Nucleotide sequence of escherichia coli pathogenicity islands

BACKGROUND OF THE INVENTION

[0005] 1. Field of the Invention

[0006] The present invention relates to novel genes located in two chromosomal regions within E. coli that are associated with virulence. These chromosomal regions are known as pathogenicity islands (PAIs).

[0007] 2. Related Background Art

[0008]

Escherichia coli
(E. coli) is a normal inhabitant of the intestine of humans and various animals. Pathogenic E. coli strains are able to cause infections of the intestine (intestinal E. coli strains) and of other organs such as the urinary tract (uropathogenic E. coli) or the brain (extraintestinal E. coli). Intestinal pathogenic E. coli are a well established and leading cause of severe infantile diarrhea in the developing world. Additionally, cases of newborn meningitis and sepsis have been attributed to E. coli pathogens.

[0009] In contrast to non-pathogenic isolates, pathogenic E. coli produce pathogenicity factors which contribute to the ability of strains to cause infectious diseases (Muihldorfer, I. and Hacker, J., Microb. Pathogen. 16:171-181 1994). Adhesions facilitate binding of pathogenic bacteria to host tissues. Pathogenic E. coli strains also express toxins including haemolysins, which are involved in the destruction of host cells, and surface structures such as O-antigens, capsules or membrane proteins, which protect the bacteria from the action of phagocytes or the complement system (Ritter, et al., Mol. Microbiol. 17:109-212 1995).

[0010] The genes coding for pathogenicity factors of intestinal E. coli are located on large plasmids, phage genomes or on the chromosome. In contrast to intestinal E. coli, pathogenicity determinants of uropathogenic and other extraintestinal E. coli are, in most cases, located on the chromosome. Id.

[0011] Large chromosomal regions in pathogenic bacteria that encode adjacently located virulence genes have been termed pathogenicity islands (“PAIs”). PAIs are indicative of large fragments of DNA which comprise a group of virulence genes behaving as a distinct molecular and functional unit much like an island within the bacterial chromosome. For example, intact PAIs appear to transfer between organisms and confer complex virulence properties to the recipient bacteria.

[0012] Chromosomal PAIs in bacterial cells have been described in increasing detail over recent years. For example, J. Hacker and co-workers described two large, unstable regions in the chromosome of uropathogenic Escherichia coli strain 536 as PAI-I and PAI-II (Hacker J., et al., Microbiol. Pathog. 8:213-25 1990). Hacker found that PAI-I and PAI-II containing virulence regions can be lost by spontaneous deletion due to recombination events. Both of these PAIs were found to encode multiple virulence genes, and their loss resulted in reduced hemolytic activity, serum resistance, mannose-resistant hemagglutination, uroepithelial cell binding, and mouse virulence of the E. coli. (Knapp, S et al., J. Bacteriol. 168:22-30 1986). Therefore, pathogenicity islands are characterized by their ability to confer complex virulence phenotypes to bacterial cells.

[0013] In addition to E. coli, specific deletion of large virulence regions has been observed in other bacteria such as Yersinia pestis. For example, Fetherston and co-workers found that a 102-kb region of the Y. pestis chromosome lost by spontaneous deletion resulted in the loss of many Y. pestis virulence phenotypes. (Fetherston, J. D. and Perry, R. D., Mol. Microbiol. 13:697-708 1994, Fetherston, et al., Mol. Microbiol. 6:2693-704 1992). In this instance, the deletion appeared to be due to recombination within 2.2-kb repetitive elements at both ends of the 102-kb region.

[0014] It is possible that deletion of PAIs may benefit the organism by modulating bacterial virulence or genome size during infection. PAIs may also represent foreign DNA segments that were acquired during bacterial evolution that conferred important pathogenic properties to the bacteria. Observed flanking repeats, as observed in Y. pestis for example, may suggest a common mechanism by which these virulence genes were integrated into the bacterial chromosomes.

[0015] Integration of the virulence genes into bacterial chromosomes was further elucidated by the discovery and characterization of a locus of enterocyte effacement (the LEE locus) in enteropathogenic E. coli (McDaniel, et al., Proc. Natl Acad. Sci. (USA) 92:1664-8 1995). The LEE locus comprises 35-kb and encodes many genes required for these bacteria to “invade” and degrade the apical structure of enerocytes causing diarrhea. Although the LEE and PAI-I loci encode different virulence genes, these elements are located at the exact same site in the E. coli genome and contain the same DNA sequence within their right-hand ends, thus suggesting a common mechanism for their insertion.

[0016] Besides being found in enteropathogenic E. coli, the LEE element is also present in rabbit diarrheal E. coli, Hafnia alvei, and Citrobacter freundii biotype 4280, all of which induce attaching and effacing lesions on the apical face of enterocytes. The LEE locus appears to be inserted in the bacterial chromosome as a discrete molecular and functional virulence unit in the same fashion as PAI-I, PAI-II, and Yersinia PAI.

[0017] Along these same lines, a 40-kb Salmonella typhimurium PAI was characterized on the bacterial chromosome which encodes genes required for Salmonella entry into nonphagocytic epithelial cells of the intestine (Mills, D. M., et al., Mol. Microbiol. 15:749-59 1995). Like the LEE element, this PAI confers to Salmonella the ability to invade intestinal cells, and hence may likewise be characterized as an “invasion” PAI.

[0018] The pathogenicity islands described above all possess the common feature of conferring complex virulence properties to the recipient bacteria. However, they may be separated into two types by their respective contributions to virulence. PAI-I, PAI-II, and the Y. pestis PAI confer multiple virulence phenotypes, while the LEE and the S. typhimurium “invasion” PAI encode many genes specifying a single, complex virulence process.

[0019] It is advantageous to characterize closely-related bacteria that contain or do not contain the PAI by the isolation of a discrete molecular and functional unit on the bacterial chromosome. Since the presence versus the absence of essential virulence genes can often distinguish closely-related virulent versus avirulent bacterial strains or species, experiments have been conducted to identify virulence loci and potential PAIs by isolating DNA sequences that are unique to virulent bacteria (Bloch, C. A., et al., J Bacteriol. 176:7121-5 1994, Groisman, E. A., EMBO J. 12:3779-87 1993).

[0020] At least two PAIs are present in E. coli J96. These PAIs, PAI IV and PAI V are linked to tRNA loci but at sites different from those occupied by other known E. coli PAIs. Swenson et al., Infect. and Immun. 64:3736-3743 (1996).

[0021] The era of true comparative genomics has been ushered in by high through-put genomic sequencing and analysis. The first two complete bacterial genome sequences, those of Haemophilus influenzae and Mycoplasma genitalium were recently described (Fleischmann, R. D., et al., Science 269:496 (1995); Fraser, C. M., et al., Science 270:397 (1995)). Large scale DNA sequencing efforts also have produced an extensive collection of sequence data from eukaryotes, including Homo sapiens (Adams, M. D., et al., Nature 377:3 (1995)) and Saccharomyces cerevisiae (Levy, J., Yeast 10:1689 (1994)).

[0022] The need continues to exist for the application of high through-put sequencing and analysis to study genomes and subgenomes of infectious organisms. Further, a need exists for genetic markers that can be employed to distinguish closely-related virulent and avirulent strains of a given bacteria.

SUMMARY OF THE INVENTION

[0023] The present invention is based on the high through-put, random sequencing of cosmid clones covering two pathogenic islands (PAIs) of uropathogenic Escherichia coli strain J96 (O4:K6; E. coli J96). PAIs are large fragments of DNA which comprise pathogenicity determinants. PAI IV is located approximately at 64 min (nearpheV) on the E. coli chromosome and is greater than 170 kilobases in size. PAI V is located at approximately 94 min (atpheR) on the E. coli chromosome and is approximately 106 kb in size. These PAIs differ in location to the PAIs described by Hacker and colleagues for uropathogenic strain 536 (PAI I, 82 minutes {selC} and PAI II, 97 minutes {leuX}).

[0024] The location of the PAIs relative to one another and the cosmid clones covering the J96 PAIs is shown in FIG. 1. The present invention relates to the nucleotide sequences of 142 fragments of DNA (contigs) covering the PAI IV and PAI V regions of the E. coli J96 chromosome. The nucleotide sequences shown in SEQ ID NOs: 1 through 142 were obtained by shotgun sequencing eleven E. coli J96 subclones, which were deposited in two pools on Sep. 23, 1996 at the American Type Culture Collection, 12301 Park Lawn Drive, Rockville, Md. 20852, and given accession numbers 97726 (includes 7 cosmid clones covering PAI (IV) and 97727 (includes 4 cosmid clones covering PAI V). The deposited sets or “pools” of clones are more fully described in Example 1. In addition, E. coli strain J96 was also deposited at the American Type Culture Collection on Sep. 23, 1996, and given accession number 98176.

[0025] Three hundred fifty-one open reading frames have been thus far identified in the 142 contigs described by SEQ ID NOs: 1 through 142. Thus, the present invention is directed to isolated nucleic acid molecules comprising open reading frames (ORFs) encoding E. coli proteins that are located in two pathogenic island regions of the chromosome of uropathogenic E. coli J96.

[0026] The present invention also relates to variants of the nucleic acid molecules of the present invention, which encode portions, analogs or derivatives of E. coli J96 PAI proteins. Further embodiments include isolated nucleic acid molecules comprising a polynucleotide having a nucleotide sequence at least 90% identical, and more preferably at least 95%, 96%, 97%, 98% or 99% identical, to the nucleotide sequence of an E. coli J96 PAI ORF described herein.

[0027] The present invention also relates to recombinant vectors, which include the isolated nucleic acid molecules of the present invention, host cells containing the recombinant vectors, as well as methods for making such vectors and host cells for E. coli J96 PAI protein production by recombinant techniques.

[0028] The invention further provides isolated polypeptides encoded by the E. coli J96 PAI ORFs. It will be recognized that some amino acid sequences of the polypeptides described herein can be varied without significant effect on the structure or function of the protein. If such differences in sequence are contemplated, it should be remembered that there will be critical areas on the protein which determine activity. In general, it is possible to replace residues which form the tertiary structure, provided that residues performing a similar function are used. In other instances, the type of residue may be completely unimportant if the alteration occurs at a non-critical region of the protein.

[0029] In another aspect, the invention provides a peptide or polypeptide comprising an epitope-bearing portion of a polypeptide of the invention. The epitope-bearing portion is an immunogenic or antigenic epitope useful for raising antibodies.

[0030] The invention further provides a vaccine comprising one or more E. coli J96 PAI antigens together with a pharmaceutically acceptable diluent, carrier, or excipient, wherein the one or more antigens are present in an amount effective to elicit protective antibodies in an animal to pathogenic E. coli, such as strain J96.

[0031] The invention also provides a method of eliciting a protective immune response in an animal comprising administering to the animal the above-described vaccine.

[0032] The invention further provides a method for identifying pathogenic E. coli in an animal comprising analyzing tissue or body fluid from the animal for one or more of:

[0033] (a) polynucleic acids encoding an open reading frame listed in Tables 1-4;

[0034] (b) polypeptides encoded for by an open reading frame listed in Tables 1-4; or

[0035] (c) antibodies specific to polypeptides encoded for by an open reading frame listed in Tables 1-4.

[0036] The invention further provides a nucleic acid probe for the detection of the presence of one or more E. coli PAI nucleic acids (nucleic acids encoding one or more ORFs as listed in Tables 1-4) in a sample from an individual comprising one or more nucleic acid molecules sufficient to specifically detect under stringent hybridization conditions the presence of the above-described molecule in the sample.

[0037] The invention also provides a method of detecting E. coli PAI nucleic acids in a sample comprising:

[0038] a) contacting the sample with the above-described nucleic acid probe, under conditions such that hybridization occurs, and

[0039] b) detecting the presence of the probe bound to an E. coli PAI nucleic acid.

[0040] The invention further provides a kit for detecting the presence of one or more E. coli PAI nucleic acids in a sample comprising at least one container means having disposed therein the above-described nucleic acid probe.

[0041] The invention also provides a diagnostic kit for detecting the presence of pathogenic E. coli in a sample comprising at least one container means having disposed therein one or more of the above-described antibodies.

[0042] The invention also provides a diagnostic kit for detecting the presence of antibodies to pathogenic E. coli in a sample comprising at least one container means having disposed therein one or more of the above-described antigens.

BRIEF DESCRIPTION OF THE FIGURES

[0043]
FIG. 1 is a schematic diagram of cosmid clones derived from E. coli J96 pathogenicity island and map positions of known E. coli PAIs (not drawn to scale). The gray bar represents the E. coli K-12 chromosome with minute demarcations of PAI junction points located above the bar. E. coli J96 overlapping cosmid clones are represented by hatched bars (overlap not drawn to scale) with positions of h/y, pap, and prs operons indicated above bar. The PAIs and estimated sizes are shown above and below the K-12 chromosome map.

[0044]
FIG. 2 is a block diagram of a computer system 102 that can be used to implement the computer-based systems of present invention.

DETAILED DESCRIPTION OF THE INVENTION

[0045] The present invention is based on high through-put, random sequencing of a uropathogenic strain of Escherichia coli. The DNA sequences of contiguous DNA fragments covering the pathogenicity islands, PAI IV (also referred to as PAIJ96(pheV)) and PAI V (also referred to as PAIJ96(pheU)) from the chromosome of the E. coli uropathogenic strain, J96 (04:K6) were determined. The sequences were used for DNA and protein sequence similarity searches of the database.

[0046] The primary nucleotide sequences generated by shotgun sequencing cosmid clones of the PAI IV and PAI V regions of the E. coli chromosome are provided in SEQ ID NOs: 1 through 142. These sequences represent contiguous fragments of the PAI DNA. As used herein, the“primary sequence” refers to the nucleotide sequence represented by the IUPAC nomenclature system. The present invention provides the nucleotide sequences of SEQ ID NOs: 1 through 142, or representative fragments thereof, in a form that can be readily used, analyzed, and interpreted by a skilled artisan. Within these 142 sequences, there have been thus far identified 351 open reading frames (ORFs) that are described in greater detail below.

[0047] As used herein, a “representative fragment” refers to E. coli J96 PAI protein-encoding regions (also referred to herein as open reading frames or ORFs), expression modulating fragments, and fragments that can be used to diagnose the presence of E. coli in a sample. A non-limiting identification of such representative fragments is provided in Tables 1 through 6. As described in detail below, representative fragments of the present invention further include nucleic acid molecules having a nucleotide sequence at least 95% identical, preferably at least 96%, 97%, 98%, or 99% identical, to an ORF identified in Tables 1 through 6.

[0048] As indicated above, the nucleotide sequence information provided in SEQ ID NOs: 1 through 142 was obtained by sequencing cosmid clones covering the PAIs located on the chromosome of E. coli J96 using a megabase shotgun sequencing method. The sequences provided in SEQ ID NOs: 1 through 142 are highly accurate, although not necessarily a 100% perfect, representation of the nucleotide sequences of contiguous stretches of DNA (contigs) which include the ORFs located on the two pathogenicity islands of E. coli J96. As discussed in detail below, using the information provided in SEQ ID NOs: 1 through 142 and in Tables 1 through 6 together with routine cloning and sequencing methods, one of ordinary skill in the art would be able to clone and sequence all “representative fragments” of interest including open reading frames (ORFs) encoding a large variety of E. coli J96 PAI proteins. In rare instances, this may reveal a nucleotide sequence error present in the nucleotide sequences disclosed in SEQ ID NOs: 1 through 142. Thus, once the present invention is made available (i.e., once the information in SEQ ID NOs: 1 through 142 and in Tables 1 through 6 have been made available), resolving a rare sequencing error would be well within the skill of the art. Nucleotide sequence editing software is publicly available. For example, Applied Biosystem's (AB) AutoAssembler can be used as an aid during visual inspection of nucleotide sequences.

[0049] Even if all of the rare sequencing errors were corrected, it is predicted that the resulting nucleotide sequences would still be at least about 99.9% identical to the reference nucleotide sequences in SEQ ID NOs: 1 through 142. Thus, the present invention further provides nucleotide sequences that are at least 99.9% identical to the nucleotide sequence of SEQ ID NOs: 1 through 142 in a form which can be readily used, analyzed and interpreted by the skilled artisan. Methods for determining whether a nucleotide sequence is at least 99.9% identical to a reference nucleotide sequence of the present invention are described below.

[0050] Nucleic Acid Molecules

[0051] The present invention is directed to isolated nucleic acid fragments of the PAIs of E. coli J96. Such fragments include, but are not limited to, nucleic acid molecules encoding polypeptides (hereinafter open reading frames (ORFs)), nucleic acid molecules that modulate the expression of an operably linked ORF (hereinafter expression modulating fragments (EMFs)), and nucleic acid molecules that can be used to diagnose the presence of E. coli in a sample (hereinafter diagnostic fragments (DFs)).

[0052] By isolated nucleic acid molecule(s) is intended a nucleic acid molecule, DNA or RNA, that has been removed from its native environment. For example, recombinant DNA molecules contained in a vector are considered isolated for the purposes of the present invention. Further examples of isolated DNA molecules include recombinant DNA molecules maintained in heterologous host cells, purified (partially or substantially) DNA molecules in solution, and nucleic acid molecules produced synthetically. Isolated RNA molecules include in vitro RNA transcripts of the DNA molecules of the present invention.

[0053] In one embodiment, E. coli J96 PAI DNA can be mechanically sheared to produce fragments about 15-20 kb in length, which can be used to generate an E. coli J96 PAI DNA library by insertion into lambda clones as described in Example 1 below. Primers flanking an ORF described in Tables 1 through 6 can then be generated using the nucleotide sequence information provided in SEQ ID NOs: 1 through 142. The polymerase chain reaction (PCR) is then used to amplify and isolate the ORF from the lambda DNA library. PCR cloning is well known in the art. Thus, given SEQ ID NOs: 1 through 142, and Tables 1 through 6, it would be routine to isolate any ORF or other representative fragment of the E. coli J96 PAI subgenomes. Isolated nucleic acid molecules of the present invention include, but are not limited to, single stranded and double stranded DNA, and single stranded RNA, and complements thereof.

[0054] Tables 1 through 6 herein describe ORFs in the E. coli J96 PAI cosmid clone library.

[0055] Tables 1 and 3 list, for PAI IV and PAI V, respectively, a number of ORFs that putatively encode a recited protein based on homology matching with protein sequences from an organism listed in the Table. Tables 1 and 3 indicate the location of ORFs (i.e., the position) by reference to its position within the one of the 142 E. coli J96 contigs described in SEQ ID NOs: 1 through 142. Column 1 of Tables 1 and 3 provides the Sequence ID Number (SEQ ID NO) of the contig in which a particular open reading frame is located. Column 2 numerically identifies a particular ORF on a particular contig (SEQ ID NO) since many contigs comprise a plurality of ORFs. Columns 3 and 4 indicate an ORF s position in the nucleotide sequence (contig) provided in SEQ ID NOs: 1 through 142 by referring to start and stop positions in the contig sequence. One of ordinary skill in the art will appreciate that the ORFs may be oriented in opposite directions in the E. coli chromosome. This is reflected in columns 3 and 4. Column 5 provides a database accession number to a homologous protein identified by a similarity search of public sequence databases (see, infra). Column 6 describes the matching protein sequence and the source organism is identified in brackets. Column 7 of Tables 1 and 3 indicates the percent identity of the protein sequence encoded by an ORF to the corresponding protein sequence from the organism appearing in parentheses in the sixth column. Column 8 of Tables 1 and 3 indicates the percent similarity of the protein sequence encoded by an ORF to the corresponding protein sequence from the organism appearing in parentheses in the sixth column. The concepts of percent identity and percent similarity of two polypeptide sequences are well understood in the art and are described in more detail below. Identified genes can frequently be assigned a putative cellular role category adapted from Riley (see, Riley, M., Microbiol. Rev. 57:862 (1993)). Column 9 of Tables 1 and 3 provides the nucleotide length of the open reading frame.

