagcag actgttggcg aacgcaaaag acatacaaca tctggtgata ctcaaaataa ctggcttgcg ttacttctaa ttgcacagac aactgacggt acgaaatcaa gcgtgaacgt agactatcad atgtgaatgg ctcaactctt caggcctgaa ttaatgccac gcgctacagg gtaacagtgc tatcaacaag tattaacagc cattaaaggc cac tgaaggc tcaggcttct atcccgtctg -actggcgaaa gattgatctg tagcggggct ggctccaggt aacatctaca aattgcaaat cgattttaca tcagttacaa SUBSTITUTE SHEET (Rule 26) (RO/AU) WO 99/61458 18 WO 9961458PCT/AU99/00385 tccttcctga tcaattgacg tttattgacg cttgatgcac attacaactg ggtgcaatca tccacaggat gatagtaaag gctgatggtt gtgaccaatg gctgaaacta tccatcgaca accaacctga gactatgcga tccgtgctgg caccaaaagc ttgtattggc tagatggtaa tgactaaaaa aagatgaaac c tgtagcaaa tcacagttga gtatagtaca cactgactac gttcaggtaa aagctgcaac aattccgttc acaacaccac ccgaagtgtc caaaagctaa gggcgatact tagcgacggt cctcactcaa ctggcataca aaccttcact tgcaagaatg tgttggagct acaacacaca cgataataca agcagtctat caccgccgat ttccctcggt tactaacctg caatatgtcg ccaggtaccg gctaacttaa aaaattaccg aacaatgctg acaggcacac ctggctggcg agtgc tgagt actggtacca ggtacaggtt accaatctgt gtttcagcgg ccactgaaag gcggtgcaaa tc tgaagcgc aaagcgcaga cagcaggttc acgttaaaat cgaaagatgg ggactgtcaa cgagtgccgt gtac tgatgc ctcttcaatc gcgcaggcga ttgaagacgc ttttgcaaaa atggtaattt ctctggacga accgtctgga agtcccgtat tcatccagca tgtctctgct tggttctacg ttcagaacta agcagccact atctacggta tactacttct ggcaac taag tattaaagtt ttacaccaaa agacggaact tactactgac agcgatcagc ttccgcagtc tcaggacgc t ggccggtaac gcagggttaa 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 <210> 28 <211> 1233 <212> DNA <213> Escherichia coli <400> 28 aacaaaaacc aacagcgcta aaaggtctga gaaggcgcac gccactaccg cgtctcgatg gcaaaagata gacctgcaaa gctctcgaaa gtgaagatgg ctgcacaacg aagagctatg acatataacg gttggtgctg gttgctaatg acaattgatg ggtgctactg ctggacaaag cgtctgagct tcccgtattc atccagcagg agtctgcgct aagatgacgc ctcaggccgc tgtctgaaat gtactaactc aaattgaccg acaccatgaa aaatcgactc ctagcgaagc atgcgtctgt taactaaagg cagcatctgt acgcagcaaa .atgcaaacaa actcattagt gtgatggtag ctgcaacatc ctatcgcatc ccgctgtaac aggacgccga caggiaactc gtcgacttct tgcgggccag acgtaacgcc caacaacaac tgattctgac cgtatccggt gattcaggtt ttctactctt gatcactcag tc tgaccgat tggtgtcgca tga tgcggga tggtgttacg tgatgcagtt caatgctaat ccttggagct agagttcgct tgttgataaa caacctgaac ctatgcgacc cgtgctgtcc atcgagcgcc gcgattgcta aacgacggta ttgcagcgtg ctgtcttcaa cagactcagt ggtgcgaacg ggtttgaacg ttgccgaacg cttaacatta acgtctactt ggtacagtaa aatgccaccc ggttttgtta ggcgctgctg aaccaggcta ggtgcttcaa ttccgttctt aacaccac ta gaagtgtcca aaa tctcttctgg accgcttcac tctctctggc ttcgtgagct t ccaggacga tcaacggcgt atggtcagac gtttctccgt gtgcgaatgc ctgatgcttc atgttgttca aactgaaiaa agattggtag ccgtgcaggg gcgctgcagc aaattgaact ccaacgatcc Ctttgggggc ccaacctgtc acatgtcgaa tctgcgcatt ttctaacatc gcagaccac t gaccgttcag aatcaaatcc gaacgtactg tatatccatc ttctaaaaat accaatcgct cgctgtttcg gtatggcgat agccgacgta tc tggttcag gaaaaactat aactagagtt tagccaaaat actgactctg ggtacagaac tgaagcgcag agcgcagatc 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1233 <210> 29 <211> 1713 <212> DNA <213> Escherichia coli SUBSTITUTE SHEET (Rule 26) (RO/AU) WO 99/61458 WO 9961458PCT/AU99/00385 19 <400> 29 atggcacaag aaccagtctg gcgaaggatg ctgactcagg gcgctgtccg accggaac ta gatgaaattg gatggctcga aaaaagatcg gttgataatg gtcgtcagtg gctaccgacg aacaatggat tattcatttg gctgatgtgc ggaagcgctc gctttatata gcaacgttgg acattcggca ggcgcaacgg gctaacggcg ggcgctaccg acagaaactt tacaaagcgg gatccgctga ggtgcggtgc ctgtctgaag tcattaatac cgctgtcgag acgccgcagg ctgcacgtaa aaatcaacaa actccacctc accgcgtatc tgaaaattca actcttctac ctaaagccac gcaacagcac tgctggcaag taggcgtagc acgcaaccgc tgaaagcc ta aggacgttaa ttggttctga acggtatttt gcggcatgac tatccgccga cacagtcttt taaatattgg atcacgaatt ctgacggttc aagcgctgga agaaccgtct cgcagtcccg caacagcctc ttctatcgag tcaggcgatt cgccaacgac caac ttacag tgacctggac cggccaaacc ggtcggcgca attgaagctg tgaagcagat ctggactaaa cgt tagcggc ggcttctact acttaccaat tgcagcaaac aat tgccagc cggcaacctg caacggtgcg Cgttgatttc agatatggac tgacgttgcc tgctgatggt tgctaacggc tctgaccact cgatgctatc ggattccgca cattcaggac gcaggccggt gctgcagggt tcgctgatca Cgtctgtctt gctaaccgtt ggtatttctg cgtattcgtg tccattcagg cagttcaacg aatgatggtg accagcttca c tgaccgctg tctactgtta ggcagcacta gcatatacct ggcgatggta ggtgataata gatggcaccc actaaaaacc aatggtaatg ac ccaggc ta actgcgttaa gc tggtgggg gaactgacga aatattctgg gaagc tac tg gcatccgtag gtcaccaacc gccgactatg aactccgtgc taa ctcaaaataa ctggcttgcg ttacttctaa t tgcacagac aactgacggt acgaaatcaa gcgtgaacgt aaaccatcac atgttaacgg cgggcttctc ctacctttaa ttagcggtta acaacgcaac ctggggccac cggctcagat tgac tgacgt aggccggcgg cagcagttga gcaaaaaagt ctgggcaggc cagtaaccgc ctgcgaccaa atgatgacgg gtaaatccga acaaattccg tgaacaacac cgaccgaagt tggcaaaagc tatcaacaag tattaacagc cattaaaggc cactgaaggc tcaggcgacg atcccgtctt actgtcaaaa gattgatctg taaaggcgct Ccaaggtgca tgcagcaaca taccggtaca cagcaagtct cactaaagtt ctccatcggc caatggtgat tccagatgcg tgcgaagatt ggatattaag ttataccgta tactacaggt caagactgtc cgcggctctg agtgaccacg ctcctccctc cactaccaac gtccaatatg caaccaggta 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1713 tcgaaagcgc agatcatcca ccgcagcagg ttctgtctct <210> <211> 1668 <212> DNA <213> Escherichia coli <400> atggcacaag aaccagtctg gctaaggatg ctgac tcagg gcgctgtccg accgggacta gacgaaattg gacggttcaa aagaaaat tg gttgctaata tcattaatac cgctgtcgag acgccgcggg C tgcacgtaa aaatcaacaa actccgattc accgcgtatc tgaaaattca actcagatac ctgcagcgac caacagcctc ttctatcgag tcaggcgatt cgccaacgac caacttacag ggatctggac tggccagacc ggttggtgcg gctggggctg taaagatgat tcgctgatca Cgtctgtctt gctaaccgtt ggtatttctg cgtatccgtg tccattcagg cagttcaacg aatgacggcc agtgggttta ttggtcgctg c tcaaaataa ctggcttgcg ttacttctaa ttgcgcagac aactgacggt acgaaatcaa gcgtgaacgt agac tatcac atgtgaatgg catcagtttc tatcaacaag tattaacagc cattaaaggc cactgaaggc tcaggcttct atcccgtctg actggcgaaa tattgatctg tggcggggc t agctgcggta SUBSTITUTE SHEET (Rule 26) (RO/AU) WO 99/61458 20 WO 9961458PCT/AU99/00385 ggtaatgaat agtctcacag gcaactggag tcaacagcgg tccgt tgaaa gcaaaagacg ggtactttag gctgtacaat gactttggaa gatgcacttc acagatggag ttaactactg tctggtaaag gccgaaacca ttccgttctt aacaccacta gaagtgtcca aaagctaacc acactgtctc atggtgcgac atgcttataa cagaactcca t tggtggcac ggtctaaact aagaagc tac ctactattac caacaaaaac tttccgccag ttgttaaatc acaataccac Ctgtgtatgc ccgccgatcc CCCtcggtgc ccaacc tgtc acatgtcgaa aggtaccgca tgctggcctg agtaac tgcg attcaatcaa atcttacctc caagcaggat ttatattgac cctcaatggc tactgcggat tgctggggct taaagcgact tggtggcaat aaaatattat tgatgcaaca cctgaaagct ggtgcaaaac cgaagcgcag agcgcagatc gcaggttctg tcgaaatcaa gctggtgtaa gcaaacaaca acgcctaagg gttgttctgg accacaggga ttagctttca ggaacttcaa attaatgtca gggtttactt gacgt ttata ttacaagatg ggaaaactaa ctggacgaag cgtctggatt tcccgtattc atccagcagg tctctgctgc ctgctgctga gcaatggttt cttttactta cgggggatac c tagtgatgg atttaaccca accactctgg tagttctagc caggagcagt Ctggcactta acaaagctga acgggtctgt ctactgacgc cgatcagctc ccgcggtcac aggacgccga ccggtaactc agggttaa tgttattgct 660 tgctgcaggg 720 caataccacc 780 cgcaactttc 840 caaaatcaca 900 aaacggtgga 960 tccagccgct 1020 aggttctggc 1080 gatcagtgct 1140 taccgtaggt 1200 cgggacggga 1260 aactaatggt 1320 tgaaactaaa 1380 catcgacaaa 1440 caacctgaac 1500 ctatgcgacc 1560 cgtgctggca 1620 1668 <210> 31 <211> 1713 <212> DNA <213> Escherichia coli <400> 31 atggcacaag aaccagtctg gcgaaggatg ctgactcagg gcgctgtccg ac cggtac ta gatgaaattg gacggttcaa aaaaaaatcg gttgataatg gttgtcagtg gctaccgatg aacaatgggt tattcatttg gctgatgttc ggtagcgctc gctttataca gcaacgttgg accttcggca ggcgcgacgg gctaacggcg ggagcgaccg acagaaactt tcattaatac cgctgtcgag acgccgcggg ctgcacgtaa aaatcaacaa actccgattc accgcgtatc tgaaaattca actcttctac caaaagccac gcaatagcac tgctggctag taggcgtagc acgcaaccgc tgaaagccta aggaagttaa ttggtgctga acggtatttt gcggcatgac tatccgccga cacagtctta taaatattgg atcacgaatt caacagcctc ttctatcgag tcaggcgatt cgccaacgac caacttacag tgacctggac tggtcagacc ggtgggcgca actgaagctg tgaagcagat ctggactaaa cgttagtggc ggcttctact acttactaat tgcagcaaac aattgccagc cggtaacctg caacggtgcg cgttgacttc agatatgaac tgacgttgcc tgc tgagggt tgctaacggc tcgctgatca cgtctgtctt gctaaccgtt ggtatttccg cgtatccgtg tc cat ccagg cagttcaatg aatgatggtg accagcttca ctgaccgctg tctactgtta ggcagcacta gcatatacct ggtgatggta ggcgataaca gatggtaccc acgaaaaacc aatggtcatg ac ccaggt ta actgcgttaa gctgatggtg gaactgacga aatattctgg ctcaaaataa ctggcttgcg ttacttctaa ttgcgcagac aactgacggt acgaaatcaa gcgtgaatgt aaaccatcac acgtcaacgg Cgggc ttctc ctacctttaa ttagcggtta acaacgcaac ctgcgggctc cggctcagat tgacggatac aggccggcgg atgcagttga gcaacaatgt ccggtcaggc cagtaactgc ctgcggccaa atgatgacgg tatcaacaag tattaacagc cattaaaggc caccgaaggc tcaggccact atctcgtctt gttgtccaaa gattgacc tg taaaggcgct ccaaagtgca tgcagcaaca tgctggcaca cagcaagtct aactaaagtt ctccatcggt taatggcgat cccagccgcg tgcgaagatt ggatattaag ttataccgta tactacaggt caagac tgtc cgcggctctg 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 SUBSTITUTE SHEET (Rule 26) (ROIAU) WO 99/61458 WO 9961458PCT/AU99/00385 21 tataaagcgg gatccgctga ggtgccgtgc ctgtccgaag tcgaaagcgc ccgcagcagg c tgacggc tc aagcgc tgga agaaccgtct cgcagtcccg agattattca ttctgtctct tctgaccact cgatgctatc ggattccgca tat tcaggac gcaggcaggt gctgcagggt gaagctacag gcatccgtag gtcaccaacc gccgactatg aac tccgtgc taa gtaaatctga acaaattccg tgaacaacac cgaccgaagt tggcaaaagc agcgaccacg ttcttccctg cac taccaac gtccaacatg taaccaggta 1440 1500 1560 1620 1680 1713 <210> 32 <211> 1188 <212> DNA <213> Escherichia coli <400> 32 aacaaaaacc aacagcgcta aaaggtctga gaaggcgcac gcgacgaccg cgtctggatg gctaaaaacg gacctgcaga gcac tgaaac aaactgagct gtacagaccc tactcagtat tataccgata ggagctgatg gctgatgctg gcaattggcg gatccactgg ggggcggtgc ctgtccgaag tcgaaagcgc agtctgcgc t aagatgacgc ctcaggccgc tgtctgaaat ggactaactc aaat tgaccg gttctatggc aaatcgactc tgagcgattc ctgttgcctc cagctggcgc ctgttgaaga ccgctaatgg cattgggtgc gcgcgctggt atattgctaa ctctgctgga agaaccgtct cgcagtcccg agatcatcca gtcgacttct tgcgggccag acgtaacgcc caacaacaac tgattctgac tgtttccggt gattcaggtt ttctactctg tatcactcag ggctctgggt agcaacagct tagctccggt cgttactacc tgctgtaggt taactccaag taaagcgaat caaagctatc gagctctgct tattcaggac gcaggcgggt atcgagcgcc gcgattgcta aacgacggta ttgcagcgtg ctgtcttcta cagacccagt ggcgcgaatg ggcctgggcg gttggtgcga gtagacgcaa aactatgttg acagttacgc ggttccatga tatgtcaccg aatgctgctg gctaacattt gcatctgttg gtaaccaacc gccgactatg aactccgtgc tctcttctgg accgcttcac tctctctggc tgcgtgagtt ttcaggacga tcaacggcgt atgggcagac gcttctccgt gtggttcac t gcactctgac tctcttctgg tgaacaccac ctggtaagta tacagggaca gtagtcagaa acactggaac ataaattccg tgaacaacac cgaccgaagt tgtc taaa tctgcgcatt ttctaacatc gcagaccac t gactgttcag aatcaaatcc gaacgtgctg catcaacatc atctaacaat ggcagatgtg tctgcacaac ttctgacaac tgatataggt cgttaaagtt aaacttcaaa tgttacttct ctcttctgca ttcttctcta cactaccaac gtccaacatg 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1188 <210> 33 <211> 1638 <212> DNA <213> Escherichia coli <400> 33 atggcacaag aaccagtctg gcgaaggatg ctgactcagg gcgctgtccg accgggacta gacgaaat tg gacggttcga tcattaatac cgctgtcgag acgccgccgg ctgcacgtaa aaatcaacaa actctgattc accgcgtatc tgaaaattca caacagcctc ttctatcgag tcaggcgat t cgccaatgac caacttacag ggatctggac cggtcagacc ggttggtgcg tcgctgatca cgtctgtctt gctaaccgtt ggtatttctg cgtattcgtg tccattcagg cagttcaacg aacgacggcc ctcaaaataa ctggcttgcg t tact tc taa ttgcacagac aactgacggt acgaaatcaa gcgtgaacgt agac tatcac tatcaacaag tattaacagc cattaaaggc cac tgaaggc tcaggcttct atcccgtctc actggcaaaa tattgatctg 120 180 240 300 360 420 480 SUBSTITUTE SHEET (Rule 26) (ROIAU) WO 99/61458 WO 9961458PCT/AU99/00385 22 aagaaaattg aaggcaagtg acttcatctg tctgatattc cttgctgata tatacggctg aaaacgacag tcgggcaata actaaaaacc gatgcgaaag gctgaaggta acgggcattg actacaaatg tactactcgc gctgatggtg cttgacgacg cgtctggatt tcccgtattc atccagcagg tctctgctgc actctgatac tcgcggcgac acggcagcac ttgcggcgct a tgcgac at c acggtgtaaa ctgcatcagt ttacttcaag aggccggcaa gtactgctgt atacaggtac catattccgc gtttcctggc aagccgatgg tctacagcac caatcagc tc ccgcggtcac aggacgccga ccggtaac tc agggctaa gc tggggc tg agctgacgga tgcatatact taagactggc gacaacttat ccaaacgaat tactattggt tgatggcgat cccgaaaaaa taaaacaacc tgtaaaaatt caacaccggt ggggga tgac c acgg tc a g agagaataaa catcgacaaa caacc tgaac ctatgcgacc cgtgctggca agtgggttta atggttaaag aaaactacag gataaaatta acttataatg gctgcagcaa gggacagcac caactttatc gcaaccgttt atcaagacag gaaggtgcta gggaatactt ttaacccagg aatagcgcag acatcgaaga ttccgttcat aacaccacta gaagtgtcca aaagctaacc acgtaaatgg acggatatat caaatactgc ccgcaacagg caaccagcaa atctcatacc agaatgtaaa tggat tcaac c tgggc ttc t aggc tggtgt ctgtttcagc atgctgttgc atgc tcaaac gcaaagaaat cgtccgatcc ccttgggtgc ccaacctgtc acatgtcgaa aggtaccgca tagcgcagat caaagggtta agcaaaagga tgcaaatagc taccttctcc tgcagcaggg tattgatgat aggtaacc tg cggaaatacg aacagttaca atctgcattt cgcaaataat tgtttcaacc ctataaagac attggctgcg tatccagaac cgaagcgcag agcgcagatc gcaggttctg 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1638 <210> 34 <211> 2145 <212> DNA <213> Escherichia coli <400> 34 aacaaatctc aacagtgcta aaaggtctga gaaggtgcgc gcaactaacg cgtctggaag gctgaaaata aatctggcaa cagaaagcaa gatgttggcg cttattgata attttgagaa tatgccgctc gggattcata ggctcataag tcttccggag agtcccactg aggttaccga cataatgcgg gtgcgtaccg agagcagaga agtcttctct aagatgacgc ctcaggcttc tgaatgaaat gtactaactc aaattgaccg atgaaatgaa aaattgatgc ctggcagtga gtgatgctta gtgttttatg cgacagcgac aattcgctgc cagcggccag acgccccagc cctgtcatac ttcgtccatt ctgcggcctg gcagttgccc ggttgagaag tagcgctgat gagctccgcc agcaggtcag ccgtaacgcg taacaacaac tgacagcgat tgtatctgag aattcaggtt gaaaactctc cc tgatt tc t tactgttaac t tcagataat ttccgtccca gtatatcgct ccatccgtca gtcgccatag gcgtaaaaca tccgcgcaga agttttttaa ggcatccaac cggtgtaagt gtccggcggt attgaacgtc gcgattgcta aatgatggta ctgcagcgtg ctttcttcta caaactcagt ggtgctaatg ggcctggacg aaatttaaag gtagatagcg gcccgatgac gccgtgccag tgctgattac tccatatcac tgcgttcacc gccagcgctg cgatgacgtc gtgacgtaaa gccattcatg gaac tgcagt gcttttgccg tctcttctgg accgttttac tttctgttgc tacgtgaact tccaggctga ttaacggcgt atggtgaaac gttttaatat cgacaggtac gagc tgggta tttgtcatgc gtgctgcctc gtgcagcttt cacgtcaaag gaatacgtgc gcgcgattta actgcccggc atcgtgttga gccatatcaa tgccatgttt ttacgcacca cctgcgtatt agcaaatatt gcagaccact gactgttcag aattactcaa gaaagtcctt catcactatc Cgatggcgcg tgataactat atgactccaa agctccaccg agattcaggt cccttcaggc ggtgacagca gcaacaaccg gccccgacat tgtatgcgcg ggccaacgcc tgattttctg tacggcagtg Ccccgtcagt 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 SUBSTITUTE SHEET (Rule 26) (RO/AU) WO 99/61458 WO 9961458PCT/AU99/00385 agctgaacag tcatacacta aatgatattt gctggtacag gataaattca aatggtgtat gacaaaaaag acgacaacca aagaaaacag aaaacgataa ggtggtagoc ccgctcgaaa gcagtacaaa tcttctgccc cgtgcgcaga gagggacagc aatcagtaag ttgttagtgc ggattgatgc cgtttaatgg attctgcaga