US20020150902A1 - Polymorphic loci that differentiate escherichia coli 0157:H7 from other strains - Google Patents

Polymorphic loci that differentiate escherichia coli 0157:H7 from other strains Download PDF

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US20020150902A1
US20020150902A1 US09/875,573 US87557301A US2002150902A1 US 20020150902 A1 US20020150902 A1 US 20020150902A1 US 87557301 A US87557301 A US 87557301A US 2002150902 A1 US2002150902 A1 US 2002150902A1
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  • the present invention relates generally to the field of microbiology and food sciences. More particularly, the inventor has discovered the gnd gene and corresponding 6-phosphogluconate dehydrogenase (6-PGD) protein from fourteen different strains of Escherichia coli and polymorphic sequences therein. Novel biotechnological tools, diagnostics, and food screening techniques are provided.
  • 6-PGD 6-phosphogluconate dehydrogenase
  • Escherichia coli O157:H7 is an exceptionally virulent food borne, human pathogen that causes a spectrum of illness, including asymptomatic and post-symptomatic carriage, mild diarrhea, bloody diarrhea/hemorrhagic colitis, and the postdiarrheal, potentially lethal, hemolytic uremic syndrome (HUS).
  • HUS hemolytic uremic syndrome
  • HUS is defined as a triad of non-immune microangiopathic hemolytic anemia, thrombocytopenia, and acute renal failure. HUS is chiefly a disorder of children under age 10, however, the elderly are also susceptible to severe complications of E. coli O157:H7 gastrointestinal infections.
  • HUS follows gastrointestinal infection with E. coli O157:H7 in approximately 10-15% of pediatric cases.
  • Talr et al. Am J Epidemiol, 129:582-586 (1989);
  • Tarr et al. Am J Epidemiol, 129:582-586 (1989)).
  • E. coli O157:H7 is often found in food and environmental vehicles that do not always undergo an efficient bacterial killing process.
  • Large outbreaks have been caused by the interstate dissemination of contaminated ground beef that was under cooked (Bell et al., JAMA, 272:1349-1353 (1994) and Riley et al., N Engl J Med, 308:681-685 (1983)); salted, fermented, but uncooked salami (Tilden et al., Am J Public Health, 86:1142-1145 (1996)); municipal (Swerdlow et al., Ann Intern Med, 117:812-819 (1992)) and swimming (Keene et al., N Engl J Med, 331:579-584 (1994)) water; unpasteurized apple juice (Anonymous, Morb Mortal Wkly Rep, 45:975 (1996)); unpasteurized milk (Keene et al., J Infect Dis, 176:815-818 (1997)); and lettuce
  • E. coli O157:H7 has not been shown to possess a capsular polysaccharide but it expresses an O side chain antigen designated 157, which consists of repeating tetrasaccharide units of variable length. These tetrasaccharide units comprise the antigenic O157 lipopolysaccaride (LPS).
  • O157:H7 fails to ferment sorbitol after overnight culture on MacConkey agar into which sorbitol rather than lactose is incorporated as the carbon source. (Wells et al., J Clin Microbiol, 18:512-520 (1983); March et al., J Clin Microbiol, 23:869-872 (1986)).
  • E. coli O157:H7 also fails to produce ⁇ -glucuronidase, another metabolic distinguishing factor.
  • Sorbitol non-fermenting E. coli almost always express the H7 flagellar antigen, though occasional sorbitol non-fermenting E. coli O157 strains recovered in the United States do not express the H7 antigen.
  • Another variant of E. coli O157:H7 has been found in Germany and Czech Republic, which expresses the O157 antigen, but are non-motile pathogens that ferment sorbitol.
  • Shiga-toxin a glycosphingolipid ligand (globotriaosylceramide) (Basta et al., J Clin Microbiol, 27:1617-1622 (1989)) (Biocarb, Gaithersburg, Md.) or employ an enzyme immunoassay (Meridian Diagnostics, Cincinnati, Ohio). (Kehl et al., J Clin Microbiol, 35:2051-2054 (1997)); Park et al., Diag Microbiol Infect Dis, 26:69-72 (1996)).
  • a glycosphingolipid ligand globotriaosylceramide
  • the inventor has discovered the gnd gene and corresponding 6-phosphogluconate dehydrogenase (6-PGD) protein of fourteen strains of E. coli . Within these genes and proteins the inventor has also found several polymorphisms that can be used to identify the presence of a particular strain of E. coli and/or differentiate one strain of E. coli from another.
  • One polymorphism in particular which involves a substitution of an isoleucine molecule for a threonine molecule at amino acid position 218, can be used to differentiate highly pathogenic strains of O157:H7 and O55:H7 from less pathogenic strains of O157:H7.
  • O55:H7 is only about 82% homologous to O157:H7
  • the highly pathogenic strains of O157:H7 can be differentiated from O55:H7 at several different loci.
  • identifying the presence and/or absence of the polymorphism at position 218 and identifying the presence or absence of a region of non-homology between O55:H7 and O157:H7 one of skill in the art can rapidly identify the presence of a highly pathogenic strain of E. coli in a sample obtained from a patient or from a food or liquid source.
  • identifying the presence or absence of other polymorphisms in the gnd locus one of skill can efficiently differentiate specific strains of E. coli allowing for a more precise diagnosis or screening.
  • Embodiments of the invention include an isolated polynucleotide encoding gnd, wherein the polynucleotide comprises one of the E. coli sequences disclosed in the sequence listing. Fragments of these sequences having least 9 consecutive bases and a polymorphism described in Table 1 are also embodiments of the invention. Other embodiments include isolated polynucleotides that encode a polypeptide that corresponds to the E.
  • a additional embodiment concerns a nucleic acid probe for detecting the presence of E. coli O157:H7 consisting of an isolated nucleic acid molecule at least 7 nucleotides in length, wherein the nucleic acid molecule hybridizes to DNA of gnd of E. coli O157:H7 and not to DNA of gnd of non-H7 E.
  • nucleic acid primer for detecting the presence of E. coli O157:H7 consisting of an isolated nucleic acid molecule at least 7 nucleotides in length, wherein the isolated nucleic acid molecule primes DNA of gnd of E. coli O157:H7 and not DNA of gnd of non-H7 E. coli O157 strains.
  • the nucleic acid probes of the invention can be provided on a substrate or in a microarray on a chip.
  • SRecombinant constructs and vectors comprising one of the sequences of the sequence listing are also embodiments of the invention.
  • a cultured cell line comprising the one of the vectors of the invention is an embodiment.
  • the proteins of the invention include an isolated protein comprising one of the sequences found in the sequence listing and an isolated polypeptide comprising at least 3 consecutive amino acids of one of the sequences of the sequence listing, wherein the polypeptide contains at least one polymorphism that can be deduced from Table 1.
  • Additional protein embodiments concern an isolated antibody capable of specifically binding to a protein having one of the sequences of the sequence listing, wherein the epitope corresponds to at least one polymorphism that can be deduced from Table 1.
  • another embodiment includes an isolated antibody capable of binding to a polypeptide comprising at least 9 consecutive amino acids of one of the sequences of the sequence listing, wherein the epitope corresponds to at least one polymorphism that can be deduced from Table 1.
  • the antibody is monoclonal.
  • Methods of detecting a polymorphism and detecting or diagnosing the presence of a highly pathogenic E. coli are also embodiments.
  • a polymorphism in a gene encoding 6-PGD is detected by obtaining a biological sample containing polynucleotides and analyzing the biological sample for the presence of a diagnostic polynucleotide having at least one polymorphism described in Table 1.
  • the presence or absence of the C653T or G653C polymorphism is analyzed and/or the analysis of the biological sample further comprises a DNA amplification step.
  • Another method concerns the identification of a pathogenic or non-pathogenic E. coli .
  • This approach is practiced by obtaining a biological sample containing polynucleotides, analyzing the biological sample for the presence of a diagnostic polynucleotide having at least one polymorphism described in Table 1, and identifying the E. coli as a pathogenic or non-pathogenic strain based on the presence or absence of at least one polymorphism described in Table 1.
  • the presence or absence of the C653T or G653C polymorphism is analyzed and/or the analysis of the biological sample further comprises a DNA amplification step.
  • Other methods of the invention include, a method of making a 6-PGD protein comprising the steps of obtaining a cDNA comprising one of the sequences of the sequence listing, inserting the cDNA in an expression vector such that the cDNA is operably linked to a promoter, and introducing the expression vector into a host cell whereby the host cell produces the protein encoded by the cDNA.
  • This method can also be used in conjunction with a step involving the isolation of the protein.
  • An additional method concerns the construction of a transformed host cell that expresses one of the sequences of the sequence listing. This method includes the steps of transforming a host cell with a recombinant DNA vector suitable for gene expression. Additionally, a method for detecting the presence of E.
  • coli O157:H7 in a sample involves the steps of: (a) contacting said sample, under hybridization conditions, with a nucleic acid probe that selectively hybridizes to a nucleic acid sequence from gnd of E. coli O157:H7 and not to nucleic acid sequence from gnd of non-H7 E. coli O157 strains, to form a hybridization complex and (b) detecting formation of said hybridization complex as an indication of the presence of E. coli O157:H7 in the sample.
  • FIG. 1 shows a graphical representation of the polymorphisms present at the gnd locus in several strains of E. coli . Bars represent the 1407 bp gnd allele and the vertical lines represent sites of polymorphisms determined by comparison to a consensus sequence.
  • FIG. 2 shows the homology between chromosomes of E. coli O55:H7 and E. coli O157:H7 observed 3916 nucleotides downstream of the 3′ terminus of gnd of E. coli O55:H7, and 52 nucleotides downstream of the 3′ terminus of gnd of E. coli O157:H7.
  • Elements of interest in the extra DNA in E. coli O55:H7 include a segment of homology to tnpA of S. enterica Typhimurium, an H-repeat protein gene with segments homologous to noncoding parts of the E. coli O157 rfb cluster, wbdJ and wbdK. Orfs are noted as homologous proteins. Loci are oriented chromosomally.
  • FIG. 3 is a representation of a chromosome having the gnd locus and flanking regions.
  • the inventor describes the discovery of the gnd gene and corresponding 6-phosphogluconate dehydrogenase (6-PGD) protein of fourteen strains of E. coli .
  • 6-PGD 6-phosphogluconate dehydrogenase
  • the inventor has also found several genetic differences or “polymorphisms” that can be used to identify the presence of a particular strain of E. coli and/or differentiate one strain of E. coli from another.
  • One polymorphism in particular involves a substitution of an isoleucine molecule for a threonine molecule at amino acid position 218. This polymorphism is referred to as “T218I” or “Thr218Iso”.
  • this form of 6-PGD or a polynucleotide encoding this form of 6-PGD i.e., an isoleucine at amino acid position 218 or a polynucleotide encoding an isoleucine at position 2178
  • Iso218 a 6-PGD molecule having a threonine at amino acid position 218 or a polynucleotide encoding a threonine at position 218
  • Thr218 a 6-PGD molecule having a threonine at amino acid position 218 or a polynucleotide encoding a threonine at position 218
  • the term “Iso218” refers to a polymorphism in a polynucleotide encoding a fragment of 6-PGD (in which case the polymorphism is with reference to codon 218 of the 6-PGD fragment-encoding polynucleotide), or to a fragment of the 6-PGD protein itself (in which case the polymorphism is with reference to amino acid position 218 of the 6-PGD polypeptide sequence provided in the sequence listing.
  • This polymorphism can also be referred to by the nucleotide differences that encode the Iso218 polymorphism.
  • the Thr218 polymorphism results from the presence of a cytosine and guanine residue at nucleotide positions 653 and 654, respectively; whereas, the Iso2I8 polymorphism has a thymine and cytosine at positions 653 and 654, respectively.