[0056] Tables 2 and 4, below, provide ORFs of E. coli J96 PAl IV and PAI V, respectively, that did not elicit a homology match with a known sequence from either E. coli or another organism. As above, the first column in Tables 2 and 4 provides the contig in which the ORF is located and the second column numerically identifies a particular ORF in a particular contig. Columns 3 and 4 identify an ORF s position in one of SEQ ID NOs: 1 through 142 by reference to start and stop nucleotides.

[0057] Tables 5 and 6, below, provide the E. coli J96 PAI IV ORFs and PAI V ORFs, respectively, identified by the present inventors that provided a significant match to a previously published E. coli protein. The columns correspond to the columns appearing in Tables 1 and 3.

[0058] Further details concerning the algorithms and criteria used for homology searches are provided in the Examples below. A skilled artisan can readily identify ORFs in the Escherichia coli J96 cosmid library other than those listed in Tables 1 through 6, such as ORFs that are overlapping or encoded by the opposite strand of an identified ORF in addition to those ascertainable using the computer-based systems of the present invention.

[0059] Isolated nucleic acid molecules of the present invention include DNA molecules having a nucleotide sequence substantially different than the nucleotide sequence of an ORF described in Tables 1 through 4, but which, due to the degeneracy of the genetic code, still encode a E. coli J96 PAI protein. The genetic code is well known in the art. Thus, it would be routine to generate such degenerate variants.

[0060] The present invention further relates to variants of the nucleic acid molecules of the present invention, which encode portions, analogs or derivatives of an E. coli protein encoded by an ORF described in Table 1 through 4. Non-naturally occurring variants may be produced using art-known mutagenesis techniques and include those produced by nucleotide substitutions, deletions or additions. The substitutions, deletions or additions may involve one or more nucleotides. The variants may be altered in coding regions, non-coding regions, or both. Alterations in the coding regions may produce conservative or non-conservative amino acid substitutions, deletions or additions. Especially preferred among these are silent substitutions, additions and deletions, which do not alter the properties and activities of the E. coli protein or portions thereof. Also especially preferred in this regard are conservative substitutions.

[0061] Further embodiments of the invention include isolated nucleic acid molecules comprising a polynucleotide having a nucleotide sequence at least 90% identical, and more preferably at least 95%, 96%, 97%, 98% or 99% identical, to the nucleotide sequence of an ORF described in Tables 1 through 6, preferably 1 through 4. By a polynucleotide having a nucleotide sequence at least, for example, 95% identical to the reference E. coli ORF nucleotide sequence is intended that the nucleotide sequence of the polynucleotide is identical to the reference sequence except that the polynucleotide sequence may include up to five point mutations per each 100 nucleotides of the ORF sequence. In other words, to obtain a polynucleotide having a nucleotide sequence at least 95% identical to a reference ORF nucleotide sequence, up to 5% of the nucleotides in the reference sequence may be deleted or substituted with another nucleotide, or a number of nucleotides up to 5% of the total nucleotides in the reference sequence may be inserted into the reference sequence. These mutations of the reference sequence may occur at the 5′ or 3′ terminal positions of the reference nucleotide sequence or anywhere between those terminal positions, interspersed either individually among nucleotides in the reference sequence or in one or more contiguous groups within the reference sequence.

[0062] As a practical matter, whether any particular nucleic acid molecule is at least 90%, 95%, 96%, 97%, 98% or 99% identical to the nucleotide sequence of an E. coli J96 PAI ORF can be determined conventionally using known computer programs such as the Bestfit program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive, Madison, Wis. 53711). Bestfit uses the local homology algorithm of Smith and Waterman, Advances in Applied Mathematics 2: 482-489 (1981), to find the best segment of homology between two sequences. When using Bestfit or any other sequence alignment program to determine whether a particular sequence is, for instance, 95% identical to a reference sequence according to the present invention, the parameters are set, of course, such that the percentage of identity is calculated over the full length of the reference nucleotide sequence and that gaps in homology of up to 5% of the total number of nucleotides in the reference sequence are allowed.

[0063] Preferred are nucleic acid molecules having sequences at least 90%, 95%, 96%, 97%, 98% or 99% identical to the nucleic acid sequence of an E. coli J96 PAI ORF that encode a functional polypeptide. By a “functional polypeptide” is intended a polypeptide exhibiting activity similar, but not necessarily identical, to an activity of the protein encoded by the E. coli J96 PAI ORF. For example, the E. coli ORF [Contig ID 84, ORF ID 3 (84/3)] encodes a hemolysin. Thus, a functional polypeptide encoded by a nucleic acid molecule having a nucleotide sequence, for example, 95% identical to the nucleotide sequence of 84/3, will also possess hemolytic activity. As the skilled artisan will appreciate, assays for determining whether a particular polypeptide is functional will depend on which ORF is used as the reference sequence. Depending on the reference ORF, the assay chosen for measuring polypeptide activity will be readily apparent in light of the role categories provided in Tables 1, 3, 5 and 6.

[0064] Of course, due to the degeneracy of the genetic code, one of ordinary skill in the art will immediately recognize that a large number of the nucleic acid molecules having a sequence at least 90%, 95%, 96%, 97%, 98%, or 99% identical to the nucleic acid sequence of a reference ORF will encode a functional polypeptide. In fact, since degenerate variants all encode the same amino acid sequence, this will be clear to the skilled artisan even without performing a comparison assay for protein activity. It will be further recognized in the art that, for such nucleic acid molecules that are not degenerate variants, a reasonable number will also encode a functional polypeptide. This is because the skilled artisan is fully aware of amino acid substitutions that are either less likely or not likely to significantly affect protein function (e.g., replacing one aliphatic amino acid with a second aliphatic amino acid).

[0065] For example, guidance concerning how to make phenotypically silent amino acid substitutions is provided in Bowie, J. U. et al., “Deciphering the Message in Protein Sequences: Tolerance to Amino Acid Substitutions,” Science 247:1306-1310 (1990), wherein the authors indicate that there are two main approaches for studying the tolerance of an amino acid sequence to change. The first method relies on the process of evolution, in which mutations are either accepted or rejected by natural selection. The second approach uses genetic engineering to introduce amino acid changes at specific positions of a cloned gene and selections or screens to identify sequences that maintain functionality. As the authors state, these studies have revealed that proteins are surprisingly tolerant of amino acid substitutions. The authors further indicate which amino acid changes are likely to be permissive at a certain position of the protein. For example, most buried amino acid residues require nonpolar side chains, whereas few features of surface side chains are generally conserved. Other such phenotypically silent substitutions are described in Bowie, J. U. et al., supra, and the references cited therein.

[0066] The present invention is further directed to fragments of the isolated nucleic acid molecules described herein. By a fragment of an isolated nucleic acid molecule having the nucleotide sequence of an E. coli J96 PAI ORF is intended fragments at least about 15 nt, and more preferably at least about 20 nt, still more preferably at least about 30 nt, and even more preferably, at least about 40 nt in length that are useful as diagnostic probes and primers as discussed herein. Of course, larger fragments 50-500 nt in length are also useful according to the present invention as are fragments corresponding to most, if not all, of the nucleotide sequence of an E. coli J96 PAI ORF. By a fragment at least 20 nt in length, for example, is intended fragments that include 20 or more contiguous bases from the nucleotide sequence of an E. coli J96 PAI ORF. Since E. coli ORFs are listed in Tables 1 through 6 and the sequences of the ORFs have been provided within the contig sequences of SEQ ID NOs: 1 through 142, generating such DNA fragments would be routine to the skilled artisan. For example, restriction endonuclease cleavage or shearing by sonication could easily be used to generate fragments of various sizes from the PAI DNA that is incorporated into the deposited pools of cosmid clones. Alternatively, such fragments could be generated synthetically.

[0067] Preferred nucleic acid fragments of the present invention include nucleic acid molecules encoding epitope-bearing portions of an E. coli J96 PAI protein. Methods for determining such epitope-bearing portions are described in detail below.

[0068] In another aspect, the invention provides an isolated nucleic acid molecule comprising a polynucleotide that hybridizes under stringent hybridization conditions to a portion of the polynucleotide in a nucleic acid molecule of the invention described above, for instance, an ORF described in Tables 1 through 6, preferably an ORF described in Tables 1, 2, 3 or 4. By “stringent hybridization conditions” is intended overnight incubation at 42° C. in a solution comprising: 50% formamide, 5×SSC (750 mM NaCl, 75 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5× Denhardt's solution, 10% dextran sulfate, and 20 g/ml denatured, sheared salmon sperm DNA, followed by washing the filters in 0.1×SSC at about 65° C.

[0069] By a polynucleotide that hybridizes to a “portion” of a polynucleotide is intended a polynucleotide (either DNA or RNA) hybridizing to at least about 15 nucleotides (nt), and more preferably at least about 20 nt, still more preferably at least about 30 nt, and even. more preferably about 30-70 nt of the reference polynucleotide. These are useful as diagnostic probes and primers as discussed above and in more detail below.

[0070] Of course, polynucleotides hybridizing to a larger portion of the reference polynucleotide (e.g., a E. coli ORF), for instance, a portion 50-500 nt in length, or even to the entire length of the reference polynucleotide, are also useful as probes according to the present invention, as are polynucleotides corresponding to most, if not all, of an E. coli J96 PAI ORF.

[0071] By “expression modulating fragment” (EMF), is intended a series of nucleotides that modulate the expression of an operably linked ORF or EMF. A sequence is said to “modulate the expression of an operably linked sequence” when the expression of the sequence is altered by the presence of the EMF. EMFs include, but are not limited to, promoters, and promoter modulating sequences (inducible elements). One class of EMFs are fragments that induce the expression of an operably linked ORF in response to a specific regulatory factor or physiological event. EMF sequences can be identified within the E. coli genome by their proximity to the ORFs described in Tables 1 through 6. An intergenic segment, or a fragment of the intergenic segment, from about 10 to 200 nucleotides in length, taken 5′ from any one of the ORFs of Tables 1 through 6 will modulate the expression of an operably linked 3′ ORF in a fashion similar to that found with the naturally linked ORF sequence. As used herein, an “intergenic segment” refers to the fragments of the E. coli J96 PAI subgenome that are between two ORF(s) herein described. Alternatively, EMFs can be identified using known EMFs as a target sequence or target motif in the computer-based systems of the present invention.

[0072] The presence and activity of an EMF can be confirmed using an EMF trap vector. An EMF trap vector contains a cloning site 5′ to a marker sequence. A marker sequence encodes an identifiable phenotype, such as antibiotic resistance or a complementing nutrition auxotrophic factor, which can be identified or assayed when the EMF trap vector is placed within an appropriate host under appropriate conditions. As described above, an EMF will modulate the expression of an operably linked marker sequence. A more detailed discussion of various marker sequences is provided below.

[0073] A sequence that is suspected as being an EMF is cloned in all three reading frames in one or more restriction sites upstream from the marker sequence in the EMF trap vector. The vector is then transformed into an appropriate host using known procedures and the phenotype of the transformed host in examined under appropriate conditions. As described above, an EMF will modulate the expression of an operably linked marker sequence.

[0074] By a “diagnostic fragment” (DF), is intended a series of nucleotides that selectively hybridize to E. coli sequences. DFs can be readily identified by identifying unique sequences within the E. coli J96 PAI subgenome, or by generating and testing probes or amplification primers consisting of the DF sequence in an appropriate diagnostic format for amplification or hybridization selectivity.

[0075] Each of the ORFs of the E. coli J96 PAI subgenome disclosed in Tables 1 through 4, and the EMF found 5′ to the ORF, can be used in numerous ways as polynucleotide reagents. The sequences can be used as diagnostic probes or diagnostic amplification primers to detect the presence of uropathogenic E. coli in a sample. This is especially the case with the fragments or ORFs of Table 2 and 4 which will be highly selective for uropathogenic E. coli J96, and perhaps other uropathogenic or extraintestinal strains that include one or more PAIs.

[0076] In addition, the fragments of the present invention, as broadly described, can be used to control gene expression through triple helix formation or antisense DNA or RNA, both of which methods are based on the binding of a polynucleotide sequence to DNA or RNA. Polynucleotides suitable for use in these methods are usually 20 to 40 bases in length and are designed to be complementary to a region of the gene involved in transcription (triple helix—see Lee et al., Nucl. Acids Res. 6:3073 (1979); Cooney et al., Science 241:456 (1988); and Dervan et al., Science 251:1360 (1991)) or to the mRNA itself (antisense—Okano, J. Neurochem. 56:560 (1991); Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, Fla. (1988)).

[0077] Triple helix-formation optimally results in a shut-off of RNA transcription from DNA, while antisense RNA hybridization blocks translation of an mRNA molecule into polypeptide. Both techniques have been demonstrated to be effective in model systems. Information contained in the sequences of the present invention is necessary for the design of an antisense or triple helix oligonucleotide.

[0078] Vectors and Host Cells

[0079] The present invention further provides recombinant constructs comprising one or more fragments of the E. coli J96 PAIs. The recombinant constructs of the present invention comprise a vector, such as a plasmid or viral vector, into which, for example, an E. coli J96 PAI ORF is inserted. The vector may further comprise regulatory sequences, including for example, a promoter, operably linked to the ORF. For vectors comprising the EMFs of the present invention, the vector may further comprise a marker sequence or heterologous ORF operably linked to the EMF. Large numbers of suitable vectors and promoters are known to those of skill in the art and are commercially available for generating the recombinant constructs of the present invention. The following vectors are provided by way of example. Bacterial: pBs, phagescript, PsiX174, pBluescript SK, pBs KS, pNH8a, pNH16a, pNH18a, pNH46a (Stratagene); pTrc99A, pKK223-3, pKK233-3, pDR540, pRIT5 (Pharmacia). Eukaryotic: pWLneo, pSV2cat, pOG44, pXT1, pSG (Stratagene) pSVK3, pBPV, pMSG, pSVL (Pharmacia).

[0080] Promoter regions can be selected from any desired gene using CAT (chloramphenicol transferase) vectors or other vectors with selectable markers. Two appropriate vectors are pKK232-8 and pCM7. Particular named bacterial promoters include lacd, lacZ, T3, T7, gpt, lambda P , and trc. Eukaryotic promoters include CMV immediate early, HSV thymidine kinase, early and late SV40, LTRs from retrovirus, and mouse metallothionein-I. Selection of the appropriate vector and promoter is well within the level of ordinary skill in the art.

[0081] The present invention further provides host cells containing any one of the isolated fragments (preferably an ORF) of the E. coli J96 PAIs described herein. The host cell can be a higher eukaryotic host cell, such as a mammalian cell, a lower eukaryotic host cell, such as a yeast cell, or the host cell can be a procaryotic cell, such as a bacterial cell. Introduction of the recombinant construct into the host cell can be effected by calcium phosphate transfection, DEAE, dextran mediated transfection, or electroporation (Davis, L. et al., Basic Methods in Molecular Biology (1986)). Host cells containing, for example, an E. coli J96 PAI ORF can be used conventionally to produce the encoded protein.

[0082] Polypeptides and Fragments

[0083] The invention further provides isolated polypeptides having the amino acid sequence encoded by an E coli PAI ORF described in Tables 1 through 6, preferably Tables 1 through 4, or a peptide or polypeptide comprising a portion of the above polypeptides. The terms “peptide” and “oligopeptide” are considered synonymous (as is commonly recognized) and each term can be used interchangeably as the context requires to indicate a chain of at least to amino acids coupled by peptidyl linkages. The word “polypeptide” is used herein for chains containing more than ten amino acid residues. All oligopeptide and polypeptide formulas or sequences herein are written from left to right and in the direction from amino terminus to carboxy terminus.

[0084] It will be recognized in the art that some amino acid sequences of E. coli polypeptides can be varied without significant effect of the structure or function of the protein. If such differences in sequence are contemplated, it should be remembered that there will be critical areas on the protein which determine activity. In general, it is possible to replace residues that form the tertiary structure, provided that residues performing a similar function are used. In other instances, the type of residue may be completely unimportant if the alteration occurs at a non-critical region of the protein.

[0085] Thus, the invention further includes variations of polypeptides encoded for by ORFs listed in Tables 1 through 6 which show substantial pathogenic activity or which include regions of particular E. coli PAI proteins such as the protein portions discussed below. Such mutants include deletions, insertions, inversions, repeats, and type substitutions (for example, substituting one hydrophilic residue for another, but not strongly hydrophilic for strongly hydrophobic as a rule). Small changes or such “neutral” amino acid substitutions will generally have little effect on activity.

[0086] Typically seen as conservative substitutions are the replacements, one for another, among the aliphatic amino acids Ala, Val, Leu and lie; interchange of the hydroxyl residues Ser and Thr, exchange of the acidic residues Asp and Glu, substitution between the amide residues Asn and Gln, exchange of the basic residues Lys and Arg and replacements among the aromatic residues Phe, Tyr.

[0087] As indicated in detail above, further guidance concerning which amino acid changes are likely to be phenotypically silent (i.e., are not likely to have a significant deleterious effect on a function) can be found in Bowie, J. U., et al., “Deciphering the Message in Protein Sequences: Tolerance to Amino Acid Substitutions,” Science 247:1306-1310 (1990).

[0088] Thus, the fragment, derivative or analog of a polypeptide encoded by an ORF described in one of Tables 1 through 6, may be (i) one in which one or more of the amino acid residues are substituted with a conserved or non-conserved amino acid residue (preferably a conserved amino acid residue) and such substituted amino acid residue may or may not be one encoded by the genetic code, or (ii) one in which one or more of the amino acid residues includes a substituent group, or (iii) one in which the mature polypeptide is fused with another compound, such as a compound to increase the half-life of the polypeptide (for example, polyethylene glycol), or (iv) one in which the additional amino acids are fused to the mature polypeptide, such as an IgG Fc fusion region peptide or leader or secretory sequence or a sequence which is employed for purification of the mature polypeptide or a proprotein sequence. Such fragments, derivatives and analogs are deemed to be within the scope of those skilled in the art from the teachings herein.

[0089] Of particular interest are substitutions of charged amino acids with another charged amino acid and with neutral or negatively charged amino acids. The latter results in proteins with reduced positive charge to improve the characteristics of said proteins. The prevention of aggregation is highly desirable. Aggregation of proteins not only results in a loss of activity but can also be problematic when preparing pharmaceutical formulations, because they can be immunogenic. (Pinckard et al., Clin Exp. Immunol. 2:331-340 (1967); Robbins et al., Diabetes 36:838-845 (1987); Cleland et al. Crit. Rev. Therapeutic Drug Carrier Systems 10:307-377 (1993)).

[0090] The replacement of amino acids can also change the selectivity of binding to cell surface receptors. Ostade et al., Nature 361:266-268 (1993) describes certain mutations resulting in selective binding of TNF-Â to only one of the two known types of TNF receptors. Thus, proteins encoded for by the ORFs listed in Tables 1, 2, 3, 4, 5, or 6, and that bind to a cell surface receptor, may include one or more amino acid substitutions, deletions or additions, either from natural mutations or human manipulation.