cttctttgat aagttgataa gaagcacgt t cggagactgc cgattctggt ctatcgacaa accgtttcga gtagccgtat tcctgcaaca tgatagaaac ttggcagcat agcagatggt tacagcactc agttgaattc aat tgatggt tacgagtgag caaggctgcc agttgttaac ttctggtaac aaacgaagat agcat tggct Ctctgctatc cgaagatgct agcgggtacc 23 agaagccac t taccgcggag tcactgacaa gcagcagcgg acaacaacaa aagtcagtga acagtttaca acactttccg ggtgcaactt aataaagtca gcagcaaaat aaagttgaca accaacc ttg gactacgcga tctgttctgg ggagcacc tc c tgt taaaga ctaaatctga ctaagaataa ctgcagcgga catttactgt aaaatagcgc atcttgatct acgatgttag tgtatctgag cgttgcaatc atctgcgttc gcaacaccgt ccgaagtgtc cgcag aaaaacacca tactacaggg cacaaacata agcacagaat tggcaatggg gacagatgct tggcctttat caatgcagct tgcagatggt caaatcagaa taccaccaac tgacctcggt aaacaacctg taacatgtct 1320 1380 1440 1500 1560 1620 1680 1740 1800 1860 1920 1980 2040 2100 2145 <210> <211> 1587 <212> DNA <213> Escherichia coli <400> aacaagaacc aacagcgcga aaaggcctga gaaggcgcgc gcaaccaccg cgtctggacg gcaaaagacg gacc tgaaga gcagcggttg ggcgttgtgg gcggcaactg ggtactggta aaatcctata ggtacttttg gtaaacatcg tccactggta gtccttactg gtcacccttg gcaatgacta gctgtagcta gctagtggta gcgaatggca ctgactacag gacgcaatca gattccgcag attcaggacg agtctgcgct aggatgacgc ctcaggctgc tgtccgaaat gtaccaactc aaattgaccg gttccatgaa agattgactc ataatgctaa attcaaatgg cagtaaacgt atggtttagg cctttgattc gtacagatac c taaaga tgg atttgactaa gtgctaattc ataaagtgaa atgagttgac ctaataacac aattaac tac atatctatga aaac tacaag gccagatcga tcaccaacct ccgactatgc gtcgagttct cgcaggtcag acgtaacgcc caacaacaac ccagtctgac cgtatccggt aattcaggtt ttctacgctg agcgacggat taatagtact actagcagca .gattgctgca tacgggggct gaatactgca gaaaattact gaacggctct agttgatgat cagcactgta agg taaggcc agtaaaaacg tgatgacaaa tgataaaggc taaatcagaa caaattccgt gaacaacacc gaccgaagtg atcgagcgtc gcgattgcta aacgacggta ttacagcgtg ctggactcta cagacccagt ggcgcgaacg aaactgactg gcaaatctga tggactaaat gttaaagatg acaagtgctt gcagtagctg aaaatcacca gatactgatg gatactttaa acaaggattg gatatcactg tataccgtag actgctgatg gccactgtta gctgctgttt gctacagcta tcatccctgg actaccaatc tccaatatgt tgtcttctgg accgttttac tttctgttgc tgcgtgaact tccaggacga tcaacggcgt atggccagac gttttaacgt ctaccgccgg caactacgac gcagcacaat atacatatca gtgccgcgtc tcgatggttc gtaaagcttt ctcaggcaac acttcgatag gcgcatctat taaatggtgc ctaaaaatgt cagaaactta atgcggcggc acccgctggc gtgctatcca tgtctgaagc cgaaagcgca cttgcgtatt ttctaacatt gcagaccacc gaccgttcag aattaaatcc gaacgtactg catcactatc gaatggcaaa ttttacacaa taatttcgat caattacacc cgatagcact cagcctgcaa tgctcaagaa atatatcgat attgaatgat cggcatgtct ttcagccgct agaatcttac ttatgttgat tcatgaattt ggatggttct cgctctggac gaaccgtctg gcagtcccgt gatcatccag 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 SUBSTITUTE SHEET (Rule 26) (ROAU.) WO 99/61458 WO 9961458PCT/AU99/00385 24 caggcaggca actccgtgct ggcaaaa 1587 <210> 36 <211> 1245 <212> DNA <213> Escherichia coli <400> 36 aacaaaaacc aacagcgcta aaaggtctga gaaggcgcac gccactaccg cgtctcgatg gcaaaagatg gatttgcaga gcagtatctg gtaaccatca ttaagtctgc aatggcggaa gcaaatgtaa ttagttcagg aaaagctttg caaccgggaa aactctgtag ccactgactc gcggtacaga tctgaagcgc aaagcgcaga agtctgcgct aagatgacgc c tcaggccgc tgtctgaaat gtactaactc aaattgaccg gctcgatgaa agattgattc ttggtgatgc agtttgatga ataacctcaa aatcttacgc cctacagcga ttggtgcaga gcattgatga catctgggac acgctgatgt tgctggacaa accgtctgag agtcccgtat ttatccagca gtcgacttct tgcgggccag acgtaacgcc caacaacaac tgattctgac cgtatccggt aattcaggtc ttctacttta tattactcaa ttcagtaaaa agatgaaaat tgctacagtc tgtcgcaaac ttctaccggt cgccgccttg aacagttgtc aacggcttcc agctatcgca ctccgctgta tcaggacgcc ggcaggtaac atcgagcgcc gcgattgcta aacgacggta ttgcagcgtg ctgtcttcaa cagactcagt ggtgcaaatg gggttaaatg ttgcctggcg actgatttaa ggtaat ttaa gctgccaatg ggtattgata acgccaaaag aagaataaca gcagcgtcaa actgaattca tctgttgata accaacctga gactatgcga tccgtgctgt tctcttctgg accgc ttcac tctctctggc ttcgtgaact tccaggacga tcaacggcgt atggtcagac gtttttctgt agacggcagc aactgaccga ctaaccagta gtaatgt tac ccgcaacgca cattcgtgtc ctggtgatgc ttcatctgag Caggtgcttc aattccgttc acaacaccac ccgaagtgtc ccaaa tctgcgcatt ttctaacatc gcagaccac t gaccgttcag aatcaaatcc gaacgtac tg aatcagcatt ttccaaaaat cgatgcacca tgcttcaggg tgttgtacag gctgaacaaa gtcaggccag tgtccaaggt taccgctact tacgggcaaa aaccaacgat ttctttgggg caccaacctg caacatgtcg 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1245 <210> 37 <211> 1185 <212> DNA <213> Escherichia coi <400> 37 aacaaaaacc aacagcgcta aaaggtc tga gaaggcgcgc gcaaccac tg cgtctggatg gctaaaaacg gacc tgcaga tccctgaaac gtggacctga gaagttcagg aactatgctg agtctgcgct aagatgacgc ctcaggctgc tgtcagagat gtaccaactc aaattgaccg gttctatggc aaatagactc tgagcgattc gcgcagtagc actctgctgg tgtctgtaga gtcgacttct tgcgggccag acgtaacgcc taacaacaac tgattccgat Cgtctctggt aattcaggtt ttctactctg tatcactacg aactaaactg tgacggtact cgcggcctct atcgagcgcc gcgattgcta aatgacggta ttgcagcgtg ctctcttcta cagacccagt ggcgcgaacg ggtctgagcg atcggcaata ggcgtgaatg ggtaccttcg ggtgcagtta tctcttctgg accgcttcac tttctctagc tgcgtgagtt ttcaggatga ttaacggcgt atggccagac gcttctctgt ctactgctgc caagcaccct ttgtttcttc acctgaacac tctgcgcatt ttctaacatc acagacagcg gaccgtgcag aattaaatct gaacgtactg tatctctatc ttctcagaac atcgaagaac gagcctgcac tggcagcgac cactgacgtc SUBSTITUT SHEET (Rule 26) (RO/AU) WO 99/61458 WO 9961458PCT/AU99/00385 25 acctatgatg gtaacttctg caggc tggtg gctgttgata actgcaactc cgttcttctc accaccacta gtgtcaaata acgc tac taa acgccaacgg cgaccggtgt ccggtactct agaacccact tgggtgcggt acctgtctga tgtctaaagc tggtgttact tgcagctgtt tgacgttctg gcaac tgagc ggcactattg acagaatcgt agcgcagtcc gcagatcgtt ggcgcgactc ggttacgtaa gcgaacagcg ggtactggtg gacaaagcta ctgagctctg cgtattcagg cagcaggccg agaacggtca ccattcaggg gtgttgcagc caactactga tcgcttctgt ctgtaaccaa atgccgacta gtaac gctgatcaaa taaaaactat tccaactaca gctgaaaggt tgataaattc cctgaataac tgcgaccgaa 780 840 900 960 1020 1080 1140 1185 <210> 38 <211> 1383 <212> DNA <213> Escherichia coli <400> 38 aacaaatctc aacagcgcaa aaaggtctga gaaggtgcgc gcaactaacg cgtctggaag gctgaaaata aatctggcaa cagaaagcaa gatgttggcg aataaagatg gtatccggtg aaaggc tcc a gctaatggta gacagtaatg aacagtgcgg ctggatctta aatgtcagcg tatctgagca ttgcaatcta ctgcgttctg aacaccgtaa gaagtgtcta cag agtcttctct aagacgatgc cccaggcttc tgaatgaaat gtactaactc aaattgaccg atgaaatgaa aaattgatgc caggcagtga gtaaaactta tttttgtaag aaagtattga ttgaatacaa aaggtgtttt cacccacggg gccagttcac atgcagccaa cagatggtaa aatcagaagg ccaccaaccc acctcggtgc acaacc tgtc acatgtctcg tagctctgct agcaggtcag ccgtaacgca taacaacaac tgacagcgat tgtatctgag aattcaggtt gaaaactctc cctgatttct taccgtgaat cgcagctgat tgcaacagaa gggcattaca gaccgcaaat tgccggcgca cactacaaaa gaaaacaggt aac gg taac t tggtagcccg gctcgaaact agtacaaaac ttctgcccgt tgcgcagatc attgagcgtc gcgattgcta aatgatggta ctgcagcgta ctttcttcta caaactcagt ggtgctaatg ggcctggacg aaatttaaag gtggagagcg ggatcgctga ctagcgaaac tttactaaca attgatggtc acaataacta gtggaaaata agcactttag gatactactc attctggtaa atcgacaagg cgtttcgact agccgtatcg ctgcaacaag tgtcttctgg accgttttac t t tctgt tgc ttcgtgaact tccaggctga ttaacggcgt atggtgaaac gttttaatat cgacaggtac gcgcggttaa cgaccagtag ttgcaataaa acactggcgc aagatgttca cagacacagc aagccgcaac ttgtaaatgg ctggtgcccc acgaagatgc cattggctaa c tgccatcac aagatgctga cgggtacctc tctgcgtatt ggcaaatatt gcagaccact ttctgttcag aattactcaa gaaagtcctzt catcactatc cgatggcgcg tgataattat gaatgatgct tgatactaaa attagctgac agagcttgat atttactatt tgtttacaaa actctctgat cgccacctac taaagtgatg agcaaaatcg agttgacaat caaccttggc ctacgcgacc tgttctggcg 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1383 <210> 39 <211> 1680 <212> DNA <213> Escherichia coli <400> 39 atggcacaag tcattaatac caacagcctc tcgctgatca ctcaaaataa tatcaacaag aaccagtctg cgctgtcgag ttctatcgag cgtctgtctt ctggcttgcg tattaacagc 120 SUBSTITUTE SHEET (Rule 26) (RO/AU) WO 99/61458 WO 9961458PCT/AU99/00385 26 gcgaaggatg ctgactcagg gcgctgtccg accgggacta gacgaaattg gacggttcaa aagaaaattg gtggctaacg gtggatacaa acaactgctg gacaccggat tattccttca ttggggacag attgccaaag ggcaac ctga gcttccgcaa gcaggtactg ctgagcggaa acgggggctg ttaacgacga atttatgatg gctaaaggga tccatcgaca accaacctga gactatgcga acgccgcagg ctgcacgtaa aaatcaacaa actctgattc accgcgtatc tgaaaattca actctgatac caaaagcgac acggaaatag acttgttatc taggggtcgc atgccaatgg gtgctaacac atggcactat Ctaagaacta ctgtaaatgc gcgcgaatat aggcgtacac taacaactac gtgccagtaa acaaaggctc aatctgaagc aattccgctc acaacaccac ccgaagtgtc tcaggcgatt cgccaacgac caacttacag ggatctggac cggccagacc ggttggtgcg tctgggtttg cgaagcagat tacttggaca gaccattaag agcagcagga tctgacgggc cgctaaaatt tacagatacg tgcgggttca cactatcaag tacaggtgca tgttgccaat agcaggtaat tacggttact gtcactgtat aaccgccgat ct ccctcggt taccaacctg caatatgtcg gctaaccgtt ggtatttctg cgtatccgtg tccattcagg cagttcaacg aatgacggcc agtggattta ttaacggggg aaatcaacca gatggctcta aattatactt gcaaataccg tctatcggtg aatggtgatg ccacc tgcag tttgatagcg tccatttctg ggtgctgagt tcacctgtgt cagacttatc aaagctgcag cccctgaaag gccgttcaaa tctgaagcgc aaagcgcaga tcacctctaa cattaaaggc 180 lztgcacagac aactgacggt acgaaatcaa gcgtgaacgt agac tatcac atgtgaatgg ctggtttctc ccaccaatta ctgttacata atgatgcgaa caactgcact gtacagagca cgctctatct caacgctgga gtatgacggt cagatgacat cttatgacgt atgccgatgc acgagtttgc atggctctct ctctggacga accgtctgga agtcccgtat tcatccagca caccgaaggc tcaggcttct atcccgtctg gctggcgaaa tattgatctg caaaggggc t tcaaggagcg ctcagctgca tgcagggaca cagtaaatct caaaggttac ggaagtgaat ggatattacc taacgtatta tgattacact ggccgcaaaa tgctgcagtt agacggtaaa taatggtaac gacttctgaa agccatcagc ttctgcggtg tcaggacgcc ggccggtaac gcagggttaa 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 tccgtgttgg caaaagctaa ccaggtaccg cagcaggttc tgtctctgct <210> <211> 1146 <212> DNA <213> Escherichia coli <400> gcgctgtcga gacgctgcgg gccgcacgta gaaa tcaac a aactctgatt gatcgtgtct ctgaatattc gactcttctg gatacagfga gcagcaacag Cacaatatc t gataattacg acttattcag gctaattctg acagatggtg cttctatcga gccaggcgat acgccaacga acaacttgca ctgacctgtc ctggtcagac aggttggcgc cccttggttt cgcaggtcgg atattggtac tagacaaaga ctgcatctgt gcggtgatat acggagaggc taaaaaacca gcgcctctct tgctaaccgc cggtatctct gcgtgttcgt ttcaatccag ccagttcaac gaa tgatggg aagtggtttt cgatggttca tgctttgggg tggtgcggca tgcagatgac taccggcgct cgttggtttc gtccactgct tctggtttgc ttcacttcta ctggcgcaga gaactgaccg gacgaaatca ggcgtgaac g cagaccatct agtgttgccg gccgcgccag caaaaggtta actgagaact gggactgtaa accaaagatg gctaccgttc gcaccaaccg gcattaacag acatcaaagg ccactgaagg ttcaggccac aatcccgctt tgctggctaa ctatcgattt gtggggcgct ttaaagtgga atgcaagttc atgttgttag c tc ttaa taa atacgttgat agggtaagaa atattgctca cgctaaagat tctgactcag cgcactgtct taccggtact ggctgaaatc aaacggttct gcagaaaata aaaattaagc tctggatgca tttaacgttg ctatggtagt aacggatatt taaagt tgc t ttatgaaatt gaccattgat SUBSTITUTE SHEET (Rule 26) (RO/AU) WO 99/61458 27 WO 9961458PCT/AU99/00385 ctggatacgg ctgatgaatt tactggggct tccactgctg atccactggc acttttagac aaagctattg cacaggttga tactttccgc tcctccctcg gtgccgttca aaaccgtctg gattccgcag tcaccaacct gaacaacact actaccaacc tgtctgaagc gcagtcccgt attcaggacg ccgactatgc gaccgaagtg tccaatatgt cgaaagcgca gatcatccag caggcc <210> 41 <211> 1506 <212> DNA <213> Escherichia coli 960 1020 1080 1140 1146 <400> 41 atggcacaag aaccagtctg gcgaaggatg ctgactcagg gcactgtctg accggaacga gaagagattg gacggcacca aaaaaaatcg atttctacga attgatatcg ttcgataata gatggtgctt gataaacaag accaccacgc ggtgctgaca gtgaccaacg gatggcacca aacagcacCa gccgctgacg aataacaccg attgcgatgg gcagtcacca gacgccgact ggtaactccg ggttaa tcattaatac cgctgtcgag acgcagcggg ctgcacgtaa aaatcaacaa actccgaatc accgcgtatc tgaaaattca actcttcaac cagcagtaac gaacggatat caacaggcaa atgaaatcca cgggtgctgc caggtacggc cgagtggcct tgggttacgg ctgtgaccac ctgcggctgt gcaaaacgta ttaaatctgt tcgacaaatt acctgaacaa at gcgac cga tgctgtccaa caacagcctc ttctatcgag tcaggcgatt cgccaatgac caacttgcag tgacctgtcc cggccagact ggtaggcgcg cctgggcctg gggggcggca tagcggtatt gtactacgca tgttaatgac tccgggtact tgttgatgtc gaaactggtt cctgcagaat gccgggcgca cacactgggt cggtgcgact tgcagacaac ccgctcctcc caccactacc agtgtccaac agctaaccag tcgc tgatca cgtctgtctt gctaaccgtt ggtatttctc cgtgtgcgtg tctatccagg cagttcaacg aacgatggtc accggttttg acgaccactt gctgctgatg cagattacca gcggatggtt ge tctgacaa actgcggcta caactgtcca gacagcggca gagac tgtga ggctctgatg gcactgaatg gctaaaccgt ctcggtgcgg aacctgtctg atgtcgaaag gttccgcagc ctcaaaataa ctggcttgcg t tact tc taa tggcgcagac aactgaccgt acgaaatcaa gcgtgaatgt agactatctc atgtttcgac atgc tga tag ctgcgttagg gtgcggccaa ccttcactgt gcggtaaagt aaactgctct acacggattc ctatctttgc cttacaaaga gcaaaaccaa gtgctgatct tggctgccct tgcaaaaccg aagcgcagtc cgcagattat aggttctgtc tatcaacaag tattaacagc tattaaaggc cactgaaggc acaggcgaca atcccgtctg gctggcaaaa tatcgatctg gaaagcgaat cgccgttgca aacgatcaat tccgggcctt agcagcgagt tcagactgca ggctgcagca cgcaggtaaa aaccgactac tgcttccggt tctggttacc gtccgatcct ggatgatgca tctggattcc ccgtattcag ccagcaggca tctgctgcag 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1506 <210> 42 <211> 950 <212> DNA <213> Escherichia coli <400> 42 aacaaaaacc aacagcgcta aaaggtc tga agtctgcgct gtcgacttct atcgagcgcc tctcttctgg tctgcgtatt aagatgacgc cgcgggccag gcgattgcta accgctttac ttctaacatc 120 ctcaggccgc acgtaacgcc aacgacggta tttctctggc gcagacggct 180 SUBSTITUTE SHEET (Rule 26) (RQ/AU) WO 99/61458 28 WO 9961458PCT/AU99/00385 gaaggcgcgc gcctctaccg cgtcttgatg tcgaaaaacg ggcttgcaac gcggatttca gtcaaggatg agtactaaat attaccggta gccgatccgt C tcggtgccg aacctgtctg atgtcgaaag tgtcagagat gcacgaactc aaattgaccg attcgatgaa aaatcgacag gcgcggcgaa ctgggggtaa atgcggtcgt cggcggcggc taaaagcac t ttcaaaaccg aagcgcagtc cgcagattat taacaacaac tgattccgac tgtatctggt gattcagatt taccactttg actgacggct acaagtcaat tgattctgca ggtaactgtt ggatgccgca t Ctggattc t ccgtattcag cc agc aggc C ttgcagcgta ctgtcttcta cagacccagt ggtgccaatg aatctgaaag gctgatggta ttactgtctt accggtaaat ggtgcagcgg atcgctaaag gcggtcacca gacgccgac t ggtaactccg ttcgtgaact ttcaggacga tcaacggtgt ataaccagac gatttaccgt cagcaattgc acactgacac acatggaagc aagtggcggg tcgacaaatt acctgaacaa atgcgaccga tgctggcaaa gaccgttcag aatcaaatcc gaacgtgc tg gatcagcatt gtccggcatg tgctgcggat cgcgtc taac cactgtagcc agccgctaca Ccgctcctcc caccaccacc agtgtccaac 240 300 360 420 480 540 600 660 720 780 840 900 950 <210> 43 <211> 1707 <212> DNA <213> Escherichia coli <400> 43 atggcacaag aaccagtctg gcgaaggatg Ctgactcagg gcgctgtccg accgggacta gacgaaattg gacggttcga aagaaaattg attgcgaaca CCttatgctg aaaaccggag gctaaaggga aatactctga gatgtgaagt Ctggacgcca tcCgatctgt acaacttata gctgatactg gcgacaatta actaatggtg accgcatctt tcaaatggcg aactctacag ctggctgccc atccagaacc gaagcgcagt gcgcagatta tcattaatac cgctgtcgag acgcagcggg ctgcacgtaa aaatcaacaa actccgattc accgcgtatc tgaaaattca actcagatac aagctgctac tgaccacaaa atacagttac acttcaccac aaccagcggc ttgatgtaga ctggtaacct ttgctagcgg actttggtgc tactgagcac agtataatac ctggtaattc acactatcaa