  • other ways of referring to the polymorphism at amino acid residue 218 include “C ⁇ T mutation at nucleotide position 653” and/or a “G ⁇ C” mutation at nucleotide position 654” or “C653T” and/or “G654C”.
  • the inventor describes the cloning, sequencing, and characterization of fourteen gnd genes and corresponding proteins from different strains of E. coli .
  • Evidence is also provided of the existence of one or more mobile DNA element(s) within the gnd-rfb region that has co-transferred among E. coli and accounts for the antigenic changes that resulted in the emergence of pathogenic E. coli that express the O55 and O157 antigens.
  • Biological tools, diagnostics, and methods of use of the foregoing are described in the sections that follow. These embodiments are useful for the rapid identification of the presence of a specific strain of E. coli , and the differentiation of one strain of E.
  • the inventor describes the cloning, sequencing, and characterization of the fourteen gnd genes and corresponding proteins of different strains of E. coli.
  • the rfb cluster of genes occurs at approximately 44 minutes on the E. coli chromosome. These clusters are generally between 8 and 14 kb in length and contain approximately 8 to 12 contiguous genes that act in concert to produce the O side chain lipopolysaccharide. (Reeves, New Compr Biochem, 27:281-314 (1994); Reeves et al., Trends Microbiol, 4:495-503 (1996)). Adjacent to the rfb cluster is the gnd allele that encodes 6-phosphogluconate dehydrogenase (6-PGD) (EC 1.1.1.44), the third enzyme in the pentose-phosphate pathway.
  • 6-PGD 6-phosphogluconate dehydrogenase
  • gnd encodes a “housekeeping” gene with critical bacterial function
  • this allele is highly polymorphic, when compared to other “housekeeping” genes in the E. coli chromosome.
  • Woodtam and Ake “Mechanisms of molecular evolution,” Sinauer, Takahata and Clark, eds., Sunderland, Mass.: 1993:223-245. It is believed by some that the polymorphisms at the gnd locus result from inter-strain or interspecies transfers and subsequent recombination with Salmonella.
  • the “hitchhiking hypothesis” the rfb region of E. coli is believed to have been acquired via horizontal transfer from other species by virtue of sequence homology and low G+C content. That is, gnd and rfb are thought to co-transfer or “hitchhike” with rfb. (Nelson and Selander, Proc Natl Acad Sci USA, 91:10227-10231 (1994)).
  • this hypothesis are the discordant electromorphic appearances of 6-PGD of E. coli O157:H7 and its closest non-O157:H7 relative, E. coli O55:H7.
  • the inventor cloned and sequenced the gnd genes of virulent strains of E. coli O157:H7, E. coli O55:H7, and E. coli that express the O157 antigen but are not as pathogenic to humans as E. coli O157:H7 and determined that, indeed, a relationship existed between polymorphisms within genes of the rfb cluster, in particular gnd and pathogenicity.
  • E. coli O157:H7 and the other E. coli strains were cloned from purified bacterial DNA.
  • bacteria were grown overnight in LB broth (Maniatis et al., Molecular cloning: a laboratory manual, Cold Spring Harbor Laboratory, (1982)) without antibiotics or with ampicillin (200 mg/mL), respectively, at 37° C.
  • LB broth Maniatis et al., Molecular cloning: a laboratory manual, Cold Spring Harbor Laboratory, (1982)
  • ampicillin 200 mg/mL
  • genomic DNA bacteria (3 ml), pelletted by centrifugation, were suspended in 50 millimolar (mM) Tris-HCl (pH8.0) and 50 mM ethylenediamine tetraacetic acid (EDTA).
  • the precipitate was centrifuged, washed once with 100% ethanol, air dried, and solubilized in 10 mM Tris-HCl (pH8.0), containing 1 mM ETDA. Plasmids were obtained and prepared using the Qiaprep Spin Miniprep Kit (Qiagen Inc., Valencia, Calif.) and manufacture's instructions.
  • the inventor To amplify gnd from E. coli expressing the O157 antigen, the inventor initially used the primer pair (1) 5′CACGGATCCGATCACACCTGACAGGAGTA3′ (SEQ. ID. No.1) (for the rfb side) and 5′CCGGAATTCCGGGCAAAAAAAAGCCCGGTGCAA3′ (SEQ. ID. No. 2) (for the his side), which were derived from published sequences (Bisercic et al., J Bacteriol, 173:3894-3900 (1991)) and were modified to contain BamHI and EcoRI sites for cloning purposes. However, these primers failed to obtain an amplicon from E. coli O55:H7 DNA.
  • the consensus oligonucleotides of primer pair (2)-5′CGGAATTCCGCGCTCAACATCGANAGCCGTGG3′ (SEQ. ID. No. 3) and 5′CGGAATTCCGCCTGGATCAGGTTAGCCGG3′ (SEQ. ID. No. 4) (derived from a computerized data base of E. coli gnd sequences and having 5′ EcoRI sites) were used to prime DNA from strain TB 182A (an E. coli O55:H7 strain). (Bokete et al., J Infect Dis, 175:1382-1389 (1997)).
  • These primers produced a PCR product of approximately 1.3 kb, consisting of the internal portion of the gnd gene. Sequence analysis of this amplicon determined that the following primer pairs would prime DNA close to the 5′ and 3′ termini, respectively, of this allele:
  • 5′TGCCCGCTACATCTCCTC3′ (SEQ. ID. No. 8) were then used to amplify DNA beyond the 5′ and 3′ termini of the E. coli O55:H7 gnd, respectively.
  • the resulting sequence data then prompted the design and use of the primer pair (8)-5′CCATCAGTAATAATGAAAAGGAATT3′ (SEQ. ID. No. 11) and 5′ATCATTAGCTCCTCTTAAGATCGC3′ (SEQ. ID. No.12) to amplify the E. coli O55 gnd allele.
  • Primer pairs (9)-5′TCGTCGCTTATGCGGTACAGAGCG3′) (SEQ. ID. No.
  • PCR was performed using either the ExpandTM Long Template PCR System (Boehringer Mannheim, Indianapolis, Ind.) (“Expand System”) or Taq DNA polymerase (Promega, Madison, Wis.). For initial pan-gnd amplifications, Taq DNA polymerase (Promega) was used. For amplifications using the Expand system, reactions were performed in 50 ⁇ 1 containing BMB buffer 1 supplied by the manufacturer. DNA polymerases used were either Taq DNA polymerase supplied by Promega, catalog number M1865 (5U/ ⁇ 1) (A) or Taq and Pwo DNA polymerases supplied by Boehringer-Mannheim (3.5U/ ⁇ 1) (B).
  • Thermocycling conditions included: 35 cycles at 94° C. (1 min), 37° C. (1 min), and 72° C. (1 min), followed by a 7 minute incubation at 72° C.); 30 cycles at 94° C. (1 min), 37° C. (1 min), and 72° C. (1 min), followed by a 7 minute incubation at 72° C.; an initial cycle at 95° C. (3 min), 55° C. (1 min), and 74° C. (1 min), followed by 35 cycles of 95° C. (1 min), 55° C. (1 min), and 74° C. (1 min), and a final incubation at 72° C. (5 min); or an initial incubation at 92° C. (2 min), followed by 10 cycles at 92° C.
  • single nucleotide polymorphisms were found in strains 13A81 and 13A83 ( E. coli O157 isolates expressing H antigens 16 and 45, respectively); strains 13A80, 7E, 3005-89, 3004-89, and G5933 ( E. coli O157 expressing H antigens 16, 43, 38, 3, and 12, respectively); and each of the non-H7 E. coli O157 strains.
  • the amino acid sequences that correspond to the polymorphisms described in Table 1 i.e., the polymorphisms expressed in terms of the amino acid
  • the T218I was discovered in pathonic O157:H7 strains and the O55:H7 strain TB182A but not any of non-pathonic O157:H7 strains.
  • the sequence data revealed that the non-pathogenic strains, except O55:H7, have a cytosine and guanine residue at nucleotide positions 653 and 654, respectively; whereas, the pathogenic strains have a thymine and cytosine at positions 653 and 654, respectively.
  • a convenient way to distinguish pathogenic O157:H7 strains from non-pathogenic O157:H7 strains involves the identification of a “C ⁇ T” mutation at nucleotide position 653 of gnd and/or a “G ⁇ C” mutation at nucleotide position 654 or the presence of an isoleucine amino acid residue at amino acid position 218. Because the gnd of E. coli O55:H7 is only about 82% homologous to the gnd of E. coli O157:H7 (e.g., strain 86-24), these strains can be easily distinguished at several different loci, as will be described in greater detail below. TABLE 1 Pos.
  • 6468 0.0 1 gb
  • E. coli O157 Three distinct allele groups were found in E. coli O157. (See Table 3). These alleles differed from one another at about 5% of their nucleotide residues.
  • the “gnd allele A” is comprised of gnds of toxigenic E. coli O157:H7 and E. coli O157:NM strains. The gnd sequences of strains 85-07 and 87-16 each differed from that of strain 86-24 at only two of their 1407 nucleotides; the remaining three were identical.
  • the “gnd allele B” is found in E.
  • E. coli O55:H7 is the closest relative to E. coli O157:H7, their gnd sequences are strikingly different.
  • the gnd sequence of E. coli O157:H7, strain 86-24 has only about 82% homology to the gnd sequence of E. coli O55:H7, strain TB182A and there appears to be no readily apparent region of conservation between these two alleles.
  • coli O157:H7 contained open reading frames (orfs) encoding UDP glucose-6-dehydrogenase and an O-antigen chain length determining protein. Sequences between positions 3′+1 and 3′+51, and 3′+1 and 3′+3915, relative to the respective E. coli O55:H7 and E. coli O157:H7 gnds, were not found to be homologous.
  • the inventor discovered an orf between positions 3′+2817 and 3′+3422 that encodes a protein of 201 amino acids, which is about 98% homologous to H-repeat protein amino acids in RhsB encoded by orf-H (Genbank number LO2370). (Zhao et al., J. Bacteriol., 175:2799-2808 (1993)). Still further, the inventor found that approximately 92% of the 114 inclusive nucleotides between positions 3′+3809 and 3′+3922 relative to gnd of E. coli O55:H7, including 7 nucleotides of the sequence common to E.
  • coli O157:H7 are identical to nucleotides adjacent to the 3′ end of tnpA of Salmonella typhimurium LT2, encoding IS200 transposase A (GenBank number AFO93749). DNA between nucleotides at the 3′+478 and 3′+1942 positions relative to gnd of E. coli O55:H7 were also found to be about 75% identical to E. coli O111 wbdJ and wbdK (Genbank number U13629).
  • the two orfs corresponding to nucleotides between positions 3′+112 and 3′+1035, and 3′+1032 and 3′+2198 relative to gnd are 67% and 80% identical to WbdJ and Wbd K, respectively. (Bastin and Reeves, Gene, 164:17-23 (1995)). Three segments between nucleotides at positions 3′+2788 and 3′+3806 relative to the E. coli O55:H7 gnd allele are 83-96% homologous to non-coding regions of the E. coli O157:H7 rfb cluster (Genbank numbers AF061251 and AB008676).
  • PCR was employed using the primers: 5′GCGTTCTTAAAGAGTCCTGC3′ (SEQ. ID. No. 13) and 5′TGCCCGCTACATCTCCTC3′ (SEQ. ID. No. 8), which correspond to the 3′ end of gnd and downstream regions, so as to obtain a 6.5 kb amplicon from the DNA of 11 E. coli O55 strains. This amplicon was not obtained when PCR was performed with these primers on DNA from E. coli O157:H7.
  • E. coli O55 strains from diverse lineages. Genomic DNA or amplicons from E. coli HB101, E. coli O157:H7 strain 86-24, E. coli O55:H7 strains TB156A, TB182A, and 5 A-E, and E. coli O55:H6 strains 1A, 1B, 2A, and 2B were produced using the primers: 5′GCGTTCTTAAAGAGTCCTGC3′ (SEQ. ID. No. 13) and 5′TGCCCGCTACATCTCCTC3′ (SEQ. ID. No. 8).