[0091] As indicated, changes are preferably of a minor nature, such as conservative amino acid substitutions that do not significantly affect the folding or activity of the protein (see Table 7).

1TABLE 7Conservative Amino Acid SubstitutionsAromaticPhenylalanineTryptophanTyrosineHydrophobicLeucineIsoleucineValinePolarGlutamineAsparagineBasicArginineLysineHistidineAcidicAspartic AcidGlutamic AcidSmallAlanineSerineGlycineThreonineMethionine

[0092] Amino acids in the proteins encoded by ORFs of the present invention that are essential for function can be identified by methods known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (Cunningham and Wells, Science 244:1081-1085 (1989)). The latter procedure introduces single alanine mutations at every residue in the molecule. The resulting mutant molecules are then tested for biological activity such as receptor binding or in vitro, or in vitro proliferative activity. Sites that are critical for ligand-receptor binding can also be determined by structural analysis such as crystallization, nuclear magnetic resonance or photoaffinity labeling (Smith et al., J. Mol. Biol. 224:899-904 (1992) and de Vos et al. Science 255:306-312 (1992)).

[0093] The polypeptides of the present invention are preferably provided in an isolated form, and preferably are substantially purified. A recombinantly produced version of the polypeptides can be substantially purified by the one-step method described in Smith and Johnson, Gene 67:31-40 (1988).

[0094] The polypeptides of the present invention include the polypeptide encoded by the ORFs listed in Tables 1-6, preferably Tables 1-4, as well as polypeptides which have at least 90% similarity, more preferably at least 95% similarity, and still more preferably at least 96%, 97%, 98% or 99% similarity to those described above, and also include portions of such polypeptides with at least 30 amino acids and more preferably at least 50 amino acids.

[0095] By “% similarity” for two polypeptides is intended a similarity score produced by comparing the amino acid sequences of the two polypeptides using the Bestfit program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive, Madison, Wis. 53711) and the default settings for determining similarity. Bestfit uses the local homology algorithm of Smith and Waterman (Advances in Applied Mathematics 2:482-489, 1981) to find the best segment of similarity between two sequences.

[0096] By a polypeptide having an amino acid sequence at least, for example, 95% “identical” to a reference amino acid sequence of a polypeptide is intended that the amino acid sequence of the polypeptide is identical to the reference sequence except that the polypeptide sequence may include up to five amino acid alterations per each 100 amino acids of the reference amino acid of said polypeptide. In other words, to obtain a polypeptide having an amino acid sequence at least 95% identical to a reference amino acid sequence, up to 5% of the amino acid residues in the reference sequence may be deleted or substituted with another amino acid, or a number of amino acids up to 5% of the total amino acid residues in the reference sequence may be inserted into the reference sequence. These alterations of the reference sequence may occur at the amino or carboxy terminal positions of the reference amino acid sequence or anywhere between those terminal positions, interspersed either individually among residues in the reference sequence or in one or more contiguous groups within the reference sequence.

[0097] As a practical matter, whether any particular polypeptide is at least 90%, 95%, 96%, 97%, 98% or 99% identical to, for instance, the amino acid sequence encoded by the ORFs listed in Tables 1, 2, 3, 4, 5, or 6 can be determined conventionally using known computer programs such the Bestfit program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive, Madison, Wis. 53711. When using Bestfit or any other sequence alignment program to determine whether a particular sequence is, for instance, 95% identical to a reference sequence according to the present invention, the parameters are set, of course, such that the percentage of identity is calculated over the full length of the reference amino acid sequence and that gaps in homology of up to 5% of the total number of amino acid residues in the reference sequence are allowed.

[0098] The polypeptide of the present invention could be used as a molecular weight marker on SDS-PAGE gels or on molecular sieve gel filtration columns using methods well known to those of skill in the art.

[0099] As described in detail below, the polypeptides of the present invention can also be used to raise polyclonal and monoclonal antibodies, which are useful in assays for detecting pathogenic protein expression as described below or as agonists and antagonists capable of enhancing or inhibiting protein function of important proteins encoded by the ORFs of the present invention. Further, such polypeptides can be used in the yeast two-hybrid system to “capture” protein binding proteins which are also candidate agonist and antagonist according to the present invention. The yeast two hybrid system is described in Fields and Song, Nature 340:245-246 (1989).

[0100] In another aspect, the invention provides a peptide or polypeptide comprising an epitope-bearing portion of a polypeptide of the invention. The epitope of this polypeptide portion is an immunogenic or antigenic epitope of a polypeptide of the invention. An “immunogenic epitope” is defined as a part of a protein that elicits an antibody response when the whole protein is the immunogen. These immunogenic epitopes are believed to be confined to a few loci on the molecule. On the other hand, a region of a protein molecule to which an antibody can bind is defined as an “antigenic epitope.” The number of immunogenic epitopes of a protein generally is less than the number of antigenic epitopes. See, for instance, Geysen et al., Proc. Natl. Acad. Sci. USA 81:3998-4002 (1983).

[0101] As to the selection of peptides or polypeptides bearing an antigenic epitope (i.e., that contain a region of a protein molecule to which an antibody can bind), it is well known in that art that relatively short synthetic peptides that mimic part of a protein sequence are routinely capable of eliciting an antiserum that reacts with the partially mimicked protein. See, for instance, Sutcliffe, J. G., Shinnick, T. M., Green, N. and Learner, R. A. (1983) Antibodies that react with predetermined sites on proteins. Science 219:660-666. Peptides capable of eliciting protein-reactive sera are frequently represented in the primary sequence of a protein, can be characterized by a set of simple chemical rules, and are confined neither to immunodominant regions of intact proteins (i.e., immunogenic epitopes) nor to the amino or carboxyl terminals. Peptides that are extremely hydrophobic and those of six or fewer residues generally are ineffective at inducing antibodies that bind to the mimicked protein; longer, peptides, especially those containing proline residues, usually are effective. Sutcliffe et al., supra, at 661. For instance, 18 of 20 peptides designed according to these guidelines, containing 8-39 residues covering 75% of the sequence of the influenza virus hemagglutinin HA1 polypeptide chain, induced antibodies that reacted with the HA1 protein or intact virus; and 12/12 peptides from the MuLV polymerase and 18/18 from the rabies glycoprotein induced antibodies that precipitated the respective proteins.

[0102] Antigenic epitope-bearing peptides and polypeptides of the invention are therefore useful to raise antibodies, including monoclonal antibodies that bind specifically to a polypeptide of the invention. Thus, a high proportion of hybridomas obtained by fusion of spleen cells from donors immunized with an antigen epitope-bearing peptide generally secrete antibody reactive with the native protein. Sutcliffe et al., supra, at 663. The antibodies raised by antigenic epitope-bearing peptides or polypeptides are useful to detect the mimicked protein, and antibodies to different peptides may be used for tracking the fate of various regions of a protein precursor which undergoes post-translational processing. The peptides and anti-peptide antibodies may be used in a variety of qualitative or quantitative assays for the mimicked protein, for instance in competition assays since it has been shown that even short peptides (e.g., about 9 amino acids) can bind and displace the larger peptides in immunoprecipitation assays. See, for instance, Wilson et al., Cell 37:767-778 (1984) at 777. The anti-peptide antibodies of the invention also are useful for purification of the mimicked protein, for instance, by adsorption chromatography using methods well known in the art.

[0103] Antigenic epitope-bearing peptides and polypeptides of the invention designed according to the above guidelines preferably contain a sequence of at least seven, more preferably at least nine and most preferably between about 15 to about 30 amino acids contained within the amino acid sequence of a polypeptide of the invention. However, peptides or polypeptides comprising a larger portion of an amino acid sequence of a polypeptide of the invention, containing about 30 to about 50 amino acids, or any length up to and including the entire amino acid sequence of a polypeptide of the invention, also are considered epitope-bearing peptides or polypeptides of the invention and also are useful for inducing antibodies that react with the mimicked protein. Preferably, the amino acid sequence of the epitope-bearing peptide is selected to provide substantial solubility in aqueous solvents (i.e., the sequence includes relatively hydrophilic residues and highly hydrophobic sequences are preferably avoided); and sequences containing proline residues are particularly preferred.

[0104] The epitope-bearing peptides and polypeptides of the invention may be produced by any conventional means for making peptides or polypeptides including recombinant means using nucleic acid molecules of the invention. For instance, a short epitope-bearing amino acid sequence may be fused to a larger polypeptide, which acts as a carrier during recombinant production and purification, as well as during immunization to produce anti-peptide antibodies. Epitope-bearing peptides also may be synthesized using known methods of chemical synthesis. For instance, Houghten has described a simple method for synthesis of large numbers of peptides, such as 10-20 mg of 248 different 13 residue peptides representing single amino acid variants of a segment of the HA1 polypeptide which were prepared and characterized (by ELISA-type binding studies) in less than four weeks. Houghten, R. A. (1985) General method for the rapid solid-phase synthesis of large numbers of peptides: specificity of antigen-antibody interaction at the level of individual amino acids. Proc. Natl. Acad. Sci. USA 82:5131-5135. This “Simultaneous Multiple Peptide Synthesis (SMPS)” process is further described in U.S. Pat. No. 4,631,211 to Houghten et al. (1986). In this procedure the individual resins for the solid-phase synthesis of various peptides are contained in separate solvent-permeable packets, enabling the optimal use of the many identical repetitive steps involved in solid-phase methods. A completely manual procedure allows 500-1000 or more syntheses to be conducted simultaneously. Houghten et al., supra, at 5134.

[0105] Epitope-bearing peptides and polypeptides of the invention are used to induce antibodies according to methods well known in the art. See, for instance, Sutcliffe et al., supra; Wilson et al, supra; Chow, M. et al., Proc. Natl. Acad. Sci. USA 82:910-914; and Bittle, F. J. et al., J. Gen. Virol. 66:2347-2354 (1985). Generally, animals may be immunized with free peptide; however, anti-peptide antibody titer may be boosted by coupling of the peptide to a macromolecular carrier, such as keyhole limpet hemacyanin (KLH) or tetanus toxoid. For instance, peptides containing cysteine may be coupled to carrier using a linker such as m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), while other peptides may be coupled to carrier using a more general linking agent such as glutaraldehyde. Animals such as rabbits, rats and mice are immunized with either free or carrier-coupled peptides, for instance, by intraperitoneal and/or intradermal injection of emulsions containing about 100 μg peptide or carrier protein and Freund's adjuvant. Several booster injections may be needed, for instance, at intervals of about two weeks, to provide a useful titer of anti-peptide antibody, which can be detected, for example, by ELISA assay using free peptide adsorbed to a solid surface. The titer of anti-peptide antibodies in serum from an immunized animal may be increased by selection of anti-peptide antibodies, for instance, by adsorption to the peptide on a solid support and elution of the selected antibodies according to methods well known in the art.

[0106] Immunogenic epitope-bearing peptides of the invention, i.e., those parts of a protein that elicit an antibody response when the whole protein is the immunogen, are identified according to methods known in the art. For instance, Geysen et al., supra, discloses a procedure for rapid concurrent synthesis on solid supports of hundreds of peptides of sufficient purity to react in an enzyme-linked immunosorbent assay. Interaction of synthesized peptides with antibodies is then easily detected without removing them from the support. In this manner a peptide bearing an immunogenic epitope of a desired protein may be identified routinely by one of ordinary skill in the art. For instance, the immunologically important epitope in the coat protein of foot-and-mouth disease virus was located by Geysen et al. supra with a resolution of seven amino acids by synthesis of an overlapping set of all 208 possible hexapeptides covering the entire 213 amino acid sequence of the protein. Then, a complete replacement set of peptides in which all 20 amino acids were substituted in turn at every position within the epitope were synthesized, and the particular amino acids conferring specificity for the reaction with antibody were determined. Thus, peptide analogs of the epitope-bearing peptides of the invention can be made routinely by this method. U.S. Pat. No. 4,708,781 to Geysen (1987) further describes this method of identifying a peptide bearing an immunogenic epitope of a desired protein.

[0107] Further still, U.S. Pat. No. 5,194,392 to Geysen (1990) describes a general method of detecting or determining the sequence of monomers (amino acids or other compounds) which is a topological equivalent of the epitope (i.e., a “mimotope”) which is complementary to a particular paratope (antigen binding site) of an antibody of interest. More generally, U.S. Pat. No. 4,433,092 to Geysen (1989) describes a method of detecting or determining a sequence of monomers which is a topographical equivalent of a ligand which is complementary to the ligand binding site of a particular receptor of interest. Similarly, U.S. Pat. No. 5,480,971 to Houghten, R. A. et al. (1996) on Peralkylated Oligopeptide Mixtures discloses linear C—C-alkyl peralkylated oligopeptides and sets and libraries of such peptides, as well as methods for using such oligopeptide sets and libraries for determining the sequence of a

\overset{1}{p} er \overset{7}{a} lkylated

[0108] oligopeptide that preferentially binds to an acceptor molecule of interest. Thus, non-peptide analogs of the epitope-bearing peptides of the invention also can be made routinely by these methods.

[0109] The entire disclosure of each document cited in this section on “Polypeptides and Peptides” is hereby incorporated herein by reference.

[0110] As one of skill in the art will appreciate, E. coli PAI polypeptides of the present invention and the epitope-bearing fragments thereof described above can be combined with parts of the constant domain of immunoglobulins (IgG), resulting in chimeric polypeptides. These fusion proteins facilitate purification and show an increased half-life in vivo. This has been shown, e.g., for chimeric proteins consisting of the first two domains of the human CD4-polypeptide and various domains of the constant regions of the heavy or light chains of mammalian immunoglobulins (EP A 394,827; Traunecker et al., Nature 331:84-86 (1988)). Fusion proteins that have a disulfide-linked dimeric structure due to the IgG part can also be more efficient in binding and neutralizing other molecules than the monomeric E. coli J96 PAI proteins or protein fragments alone (Fountoulakis et al., J. Biochem 270:3958-3964 (1995)).

[0111] Vaccines

[0112] In another embodiment, the present invention relates to a vaccine, preferably in unit dosage form, comprising one or more E. coli J96 PAI antigens together with a pharmaceutically acceptable diluent, carrier, or excipient, wherein the one or more antigens are present in an amount effective to elicit a protective immune response in an animal to pathogenic E. coli. Antigens of E. coli J96 PAI IV and V may be obtained from polypeptides encoded for by the ORFs listed in Tables 1-6, particularly Tables 1-4, using methods well known in the art.

[0113] In a preferred embodiment, the antigens are E. coli J96 PAI IV or PAI V proteins that are present on the surface of pathogenic E. coli. In another preferred embodiment, the pathogenic E. coli J96 PAI IV or PAI V protein-antigen is conjugated to an E. coli capsular polysaccharide (CP), particularly to capsular polypeptides that are more prevalent in pathogenic strains, to produce a double vaccine. CPs, in general, may be prepared or synthesized as described in Schneerson et al. J. Exp. Med. 152:361-376 (1980); Marburg et al. J. Am. Chem. Soc. 108:5282 (1986); Jennings et al., J. Immunol. 127:1011-1018 (1981); and Beuvery et al., Infect. Immunol. 40:39-45 (1983). In a further preferred embodiment, the present invention relates to a method of preparing a polysaccharide conjugate comprising: obtaining the above-described E. coli J96 PAI antigen; obtaining a CP or fragment from pathogenic E. coli; and conjugating the antigen to the CP or CP fragment.

[0114] In a preferred embodiment, the animal to be protected is selected from the group consisting of humans, horses, deer, cattle, pigs, sheep, dogs, and chickens. In a more preferred embodiment, the animal is a human or a dog.

[0115] In a further embodiment, the present invention relates to a prophylactic method whereby the incidence of pathogenic E. coli-induced symptoms are decreased in an animal, comprising administering to the animal the above-described vaccine, wherein the vaccine is administered in an amount effective to elicit protective antibodies in an animal to pathogenic E. coli. This vaccination method is contemplated to be useful in protecting against severe diarrhea (pathogenic intestinal E. coli strains), urinary tract infections (uropathogenic E. coli) and infections of the brain (extraintestinal E. coli). The vaccine of the invention is used in an effective amount depending on the route of administration. Although intra-nasal, subcutaneous or intramuscular routes of administration are preferred, the vaccine of the present invention can also be administered by an oral, intraperitoneal or intravenous route. One skilled in the art will appreciate that the amounts to be administered for any particular treatment protocol can be readily determined without undue experimentation. Suitable amounts are within the range of 2 micrograms of the protein per kg body weight to 100 micrograms per kg body weight.

[0116] The vaccine can be delivered through a vector such as BCG. The vaccine can also be delivered as naked DNA coding for target antigens.

[0117] The vaccine of the present invention may be employed in such dosage forms as capsules, liquid solutions, suspensions or elixirs for oral administration, or sterile liquid forms such as solutions or suspensions. Any inert carrier is preferably used, such as saline, phosphate-buffered saline, or any such carrier in which the vaccine has suitable solubility properties. The vaccines may be in the form of single dose preparations or in multi-dose flasks which can be used for mass vaccination programs. Reference is made to Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., Osol (ed.) (1980); and New Trends and Developments in Vaccines, Voller et al. (eds.), University Park Press, Baltimore, Md. (1978), for methods of preparing and using vaccines.

[0118] The vaccines of the present invention may further comprise adjuvants which enhance production of antibodies and immune cells. Such adjuvants include, but are not limited to, various oil formulations such as Freund's complete adjuvant (CFA), the dipeptide known as MDP, saponins (ex. Quillajasaponin fraction QA-21, U.S. Pat. No. 5,047,540), aluminum hydroxide, or lymphatic cytokines. Freund's adjuvant is an emulsion of mineral oil and water which is mixed with the immunogenic substance. Although Freund's adjuvant is powerful, it is usually not administered to humans. Instead, the adjuvant alum (aluminum hydroxide) may be used for administration to a human. Vaccine may be absorbed onto the aluminum hydroxide from which it is slowly released after injection. The vaccine may also be encapsulated within liposomes according to Fullerton, U.S. Pat. No. 4,235,877.

[0119] Protein Function

[0120] Each ORF described in Tables 1 and 3 possesses a biological role similar to the role associated with the identified homologous protein. This allows the skilled artisan to determine a function for each identified coding sequence. For example, a partial list of the E. coli protein functions provided in Tables 1 and 3 includes many of the functions associated with virulence of pathogenic bacterial strains. These include, but are not limited to adhesins, excretion pathway proteins, O-antigen/carbohydrate modification, cytotoxins and regulators. A more detailed description of several of these functions is provided in Example 1 below.

[0121] Diagnostic Assays

[0122] In another preferred embodiment, the present invention relates to a method of detecting pathogenic E. coli nucleic acid in a sample comprising:

[0123] (a) contacting the sample with the above-described nucleic acid probe, under conditions such that hybridization occurs, and

[0124] (b) detecting the presence of the probe bound to pathogenic E. coli nucleic acid.

[0125] In another preferred embodiment, the present invention relates to a diagnostic kit for detecting the presence of pathogenic E. coli nucleic acid in a sample comprising at least one container means having disposed therein the above-described nucleic acid probe.

[0126] In another preferred embodiment, the present invention relates to a diagnostic kit for detecting the presence of pathogenic E. coli antigens in a sample comprising at least one container means having disposed therein the above-described antibodies.

[0127] In another preferred embodiment, the present invention relates to a diagnostic kit for detecting the presence of antibodies to pathogenic E. coli antigens in a sample comprising at least one container means having disposed therein the above-described antigens.