caggtgcaac gcaaactgac tggatgaagc gtctggattc cccgtattca tccagcaggc caacagcctc ttctatcgag tcaggcgatt Cgccaacgac caacttacag ggatctggac cggtcaaacc ggt tggtgcg gctggggctg agtcagcgat caatacagca tactactggc tCaagcaaca tggcactact tgctaatggc atctacaaac tagtacctta agcggcaact agtgcagagt aggtattcag gaatgacacc ctacaacgtc tggtaaatt~t cac tgaaacc tatcagctcc cgcggttacc ggacgccgac cggtaactcc tcgctgatca cgtctgtctt gctaaccgtt ggtatttctg cgtatccgtg tccattcagg cagttcaacg aatgacggcc aatggtttca ctgaccgctg ctcagcgcta tcgagtgctg gttgcagatg gcatcaggtg gatgtgacca aaccccggca gcgacaactg tctggcgtaa gctgcaacgg tctgcaacgg tatactgatg gataaggata gcagctactg accagtgcag atcgacaaat aacc tgaaca tatgcgaccg gtgctggcaa c tcaaaataa ctggcttgcg ttacctctaa ttgcacagac aactgacggt acgaaatcaa gtgtgaacgt agactatcac acgttaatgg ctggtgcaac gcgatgcact cgatctatac gcgatgttgt tttatactcg tcggtggtaa ttgcatcttc gttctatcca cctacaccaa Ctaacacagc cgtccttcgg cagacaaaga ccggtacagt t tggggcaca gcactgcaac tccgttcatc aCaccactac aagtgtccaa aagccaacca tatcaacaag tattaacagc cattaaaggt cactgaaggc tcaggcttct atcccgtctg actggcgaaa gattgatctg caaaggcac t gggaacaggt gtctcgcctg ttatgatgcg taactttgcg tagtactggt agccgcgtac agcgaaat tg gctgtctggc aactgtaagc agttactggt tggtgtgaat gctcaccaca aactgtag-t ggcttatgtt caaagatcct CCtgggtgct caacctgtcc Ca tgtcgaaa ggtaccgcag 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 SUBSTITUTE SHEET (Rule 26) (RO/AU) WO 99/61458 WO 9961458PCT/AU99/00385 29 caggttctgt ctctgctgca gggttaa 1707 <210> 44 <211> 1720 <212> DNA <213> Escherichia coli <400> 44 atggcacaag aaccagtctg gcgaaggatg ctgactcagg gcgctgtccg accggtacca gacgaaattg gacggttcca aagaagattg gtggcgaata actcctgctg gccacagcag tccaacggtt ttcactttca ctgcaatcct acgacatctg gctctgtata gcaaccc tgg acgaccgcag caatcaggtg gcgaaaggta gggtgtcgtt aaccaacacc aggggtttac caccaccgat atccctgggt taccaacctg caatatgtcg ccaggtaccg tcattaatac cgctgtcgag acgccgcagg c tgcacgtaa aaatcaacaa actcccagtc accgcgtatc tgaaaattca actcttctac ctgcggcgac tcggtactga caaacgtgtt ttggtgcagc atgccagcat tcctgactcc ttaatgttgt tcgactcaac atggactgac atggcgcaac caattacact ctgttatcaa actaccggag gttgattatt accgatgcag ccgctgaaag gctatccaga tccgaagcgc aaagcgcaga cagcaggttc caacagcctc ttctatcgag tcaggcgatt cgccaatgac caacttacag tgatctggac cggtcagact ggttggcgcg gttgaaactg taaagacgaa cggcgtgacc tgcaaacctt agcagccaca tgctgctggt aaaagcaggt tctggcgagc gggtaacctg caaaaaccat tatctctctg gaaaaatgtt tgttgataat gtgcgcctac tcctgcaaac ctggtaaatt cccttgatga accgtctgga agtcccgtat tcatccagca tgtctctgct tcgctgatca cgtctgtctt gctaaccgtt ggtatttctg cgtgtgcgtg tctatccagg cagttcaacg aatgatggcc actggtttta ctggctgctg aaatataccg gcagatggtg gactacacct gctgcggccg gatacagcta gatggcaaaa actcagaaca gatgcgacag gcaggctctg cgtatcagtg ggtgctgatg ttatactgat tgatggcagc cactaccgac cgcaatcagc ttccgcggtt tcaggacgcc ggccggtaac gcagggttaa ctcaaaataa ctggcttgcg ttacttctaa ttgcacagac aactgaccgt acgaaatcaa gcgtgaacgt agaccatcac acgtgaatgg ctgctgcggc tagacgcagg ctgttgttga acaataaagc gtgatagtaa acctgagcgt t tacagcgaa gcgcaggcac gagctgttgg ctaacgcggc ctgatgctct atatttctgt gctgatggta gtaaccaatg gctgcaacca cagatcgata accaacctga gactatgcga tccgtgttgg tatcaacaag tattaacagc tattaaaggc cac tgaaggc tcaggcgacc atcccgtctg actggcaaaa tatcgacctg ttctggttct ggcgggtaca gcttaacaaa tgctagcatt tacaaatgat cagcgcagc t caaaatcggt agatggctca tgtaacagca tgttgatatc aacaggtact gcagtctgct tagtaaaacc aattaacgac gttctggtaa aagccgcaac agttccgttc acaacaccac ccgaagtgtc caaaagctaa 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1720 <210> <211> 14516 <212> DNA <213> Escherichia coli <400> gatctgatgg ccgtagggcg ctacgtgctt tctgctgata actgctccag gtgcctgggg acgtattcaa ctgactgatg aaacagtctg ttgatgccat gctgatgacc ggcgacagct ggctatatgc aggcattcgt taagtatggg ctgcgcaacc tctgggctga ctattgcaga acgactgcgg t taaagaagg gttggaaaaa gttggctaaa 120 taagaagatg 180 ggcgaagttc 240 SUBSTITUJTE SHEET (Rule 26) (RO/AU) WO 99/61458 WO 9961458PCT/AU99/00385 cgtaagagca ccagaaaaaa tgccgttttt ttttgttgga gagagttttg tagctgttaa tgccgcttat gtttgtagtc cgatttagat t tagaatgtg ttcaggaaag aatgattttt ttctctctta aaaaaaattc atatttggaa ggtctaggta aagtggatac ttaaatcatg gtgttaggtg aaaattacct gaagccgcaa tttgagagta catgatctta gtttttgttt gctattggta ggggtcaatg tattttattg gaaaaaaac t aatgattttt t tatgaataa aattattgtt atactgagcg tacactatgg cagatgaagt aatacactgc tggggctgga ttcaagaaat caaaattata gcaatggtat aaataacacg atatggattc tgctgcagca gtgagtttgt gagtaaatga cgggcgatgt tgcttggcga gtgaaatggt tgaaagctaa tcaagaagct tagcggcagt tatgaaaaat ttttttcttg cgggatctcg gccaggggcg agcaaataaa gctcagggaa ggcagttgat atatgaaaaa aattaatcat ccactgaaga actcaaaggg ttaaggatat ctattgcttc atgccttcac aaattctttt atgataaaaa ggattggatt ttatttttat agttcgttaa ataatccttt tttatcctgg tacctacatc gacc tgtaat gctttttgat agaataaaga ttgatgcttt gacttgagca agtggcatta agaaaaaggt agtggatcac cgatttgaca ttacaatttg tgatgttgat aaaaaagaca tccacaaaaa tgcctattgg tctctttaac cgggatagca tctgcgtgat agaaac tcca cacaatggcg aaaaggtgt t aattatatct tcctactaat aaaagaaatg taacattgcc actgagtgag taacatccag gaccaataat tttctggtcg cggaactgct gtagcttgcc gtcaatcgga attggttttg gttactatgc aatagtgatc gaatttagtg tcttaaagta tattaatcct ttccccagat aaagttgtca ttattataag atataagtta agatttaatc ggatcttaat agcaaggtta gacaacttat ctcattacaa tcatgtggaa atatcgagaa aacaactaat acctccattt taaagtactc tgaaaaaaat gaaattattt attactggta tatgaagt tc atctatcagg gatacttcca ggggcgatga gcgataggaa aaattttatc gagactacgc atcactgtta cacgaatcac aatattgctc tggggacatg gaagattttg gcagagcaag gttgtttcgg gtagatccaa gcgcataaaa gtttccagcg actaatattc 30 tagagattta gcttctatgc aacaagttaa catttggtaa cacatctttg taattaattt ttaaacttct gtagtagtac tgcatacata ataggcaatg aggcaaggtg ct ttcgtca t tttaaggata attgtatttt aaagtgccaa ggaaagcaga gcattaccga gatcagtata gagttttcat ttaagagaga ccaagttctg aaaaatgaaa aatgttcaag ggcgtaccaa gtacctgggt gaaattaatt gaaa tggggc aatagactag atatttcaat tcactgggca atggtattaa attcacattt atctgacccg gccatgtagc cattgcgtct aggcttcaac cattttatcc attatcgtga Ctcgccgtgg aaggtcttga ctaaggatta taattgctac taggcataga tcaatggcac ggtattttag aattaggatg atttagcaat cgcaagaata cacgtctttg tttaagcaat cctaccaagt gacaattagc gcatttagtt t taacgtata tttccattag ttttcaaatt tcaatgtata tagcgtcaat ataatgtata ggggcgttaa taattgctgt catattttgt ggattgttgg ccacaaaaac tgcttgatga atattaaagc ataaagagcc aagggatatt aatttgtaat ttgaatcgct attggttaga gggtgatcca gtagggatat tactggcaga ttgctggaag catcaataat c tgaaaaata agatggctcc acgccgtgca agctaatcct tattttaaaa ggtatcattt tcttgaagct ttcagagctt acgttcgcct gtcttatggt cgagaccttt taaatgctta tgtcaaaatg aggaattcaa gttagcattt tgatgctaaa gcc tgcagaa gagccctgaa agcgaaaaag aaaaagataa tgacgataag ggaatgttac t taa tc tgc t gtgagtttta agtgcactgg catttattct gtaaaagagt atccattttc ttatttactt gatgttaagg ttgtctagca gggggttaaa ttatgaacta aaagccagta aatgattgaa taaaatgata tttgattcta taaggtaaca accgaaagag tgagtttatt t ttaggaggt aagaaaagaa gaaaagttct agaagctatg aataaatgat aaaaatgaaa gaagtttgca aaaatcaaat aaggctgtta tatttggcag tcttcattta aaactttttc gaagttcaac gagtcaccag atcaggatat tatggtttgg tatgctgttg atgtttgcct gttactcgta tacttgggaa caatggatga tattctgtcc gaaggtgagg gctgtaaacc gttgaaacct attacattgc aacgtcttgc tacattaaat 300 360 420 480 540 600 660 720 78.0 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 1860 1920 1980 2040 2100 2160 2220 2280 2340 2400 2460 2520 2580 2640 2700 2760 2820 2880 2940 3000 3060 3120 SUBSTITUTE SHEET (Rule 26) (RO/AU) WO 99/61458 WO 9961458PCT/AU99/00385 aattaaaaat ttacatcaga taat tgaaaa attatttttt aaagaatatc gtgtatggga tagtgctttg atcgtgaata attcaaaaaa Ctcaatgtct ctcgagtatt caacaattac atcaccgatt tacttgagcc agaagaataa ataatgaaaa agt tagtaac gaagttcttc aaaaaccaga gcggaatgtt atatttatca gaattaacga aaaaaacaaa ggtcatcact tgctcaatca tcggagtaag ataaagtcca actacatgca atagatatag atcatcatag gtgaagttaa gtcttgagaa ttgaatcaga gagattgata tggtgctgaa taaacctcaa atcactctca tactgaagag tgcaagccat aatcagcagt agagctcaac aaatcatttg ttctgggaat caatattccg tatccctaat tagtgctgat aaatggacaa gaaatatcca ggtgctagat taaatttaga cgagaatggt tgttccaggt atctatggaa acatttctat ttacacactg Cctttggcta ttattttttg ttaccctgta ataccctaaa gcgtttggat tat tgtagca gaaaggccgt tcctaatgac agcatttcga atttggaatt agctgatcct tgttaaaaca tttatttcgc tagctgtgaa ggctgagttt agacgctgta ttgggatata tgatggagaa taatttagta aaatgttaaa tcgtgcagtt agtaaaaaaa ggcagagcat actattagtt tccaggcgta tgatatagtg aatatgaata ttgaatgatg actgtagttg aatgggctat atatattttt aatccaattg gacacaggtc ctagaaaaaa atgggctatg ggtgcagctg attcagtttg ccattactac tttggtattg tttattgaag aacgcaaaaa ttattagtac gaaattatca gaatatttat ggtaggattc ttaggtaaag gatgatggtt aaagttctta actaaacacc taatttttat attattgccg caatttttaa ggcatcgaat gagcaattac aatactgcac gaccctttat gagtcaataa attccggaca aataaagaat gcacaggaat gccagtaaat tgtgcaaccg attaattgtc gttcttccga agccaaaagg aatagtttta attgtccaaa aacatagttg tttcgccctt ataatagtta tggattgttg tctaatgagt atacctttgc cgttttactg aaataacttg aaatagcata tgggaggaga gtgatgcagg zcacttggta attataatgg tcaaagatat aagggaatat ctaatctgca gtcctgtctat taaaaataaa ctgagtgcag :atttgatgg jatactacat tcattcatga 31 cattattttt agttagttcc ttggtcttag gcaaaaatga agtatggtat ttttttctga aaagtgaatt aaataaatgc taaaaattaa gaggaaccgg atttagttgg gcgaaaatcc gacagattgg ctgccatagc tattagtact taaaagctat cggcaaatac tcccagcata atatttcgag atcttgatga ctacagcaaa ctgaagagtc tagatattgg attgccatgg tttactctga ccaaggatgc acgatctaaa ggggtaaatt aaccaggaga tatccggtac ctatatatat atctaattga acagatataa cttcaaagca tagaattggt tgctcgctta cgtaaatgtc tttaggaatt aatgaaatta acaacaatta taccaaatat aaaaataaaa tgatgctatt taatacaccc agaagatacc tgattttgat tgttggttta tcctcgcctt ttttgggtga attagtatca gaataatcga atctattaaa t tcaggaagt aggcgaggca gaatctccca taaacaagat tatgcgagag aagccgtcta ggattctaca aattgttatc taagc taacc tttagctgct tgcggcagac gccgtatgca tggttatgga taatgttgcg tgggaattat actacggaaa tatagatatg tatcgattat ctggaatgac taatgtgtgc gtcaagtctg tgtactggtt aaagagaaaa cgatgcaata agggttagat tgctaaagtt ccctcaggga agtaagttct cagtaaacaa taigatatac cgcgcttatg acaagtgaga ttagatcttg gatggtggaa gtaaccaaag gtagagagta tccacccgag aaaatcaaaa gaggaatgct gatggtaatt agcagtgcgg aggtgttttt ttagcggaag atatggaata ctaatgttta 3180 attgatctgc 3240 ccggccaaaa 3300 aatgctttta 3360 gtttttaatg 3420 acacattata 3480 gatgatcaac 3540 gttcataact 3600 aattgtatgt 3660 tggccgttgt 3720 atgttgcaaa 3780 tgcaatgaag 3840 aagaatatta 3900 tttatcgctc 3960 cactctataa 4020 acttctggga 4080 tatattaaga 4140 gagtttgtag 4200 tactggaata 4260 tttagaccag 4320 gactttgtcc 4380 gctgtgatgg 4440 gtgggttctt 4500 catggggatg 4560 gttgcgacag 4620 gcggaccgtg 4680 cgtgctgaat 4740 gaccaaggcg 4800 ttaaggatgc 4860 tcactaggta 4920 gcaaaatata 4980 ggtgattacc 5040 ttcctaaagc 5100 gtgggcgtct 5160 gtgagttttt 5220 gtttaaagaa 5280 gaatgtgtgg 5340 tcgaggtaac 5400 gtgctcgacc 5460 ataattttga 5520 atgcctacat 5580 tagttgtgaa 5640 ttttacggaa 5700 ttccacatgg 5760 ttataagaca 5820 tctttgatga 5880 tttttttagg 5940 ctattgatat 6000 SUBSTITUTE SHEET (Rule 26) (ROIAU) WO 99/61458 WO 9961458PCT/AU99/00385 32 cgtagaaagt aagaatgcgt aga t tttgca tctgacaaat tggagaaata ttacaaagat t tggcgt t tt taggaataat ataaatttgc ctcaactggc acttactcca gcttatcaac aaatataagc ttccgtcgca ccccaaaaac aactaatgag aagagaaaac tgataaattt tgcgaaaatt gtttatgtat catgcctaat gataattgca aggaatgaaa ctttgaactc atgattacat tcagtaattg tttgctgatt ctgt taatga ataatagtac aaggtgaagt aatgctattt gattttgcaa tgtgagtcga acctttagtt gatgatgaag ttaccaaaag aagtttgttt gagcaactta gtagataaat tggtttggtt aataatctga aatgaacgtg tatatagata gattatctac ttaagatggt cggt tat tga accacctggg ctcagttaat catggtggta gaagaggatg tactgcgata aaaaaattag aactgtacac agtgccttag aatgttagat gctattctta acctgagttc atggttggta accagctctg atgccagact aggccgtttg aaaaaac tag tttgaagagg ggatatgcta tctaattatt gatgataact aataatgtcc gcagaagatt atggtaaatg gaagaaattg cggaagctag agagatggca acccacttgc actcaaaaat tgtttggtag ttgctgccct ctgcagtgtc ttgtcgatat cagataaaac aaataaatga tgggcgcggt ctttttactc agctgtatca agaatatggt taccaggata aaaagttacc ttaaagatca tttccttcgt tctcagcagg ttttgagtta agaatggttt gaaaagtatt attaacagtg acagtttgtc taagcaaagc atgtggagga tacctataat ccgtatatgg gtggaatgat gtgaactggt tagacaatcc ctgttgatta gc tcaaatac tgcaggaaaa ataatgggaa agaatatatt atttgaattt gtattataca attttctgga gtatcaagaa ctattcctga ttgcgaaaat tttataaaac cgtcacatat cagagatcga tagctgatct ctggtttagg gttatactgg tagatatttc tcagaaagac tagtaatact gtttaccatg caaatatgcc tttcttcact atggtc tacg caataaagaa aaaagcaata gataataaat ctttcaaaat tcatcatata tgtattgttg tacaggcact caatgttcgc aggttttata tccattcctt taiaaaggag cattgaatgc ttttgattac ttttgtcgga aaaataac ta aaaaaaattt aatccaattt tatggtaatt tgttccgcat gactaaaacc cggcgaaatg tccttggatt ttgtggttgt gcaaaatgaa cactgatgga agaacctgta aacagaagaa caagaaatat agagcatgat attagataaa tgcagcggga aaaaaatttg agtgcttaac gaaagctctg tgctgtagca aattatccca gattccggca aatttggggg tattttttat ttacgattat gagtttttct attgcttaat ctataattat tgggatgaat ggtaaaaagg gtaatggtta aataaaccaa acatattacc actttaaata ttgacagtaa aatagggata aagcaggcag gctacaatgg tgccttcgag aagagtgatg ccacttgaaa tccaccagac gatatacaaa ggagctgcta cgaccaattg tctgtacatg aaccaccaga acgaggcact tagcgtttgg gcatcttcat ggattttatt ggattgcaaa ggtcatgctt agtgcgcatc ttaatttgtg ataaacgact atagataaat ttaactatgg gtacgattga attctgaatt atgaaagtac agtgcaagta aatgaaatag ttagttggag cagatgggtt ttgggtagtt ttaactggtg aaagcatgcg tgtaatttat gttataaaaa gatggtaatt gttattccta tcaattaatg catgatttaa aaaattggtt tacttggaga atgagtatgc ttgagttata gctctggttc aacttaaaag ctctgcaaca ttgatatcga atttattagg ttatcttact gcacattcgg aagggggctg ctcatggttg atattttcga tgagtggtgc gttccaatgc aagaagttgg t tgagaggaa ttactgggaa atacggtagc tacctttgtt ctatttcgaa c tat t ctaaa tatcacacca aattactatt aatcattgca acat taagca attattttaa aacttttgag ggccggcaag tatttaatcg agttctctga atgtagaatc ttatatcaaa ttctgactgg aatataatat aaaaattcat gcattcatgc taaatttagt catgcatgta agctagaaga aatatatatc atgggaaata aaatccatca cgcgccgtga aaatagaatt actattataa caaaaccaac ggtcaagtca atcaaaataa agcaatacag tgagaaaaat tacagctaat aggtgatgaa gtatggctta tagtttgaaa taatcctaat agaagataac agttatgggt cgtagttact gacaagaaat agagtcgtt t tattgggata acaatatttt tgaaagtagc gagt ttagta ttttctcaaa aaaligccgaa taatgaaata tagagtgcc t gtac taccac ctaatactca gtatcttttt gaacagagaa 6060 6120 6180 6240 6300 6360 6420 6480 6540 6600 6660 6720 6780 6840 6900 6960 7020 7080 7140 7200 7260 7320 7380 7440 7500 7560 7620 7680 7740 7800 7860 7920 7980 8040 8100 8160 8220 8280 8340 8400 8460 8520 8580 8640 8700 8760 8820 8880 SUBSTITUTE SHEET (Rule 26) (RO/AU) WO 99/61458 WO 9961458PCT/AU99/00385 33 ttcttagtga ttattatcgc ttgcgaggaa t tga taat tt ttgaagtaat cactcttata tatgtctgaa atttgttaaa tgtttgatag cttgccttca taccaatgtt ttctgcttgt atatattatc ttttagctat gaattggtaa cttcaacgtt atccggcttt