  • the amplicon probe was labeled with the Megaprime DNA system (Amersham) and [ ⁇ 32 P]dATP (New England Nuclear Research Products). This experiment showed a strong signal in the lanes loaded with DNA from an O55 strain but not from a lane loaded with DNA from an O157 strain or the HB101 control.
  • the study above not only provides strong evidence that the region 3′ to gnd in E. coli O55 strains contains a conserved element with sequences that are involved in DNA mobility but also teach a rapid method to differentiate E. coli O55:H7 from O157:H7.
  • E. coli O55 and O111 O-side chains each contain colitose (Keene et al., Carbohydr. Res., 111:289-296 (1983)), an unusual residue among known bacterial LPS sugars.
  • the rfb regions specifying these two serogroups have genes encoding WbdK and WbdJ homologues, though on different sides of gnd.
  • WbdK is homologous to RfbH of Yersinia pseudotuberculosis, a CDB- 4-keto-6-deoxy-D-glucose-3-dehydrase in the CDP-abequose pathway.
  • WbdK is a putative pyridoxamine 5-phosphate-dependent dehydrase at a corresponding step in the synthesis of the O111 antigen. (Bastin and Reeves, Gene, 164:17-23 (1995)). WbdJ is homologous to Orf1.9 encoded by the E. coli capsular polysaccharide gene cluster, and is believed to perform a related function in the synthesis of the E. coli O111 LPS antigen.
  • a computer readable medium having the gnd sequences and/or corresponding proteins of SEQ. ID. Nos. 16-43 are useful for the determination of homologous sequences, design of probes and primers, epitope analysis, elucidation of structural and functional domains, and the construction of protein models for rational drug design.
  • the gnd sequences and/or corresponding proteins of SEQ. ID. Nos. 16-43 can be stored, recorded, and manipulated on any medium that can be read and accessed by a computer.
  • the words “recorded” and “stored” refer to a process for storing information on computer readable medium.
  • a skilled artisan can readily adopt any of the presently known methods for recording information on computer readable medium to generate manufactures comprising the nucleotide or polypeptide sequence information of this embodiment of the invention.
  • a variety of data storage structures are available to a skilled artisan for creating a computer readable medium having recorded thereon a nucleotide or polypeptide sequence. The choice of the data storage structure will generally be based on the component chosen to access the stored information.
  • Computer readable media include magnetically readable media, optically readable media, or electronically readable media.
  • the computer readable media may be a hard disc, a floppy disc, a magnetic tape, CD-ROM, RAM, or ROM as well as other types of other media known to those skilled in the art.
  • the computer readable media on which the sequence information is stored may be in a personal computer, a network, a server or other computer systems known to those skilled in the art.
  • Embodiments of the invention include systems, particularly computer-based systems that contain the sequence information described herein.
  • a computer-based system refers to the hardware, software, and database used to analyze the gnd sequences and/or corresponding proteins of SEQ. ID. Nos. 16-43, or fragments thereof.
  • the computer-based system preferably includes the storage media described above, and a processor for accessing and manipulating the sequence data.
  • the hardware of the computer-based systems of this embodiment comprise a central processing unit (CPU) and one or more databases.
  • CPU central processing unit
  • a skilled artisan can readily appreciate that any one of the currently available computer-based systems are suitable.
  • the computer system includes a processor connected to a bus which is connected to a main memory (preferably implemented as RAM) and a variety of secondary storage devices, such as a hard drive and removable medium storage device.
  • the removable medium storage device may represent, for example, a floppy disk drive, a compact disk drive, a magnetic tape drive, etc.
  • a removable storage medium, such as a floppy disk, a compact disk, a magnetic tape, etc. containing control logic and/or data recorded therein (e.g., the gnd sequences and/or corresponding proteins of SEQ. ID. Nos. 16-43) may be inserted into the removable storage device.
  • the computer system includes appropriate software for reading the control logic and/or the data from the removable medium storage device once inserted in the removable medium storage device.
  • the gnd sequences and/or corresponding proteins of SEQ. ID. Nos. 16-43 may be stored in a well known manner in the main memory, any of the secondary storage devices, and/or a removable storage medium.
  • Software for accessing and processing the gnd sequences and/or corresponding proteins of SEQ. ID. Nos. 16-43 (such as search tools, compare tools, and modeling tools etc.) reside in main memory during execution.
  • a database refers to memory that can store nucleotide or polypeptide sequence information, and protein model information. Additionally, a “database” refers to a memory access component which can access manufactures having recorded thereon nucleotide or polypeptide sequence information, and/or protein model information. In other embodiments, a database stores an “ E. coli pathogen profile” that comprises nucleotide and/or polypeptide sequence information, and/or protein model information on gnd genes and 6-PGD proteins and the polymorphisms therein.
  • an E. coli pathogen profile has recorded or stored in a database a plurality of polymorphisms associated with highly pathogenic and/or less pathogenic E.
  • the pathogen profile can be stored such that the sequences therein that correspond to specific organisms are fully searchable by sequence, organism, and/or restriction map and homology, identity and matches to queried sequences can be determined.
  • a preferable organization of the database is as provided by NCBI, which allows BLAST-type searching, protein model searching, key word searches, and an interface with Medline. Many other types of databases and organizations are known to those of skill in the art and several will be discussed below.
  • the gnd sequences and/or corresponding proteins of SEQ. ID. Nos. 16-43 may be stored and manipulated in a variety of data processor programs in a variety of formats.
  • the sequence data may be stored as text in a word processing file, such as MicrosoftWORD or WORDPERFECT or as an ASCII file in a variety of database programs familiar to those of skill in the art, such as DB2, SYBASE, or ORACLE.
  • a “search program” refers to one or more programs that are implemented on the computer-based system to compare a nucleotide or polypeptide sequence with other nucleotide or polypeptide sequences stored within the database.
  • a search program also refers to one or more programs that compare one or more protein models to several protein models that exist in a database.
  • a search program is used, for example, to compare regions of the gnd sequences and/or corresponding proteins of SEQ. ID. Nos. 16-43 that match sequences in nucleic acid and/or protein data base so as to identify homologies and structural or functional motifs.
  • a search program is used to compare an E. coli pathogen profile to a queried sequence so as to identify the presence of one or more polymorphisms in the queried sequence and determine the strain of the bacteria from which the queried sequence was derived.
  • a “retrieval program” refers to one or more programs that are implemented on the computer based system to identify a homologous nucleic acid sequence, a homologous protein sequence, or a homologous protein model. Further a retrieval program can be used to identify an E. coli pathogen profile that matches a queried sequence, keyword, disease characteristic, or restriction map. Preferably, the retrieval program interfaces with a display format that presents the data from the E. coli pathogen profile in a form that can be rapidly discerned. For example, the “bar code” shown in FIG. 1 is one format that can be obtained by a retrieval program that provides information on the position of polymorphisms that can be used to identify or distinguish a particular strain of E. coli.
  • one of the novel sequences disclosed in is compared to a queried sequence and the percent sequence identity is determined.
  • Standard methods that are commonly used to compare the similarity and position of the amino acid of two polypeptides can be used to make these comparisons.
  • two polypeptides can be aligned for optimal matching of their respective amino acids (either along the full length of one or both sequences, or along a predetermined portion of one or both sequences).
  • Such programs provide “default” opening penalty and a “default” gap penalty, and a scoring matrix such as PAM 250 (a standard scoring matrix; see Dayhoff et al., in: Atlas of Protein Sequence and Structure, Vol. 5, Supp. 3 (1978)) can be used in conjunction with the computer program.
  • the percent identity can then be calculated as:
  • Polypeptides that are at least 70% identical will typically have one or more amino acid substitutions, deletions and/or insertions. Usually, the substitutions will be conservative so as to have little or no effect on the overall net charge, polarity, or hydrophobicity of the protein but optionally may increase the activity of 6-PGD.
  • Blast 2 (BlastP 2.0.9) searches on the NCBI data base using the BLOSUM matrix with an opening penalty of 11, a gap extension of 1, and an x_-dropoff of 50. These later search parameters were used to compare 6-PGD encoded by O157:H7, strains 86-24, H8, ADAL233, and 2755 to:
  • ORFs encoded by the gnd sequences and/or corresponding proteins of SEQ. ID. Nos. 16-43 and regions within the gnd/rfb gene cluster were also compared to known amino acid sequences found in Swissprot.
  • Many computer programs and databases may be used with embodiments of the invention. The following list is intended not to limit the invention but to provide guidance to programs and databases that are useful with the nucleic acid and protein sequence embodiments of the invention.
  • the programs and databases that can be used include, but are not limited to: MacPattern (EMBL), DiscoveryBase (Molecular Applications Group), GeneMine (Molecular Applications Group), Look (Molecular Applications Group), MacLook (Molecular Applications Group), BLAST and BLAST2 (NCBI), BLASTN and BLASTX (Altschul et al, J. Mol. Biol. 215: 403 (1990)), FASTA (Pearson and Lipman, Proc. Natl. Acad. Sci.
  • aspects of the invention include recombinant vectors, probes, and primers comprising the gnd sequences and/or corresponding proteins of SEQ. ID. Nos. 16-43 and fragments thereof, in particular portions of the gnd gene or corresponding protein that contain a polymorphism described in Table 1.
  • the discussion below describes these aspects of the invention.
  • Several embodiments of the invention include recombinant vectors, probes, and primers comprising the gnad sequences of SEQ. ID. Nos. 22, 16, 18, 24, 26, 20, 42, 28, 30, 40, 32, 36, 38, and 34 and fragments thereof.
  • preferred nucleic acid embodiments include fragments of any gnd gene that have a polymorphism described in Table 1.
  • full-length refers to either the entire sequence of genomic gnd or cDNA gnd depending on the context. Further embodiments include nucleic acids that complement the full-length gnd described in SEQ.
  • nucleic acid embodiments of the invention can have from 9 to approximately 1,406 consecutive nucleotides of SEQ. ID. Nos.: 22, 16, 18, 24, 26, 20, 42, 28, 30, 40, 32, 36, 38, and 34 or a complement to these sequences of virtually any length so long as the nucleic acid includes at least one polymorphism described in Table 1.
  • gnd nucleic acids of the invention can be joined to an exogenous nucleic acid so as create a fusion product, which is within the scope of the invention, having virtually any length.
  • a nucleic acid having a portion (i.e., about 9 to about 1,406 consecutive nucleotides) of SEQ. ID. Nos.: 22, 16, 18, 24, 26, 20, 42, 28, 30, 40, 32, 36, 38, and 34 or a complement to these sequences or a full-length gnd of the invention (either genomic or cDNA) are embodiments.
  • embodiments include a nucleic acid having at least one polymorphism described in Table 1 and less than or equal to 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450,
  • the nucleic acid embodiments comprise at least 12, 13, 14, 15, 16, 17, 18, or 19 consecutive nucleotides from SEQ. ID. Nos.: 22, 16, 18, 24, 26, 20, 42, 28, 30, 40, 32, 36, 38, and 34 or a complement to these sequences, as conditions dictate, so long as the fragment has at least one polymorphism described in Table 1. More preferably, the nucleic acid embodiments comprise at least 20-30 consecutive nucleotides. These nucleic acid oligomers have biotechnological and diagnostic use, e.g., in nucleotide acid hybridization assays, Southern and Northern Blot analysis, etc. and the prognosis of E. coli infection.
  • Some embodiments comprise recombinant constructs having all or part of the gnd genes disclosed in SEQ. ID. Nos. 22, 16, 18, 24, 26, 20, 42, 28, 30, 40, 32, 36, 38, and 34 or complements thereof
  • a recombinant construct can be capable of replicating autonomously in a host cell. Alternatively, the recombinant construct can become integrated into the chromosomal DNA of the host cell.