[0128] The present invention provides methods to identify the expression of an ORF of the present invention, or homolog thereof, in a test sample, using one of the antibodies of the present invention. Such methods involve incubating a test sample with one or more of the antibodies of the present invention and assaying for binding of the antibodies to components within the test sample.

[0129] In a further embodiment, the present invention relates to a method for identifying pathogenic E. coli in an animal comprising analyzing tissue or body fluid from the animal for a nucleic acid, protein, polypeptide-antigen or antibody specific to one of the ORFs described in Tables 1-4 herein from E. coli J96 PAI IV or V. Analysis of nucleic acid specific to pathogenic E. coli can be by PCR techniques or hybridization techniques (cf. Molecular Cloning: A Laboratory Manual, second edition, edited by Sambrook, Fritsch, & Maniatis, Cold Spring Harbor Laboratory, 1989; Eremeeva et al., J. Clin. Microbiol. 32:803-810 (1994) which describes differentiation among spotted fever group Rickettsiae species by analysis of restriction fragment length polymorphism of PCR-amplified DNA).

[0130] Proteins or antibodies specific to pathogenic E. coli may be identified as described in Molecular Cloning: A Laboratory Manual, second edition, Sambrook et al., eds., Cold Spring Harbor Laboratory (1989). More specifically, antibodies may be raised to E. coli J96 PAI proteins as generally described in Antibodies: A Laboratory Manual, Harlow and Lane, eds., Cold Spring Harbor Laboratory (1988). E. coli J96 PAI-specific antibodies can also be obtained from infected animals (Mather, T. et al., JAMA 205:186-188 (1994)).

[0131] In another embodiment, the present invention relates to an antibody having binding affinity specifically to an E. coli J96 PAI antigen as described above. The E. coli J96 PAI antigens of the present invention can be used to produce antibodies or hybridomas. One skilled in the art will recognize that if an antibody is desired, a peptide can be generated as described herein and used as an immunogen. The antibodies of the present invention include monoclonal and polyclonal antibodies, as well as fragments of these antibodies. The invention further includes single chain antibodies. Antibody fragments which contain the idiotype of the molecule can be generated by known techniques, for example, such fragments include but are not limited to: the F(ab ) fragment; the Fab fragments, Fab fragments, and Fv fragments.

[0132] Of special interest to the present invention are antibodies to pathogenic E. coli antigens which are produced in humans, or are “humanized” (i.e. non-immunogenic in a human) by recombinant or other technology. Humanized antibodies may be produced, for example by replacing an immunogenic portion of an antibody with a corresponding, but non-immunogenic portion (i.e. chimeric antibodies) (Robinson, R. R. et al., International Patent Publication PCT/US86/02269; Akira, K. et al., European Patent Application 184,187; Taniguchi, M., European Patent Application 171,496; Morrison, S. L. et al., European Patent Application 173,494; Neuberger, M. S. et al., PCT Application WO 86/01533; Cabilly, S. et al., European Patent Application 125,023; Better, M. et al., Science 240:1041-1043 (1988); Liu, A. Y. et al., Proc. Natl. Acad. Sci. USA 84:3439-3443 (1987); Liu, A. Y. et al., J. Immunol. 139:3521-3526 (1987); Sun, L. K. et al., Proc. Natl. Acad. Sci. USA 84:214-218 (1987); Nishimura, Y. et al., Canc. Res. 47:999-1005 (1987); Wood, C. R. et al., Nature 314:446-449 (1985)); Shaw et al., J. Natl. Cancer Inst. 80:1553-1559 (1988). General reviews of “humanized” chimeric antibodies are provided by Morrison, S. L. (Science, 229:1202-1207 (1985)) and by Oi, V. T. et al., BioTechniques 4:214 (1986)). Suitable “humanized” antibodies can be alternatively produced by CDR or CEA substitution (Jones, P. T. et al., Nature 321:552-525 (1986); Verhoeyan et al., Science 239:1534 (1988); Beidler, C. B. et al., J. Immunol. 141:4053-4060 (1988)).

[0133] In another embodiment, the present invention relates to a hybridoma which produces the above-described monoclonal antibody. A hybridoma is an immortalized cell line which is capable of secreting a specific monoclonal antibody.

[0134] In general, techniques for preparing monoclonal antibodies and hybridomas are well known in the art (Campbell, “Monoclonal Antibody Technology: Laboratory Techniques in Biochemistry and Molecular Biology,” Elsevier Science Publishers, Amsterdam, The Netherlands (1984); St. Groth et al., J. Immunol. Methods 35:1-21 (1980)).

[0135] In another embodiment, the present invention relates to a method of detecting a pathogenic E. coli antigen in a sample, comprising: a) contacting the sample with an above-described antibody, under conditions such that immunocomplexes form, and b) detecting the presence of said antibody bound to the antigen. In detail, the methods comprise incubating a test sample with one or more of the antibodies of the present invention and assaying whether the antibody binds to the test sample.

[0136] Conditions for incubating an antibody with a test sample vary. Incubation conditions depend on the format employed in the assay, the detection methods employed, and the type and nature of the antibody used in the assay. One skilled in the art will recognize that any one of the commonly available immunological assay formats (such as radioimmunoassays, enzyme-linked immunosorbent assays, diffusion based Ouchterlony, or rocket immunofluorescent assays) can readily be adapted to employ the antibodies of the present invention. Examples of such assays can be found in Chard, An Introduction to Radioimmunoassay and Related Techniques, Elsevier Science Publishers, Amsterdam, The Netherlands (1986); Bullock et al., Techniques in Immunocytochemistry, Academic Press, Orlando, Fla. Vol. 1 (1982), Vol. 2 (1983), Vol. 3 (1985); Tijssen, Practice and Theory of Enzyme Immunoassays: Laboratory Techniques in Biochemistry and Molecular Biology, Elsevier Science Publishers, Amsterdam, The Netherlands (1985); and Antibodies: A Laboratory Manual, Harlow and Lane, eds., Cold Spring Harbor Laboratory (1988).

[0137] The immunological assay test samples of the present invention include cells, protein or membrane extracts of cells, or biological fluids such as blood, serum, plasma, or urine. The test sample used in the above-described method will vary based on the assay format, nature of the detection method and the tissues, cells or extracts used as the sample to be assayed. Methods for preparing protein extracts or membrane extracts of cells are well known in the art and can be readily be adapted in order to obtain a sample which is capable with the system utilized.

[0138] In another embodiment, the present invention relates to a method of detecting the presence of antibodies to pathogenic E. coli in a sample, comprising: a) contacting the sample with an above-described antigen, under conditions such that immunocomplexes form, and b) detecting the presence of said antigen bound to the antibody. In detail, the methods comprise incubating a test sample with one or more of the antigens of the present invention and assaying whether the antigen binds to the test sample.

[0139] In another embodiment of the present invention, a kit is provided which contains all the necessary reagents to carry out the previously described methods of detection. The kit may comprise: i) a first container means containing an above-described antibody, and ii) second container means containing a conjugate comprising a binding partner of the antibody and a label. In another preferred embodiment, the kit further comprises one or more other containers comprising one or more of the following: wash reagents and reagents capable of detecting the presence of bound antibodies. Examples of detection reagents include, but are not limited to, labeled secondary antibodies, or in the alternative, if the primary antibody is labeled, the chromophoric, enzymatic, or antibody binding reagents which are capable of reacting with the labeled antibody. The compartmentalized kit may be as described above for nucleic acid probe kits.

[0140] One skilled in the art will readily recognize that the antibodies described in the present invention can readily be incorporated into one of the established kit formats which are well known in the art.

[0141] Screening Assay for Binding Agents

[0142] Using the isolated proteins described herein, the present invention further provides methods of obtaining and identifying agents that bind to a protein encoded by an E. coli J96 PAI ORF or to a fragment thereof.

[0143] The method involves:

[0144] (a) contacting an agent with an isolated protein encoded by a E. coli J96 PAI ORF, or an isolated fragment thereof; and

[0145] (b) determining whether the agent binds to said protein or said fragment.

[0146] The agents screened in the above assay can be, but are not limited to, peptides, carbohydrates, vitamin derivatives, or other pharmaceutical agents. The agents can be selected and screened at random or rationally selected or designed using protein modeling techniques. For random screening, agents such as peptides, carbohydrates, pharmaceutical agents and the like are selected at random and are assayed for their ability to bind to the protein encoded by an ORF of the present invention.

[0147] Alternatively, agents may be rationally selected or designed. As used herein, an agent is said to be “rationally selected or designed” when the agent is chosen based on the configuration of the particular protein. For example, one skilled in the art can readily adapt currently available procedures to generate peptides, pharmaceutical agents and the like capable of binding to a specific peptide sequence in order to generate rationally designed antipeptide ligands, for example see Hurby et al., Application of Synthetic Peptides: Antisense Peptides, In Synthetic Peptides, A User's Guide, W.H. Freeman, NY (1992), pp. 289-307, and Kaspczak et al., Biochemistry 28:9230-8 (1989).

[0148] In addition to the foregoing, one class of agents of the present invention, can be used to control gene expression through binding to one of the ORFs or EMFs of the present invention. As described above, such agents can be randomly screened or rationally designed and selected. Targeting the ORF or EMF allows a skilled artisan to design sequence specific or element specific agents, modulating the expression of either a single ORF or multiple ORFs that rely on the same EMF for expression control.

[0149] One class of DNA binding agents are those that contain nucleotide base residues that hybridize or form a triple helix by binding to DNA or RNA. Such agents can be based on the classic phosphodiester, ribonucleic acid backbone, or can be a variety of sulfhydryl or polymeric derivatives having base attachment capacity.

[0150] Agents suitable for use in these methods usually contain 20 to 40 bases and are designed to be complementary to a region of the gene involved in transcription (triple helix—see Lee et al., Nucl. Acids Res. 6:3073 (1979); Cooney et al., Science 241:456 (1988); and Dervan et al., Science 251: 1360 (1991)) or to the mRNA itself (antisense—Okano, J. Neurochem. 56:560 (1991); Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, Fla. (1988)). Triple helix-formation optimally results in a shut-off of RNA transcription from DNA, while antisense RNA hybridization blocks translation of an mRNA molecule into polypeptide. Both techniques have been demonstrated to be effective in model systems. Information contained in the sequences of the present invention is necessary for the design of an antisense or triple helix oligonucleotide and other DNA binding agents.

[0151] Computer Related Embodiments

[0152] The nucleotide sequence provided in SEQ ID NOs: 1 through 142, representative fragments thereof, or nucleotide sequences at least 99.9% identical to the sequences provided in SEQ ID NOs: 1 through 142, can be “provided” in a variety of media to facilitate use thereof. As used herein, “provided” refers to a manufacture, other than an isolated nucleic acid molecule, that contains a nucleotide sequence of the present invention, i.e., the nucleotide sequence provided in SEQ ID NOs: 1 through 142, a representative fragment thereof, or a nucleotide sequence at least 99.9% identical to SEQ ID NOs: 1 through 142. Such a manufacture provides the E. coli J96 PAI subgenomes or a subset thereof (e.g., one or more E. coli J96 PAI open reading frame (ORF)) in a form that allows a skilled artisan to examine the manufacture using means not directly applicable to examining the E. coli J96 PAI subgenome or a subset thereof as it exists in nature or in purified form.

[0153] In one application of this embodiment, one or more nucleotide sequences of the present invention can be recorded on computer readable media. As used herein, “computer readable media” refers to any medium that can be read and accessed directly by a computer. Such media include, but are not limited to: magnetic storage media, such as floppy discs, hard disc storage medium, and magnetic tape; optical storage media such as CD-ROM; electrical storage media such as RAM and ROM; and hybrids of these categories such as magnetic/optical storage media. A skilled artisan can readily appreciate how any of the presently known computer readable mediums can be used to create a manufacture comprising computer readable medium having recorded thereon a nucleotide sequence of the present invention.

[0154] As used herein, “recorded” refers to a process for storing information on computer readable medium. A skilled artisan can readily adopt any of the presently know methods for recording information on computer readable medium to generate manufactures comprising the nucleotide sequence information of the present invention. A variety of data storage structures are available to a skilled artisan for creating a computer readable medium having recorded thereon a nucleotide sequence of the present invention. The choice of the data storage structure will generally be based on the means chosen to access the stored information. In addition, a variety of data processor programs and formats can be used to store the nucleotide sequence information of the present invention on computer readable medium. The sequence information can be represented in a word processing text file, formatted in commercially-available software such as WordPerfect and MicroSoft Word, or represented in the form of an ASCII file, stored in a database application, such as DB2, Sybase, Oracle, or the like. A skilled artisan can readily adapt any number of dataprocessor structuring formats (e.g. text file or database) in order to obtain computer readable medium having recorded thereon the nucleotide sequence information of the present invention.

[0155] By providing the nucleotide sequence of SEQ ID NOs: 1 through 142, representative fragments thereof, or nucleotide sequences at least 99.9% identical to SEQ ID NOs: 1 through 142, in computer readable form, a skilled artisan can routinely access the sequence information for a variety of purposes. Computer software is publicly available which allows a skilled artisan to access sequence information provided in a computer readable medium. The examples which follow demonstrate how software which implements the BLAST (Altschul et al., J. Mol. Biol. 215:403-410 (1990)) and BLAZE (Brutlag et al., Comp. Chem. 17:203-207 (1993)) search algorithms on a Sybase system can be used to identify open reading frames (ORFs) within the E. coli J96 PAI subgenome that contain homology to ORFs or proteins from other organisms. Such ORFs are protein-encoding fragments within the E. coli J96 PAI subgenome and are useful in producing commercially important proteins such as enzymes used in modifying surface O-antigens of bacteria. A comprehensive list of ORFs encoding commercially important E. coli J96 PAI proteins is provided in Tables 1 through 6.

[0156] The present invention provides a DNA sequence—gene database of pathogenicity islands (PAIs) for E. coli involved in infectious diseases. This database is useful for identifying and characterizing the basic functions of new virulence genes for E. coli involved in uropathogenic and extraintestinal diseases. The database provides a number of novel open reading frames that can be selected for further study as described herein.

[0157] Selectable insertion mutations in plasmid subclones encoding PAI genes with potentially significant phenotypes for E. coli uropathogenesis and sepsis can be isolated. The mutations are then crossed back into wild type, uropathogenic E. coli by homologous recombination to create wild-type strains specifically altered in the targeted gene. The significance of the genes to E. coli pathogenesis is assessed by in vitro assays and in vivo murine models of sepsis/peritonitis and ascending urinary tract infection.

[0158] New virulence genes and PAI sites in uropathogenic E. coli may be identified by the transposon signature-tagged mutagenesis system and negative selection of E. coli mutants avirulent in murine models of ascending urinary tract infection or peritonitis.

[0159] Epidemiological investigations of new virulence genes and PAIs may be used to test for their occurrence in the genomes of other pathogenic and opportunistic members of the Enterobacteriaceae.

[0160] One can choose from the ORFs included in SEQ ID NOs: 1 through 142, using Tables 1 through 6 as a useful guidepost for selecting, as candidates for targeted mutagenesis, a limited number of candidate genes within the PAIs based on their homology to virulence, export or regulation genes in other pathogens. For the large number of apparent genes within the PAIs that do not share sequence similarity to any entries in the database, the transposon signature-tagged mutagenesis method developed by David Holden's laboratory can be employed as an independent means of virulence gene identification.

[0161] Allelic knock-outs are constructed using different pir-dependent suicide vectors (Swihart, K. A. and R. A. Welch, Infect. Immun. 58:1853-1869 (1990)). In addition, two different animal model systems can be employed for assessment of pathogenic determinants. The initial identification of E. coli hemolysin as a virulence factor came from the construction of isogenic E. coli strains that were tested in a rat model of intra-abdominal sepsis (Welch, R. A. et al., Nature (London) 294:665-667 (1981)). The ascending UTI (Urinary Tract Infection) mouse model was also successfully performed with allelic knock-outs of the hpmA hemolysin of Proteus mirabilis (Swihart, K. A. and R. A. Welch, Infect. Immun. 58:1853-1869 (1990)).

[0162] The present invention further provides systems, particularly computer-based systems, which contain the sequence information described herein. Such systems are designed to identify commercially important fragments of the E. coli J96 PAI subgenome. As used herein, “a computer-based system” refers to the hardware means, software means, and data storage means used to analyze the nucleotide sequence information of the present invention. The minimum hardware means of the computer-based systems of the present invention comprises a central processing unit (CPU), input means, output means, and data storage means. A skilled artisan can readily appreciate that any one of the currently available computer-based system are suitable for use in the present invention.

[0163] As indicated above, the computer-based systems of the present invention comprise a data storage means having stored therein a nucleotide sequence of the present invention and the necessary hardware means and software means for supporting and implementing a search means. As used herein, “data storage means” refers to memory that can store nucleotide sequence information of the present invention, or a memory access means which can access manufactures having recorded thereon the nucleotide sequence information of the present invention. As used herein, “search means” refers to one or more programs which are implemented on the computer-based system to compare a target sequence or target structural motif with the sequence information stored within the data storage means. Search means are used to identify fragments or regions of the E. coli genome that match a particular target sequence or target motif. A variety of known algorithms are disclosed publicly and a variety of commercially available software for conducting search means are available and can be used in the computer-based systems of the present invention. Examples of such software include, but are not limited to, MacPattern (EMBL), BLASTN and BLASTX (NCBIA). A skilled artisan can readily recognize that any one of the available algorithms or implementing software packages for conducting homology searches can be adapted for use in the present computer-based systems.

[0164] As used herein, a “target sequence” can be any DNA or amino acid sequence of six or more nucleotides or two or more amino acids. A skilled artisan can readily recognize that the longer a target sequence is, the less likely a target sequence will be present as a random occurrence in the database. The most preferred sequence length of a target sequence is from about 10 to 100 amino acids or from about 30 to 300 nucleotide residues. However, it is well recognized that during searches for commercially important fragments of the E. coli J96 PAI subgenome, such as sequence fragments involved in gene expression and protein processing, may be of shorter length.

[0165] As used herein, “a target structural motif,” or “target motif,” refers to any rationally selected sequence or combination of sequences in which the sequence(s) are chosen based on a three-dimensional configuration which is formed upon the folding of the target motif. There are a variety of target motifs known in the art. Protein target motifs include, but are not limited to, enzymic active sites and signal sequences. Nucleic acid target motifs include, but are not limited to, promoter sequences, hairpin structures and inducible expression elements (protein binding sequences).

[0166] Thus, the present invention further provides an input means for receiving a target sequence, a data storage means for storing the target sequence and the homologous E. coli J96 PAI sequence identified using a search means as described above, and an output means for outputting the identified homologous E. coli J96 PAI sequence. A variety of structural formats for the input and output means can be used to input and output information in the computer-based systems of the present invention. A preferred format for an output means ranks fragments of the E. coli J96 PAI subgenome possessing varying degrees of homology to the target sequence or target motif. Such presentation provides a skilled artisan with a ranking of sequences which contain various amounts of the target sequence or target motif and identifies the degree of homology contained in the identified fragment.

[0167] A variety of comparing means can be used to compare a target sequence or target motif with the data storage means to identify sequence fragments of the E. coli J96 PAI subgenomes. For example, implementing software which implement the BLAST and BLAZE algorithms (Altschul et al., J. Mol. Biol. 215:403-410 (1990)) can be used to identify open reading frames within the E. coli J96 PAI subgenome A skilled artisan can readily recognize that any one of the publicly available homology search programs can be used as the search means for the computer-based systems of the present invention.