atgtctattg tacatattta cttttatgct ttggtcgatt ccgaaattat ttatatttaa ttttacttgt gcgttgattt gggcagattt aaaaagattc attggtttta gaacgtacgg atggggacat tcattaattc ataggagcat ttatatgaga ttaatttata ggagttgttt tccacttgtt gcaatcggta ttctgataaa agagaaagat ttgggtagca gatgaagggg aggtcccgat taagtttaat tgatttcttc gtttctgaga gacccctatt aactacgcta gctgattctt tttatcaaaa atttgcggta taattcttca atttagtggt tacaagagtg ttttacatgt tattaccggt tgagtcaaaa gctcgtaata actagctcaa tgctagaatg atcactaatt aatatggata aagt tacat t atctaagctt aatcgcagct tcaattttat atttactttt ctcaatgttt tgctcttttt ctttaaagtt gctztgtgggt agacattcga ttagattggg ttctctttat tatattccgt catacttttt attatttcaa ggatgatata taaatttctt ctaatgaccc tggtttatca gaaatgctcc cattaaaccc atggatttgc tcaataatca actaatcaat acgc ttgata cgtggaattt tttattggtt gttatggtat aggaacatat tattacaaat agaaacgaac tgtttcatca gttagaatgg gaaagtttca agatatgcta aaaaatgctt ttgatttctt ttcttattcg gcgctaaaag ct Ctgggct t ttagccttta tgtttcatca tggatgtttc ccattgattt ttgatgttca aaagcatc ta tctgttttgc aaccctacat ttattgtcaa gttgcaaatt cattatggcc tacctttatg ttcaattact gttaatgcgg tttagtaatc atcactgcct tattattgca taagtttagc aattggtaat attaatagac aatttgcatc tttaatgctg taaaggcgta ttatttggtt aaataacggc tcatgatttt ttctatattt tttcattgtt ttttgatgct aaaaattaga ttgcaactta cttataagaa ttgcaaaatc atgatgcctg cagatgatgt ctaatggcgc gtaatttacc gttttgatat aatggagtaa gatatggtgg ttgtacgcaa aaattctggt tacgtcaaat ttcttatatt cgattattat gttttgaaaa ctatagtaat ccat taaagg atcctaattt ttcaattaac tatctgtcag ctctttctgc acacatttcc cttgtcttgc ttgcaactga tgatgacatc taaatctggt tttatgcaat tagcttttgt gaaa t cgcaa aggatctatt attcaacttc ttattgatgg ttgttgtttt tcatttcctt ttaccggttt ataatgcagt ctgatacttg ccagttttat actttttttg tcacagcttg ggacaatata ttattacggt tttgtttttt gtaagttgtt tttaatcgat taagtatacc taattctgaa tattgaaatc gtttaaagac gaataaagca ttactatcat ccctgtggtt aggcagtgaa attgcccatt taaat taaaa agagaagtca ggtttcgact aaagaataat gatggtagcg ttcctataat aagtatttgt ttgtggtttt atttaatgta atatggcatt tatgctaaaa taatagagtt tggtggcgtc taaactggct gtctgcaaat ctctaattgt gttattcttt aaattataaa ttttcatttc tgcagggctc atctatggta ctattttaat ttgttttttc tagataaaag ctgagcttaa tctttatcgc tgaactctgc ttactttttt ataaaaataa cattgttaat tgtttaaagt atgtaattat aacc tacagc gtgattatat agacgttata tctttataag t taggagaat tgttactgtt tgatttgcgg tgtaatggaa cttgatatag ataataatgg gaccgaataa gttgatttat acagatgcaa tatgggagga tggtacaacc atgagcgcaa attgttgctg ccttatttta gatatttctt atatcctgtt atcaaaaata ttttcaattg ttcttcgatg tttcaggaag tcatgttttt tacggaaatg tatattcttg gggattgtta tcacttagtt tcttatgtcc caaatattac ttttttaaaa t ttggtcgtg ttaatgcaaa ttgttattag gcacttgctg aaaataatat agagcgaaaa ttgcactatt tattttaatt tgtaaacggt ttttcaaaaa atttaatttt tatattgctg aaaattttac aaattatagg taatttaaga tatggcttat aagtaatatt attccatggt tggacttcca tattggtgtg atctttctta acaagttgtg gcttacagtt atttagacgc ctaagtgttt atggaggatc aaatagtttc ccattggtga ttgcttcatt cagcgcacga taacaggat t tatattctcg acgctgattg ttaatgacac ctcaagttaa taaacataca tttttggcaa 8940 9000 9060 9120 9180 9240 9300 9360 9420 9480 9540 9600 9660 9720 9780 9840 9900 9960 10020 10080 10140 10200 10260 10320 10380 10440 10500 10560 10620 10680 10740 10800 10860 10920 10980 11040 11100 11160 11220 11280 11340 11400 11460 11520 11580 11640 11700 11760 SUBSTITUTE SHEET (Ride 26) (RO/AU) WO 99/61458 WO 9961458PCT/AU99/00385 34 taatgtttat atgaagattg cagt tagcag atatctgagg tttagaagct gatataatac attccagcgg atgttttgtt gctgttcaag aattttatta gcttttaatt aaagactatc tgtaatgat t ttggtttgtc aaagaattaa ctcgttgttg gttaaagaag gcagagaaaa aaaaatagag cgcttatatt ctctggatgc ctggattttg cgcgcatgcg cggcgtcgtc ttataccgtc cccggataag cccacgtcgt cctgaagccg ggacactatc cgtgtccggc agaagcgtat cgaaccatgt caacggtatc cggccttaat gctaagtagc taaatacctg cagccagagc tcgctacatc gcaggctaaa cctgggtaaa atacaactgg tc gtgcgcag cctgttgctg tgtagtggct ctactacgac ttacttcggt aaattaatgc tttatataat accaaatgtt taaagccaac tttttagagc atagtcatat tgcccctgat atcgactgag agtttatagc atacaaataa tgaaagacag cgaacttatt ttattttgct aattgaatct tgtgtgctgc cagaagctat tcgttggacc tcgctgagac aatatattgt ttaaatattc aatagtttct ttatatatca tcgcgcggtg ggtatggcag tccatcttca aaactggttc atcctgttaa tatctggata cgtcgtaacc ggtgaagagg gagctggttg ataacttaca gaatatggcg ctgtctaacg tacctgattg gttgatgtga tctctggatc tcttctctga ctggctggtg atcgtctctt gatc tgaac t ttcctgcaga gctccgtact tatgctgtgc agctaccgtt gcgcacacgt ataacgggta aaccgggctt tatacgcggg acaaaatatt tttatttcaa gtttcatgct atgtaccgca tgatttttta aagaaaggc t atttgatttt tacagcagta aaatgcaata tattgctggc tgtggataaa agatc tt ttt ggcgtgtgaa tcataatgat acttaaaata ttccaatttt caagcgtaat ctatgctgtt gtattaatct actacacctg tgatggggcg accgctcccg cttattacac tggtaaaagc aaggcgacat gtgaactgtc gcgccctgaa cgcctatcct tcggtgctga atatgcagct aagagctggc acatcaccaa tcctggacga tgggtgaacc aagaccagcg ataaagcaga atgcccaagg acggcgaaat aaattactga tcaaaaatat agaacggtat ctgcggtact ataaacgcac tcattcccta acttgtggtg catgatgtta aatattcatt gtaaaaaaaa aatattttta cacaacaaaa gcttctatta acacc taaaa gatattaatg ctgctcgcag aatcatttga gatggcgcat gttttcttct gttttgagtt Cgtcccgttg gttatccctg gatga taacg tcaattaaaa aatataattg tttttactgg gtctcaactt acaggagtat caacctggcg cgagaaaac t ggtgaaagag aggggcggga cattattgat cgcggaaggc aggcccatct gaccaagatt cggtgcgggt gattgctgaa aaccactttt agacatcttc agctgcgaac gctgtcgctg cattgcggca gttcgttgag cttctctcaa cgcgaagatc cgcgtatgct cgctgatgaa tccggtaccg gccggctaat tgataaagaa aagaaaatca gagccgaaca atattatttg atgttaatat taattgtcgc gtcgttttat atgaaggtgg ctacaaatgt ataaaatagt tcagaaagaa taggaagac t ttctttcaaa taagaaataa tggggcaaag c tgagtggga t tgc taccga tcagtaatca caagaaaaat cgatagtgag attgaaaata ctccgtattt catctagact gtaatgtcca ctcaacatcg gaagaagt tg ttcgtcgagt actgatgctg ggtggcaaca tttaacttca at ca tgccag gctgcggttg cactacgtga gcctattctc accgagtgga accaaaaaag aaaggcaccg atcaccgaat tctaaagtgc aaagtccgtc ctgcgtgccg ttccgcgcgg gaaaacaaag tatcagcaag accttctctg ctgattcagg ggtgtgttcc agaataaaat ccttatgacg tctaactggt ggataaaaat cttaaagcca taggatgctg caatgcaagg aagtaaagag agagattccg aacgcgagat tgt tgaagca aacatcaaat attattggat aagtgatatt aggttttggt ttctggtgga tattctgttg aataggtatg tgagtgggag taagtttgta ttacttatag acat tcaagc agcaacagat aaagccgcgg t tgccgagaa Ctcttgaaac ctatcgattc ccttcttcca tcggtaccgg gtggccagaa ctgaagatgg agatggtgca tgcttaaagg atgaaggcga atgaagaggg gtaaatggac ccgtattcgc tgtctggtcc gcgcgctgta cgtctgacga gctgcatcat gcattgctaa cgctgcgtga cagcggtagc cacagcgtga acaccg 11820 11880 11940 12000 12060 12120 12180 12240 12300 12360 12420 12480 12540 12600 12660 12720 12780 12840 12900 12960 13020 13080 13140 13200 132 13320 13380 13440 13500 13560 13620 13680 13740 13800 13860 13920 13980 14040 14100 14160 14220 14280 14340 14400 14460 14516 <210> 46 SUBSTITUTE SHEET (Rule 26) (RO/AU) WO 99/61458 WO 9961458PCT/AU99/00385 35 <211> 1380 <212> DNA <213> Escherichia coli <400> 46 aacaaatctc aacagcgcaa aaaggtctga gaaggtgcgc gcaac taacg cgtctggaag gctgaaaata aatctggcaa cagaaagcaa caaattaacg gatggcaaac caattcaaga aagattgtct ggtaatggtg agtgctgata agtgcagggc gatctgaacg gttagtgcag ctgagcaaat caatctacca cgttctgacc accgtaaaca gtgtctaaca agtcttctct aagacgatgc cccaggcttc tgaa tgaaat gtactaactc aaattgaccg atgaaatgaa aaattgatgc ccggcagtga gtactgataa aagtttatgt ttgatgcaac acgaaggtat aattaaccgc catcaggtac aattgactgc ctgccaagaa atggtaaaac cagaaggtgg ccaacccgct tcggtgcagt acctgtcttc tgtctcgtgc tagctctgct agcaggtcag ccgtaacgcg taacaacaac tgacagcgat tgtatctgag aattcaggtt gaaaactctc cctgatttct ctatactgtt gagtgctgcg taagcttgca cgaatttaca caatgttgat tagtgcaacc aacaaaagtt aacaggaagc gataacggag tagcccgatt cgaaactatc acaaaaccgt tgcccgtagc gcagatcc tg attgagcgtc gcgattgcta aatgatggta ctgcagcgta ctttcttcta caaactcagt ggtgctaatg ggcctggacg aaatttaaag aatgtagata gatggttcac gtggctgcta aataccggca ggtaaggctg gttgccccta gaaaataaag acgttagttg actgcttctg ctggtaaacg gacaaagcat ttcgactctg cgtatcgaag caacaagcgg tgtcttctgg accgttttac tttctgttgc ttcgtgaact tccaggctga ttaacggcgt atggtgaaac gttttaatat cgacaggtac gtggagtagt ttacgaccag aagatttagc ctggcgctat ttgaattcac cgacagccct cagcgacact ttaacggtgc gtaacaataa aagatgcagc tggctaaagt ccatcaccaa atgctgacta gtacctctgt tctgcgtatt ggcaaatatt gcagaccact ttctgttcag aattactcaa gaaagtcctt catcactatc cgatggcgcg tgataattat acaggataaa cagtgatact tcaaggtaat acctgccaca tatttcgggg atacaaaaat atc tga tct t aacttacgat agtcatgtat aaaatcgttg tgacaatctg ccttggcaac cgcgaccgaa tctggcacag 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 <210> 47 <211> 1497 <212> DNA <213> Escherichia coli <400> 47 atggcacaag aaccagtctg gcgaaggatg c tgac tcagg gcgctgtccg accggtacta gatgaaattg aatggctcca aagcagattg gttaccacta actggaatta attgagggtg aacgatggga tcattaatac cgctgtcgag acgcagcggg cggcccgtaa aaatcaacaa actctgagtc accgcgtatc tgaaaatcca atgctaaaac gtgctccagt ccctttctac tttatactga agtattacgc caacagcctc ttc tatcgag tcaggcgatt cgccaacgac caacttacag tgatctgtct tggtcagacc ggt tggcgca tcttggcctt aactgctttt ggaagcagcc taatggtaat agtaacagtt tcgctgatca cgtctgtctt gctaaccgtt ggtatc tccg cgtgtgcgtg tctatccagg cagttcaacg aatgataacc gatggtttta ggtgctacca actgatactg gattactatg gctaatgatg ctcaaaataa ctggcttgcg tcacc tctaa ttgcgcagac aactgacggt acgaaattaa gcgtgaacgt agactatcac gcgttaaaaa ccacaaacaa gcggaactaa cgaaaatcac gtacagtgac tatcaacaag tattaacagc cattaaaggc caccgaaggc acaggccact atcccgtctg gctggcaaaa tatcgatctg taacgataca tattaaactt cccagcttca cggtggtgat aatggcgact 120 180 240 300 360 420 480 540 600 660 720 780 SUBSTITUTE SHEET (Rule 26) (ROIAU) WO 99/61458 WO 9961458PCT/AU99/00385 36 ggagcaacgg tcaggcggta gctgttagct aaagatacaa gttaaaacta ctgggcggag gccgatttaa gctgttgcaa gtagacaaat aacc tgaaca tatgcgaccg gtgttggcaa caaatgcaac cacctgttca tagtaaaact atggcaatct ttacctatac atgatggcaa atggcggtaa atggtaaaac tccgttcttc acaccactac aagtgtccaa aagctaacca tgtaactgat gattgataat gcaggattcc ttacgctgcg tgactcttcc aacagaagtg tctgcaaaca cacggatccg cctcggtgcg caacctgtct tatgtcgaaa ggtaccgcag gcaaatacta actgcaggtt aagggtaatg gatgtgaatg ggtgccgcca gtcgatattg ggtttgactg ctgaaagcgc gtgcaaaacc gaagcgcagt gcgcagatca caggttctgt ctaaagctac ccgcaac tgc ataccgatac aaac tactgg gttctccaac atggtaaaac ctggtggtga tggacgatgc gtctggattc cccgtattca tccagcaggc ctctgctgca aactatcact caaccttggt atatgcgctt tgctgtttct cgcggtcaaa atacgattct ggctctgact tatcgcatct cgcggttacc ggacgccgac cggtaactcc gggttaa 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1497 <210> 48 <211> 1695 <212> DNA <213> Esoherichia coli <400> 48 atggcacaag aaccagtctg gcgaagga tg ctgactcagg gcgctgtctg accgggacta gacgaaattg gacggttcga aagaaaattg attgcgaaca tcaagcaata aaactcaaag accaatgatt gctactctga aaagttaact ttggttggtg tccctgttta aaaaaatacg acggtgtctt aaaatcacc t ggctccgcgg tcctacaaca agcggtacgg aaattaacca gatgaggcaa ctggattccg cgtattcagg cagcaggccg ctgctgcagg tcattaatac cgctgtcgag acgccgcagg ctgcacgtaa aaatcaacaa actctgattc accgcgtatc tgaaaattca actctgatac aagcggcaac ttgttgtcac atggtgattc ttacgacaga aggctgctgc ttgatgttga gagcgctgac aggccgcgga aatttgctgg ctgacgcgct t taacaatgg catcgaatgc caaattattc gtaaatacgc cgaatactac ttgcatccat cagtcaccaa acgccgacta gtaactccgt gttaa caacagcctc ttctatcgag tcaggcgatt cgccaacgac caacttacag ggatctggac cggtcaaacc ggttggtgcg gctggggctg cattagtgat gacaaagttc tgttgccgtt aaatacagta tgggcagagt tgcaagcggt tactaacgat tgacaaagat tggcaattct tttggctcag tcctctgtca agcctacatt cgtagacaaa cgcaaacgtg tagtaccggc cgacaaattc cc tgaacaac tgcgaccgaa gttggcaaaa tcgctgatca cgtctgtctt gctaaccgtt ggtatttctg cgtattcgtg tccattcagg cagt tcaacg aatgacggcc aatggtttta ctggcggcga aatgccttgg gctgctcaga gcgacaggca caatcaggta aatatcacta cccaccggct gccgctcaat actaatggtg gt taaagcgg ttcactgcat gatagcgaag gacacggggg ggtgctcagg tctgcaacca cgttcttccc accactacca gtgtccaaca gc taaccagg ctcaaaataa ctggcttgcg ttacttctaa ttgcgcagac aactgacggt acgaaatcaa gtgtgaacgt agactatcac acgt taacgg cgggggcgaa atgcagcgac aatatactta ctgcaacgac catatacctt ttggcggcga ccactccagc cctcgattga gcggcgttaa atagtactgc cgttccaaaa gcgaactgac ctgtaagtgt cttatgtagg aagatccact tgggggctat acctgtctga tgtcgaaagc taccgcagca tatcaacaag tattaacagc cat taaaggc caccgaaggc tcaggcttct atcccgtctg actggcgaaa tattgatctg caaaggtact tgttactaac tgcatttagc taacgcatcg agatcttggc tgcaaatggt aaaggctttc aacgatgtct ttttggcggg attcaaagac taataatgta tggtgtatc t aactactgaa t-acagggggg itgcagatggt aaatgcgctg 'Zcagaaccgt agcgcagtcc gcagatcatc ggttctgtct 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1695 SUBSTITUTE SHEET (Rule 26) (RQ/AU) WO 99/61458 WO 9961458PCT/AU99/00385 37 <210> 49 <211> 1164 <212> DNA <213> Escherichia coli <400> 49 aacaagaacc aacagcgcga aaaggcc tga gaaggcgcgc gcgaccaccg cgcc tggaag gctaaagatg gatctgaaaa ggcaccagtg tttaaagata tactatgtaa ggtgaaat La gtaactactg cttgtcgtgt acaactaatc ggtgcatcta gcaattgcat tctgccattg caggacgctg gccggtaact agtctgcgct aggatgacgc ctcaggctgc tgtccgaaat gtactaactc agattgatcg ggaaaatgaa agatcgattc ttaaagatgg aagcttcagg atgacactaa acttcaactc ttggccgcga ataaagataa aatcaacttt caaccgcgcc ctgttgataa ccaacctgaa actatgcgac ccgtgctggc gtcgagttct cgcgggtcag acgtaacgcc taacaacaac tgagtctgac tgtttcaagt cattcaggtt atctacacta ggccaccatc atcgt taggt aagtagtaag tacaaatgaa tgtaaaattg aagcggcaat caatgccgct aagcaattta attccgctct caacaccact cgaagtgtcc aaaa atcgagcgtc gcgattgcta aacgacggta ttacagcgtg ctgtcttcta cagactcaat ggggcaagtg aacctctcca aataagcaag accctaaaat tactacgatg agtggaacta gatgcttctg gatgcttata aatatcagtg acagctgacc tctctcggtg accaacctgt aacatgtcga tgtcttctgg accgttttac tttctgttgc tgcgtgagct tccaggacga ttaacggcgt atggacagac gttttgatgc tggcagtaga tagttgagaa ccgaagtaga ctcc tactgc cacttaaagc tcattcagac atgctggtgt cgcttaaggc ccgt tcagaa c tgaagcgca aagcgcagat cttgcgtatt ttctaacatt gcagaccacc gactgttcag aatcaaatct gaatgttttg tatcactatt tacaaac ttg tgctggcgac agacggtaag tactagtaag agcgacggaa caaccaatcg caaagatgta tttatctatt tcttgatgat ccgtctggat gtcccgtat t tatccagcag 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1164 <210> <211> 1818 <212> DNA <213> Escherichia coli <400> atggcacaag aaccagtctg gcgaaggatg ctgactcagg gcactgtctg accggtacta tctgaaattg gatggctcc c aagaaaat tg attgcaaaca aatggtactt aagctgagtg gttagttata acaagtgc tg tcattaatac cgctgtcgag acgcagcggg ctgcacgtaa agattaacaa actctgattc accgcgtatc tgaaaattca actctgatac aagcggccac ataaagt tac atggcgatac aatacgacgc cggctggaac caacagcctc ttctatcgag tcaggcgatt cgctaacgat caacttacaa tgacctggct cgggcagacc ggrttggcgca tctgggtttg aatcagtgac aac tagcaac tgtagatatt agatgcaggt tctggcagat tcgctgatca cgtctgtctt gctaaccgtt ggtatctctc cgtgtgcgtg tctattcagg cagttcaacg aatgatggtc aatggtttca ttgactgctc gctgcactta gcaacctatg aacttcagtt actcttctcc ctcaaaataa ctggc ttgcg tcacctctaa tggcgcagac agttgactgt acgaaatcaa gcgtgaacgt agactatctc acgttaatgg