  • Such a recombinant polynucleotide comprises a polynucleotide of genomic or cDNA, of semi-synthetic or synthetic origin by virtue of human manipulation. Therefore, recombinant nucleic acids comprising sequences otherwise not naturally occurring are provided by embodiments of this invention.
  • nucleic acid embodiments of this invention can also be altered by mutation such as substitutions, additions, or deletions that provide for sequences encoding functionally equivalent molecules. Due to the degeneracy of nucleotide coding sequences, other DNA sequences that encode substantially the same 6-PGD amino acid sequence as depicted in SEQ. ID. Nos.: 23, 17, 19, 25, 27, 21, 43, 29, 31, 41, 33, 37, 39, and 35 can be used in some embodiments of the invention. These include, but are not limited to, nucleic acid sequences comprising all or portions of gnd depicted in SEQ. ID. Nos.: 22, 16, 18, 24, 26, 20, 42, 28, 30, 40, 32, 36, 38, and 34 or complements thereof that have been altered by the substitution of different codons that encode a functionally equivalent amino acid residue within the sequence, thus producing a silent change.
  • gnd-encoding nucleic acid sequences and their complementary sequences can be engineered so as to modify processing or expression.
  • the gnd genes depicted in SEQ. ID. Nos.: 22, 16, 18, 24, 26, 20, 42, 28, 30, 40, 32, 36, 38, and 34 can be combined with a promoter sequence and/or ribosome binding site, or a signal sequence may be inserted upstream of 6-PGD-encoding sequences to permit secretion of 6-PGD and thereby facilitate harvesting or bioavailability.
  • a given gnd nucleic acid can be mutated in vitro or in vivo, to create and/or destroy translation, initiation, and/or termination sequences, or to create variations in coding regions and/or form new restriction sites or destroy preexisting ones, or to facilitate further in vitro modification.
  • Any technique for mutagenesis known in the art can be used, including but not limited to, in vitro site-directed mutagenesis. (Hutchinson et al., J. Biol. Chem. 253:6551 (1978)).
  • nucleic acids encoding other proteins or domains of other proteins can be joined to nucleic acids encoding 6-PGD so as to create a fusion protein.
  • the resulting fusion proteins can be used as biotechnological tools to investigate the mobility of regions of the gnd/rfb cluster, for example, or to develop strain specific antibodies.
  • the nucleic acid embodiments can also be used as biotechnological tools for isolation procedures and diagnostic assays.
  • probes that complement these sequences can be designed and manufactured by oligonucleotide synthesis.
  • Preferred hybridization probes comprise at least one polymorphism found in Table 1. These probes can be used to screen cDNA or genomic libraries so as to isolate natural sources of the nucleic acid embodiments of the invention or can be used to identify specific strains or classes of strains of E. coli .
  • Nos.: 22, 16, 18, 24, 26, 20, 42, 28, 30, 40, 32, 36, 38, and 34 can be used to make oligonucleotide primers by conventional oligonucleotide synthesis for use in amplification strategies, such as PCR.
  • oligonucleotide primers can be used, for example, to isolate the nucleic acid embodiments of this invention by amplifying the sequences resident in genomic DNA or biological samples by using PCR or other enzyme-mediated nucleic acid amplification techniques. Such diagnostic and food or water screening techniques are discussed in greater detail below.
  • nucleic acids encoding the gnd sequences disclosed in SEQ. ID. Nos.: 22, 16, 18, 24, 26, 20, 42, 28, 30, 40, 32, 36, 38, and 34, or fragments thereof are manipulated using conventional techniques in molecular biology to create recombinant constructs that express 6-PGD or fragments of 6-PGD.
  • the discussion that follows describes some of these expression constructs and protein embodiments.
  • the 6-PGD polypeptide embodiments or derivatives thereof include but are not limited to, those molecules having as a primary amino acid sequence all of the amino acid sequence substantially as depicted in SEQ. ID. Nos.: 23, 17, 19, 25, 27, 21, 43, 29, 31, 41, 33, 37, 39, and 35 and fragments of these sequences at least three amino acids in length including altered sequences in which functionally equivalent amino acid residues are substituted for residues within the sequence resulting in a silent change.
  • Preferred fragments include at least one of the polymorphisms that can be deduced from Table 1, as described previously. It is to be understood that in the following discussion in this section, references made to 6-PGD in a general sense are intended to encompass the proteins and fragments thereof found in SEQ. ID. Nos. 23, 17, 19, 25, 27, 21, 43, 29, 31, 41, 33, 37, 39, and 35.
  • one or more amino acid residues within the 6-PGD polypeptide of SEQ. ID. Nos.: 23, 17, 19, 25, 27, 21, 43, 29, 31, 41, 33, 37, 39, and 35 or fragments thereof can be substituted by another amino acid of a similar polarity that acts as a functional equivalent, resulting in a silent alteration.
  • Substitutes for an amino acid within the sequence can be selected from other members of the class to which the amino acid belongs.
  • the non-polar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine.
  • the polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine and glutamine.
  • the positively charged (basic) amino acids include arginine, lysine, and histidine.
  • the negatively charged (acidic) amino acids include aspartic acid and glutamic acid.
  • the aromatic amino acids include phenylalanine, tryptophan, and tyrosine.
  • the 6-PGD fragments of the invention can be less than or equal to 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, and 468
  • the 6-PGD polypeptide of SEQ. ID. Nos.: 23, 17, 19, 25, 27, 21, 43, 29, 31, 41, 33, 37, 39, and 35 or fragments thereof or derivatives thereof are differentially modified during or after translation, e.g., by phosphorylation, glycosylation, cross-linking, acylation, proteolytic cleavage, linkage to an antibody molecule, membrane molecule, or other ligand. (Ferguson et al., Ann. Rev. Biochem. 57:285-320 (1988)).
  • the 6-PGD polypeptide of SEQ. ID. Nos.: 23, 17, 19, 25, 27, 21, 43, 29, 31, 41, 33, 37, 39, and 35 or fragments thereof are expressed in a cell line.
  • the sequences, constructs, vectors, clones, and other materials comprising the present invention can advantageously be in enriched or isolated form.
  • “enriched” means that the concentration of the material is at least about 2, 5, 10, 100, or 1000 times its natural concentration (for example), advantageously 0.01%, by weight, preferably at least about 0.1% by weight.
  • Enriched preparations from about 0.5%, 1%, 5%, 10%, and 20% by weight are also contemplated.
  • isolated requires that the material be removed from its original environment (e.g., the natural environment if it is naturally occuriang).
  • a naturally-occuring polynucleotide or polypeptide present in a living animal is not isolated, but the same polynucleotide or polypeptide, separated from some or all of the coexisting materials in the natural system, is isolated. It is also advantageous that the sequences be in purified form.
  • purified does not require absolute purity; rather, it is intended as a relative definition. Purification of starting material or natural material to at least one order of magnitude, preferably two or three orders, and more preferably four or five orders of magnitude is expressly contemplated.
  • nucleic acids containing the coding sequence for 6-PGD or fragments of 6-PGD are obtained and cloned into a suitable expression vector such that the coding region is operably linked to a heterologous promoter.
  • the nucleic acid encoding the protein or polypeptide to be expressed is operably linked to a promoter in an expression vector using conventional cloning technology.
  • the expression vector can be in any of the mammalian, yeast, amphibian, insect, parasite, or bacterial expression systems known in the art.
  • the following is provided as one exemplary method to express the proteins encoded by the nucleic acids described above.
  • the methionine initiation codon for the gene and the poly A signal of the gene are identified. If the nucleic acid encoding the polypeptide to be expressed lacks a methionine to serve as the initiation site, an initiating methionine can be introduced next to the first codon of the nucleic acid using conventional techniques.
  • this sequence can be added to the construct by, for example, splicing out the Poly A signal from pSG5 (Stratagene) using BglI and SalI restriction endonuclease enzymes and incorporating it into the mammalian expression vector pXT1 (Stratagene).
  • the vector 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 nucleic acid encoding the polypeptide to be expressed can be obtained by PCR from the bacterial vector using oligonucleotide primers complementary to the nucleic acid and containing restriction endonuclease sequences for Pst I incorporated into the 5′ primer and BglII at the 5′ end of the corresponding cDNA 3′ primer, taking care to ensure that the nucleic acid is positioned in frame with the poly A signal.
  • the purified fragment obtained from the resulting PCR reaction is digested with PstI, blunt ended with an exonuclease, digested with Bgl II, purified and ligated to pXT1, now containing a poly A signal and digested with BglII.
  • the ligated product is transfected into a suitable cell line, e.g., 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 ⁇ g/ml G418 (Sigma, St. Louis, Mo.). Preferably the expressed protein is released into the culture medium, thereby facilitating purification.
  • a suitable cell line e.g., mouse NIH 3T3 cells
  • Lipofectin Life Technologies, Inc., Grand Island, N.Y.
  • Positive transfectants are selected after growing the transfected cells in 600 ⁇ g/ml G418 (Sigma, St. Louis, Mo.).
  • the expressed protein is released into the culture medium, thereby facilitating purification.
  • Another embodiment utilizes the “Xpress system for expression and purification” (Invitrogen, San Diego, Calif.).
  • the Xpress system is designed for high-level production and purification of recombinant proteins from bacterial, mammalian, and insect cells.
  • the Xpress vectors produce recombinant proteins fused to a short N-terminal leader peptide that has a high affinity for divalent cations.
  • a nickel-chelating resin Invitrogen
  • the recombinant protein can be purified in one step and the leader can be subsequently removed by cleavage with enterokinase.
  • One preferred vector for the expression of 6-PGD and fragments of 6-PGD is the pBlueBacHis2 Xpress.
  • the pBlueBacHis2 Xpress vector is a Baculovirus expression vector containing a multiple cloning site, an ampicillin resistance gene, and a lac z gene.
  • the gnd nucleic acid, or portion thereof is cloned into the pBlueBacHis2 Xpress vector and SF9 cells are infected.
  • the expression protein is then isolated or purified according to the maufacturer's instructions.
  • Several other cultured cell lines having recombinant constructs or vectors comprising gnd or portions thereof are embodiments of the present invention and their manufacture would be routine given the present disclosure.
  • Proteins in the culture medium can also be separated by gel electrophoresis. The separated proteins are then detected using techniques such as Coomassie or silver staining or by using antibodies against the protein. Coomassie, silver staining, and immunolabeling of proteins are techniques familiar to those skilled in the art. If desired, the proteins can also be ammonium sulfate precipitated or separated based on size or charge prior to electrophoresis.
  • the protein encoded by gnd or portion thereof can also be purified using standard immunochromatography techniques.
  • a solution containing the protein such as the culture medium or a cell extract, is applied to a column having antibodies against the protein attached to the chromatography matrix.
  • the protein is allowed to bind the immunochromatography column. Thereafter, the column is washed to remove non-specifically bound proteins.
  • the specifically bound protein is then released from the column and recovered using standard techniques.
  • gnd or portion therof can be incorporated into expression vectors designed for use in purification schemes employing chimeric polypeptides.
  • the coding sequence of gnd or portion therof is inserted in frame with the gene encoding the other half of the chimera.
  • the other half of the chimera may be ⁇ -globin or a nickel binding polypeptide encoding sequence.
  • a chromatography matrix having antibody to ⁇ -globin or nickel attached thereto is then used to purify the chimeric protein.
  • Protease cleavage sites can be engineered between the ⁇ -globin gene or the nickel binding polypeptide and the gnd cDNA such as enterokinase.
  • the two polypeptides of the chimera can be separated from one another by protease digestion.