[0168] One application of this embodiment is provided in FIG. 2. FIG. 2 provides a block diagram of a computer system 102 that can be used to implement the present invention. The computer system 102 includes a processor 106 connected to a bus 104. Also connected to the bus 104 are a main memory 108 (preferably implemented as random access memory, RAM) and a variety of secondary storage devices 110, such as a hard drive 112 and a removable medium storage device 114. The removable medium storage device 114 may represent, for example, a floppy disk drive, a CD-ROM drive, a magnetic tape drive, etc. A removable storage medium 116 (such as a floppy disk, a compact disk, a magnetic tape, etc.) containing control logic and/or data recorded therein may be inserted into the removable medium storage device 114. The computer system 102 includes appropriate software for reading the control logic and/or the data from the removable medium storage device 114 once inserted in the removable medium storage device 114.

[0169] A nucleotide sequence of the present invention may be stored in a well known manner in the main memory 108, any of the secondary storage devices 110, and/or a removable storage medium 116. Software for accessing and processing the genomic sequence (such as search tools, comparing tools, etc.) reside in main memory 108 during execution.

[0170] Having generally described the invention, the same will be more readily understood by reference to the following examples, which are provided by way of illustration and are not intended as limiting.

Experimental

EXAMPLE 1

High Through-put Sequencing of Cosmid Clones Covering PAI IV and PAI V in E. coli J96

[0171] The complete DNA sequence of the pathogenicity islands, PAI IV and PAI V (respectively >170 kb and ˜110 kb), from uropathogenic E. coli strain, J96 (04:K6) was determined using a strategy, cloning and sequencing method, data collection and assembly software essentially identical to those used by the TIGR group for determining the sequence of the Haemophilus influenzae genome (Fleischmann, R. D., et al., Science 269:496 (1995)). The sequences were then used for DNA and protein sequence similarity searches of the databases as described in Fleischmann, Id.

[0172] The analysis of the genetic information found within the PAIs of E. coli J96 was facilitated by the use of overlapping cosmid clones possessing these unique segments of DNA. These cosmid clones were previously constructed and mapped (as further described below) as an overlapping set in the laboratory of Dr. Doug Berg (Washington University). A gap exists between the left portion of cosmid 2 and the end of the PAI IV that would represent the pheV junction to the E. coli K-12 genome.

[0173] Uropathogenic strain E. coli J96 (O4:K6) was used as a source of chromosomal DNA for construction of a cosmid library. E. coli K-12 DH5Â and DH12 (Gibco/BRL, Gaithersburg, Md.) were used as hosts for maintaining cosmid and plasmid clones. The cosmid library of E coli J96 DNA was constructed essentially as described by Bukanow & Berg (Mol. Microbiol 11:509-523 (1994)). DNA was digested with Sau3AI under conditions that generated fragments with an average size of 40 to 50 kb and electrophoresed through 1% agarose gels. Fragments of 35 to 50 kb were isolated and cloned into Lorist 6 vector that had been linearized with BamIII and treated with bacterial alkaline phosphatase to block self-ligation. (Lorist 6 is a 5.2-kb moderate-copy-number cosmid vector with T7 and SP6 promoters close to the cloning site.) Cloned DNA was packaged in lambda phage particles in vitro by using a commercial kit (Amersham, Arlington Heights, Ill.) and cosmid-containing phage particles were used to transduce E. coli DH5a. Transductant colonies were transferred to 150 mL of Luria-Bertani broth supplemented with kanamycin in 96-well microtiter plates and grown overnight at 37° C. with shaking. Two sets of clones, one for each PAI were ultimately assembled, as previously described (Swenson et al., Infection and Immunity 64:3736-3743 (1996)), fully incorporated by reference herein).

[0174] The two sets of clones contain eleven sub-clones that were employed in the sequencing method described below. One set of four overlapping cosmid clones covers the prs-containing PAI V, ATCC Deposit No. 97727, deposited Sep. 23, 1996. A second set of seven subclones covers much of the pap-containing PAI V, ATCC Deposit No. 97726, deposited Sep. 23, 1996. See FIG. 1.

[0175] A high throughput, random sequencing method (Fleischmann et al., Science 269:496 (1995); Fraser et al., Science 270:397 (1995)) was used to obtain the sequences for 142 (contigs) fragments of E. coli J96 PAIs. All clones were sequenced from both ends to aid in the eventual ordering of contigs during the sequence assembly process. Briefly, random libraries of 2 kb clones covering the two J96 PAIs were constructed, 2,800 clones were subjected to automated sequencing (450 nt/clone) and preliminary assemblies of the sequences accomplished which result in 142 contigs for each of the two PAIs that total 95 and 135 kb respectively. The estimated sizes of the PAI IV and PAI V based on the overlapping cosmid clones are 1.7×105 and 1.1×105 bp respectively. The 142 sequences were assembled by means of the TIGR Assembler (Fleischmann et al.; Fraser et al.); Sutton et al., Genome Sci. Tech. 1:9 (1995)). Sequence and physical gaps were closed using a combination of strategies (Fleischmann et al.; Fraser et al.). Presently the average depth of sequencing for each base assembled in the contigs is 6-fold. The tentative identity of many genes based on sequence homology is covered in Tables 1, 3, 5 and 6.

[0176] Open reading frames (ORFs) and predicted protein-coding regions were identified as described (Fleischmann et al.; Fraser et al.) with some modification. In particular, the statistical prediction of uropathogenic E. coli J96 pathogenicity island genes was performed with GeneMark (Borodovsky, M. & McIninch, J. Comput. Chem. 17:123 (1993)). Regular GeneMark uses nonhomogeneous Markov models derived from a training set of coding sequences and ordinary Markov models derived from a training set of noncoding sequences. The ORFs in Tables 1-6 were identified by GeneMark using a second-order Markov model trained from known E. coli coding regions and known E. coli non-coding regions. Among the important genes that are implicated in the virulence of E. coli J96 PAIs are adhesins, excretion pathway proteins, proteins that participate in alterations of the O-antigen in the PAIs, cytotoxins, and two-component (membrane sensor/DNA binding) proteins.

[0177] Adhesins.

[0178] It is believed that the principal adhesin determinants involved in uropathogenicity that are present within PAIs of uropathogenic E. coli are the pili encoded by the pap-related operons (Hultgren et al., Infect. Immun. 50:370-377 (1993), Stromberg et al., EMBO J 9:2001-2010 (1990), High et al., Infect. Immun. 56:513-517 (1988)) and the distantly related afimbrial adhesins (Labigne-Roussel et al., Infect. Immun. 46:251-259 (1988)). The presence of two of these (pap, and prs) has been confirmed. In addition potential genes for five other adhesins including sla (described above), AIDA-I (diffuse adherence-DEAC), hra (heat resistant hemagglutinin-ETEC), fha (filamentous hemagglutinin—Bordetella pertussis) and the arg-gingipain proteinase of Porphyromonas gingivalis have been found.

[0179] Type II Exoprotein Secretion Pathway.

[0180] Highly significant statistics support the presence of multiple genes involved in the type II exoprotein pathway. Curiously, perhaps two different determinants appear to be present in PAI IV where one set of genes has the highest sequence similarity to eps-like genes (Vibrio cholerae Ctx export) and the other has greatest similarity to exe genes (Aeromonas hydophilia aerolysin and protease export). At present, the assembly of contigs involving these potential genes is incomplete. Thus, it is uncertain if two separate and complete determinants are present. However, it is clear that these genes are newly discovered and novel to pathogenic E. coli because the derived sequences do not have either the bfp or hop genes as the highest matches. The gene products that are the target of the type II export pathway are not evident at this time.

[0181] Within PAI IV there are sequences which suggest genes very similar to secD and secF. These two linked genes encode homologous products that are localized to the inner membrane and are hypothesized to play a late role in the translocation of leader-peptide containing proteins across the inner membrane of gram-negative bacteria. In addition, in each PAI, sequences are found that are reminiscent of the heat-shock htrA/degA gene that encodes a piroplasmic protease. They may perform endochaperone-like function as Pugsley et al. have hypothesized for different exoprotein pathways.

[0182] O-Antigen/Capsule/Carbohydrate Modification (Nod Genes).

[0183] J96 has the O4. The O-antigen portion of lipopolysaccharide is encoded by rfb genes that are located at 45 min. on the E. coli chromosome. We have found in both PAIs a cumulative total of five possible rfb-like genes which could participate alterations of the O-antigen in the PAIs. Overall these data suggest that PAIs provide the genetic potential for greater change of the cell surface for uropathogenic E. coli strains than what was previously known.

[0184] The apparent capsule type for strain J96 is a non-sialic acid K6-type. Sequence similarity “hits” were made in PAI TV region to two region-1 capsule genes, kpsS and kpsE involved in the stabilization of polysaccharide synthesis and polysaccharide export across the inner membrane. This is not altogether surprising based on the genetic mapping of the kps locus to serA at 63 minutes on the genome of the K1 capsular type of E. coli. This suggests that these kps-like genes either are participating in the K6-biosynthesis or perhaps are involved in complex carbohydrate export for other purposes.

[0185] An intriguing discovery are the hits made on genes involved in bacteria-plant interactions by Rhizobium, Bradyrhizobium and Agrobacterium. Four potential genes identified thus far share significant sequence similarity to genes encoding products that modify lipo-oligosaccharides that influence nodule morphogenesis on legume roots. These are: ORF140, carbamyl phosphate synthetase; modulation protein 1265; phosphate-regulatory protein; and an ORF at a plant-inducible locus in Agrobacterium. To date there are no descriptions in the literature of such gene products being utilized by human or animal bacterial pathogens for the purposes of modification or secretion of extracellular carbohydrate. However, the sequence similarity to the capsular region-2 genes and to lipooligosaccharide biosynthetic genes in Rhizobium spp has been recently noted by Petit (1995).

[0186] Cytotoxins.

[0187] Besides the previously known hemolysin and CNF toxins in the PAIs, in each PAI sequences similar to the shlBA operon (cosmid 5 and 12) were found for a cytolytic toxin from Serratia marcescens and Proteus mirabilis. Ironically, the P. mirabilis hemolysin (HpmA) member of this family of toxins was discovered by Uphoff and Welch (1990), but not thought to exist in other members of the Enterobacteriaceae (Swihart (1990)). A shlB-like transporter does also appear to be involved in the export of the filamentous hemagglutinin of Bordetella pertussis which was described above and a cell surface adhesin of Haemophilus influenzae. It has been demonstrated that cosmid #5 of E. coli J96 encodes an extracellular protein that is 1 80 kDa and cross-reactive to polyclonal antisera to the P. mirabilis HpmA hemolysin. Thus, there is evidence suggesting there is new member of this family of proteins in extraintestinal E. coli isolates. In addition, there is also a hit on the FhaC hemolysin-like gene within the PAI V although its statistical significance for the sequence thus far available is only 0.0043.

[0188] Regulators.

[0189] A common regulatory motif in bacteria are the two-component (membrane sensor/DNA binding) proteins. In numerous instances in pathogenic bacteria, external signals in the environment cause membrane-bound protein kinases to phosphorylate a cytoplasmic protein which in turn acts as either a negative or positive effector of transcription of large sets of operons. On cosmid 11 representing PAI V were found, in two different PstI clones, sequences for two-component regulators (similar probabilities for OmpR/ AIGB and separately RcsC, probabilities at the 10−22 level).

[0190] In addition, the phosphoglycerate transport system (pgtA, pgtC, and pgtP) including the pgtB regulator is present in PAI IV. This transport system which was originally described in S. typhimurium is not appreciated as a component of any pathogenic E. coli genome. The operon had been previously mapped at 49 minutes near or within one of the S. typhimurium chromosome specific-loops not present in the K-12 genome. It should be noted that the E. coli K-12 glpT gene product is similar to pgtP gene product (37% identity), but the E. coli J96 genes are clearly homologs to the pgt genes and their linkage within the middle of PAI IV element (cosmid #4) is suspicious.

[0191] Mobile Genetic Elements.

[0192] There are numerous sequences that share similarity to genes found on insertion elements, plasmids and phages. The temperate bacteriophage P4 inserts within tRNA loci in the E. coli chromosome. The hypothesis was made that PAIs are the result of bacteriophage P4-virulence gene recombination events (Blum et al., Infect. Immun. 62:606-614 (1994). Data supporting this hypothesis was found during our sequencing with the identification of P4-like sequences in each of the PAIs (cosmids 7 and 9). This is a very important preliminary result which supports the hypothesis that PAIs can be identified by common sequence or genetic elements. However, there are indications that multiple mobile genetic elements involved in the evolution of the J96 PAIs. Conjugal plasmid-related sequences may also be present at two different locations (F factor and R1 plasmid). Sequences for multiple transposable elements are present that are likely to have originated from different bacterial genera (Tn1000, IS630, IS911, IS 100, IS21, IS 1203, IS5376 (B. stearothermophflus) and RHS). Of particular interest is IS100, which was originally identified in Yersinia pestis (Fetherston et al., Mol. Microbiol. 6:2693-2704 (1992)). The presence of IS106 is significant because it has been associated with the termini of a large chromosomal element encoding pigmentation and some aspect of virulence in Y. pestis. This element undergoes spontaneous deletions similar to the PAIs from E. coli 536 (Fetherston et al., Mol. Microbiol. 6:2693-2704 (1992)) and appears to participate in plasmid-chromosome rearrangements. This element was not previously known to be in genera outside of Yersinia.

[0193] The discovery of the apparent att site for bacteriophage P2 in the PAIs is interesting. P2 acts as a helper phage for the P4 satellite phage. The P2 att site is at 44 min in the K-12 genome. The significance of this hit is unknown at present, but may be explained as either a cloning artifact (some K-12 fragments in the Pst I library of cosmid 5) or evidence of some curious chromosomal-P4/P2 phage history. It may indicate that the J96 PAIs are composites of multiple smaller PAIs.

EXAMPLE 2

Preparation of PCR Primers and Amplification of DNA

[0194] Various fragments of the sequenced E. coli J96 PAIs, such as those disclosed in Tables 1 through 6 can be used, in accordance with the present invention, to prepare PCR primers. The PCR primers are preferably at least 15 bases, and more preferably at least 18 bases in length. When selecting a primer sequence, it is preferred that the primer pairs have approximately the same G/C ratio, so that melting temperatures are approximately the same. The PCR primers are useful during PCR cloning of the ORFs described herein.

EXAMPLE 3

Gene Expression from DNA Sequences Corresponding to ORFs

[0195] A fragment of an E. coli J96 PAIs (preferably, a protein-encoding sequence provided in Tables 1 through 6) is introduced into an expression vector using conventional technology (techniques to transfer cloned sequences into expression vectors that direct protein translation in mammalian, yeast, insect or bacterial expression systems are well known in the art). Commercially available vectors and expression systems are available from a variety of suppliers including Stratagene (La Jolla, Calif.), Promega (Madison, Wis.), and Invitrogen (San Diego, Calif.). If desired, to enhance expression and facilitate proper protein folding, the codon context and codon pairing of the sequence may be optimized for the particular expression organism, as explained by Hatfield et al., U.S. Pat. No. 5,082,767, which is hereby incorporated by reference.

[0196] The following is provided as one exemplary method to generate polypeptide(s) from a cloned ORF of an E. coli J96 PAI whose sequence is provided in SEQ ID NOs: 1 through 142. A poly A sequence can be added to the construct by, for example, splicing out the poly A sequence from pSG5 (Stratagene) using BglI and SalI restriction endonuclease enzymes and incorporating it into the mammalian expression vector pXT1 (Stratagene) for use in eukaryotic expression systems. pXT1 contains the LTRs and a portion of the gag gene from Moloney Murine Leukemia Virus. The position of the LTRs in the construct allow efficient stable transfection. The vector includes the Herpes Simplex thymidine kinase promoter and the selectable neomycin gene. The E. coli J96 PAI DNA is obtained by PCR from the bacterial vector using oligonucleotide primers complementary to the E. coli J96 PAI DNA and containing restriction endonuclease sequences for PstI incorporated into the 5 primer and BglII at the 5 end of the corresponding E. coli J96 PAI DNA 3 primer, taking care to ensure that the E. coli J96 PAI DNA is positioned such that its followed with the poly A sequence. The purified fragment obtained from the resulting PCR reaction is digested with PstI, blunt ended with an exonuclease, digested with BglII, purified and ligated to pXT1, now containing a poly A sequence and digested BglII.

[0197] The ligated product is transfected into mouse NIH 3T3 cells using Lipofectin (Life Technologies, Inc., Grand Island, N.Y.) under conditions outlined in the product specification. Positive transfectants are selected after growing the transfected cells in 600 ug/ml G418 (Sigma, St. Louis, Mo.). The protein is preferably released into the supernatant. However if the protein has membrane binding domains, the protein may additionally be retained within the cell or expression may be restricted to the cell surface.

[0198] Since it may be necessary to purify and locate the transfected product, synthetic 15-mer peptides synthesized from the predicted E. coli J96 PAI DNA sequence are injected into mice to generate antibody to the polypeptide encoded by the E. coli J96 PAI DNA.

[0199] If antibody production is not possible, the E. coli J96 PAI DNA sequence is additionally incorporated into eukaryotic expression vectors and expressed as a chimeric with, for example, 1-globin. Antibody to 1-globin is used to purify the chimeric. Corresponding protease cleavage sites engineered between the 8-globin gene and the E. coli J96 PAI DNA are then used to separate the two polypeptide fragments from one another after translation. One useful expression vector for generating β-globin chimerics is pSG5 (Stratagene). This vector encodes rabbit β-globin. Intron II of the rabbit β-globin gene facilitates splicing of the expressed transcript, and the polyadenylation signal incorporated into the construct increases the level of expression. These techniques as described are well known to those skilled in the art of molecular biology. Standard methods are available from the technical assistance representatives from Stratagene, Life Technologies, Inc., or Promega. Polypeptides may additionally be produced from either construct using in vitro translation systems such as In vitro Express™ Translation Kit (Stratagene).

EXAMPLE 4

E. coli
Expression of an E. coli J96 PAI ORF and Protein Purification

[0200] An E. coli J96 PAI ORF described in Tables 1 through 6 is selected and amplified using PCR oligonucleotide primers designed from the nucleotide sequences flanking the selected ORF and/or from portions of the ORF s NH- or COOH-terminus. Additional nucleotides containing restriction sites to facilitate cloning are added to the 5′ and 3′ sequences, respectively. 2

[0201] The restriction sites are selected to be convenient to restriction sites in the bacterial expression vector pQE60. The bacterial expression vector pQE60 is used for bacterial expression in this example. (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, Calif., 91311). pQE60 encodes ampicillin antibiotic resistance (“Ampr”) and contains a bacterial origin of replication (“ori”), an IPTG inducible promoter, a ribosome binding site (“RBS”), six codons encoding histidine residues that allow affinity purification using nickel-nitrilo-tri-acetic acid (“Ni-NTA”) affinity resin sold by QIAGEN, Inc., supra, and suitable single restriction enzyme cleavage sites. These elements are arranged such that a DNA fragment encoding a polypeptide may be inserted in such as way as to produce that polypeptide with the six His residues (i.e., a “6×His tag”) covalently linked to the carboxyl terminus of that polypeptide.

[0202] The DNA sequence encoding the desired portion of an E. coli J96 PAI is amplified from the deposited cDNA clone using PCR oligonucleotide primers which anneal to the amino terminal sequences of the desired portion of the E. coli protein and to sequences in the deposited construct 3′ to the cDNA coding sequence. Additional nucleotides containing restriction sites to facilitate cloning in the pQE60 vector are added to the 5′ and 3′ sequences, respectively.