agaaagccgt ctgcatctca c tggtggtac ataacaatac cggcagc tgg tatcaacaag tattaacagc cattaaaggc cactgaaggc acaggcgacc atcccgtttg attgtctaaa tatcgacctg ttctggtacc tgacaacggt ggcattaagt aagt tcaaca tgcaaacaaa ccagactaaa SUBSTITUTE SHEET (Rule 26) (RO/AU) WO 99/61458 38 WO 9961458PCT/AU99/00385 accggtactt ctgacaattg tccggtggtg tctctgggga gctgctgcga gccactgttt accgctaccg tccgctacct gtcgtagcta gcatcttacg ggtactggta ggcaaattaa ctggatgctg cgtctggatt tcccgtattc atccagcagg tctctgctgc acaaggc tgc gcggacagca cggctactgc acggcggtac acgttactga ctaaagatgt acggtgatac ttaccaatgg caggtgctaa atacgactta ctggtaaatt cgacagaaac ctatcagctc ccgcagtcac aggacgccga ccggtaactc agggttaa tactggtgat agcctacctg aactcttaaa tgc taccgtt tggtgctggt cattctggca tgtcgcaacg taaaggtact agctgtatat ctctgtcaac tgaagctgtt caccagtgca catcgacaaa caacc tgaac ctatgcgacc tgtgttggca gttaacttta actactgatg gagctgttta actctggata gtcatcgctg caactgcaat atcaactata gccgatggta gt tgatggca gcagatacag gctggtgcgg ggcactgcaa ttccgttcct aacaccacta gaagtgtcca aaagctaacc atgttgacgc gtaaccttac ctcttgctgg atactacgta ctgctggtgt ctgcaagtca aatc tggtgt tgacttctgg acaatgaact gcgcagtaaa atgcttatgt ccaaagatcc ccctgggtgc ctaacctgtc atatgtcgaa aggtaccgca aactggtaat aacaaacaac cgatggtaaa taatttcaaa aacttataca ggcagcagca catgatcggt tacaactcca gacttccact agtggtatca aagcaaagat tttggctgcc tatccagaac tgaagcgcag agcgcagatc gcaggttctg 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 1818 <210> 51 <211> 1344 <212> DNA <213> Escherichia coli <400> 51 atggcacaag aaccagtctg gcgaaggatg ctgactcagg gcgctgtccg accgggacta gacgaaat tg gacggttcga cagaaaattg aaagttagcg gttaaatttg gatacatcag tatgttgttc acgcttaata cagacaggtc actattcaag ggtgacgctg ggt tc tgtaa gcacttttag caaaaccgtc gcgcagtccc cagatcatcc gttctgtctc tcattaatac cgctgtcgag acgccgcagg ctgcacgtaa aaatcaacaa actctgattc accgcgtttc tgaagattca attcttcaac atgcgataac gtgcgaacga gcttgtccct aatctggtaa ccaccaatgt agcctatcaa gcaaagatta caacaaatga aaacagcggc acaaagc tat tggattctgc gtattcagga agcaggcggg tgctgcaggg caacagcctc ttctatcgag tcaggcgatt cgccaacgac caacttacag ggatctggac cggtcagacc ggttggcgcg gctgggattg tacagttcct taccgctgct acataacgta tgacttctat tactttcact ggtcacgacg ccttgctggt agacacaaaa aacagcaaca ctcgcaagtt ggtcaccaac cgccgactat taactctgtg ttaa tcgctgatca cgtctgtctt gctaaccgtt ggtatttctg cgtattcgtg tccattcagg cagttcaacg aatgacgggc aaaggtttct ggtgctaatg gccgcaatgg caaagcgcgg tcggcttccg gatcctgcga aatagtgctg gcagacggta atccaactta ttttctggta gatactttcc ctgaataaca gcgaccgaag ctgtctaaag c tcaaaataa ctggcttgcg ttacttctaa ttgcacagac aactgacggt acgaaatcaa gcgtgaacgt agaccatctc cggtatcagg ctggcgatgc ctaaaacatt atggtaaagc ttaatgctgg acggtgttac gcgcggctgt aggatgcaat ccgatgaact ctgcaaccaa gctcctccct ccaccaccaa tgtccaacat ctaaccaggt tatcaacaag tattaacagc cattaaaggc cactgaaggc tcaggcttct atcccgtctc gc tggcgaaa tatcgatttg gaacgcatta cccggttacg gggaataagt gacaggaacc tggcgttgtt cacagcaaca tggctatgtt tgaaaacggt cgatgttgat cgatccgctg cggtgccgta cctgtctgaa gtcgaaagcg accgcagcag 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1344 SUBSTITUTE SHEET (Rule 26) (ROIAU) WO 99161458 WO 9961458PCT/AU99/00385 <210> 52 <211> 2599 <212> DNA <213> Escherichia coli <400> 52 39 cttctcttag acgatgcagc aggcttcccg atgaaattaa ctaactctga ttgaccgtgt aaatgaaaat atacaacact cgctgcaaat atcc tgccgc attcaaacat cttgcccggc acggcaagtg tgcaatgctg cgtcaggcat ggaaagtgcg cagcggcaga actgaggtga caactgccaa ccctgataaa taaacctgta cacggcttcc tctttatcca gacttcaggc aacgtggcat gcttcgcgga tcatgtggct accatcacta atcgatggcg actgataatt gtacaggata agcagtgata gctcaaggta atacctgcca actatttcgg ctatacaaaa ctatctgatc gcaacttacg aaagtcatgt gcaaaatcgt gttgacaatc aaccttggca ctctgctatt aggtcaggcg taacgcgaat caacaacctg cagcgatctt atctgagcaa tcaggttggt cagttagtaa tcagacggtg cagtcattaa tcatcgcgaa tggattaagc aactccggcc t cc agaa tac tcctttgtga tccatgacga cgttctgttg taaagccggt atacgacggt tgcgcatcag agcctgcagg cgcttctggt gcatggcttc gcttaacttc cggaaatggc gaatactgat tatgaagaca tcaatctggc cgcagaaagc atcaaattaa aagatggcaa ctcaattcaa ataagattgt caggtaatgg ggagtgc tga atagtgcagg ttgatctgaa atgttagtgc atctgagcaa tgcaatctac tgcgttctga acaccgtaaa gagcgtctgt attgctaacc gatggtattzt cagcgtattc tcttctatcc actcagttta gctaatgatg cgtcggaatc tctgataatt taattttcct atcgtccgtt gcaac tcaac cctggtcagt gcgtgacctg aatcatcgac aatccatcga ccagcccttt acacgcgctt agccaaaacg cagccggacg cacgacgttg ctgtcgtcag ggcaagcagc aggcacctcc atgcttgcgg gatctgttcg ttactaacat aaaaattgat aaccggcagt cggtactgat acaagtttat gattgatgca ctacgaaggt taaattaacc tacatcaggt gcaattgact cgctgccaag agatggtaaa atcagaaggt caccaacccg cc tcggtgca caacctgtct ct tc tggtc t gttttacggc ctgttgcgca gtgaactttc aggctgaaat acggcgtgaa gtgaaaccat ttcattctca cagcgtggag ggcatgaacg aaagctctca accatgctca tcttatcgtc aacgcc tgaa gcaggtaaga ccaggtcaga acgacgtctt atgattaaca cctgcgctcc gtgagcctca cgacagaccg tactttcgcc ttcttgagtc ataccgccat cagagttcac tcggaaaaac cggggtgtac gcgaaaactc gacctgattt aactatactg gtgagtgctg actaagcttg atcgaattta gccaatgttg ac tagtgcaa gcaacaaaag aaaacaggaa acgataacgg ggtagcccga ctcgaaacta g tac aaaac c tctgcccgta gcgtattaac aaatattaaa gaccactgaa tgttcaggca tactcaacgt agtccttgct tgacctgccc gaatgaccct tgcgggcggc atatcgctga ataaatccgt aaggcccatt gccggatagc atcccaaagg cacttgatcc t tgggcgccg ctgcgtttta tgaagccctt agtgccagct tagcggcagg gtcgcatcac caagagccac tggtgttc tc acttcttacg gggcgggtac gcttcttcat taatcaacgg tcggcctgga ctaaatttaa ttaatgtaga cggatggttc cagtggctgc caaataccgg atggtaaggc ccgttgcccc ttgaaaataa gcacgttagt agactgcttc ttctggtaaa tcgacaaagc gtttcgactc gccgtatcga agcgcaaaag ggtctgaccc ggtgcgctga actaacggta c tggaagaaa gaaaataatg ccacgattag ttctccagcc attcgttata accagtgctc tctgcgttgg gatccagtgc ctcgaaacag caacagtgac tgcgaccggt ccggacggag cgcccaggcc cacggcgcag cagtgatgcg tcgacaggga acatcaacat ctgaagcgcc ttcctcaagc ccaggtgtaa cccagcttcg ggggatgtcc ggagcaggtc cggttttaat agcgacaggt tagtggagta acttacgacc taaagat tta cactggcgct tgttgaattc tacgacagcc agcagcgaca tgttaacggt tggtaacaat cgaagatgca attggctaaa tgccatcacc agatgctgac 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 1860 1920 1980 2040 2100 2160 2220 2280 2340 2400 2460 2520 SUBSTITUTE SHEET (Rule 26) (RO/AU) WO 99/61458 PCT/AU99/00385 tacgcgaccg aagtgtctaa catgtctcgt gcgcagatcc tgcaacaagc gggtacctct 2580 gttctggcac aggctaacc 2599 <210> 53 <211> 1245 <212> DNA <213> Escherichia coli <400> 53 aacaaaaacc aacagcgcta aaaggtctga gaaggcgcac gccactaccg cgtc tcgatg gcaaaagatg gat ttgcaga gcagtatctg gtaaccatca ttaagtctgc aatggcggaa gcaaatgtaa ttagttcagg aaaagctttg ccaccgggaa aactctgtag ccactgactc gcggtacaga tctgaagcgc aaagcgcaga agtctgcgct aagatgacgc ctcaggccgc tgtctgaaat gtactaactc aaattgaccg gctcgatgaa agattgattc ttggtgatgc agtttgatga ataacctcaa aatcttacgc cctacagcga ttggtgcaga gcattgatga catctgggac acgctgatgt tgctggacaa accgtctgag agtcccgtat ttatccagca gtcgacttct tgcgggccag acgtaacgcc caacaacaac tgattctgac cgtatccggt aattcaggtc ttctacttta tattactcaa ttcagtaaaa agatgaaaat tgctacagtc tgtcgcaaac ttctaccggt cgccgccttg aacagttgtc aacggcttcc agctatcgca ctccgctgta tcaggacgcc ggcaggtaac atcgagcgcc gcgattgcta aacgacggta ttgcagcgtg ctgtcttcaa cagactcagt ggtgcaaatg gggttaaatg ttgcctggcg actgatttaa ggtaat ttaa gctgccaatg ggtat tgata acgccaaaag aagaataaca gcagcgtcaa actgaattca tctgttgata accaacctga gactatgcga tcCgtgctgt tctcttctgg accgcttcac tctctctggc ttcgtgaact tccaggacga tcaacggcgt atggtcagac gtt tt tctgt agacggcagc aactgaccga c taaccagta gtaatgttac ccgcaacgca cattcgtgtc ctggtgatgc ttcatctgag caggtgcttc aattccgttc acaacaccac ccgaagtgtc ccaaa tctgcgcatt ttctaacatc gcagaccact gaccgttcag aatcaaatcc gaacgtactg aatcagcatt ttccaaaaat cgatgcacca tgcttcaggg tgttgtacag gctgaacaaa gtcaggccag tgtccaaggt taccgctact tacgggcaaa aaccaacgat ttctttgggg caccaacctg caacatgtcg 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1245 <210> 54 <211> 1212 <212> DNA <213> Escherichia coli <400> 54 aacaaaaacc aacagtgcga aaaggcctga gaaggtgcgt gcgacgac cg cgtctggatg gcaaaagacg gat ttacaga tcacttaacg ggtgttgatt gtttccagcc agtctgcgct aagatgacgc Ctcaggctgc tgtctgaaat gtactaactc agattgaccg gttcgatgaa aaattgactc ttggtgattc tcactgctgt tgacgt taca gtcgacttct tgccggtcag gcgtaacgcc caacaacaac tgattctgac tgtttccggt gattcaggtt ttctacatta aattactcaa tgcgaaagat caacaccc tg atcgaacgcc gcgatagcta aacgacggta ttgcaacgtg ctgtcatcta cagacccagt ggcgcgaatg gggttgaatg attacaggag ctgactactg gatgcgaaag tctcttctgg accgtttcac tttctctggc tgcgtgagtt lftcaggacga tcaacggcgt atggccagac gtttctccgt ccgctgggac cgacaggtaa gggctgccac cctgcgtatt Ctctaacatt gcagaccaca gaccgttcag aatcaaatcc gaatgtactg tattagcatt ttctgctcaa aaaacctgtt aactgtcgat cgcacagttc 120 180 240 300 360 420 480 540 600 660 SUBSTITUTE SHEET (Rule 26) (RO/AU) WO 99/61458 41 WO 9961458PCT/AU99/00385 gtcgttcaat acgt tgaata actcagaaag gtaacattcc acggcaacag cagttctcag gttgatactt aacctgaaca tatgcgaccg gtgctgtcta ccggtagtga aagccgatgt atcagctgat agggtaaaaa ccacagcgaa gggcttcttc tccgctcctc acaccaccac aagtgtctaa aa tttctactcc cgaatacaaa taaagttgcc ctacgctaca caaagttgtt tgctgatcca cctcggtgcc caacctgtct catgtcgaaa gcgtccattg gacaccgata gctgactctg acggc tccag gttgaattat ctggcacttt gttcaaaacc gaagcgcagt gcgcagatca accatgcaag atggactaac acggcgcggc cggcgcttaa ctacagcaac tagacaaagc gtctggactc cccgtattca tccagcaggc tggtgaagtg gactgcagct tgcgggatat tgatgacact tccgactgcg cattgcacag tgcggtaacc ggacgccgac gggtaactct 720 780 840 900 960 1020 1080 1140 1200 1212 <210> <211> 1758 <212> DNA <213> Escherichia coli <400> atggcacaag aaccagtctg gcgaaggatg ctgactcagg gcgctgtccg accgggacta gacgaaattg gacggttcga aagaaaatcg attaccaaca acgacaggtc gataaattag aaatctggtg caggctactg ggtaaatctg tcagcaggta acgactaaca agcgaaggta gcaactcctg gctacagtga attaccttta gatgctgcga gtctcttaca actgatacca ggtaaaatca ctggacgacg cgtctggatt tcccgtattc atccagcagg tctctgctgc tcattaatac cgctgtcgag acgccgcggg ctgcacgtaa aaatcaacaa actctgattc accgcgtatc tgaaaattca attctgatac aagc tgcaac tttatgatct ggaatggcga attttactac attcagctaa ttaatggttc atatcaccat acgctggtag gtgacggtgc cgacaacctc gtaaagatgt attccggtgt agtcttatgt gcgttaacaa ataaagatta ct Lctgagac ctatcagctc ccgcagtcac aggacgccga ccggtaactc agggttaa caacagcctc ttctatcgag tcaggcgatt cgccaacgac caacttacag ggatctggac cggtcagacc ggttggtgcg tctgggtctg ggtaagtgat gaaaaccgaa taaagtcacc caccaaatct aaaacgtgat ttacaccaca cggtggaagc cgcagctaaa ctctctgaca tccagtagct agtattgagc actgagcaaa ggatgataaa ggataacggc tgctccagca taccagtgct cat cgac aaa caacc tgaac ctatgcgacc cgtgctggca tcgc tgatca cgtctgtctt gctaaccgtt ggtatttctg cgtatccgtg tccattcagg cagttcaacg aatgacggtg aatggtttta ttaacttctg aataccttgt gttggcggcg actgctggta gcgttagctg aaagatggta caggcatacg gctgatatga ttcaatggca ccgttaatcc gaaaccaaag actattgggt ggtggtatta tctgtgactg attggtactg ggttctgcaa ttccgttctt aacaccacta gaagtgtcca aaagccaacc ctcaaaataa ctggcttgcg ttacttctaa ttgcacagac agc tgacggt acgaaatcaa gcgtgaacgt aaactatcac acgtaaatgg ctggcgcgaa taactaccga tagattatac cgggtgtaga ccacccttca ctgtttcttt tagacgatgc aagcgctgct ctgaatatac ctggtgggat cggctgccgc ttaccgcggg ctaacgttgc ttgccgggta ctgtaaatgt cgaccaaccc ccc tgggtgc ccaacctgtc acatgtcgaa agg ta ccgc a tatcaacaag tattaacagc cat taaaggc cactgaaggc tcaggcttct atcccgtctc actggcaaaa tatcgacctg taaaggtact gttaaacacc tgctgcattc ttacaacgct cgccgcggcg tgctgatgtg cgaaacggat aggcaacttg taaagccgcg tatcgcaaaa tacttatcag gacatcttca tgaatccagt c gac tatac a tgcttcagcg gaactccgcg gcttgctgcc tatccagaac tgaagcgcag agcgcagatt gcaggttctg 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1758 SUBSTITUTE SHEET (Rule 26) (RO/AU) WO 99/61458 WO 9961458PCT/AU99/00385 42 <210> 56 <211> 14024 <212> DNA <213> Escherichia coli <400> 56 gtaaccaagg tatataagaa ggggccaaaa gttttgtata tacaaaaaca agtcgaaatt gatttgtggc gatgtggtat aatgctcctc acaggaatct gattatttga aatctggcgt aaatggacat tattttttat agaaaggaga tttatagtct aggtattaac cgattataaa tactcgtttt gtattcctat gactttacat t ttatga tga ctttatttgg atatcctgtc tggtaggggt actcatataa ttccatatgt aggaattaaa gagaacggt t cgtatggagc ggatatatat gcgcattcta cggccttctt tgcatgatac aataatgctg tttgagatta tttactagta aataagggcc gtaattggag gaaaatgata caaggggtga gattataagc gcggtacgtg attctcaaat ctataatctc tcattgacga atcagaaaat atggaataga acgagaaaaa gttctaatta atgtgataaa ataatgccaa tgtggctgga attacatgcg ggagtatata tatatgcttc ctaaaaagtg ccagttgtat tagtattata tgaaagaaaa cctttttgtg atttgatgat tagatatatt gcataaattc ttatcgttct aaaggataac tgtatgctcg taatatgtta tgtttctgaa agcaataata acataaacaa agaactgtta acctttttat ttcatatatc ttttggtcct gt taaagaga aagggttaga ttatagttga tgaatattac gaatgttggc atatatataa aacttctggg ttttcccaaa ttattcaaga cataaatttt gaacaaagaa atcagtagaa ttgtagcacc aagaatattg aatggccacg attagagcgt ttatgttata ttatagaaaa caaattgggt aataattaat ttcaaataat tagagaacat aaatggagtc aagtcagcgg ggggttatca attatatttc cagcagaaaa actatagaaa gatccagggg aagtattttg aaacagagga gaattggtgt cgtaatccta ttgttttatc aggataat ta tctattaaga aatagaatac gtatttggag gttttttttg cttttaaaga attatgattt tttctctccg ttatcacgaa aaaaacttta tggcggctct acatgtttat caaaggcgac aaatatcaaa attttcttct gaatcattca ggtgtttcct aatgcttatc accgtttcaa tcaattatac gatgatacat cgtaacaaga gggaaatata caaatcgaag gataacaata atgctcatga aagttttatc aaaacaaatg tcactatcgg ttacatttgt atgaaaaaaa ctaagttgat tagatgatcg agattttttt aatttatcgt ttgctgctgt gggctaaact gtaatatagt ccatcatatt tgctcattct aaataaaaag taagtttagg ataggttaac acgatttctt agggaataaa acatgggagc gttttctctg gaatgaaaaa tattgcaata tattgataat atgaaaatat agtagtttat acagatggaa gaaaaagatg ttaatacatt gagccatgtt ataacccatt gaatatgatc gaagggt taa aaaactatta taattatgcc atcaatctta tttcattaat caaatttagg tttctttttg tgttaaataa gaaatattgt aaaactacat aaaaaaagat gtgctatttg gtaataaaat cctttccaaa taacacattc ttttttattc tataacaaat tgttttatta gaactgggag agttttattt tagaatagct tgtgtttgca tgtatatttt tcaatatata aataataggg acaagacgga aatagagcaa tccgacacca gcatcaagac aaatttttta tgtattcatt aacccatagc cttagtggct gtgtactcct cagttataac caattttaaa cgaatcgtgt aagggatata atttaaacgc tgattaaagt caaatacagg taaggtataa gatctctatc gcattaaaaa cgtttacaat 120 tcaagatttt 180 caacagtcga 240 tgttgcagaa 300 tgatgcggat 360 tgaatgtgta 420 tggcgaagtt 480 agggaatttg 540 tggtcacgag 600 tattcaagat 660 taaagctgca 720 aacattatat 780 actattaagg 840 ctatttacac 900 tttgatacaa 960 ttttatctaa 1020 ctaaagttaa 1080 cttaaagaag 1140 gaaggtaatg 1200 ttaattattc 1260 acaacgattg 1320 ttgattacca 1380 tattttttat 1440 gaacaaaatg 1500 gttgaaggtg 1560 gagttagaaa 1620 ttattttatg 1680 tcagttacta 1740 atccctttag 1800 tcgataaatt 1860 gggaatgcat 1920 ctgatcttat 1980 tgtgacttat 2040 aataaaacct 2100 cattagtaga 2160 tgatgcgatg 2220 cggcgatagc 2280 tggccttttc 2340 gtattgtcat 2400 aatatgtgct 2460 tttgattact 2520 SUBSTITUTE SHEET (Rule 26) (RO/AU) WO 99/61458 tcgggttatg taaaatatga tatgggggg.