  • One useful expression vector for generating ⁇ -globin chimerics is pSG5 (Stratagene), which encodes rabbit ⁇ -globin. Intron II of the rabbit ⁇ -globin gene facilitates splicing of the expressed transcript, and the polyadenylation signal incorporated into the construct increases the level of expression.
  • these molecules can be prepared by chemical synthesis methods (such as solid phase peptide synthesis) using methods known in the art such as those set forth by Merrifield et al., J. Am. Chem. Soc. 85:2149 (1964), Houghten et al., Proc. Natl. Acad. Sci. USA, 82:51:32 (1985), and Stewart and Young (solid phase peptide synthesis, Pierce Chem Co., Rockford, Ill. (1984).
  • Such polypeptides can be synthesized with or without a methionine on the amino terminus.
  • 6-PGD and fragments of 6-PGD can be oxidized using methods set forth in these references to form disulfide bridges.
  • 6-PGD and fragments of 6-PGD can be employed as biologically active or immunological substitutes for natural, purified 6-PGD and fragments of 6-PGD.
  • Analogs of 6-PGD or fragments of 6-PGD include small molecules modeled on the peptides. These small molecules are also known as peptidomimetics.
  • a peptidomimetic is a molecule that has the same effect as a peptide, usually because it has the same critical ‘shape’, but is not itself a peptide and hence is not broken down by proteases and is cheaper to produce.
  • peptidomimetics that structurally and/or functionally resemble 6-PGD or fragments of 6-PGD can be made and evaluated for their ability to interact with 6-PGD in a 6-PGD characterization assay (e.g., inhibit the function of natural 6-PGD or fragment thereof) or induce an immune response in a subject.
  • 6-PGD characterization assay e.g., inhibit the function of natural 6-PGD or fragment thereof
  • Several approaches to make peptidomimetics that resemble polypeptides are described in the art. A vast number of methods, for example, can be found in U.S. Pat. Nos. 5,288,707; 5,552,534; 5,811,515; 5,817,626; 5,817,879; 5,821,231; and 5, 874,529, herein incorporated by reference in their entirety.
  • the isolated or purified proteins can be used to generate antibodies and tools for identifying agents that interact with 6-PGD and fragments of 6-PGD.
  • Antibodies that recognize 6-PGD and fragments of 6-PGD have many uses including, but not limited to, biotechnological applications, therapeutic/prophylactic applications, and diagnostic applications.
  • Such antibodies include, but are not limited to, polyclonal, monoclonal, chimeric, single chain, Fab fragments and fragments produced by a Fab expression library.
  • various hosts including goats, rabbits, rats, mice, etc can be immunized by injection with 6-PGD or any portion, fragment or oligopeptide that retains immunogenic properties.
  • various adjuvants can be used to increase immunological response.
  • adjuvants include but are not limited to Freund's, mineral gels such as aluminum hydroxide, and surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, and dinitrophenol.
  • BCG Bacillus Calmette-Guerin
  • Corynebacterium parvum are potentially useful adjuvants.
  • Peptides used to induce specific antibodies can have an amino acid sequence consisting of at least three amino acids, preferably at least 10 or 15 amino acids that include a polymorphism as can be deduced from Table 1.
  • Preferred antibodies include ones that specifically bind to a polypeptide having the T218I polymorphism or a nucleic acid having either the C653T or G654C but not 6-PGD or gnd that has the Thr218 or thymine or cytosine polymorphisms at nucleic acid positions 653 and 654, respectively.
  • preferred antibodies recognize an epitope that uniquely identifies the Iso218 polymorphism but not the Thr218 polymorphism or vice versa or the antibodies recognize an epitope that uniquely identifies a cytosine at nucleic acid position 653 and/or a guanine at nucleic acid position 654 or a thymine at position 653 and/or a cytosine at nucleic acid position 654.
  • short stretches of amino acids encoding fragments of 6-PGD are fused with those of another protein such as keyhole limpet hemocyanin and antibody is produced against the chimeric molecule.
  • While antibodies capable of specifically recognizing 6-PGD can be generated by injecting into mice synthetic 3-mer, 10-mer, and 15-mer peptides that correspond to a protein sequence of 6-PGD, a more diverse set of antibodies can be generated by using recombinant or purified 6-PGD and fragments of 6-PGD.
  • substantially pure 6-PGD or a fragment of 6-PGD is isolated from a transfected or transformed cell.
  • concentration of the polypeptide 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 polypeptide of interest can then be prepared as follows:
  • Monoclonal antibodies to 6-PGD or a fragment of 6-PGD can be prepared using any technique that provides for the production of antibody molecules by continuous cell lines in culture. These include but are not limited to the hybridoma technique originally described by Koehler and Milstein (Nature 256:495-497 (1975), the human B-cell hybridoma technique (Kosbor et al. Immunol Today 4:72 (1983); Cote et al Proc Natl Acad Sci 80:2026-2030 (1983), and the EBV-hybridoma technique Cole et al. Monoclonal Antibodies and Cancer Therapy, Alan R. Liss Inc, New York N.Y., pp 77-96 (1985).
  • Antibodies can also be produced by inducing in vivo production in the lymphocyte population or by screening recombinant immunoglobulin libraries or panels of highly specific binding reagents as disclosed in Orlandi et al., Proc Natl Acad Sci 86: 3833-3837 (1989), and Winter G. and Milstein C; Nature 349:293-299 (1991).
  • Antibody fragments that contain specific binding sites for 6-PGD can also be generated.
  • fragments include, but are not limited to, the F(ab′) 2 fragments that can be produced by pepsin digestion of the antibody molecule and the Fab fragments that can be generated by reducing the disulfide bridges of the F(ab′) 2 fragments.
  • Fab expression libraries can be constructed to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity. (Huse W. D. et al. Science 256:1275-1281 (1989)).
  • monoclonal antibodies to 6-PGD of fragments thereof are made as follows. Briefly, a mouse is repetitively inoculated with a few micrograms of the selected protein or peptides derived therefrom 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 in the presence 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.
  • HAT media aminopterin
  • 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 derivative 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.
  • Polyclonal antiserum containing antibodies to heterogenous epitopes of a single protein can be prepared by immunizing suitable animals with the expressed protein or peptides derived therefrom 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 others and may require the use of carriers and adjuvant.
  • 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 intraderinal 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).
  • 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, for example, Ouchterlony, O. et al., Chap. 19 in: Handbook of Experimental Immunology D. Wier (ed) Blackwell (1973). Plateau concentration of antibody is usually in the range of 0.1 to 0.2 mg/ml of serum (about 12 ⁇ M). 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, 2d Ed.
  • Antibody preparations prepared according to either protocol are useful in quantitative immunoassays that determine concentrations of antigen-bearing substances in biological samples; they are also used semi-quantitatively or qualitatively (e.g., in diagnostic embodiments that identify the presence of 6-PGD in biological samples).
  • the diagnostics and screening methods of the invention can be classified according to whether the embodiment is a nucleic acid or protein based assay.
  • These assays preferably identify and distinguish the strain and extent of pathogenicity of an E. coli present in a biological sample (e.g., a sample from a patient, food source, or liquid source) by detecting the presence of one or more polymorphisms at the gnd locus. That is, several of the diagnostic and screening embodiments focus on the detection of one or more polymorphisms provided in Table 1 or that can be deduced from Table 1 in a nucleic acid or protein sample.
  • kits that incorporate the reagents and methods described in the following embodiments so as to allow for the rapid detection and identification of highly pathogenic O157:H7 E. coli are contemplated.
  • the diagnostic kits can include a nucleic acid probe or an antibody or combinations thereof, which specifically detect the one or more polymorphisms described in Table 1 or that can be deduced from Table 1.
  • the detection component of these kits will typically be supplied in combination with one or more of the following reagents.
  • a support capable of absorbing or otherwise binding DNA, RNA, or protein will often be supplied. Available supports include membranes of nitrocellulose, nylon or derivatized nylon that can be characterized by bearing an array of positively charged substituents.
  • One or more restriction enzymes, control reagents, buffers, amplification enzymes, and non-human polynucleotides like calf-thymus or salmon-sperm DNA can be supplied in these kits.
  • Useful nucleic acid-based diagnostic techniques include, but are not limited to, direct DNA sequencing, Southern Blot analysis, single-stranded confirmation analysis (SSCA), RNase protection assay, dot blot analysis, nucleic acid amplification, and combinations of these approaches.
  • the starting point for these analysis is isolated or purified DNA from a biological sample. Most simply, fecal material is obtained from a subject to be tested or a food or water sample is provided.
  • the bactrerial DNA is extracted from the sample and amplified by a DNA amplification technique such as PCR using primers that correspond to regions of the gnd locus and/or the gnd/rfb cluster, preferably regions having a polymorphism listed in Table 1.
  • SSCA single-stranded confirmation polymorphism assay
  • CDGE clamped denaturing gel electrophoresis
  • HA heteroduplex analysis
  • CMC chemical mismatch cleavage
  • a rapid preliminary analysis to detect polymorphisms and DNA sequences can be performed by looking at a series of Southern Blots of DNA cut with one or more restriction enzymes preferably with a large number of restriction enzymes. Each block contains lanes of DNA from uninfected individuals and the DNA to be tested. Southern Blots displaying hybridizing fragments when probed with sequences corresponding to one or more polymorphisms described in Table 1 indicate the presence of the specific E. coli strain.
  • the detection of point mutations can also be accomplished by amplifying the DNA directly from the sample using primers corresponding to the regions flanking one or more polymorphisms described in Table 1 by standard PCR techniques and sequencing the amplicons, as will be discussed in greater detail below.
  • nucleic acid-based methods for confirming the presence of one or more polymorphisms described in Table 1 are provided below. Provided for exemplary purposes only and not intended to limit any aspect of the invention, these methods include:
  • DGGE denaturing gradient gel electrophoresis
  • TTGE temporal temperature gradient gel electrophoresis
  • SSCA SSCA
  • TTGE TTGE
  • RNase protection involves cleavage of the mutant polynucleotide into two or more smaller fragments.
  • DGGE detects differences in migration rates of sequences compared to less pathogenic strain gnd sequences, using a denaturing gradient gel.
  • ASOs allele-specific oligonucleotide assay
  • an oligonucleotide is designed that detects a specific sequence, and an assay is performed by detecting the presence or absence of a hybridization signal.
  • the protein binds only to sequences that contain a nucleotide mismatch in a heteroduplex between polymorphic and non-polymorphic sequences.
  • Mismatches in this sense of the word refers to hybridized nucleic acid duplexes in which the two strands are not 100% complementary.
  • the lack of total homology results from the presence of one or more polymorphisms in an amplicon obtained from a biological sample, for example, that has been hybridized to a non-polymorphic strand.
  • Mismatched detection can be used to detect point mutations in the gnd gene or in its mRNA product. While these techniques are less sensitive than sequencing, they are easily performed on a large number of biological samples and are amenable to array technology.
  • the nucleic acid embodiments of the present invention are attached to a support in an ordered array wherein a plurality of nucleic acid probes are attached to distinct regions of the support that do not overlap with each other.
  • such an ordered array is designed to be “addressable” where the distinct locations of the probe are recorded and can be accessed as part of an assay procedure.
  • addressable nucleic acid arrays comprise a plurality of nucleic acid probes that complement a plurality of polymorphisms listed in Table 1. These probes are joined to a support in different known locations. The knowledge of the precise location of each nucleic acid probe makes these “addressable” arrays particularly useful in binding assays.
  • the nucleic acids from a preparation of several biological samples are then labeled by conventional approaches (e.g., radioactivity or fluorescence) and the labeled samples are applied to the array under conditions that permit hybridization. If a nucleic acid in the samples hybridizes to a probe on the array, then a signal will be detected at a position on the support that corresponds to the location of the hybrid. Since the identity of each labeled sample is known and the region of the support on which the labeled sample was applied is known, an identification of the presence and polymorphic variant (i.e., the strain of E. coli ) can be rapidly determined. Conventional methods in DNA amplification, as will be discussed below, can also be incorporated so as to detect the presence of less than 10 bacterial cells. These approaches are easily automated using technology known to those of skill in the art of high throughput diagnostic or detection analysis.