[0203] The amplified E. coli J96 PAI DNA fragments and the vector pQE60 are digested with one or more appropriate restriction enzymes, such as SalI and XbaI, and the digested DNAs are then ligated together. Insertion of the E. coli J96 PAI DNA into the restricted pQE60 vector places the E. coli J96 PAI protein coding region, including its associated stop codon, downstream from the IPTG-inducible promoter and in-frame with an initiating AUG. The associated stop codon prevents translation of the six histidine codons downstream of the insertion point.

[0204] The ligation mixture is transformed into competent E. coli cells using standard procedures such as those described in Sambrook et al., Molecular Cloning: a Laboratory Manual, 2nd Ed.; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989). E. coli strain M15/rep4, containing multiple copies of the plasmid pREP4, which expresses the lac repressor and confers kanamycin resistance (“Kanr”), is used in carrying out the illustrative example described herein. This strain, which is only one of many that are suitable for expressing an E. coli J96 PAI protein, is available commercially from QIAGEN, Inc., supra. Transformants are identified by their ability to grow on LB plates in the presence of ampicillin and kanamycin. Plasmid DNA is isolated from resistant colonies and the identity of the cloned DNA confirmed by restriction analysis, PCR and DNA sequencing.

[0205] Clones containing the desired constructs are grown overnight (“O/N”) in liquid culture in LB media supplemented with both ampicillin (100 μg/ml) and kanamycin (25 μg/ml). The O/N culture is used to inoculate a large culture, at a dilution of approximately 1:25 to 1:250. The cells are grown to an optical density at 600 nm (“OD600”) of between 0.4 and 0.6. isopropyl-β-D-thiogalactopyranoside (“IPTG”) is then added to a final concentration of 1 mM to induce transcription from the lac repressor sensitive promoter, by inactivating the laci repressor. Cells subsequently are incubated further for 3 to 4 hours. Cells then are harvested by centrifugation.

[0206] The cells are then stirred for 3-4 hours at 4° C. in 6M guanidine-HCl, pH8. The cell debris is removed by centrifugation, and the supernatant containing the E. coli J96 PAI protein is dialyzed against 50 mM Na-acetate buffer pH6, supplemented with 200 mM NaCl. Alternatively, the protein can be successfully refolded by dialyzing it against 500 mM NaCl, 20% glycerol, 25 mM Tris/HCl pH7.4, containing protease inhibitors. After renaturation the protein can be purified by ion exchange, hydrophobic interaction and size exclusion chromatography. Alternatively, an affinity chromatography step such as an antibody column can be used to obtain pure E. coli J96 PAI protein. The purified protein is stored at 4° C. or frozen at −80° C.

EXAMPLE 5

Cloning and Expression of an E. coli J96 PAI Protein in a Baculovirus Expression System

[0207] An E. coli J96 PAI ORF described in Tables 1 through 6 is selected and amplified as above. The plasmid is digested with appropriate restriction enzymes and optionally, can be dephosphorylated using calf intestinal phosphatase, using routine procedures known in the art. The DNA is then isolated from a 1% agarose gel using a commercially available kit (“Geneclean” BIO 101 Inc., La Jolla, Calif.). This vector DNA is designated herein “V1”.

[0208] Fragment F1 and the dephosphorylated plasmid V1 are ligated together with T4 DNA ligase. E. coli HB101 or other suitable E. coli hosts such as XL-1 Blue (Stratagene Cloning Systems, La Jolla, Calif.) cells are transformed with the ligation mixture and spread on culture plates. Bacteria are identified that contain the plasmid with the E. coli J96 PAI gene by digesting DNA from individual colonies using appropriate restriction enzymes and then analyzing the digestion product by gel electrophoresis. The sequence of the cloned fragment is confirmed by DNA sequencing. This plasmid is designated herein pBac E. coli J96.

[0209] Five μg of the plasmid pBac E. coli J96 is co-transfected with 1.0 μg of a commercially available linearized baculovirus DNA (“BaculoGold baculovirus DNA”, Pharmingen, San Diego, Calif.), using the lipofection method described by Felgner et al., Proc. Natl. Acad. Sci. USA 84:7413-7417 (1987). 1 μg of BaculoGold virus DNA and 5 μg of the plasmid pBac E. coli J96 are mixed in a sterile well of a microliter plate containing 50 μl of serum-free Grace's medium (Life Technologies Inc., Gaithersburg, Md.). Afterwards, 10 μl Lipofectin plus 90 μl Grace's medium are added, mixed and incubated for 15 minutes at room temperature. Then the transfection mixture is added drop-wise to Sf9 insect cells (ATCC CRL 1711) seeded in a 35 mm tissue culture plate with 1 ml Grace's medium without serum. The plate is rocked back and forth to mix the newly added solution. The plate is then incubated for 5 hours at 27° C. After 5 hours the transfection solution is removed from the plate and 1 ml of Grace's insect medium supplemented with 10% fetal calf serum is added. The plate is put back into an incubator and cultivation is continued at 27° C. for four days.

[0210] After four days the supernatant is collected and a plaque assay is. performed, as described by Summers and Smith, supra. An agarose gel with “Blue Gal” (Life Technologies Inc.) is used to allow easy identification and isolation of gal-expressing clones, which produce blue-stained plaques. (A detailed description of a “plaque assay” of this type can also be found in the user's guide for insect cell culture and baculovirology distributed by Life Technologies Inc., page 9-10). After appropriate incubation, blue stained plaques are picked with the tip of a micropipettor (e.g., Eppendorf). The agar containing the recombinant viruses is then resuspended in a microcentrifuge tube containing 200 μl of Grace's medium and the suspension containing the recombinant baculovirus is used to infect Sf9 cells seeded in 35 mm dishes. Four days later the supernatants of these culture dishes are harvested and then they are stored at 4° C. The recombinant virus is called V-E. coli J96.

[0211] To verify the expression of the E. coli gene Sf9 cells are grown in Grace's medium supplemented with 10% heat inactivated FBS. The cells are infected with the recombinant baculovirus V-E. coli J96 at a multiplicity of infection (“MOI”) of about 2. Six hours later the medium is removed and is replaced with SF900 II medium minus methionine and cysteine (available from Life Technologies Inc.). If radiolabeled proteins are desired, 42 hours later, 5 μCi of 35S-methionine and 5 μCi 35S-cysteine (available from Amersham) are added. The cells are further incubated for 16 hours and then they are harvested by centrifugation. The proteins in the supernatant as well as the intracellular proteins are analyzed by SDS-PAGE followed by autoradiography (if radiolabeled). Microsequencing of the amino acid sequence of the amino terminus of purified protein may be used to determine the amino terminal sequence of the mature protein and thus the cleavage point and length of the secretary signal peptide.

EXAMPLE 6

Cloning and Expression in Mammalian Cells

[0212] Most of the vectors used for the transient expression of an E. coli J96 PAI gene in mammalian cells should carry the SV40 origin of replication. This allows the replication of the vector to high copy numbers in cells (e.g., COS cells) which express the T antigen required for the initiation of viral DNA synthesis. Any other mammalian cell line can also be utilized for this purpose.

[0213] A typical mammalian expression vector contains the promoter element, which mediates the initiation of transcription of mRNA, the protein coding sequence, and signals required for the termination of transcription and polyadenylation of the transcript. Additional elements include enhancers, Kozak sequences and intervening sequences flanked by donor and acceptor sites for RNA splicing. Highly efficient transcription can be achieved with the early and late promoters from SV40, the long terminal repeats (LTRS) from Retroviruses, e.g., RSV, 1HTLVI, HIVI and the early promoter of the cytomegalovirus (CMV). However, cellular elements can also be used (e.g., the human actin promoter). Suitable expression vectors for use in practicing the present invention include, for example, vectors such as PSVL and PMSG (Pharmacia, Uppsala, Sweden), pRSVcat (ATCC 37152), pSV2dhfr (ATCC 37146) and pBC12MI (ATCC 67109). Mammalian host cells that could be used include, human Hela, 293, H9 and Jurkat cells, mouse NIH3T3 and C127 cells, Cos 1, Cos 7 and CV I, quail QC1-3 cells, mouse L cells and Chinese hamster ovary (CHO) cells.

[0214] Alternatively, the gene can be expressed in stable cell lines that contain the gene integrated into a chromosome. The co-transfection with a selectable marker such as dhfr, gpt, neomycin, hygromycin allows the identification and isolation of the transfected cells.

[0215] The transfected gene can also be amplified to express large amounts of the encoded protein. The DHFR (dihydrofolate reductase) marker is useful to develop cell lines that carry several hundred or even several thousand copies of the gene of interest. Another useful selection marker is the enzyme glutamine synthase (GS) (Murphy et al., Biochem J. 227:277-279 (199 1); Bebbington et al., Bio/Technology 10:169-175 (1992)). Using these markers, the mammalian cells are grown in selective medium and the cells with the highest resistance are selected. These cell lines contain the amplified gene(s) integrated into a chromosome. Chinese hamster ovary (CHO) and NSO cells are often used for the production of proteins.

[0216] The expression vectors pC1 and pC4 contain the strong promoter (LTR) of the Rous Sarcoma Virus (Cullen et al., Molecular and Cellular Biology, 438447 (March, 1985)) plus a fragment of the CMV-enhancer (Boshart et al., Cell 41:521-530 (1985)). Multiple cloning sites, e.g., with the restriction enzyme cleavage sites BamHI, Xbal and Asp718, facilitate the cloning of the gene of interest. The vectors contain in addition the 3′ intron, the polyadenylation and termination signal of the rat preproinsulin gene.

EXAMPLE 6(a)

Cloning and Expression in COS Cells

[0217] The expression plasmid, p E. coli J96HA, is made by cloning a cDNA encoding E. coli J96 PAI protein into the expression vector pcDNAI/Amp or pcDNAIII (which can be obtained from Invitrogen, Inc.).

[0218] The expression vector pcDNAI/amp contains: (1) an E. coli origin of replication effective for propagation in E. coli and other prokaryotic cells; (2) an ampicillin resistance gene for selection of plasmid-containing prokaryotic cells; (3) an SV40 origin of replication for propagation in eukaryotic cells; (4) a CMV promoter, a polylinker, an SV40 intron; (5) several codons encoding a hemagglutinin fragment (i.e., an “HA” tag to facilitate purification) followed by a termination codon and polyadenylation signal arranged so that a cDNA can be conveniently placed under expression control of the CMV promoter and operably linked to the SV40 intron and the polyadenylation signal by means of restriction sites in the polylinker. The HA tag corresponds to an epitope derived from the influenza hemagglutinin protein described by Wilson et al., Cell 37:767 (1984). The fusion of the HA tag to the target protein allows easy detection and recovery of the recombinant protein with an antibody that recognizes the HA epitope. pcDNAIII contains, in addition, the selectable neomycin marker.

[0219] A DNA fragment encoding the E. coli J96 PAI protein is cloned into the polylinker region of the vector so that recombinant protein expression is directed by the CMV promoter. The plasmid construction strategy is as follows. The E. coli cDNA of the deposited clone is amplified using primers that contain convenient restriction sites, much as described above for construction of vectors for expression of E. coli J96 PAI protein in E. coli.

[0220] The PCR amplified DNA fragment and the vector, pcDNAI/Amp, are digested with appropriate restriction enzymes for the chosen primer sequences and then ligated. The ligation mixture is transformed into E. coli strain SURE (available from Stratagene Cloning Systems, La Jolla, Calif. 92037), and the transformed culture is plated on ampicillin media plates which then are incubated to allow growth of ampicillin resistant colonies. Plasmid DNA is isolated from resistant colonies and examined by restriction analysis or other means for the presence of the E. coli J96 PAI protein-encoding fragment.

[0221] For expression of recombinant E. coli J96 PAI protein, COS cells are transfected with an expression vector, as described above, using DEAE-DEXTRAN, as described, for instance, in Sambrook et al., Molecular Cloning: a Laboratory Manual, Cold Spring Laboratory Press, Cold Spring Harbor, N.Y. (1989). Cells are incubated under conditions for expression of E. coli J96 PAI protein by the vector.

[0222] Expression of the E. coli J96 PAI-HA fusion protein is detected by radiolabeling and immunoprecipitation, using methods described in, for example Harlow et al., Antibodies: A Laboratory Manual, 2nd Ed.; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1988). To this end, two days after transfection, the cells are labeled by incubation in media containing 35S-cysteine for 8 hours. The cells and the media are collected, and the cells are washed and the lysed with detergent-containing RIPA buffer: 150 mM NaCl, 1% NP-40, 0.1% SDS, 1% NP-40, 0.5% DOC, 50 mM TRIS, pH 7.5, as described by Wilson et al. cited above. Proteins are precipitated from the cell lysate and from the culture media using an HA-specific monoclonal antibody. The precipitated proteins then are analyzed by SDS-PAGE and autoradiography. An expression product of the expected size is seen in the cell lysate, which is not seen in negative controls.

EXAMPLE 6(b)

Cloning and Expression in CHO Cells

[0223] The vector pC4 is used for the expression of an E. coli J96 PAI protein. Plasmid pC4 is a derivative of the plasmid pSV2-dhfr (ATCC Acc. No. 37146). The plasmid contains the mouse DHFR gene under control of the SV40 early promoter. Chinese hamster ovary- or other cells lacking dihydrofolate activity that are transfected with these plasmids can be selected by growing the cells in a selective medium (alpha minus MEM, Life Technologies, Inc.) supplemented with the chemotherapeutic agent methotrexate. The amplification of the DHFR genes in cells resistant to methotrexate (MTX) has been well documented (see, e.g., Alt, F. W. et al., 1978, J. Biol. Chem. 253:1357-1370, Hamlin, J. L. and Ma, C. 1990, Biochim. et Biophys. Acta, 1097:107-143, Page, M. J. and Sydenham, M. A. 1991, Biotechnology 9:64-68). Cells grown in increasing concentrations of MTX develop resistance to the drug by overproducing the target enzyme, DHFR, as a result of amplification of the DHFR gene. If a second gene is linked to the DHFR gene, it is usually co-amplified and over-expressed. It is known in the art that this approach may be used to develop cell lines carrying more than 1,000 copies of the amplified gene(s). Subsequently, when the methotrexate is withdrawn, cell lines are obtained which contain the amplified gene integrated into one or more chromosome(s) of the host cell.

[0224] Plasmid pC4 contains for expressing the gene of interest the strong promoter of the long terminal repeat (LTR) of the Rouse Sarcoma Virus (Cullen, et al., Molecular and Cellular Biology, March 1985:438-447) plus a fragment isolated from the enhancer of the immediate early gene of human cytomegalovirus (CMV) (Boshart et al., Cell 41:521-530 (1985)). Downstream of the promoter is BamHI restriction enzyme site that allows the integration of the gene. Behind these cloning sites the plasmid contains the 3′ intron and polyadenylation site of the rat preproinsulin gene. Other high efficiency promoters can also be used for the expression, e.g., the human-actin promoter, the SV40 early or late promoters or the long terminal repeats from other retroviruses, e.g., HIV and HTLVI. Clontech's Tet-Off and Tet-On gene expression systems and similar systems can be used to express the E. coli protein in a regulated way in mammalian cells (Gossen, M., & Bujard, H. 1992, Proc. Natl. Acad. Sci. USA 89: 5547-5551). For the polyadenylation of the mRNA other signals, e.g., from the human growth hormone or globin genes can be used as well. Stable cell lines carrying a gene of interest integrated into the chromosomes can also be selected upon co-transfection with a selectable marker such as gpt, G418 or hygromycin. It is advantageous to use more than one selectable marker in the beginning, e.g., G418 plus methotrexate.

[0225] The plasmid pC4 is digested with appropriate restriction enzymes and then dephosphorylated using calf intestinal phosphates by procedures known in the art. The vector is then isolated from a 1% agarose gel.

[0226] The DNA sequence encoding the complete E. coli J96 PAI protein including its leader sequence is amplified using PCR oligonucleotide primers corresponding to the 5′ and 3′ sequences of the gene.

[0227] The amplified fragment is digested with appropriate endonucleases for the chosen primers and then purified again on a 1% agarose gel. The isolated fragment and the dephosphorylated vector are then ligated with T4 DNA ligase. E. coli HB 101 or XL-1 Blue cells are then transformed and bacteria are identified that contain the fragment inserted into plasmid pC4 using, for instance, restriction enzyme analysis.

[0228] Chinese hamster ovary cells lacking an active DHFR gene are used for transfection. 5 μg of the expression plasmid pC4 is cotransfected with 0.5 μg of the plasmid pSVneo using lipofectin (Felgner et al., supra). The plasmid pSV2neo contains a dominant selectable marker, the neo gene from Tn5 encoding an enzyme that confers resistance to a group of antibiotics including G418. The cells are seeded in alpha minus MEM supplemented with 1 mg/ml G418. After 2 days, the cells are trypsinized and seeded in hybridoma cloning plates (Greiner, Germany) in alpha minus MEM supplemented with 10, 25, or 50 ng/ml of methothrexate plus 1 mg/ml G418. After about 10-14 days single clones are trypsinized and then seeded in 6-well petri dishes or 10 ml flasks using different concentrations of methotrexate (50 nM, 100 nM, 200 nM, 400 nm, 800 nM). Clones growing at the highest concentrations of methotrexate are then transferred to new 6-well plates containing even higher concentrations of methotrexate (1 μM, 2 μM, 5 μM, 10 mM, 20 mM). The same procedure is repeated until clones are obtained which grow at a concentration of 100-200 μM. Expression of the desired gene product is analyzed, for instance, by SDS-PAGE and Western blot or by reversed phase HPLC analysis.

EXAMPLE 7

Production of an Antibody to an E. coli J96 Pathogenicity Island Protein

[0229] Substantially pure E. coli J96 PAI protein or polypeptide is isolated from the transfected or transformed cells described above using an art-known method. The protein can also be chemically synthesized. Concentration of protein in the final preparation is adjusted, for example, by concentration on an Amicon filter device, to the level of a few micrograms/ml. Monoclonal or polyclonal antibody to the protein can then be prepared as follows:

[0230] Monoclonal Antibody Production by Hybridoma Fusion

[0231] Monoclonal antibody to epitopes of any of the peptides identified and isolated as described can be prepared from murine hybridomas according to the classical method of Kohler and Milstein, Nature 256:495 (1975) or modifications of the methods thereof. Briefly, a mouse is repetitively inoculated with a few micrograms of the selected protein over a period of a few weeks. The mouse is then sacrificed, and the antibody producing cells of the spleen isolated. The spleen cells are fused by means of polyethylene glycol with mouse myeloma cells, and the excess unfused cells destroyed by growth of the system on selective media comprising aminopterin (HAT media). The successfully fused cells are diluted and aliquots of the dilution placed in wells of a microtiter plate where growth of the culture is continued. Antibody-producing clones are identified by detection of antibody in the supernatant fluid of the wells by immunoassay procedures, such as ELISA, as originally described by Engvall, E., Meth. Enzymol. 70:419 (1980), and modified methods thereof. Selected positive clones can be expanded and their monoclonal antibody product harvested for use. Detailed procedures for monoclonal antibody production are described in Davis, L. et al. Basic Methods in Molecular Biology Elsevier, New York. Section 21-2 (1989).