aaagagc ttc atttcaataa tatatttgtc caaaaaaata agaacaggaa actctCacta gagatttggt aggtatagga aaagatgagt aactgcaata aaacgagaat acttggaatt aagtaatat t gaaactacat gggaatc tat catacatgct acagttaggc tgttacttat tcttacgatt tactcaattt cttattggcc aaagattcag tttttcgaat tgctgttgta tgggttaact gtataaatgt aaatatatac tgtctggact tttgcggaac catttagctt acatatatag tcagataatg actaaagcta gaactggcca aaatataaag aataaaacta gaccgttgtt gttataggct gaacaagctg atcaacagtc gtc tcaatt c aaactcatcg aaatatcaaa ttccccagcc agtgataaat ggatt ttacg acgaaaccag rccaaaattat itcaaaatttt :taactttatt i cttaaatttt *aggtacagga *ctttctctta gtatggatga aatgcattaa cggcaaatta tgctatatta gtgtatgcag tattcaaatg gttaatgcca gcgggactac ttaacaatta aaaagagaag actctggcaa gttgctgttt tataacatcc ataaaaaaga ttcatattag gtacctcgaa acatttgcaa acattgatat gttatgttgt agttttaaaa cagtttacca caacgtggat aaaaccatgt tgttagcgtt ca tcagt taa aaacttggca ttatgtgtgt aaagtagaaa gtaaatatgt ttactacagg tacattttaa acaattatag atgattttat ttgtacaagt taac taggac aaacaaggcc agcaccctat tatcgaatga agcc taaaa t agtggggcgg aaatatgtat gtttgaaaaz attccctgai cttcatgaac gcactttaaw L ttatatcctt L tattaactgt tattaccac *caaacaggat gtggtgggct cttctggcat agttacaaca gtgtgcaaaa tatttatatt cagttttaat atcttattat Ctccatattt catggagtgg ttagcattac cgttatgggc cgctcagaac tagtgt tcgg cattcataat gctttttaaa tgatcgcaat actgcttcat aacatatcga accgtcattg ttcatcaaaa gcaatatgca aggtatatcg tgctataaaa aatgtctgtt ccatttatac t ttgt ttgta gggaacattt tgaaggtgga aggccaagga gatgacaaat atcacgaaaa ccacaaggaa cgcagaggaa agttttttac agctgaggat gcaagttatt attgtaaagg tggaattgat cgatattctt 43 a aataaaaaaz a cgtttaagae I aatagttcac I aaaatatgat :gtctgttatt aagtttaact Ttcttggtgct *cgcacattca *cactttgctg *gattgattgg Lctcaattaaa L agtttatatg *gttatctatt tgtcagcact aaagcgatta gatattaaac tgataacttt acagagatta tgcttatgca atcattgaaa tagtgaagtc agcttatgct tggtttgaac tccagcaaaa ttttatatat tagacagt ta acaggaaaag ggaaa ctat a actactgtaa gaaggagatg tacacaggag agtgacatag ggacatccat attgaagatt ggagatattt atggttgtca ttagctgtac atctgcgctg cgtgaaattg agtaaagatg agagaggaat cctgtccaca cttggttggc tatatttgtg tcattcatga tttattaaat ttaccgagaa iaaaaaagaat i accttattaa igagtattgcg catatgataa acatcaagtg tcaagtttga ttaccttatt *gctctgacat *tttgcgtgtg gctggattat *caactagtaa gtctttgtaa ggaatacaaa attactctag cttggtattc ataaagttta ggttttttct ataatatcta tttcaaatat gatgctcatg atagtgggta gttaatattt ttatggtctg atagttaaac tacatcatag attgtaaatt aatataagag aaaaagaata ttcagaagtt gtaatggaac aagttattgt Ccacccccat aacaaaaaat gtgatatgga gcgctgaagc ctacttttag cgaatgacaa ataggcaata ctataggatt ctgatattta tttttcacac1 taaggaatca cgatgccaat gtggaattaa aatctattaa aaattgcgtt atattctgtcz atgatataca PCT/AU99/00385 tttaagagat 2580 gtttattcca 2640 gaaaatgcaa 2700 tgaataaaat 2760 ctttaggtag 2820 ttagtaaaat 2880 taggacaaga 2940 ttttggactt 3000 gcaaaaattt 3060 cgtttgtcat 3120 taaaaggtat 3180 tcatatttgg 3240 aagcctatat 3300 taatatcgtc 3360 aatacatatc 3420 caaaagttaa 3480 tttttatttt 3540 taacattggg 3600 ctacggtccc 3660 cacgcaatga 3720 tttcatcatt 3780 ggacagaagg 3840 ttattgatgc 3900 aacaaatgct 3960 ttagccattt 4020 actttatatg 4080 gatgaaaatg 4140 tgtaaatgaa 4200 tgaaaataaa 4260 ggttgctctt 4320 tccaacactg 4380 tttcgttgat 4440 cactaataaa 4500 acaaattgta 4560 ctttggttct 4620 cttttttgga 4680 aacactttat 4740 ttggcatgac 4800 agcccagtta 4860 :aaaaaaaat 4920 :tattggatg 498 0 ::cttgcagat 504=0 itactcggaa 5100 :ttacctagt 5160 7gaattttat 5220 laattcagat 5280 ~atattagaa 5340 :tctcttata 5400 SUBSTITUTE SHEET (Rule 26) (RO/AU) WO 99/61458 PCT/AU99/00385 44 catttccttt taaaagtata ttaaaagcaa ttttaaagag ggaaaggcct 5460 agagaaaaag cgatggattt aatatagaga gatagtagcg aaaaaagca t agtaagcgag aatattattg tctgcaaaac tactctggta ttttggaaac aatcctgatg tttaatgaat gggcatatac ccaaccttat aaaaaagt ta ttctttcagg aaaatttttt gcagattttc gaggtatata tctagctgag atcttttaat ttttcacttg ggtacagcca gtctccagaa tcgcttttta tggacttgtt tgcagttgca ttatgcatgt aacaaggaaa tttagggaat atggttgatg ctcagtccgt tggtaaagga tgt gaaac ca tgatactttg tactcttgct ttctctttta caacgtattt aaacaacgtg agtagcgctg gcaaaagtcg ataatgatag ttcctcggtt ttattacaag attaaattat accaatcttt Cttttgcgcc agtggtactc cattaaatag cgaatcttac attgtgatgt ggattggtta aaatagatca ttaatgcaca tacattctct atattgccaa ataaagatca tttatttagt tgatggtttt ctaaacttga t tgaaggcgg tactatctga caaaaaacca atgaacctac ttttagatgt atgactaaag t tt ttgc ttg acagaacgca cactatggag gatgaagtat tatacagccg ggat tggaaa caggaaatcc aaac tt tacg aatggtatat at tactcgag atggattcgt ttacaacagg cagtttgtcg atcgaagaaa ggtgatgtca cttggagatc gaaatgattt aaatcgcatg ttattgctgg atgatgttga ttttggattt gaggtatttt aggcgaatgt cgtcgtgtat ggaaacttga gtgaatctta atggtccaaa gctttcatga caaagcgtga atttaatgag c t ttat taaa tattcttcct tatttatgac aaggaatgtg ttcagttatt tccatttagt atataatatt tgcaactgct atgcacggga ggcaaaaaag acaaattgaa gcc tggaggg catagatgtc ttattcatta aactctgata tgtgaaacaa tcgctcttat ataaagggta tagaccatat atctgactga ataatttagc atgtcgatgc acaaaacgcg ctcaaaaaga catattggat tgttcaatca gacttgcaaa tacgagattg agcaacccga aaatggcagc aaggcattgt ttgttgctgt cgagcaaagc ctgaaatggt gtttttctgt tc accaagga gttggtttta tttttcttca agctaacagt cattcatgct ttatcctaag gccaacaaat taaccgtcag tgacaatttt tgctgtggaa attcttacat caatactata agtaagagct tgcatgcgtg tttcaacact ttttttaaat accgatgcaa ccatgccctc gacaaggatt tttagggcat gctactcaag ctcggaattg ttaatcaaaa ggggtaacat aataaagaag aatgacgcga gaattgggtc gaaattgaat tacaggtgta tgaagttcat ttatcaagat t tcatctaac tgctatgagt aattggtaca tttctatcaa atccacccct cacggtaaat tgaatctcca tattgcacaa gggacatgca agattttgtg agcacaactt agattcggtt tgatcctcgt taatctcaaa tgccaaagat aagcttagct atggttggat cgtactcggg cagaaaatcg tcttatcctg gccdacaaaa ttagcacacc gaaccttatg tttgggcgtg catccaagta aacaattctc gtagatgata gagatgcctt ctgcctttta ttccttcggg gttactatcc tgatgctcaa ataaatatgt aattaaaatg attttataat ttaaaattta attatcgatt aatcgaaaat attgcattgc ttgacgctat ttaattgcgg tggtaaaagc tcaaaagacg cccgatctta actggacaag ggtatcaaac ccacatggtt ctcactagaa cacgtagcag ttacgtttac gcttcaacct ttttatcctc tatcgagagt cgccgtggag ggcttggaat aaagattatg attgcaacag ggtattaaga gaaggacagg tatttccgac cttggttgga cttgaagccg ctggaatgat cagctattac atgaat tgaa accaggttta ccgat t ttat ataatgtaaa aaccgattat ctatcgcaaa attaccgttc attctcatgt cgaatgttgt tggcttctgc tacacaaaat 5520 ttcatatttt 5580 aaatttgaat 5640 ttcatttttt 5700 ttgcgctaac 5760 tgggaattat 5820 gttcgctgat 5880 ttgcaatcaa 5940 tactgaatat 6000 ccctggatat 6060 taagatatta 6120 tgtaatacaa 6180 tgcattaggg 6240 tgatgtatat 6300 tgatgaatct 6360 caatgcgtgt 6420 atatattcaa 6480 atggatctta 6540 gccgagcctc 6600 ctaacccaaa 6660 ttctaaagga 6720 tttcttttga 6780 tggaagcaat 6840 cagaattata 6900 gttcccctta 6960 catatggtat 7020 aaacgtttgt 7080 catgtttgta 7140 ttagaatgca 7200 gagtccaata 7260 tgagctttgt 7320 atgctccagg 7380 cagctgaagt 7440 gaccagaaat 7500 ctaaaaaaca 7560 gatgaataag 7620 ccgacgcctc 7680 cttgttggat 7740 tttggcagca 7800 atatgagaat 7860 taaactgctt 7920 ggaagacgaa 7980 aattgcaggt 8040 agtaatgcca 8100 gattccggcg 8160 tgtttgggga 8220 aagcatttat 8280 SUBSTITUJTE SHEET (Rule 26) (RO/AU) WO 99/61458 WO 9961458PCT/AU99/00385 45 gtcatggaga aata ttggaa gtaggttata ctacttgatg ggtcttgaaa tgtttttaca atttgattgt cacagggcta cctttgaacg tttatggtat attatatagt cacaacataa attataattg aggatataat tcgtctttgg taacaccttg agtgataaca attaaatggt atctgcgttt ggcagaccac catcgctaat ttatgggtat tttttattat gacttctggt tgaggaattg ctcatacatt tgaatctatt tattggttgg aacaggagat ctctgagtca agatgccgt t gcttaaattg aaaatttgat tggtgagggg tggtacagca atacattccc tattgaagtc ttacaaacat cgggaaatta cgaatttctc gttaaaactg tatgtacggc cgaagttacc ggctcgcccg tgacttccct cgcttacgtt ggtgatcaac taaagccctc tgccatacga caggtattga aagggcatat taacgcttct atacatacaa ttcccaagac ggaaaacgag ttggttcgtt attgacagaa ctggcagcac tatagcattc tgcttattgc tcgcgcatat atgtctgatg ccactttctc ttacaaacga aatgaacagc aatattattc catgcgttaa gttatagcta caaggtaaaa attgagagag gtaaataagt aatcactatt agaaaattta gatctagatt gattttgctg agtgacgttg gtatgtaaag gcgttggtag cttgtgtcta cagcaacgta tcgattgacc ctttctttaa aaagtaaccc cttggcgcag agttcagggg gaagattaac ggcgaagaac aaaccgaaaa gcgcttgcga accgaagaga gccagccata atcagcggtg cctgtcgatg gatcacctgt tccgggaacg ggcgcaccgg tatatggcaa ctgcacgatt tacgttcgat tcatcaacta ctggtttctt tttgccacaa tttggcgaaa cctggtggta attgaactag ttctacgaag cttcttaaat tggctatcgc tttaacaata cgccaataat gtgaactata ctttgctacg atcgctttgt tagaaccatg aacgtaatcc aagaaagtgt ttgtaacgtt gtgaactatc ttgtcgaaaa ggaatagtgg gacctgacat ttattcgatt taatggaaaa gatcttggca gtgatatatt ccgccattgg aaaagagtga cagagtatat aaggtgagcg ggatgcatca ttggcgataa cgtatagtct attatttggg atatgaaatc tgaatgaaga ccattgtttt aaggtttaca tctatttcgc acccgatgga ataccggact aaaccaaacg tcggttatat gcgcagcggg tggaattaat aaaaatacta tgtgagcttg acaacaaagc ggttggaatc gaaaaccaac ttgtaaggtc ttttgctagg gggtgttgaa gaattcgttt acaatagtat tacaaccaaa gagcaaagct aaacaaatga tgctgtagtt tccaaagcag actttcaggc tgtggctgaa cgggcgaaat tcaggaagat tttctgtgat tggaat tata tgtaccgctt gcctaatcgt aatattcatg ttacaatgtt at caaaaga a aacagaaaaa atcgttatgg aacctatgat aattgaagat tgtacagcat tagtcatcgt atacaaagtc ccatcgttct aactaaacta tgagaatccg, agaggatgat tttaacctgc tattgcctgg aggeggtgat ggatgcgggc cacgttccat ttacaacggc gcgcgatgtc tggtcgctat caacgtcaaa tccggtggtg caaagtacac aagtaatgtt cggaaacaat ccga tggagc ataaaattac ttcaatatcg tactcctctt aaaacgaatc agatgaaaaa gcctctctct ggggggagac cattttgaaa gataaatgat tgcgattggc atggccggtg tttttacaac ctatcatgtc cagttaaggg actgcaccag ccattgcttc gctattaaaa ccagaatatg caagggcatg gaaaccgcag tttaaggcat tgtgaacagg caatttcaag tgtgttgtat gacattagtc ac taagaa ta atggttatcg gtaaaaaaaa gaagttttcc aagaaaatta gaacattgga gtcaccgcaa ggcataatcc attataagac tttaaagcct cgcattgggc gtccgcctca gtcgatgtgc ctcggagtgg atgaagctgg cagcgtctgg cagcaaatca aacctcacgc gacgccattg aacacgccgg gtctcatatc agcaaaagt t ccctcgaaaa ccttcacaag ggggtaataa at ttc tatag aac cgc ccgg ttgcagacag gtgggtaagt ttttcaacgc ttaccgaagt gacgatgtgc ttagataata gtacaggcag tctctggtga aaaaaccatt aaataaataa caatagcgat tagttcttgc atgcaactcc ctgaaactgg aaaatactgg aattgtatat ctgtttatct ttgcctcatc attgtcctgc gccctgttga taaaatcgaa attatatcta tgcaaactaa tagtcgaaat gaccatgggg t tgtgaaacc tcgtgctttc atgaatcgat ctcttaatct agaaagaacg atgatattcg gtgcctatgg ccagcgaagc tggatatcgg atggcggcat tgcgcgaagg cagaagccaa atctgcgtga cgctcaagct aagcccgat t acggcaattt 8340 8400 8460 8520 8580 8640 8700 8760 8820 8880 8940 9000 9060 9120 9180 9240 9300 9360 9420 9480 9540 9600 9660 9720 9780 9840 9900 9960 10020 10080 10140 10200 10260 10320 10380 10440 10500 10560 10620 10680 10740 10800 10860 10920 10980 11040 11100 11160 SUBSTITUTE SHEET (Rule 26) (RO/AU) WO 99/61458 WO 9961458PCT/AU99/00385 46 ccccaacggt catcaaacac gtttgacgaa gttcctcgaa cgttgatgtg tat taaagaa ttacttccgt actggtgtgc tccggcaagc ggaacagcat ctttgccgac tgtggaatca gctaagtgag gttattgcgg ttatctagga acgattgaga ccttatataa atccctgttt gagtgctttg attgatggta gtcattagtg aacctaatgc t aa t cataac gatgtgatta agatatt tac agaggcacgg ttgatttcac caataatagc ctgctttaac agttgcacag tgcattcgaa tattcaaggc cgtcaggcat tttggagatg acaataccca tttgtggatg tgctttctaa atacgacaga catccaaata tttgttacaa tacgttgcac aatggatatg ggtgttccta gtcac taaaa agatcgccaa aaaatatata ttagttatta caagccgcat attcctaacc ggcgcggata aaagggcagt aaaaatcccg gtgactgccg cgtatgcgca gatttcgctt c tgaaaggaa ggtgagatca tttagccgcg tggcgcttta cgcggtgatg tgattattta tatatgatga aaagattaat cgttcctttg aaaggagaac acgatattat ttaactgaat aaacacttcg cgtttcagca gttcacagtt tgatgcgatg tttattcgct gc tgaaagaa cagagacgat gtttgaatgg agagcaaaag cgcagagaag tagcctgatg tgattttcta aggattatca tgcagcgtgc ggaaaatgaa ttctaaatca atgttttggc aagcccctta gagtgtctga gcgttgttta tatctactaa atgattgtca ttgttattga atcgcccact gtgttcctgc catctattta taatttcgct ctgatacagc atctagcggt cgctgctgcc *tgggcattgc ttatcgaggg *gcgcgaagat caggcggcac aggaagacgc actgcgacag aaacgctggg acagcaaact aggcgc tggc acctgcgctc taaagctaat cattaatcat atatgtgggc agaaataaag cttaagacat aaaatggaac agccaaagtt acatgtctgc gcacacttat atgcacagtc attcatgaac gcttgccaga gtaaaaggaa ttaaataatc gtccgtcttc aaagggctgc aaagaatccg ttcgccacag tggtaatgaa gaatgcggca tgtaagatgc gggctttcat tttgtatggt acaaataaag aggaaagaaa tttaaaaaag gtcaatatta tgatcatact tactcatata aataggagac agacaatgca tattattggc cggtataac t atgggaatgc aatttactaa atgaaattta aacccc tgac ggaatgccgc gacgacaccc gtaatgcggt 11220 ctttgatggc ctactacatt catccacgat Cccggtaatg catc tacggt cggcatgatc cgaaatggtg ggcgcaaccc ggtggatcgc ctccaacacc ggaaaagaaa taagcgtatt ttttttatgt ttttgtactg tttttcatcg t taaaataat gtatcctgca cattttccag gacaagagtc tggtcctcgg tttctaaaat aagatattgc ataagagtcg caaaacatga atattgtttg agaatttatg aaatgacgat taaatcgaaa tgaaatcgac catcgctatt gaaaagcagt aatctztgcat atttttggtg caagaatgtg gttaatggtt tattttaatg ttacacgggg atgataggta gggaggtttt tatgttacat tatataggct gcgggagcca gtgtgcggaa gaaaacacgt attatggctt taatactctg aggagtaaac gattttgacc gtcggcctgc ccacgtctct tcgaaaaccg ggcgaaatga ccgtggctgc cgcgaccgga gttgaggcaa accgatggca gaaccggtgg actaaagctc taagattata ataacgacta accaatttgc cttatgtaat tgagacaata tcagtcctgc atgataacga gtcgcagagg atagatcaag gatgggtatt agagaaga ta gcttaataga cagttacgca agatgctcct aatggcagtc caaatattat tcactggcgc tataatataa aatatctgac catggacaga tggttttgat ctggaagtta gttctgacta t tgaagtgc t ttgctattgc ttgaaccaat gtggcgctat ttcatgcaaa ttgctcctgg cgggtgcagt ttataggtat atccagcaag tccaaatggg ctttttaagc ataca tt tt t aatg gctgtttcct 11280 tggcagaagc 11340 cctggaacac 11400 gacacgcctt 11460 gcgctcacca 11520 tggtcgccga 11580 tggcggcgtt 11640 ttaatcgcgt 11700 tcagcatgac 11760 tgcggttgaa 11820 ttcttaaatt 11880 ttaaagtaat 11940 taccgcaact 12000 atttcacgtc 12060 aacaaatgtg 12120 gatttttatt 12180 aatatttcac 12240 cgtcatcgca 12300 agtggttcat 12360 acggatgaga 12420 aaaggaaaaa 12480 taaaaacaga 12540 gtctttgagg 12600 attagtgaaa 12660 gatgagctta 12720 cactttctct 12780 attagatctg 12840 atggagaata 12900 gaaggcgagt 12960 aatgataatg 13020 aacttcctag 13080 aagatatttc 13140 tggtagagaa 13200 tgctctggtt 13260 ttcaaccaac 13320 taatgataag 13380 aactataaaa 13440 tatttctccc 13500 catatactca 13560 ggctaaatgt 13620 aattaagcag 13680 gggggctgtt 13740 agaaatgaaa 13800 actaatgttt 13860 tatcctttac 13920 atacgttatt 13980 14024 SUBSTITUTE SHEET (Rule 26) (RO/AU) WO 99/61458 WO 9961458PCT/AU99/00385 47 <210> 57 <211> 1758 <212> DNA <213> Escherichia coli <400> 57 atggcacaag aaccagtctg gcgaagga tg ctgactcagg gcgctgtccg acagggacta gatgaaattg gacggttcaa aaaaaaatcg attaccaaca acgacaggtc gataaattag aaatctggtg caggctgctg ggtaaatctg tcagcaggta acgactaaca agcgaaggta gcaactcctg gctacagtga attaccttta gatgctgcga gtctcttaca actgatacca ggtaaaatca ctggacgacg cgtctggatt tcccgtattc attcagcagg tctctgctgc tcattaatac cgctgtcgag acgccgcagg cggcccgtaa aaatcaacaa aCtccgattc accgcgtatc tgaaaattca attctgatac aagctgcaac tttatgatct ggaatggcga attttactac attcagcttc ttaatggttc atatcaccat acgctggtag gtgacggtgc cgacaaccac gtaaagatgt attccggtgt agtcttatgt gcgttaacaa ataaagatta ctactgagac caatcagctc ccgcagtcac aggacgccga ccggtaactc agggttaa caacagcc tc ttctatcgag tcaggcgatt cgccaacgac caacttacag tgacctggac cggccagacc ggttggtgcg tctgggtctg ggtaagtgat gaaaaccgaa taaagtcaca cactaaatct aaaacgtgat ttacaccaca cggtggaagc cgcagctaaa ctctctgaca tccagtagct agtattgagc actgagcaaa ggatgataaa ggataacggc tgctccagca taccagtgct catcgacaaa caacc tgaac ctatgcgacc cgtgctggca tcgctgatca cgtctgtctt gctaaccgtt ggtatttctg cgtattcgtg tccatccagg cagttcaacg aatgacggcg aatggcttta ttaacttctg aataccttgt gttggcggcg actgctggta gcgttagctg aaagatggta caggcatacg gctgatatga ttcaatggca ccgttaatcc gaaac caaag actattgggt ggtggtatca tCtgtgactg attggtactg ggttc tgcaa ttccgttctt aacaccacta gaagtgtcca aaagc taacc ctcaaaataa ctggcttgcg ttacttctaa ttgcgcagac aactgacggt acgaaatcaa gcgtgaacgt aaaccatcac acgtaaatgg ctggcgcgaa taactaccga tagattatac cgggtgtaga ccacccttca c tgt ttc tt t tagacgatgc aagcgctgct cagaatatac c tggtgggat cggctgccgc ttaccgcggg ctaacgttgc ttgccgggta c tgtaaatgt cgaccaaccc ccctgggtgc CCaacctgtc acatgtcgaa aggtaccgca tatcaacaag tattaacagc cattaaaggc caccgaaggc tcaggccact atctcgtctt gctggcgaaa gatcgacctg taaaggtac t gttaaacacc tgctgcattc ttacaacgct cgccgcggcg tgctgatgtg cgaaacggat aggcaacttg caaagcagcg catcgcaaaa tacttatcag gacatcttca tgaatccagt cgactataca tgcttcagcg gaactccgcg gcttgctgcc tatccagaac cgaagcgcag agcgcagatc gcaggttctg 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1758 <210> 58 <211> 1758 <212> DNA <213> Escherichia coli <400> 58 atggcacaag aaccagtctg gcgaaggatg ctgac tcagg tcattaatac caacagcctc cgctgtcgag ttctatcgag acgcagcggg tcaggcgatt ctgcacgtaa cgccaacgac tcgctgatca Cgtctgtctt gctaaccgtt ggtatttctg c tcaaaataa ctggcttgcg ttacttctaa ttgcgcagac tatcaacaag tattaacagc 120 cattaaaggc 180 caccgaaggc 240 SUBSTITUTE SHEET (Rule 26) (RO/AU) WO 99/61458 WO 9961458PCT/AU99/00385 48 gcgctgtccg acagggacta gatgaaattg gacggt tcaa aaaaaaatcg attaccaaca acgacaggtc gataaattag aaatctggtg caggctgctg ggtaaatctg tcagcaggta acgactaaca agcgaaggta gcaactcctg gctacagtga attaccttta gatgctgcga gtctcttaca actgatacca ggtaaaatca ctggacgacg cgtctggatt tcccgtattc at ccagc agg tctctgctgc aaatcaacaa actccgattc accgcgtatc tgaaaattca attctgatac aagctgcaac tttatgatct ggaatggcga attttactac attcagcttc ttaatggttc atatcaccat acgc tggtag gtgacggtgc cgacaaccac gtaaagatgt attccggtgt agtcttatgt gcgttaacaa ataaagatta ctactgagac caatcagctc ccgcggtcac aggacgccga ccggtaactc agggttaa caact tacag tgacctggac cggccagacc ggttggtgcg tctgggtctg ggtaagtgat gaaaaccgaa taaagtcaca cactaaatct aaaacgtgat ttacaccaca cggtggaagc cgcagctaaa ctctctgaca tccagtagct agtattgagc actgagcaaa ggatgataaa ggataacggc tgctccagca taccagtgct catcgacaaa caacc tgaac c tatgcgacc cgtgctggca cgtattcgtg tccatccagg cagttcaacg aatgacggcg aatggcttta ttaacttctg aataccttgt gttggcggcg actgctggta gcgttagctg aaagatggta caggcatacg gctgatatga ttcaatggca ccgttaatcc gaaaccaaag actattgggt ggtggtatca tctgtgactg attggcactg g gttctgcaa ttccgttctt aacaccacta gaagtgtcca aaagctaacc aac tgacggt acgaaatcaa gcgtgaacgt aaaccatcac acgtaaatgg ctggcgcgaa taactaccga tagattatac cgggtgtaaa ccacccttca ctgtttcttt tagacgatgc aagcgctgct cagaatatac ctggtgggat cggctgccgc ttaccgcggg ctaacgttgc ttgccgggta c tgtaaatgt cgaccaaccc Ccctgggtgc ccaacctgtc acatgtcgaa agg ta ccgc a tcaggccact atctcgtctt gc tggcgaaa gatcgacctg taaaggtact gttaaacacc tgctgcattc ttacaacgct cgccgcggcg tgctgatgtg cgaaacggat aggcaac ttg caaagcagcg catcgcaaaa tacttatcag gacatcttca tgaatccagt cgactataca tgcttcagcg gaactccgcg gcttgctgcc tat ccagaac cgaagcgcag agcgcagatc gcaggttctg 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1758 <210> 59 <211> 1758 <212> DNA <213> Escherichia coi <400> 59 atggcacaag aaccagtctg gcgaaggatg ctgactcagg gcgctgtccg accgggacta gacgaaattg gacggttcga aagaaaatcg attaccaaca acgacaggtc gataaattag aaatctggtg caggctactg ggtaaatctg tcattaatac cgctgtcgag acgccgcggg Ctgcacgtaa aaatcaacaa actctgattc accgcgtatc tgaaaattca attctgatac aagctgcaac tttatgatct ggaatggcga attttactac attcagctaa ttaatggttc caacagcctc ttctatcgag tcaggcgatt cgccaacgac caacttacag ggatctggac cggtcagacc ggttggtgcg tctgggtctg ggtaagtgat gaaaaccgaa taaagtcacc caccaaatct aaaacgtgat ttacaccaca tcgctgatca cgtctgtctt gctaaccgtt ggtatttctg cgtatccgtg tccattcagg cagttcaacg aatgacggtg aatggtttta ttaacttctg aataccttgt gt tggcggcg actgctggta gcgttagctg aaagatggta ctcaaaataa ctggcttgcg ttacttctaa ttgcacagac agctgacggt acgaaatcaa gcgtgaacgt aaactatcac acgtaaatgg ctggcgcgaa taactaccga tagattatac cgggtgtaga ccacccttca ctgtttcttt tatcaacaag tattaacagc cattaaaggc cactgaaggc tcaggcttct atcccgtctc actggcaaaa tatcgacctg taaaggtact gttaaacacc tgctgcattc ttacaacgct cgccgcggcg tgctgatgtg cgaaacggat 120 180 240 300 