  • conventional approaches e.g., radioactivity or fluorescence
  • Nucleic acids present in biological samples can be disposed on a support so as to create an addressable array.
  • the samples are disposed on the support at known positions that do not overlap.
  • the presence of nucleic acids having a desired polymorphism in each sample is determined by applying labeled nucleic acid probes that complement nucleic acids that encode the polymorphism and detecting the presence of a signal at locations on the array that correspond to the positions at which the biological samples were disposed. Because the identity of the biological sample and its position on the array is known, the identification of the polymorphic variant can be rapidly determined.
  • conventional methods in DNA amplification can be incorporated so as to detect the presence of very few bacterial cells.
  • any addressable array technology known in the art can be employed with this aspect of the invention.
  • One particular embodiment of polynucleotide arrays is known as GenechipsTM, and has been generally described in U.S. Pat. No. 5,143,854; PCT publications WO 90/15070 and 92/10092. These arrays are generally produced using mechanical synthesis methods or light directed synthesis methods, which incorporate a combination of photolithographic methods and solid phase oligonucleotide synthesis. (Fodor et al., Science, 251:767-777, (1991)).
  • VLSIPSTM Very Large Scale Immobilized Polymer Synthesis
  • a wide variety of labels and conjugation techniques are known by those skilled in the art and can be used in various nucleic acid assays.
  • There are several ways to produce labeled nucleic acids for hybridization or PCR including, but not limited to, oligolabeling, nick translation, end-labeling, or PCR amplification using a labeled nucleotide.
  • a nucleic acid encoding 6-PGD, or any portion of it can be cloned into a vector for the production of an mRNA probe.
  • RNA polymerase such as T7, T3 or SP6 and labeled nucleotides.
  • RNA polymerase such as T7, T3 or SP6 and labeled nucleotides.
  • Suitable reporter molecules or labels include those radionuclides, enzymes, fluorescent, chemiluminescent, or chromogenic agents, as well as, substrates, cofactors, inhibitors, magnetic particles and the like.
  • RNA mismatch cleavage technique that is amenable to array technology
  • the method involves the use of a labeled riboprobe which is complementary to a gnd sequence having a polymorphism (e.g., the C653T and G654C polymorphism that distinguishes highly pathogenic O157:H7 and O55:H7 from less pathogenic E. coli strains).
  • the riboprobe and either mRNA or DNA isolated and amplified from a biological sample are annealed (hybridized) and subsequently digested with the enzyme RNase A, which is able to detect mismatches in a duplex RNase structure.
  • RNA product will be seen which is much smaller than the full length duplex RNA for the riboprobe and the mRNA or DNA.
  • complements to the riboprobe can be dispersed on an array and stringently probed with the products from the Rnase A digestion after denaturing any remaining hybrids.
  • RNA probes can be used to detect mismatches, through enzymatic or chemical cleavage. See, e.g., Cotton, et al., Proc. Natl. Acad. Sci.
  • mismatches can be detected by shifts in the electrophoretic ability of mismatched duplexes relative to matched duplexes.
  • riboprobes or DNA probes the cellular mRNA or DNA that corresponds to regions of gnd containing polymorphisms can be amplified by PCR before hybridization. DNA sequences isolated from biological samples which have been amplified by use of PCR can then be screened using allele-specific probes. These probes are nucleic acid oligomers, each of which contains a region including one or more polymorphisms present in Table 1.
  • one oligomer may be about 30 nucleotides in length and corresponds to the C653T and G654C polymorphism.
  • PCR amplification products can be screened to identify the presence of specific polymorphisms.
  • the most definitive test for the presence of a highly pathogenic E. coli in a sample is to directly compare nucleotide or protein sequences isolated from a biological sample with one or more of the polymorphisms present in Table 1.
  • PCR techniques are familiar to those skilled in the art. For a review of PCR technology, see Molecular Cloning to Genetic Engineering White, B. A. Ed. in Methods in Molecular Biology 67: Humana Press, Totowa (1997), the disclosure of which is incorporated herein by reference in its entirety and the publication entitled “PCR Methods and Applications” (1991, Cold Spring Harbor Laboratory Press), the disclosure of which is incorporated herein by reference in its entirety.
  • RT-PCR reverse transcribe mRNA into cDNA followed by PCR
  • primers on either side of the sequence to be amplified are added to a suitably prepared nucleic acid sample along with dNTPs and a thermostable polymerase such as Taq polymerase, Pfu polymerase, or Vent polymerase.
  • a thermostable polymerase such as Taq polymerase, Pfu polymerase, or Vent polymerase.
  • the nucleic acid in the sample is denatured and the primers are specifically hybridized to complementary nucleic acid sequences in the sample.
  • the hybridized primers are extended. Thereafter, another cycle of denaturation, hybridization, and extension is initiated. The cycles are repeated multiple times to produce an amplified fragment containing the nucleic acid sequence between the primer sites.
  • PCR has further been described in several patents including U.S. Pat. Nos. 4,683,195, 4,683,202 and 4,965,188, the disclosure of which is incorporated herein by reference in its entirety.
  • the primers are selected to be substantially complementary to a portion of the sequence of gnd DNA or mRNA and a portion of the sequence that complements the sequence of gnd DNA or mRNA, thereby allowing the sequences between the primers to be amplified.
  • the length of the primers for use with this aspect of the invention is identical to most of the lengths of the nucleic acid embodiments provided previously.
  • primer length can be less than or equal to 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, 6
  • primers are 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 and 30 nucleotides in length. Shorter primers tend to lack specificity for a target nucleic acid sequence and generally require cooler temperatures to form sufficiently stable hybrid complexes with the template. Longer primers are expensive to produce and can sometimes self-hybridize to form hairpin structures.
  • the formation of stable hybrids depends on the melting temperature (Tm) of the DNA. The Tm depends on the length of the primer, the ionic strength of the solution and the G+C content. The higher the G+C content of the primer, the higher is the melting temperature because G:C pairs are held by three H bonds whereas A:T pairs have only two.
  • the G+C content of the amplification primers of the present invention preferably ranges between 10 and 75%, more preferably between 35 and 60%, and most preferably between 40 and 55%.
  • the appropriate length for primers under a particular set of assay conditions may be empirically determined by one of skill in the art.
  • the spacing of the primers determines the length of the segment to be amplified.
  • amplified segments carrying nucleic acid sequence encoding fragments of 6-PGD can range in size from at least about 25 bp to 35 kb.
  • Amplification fragments from 25-1407 bp are typical, fragments from 50-1000 bp are preferred and fragments from 100-600 bp are highly preferred.
  • amplification primers for the gnd genes of the invention can be of any sequence that allows for specific amplification of a region of the gnd genes disclosed in SEQ. ID. Nos. 22, 16, 18, 24, 26, 20, 42, 28, 30, 40, 32, 36, 38, and 34 and can, for example, include modifications such as restriction sites to facilitate cloning.
  • highly pathogenic O157:H7 E. coli are identified and differentiated from less pathogenic E. coli by employing PCR amplification with two sets of primers.
  • a first set of primers is designed to produce an amplicon containing at least the C653T and G654C polymorphisms, which distinguish the highly pathogenic O157:H7 and O55:H7 E. coli from less pathogenic strains.
  • a second set of primers is designed to produce an amplicon that is unique to the O55:H7 parasite, e.g., the primer pair: 5′GCGTTCTTAAAGAGTCCTGC3′ (SEQ. ID. No. 13) and 5′TGCCCGCTACATCTCCTC3′ (SEQ.
  • the presence of a 6-PGD protein of the invention can also be detected by using conventional assays.
  • monoclonal antibodies immunoreactive with a polymorphism found on a specific 6-PGD sequence can be used to screen biological samples for the presence of a particular strain of E. coli and can be used to distinguish one strain from another. Because the T218I polymorphism can distinguish highly pathogenic O157:H7 and O55:H7 from less pathogenic O157:H7, diagnostic and screening assays that comprise reagents and methods that involve the detection of the presence or absence of the T218I polymorphism are preferred embodiments.
  • diagnostic assays can also include a reagent that specifically differentiates the O55:H7 and O157:H7 parasites, for example, an antibody directed to an epitope found in a region of the O55:H7 6-PGD protein that is not homologous to the 6-PGD protein from an O157:H7 parasite.
  • a reagent that specifically differentiates the O55:H7 and O157:H7 parasites for example, an antibody directed to an epitope found in a region of the O55:H7 6-PGD protein that is not homologous to the 6-PGD protein from an O157:H7 parasite.
  • Such immunological assays can be done in many convenient formats.
  • antibodies are used to immunoprecipitate the 6-PGD of the invention from solution and, in another embodiment, antibodies are used to react with 6-PGD on Western or Immuneblots of a polyacrylamide gel.
  • ELISA enzyme-linked immunosorbant assays
  • RIA radioimmunoassays
  • IRMA immunoradiometric assays
  • IEMA immunoenzymatic assays
  • Exemplary sandwich assays are described by David et al., in U.S. Pat. Nos. 4,376,110 and 4,486,530, hereby incorporated by reference.
  • antibodies of the present invention are attached to a support in an ordered array wherein a plurality of antibodies are attached to distinct regions of the support that do not overlap with each other.
  • the protein-based arrays are ordered arrays that are designed to be “addressable” such that the distinct locations are recorded and can be accessed as part of an assay procedure.
  • addressable antibody arrays comprise a plurality of antibodies that recognize the 6-PGD polymorphisms that can be deduced from Table 1. These probes are joined to a support in different known locations. The knowledge of the precise location of each probe makes these “addressable” arrays particularly useful in binding assays.
  • an addressable array can comprise a support having several regions to which are joined a plurality of antibody probes that recognize the 6-PGD polymorphisms that can be deduced from Table 1. Proteins obtained from biological samples are labeled by conventional approaches (e.g., radioactivity, calorimetrically, or fluorescently) and the labeled samples are applied to the array under conditions that permit binding.
  • a protein in the sample binds to an antibody probe on the array, then a signal will be detected at a position on the support that corresponds to the location of the antibody-protein complex. Since the identity of each labeled sample is known and the region of the support on which the labeled sample was applied is known, an identification of the presence, concentration, and/or expression level is rapidly determined. That is, by employing labeled standards of a known concentration of 6-PGD, an investigator can accurately determine the protein concentration of 6-PGD in a sample and from this information can assess the expression level of 6-PGD. Conventional methods in densitometry can also be used to more accurately determine the concentration or expression level of 6-PGD. These approaches are easily automated using technology known to those of skill in the art of high throughput diagnostic analysis.
  • Proteins present in biological samples can be disposed on a support so as to create an addressable array.
  • the protein samples are disposed on the support at known positions that do not overlap.
  • the presence of a protein encoding a specific form of 6-PGD in each sample is then determined by applying labeled antibody probes that recognize epitopes of 6-PGD that correspond to the polymorphisms that can be deduced from Table 1 and detecting a signal at locations on the array that correspond to the positions at which the biological samples were disposed. Because the identity of the biological sample and its position on the array is known, an identification of the presence, concentration, and/or expression level of a particular 6-PGD can be rapidly determined.
  • an investigator can accurately determine the concentration of 6-PGD in a sample and from this information can assess the expression level of 6-PGD.
  • Conventional methods in densitometry can also be used to more accurately determine the concentration or expression level of 6-PGD.
  • These approaches are also easily automated using technology known to those of skill in the art of high throughput diagnostic analysis.
  • any addressable array technology known in the art can be employed with this aspect of the invention and display the protein arrays on the chips in an attempt to maximize antibody binding patterns and diagnostic information.