[0232] Polyclonal Antibody Production by Immunization

[0233] Polyclonal antiserum containing antibodies to heterogenous epitopes of a single protein can be prepared by immunizing suitable animals with the expressed protein described above, which can be unmodified or modified to enhance immunogenicity. Effective polyclonal antibody production is affected by many factors related both to the antigen and the host species. For example, small molecules tend to be less immunogenic than other molecules and may require the use of carriers and adjuvant. Also, host animals vary in response to site of inoculations and dose, with both inadequate or excessive doses of antigen resulting in low titer antisera. Small doses (ng level) of antigen administered at multiple intradermal sites appears to be most reliable. An effective immunization protocol for rabbits can be found in Vaitukaitis, J. et al., J. Clin. Endocrinol. Metab. 33:988-991 (1971).

[0234] Booster injections can be given at regular intervals, and antiserum harvested when antibody titer thereof, as determined semi-quantitatively, for example, by double immunodiffusion in agar against known concentrations of the antigen, begins to fall (See Ouchterlony, O. et al., Chap. 19 in: Handbook of Experimental Immunology, Wier, D., ed, Blackwell (1973)). Plateau concentration of antibody is usually in the range of 0.1 to 0.2 mg/ml of serum (about 12 μM). Affinity of the antisera for the antigen is determined by preparing competitive binding curves, as described, for example, by Fisher, D., Chap. 42 in: Manual of Clinical Immunology, 2nd ed., Rose and Friedman, (eds.), Amer. Soc. For Microbio., Washington, D.C. (1980).

[0235] Antibody preparations prepared according to either protocol are useful in quantitative immunoassays which determine concentrations of antigen-bearing substances in biological samples; they are also used semi-quantitatively or qualitatively to identify the presence of antigen in a biological sample.

[0236] While the present invention has been described in some detail for purposes of clarity and understanding, one skilled in the art will appreciate that various changes in form and detail can be made without departing from the true scope of the invention.

[0237] All patents, patent applications and publications recited herein are hereby incorporated by reference.

2TABLE 1(PAI IV)Putative coding regions of novel E. coli PAI IV proteins similar to known proteinsORFStartStopmatchlengthContig IDID(nt)(nt)acessionmatch gene name% sim% Ident(nt)65219021042gi|1655838ORPB, putative transposase [Yersinla pestis]10010086165320961821gi|467612ORPI [Yersinla pestis]100100276631178569238gi|154262transporter protein pgtP [Salmonella typhimurium]9893138365428891915gi|1655837ORFA, putative transposase [Yersinla pestis]979697513812172gi|1208992unknown [Escherichia coli]977817164640754338gi|1143207Description, IS630 Insertion element, ORF5 protein, Method;9292264conceptual translation supplied by author [Shigella sonnel]6711273gi|809648Exef gene product [Aeromonas hydrophila]927127373430292511gi|799234glucose-1-phosphate thymidylyltransferase [Escherichia coli]928651573531392996gi|454900rfbC gene product [Shigella flexnerl]929214464537414088gi|47542ORF (343 AA) [Shigella sonnel]918534873326132242gi|46985glucose-1-phosphate thymidylyltransferase [Salmonella enterica]91823729011366gi|38826ExeE gene product [Aeromonas hydrophila]9177366912604248gi|609625putative [Vibrio cholerae]916735763963015234gi|858753regulatory protein pgtB [Salmonella typhimurium]8984106073221791811gi|294899dTDP-6-deoxy-L-mannose-dehydrogenase [Shigella flexnerl]8984369902201689gi|38826ExeE gene product [Aeromonas hydrophila]89804899521519413gi|581654dTDP-glucose 4,6-dehydratase [Salmonella enterica]88811107961729457plr|S43483|S434Orf104 homolog - Escherichia coli887227363642813019gi|154755phosphoglyoarate transport system activator protein [Salmonella87751363typhimurium]672251745gi|609628putative [Vibrio cholerae]8772495821252544406gi|1208992unknown [Escherichia coli]87748496016934gi|609625putative [Vibrio cholerae]86576909514283gi|508238dTDP-6-deoxy-L-mannose-dehydrogenase [Escherichia coli]857442664743364731gi|47542ORF [343 AA] [Shigella sonnel]848139680828002582gi|38832ExaK gene product [Aeromonas hydrophila]8453219821043803829gi|1033137ORF_o157 [Escherichia coli]847256263853994830ap|P37433|PGTB—PHOSPHOGLYCERATE TRANSPORT SYSTEM8375570SENSOR PROTEIN PGTB [EC 2.7.3,—.]631075726259gi|154258regulatory protein pgtC [Salmonalls typhimurium]8378131465733513100gi|1196999unknown protein [Transporon Tn3411]828025210013372gi|41004URF 2 [Escherichia coli]82643361382109429gi|1033128ORF_o273 [Escherichia coli]80623217441332831gi|38826ExeE gene product [Aeromonas hydrophlia]796250163748734256sp|p37433|PGTB—PHOSPHOOLYCERAATE TRANSPORT SYSTEM SENSOR7872618PROTEIN PGTB [EC 2.7.3,—.]701357595529gi|1773143Hha protein [Escherichia coli]78582319131154534gi|609625putative [Vibrio cholerae]776562175535243255gi|463911heat reaistant agglutinin 1 [Escherichia coli]76622706312667gi|1574313H. influenzas predicted coding region HI14727556666[Haemophilus influenzas]1042485315gi|530438arabinose transport protein [Hycoplasma capricolum]724117163321801629gi|622948transposase [Escherichia coli]72605526312968810005sp|P39213|YI91—INSERTION ELEMENT I6911 HYPOTHETICAL715731812.7 KD PROTEIN.6131283876gi|581535ORF140 gene product [Rhirobium sp.]705440884323613437gi|1772623HecA [Erwinia chrysanthami]706010779113004gi|295430spsE [Vibrio choleras]70492977415412gi|609627putativa [Vibrio choleras]695454067412971581gi|151469piID-depandent protein [Pseudomonas aeruginose]68502858415781741gi|1772622HecB [Erwinia chryasnthemi]6854116484216982363gi|1772622HecB [Erwinia chryasnthemi]674866663217341393gi|1323798transposase [Plasmid pRL1063a]654634271111344gi|397405KpaE gene product [Escherichia coli]6536113164228281839gi|310632hydrophobio membrane protein [Streptococcus gordonil]6438990742861355gi|148436secretory component [Erwinia chrysanthami]64545076615562gi|1235662Rfbc[Hyxococcus xanthus]623955570630172814gi|1657478similar to E. coli ORF_o208 [Escherichia coli]624120485127866pir|A45253|A452activator 1 37 K chain - human625621312613323gi|1778562hypothetical protein [Escherichia coli]62453217317733pri|S32879|S328lipA protein - Heisseria meningitidis6146771962796644gnl|PID|e276217T03P6.1 [Caenorhabditis elegans]61461536737431312gi|609629putative [Vibrio cholorae]6043570701046664292gi|1657478similar to E. coli ORP_o208 [Escherichia coli]604537581111179gi|1591717spore coat polysaccharide biosynthesis60441179protein E [Methanococcus jannaschil]80525631790gi|609632putative [Vibrio cholorae]5941774137173528gi|1736670Adhesin AIDA-I precursor. [Escherichia coli]59454566117733gi|1196968unknown protein [Insertion sequence IS66]584177163528312178gi|622948transposase [Escherichia coli]584165464335682690gi|1335913unknown [Eryslpelothrix rhoslopathlae]57368796411819917gi|153826adhesin B [Streptococcus sanguis]553090364970086685gi|152259lcrB gene product [Rhizobium sp.]5542324701464816753pir|G42465|G424hypothetical protein 86 - phage phi-R73533027385593171530gi|144048filamentous hemagglutinin [Bordetella pertussis]5237778864850634806gnl|PID|e264304P53C11.6 [Caenorhabditis elegans]512725880934112761gi|149309pulJ [Klebsiella pneumoniae]504065188198388gi|156087[Brugia malayl myosin heavy chain gene, complete cds.],5032291gene product [Brugia malayl]9631127687gi|1196964unknown protein [Plasmid Ti]50384418919814gi|57633neuronal myosin heavy chain [Rattus rattus]48229781131657199gi|147899extragenic suppressor [Escherichia coli]48254591181654145pir|S27564|S275polysaccharide translocation-related protein - Escherichia coli482551058221014245gi|1235662RfbC [Hyxococcus xanthus]47352145871595134gi|1235662RfbC [Hyxococcus xanthus]42284628521018515bbs/117606glycine-rich protein, atGRP [clone atGRP-1]3636504[Arabidopsis thaliana, C24, Peptide Partial,210 aa] [Arabidopsis thaliana]8531779973bbs|157676silk fibroin heavy chain [C-terminal] [Bombyx mori-silkworms,3429807Peptide Partial, 633 aa] [Bombyx mori]

[0238]

3

TABLE 2

(PAI IV)

Putative coding regions of novel E. coli PAI IV

proteins not similar to known proteins

Contig
ORP
Start
Stop

ID
ID
(nt)
(nt)

58
1
1176
2120

61
2
54
560

63
4
1875
2639

64
4
3911
3627

65
6
3009
3239

65
12
6027
6683

66
2
1289
978

70
2
1418
861

70
3
1886
1476

70
4
2124
1900

70
5
2795
2220

70
7
3645
3259

70
8
4078
3680

70
9
4220
4513

70
11
4950
4498

70
12
4594
4866

70
15
6805
7449

70
16
9520
10806

73
7
3247
3666

74
3
720
1301

75
1
1
165

79
1
719
354

80
6
2108
2575

80
7
2831
2469

80
10
3223
3387

80
11
3541
3362

82
8
3313
4260

82
11
4340
5218

82
13
6090
5614

84
4
3487
3281

85
4
1485
2285

85
6
8373
9320

104
1
358
2

112
1
677
105

142
1
3
143

142
2
119
328

[0239]

4

TABLE 3

(PAI V)

Putative coding regions of novel E. coli PAI V proteins similar to known proteins

Contig
ORF
Start
Stop
match

length

ID
ID
(nt)
(nt)
acession
match gene name
% sim
% Ident
(nt)

14
3
2826
3686
gi|1655838
(ORFB1 putative transposase [Yersinia pestis]
100
100
861

14
2
1837
2907
gi|1655837
(ORFA1 putative transposase [Yersinia pestis]
99
99
1071

3
9
7927
7595
gi|1657499
putative transposase for insertion sequence IS3 [Escherichia coli]
89
85
333

20
6
3462
4304
gi|1208992
unknown [Escherichia coli]
87
73
843

6
6
3541
3263
pir|S43483|S434
Orf104 homolog - Escherichia coli
81
62
279

20
3
1616
2332
gi|1033129
ORF_o233 [Escherichia coli]
80
61
717

9
1
1
681
gi|537112
ORF_o396 [Escherichia coli]
77
55
681

15
3
1899
1672
pir|S43483|S434
Orf104 homolog - Escherichia coli
75
55
228

20
9
4302
4880
gi|1552816
similar to E. coli ORF_o152 [Escherichia coli]
74
60
579

14
13
12972
15359
gi|1772623
HecA [Erwinia chrysanthami]
70
60
2388

5
3
4112
1570
gi|1001717
regulatory components of sensory transduction system
68
45
459

[Synechocystis sp.]

3
1
2572
1373
gi|849022
Lactate oxidase [Aerococcus viridans]
66
46
1200

3
8
6869
6498
gi|581535
ORF140 gene product [Rhizobium sp.]
66
45
372

6
5
3265
2951
gi|642184
F19C6.1 [Caenorhabditis elegans]
66
44
315

14
12
11775
12974
gi|1772622
HecB [Erwinia chrysanthami]
66
50
1200

20
1
545
1450
gi|1033127
ORF_o289 [Escherichia coli]
66
45
906

57
1
696
124
gi|1772622
HecB [Erwinia chrysanthami]
66
47
573

3
3
3320
3700
gi|431950
similar to a B. subtilis gene (GB1 BACHEHEITY_5)
65
34
381

[Clostridium pasteurianum]

5
7
4565
4239
sp|p39213|Y191—
INSERTION ELEMENT IS911 HYPOTHETICAL
65
38
327

12.7 KD PROTEIN.

22
2
1651
557
gI|290430
adhesin [Escherichia coli]
64
48
1095

5
4
1455
1841
gi|1575577
DNA-binding response regulator [Thermotoga maritims]
61
47
387

14
11
11161
11937
gi|1772622
HecB [Erwinia chrysanthemi]
60
39
227

14
1
930
1700
gi|1657478
similar to E. coli ORF_o208
58
47
721

[Escherichia coli]

5
6
3834
3391
gi|155032
ORF B [Plasmid pEa34]
56
36
444

3
5
6500
5982
gi|1633572

Harpeavirus salmiri
ORF73 homolog [Kaposl's sercoma-
54
25
519

associated herpas-like virus]

14
7
8429
8809
gi|1196729
unknown protein [Bactariophage P4]
54
41
381

14
14
15191
21793
gi|144048
filamentous hemagglutinin [Bordetella pertussis]
52
37
6603

14
16
21427
22671
bbs|117613
glycine-rich protein, atGRP (clone atGRP-4)
52
39
1245

(Arabidopsis thaliana, C24, Peptide Partial, 112 aa)

[Arabidopsis thaliana]

5
2
1004
381
gi|48518
HydC [Wolineila succinogenes]
51
34
624

5
5
1941
3311
gi|143331
alkaline phosphatase regulatory protein [Bacillus subtilis]
51
21
1371

14
4
3968
5431
gi|1033120
ORF_o469 [Escherichia coli]
51
29
1464

32
1
481
227
gi|1673731
(AE000010) Hycoplasma pneumoniae, fructose-permease
50
41
255

IIBC component, similar to Swiss-Prot Accession Number P20966,

from E. coli [Hycoplasma pneumoniae]

20
17
7039
7284
gi|1123054
coded for by C. elegans cDNA CEESN53F1similar to
48
28
246

protein kinases including CDC15 in yeast

[Caenorhabditis elegans]

[0240]

5

TABLE 4

(PAI V)

Putative coding regions of novel E. coli PAI V

proteins not similar to known proteins

Contig
ORP
Start
Stop

ID
ID
(nt)
(nt)

1
1
809
1165

3
2
3275
2640

3
6
6006
6425

3
7
6423
6833

4
1
3
455

5
1
501
4

6
1
2168
1749

6
2
2527
2114

6
3
2648
2331

6
4
3099
2626

14
5
7112
7699

14
6
7800
8507

14
8
9040
9624

14
10
10586
10846

14
15
21721
20921

15
1
575
826

15
2
850
1365

20
2
904
605

20
4
2330
3157

20
5
3139
3396

20
7
3812
3492

20
8
4373
3828

20
18
7282
7950

22
1
356
3

24
1
492
4

[0241]

6

TABLE 5

(PAI IV)

Putative coding regions of novel E. coli PAI IV containing known E. coli sequences

Config
ORF
Start
Stop
match

percent
HSP nt
ORF nt

ID
ID
(nt)
(nt)
acession
match gene name
ident
length
length

59
1
968
54
emb|X61239|ECPA

E. coli
papABCDEFGHIJK genes for F13 P-p111 proteins
99
790
915

59
2
1551
805
emb|Y00529|ECPA

E. coli
papC gene involved in formation of pap p111
99
518
747

59
3
1742
1494
emb|Y00529|ECPA

E. coli
papC gene involved in formation of pap p111
99
182
249

61
4
1975
1220
emb|X61239|ECPA

E. coli
papABCDEFGHIJK genes for F13 P-p111 proteins
100
69
756

63
13
10097
10480
gb|AE000133|

Escherichia coli
from bases 263572 to 274477
91
216
384

(section 23 of 400) of the complete genome

65
1
886
671
gb|U06468|

Escherichia coli
01111H- insertion sequence IS1203
93
164
216

12.7 kDa protein and putative transposase genes, complete cds

65
5
3218
2868
gb|U06468|

Escherichia coli
01111H- insertion sequence IS1203
85
285
351

12.7 kDa protein and putative transposase genes, complete cds

65
8
4064
3216
gb|U06468|

Escherichia coli
01111H- insertion sequence IS1203
86
145
849

12.7 kDa protein and putative transposase genes, complete cds

65
9
4939
4337
emb|Y00976|ECHN

E. coli
hns gene for DNA-binding protein H-HS (5′-region)
96
53
603

65
10
4919
5266
emb|Y00976|ECHN

E. coli
hns gene for DNA-binding protein H-HS (5′-region)
98
310
348

65
11
5206
5781
gb|AE000133|

Escherichia coli
from bases 263572 to 274477
89
431
576

(section 23 to 400) of the complete genome

68
1
1575
1315
emb|X61239|ECPA

E. coli
papABCDEFGHIJK genes for P13 P-p111 proteins
100
186
261

68
2
2468
1848
emb|X51704|ECPA

Escherichia coli
papJ gene for PapJ protein
99
621
621

68
3
2232
2594
emb|X61239|ECPA

E. coli
papABCDEFGHIJK genes for F13 P-p111 proteins
99
363
363

68
4
3212
2466
emb|X61239|ECPA

E. coli
papABCDEFGHIJK genes for F13 P-p111 proteins
100
747
747

69
1
300
4
gb|H14040|

E. coli
apt gene encoding adenine phosphorlbosyl-transferase
98
225
297

(APRT), complete cds

69
2
383
117
gb|H14040|

E. coli
apt gene encoding adenine phosphorlbosyl-transferase
95
162
267

(APRT), complete cds

70
1
832
149
gb|U09857|

Escherichia coli
4787 o115,v165,f165 flmbrial regulatory
89
225
684

f16521, f16528 and f1652 A genes, complete cds

70
17
10799
11767
gb|AE000291|

Escherichia coli
, nV, erfK, cobT, cobS, cobU, y152_6,
95
553
969

y122_3, y121_3 genes from bases 2060089 to 2072765

(section 181 of 400) of the complete genome

70
18
11809
11045
gb|AE000291|

Escherichia coli
, nV, erfK, cobT, cobS, cobU, y152_6,
94
595
765

y122_3, y121_3 genes from bases 2060089 to 2072765

(section 181 of 400) of the complete genome

70
19
12022
15772
dbj|D90838|D908

E. coli
genomic DNA, Kohara clone I348(44.5-44.9 min.)
89
2667
3201

70
20
15316
16836
gb|AE000292|

Escherichia coli
, yeeA, abmC, yeeC, abcB, yeeD, yeeE genes
96
1488
1521

from bases 2072708 to 2083664 (section 182 of 400) of the

complete genome

70
21
16722
17711
gb|AE000292|

Escherichia coli
, yeeA, abmC, yeeC, abcB, yeeD, yeeE genes
96
82
990

from bases 2072708 to 2083664 (section 182 of 400)

of the complete genome

70
22
17426
16776
gb|AE000292|

Escherichia coli
, yeeA, abmC, yeeC, abcB, yeeD,
96
63
651

yeeE genes from bases 2072708 to 2083664 (section 182 of 400)