360 420 480 540 600 660 720 780 840 900 SUBSTITUTE SHEET (Rule 26) (RQ/AU) WO 99/61458 WO 9961458PCT/AU99/00385 tcagcaggta acgactaaca agcgaaggta gcaac tcc tg gctacagtga attaccttta gatgc tgcga gtctcttaca actgatacca ggtaaaatca ctggacgacg cgtctggatt tcccgtattc atccagcagg tctctgctgc atatcaccat acgc tggtag gtgacggtgc cgacaacctc gtaaagatgt at tccggtgt agtcttatgt gcgttaacaa ataaagatta ctactgagac ctatcagctc ccgcagtcac aggacgccga ccggtaactc agggttaa cggtggaagc cgcagctaaa ctctctgaca tccagtagct agtattgagc actgagcaaa ggatgataaa ggataacggc tgctccagca taccagtgct catcgacaaa caacctgaac ctatgcgacc cgtgctggca 49 caggcatacg gctgatatga ttcaatggca ccgttaatcc gaaaccaaag actattgggt ggtggtatta tctgtgactg attggtactg ggttctgcaa ttccgttctt aacaccacta gaagtgtcca aaagccaacc tagacgatgc aagcgctgct ctgaatatac ctggtgggat cggctgccgc ttaccgcggg ctaacgttgc ttgccgggta ctgtaaatgt cgaccaaccc ccctgggtgc ccaacctgtc acatgtcgaa aggtaccgca aggcaacttg taaagccgcg tat cgc aaaa tacttatcag gacatcttca tgaatccagt cgactataca tgcttcagcg gaactccgcg gcttgctgcc tatccagaac tgaagcgcag agcgcagatt gcaggttctg 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1758 <210> <211> 1758 <212> DNA <213> Escherichia coli <400> atggcacaag aaccagtctg gcgaaggatg ctgactcagg gcgctgtccg acagggac ta gatgaaattg gacggttcaa aaaaaaatcg attaccaaca acgacaggtc gataaattag aaatctggtg caggctgc tg ggtaaatc tg tcagcaggta acgactaaca agcgaaggta gcaactcctg g~tacagtga attaccttta gatgctgcga gtctcttaca actgatacca ggtaaaatca c tggacgacg tcattaatac cgctgtcgag acgccgcagg cggcccgtaa aaatcaacaa act ccga tt c accgcgtatc tgaaaattca attctgatac aagctgcaac tttatgatct ggaatggcga attttactac attcagcttc ttaatggttc atatcaccat acgctggtag gtgacggtgc cgacaaccac gtaaagatgt attccggtgt agtcttatgt gcgttaacaa ataaagatta ctactgagac caatcagctc caacagcctc ttctatcgag tcaggcgatt cgccaacgac caacttacag tgacctggac cggccagacc ggttggtgcg tctgggtctg ggtaagtgat gaaaaccgaa taaagtcaca cactaaatct aaaacgtgat ttacaccaca cggtggaagc cgcagctaaa ctctctgaca tccagtagct agtattgagc ac tgagcaaa ggatga taaa ggataacggc tgctccagca taccagtgct catcgacaaa tcgctgatca cgtctgtctt gctaaccgtt ggtatttctg cgtattcgtg tccatccagg cagttcaacg aatgacggcg aatggcttta ttaacttctg aataccttgt gttggcggcg actgctggta gcgttagctg aaagatggta caggcatacg gctgatatga ttcaatggca ccgttaatcc gaaaccaaag actattgggt ggtggtatca tctgtgactg attggtactg ggttctgcaa ttccgttctt ctcaaaataa ctggcttgcg t tact tc taa ttgcgcagac aactgacggt acgaaatc aa gcgtgaacgt aaaccatcac acgtaaatgg c tggcgcgaa taactaccga tagat tatac cgggtgtaga ccacccttca ctgtttcttt tagacgatgc aagcgc tgc t cagaatatac ctggtgggat cggctgccgc ttaccgcggg ctaacgttgc ttgccgggta ctgtaaatgt cgaccaaccc ccctgggtgc tatcaacaag tattaacagc cattaaaggc caccgaaggc tcaggccact atctcgtctt gc tggcgaaa gatcgacctg taaaggtact gttaaacacc tgctgcattc ttacaacgct cgccgcggcg tgctgatgtg cgaaacggat aggcaacttg caaagcagcg catcgcaaaa tacttatcag gacatcttca tgaatccagt cgac tataca tgcttcagcg gaactccgcg gcttgctgcc tatccagaac 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 SUBSTITUTE SHEET (Rule 26) (RO/AU) III. f IlAf VV. 7IIy 50 Y%-I /AI Cgtctggatt ccgcagtcac caacctgaac aacaccacta ccaacctgtc cgaagcgcag tcccgtattc aggacgccga ctatgcgacc gaagtgtcca acatgtcgaa agcgcagatc attcagcagg ccggtaactc cgtgctggca aaagctaacc aggtaccgca gcaggttctg tctctgctgc agggttaa <210> 61 <211> 1758 <212> DNA <213> Escherichia coli UJ99/00385 1620 1680 1740 1758 <400> 61 atggcacaag aaccagtctg gcgaaggatg ctgactcagg gcgctgtccg acagggacta gatgaaattg gacggttcaa aaaaaaatcg attaccaaca acgacaggtc gataaattag aaatctggtg caggctgctg ggtaaatctg tcagcaggta acgactaaca agcgaaggta gcaactcctg gctacagtga attaccttta gatgctgcga gtctcttaca actgatacca ggtaaaatca ctggacgacg cgtctggatt tcccgtattc atccagcagg tctctgctgc tcattaatac cgctgtcgag acgccgcagg o tgcacgtaa aaatcaacaa actccgattc accgcgtatc tgaaaat tca attctgatac aagctgcaac tttatgatct ggaatggcga attttactac attcagcttc ttaatggttc atatcaccat acgctggtag gtgacggtgc, cgacaaccac gtaaagatgt attccggtgt agtcttatgt gcgttaacaa ataaagatta ctactgagac caatcagctc ccgcggtcac aggacgccga ccggtaactc agggttaa caacagcc to ttctatcgag tcaggcgatt cgccaacgac caacttacag tgacctggac cggccagacc ggttggtgcg tc tgggtctg ggtaagtgat gaaaaccgaa taaagtcaca cactaaatct aaaacgtgat ttacaccaca cggtggaagc cgcagctaaa ctctctgaca tccagtagct agtattgagc actgagcaaa ggatgataaa ggataacggc tgctccagca taccagtgct catcgacaaa caacctgaac ctatgcgacc cgtgctggca tcgctgatca cgtctgtctt gctaaccgtt ggtatttctg cgtattcgtg tccatccagg cagttcaacg aatgacggcg aatggcttta ttaacttctg aatacc ttgt gttggcggcg actgctggta gcgttagctg aaagatggta caggcatacg gctgatatga ttcaatggca ccgt taatcc gaaaccaaag actat tgggt ggtggtatca tctgtgactg attggcactg ggttctgcaa ttccgttctt aacaccacta gaagtgtcca aaagctaacc Ctcaaaataa Ctggcttgcg ttacttctaa ttgcgcagac aactgacggt acgaaatcaa gcgtgaacgt aaaccatcac acgtaaatgg o tggcgcgaa taac taccga tagattatac cgggtgtaga ccacccttca o tgt tto ttt tagacgatgc aagcgctgct cagaatatac ctggtgggat cggctgccgc ttaccgcggg ctaacgttgc t tgccgggta ctgtaaatgt cgaccaaccc ccctgggtgc ccaacctgtc acatgtcgaa aggt accgca tatcaacaag tattaacagc cattaaaggc caccgaaggc tcaggccact atctcgtctt gctggcgaaa gatcgacctg taaaggtact gttaaacacc tgctgcattc ttacaacgct ogoogoggog tgctgatgtg cgaaacggat aggcaacttg caaagcagcg catcgcaaaa tacttatcag gacatcttca tgaatccagt cgactataca tgcttcagcg gaactccgcg gcttgctgcc tatccagaac cgaagcgcag agcgcagatc gcaggttctg 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1758 <210> 62 <211> 1758 <212> DNA <213> Escherichia coli <400> 62 SUBSTITUTE SHEET (Rule 26) (ROIATJ) WO 99/61458 51 WO 9961458PCT/AU99/00385 atggcacaag tcattaatac caacagcctc aaccagtc tg gcgaaggatg ctgactcagg gcgctgtccg accgggacta gacgaaattg gacggttcga aagaaaatcg attaccaaca acgacaggtc gataaattag aaatctggtg caggctactg ggtaaatctg tcagcaggta acgactaaca agcgaaggta gcaactcctg gctacagtga attaccttta gatgctgcga gtctcttaca actgatacca ggtaaaatca ctggacgacg cgtctggatt tcccgtattc atccagcagg tctctgctgc cgctgtcgag acgccgcggg ctgcacgtaa aaatcaacaa actctgattc accgcgtatc tgaaaattca attctgatac aagctgcaac tttatgatct ggaatggcga attttactac attcagctaa ttaatggttc atatcaccat acgctggtag gtgacggtgc cgacaacctc gtaaagatgt attccggtgt agtcttatgt gcgttaacaa ataaagatta ctactgagac ctatcagctc ccgcagtcac aggacgccga CCggtaactc agggttaa ttctatcgag tcaggcgatt cgccaacgac caacttacag ggatctggac cggtcagacc ggt tggtgcg tctgggtctg ggtaagtgat gaaaaccgaa taaagtcacc caccaaatct aaaacgtgat ttacaccaca cggtggaagc cgcagctaaa ctctctgaca tccagtagct agtattgagc actgagcaaa ggatgataaa ggataacggc tgctccagca taccagtgct catcgacaaa caacctgaac ctatgcgacc cgtgctggca tcgc tgatca cgtctgtctt gctaaccgt t ggtatttctg cgtatccgtg tccattcagg cagt tcaacg aatgacggtg aatggtttta ttaacttctg aataccttgt gttggcggcg actgctggta gcgt tagctg aaagatggta caggcatacg gctgatatga ttcaatggca ccgttaatcc gaaaccaaag actattgggt ggtggtatta tctgtgactg attggtactg ggttctgcaa ttccgttctt aacaccacta gaagtgtcca aaagccaacc ctcaaaataa ctggcttgcg ttacttctaa t tgc acagac agctgacggt acgaaatcaa gcgtgaacgt aaactatcac acgtaaatgg ctggcgcgaa taactaccga tagattatac cgggtgtaga Ccacccttca Ctgtttcttt tagacgatgc aagcgctgc t ctgaatatac Ctggtgggat cggctgccgc ttaccgcggg ctaacgttgc ttgccgggta ctgtaaatgt cgaccaaccc Ccctgggtgc Ccaacctgtc acatgtcgaa aggtaccgca tatcaacaag tattaacagc cat taaaggc cactgaaggc tcaggcttct atcccgtctc actggcaaaa tatcgacctg taaaggtac t gttaaacacc tgctgcattc ttacaacgc t cgccgcggcg tgctgatgtg cgaaacggat aggcaacttg taaagccgcg tatcgcaaaa ttcttatcag gacatcttca tgaatccagt cgactataca tgcttcagcg gaactccgcg gcttgctgcc tatccagaac tgaagcgcag agcgcagatt gcaggttctg 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1758 <210> 63 <211> 1758 <212> DNA <213> Escherichia coli <400> 63 atggcacaag aaccagtctg gcgaaggatg ctgactcagg gcgctgtccg ac agggac ta gatgaaattg gacggttcaa aaaaaaatcg attaccaaca acgacaggtc tcattaatac cgctgtcgag acgccgcagg cggcccgtaa aaatcaacaa actccgattc accgcgtatc tgaaaattca attctgatac aagctgcaac tttatgatct caacagcctc ttctatcgag tcaggcgatt cgccaacgac caacttacag tgacc tggac cggccagacc ggt tggtgcg tctgggtctg ggtaagtgat gaaaaccgaa tcgctgatca cgtctgtctt gctaaccgtt ggtatttctg cgtattcgtg tccatccagg cagttcaacg aatgacggcg aatggcttta ttaacttctg aataccttgt ctcaaaataa ctggcttgcg ttacttctaa t tgcgcagac aactgacggt acgaaatcaa gcgtgaacgt aaaccatcac acgtaaatgg ctggcgcgaa taactaccga tatcaacaag tattaacagc cattaaaggc caccgaaggc tcaggbcact atctcgtctt gctggcgaaa gatcgacctg taaaggtact gt taaacacc tgctgcattc 120 180 240 300 360 420 480 540 600 660 SUBSTITUTE SHEET (Rule 26) (RO/AU) WO 99161458 WO 9961458PCT/AU99/00385 52 gataaat tag aaatctggtg caggctgctg ggtaaatctg tcagcaggta acgactaaca agcgaaggta gcaactcctg gctacagtga attaccttta gatgctgcga gtctcttaca actgatacca ggtaaaatca ctggacgacg cgtctggatt tcccgtattc attcagcagg tctctgctgc ggaatggcga attttactac attcagcttc ttaatggttc atatcaccat acgctggtag gtgacggtgc cgacaaccac gtaaagatgt attccggtgt agtcttatgt gCgttaacaa ataaagatta Ctactgagac caatcagctc ccgcagtcac aggacgccga ccggtaactc agggttaa taaagtcaca cactaaatct aaaacgtgat ttacaccaca cggtggaagc cgcagctaaa ctctctgaca tccagtagc t agtattgagc actgagcaaa ggatgataaa ggataacggc tgc tccagca taccagtgct catcgacaaa caacc tgaac ctatgcgacc cgtgctggca gt tggcggcg actgctggta gcgttagctg aaagatggta caggcatacg gctgatatga ttcaatggca ccgttaatcc gaaaccaaag aotattgggt ggtggtatca tctgtgactg attggtactg ggttc tgcaa ttccgttctt aacaccacta gaagtgtcca aaagc taacc tagattatac cgggtgtaga ccacccttca ctgtttcttt tagacgatgc aagcgctgct cagaatatac c tggtgggat cggc tgccgc ttaccgcggg ctaacgt tgc t tgccgggta ctgtaaatgt cgaccaaccc ccctgggtgc ccaacctgtc acatgtcgaa aggtaccgca ttacaacgct cgccgcggcg tgctgatgtg cgaaacggat aggcaacttg caaagcagcg catcgcaaaa tacttatcag gacatcttca tgaatccagt cgactataca tgcttcagcg gaac tccgcg gcttgctgcc tatccagaac cgaagcgcag agcgcagatc gcaggttctg 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1758 <210> 64 <211> 1758 <212> DNA <213> Escherichia coli <400> 64 atggcacaag aaccagtctg gcgaaggatg ctgactcagg gcgctgtctg accggaacta gatgaaattg gacggttcga aagaaaatcg attaccaaca acgacaggtc gataaattag aaatctggtg caggctactg ggtaaatctg tcagcaggta acgactaaca agcgaaggta gcaactcctg gctacagtga attaccttta gatgc tgcga tcattaatac cgctgtcgag acgccgcggg ctgcacgtaa aaatcaacaa actctgattc accgcgtatc tgaaaattca attctgatac aagc tgcaac tttatgatct ggaatggcga attttactac attcagctaa ttaatggttc atatcaccat acgctggtag gtgacggtgc cgacaacctc gtaaagatgt attccggtgt agtcttatgt caacagcctc ttctatcgag tcaggcgatt cgccaacgac caacttacag ggatctggac cggccagacc ggttggtgcg tctgggtctg ggtaagtgat gaaaaccgaa taaagtcacc caccaaatct aaaacgtgat ttacacca-ca cggtgga-agc cgcagctaaa ttctctgaca tccagtagct agtat tgagc actgagcaaa ggatgataaa tcgctgatca cgtctgtctt gctaaccgtt ggtatttctg cgtatccgtg tccat tcagg cagttcaacg aatgacggtg aatggtttta ttaacttctg, aataccttgt gttggcggcg actgctggta gcgttagctg aaagatggta caggcatacg gctgatatga ttcaatggca ccgttaatcc gaaaccaaag actattgggt ggtggtatta ctcaaaataa ctggcttgcg ttacttctaa ttgcacagac agctgacggt acgaaatcaa gcgtgaacgt aaac tat cac acgtaaatgg ctggcgcgaa taactaccga tagattatac cgggtgtaga ccacccttca ctgtttcttt tagacgatgc aagcgctgct ctgaatatac ctggtgggat cggctgccgc ttaccgcggg ctaacgttgc tatcaacaag tattaacagc cattaaaggc caccgaaggc tcaggcttct atcccgtctt actggcaaaa tatcgacc tg taaaggtact gttaaacacc tgctgcattc ttacaacgct cgccgcggcg tgctgatgtg cgaaacggat aggcaacttg taaagccgcg tatcgcaaaa tacttatcag gacatcttca tgaatccagt cgactataca 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 SUBSTITUTE SHEET (Rule 26) (ROIAU) WO 99/61458 53 WO 9961458PCT/AU99/00385 gtctcttaca actgatacca ggtaaaatca ctggacgacg cgtctggatt tcccgtattc atccagcagg tctctgctgc gcgttaacaa ataaagatta ctactgagac ctatcagctc ccgcagtcac aggacgccga ccggtaactc agggttaa ggataacggc tgctccagca taccagtgct catcgacaaa caacctgaac ctatgcgacc cgtgctggca tctgtgactg attggtactg ggttctgcaa ttccgttctt aacaccacta gaagtgtcca aaagccaacc ttgccgggta ctgtaaatgt cgaccaaccc ccctgggtgc ccaacctgtc acatgtcgaa aggtaccgca tgcttcagcg gaac tccgcg gcttgctgcc tatccagaac tgaagcgcag agcgcagatt gcaggttctg 1380 1440 1500 1560 1620 1680 1740 1758 <210> <211> 1758 <212> DNA <213> Escherichia coli <400> atggcacaag aaccagtctg gcgaaggatg ctgactcagg gcgctgtccg accgggacta gacgaaat tg gacggttcga aagaaaatcg attaccaaca acgacaggtc gataaattag aaatctggtg caggctactg ggtaaatctg tcagcaggta acgactaaca agcgaaggta gcaactcctg gctacagtga attaccttta gatgctgcga gtctcttaca actgatacca ggtaaaatca -ctggacgacg cgtctggatt tcccgtattc atccagcagg tctctgctgc <210> 66 <211> 1788 tcat taatac cgctgtcgag acgccgcggg ctgcacgtaa aaatcaacaa actctgattc accgcgtatc tgaaaattca attctgatac aagctgcaac tttatgatct ggaatggcga attttactac attcagctaa ttaatggttc atatcaccat acgctggtag gtgacggtgc cgacaacctc gtaaagatgt attccggtgt agtc ttatgt gcgttaacaa ataaagatta ctactgagac ctatcagctc ccgcagtcac aggacgccga ccggtaactc agggttaa caacagcc tc ttctatcgag tcaggcgatt cgccaacgac caacttacag ggatctggac cggtcagacc ggttggtgcg tctgggtctg ggtaagtgat gaaaaccgaa taaagtcacc caccaaatct aaaacgtgat ttacaccaca cggtggaagc cgcagctaaa ctctctgaca tccagtagct agtattgagc actgagcaaa ggatgataaa ggataacggc tgctccagca taccagtgct catcgacaaa caacctgaac ctatgcgacc cgtgc tggca tcgctgatca cgtctgtctt gctaaccgtt ggtatttctg cgtatccgtg tccattcagg cagttcaacg aatgacgg tg aatggtttta ttaacttctg aataccttgt gttggcggcg actgctggta gcgttagctg aaagatggta caggcatacg gctgatatga ttcaatggca ccgttaatcc gaaaccaaag actattgggt ggtggtatta tctgtgactg attggtactg ggttctgcaa ttccgttctt aacaccacta gaagtgtcca aaagccaacc ctcaaaataa ctggcttgcg t tact tc taa ttgcacagac agctgacggt acgaaatcaa gcgtgaacgt aaactatcac acgtaaatgg ctggcgcgaa taactaccga tagattatac cgggtgtaga ccacccttca ctgtttcttt tagacgatgc aagcgctgct ctgaatatac c tggtgggat cggctgccgc ttaccgcggg ctaacgttgc ttgccgggta ctgtaaatgt cgaccaaccc ccctgggtgc ccaacctgtc acatgtcgaa aggtaccgca tatcaacaag tattaacagc cattaaaggc cactgaaggc tcaggcttct atcccgtctc actggcaaaa tatcgacctg taaaggtact gttaaacacc tgctgcattc ttacaacgct cgccgcggcg tgctgatgtg cgaaacggat aggcaacttg taaagccgcg tatcgcaaaa ttcttatcag gacatcttca tgaatccagt cgactataca tgcttcagcg gaac tccgcg gcttgctgcc tatccagaac tgaagcgcag agcgcagatt gcaggttctg 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1758 SUBSTITUTE SHEET (Rule 26) (RO/AU) WO 99/61458 WO 9961458PCT/AU99/00385 54 <212> DNA <213> Escherichia coli <400> 66 atggcacaag aaccagtctg gcgaaggatg ctgactcagg gcgctgtccg accgggacta gacgaaattg gacggttcga aagaaaattg atagocaata aatactataa gatggtgaca aatgcatc tg ggtgatgtag gcagcaagcg oaggaagco t gcggctacgc aagactgcat ggtgctgcaa t taaa taaag gcaacaatta ggtactgcta acagatgctg gctaacaacg gtcggggctg ggcacagtaa ttccgttcat aacaccacta gaagtgtcca aaagccaacc tcattaatac cgctgtcgag acgoogcggg otgcaogtaa aaatcaacaa actctgattc accgcgtatc tgaaaattca actctgatac aagcggogac ctacaacaaa ctgttactat ctggtaactt cagctagcct gtgaagtgaa atttaactag ttgatggttt cagtcacgat ctgctaacgc tggctacago cctataaatc gcgcaaaata atggtgaaat gcaaggtaac aagtatatgt caaaagatoc coctgggggc ccaacctgtc acatgtcgaa aggtaccgca oaacagcctc ttctatcgag tcaggcgatt cgccaacgac caacttacag gga tc tggac cggtcagaoc ggtaggtgog gotggggotg cattagogac taatgcgctg caaagcagat otcattcagt tctcccgccg ctttgatgtt tgatggtaac attcaagaaa ggggggaaca aggggtatcg taaacaaggc tggcgttcag tgccgataat gactacaatt tgttgattct tagtgctaat actgaaagct tatccaaaac tgaagcgcag agcgcagat t gcaggt totg tcgotgatca cgtctgtctt gctaacogtt ggtatttctg cgtatccgtg tccattcagg cagttcaacg aaogacggcc aatggtttta ctgacagcag actgoatcaa gcagctcaaa aatgtatcga go tgggcaaa gatgcgaatg ttaaotacaa gctggtgatg acttataact ttcactgata acagcagttg acgtatcagg actgacgttt ggttcataca ggaactggtt ggtactttaa ctggatgaag cgtttggatt tcocgtattc atccagcagg tctctactgc o tcaaaataa otggcttgcg ttacttctaa ttgoacagac agctgacggt acgaaatcaa gcgtgaacgt agac tatoac acgtgaatgg cgaaaatgga aggcccttga ctgccacggt ataatacttc ctgctagtgg gtaaaattao acgatgctgg gtcaatcaat ttaaaacggg cagctagcaa cagctaacgg cggtatttgc ctaatgcaac ccacgaagta cgggtaaata caacagatgc ctatcagctc ccgccgtcac aggacgccga ccggtaactc agggttaa tatcaacaag tattaacagc cattaaaggc cactgaaggo tcaggcttct atcccgtcto actggcaaaa tattgatctg t tccggtacg tgctgcaao t tcaactgaaa ctatacatac agcaaaagca tgtttacaaa aatcggagga tggtgogact cgggtttaat tgctgatgct agaaaccgtt tgatacatoc cgcaggtgac agcaacatac ttcaatcgat tgcgccgaaa aactagcgaa catcgacaaa caacctgaao ctatgcgacc cgtgctggca 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1788 <210> 67 <211> 1398 <212> DNA <213> Escherichia coli <400> 67 aacaaatctc aacagcgcaa aaaggtctga gaaggtgcgo gcaactaacg cgtctgagtg gcttctgatc agtcttctct aagacgatgc coaggcttc tgaatgaaat gtactaactc aaattgaccg aggatatgac tagctctgct agcaggtcag ccgtaacgca taacaacaac tgaoagtgac tgtttctggt tattcaggtt attgagcgtc gcgattgcta aatgatggta o tgcagcgta ctgacctcca cagactcagt ggtgcaaacg tgtcttctgg accgttttac tttctgttgc ttcgtgaact tccagtccga ttaacggcgt acggogaaac tctgcgtatt ggcaaatatt goagaccact ttctgttcag aatccagcag taaagtgctg aattactatt 120 180 240 300 360 420 SUBSTITUTE SHEET (Rule 26) (RO/AU) WO 99/61458 WO 9961458PCTIAU99/00385 55 aaactgcagg actaaattaa gacgctaata caacgtgacg tcagataaac cctgatgcag ttaaaggaag accgcaaccg gatactggtt cagaaaacag cacgacaatg accgatggca attgcagcaa tctcaggttg atcaccaacc gctgactacg acctctgttc aaattaattc aagccgcaac cacttgatgc gtaatattat ccgctgaaaa gtaagctgaa tcacaacggt cttctatcac catttgcgtt gagcagtttc ttaaagttga aaac ttacag tttctacgca actcgttgcg ttggcaacac cgaccgaagt tggcgcag cgacacactg ggctgaaaca agatattaca gtctgatgct tggttattat gc tgggggc t gaaagggaag aggtgcaaaa gattggtgat cgttaaaaca actgggtgga tgttagtgat gaaaacagaa ttctaaccta cgtaaacaac gtctaacatg ggattatctg acc tatt ttg gctacagtta aacggtaagt gaagttactg ctagcgggta ggggctattg ctctttaagt gacggtaaac atgtc ttaca agcgatggca ttacaaggta gatcctttgg ggtgcaattc ctgtcttctg tctcgtgcgc gttttggtat gatcgacagt aaggcactac tgtacgt taa tggaggatga cccagcctca atgttcagtt tagaagacgc agtatgcagc ctgatgctga aaaccgaagt agagcctgaa ctgctatcga aaaatcgttt cccgtagccg agatcctgca taaagatcct taagcttgct gactccgggc agt tgccggt tccgacatct agctggtaat gggtac tgat caatggcaaa gaatgttgat cggtgtcaaa tgtaac tgca aactgattct taaagcactg cgactctgcc tatcgaagat acaagcgggt 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1398 <210> 68 <211> 1479 <212> DNA <213> Escherichia coli <400> 68 aacaaatc tc aacagtgcta aaaggtctga gaaggtgcgc gccactaatg cgccttagtg gcttctgatc gcccttgata aaagttccaa gtaaatgcaa gctgatgttg gcgaccggta gttggtcaag ggt tatgaaa gtaactgcat atgggtaaag ctagatgtcc aaaacatcag gttattactg caggcaatta accaaaggtg tccacattag caaaaccgtt gcccgtagcc agtc ttc tot aagatgacgc ctcaggcttc tttctgaaat gtacaaactc aaattgatcg agactatgaa aaattgatgc tgtcctctgc ctgatggtag aaacttattt ctgcaggaac ataataatac aagttcaggt ttgttgaaga cattagctta accaagtaca acggcaccta atocctaatgg tgcgtgaaga ctgaactcag acgaagcttt tcgac tc tgc gtatagaaga gagctccgcc agcaggtcag ccgtaacgcg caacaataac tgactccgac tgtttctaac aattcaagta taaaaccttg ggttgcactt tgtgggaggt tggtaccggt aaaagtttat caacacgaac gggtggtaag taatggttct taatgatgca agatacccaa cattgcagta taaggcagtt tgataaggtt tgcctcagat ggcaaaagt t catcaccaac tgctgactac attgaacgtc gcgattgcta aatgatggta ttacagcgta ct gaa t tcaa cagac acaa t ggtgcgaacg gggcttgata aagagcgaag gctaaagcat aatgtacaag caagtacagg ggttttacat gatgttcagt gccacatcag ccaatgtctg gggaatcctg aatgtagatg gaatgggcag tatacagcca ttgaaagcct gataagttgc cttggcaaca gcaaccgaag tctcttctgg accgttttac tttctgttgc ttcgtgaatt ttcaggatga ttaatggtgt atggtgaaac actttagcgt ccgctcctga tcggtagcaa atacaaagga tggaagggca tattgaaaca tagcaaactt ttgatttagc tttattttgg tacctaattc ccgo tacag4 taaaaaatga atatcacgaa tagcaaccac gcagttcttt ccgtaaacaa tgtctaacat cctgcgtatt agcaaatatt gcagaccact gtcagtacag aattacacaa aaaagttctg cattgagatt agcaccagga cttaactaag.