  • the presence or detection of one or more polymorphisms in 6-PGD can provide a diagnosis of a subject's disease or indicate the contamination of a food or water supply.
  • Additional embodiments include the preparation of diagnostic kits comprising detection components such as antibodies specific for one or more polymorphisms of 6-PGD.
  • the detection component will typically be supplied in combination with one or more of the following reagents.
  • a support capable of absorbing or otherwise binding RNA or protein will often be supplied. Available supports for this purpose include, but are not limited to, membranes of nitrocellulose, nylon or derivatized nylon that can be characterized by bearing an array of positively charged substituents, and GenechipsTM or their equivalents.
  • One or more enzymes such as Reverse Transcriptase and/or Taq polymerase, can be furnished in the kit, as can dNTPs, buffers, or non-human polynucleotides like calf-thymus or salmon-sperm DNA. Results from the kit assays can be interpreted by a healthcare provider or a diagnostic laboratory. Alternatively, diagnostic kits are manufactured and sold to private individuals for self-diagnosis.
  • restriction site “A” is present in fragment “B-G” (i.e., “A 1 , A 2 , and A 3 ”), defined below.
  • B corresponds to the left-hand border of a pathogenicity or antigenicity island and “G” corresponds to the right hand border of this element.
  • a 1 is the first restriction site A site to the left of B
  • a 2 is the first restriction site A site to the right of G.
  • the BG island flanking this sequence can be determined by using inverse PCR.
  • Primers “C”, “D”, “E”, and “F” are derived from the sequence of the unique pathogenicity/antigenicity island. Actual sequence is derived from the raw data, depicted in the 5′ to 3′ direction, as indicated under the line shown in FIG. 3. The primers are in the same (primer D, primer F) or opposite orientation (primer C and primer E).
  • E. coli DNA is digested to completion with enzyme A. Ligase is then added and the resulting fragments are re-circularized. Primers are added in separate tubes with a heat stable polymerase and PCR is conducted to obtain amplicons. The amplicons are cloned and sequenced. This approach identifies sequences beyond the 5′ and 3′ ends of the known pathogenicity/antigenicity islands and primers derived from these sequences are used to amplify this region in a variety of pathogens and non-pathogens, as was performed for the gnd allele. The resulting amplicons are then sequenced to identify differentiating polymorphisms.

Abstract

The present invention relates generally to the field of microbiology and food sciences. More particularly, the inventor has discovered several polynucleotide sequences encoding the gnd gene and corresponding 6-phosphogluconate dehydrogenase (6-PGD) proteins from different strains of Escherichia Coli and polymorphic sequences therein. Novel biotechnological tools, diagnostics, and food screening techniques are provided.

Description

    CROSS-REFERENCE TO RELATED APPLICATIONS
  • This application is a continuation of International Application number PCT/US99/29149 and claims priority to said International Application and U.S. Provisional Patent Application No. 60/111,493, filed Dec. 8, 1998, both of which are hereby expressly incorporated by reference in their entireties.[0001]
    • FIELD OF THE INVENTION
  • The present invention relates generally to the field of microbiology and food sciences. More particularly, the inventor has discovered the gnd gene and corresponding 6-phosphogluconate dehydrogenase (6-PGD) protein from fourteen different strains of [0002] Escherichia coli and polymorphic sequences therein. Novel biotechnological tools, diagnostics, and food screening techniques are provided.
    • BACKGROUND OF THE INVENTION
  • [0003] Escherichia coli O157:H7 is an exceptionally virulent food borne, human pathogen that causes a spectrum of illness, including asymptomatic and post-symptomatic carriage, mild diarrhea, bloody diarrhea/hemorrhagic colitis, and the postdiarrheal, potentially lethal, hemolytic uremic syndrome (HUS). (Wilson et al., J Infect Dis, 174:1021-1027 (1996); (Karch et al., J Clin Microbiol, 33:1602-1605 (1995); (Rodrigue et al., J Infect Dis, 172:1122-1125 (1995); (Riley et al., N Engl J Med, 308:681-685 (1983); (Karmali et al., Lancet, 1:619-620 (1983); Neill et al., Arch Intern Med, 145:2215-2217 (1985); Neill et al., Pediatrics, 80:37-40 (1987); and Tarr et al., J Infect Dis, 162:553-556 (1990)). While other E. coli strains are considered in some contexts to be pathogens, the excessive pathogenicity of E. coli O157:H7 is a well recognized distinguishing feature.
  • HUS is defined as a triad of non-immune microangiopathic hemolytic anemia, thrombocytopenia, and acute renal failure. HUS is chiefly a disorder of children under age 10, however, the elderly are also susceptible to severe complications of [0004] E. coli O157:H7 gastrointestinal infections. (Martin et al., N Engl J Med, 323:1161-1167 (1990); Siegler et al., Pediatrics, 94:35-40 (1994); Tarr and Hickman, Pediatrics, 80:41-45 (1987); Tarr et al., Am J Epidemiol, 129:582-586 (1989); Tarr et al., J Infect Dis, 162:553-556 (1990); (Carter et al., N Engl J Med, 317:1496-1500 (1987); and Ryan et al., J Infect Dis, 154:631-638 (1986)).
  • HUS follows gastrointestinal infection with [0005] E. coli O157:H7 in approximately 10-15% of pediatric cases. (Bell et al., JAMA, 272:1349-1353 (1994) and Bell et al., Pediatrics, 100:E12 (1997)). Approximately three-quarters of children with HUS require blood transfusions and approximately one-half require dialysis. (Tarr et al., Am J Epidemiol, 129:582-586 (1989); (Brandt et al., J Pediatr, 125:519-526 (1994); and Tarr et al., Am J Epidemiol, 129:582-586 (1989)). Despite recognition of O157:H7 infection and the use of modem pediatric intensive care, about 5-10% of those infected die. (Brandt et al., J Pediatr, 125:519-526 (1994) and Tarr et al., Am J Epidemiol, 129:582-586 (1989)). Investigation of O157:H7 outbreaks have provided evidence that the infectious dose is low. For example, limited exposure to a municipal lake in Portland, Oreg., wherein the levels of E. coli O157:H7 were undetectable, was sufficient to produce disease in visitors. (Keene et al., N Engl J Med, 331:579-584 (1994)) and during a salami-associated outbreak in the Pacific Northwest in 1994, investigators concluded that the people who became ill had consumed between 2 and 45 viable E. coli O157:H7 organisms. (Tilden et al., Am J Public Health, 86:1142-1145 (1996)).
  • [0006] E. coli O157:H7 is often found in food and environmental vehicles that do not always undergo an efficient bacterial killing process. Large outbreaks have been caused by the interstate dissemination of contaminated ground beef that was under cooked (Bell et al., JAMA, 272:1349-1353 (1994) and Riley et al., N Engl J Med, 308:681-685 (1983)); salted, fermented, but uncooked salami (Tilden et al., Am J Public Health, 86:1142-1145 (1996)); municipal (Swerdlow et al., Ann Intern Med, 117:812-819 (1992)) and swimming (Keene et al., N Engl J Med, 331:579-584 (1994)) water; unpasteurized apple juice (Anonymous, Morb Mortal Wkly Rep, 45:975 (1996)); unpasteurized milk (Keene et al., J Infect Dis, 176:815-818 (1997)); and lettuce (Ackers et al., J Infect Dis, 177:1588-1593 (1998)). Improper food handling has been reported to be a significant factor associated with human infection. (Mead et al., Arch Intern Med, 157:204-208 (1997)).
  • [0007] E. coli O157:H7 has not been shown to possess a capsular polysaccharide but it expresses an O side chain antigen designated 157, which consists of repeating tetrasaccharide units of variable length. These tetrasaccharide units comprise the antigenic O157 lipopolysaccaride (LPS). In contrast to other E. coli strains, O157:H7 fails to ferment sorbitol after overnight culture on MacConkey agar into which sorbitol rather than lactose is incorporated as the carbon source. (Wells et al., J Clin Microbiol, 18:512-520 (1983); March et al., J Clin Microbiol, 23:869-872 (1986)). E. coli O157:H7 also fails to produce β-glucuronidase, another metabolic distinguishing factor. (Ratnam et al., J Clin Microbiol, 26:2006-2012 (1988)). Sorbitol non-fermenting E. coli almost always express the H7 flagellar antigen, though occasional sorbitol non-fermenting E. coli O157 strains recovered in the United States do not express the H7 antigen. (Slutsker et al., Ann Intern Med, 126:505-513 (1997)). Another variant of E. coli O157:H7 has been found in Germany and Czech Republic, which expresses the O157 antigen, but are non-motile pathogens that ferment sorbitol. (Bielaszewska et al., J Clin Microbiol, 36:2135-2137 (1998); Gunzer et al., J Clin Microbiol, 30:1807-1810 (1992)). Such sorbitol non-fermenting E. coli O157 variants are difficult to identify by using the sorbitol MacConkey agar screening technique.
  • Current diagnostic approaches involve monitoring the growth characteristics of cultured [0008] E. coli on MacConkey agar, as described above, and utilizing a seriological agent specific for O157 LPS. That is, organisms with an appearance typical of E. coli on sorbitol MacConkey agar, that fail to ferment sorbitol, react with a serologic reagent specific for the O157 LPS side chain but fail to react with a control (negative) reagent are considered to be Shiga-toxigenic, and, presumably, pathogenic, E. coli O157:H7. The identification of the H7 antigen and the toxinogenic phenotype are not necessary for clinical purposes because sorbitol non-fermenting E. coli that are non mucoid, react with a specific O157 antigen determining reagent and do not react with a negative control reagent are almost always toxigenic. (Strockbine et al., “Overview of detection and subtyping methods,” Escherichia coli O157:H7 and other Shiga toxin-producing E. coli, Chapter 33, Kaper and O'Brien, eds., Washington, D.C.: ASM Press, 1998:331-356 and Tarr, “Shiga toxin-producing Escherichia coli infections: challenges and opportunities,” Escherichia coli O157:H7 and other Shiga toxin-producing E. coli, Chapter 39, Kaper and O'Brien, eds., Washington, D.C.: ASM Press, 1998:393-402).
  • Alternate diagnostic approaches have been recently developed. One approach involves the detection of the presence of released Shiga-toxin. These tests either exploit the ability of Shiga-toxins to bind to a glycosphingolipid ligand (globotriaosylceramide) (Basta et al., [0009] J Clin Microbiol, 27:1617-1622 (1989)) (Biocarb, Gaithersburg, Md.) or employ an enzyme immunoassay (Meridian Diagnostics, Cincinnati, Ohio). (Kehl et al., J Clin Microbiol, 35:2051-2054 (1997)); Park et al., Diag Microbiol Infect Dis, 26:69-72 (1996)). These tests have the advantage that they detect Shiga toxigenic E. coli besides E. coli O157:H7. Several diagnostic tests also involve the use of probes or primers to detect sequences of O157:H7 through hybridization, enzyme cleavage, or Polymerase Chain Reaction (PCR). (See e.g., U.S. Pat Nos. 5,738,995; 5,747,257; and 5,756,293).