of the complete genome

72
1
12
1061
gb|H10133|

E. coli
(J96) hlyC, hlyA, hlyB, and hlyD genes coding for
99
1024
1050

chromosomal hemolysins C, A, B and D

72
2
947
1285
gb|H10133|

E. coli
(J96) hlyC, hlyA, hlyB and hlyD genes coding for
96
261
339

chromosomal hemolysins C, A, B and D

73
6
4437
3205
gb|AE000379|

Escherichia coli
from bases 3107169 to 3112339
95
392
1233

(section 269 of 400) of the complete genome

73
8
6177
4555
gb|028377|

Escherichia coli
K-12 genome,
90
1133
1623

approximately 65 to 68 minutes

73
9
6835
6128
gb|AE000380|

Escherichia coli
, glcB, glcO, glcD genes from bases 3112500
93
703
708

to 3126189 (section 270 of of 400) of the complete genome

75
2
1553
1059
gb|AE000498|

Escherichia coli
from bases 4493507 to 4503769 (section 388
90
385
495

of 400) of the complete genome

75
3
2579
1566
gb|AE000498|

Escherichia coli
from bases 4493507 to 4503769 (section 388
92
464
1014

of 400) of the complete genome

75
4
3297
2743
gb|O07174|

Escherichia coli
091H101K99 heat resistant agglutinin 1 gene,
81
283
555

complete cds

76
1
698
3
gb|H10133|

E. coli
(J96) hlyC, hlyA, hlyB and hlyD genes coding for
99
693
696

chromosomal hemolysins C, A, B and D

78
1
382
59
gb|AE000360|

Escherichia coli
from bases 2885166 to 2897277
99
315
324

(section 250 of 400) of the complete genome

79
2
2620
1529
gb|H10133|

E. coli
(J96) hlyC, hlyA, hlyB and hlyD genes coding for
99
1084
1092

chromosomal hemolysins C, A, B and D

79
3
2925
2587
gb|H10133|

E. coli
(J96) hlyC, hlyA, hlyB and hlyD genes coding for
97
322
339

chromosomal hemolysins C, A, B and D

79
4
3576
2923
gb|H10133|

E. coli
(J96) hlyC, hlyA, hlyB and hlyD genes coding for
99
654
654

chromosomal hemolysins C, A, B and D

80
1
376
83
gb|005251|

Escherichia coli
polysialic acid gene cluster region 3,
93
210
294

promoter region

80
2
638
210
gb|AE000379|

Escherichia coli
from bases 3102169 to 3112339 (section 269
95
347
429

of 400) of the complete genome

80
3
1246
710
gb|AE000379|

Escherichia coli
from bases 3102169 to 3112339 (section 269
96
388
537

of 400) of the complete genome

80
4
1796
1182
gb|AE000379|

Escherichia coli
from bases 3107169 to 3112339 (section 269
94
397
615

of 400) of the complete genome

82
1
1
567
emb|X74567|ECKP

E. coli
K5 antigen gene cluster region 1 kpeE, kpsD, kpeU,
87
551
567

kpsC and kpeS genes

82
2
549
1157
emb|X74567|ECKP

E. coli
K5 antigen gene cluster region 1 kpsE, kpeD, kpsU,
88
554
609

kpsC and kpsS genes

82
3
1500
1180
gb|AE000292|

Escherichia coli
, yaeA, sbmC, yeeC, abcB, yeeD,
90
62
321

yeeE genes from bases 2072708 to 2083664 (section 182 of 400)

of the complete genome

82
4
2163
1519
gb|AE000292|

Escherichia coli
yaeA, sbmC, yaeC, sbcB, yaeD,
89
143
645

yaeE genes from bases 2072708 to 2083664 (section 182 of 400)

of the complete genome

82
5
2594
2139
gb|AE000292|

Escherichia coli
, yaeA, sbmC, yaeC, sbcB, yaeD,
97
456
456

yeaE genes from bases 2072708 to 2083664 (section 182 of 400)

of the complete genome

82
6
3000
2605
gb|AE000292|

Escherichia coli
, yaeA, abmC, yaeC, sbcB, yeeD,
98
396
396

yaeE genes from bases 2072708 to 2083664 (section 182 of 400)

of the complete genome

82
7
3463
3047
gb|AE000292|

Escherichia coli
, yaeA, sbmC, yaeC, sbcB, yaeD,
96
283
417

yaeE genes from bases 2072708 to 2083664 (section 182 of 400)

of the complete genome

82
9
3831
3337
gb|AE000292|

Escherichia coli
, yaeA, sbmC, yaeC, sbcB, yaaD,
96
453
495

yaeE genes from bases 2072708 to 2083664 (section 182 of 400)

of the complete genome

83
1
3
311
gb|AE000151|

Escherichia coli
, ybaE, cof, mdlA, mdlB, glnK, amtB,
99
207
309

tasB, ffa genes from bases 464774 to 475868 (section 41 of 400)

of the complete genome

83
2
176
433
gb|AE000151|

Escherichia coli
, ybaE, cof, mdlA, mdlB, glnK, amtB,
100
223
258

tesB, faa genes from bases 464774 to 475868 (section 41 of 400)

of the complete genome

86
1
529
2
gb|AE000379|

Escherichia coli
, from bases 3102169 to 3112339 (section 269
93
398
528

of 400) of the complete genome

93
1
440
3
gb|H10133|

E. coli
(396) hlyC, hlyA, hlyB and hlyD genes coding for
95
351
438

chromosomal hemolysins C, A, B and D

94
1
368
72
emb|X14180|ECGL

Escherichia coli
glutamine permease glnHPQ operon
100
229
297

99
1
161
586
gb|AE000379|

Escherichia coli
from bases 3102169 to 3112339 (section 269
98
426
426

of 400) of the complete genome

99
2
643
476
gb|AE000379|

Escherichia coli
from bases 3102169 to 3112339 (section 269
99
168
168

of 400) of the complete genome

99
3
532
1092
gb|AE000379|

Escherichia coli
from bases 3102169 to 3112339 (section 269
95
537
561

of 400) of the complete genome

99
4
1094
1396
gb|AE000379|

Escherichia coli
from bases 3102169 to 3112339 (section 269
94
274
303

of 400) of the complete genome

102
1
527
3
emb|Y00529|ECPA

E. coli
papC gene involved in formation of pap p111
100
427
525

102
2
762
373
amb|Y00529|ECPA

E. coli
papC gene involved in formation of pap p111
99
333
390

105
1
377
3
gb|AE000480|

Escherichia coli
from bases 4277211 to 4288813 (section 370
100
343
375

of 400) of the complete genome

107
1
2
397
gb|M10133|

E. coli
(J96) hlyC, hlyA, hlyB and hlyD genes coding for
99
390
396

chromosomal hemolysins C, A, B end D

107
2
406
966
gb|M10133|

E. coli
(J96) hlyC, hlyA, hlyB and hlyD genes coding for
99
549
561

chromosomal hemolysins C, A, B end D

110
1
148
2
emb|X56175|ECSE

Escherichia coli
secD and secF genes for membrane proteins
99
143
147

involved in protein export

110
2
312
40
gb|M63939|

E. coli
tRNA-guanine-transglycosylase (tgt) gene,
100
125
273

complete cde

115
1
501
325
gb|AE000459|

Escherichia coli
from bases 4013123 to 4024654 (section 349
98
177
177

of 400) of the complete genome

117
1
3
302
gb|AE000506|

Escherichia coli
from bases 4584059 to 4594314 (section 396
100
263
300

of 400) of the complete genome

121
1
2
250
gb|M16202|

E. coli
papH gene encoding a pilin-like protein
98
148
249

123
1
361
2
gb|AE000379|

Escherichia coli
from bases 3102169 to 3112339 (section 269
99
113
360

of 400) of the complete genome

127
1
2
227
gb|AE000233|

Escherichia coli
, racC, ydaD, sieB, trkG genes from bases
100
200
228

1415432 to 1425731 (section 123 of 400) of

the complete genome

127
2
227
382
gb|AE000233|

Escherichia coli
, racC, ydaD, sieB, trkG genes from bases
97
113
156

1415432 to 1425731 (section 123 of 400) of

the complete genome

130
1
337
2
emb|X60200|ECTH

E. coli
transposon Tn1000 (gamma delta) tnpR and tnpA genes
99
335
336

for resolvase and transposase

131
1
510
79
gb|M30198|

E. coli
recQ gene complete cds, end pidA gene, 3′ end
98
304
432

131
2
743
270
gb|M30198|

E. coli
recQ gene complete cds, and pidA gene, 3′ end
99
314
474

133
1
1
258
gb|AE000115|

Escherichia coli
, yabP, kefC, folA, spaH, spaG, ksgA,
98
237
258

pdxA, surA, lmp genes from bases 47163 to 57264

(section 5 of 400) of the complete genome

133
2
192
350
gb|AE000115|

Escherichia coli
, yabF, kefC, folA, spaH, spaG, ksgA,
99
115
159

pdxA, surA, lmp genes from bases 47163 to 57264

(section 5 of 400) of the complete genome

135
1
103
327
emb|X02143|ECPL

Escherichia coli
K-12 pldA gene for DR-phospholipase A
97
178
225

135
2
152
409
emb|X02143|ECPL

Escherichia coli
K-12 pldA gene for DR-phospholipase A
98
157
258

136
1
122
532
gb|AE000459|

Escherichia coli
from bases 4013123 to 4024654
97
237
411

(section 349 of 400) of the complete genome

140
1
576
244
gb|AE000291|

Escherichia coli
, nV, erfK, cobT, cobS, cobU, y152_6,
89
329
333

y122_3, y121_3 genes from bases 2060089 to 2072765

(section 181 of 400) of the complete genome

141
1
445
2
gb|AE000291|

Escherichia coli
, nV, erfK, cobT, cobS, cobU, y152_6,
77
432
444

y122_3, y121_3 genes from bases 2060089 to 2072765

(section 181 of 400) of the complete genome

[0242]

7

TABLE 6

(PAI V)

Putative coding regions of novel E. coli PAI V containing known E. coli sequences

Contig
ORF
Start
Stop
match

percent
HSP nt
ORF nt

ID
ID
(nt)
(nt)
acession
match gene name
Ident
length
length

3
4
6150
4855
gb|AE000292|

Escherichia coli
, yeeA, sbmC, yeeC, sbcB, yeeD, yaaE,
91
129
1296

genee from bases 2072708 to 2083664 (section 182 of 400)

of the complete genome

3
10
8214
7723
emb|X02311|ECIS

E. coli
insertion sequence IS3
76
274
492

3
11
7867
8319
emb|Z11606|ECIS

E. coli
DNA for insertion sequence IS3
80
378
453

3
12
8462
8157
emb|Z11606|ECIS

E. coli
DNA for insertion sequence IS3
90
267
306

3
13
8487
8663
gb|L19084|

Escherichia coli
RhsD genetic element, core protein (rheD)
96
112
177

gene, complete cds, complete ORF-D2, complete ORF-D3

4
2
1441
815
gb|AE000498|

Escherichia coli
from bases 4493507 to 4503769 (section 388
91
577
627

of 400) of the complete genome

4
3
923
1372
gb|AE000498|

Escherichia coli
from bases 4493507 to 4503769 (section 388
92
448
450

of 400) of the complete genome

4
4
2343
1324
gb|AE000498|

Escherichia coli
from bases 4493507 to 4503769 (section 388
92
244
1020

of 400) of the complete genome

7
1
3
743
emb|X61239|ECPA

E. coli
papABCDEFGHIJK genes for P13 P-pill proteins
100
741
741

7
2
977
615
emb|X61239|ECPA

E. coli
papABCDEFGHIJK (genes for F13 P-pill proteins
99
363
363

7
3
741
1214
emb|X51704|ECPA

Escherichia coli
papJ gene for PapJ protein
98
459
474

8
1
438
4
emb|X60200|ECTH

E. coli
transposon Tn1000 (gamma delta) tnpR and tnpA genes
99
435
435

for resolvase and transposase

10
1
1932
2426
emb|X61238|ECPR

E. coli
prsEFG genes for F13 pill tip proteins
97
462
495

11
1
903
1550
gb|H10133|

E. coli
(J96) hlyC, hlyA, hlyB and hlyD genes coding for
99
452
648

chromosomal hemolysins C, A, B and D

12
1
2559
1531
gb|U82598|

Escherichia coli
genomic sequence of minutes 9 to 12
100
1029
1029

12
2
1594
1860
emb|X13668|ECIS

E. coli
insertion element 5 (IS5) DNA
100
267
267

12
3
1858
2235
gb|U95365|

Escherichia coli
transposon IS5, transposase (is5B) gene,
99
354
378

complete cds

13
1
93
1424
emb|X61239|ECPA

E. coli
papABCDEFGHIJK genes for P13 P-pill proteins
99
885
1332

14
9
9832
10515
gb|U09857|

Escherichia coli
4787 o115,v165,f265 fimbrial regulatory
92
225
684

f1652I, f1652B and f1652 A genes, complete cds

16
1
1
375
gb|U07174|

Escherichia coli
O9,H10,K99 heat resistent agglutinin 1 gene,
94
320
375

complete cds

16
2
263
616
gb|U07174|

Escherichia coli
O9,H10,K99 heat resistant agglutinin 1 gene,
98
283
354

complete cds

17
1
282
4
emb|Y00529|ECPA

E. coli
papC gene involved in formation of pap pill
98
240
279

17
2
410
174
emb|Y00529|ECPA

E. coli
papC gene involved in formation of pap pill
100
168
237

19
1
1
369
gb|AE000418|

Escherichia coli
from bases 3550279 to 3561054 (section 308
99
347
369

of 400) of the complete genome

20
10
5401
4829
gb|AE000292|

Escherichia coli
, yeeA, sbmC, yeeC, sbcB, yeeD,
96
468
573

yeeE genes from bases 2072708 to 2083664 (section 182 of 400)

of the complete genome

20
11
4874
5371
gb|AE000292|

Escherichia coli
, yeeA, sbmC, yeeC, sbcB, yeeD,
96
453
498

yeeE genes from bases 2072708 to 2083664 (section 182 of 400)

of the complete genome

20
12
5245
5679
gb|AE000292|

Escherichia coli
, yeeA, sbmC, yeeC, sbcB, yeeD,
89
235
435

yeeE genes from bases 2072708 to 2083664 (section 182 of 400)

of the complete genome

20
13
5732
6139
gb|AE000292|

Escherichia coli
, yeeA, sbmC, yeeC, sbcB, yeeD,
93
329
408

yeeE genes from bases 2072708 to 2083664 (section 182 of 400)

of the complete genome

20
14
6316
5822
gb|AE000292|

Escherichia coli
, yeeA, sbmC, yeeC, sbcB, yeeD,
95
239
495

yeeE genes from bases 2072708 to 2083664 (section 182 of 400)

of the complete genome

20
15
6048
6590
gb|AE000292|

Escherichia coli
, yeeA, sbmC, yeeC, sbcB, yeeD,
87
406
543

yeeE genes from bases 2072708 to 2083664 (section 182 of 400)

of the complete genome

20
16
6569
7075
gb|AE000292|

Escherichia coli
, yeeA, sbmC, yeeC, sbcB, yeeD,
87
136
507

yeeE genes from bases 2072708 to 2083664 (section 182 of 400)

of the complete genome

20
19
8686
9915
gb|H67452|

Escherichia coli
lysine decarboxylase (cadB, and cadC,
98
1205
1230

complete cds, and cadA, 5′ end) genes

20
20
10604
11938
gb|U14003|

Escherichia coli
K-12 chromosomal region from 92.8 to
98
1308
1335

00.1 minutes

20
21
11940
12368
gb|H76411|

E. coli
cadA gene, 5′ cds and cadB and cadC genes,
100
363
429

complete cds

21
1
369
4
emb|x03391|ECPA

E. coli
major pilu subunit genes genes papI, papB, papA, and
98
201
366

papH 5′-region

23
1
1
879
gb|U14003|

Escherichia coli
K-12 chromosomal region from 92.8 to
98
879
879

00.1 minutes

23
2
900
16
gb|U14003|

Escherichia coli
K-12 chromosomal region from 92.8 to
98
885
885

00.1 minutes

23
3
953
1186
emb|X77707|ECCY

E. coli
ORF112, DIPZ and ORF191 genes
99
225
234

23
4
1223
2677
emb|X77707|ECCY

E. coli
ORF112, DIPZ and ORF191 genes
97
1454
1455

25
1
536
171
emb|X60200|ECTII

E. coli
transposon Tn1000 (gamma delta) tnpR and tnpA
100
164
366

genes for resolvase and transposase

25
2
1128
562
emb|X60200|ECTII

E. coli
transposon Tn1000 (gamma delta) tnpR and tnpA
99
459
567

genes for resolvase and transposase

27
1
708
436
emb|X61239|ECPA

E. coli
papABCDEFGHIJK genes for F13 P-pill proteins
100
252
273

28
1
309
4
emb|X77707|ECCY

E. coli
ORF112, DIPZ and ORF191 genes
98
278
306

28
2
131
213
emb|X77707|ECCY

E. coli
ORF112, DIPZ and ORF191 genes
96
150
219

30
1
399
4
gb|H26893|

E. coli
amldophoaphoribosyltransferase (purF) gene,
98
295
396

complete cds

31
1
706
170
emb|X56780|ECRR

E. coli
terminator sequence of RNA operon gene
99
513
537

37
1
2
400
gb|H63703|

E. coli
pyruvate kinase type II (pykA) gene, complete cds
98
399
399

38
1
463
2
emb|X13463|ECGU

Escherichia coli
gutH gene and gutR gene for activator and
99
363
462

repressor proteins

42
1
413
3
gb|H64367|

Escherichia coli
DNA recombinase (recG) gene, complete cds,
97
316
411

spoU gene, 3′ end; and gltS gene, 3′end

42
2
115
591
gb|H64367|

Escherichia coli
DNA recombinase (recG) gene, complete cds,
98
266
477

spoU gene, 3′ end; and gltS gene, 3′end

46
1
2
277
emb|X77707|ECCY

E. coli
ORF112, DIPZ and ORF191 genes
98
187
276

48
1
1
171
gb|AE000491|

Escherichia coli
from bases 4413548 to 4424699 (section 381
98
162
171

of 400) of the complete genome

48
2
105
464
gb|AE000491|

Escherichia coli
from bases 4413548 to 4424699 (section 381
98
144
360

of 400) of the complete genome

49
1
2
172
gb|U00800|

Escherichia coli
cloning vactor pk184, complete sequence,
98
167
171

kanamycin phosphotransfarase (kan) and (lacZalpha) genes,

complete cds

50
1
414
4
gb|AE000341|

Escherichia coli
, glyA, hmpA, glnB, yfhA, yfhG genes from
99
411
411

bases 2677406 to 2687636 (section 231 of 400)

of the complete genome

52
1
2
307
emb|X60200|ECTH

E. coli
transposon Tn1000 (gamma delta) tnpR and tnpA
100
284
306

genes for resolvase and transposase

53
1
280
41
gb|H36536|

E. coli
htrA gene, complete cds
100
131
240

53
2
558
214
gb|H36536|

E. coli
htrA gene, complete cds
99
315
345

54
1
9
263
gb|AE000381|

Escherichia coli
from bases 3125914 to 3136425 (section 271
94
111
255

of 400) of the complete genome

55
1
1
675
gb|AE000179|

Escherichia coli
, modA, modB, modC, ybhA, ybhE,
98
332
675

ybhD, genes from bases 794199 to 805132 (section 69 of 400)

of the complete genome

[0243]

	Number	Date	Country
	60061953	Oct 1997	US
	60031626	Nov 1996	US

	Number	Date	Country
Parent	09956004	Sep 2001	US
Child	10808570	Mar 2004	US
Parent	08976259	Nov 1997	US
Child	09956004	Sep 2001	US

Nucleotide sequence of escherichia coli pathogenicity islands

Information

Publication Number

Date Filed

Date Published

Inventors

Original Assignees

CPC

US Classifications

International Classifications

Abstract

Description

Claims

CROSS-REFERENCE TO RELATED APPLICATIONS

STATEMENTS AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY-SPONSORED RESEARCH AND DEVELOPMENT

Provisional Applications (2)

Divisions (2)