ttataaaaat tacaactgat gacttatttt aaactctaca tggtggtcgt tgcgggtaaa gggaaaaaac atttgctgct taacacgtct tggttctgca taagacggca aaatccatta gggtgcagta cctgtcttct gtctcgtgcg 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 SUBSTITUTE SHEET (Rule 26) (RO/AU) WO 99/61458 -5 cagatcctgc aacaagcggg tacctctgtt ctggcacag PCT/AU99/00385 1479 SUBSTITUTE SHEET (Rule 26) (RO/AU)

Claims (22)

1. A nucleic acid molecule which encodes all or part of an E. coli flagellin protein, the molecule being capable of identifying the H serotype of an E. coli when hybridised to a gene of the E. coli which encodes a flagellin protein, provided that the molecule does not encode a flagellin protein expressed by the E. coli H1, H7, H12 or H48 type strains.

2. A nucleic acid molecule according to claim 1 wherein the molecule is derived from a fliC gene.

3. A nucleic acid molecule according to claim 1 including all or part of a sequence according to any one of SEQ ID NOs:l to 68.

4. A nucleic acid molecule according to claim 1 consisting of all or part of a sequence according to any one of SEQ ID NOs: 1 to 68. A nucleic acid molecule according to claim 4 wherein the molecule is from about 10 to 20 nucleotides in length.

6. A primer selected from the group of primers shown in Table 3.

7. A method of detecting the H serotype of E. coli in a sample, the method comprising the following steps: contacting a gene of an E. coli in the sample with a nucleic acid molecule according to claim 1 in conditions sufficient to allow the nucleic acid molecule to hybridise to the gene; and detecting a nucleic acid molecule which is hybridised to the gene, to detect the H serotype of the E. coli in the sample. z IPEi-U PCT/AU99/0385 Received 1 May 2000 -84-

8. A method according to claim 7 wherein the hybridised nucleic acid molecules are detected by Southern Blot analysis.

9. A method of detecting the H serotype of E. coli in a sample, the method comprising the following steps: contacting a gene of an E. coli in the sample with a pair of nucleic acid molecules according to claim 1 in conditions sufficient to allow the pair of nucleic acid molecules to hybridise to the gene; and detecting a pair of nucleic acid molecules which is hybridised to the gene, to detect the H serotype of the E. coli in the sample. A method according to claim 9 wherein the hybridised pairs of nucleic acid molecules are detected by the polymerase chain reaction.

11. A method for detecting the H and 0 serotype of E. coli in a sample, the method comprising the following steps: contacting a gene of the E. coli with a nucleic acid molecule derived from a gene encoding a transferase or a gene encoding an enzyme for the transport or processing of a polysaccharide or oligosaccharide unit, the gene being involved in the synthesis of a E. coli 0 antigen, in conditions sufficient to allow the nucleic acid molecule to hybridise to the gene; contacting a gene of an E. coli in the sample with a nucleic acid molecule according to claim 1 in conditions sufficient to allow the nucleic acid molecule to hybridise to the gene; and detecting nucleic acid molecules which are hybridised to the genes, to detect the H and 0 serotype of the E. coli in the sample. S 7- PCT/AU99/0385 Received 1 May 2000

12. A method according to claim 11 wherein the nucleic acid molecule of step is selected from the group consisting of: wbdH (nucleotide position 739 to 1932 of Figure wzx (nucleotide position 8646 to 9911 of Figure wzy (nucleotide position 9901 to 10953 of Figure wbdM (nucleotide position 11821 to 12945 of Figure wbdN (nucleotide position 79 to 861 of Figure 6), wbdO (nucleotide position 2011 to 2757 of Figure 6), wbdP (nucleotide position 5257 to 6471 of Figure 6), wbdR (nucleotide position 13156 to 13821 of Figure 6), wzx (nucleotide position 2744 to 4135 of Figure 6) and wzy (nucleotide position 858 to 2042 of Figure 6).

13. A method according to claim 12 wherein the nucleic acid molecule of step is a.primer selected from the group of primers shown in Tables 8, 8A, 9 and 9A.

14. A method according to claim 11 wherein the hybridised nucleic acid molecules are detected by Southern Blot analysis. A method for detecting the H and O serotype of E. coli in a sample, the method comprising the following steps: contacting a gene of the E. coli with a pair of nucleic acid molecules derived from a gene encoding a transferase or a gene encoding an enzyme for the transport or processing of a polysaccharide or oligosaccharide unit, the gene being involved in the synthesis of a E. coli 0 antigen, in conditions sufficient to allow the pair of nucleic acid molecules to hybridise to the gene; contacting a gene of an E. coli in the sample with a pair of nucleic acid molecules according to claim 1 in condit.ions sufficient to allow the pair of nucleic acid molecules to hybridise to the gene; and detecting pairs of nucleic acid molecules which SAMLEND z PCT/AU99/0385 Received 1 May 2000 -86- are hybridised to the genes, to detect the H and O serotype of the E. coli in the sample.

16. A method according to claim 15 wherein the pair of nucleic acid molecules of step is selected from the group consisting of: wbdH (nucleotide position 739 to 1932 of Figure wzx (nucleotide position 8646 to 9911 of Figure wzy (nucleotide position 9901 to 10953 of Figure wbdM (nucleotide position 11821 to 12945 of Figure wbdN (nucleotide position 79 to 861 of Figure 6), wbdO (nucleotide position 2011 to 2757 of Figure 6), wbdP (nucleotide position 5257 to 6471 of Figure 6), wbdR (nucleotide position 13156 to 13821 of Figure 6), wzx (nucleotide position 2744 to 4135 of Figure 6) and wzy (nucleotide position 858 to 2042 of Figure 6).

17. A method according to claim 15 wherein the nucleic acid molecules of the pair of step are primers selected from the group of primers shown in Tables 8, 8A, 9 and 9A.

18. A method according to claim 15 wherein the hybridised pairs of nucleic acid molecules are detected by the polymerase chain reaction.

19. A method for detecting the H and O serotype of E. coli in a sample, the method comprising the following steps: contacting a gene of an E. coli in the sample with a nucleic acid molecule according to claim 1, in conditions sufficient to allow the nucleic acid molecule to hybridise to the gene; and detecting a nucleic acid molecule which is hybridised to the gene, to detect the H and O serotype.of coli in the sample. IiA PCT/AU99/0385 Received 1 May 2000 -87- A method according to claim 19 wherein the nucleic acid molecule is according to any one of SEQ ID NOS: 9, 57 to

21. A method according to any one of claims 8, 9, 11, or 19 wherein the sample is selected from the group consisting of a sample derived from food, a sample derived from faeces and a sample derived from a patient or animal.

22. A kit for identifying the H serotype of E. coli, the kit comprising at least one nucleic acid molecule according to any one of claims 1 to 6.

23. A kit for identifying the H and O serotype of E. coli, the kit comprising: at least one nucleic acid molecule according to any one of claims 1 to 6; and at least one nucleic acid molecule derived from and specific for a gene encoding a transferase or a gene encoding an enzyme for the transport or processing of a polysaccharide or oligosaccharide unit, the gene being involved in the synthesis of a particular E. coli 0 antigen.

24. A kit according to claim 23 wherein the at least one nucleic acid molecule of is selected from the group consisting of: wbdH (nucleotide position 739 to 1932 of Figure wzx (nucleotide position 8646 to 9911 of Figure wzy (nucleotide position 9901 to 10953 of Figure wbdM (nucleotide position 11821 to 12945 of Figure wbdN (nucleotide position 79 to 861 of Figure 6), wbdO (nucleotide position 2011 to 2757 of Figure 6), wbdP (nucleotide position 5257 to 6471 of Figure 6), wbdR (nucleotide position 13156 to 13821 of Figure 6), wzx (nucleotide position 2744 to 4135 of Figure 6) and- wzy (nucleotide position 858 to 2042 of Figure 6). ,s -7AEDSJT -88- A kit according to claim 24 wherein the nucleic acid molecule of is a primer selected from the group of primers shown in Tables 8, 8A, 9 and 9A.

26. A nucleic acid according to claim 1 substantially as hereinbefore described.

27. A method for detecting the H and O serotype of E. coli in a sample according to claim 15, substantially as hereinbefore described. Dated this 6 th day of February 2003 THE UNIVERSITY OF SYDNEY By their Patent Attorneys GRIFFITH HACK