  • A variety of techniques to identify excessively pathogenic [0010] E coli in food have also been developed. (Bennett et al., Lett Appl Microbiol, 22:237-243 (1996); Bennett et al., Lett Appl Microbiol, 20:375-379 (1995); Blanco et al., Microbiologia, 12:385-394 (1996); Bolton et al., Lett Appl Microbiol, 23:317-321 (1996); Doyle and Schoeni, Appl Environ Microbiol, 53:2394-2396 (1987); Feldsine et al., J AOAC Int, 80:517-529 (1997); Feldsine et al., J AOAC Int, 80:530-543 (1997); Feldsine et al., J AOAC Int, 80:43-48 (1997); Feldsine et al., J AOAC Int, 80:37-42 (1997); Jinneman et al., J Food Protect, 58:722-726 (1995); Johnson et al., Appl Environ Microbiol, 61:386-388 (1995); Kim and Doyle, Appl Environ Microbiol, 58:1764-1767 (1992); Notermans et al., Int J Food Microbiol, 13:31-40 (1991); Okrend et al., J Food Protect, 53:936-940 (1990); Padhye and Doyle, Appl Environ Microbiol, 57:2693-2698 (1991); Pawelzik, Acta Microbiol Hung, 38:315-320 (1991); Ratnan and March, Can Med Assoc J, 134:43-46 (1986); Read et al., Epidemiol Infect, 105:11-20 (1990); Sequel, Can Med Assoc J, 143:519-521 (1990); Tortorello and Stewart, Appl Environ Microbiol, 60:3553-3559 (1994); Vernozy-Rozand et al., Revue de Medecine Veterinaire, 149:239-244 (1998); Vernozy-Rozand et al., Revue de Medecine Veterinaire, 148:879-882 (1997); Vemozy-Rozand et al., Lett Appl Microbiol, 25:442-446 (1997); Willshaw et al., J Appl Bacteriol, 75:420-426 (1993); Yu and Bruno, Appl Environ Microbiol, 62:587-592 (1996)). Many of these techniques include a hydrophobic grid membrane filter (Doyle and Schoeni, Appl Environ Microbiol, 53:2394-2396 (1987)), a dipstick immunoassay (Padhye and Doyle, Appl Environ Microbiol, 57:2693-2698 (1991)), multiplex polymerase chain reaction (Jinneman et al., J Food Protect, 58:722-726 (1995)), standard microbiologic techniques, immunomagnetic bead separation (Bennett et al., Lett Appl Microbiol, 22:237-243 (1996); Blanco et al., Microbiologia, 12:385-394 (1996); Karch et al., J Clin Microbiol, 34:516-519 (1996); Vemozy-Rozand et al., Lett Appl Microbiol, 25:442-446 (1997); and (Yu and Bruno, Appl Environ Microbiol, 62:587-592 (1996)) or combinations thereof. There remains a need for a better understanding of the origin of virulent strains of E. coli, in particular O157:H7, and novel approaches to rapidly detect the presence of these organisms in infected individuals and vehicles including, but not limited to, food and water supplies.
    • SUMMARY OF THE INVENTION
  • In the present invention the inventor has discovered the gnd gene and corresponding 6-phosphogluconate dehydrogenase (6-PGD) protein of fourteen strains of [0011] E. coli. Within these genes and proteins the inventor has also found several polymorphisms that can be used to identify the presence of a particular strain of E. coli and/or differentiate one strain of E. coli from another. One polymorphism in particular, which involves a substitution of an isoleucine molecule for a threonine molecule at amino acid position 218, can be used to differentiate highly pathogenic strains of O157:H7 and O55:H7 from less pathogenic strains of O157:H7. Since O55:H7 is only about 82% homologous to O157:H7, the highly pathogenic strains of O157:H7 can be differentiated from O55:H7 at several different loci. By identifying the presence and/or absence of the polymorphism at position 218 and identifying the presence or absence of a region of non-homology between O55:H7 and O157:H7, one of skill in the art can rapidly identify the presence of a highly pathogenic strain of E. coli in a sample obtained from a patient or from a food or liquid source. Further, by identifying the presence or absence of other polymorphisms in the gnd locus, one of skill can efficiently differentiate specific strains of E. coli allowing for a more precise diagnosis or screening.
  • Embodiments of the invention include an isolated polynucleotide encoding gnd, wherein the polynucleotide comprises one of the [0012] E. coli sequences disclosed in the sequence listing. Fragments of these sequences having least 9 consecutive bases and a polymorphism described in Table 1 are also embodiments of the invention. Other embodiments include isolated polynucleotides that encode a polypeptide that corresponds to the E. coli nucleic acid sequences disclosed in the sequence listing and polynucleotides of at least 9 bases that hybridize to a nucleotide sequence found in the sequence listing under the following conditions: 7% sodium dodecyl sulfate (SDS), 0.5M NaPO4 pH 7.0, 1 mM EDTA at 50° C.; and washing with 1% SDS at 42° C. A additional embodiment concerns a nucleic acid probe for detecting the presence of E. coli O157:H7 consisting of an isolated nucleic acid molecule at least 7 nucleotides in length, wherein the nucleic acid molecule hybridizes to DNA of gnd of E. coli O157:H7 and not to DNA of gnd of non-H7 E. coli O157 strains. Another aspect involves a nucleic acid primer for detecting the presence of E. coli O157:H7 consisting of an isolated nucleic acid molecule at least 7 nucleotides in length, wherein the isolated nucleic acid molecule primes DNA of gnd of E. coli O157:H7 and not DNA of gnd of non-H7 E. coli O157 strains. The nucleic acid probes of the invention can be provided on a substrate or in a microarray on a chip.
  • SRecombinant constructs and vectors comprising one of the sequences of the sequence listing are also embodiments of the invention. Further, a cultured cell line comprising the one of the vectors of the invention is an embodiment. The proteins of the invention include an isolated protein comprising one of the sequences found in the sequence listing and an isolated polypeptide comprising at least 3 consecutive amino acids of one of the sequences of the sequence listing, wherein the polypeptide contains at least one polymorphism that can be deduced from Table 1. Additional protein embodiments concern an isolated antibody capable of specifically binding to a protein having one of the sequences of the sequence listing, wherein the epitope corresponds to at least one polymorphism that can be deduced from Table 1. Further, another embodiment includes an isolated antibody capable of binding to a polypeptide comprising at least 9 consecutive amino acids of one of the sequences of the sequence listing, wherein the epitope corresponds to at least one polymorphism that can be deduced from Table 1. In some embodiments, the antibody is monoclonal. [0013]
  • Methods of detecting a polymorphism and detecting or diagnosing the presence of a highly pathogenic [0014] E. coli are also embodiments. By one approach, a polymorphism in a gene encoding 6-PGD is detected by obtaining a biological sample containing polynucleotides and analyzing the biological sample for the presence of a diagnostic polynucleotide having at least one polymorphism described in Table 1. In some aspects, the presence or absence of the C653T or G653C polymorphism is analyzed and/or the analysis of the biological sample further comprises a DNA amplification step. Another method concerns the identification of a pathogenic or non-pathogenic E. coli. This approach is practiced by obtaining a biological sample containing polynucleotides, analyzing the biological sample for the presence of a diagnostic polynucleotide having at least one polymorphism described in Table 1, and identifying the E. coli as a pathogenic or non-pathogenic strain based on the presence or absence of at least one polymorphism described in Table 1. In some aspects of this embodiment, the presence or absence of the C653T or G653C polymorphism is analyzed and/or the analysis of the biological sample further comprises a DNA amplification step.
  • Other methods of the invention include, a method of making a 6-PGD protein comprising the steps of obtaining a cDNA comprising one of the sequences of the sequence listing, inserting the cDNA in an expression vector such that the cDNA is operably linked to a promoter, and introducing the expression vector into a host cell whereby the host cell produces the protein encoded by the cDNA. This method can also be used in conjunction with a step involving the isolation of the protein. An additional method concerns the construction of a transformed host cell that expresses one of the sequences of the sequence listing. This method includes the steps of transforming a host cell with a recombinant DNA vector suitable for gene expression. Additionally, a method for detecting the presence of [0015] E. coli O157:H7 in a sample is provided, which involves the steps of: (a) contacting said sample, under hybridization conditions, with a nucleic acid probe that selectively hybridizes to a nucleic acid sequence from gnd of E. coli O157:H7 and not to nucleic acid sequence from gnd of non-H7 E. coli O157 strains, to form a hybridization complex and (b) detecting formation of said hybridization complex as an indication of the presence of E. coli O157:H7 in the sample.
    •  BRIEF DESCRIPTION OF THE DRAWINGS
  • FIG. 1 shows a graphical representation of the polymorphisms present at the gnd locus in several strains of [0016] E. coli. Bars represent the 1407 bp gnd allele and the vertical lines represent sites of polymorphisms determined by comparison to a consensus sequence.
  • FIG. 2 shows the homology between chromosomes of [0017] E. coli O55:H7 and E. coli O157:H7 observed 3916 nucleotides downstream of the 3′ terminus of gnd of E. coli O55:H7, and 52 nucleotides downstream of the 3′ terminus of gnd of E. coli O157:H7. Elements of interest in the extra DNA in E. coli O55:H7 include a segment of homology to tnpA of S. enterica Typhimurium, an H-repeat protein gene with segments homologous to noncoding parts of the E. coli O157 rfb cluster, wbdJ and wbdK. Orfs are noted as homologous proteins. Loci are oriented chromosomally.
  • FIG. 3 is a representation of a chromosome having the gnd locus and flanking regions.[0018]
    • DETAILED DESCRIPTION OF THE INVENTION
  • Herein the inventor describes the discovery of the gnd gene and corresponding 6-phosphogluconate dehydrogenase (6-PGD) protein of fourteen strains of [0019] E. coli. Within these genes and proteins the inventor has also found several genetic differences or “polymorphisms” that can be used to identify the presence of a particular strain of E. coli and/or differentiate one strain of E. coli from another. One polymorphism in particular involves a substitution of an isoleucine molecule for a threonine molecule at amino acid position 218. This polymorphism is referred to as “T218I” or “Thr218Iso”. In some contexts, this form of 6-PGD or a polynucleotide encoding this form of 6-PGD (i.e., an isoleucine at amino acid position 218 or a polynucleotide encoding an isoleucine at position 218) is referred to as “Iso218”, whereas a 6-PGD molecule having a threonine at amino acid position 218 or a polynucleotide encoding a threonine at position 218 is referred to as “Thr218”. In other contexts, the term “Iso218” refers to a polymorphism in a polynucleotide encoding a fragment of 6-PGD (in which case the polymorphism is with reference to codon 218 of the 6-PGD fragment-encoding polynucleotide), or to a fragment of the 6-PGD protein itself (in which case the polymorphism is with reference to amino acid position 218 of the 6-PGD polypeptide sequence provided in the sequence listing. This polymorphism can also be referred to by the nucleotide differences that encode the Iso218 polymorphism. That is, the Thr218 polymorphism results from the presence of a cytosine and guanine residue at nucleotide positions 653 and 654, respectively; whereas, the Iso2I8 polymorphism has a thymine and cytosine at positions 653 and 654, respectively. Thus, other ways of referring to the polymorphism at amino acid residue 218 include “C→T mutation at nucleotide position 653” and/or a “G→C” mutation at nucleotide position 654” or “C653T” and/or “G654C”.
  • In the following disclosure, the inventor describes the cloning, sequencing, and characterization of fourteen gnd genes and corresponding proteins from different strains of [0020] E. coli. Evidence is also provided of the existence of one or more mobile DNA element(s) within the gnd-rfb region that has co-transferred among E. coli and accounts for the antigenic changes that resulted in the emergence of pathogenic E. coli that express the O55 and O157 antigens. Biological tools, diagnostics, and methods of use of the foregoing are described in the sections that follow. These embodiments are useful for the rapid identification of the presence of a specific strain of E. coli, and the differentiation of one strain of E. coli from another, for example, the excessively virulent strains of O157:H7 from less pathogenic strains of E. coli. In the section below, the inventor describes the cloning, sequencing, and characterization of the fourteen gnd genes and corresponding proteins of different strains of E. coli.
  • Cloning, sequencing, and characterization of gnd genes and corresponding proteins of different [0021] E. coli strains Recently, research has focussed on the use of the rfb region (a cluster of genes that encodes the enzyme necessary for the production of the E. coli O157 O side chain antigen) of E. coli O157:H7 as a potential target for DNA based detection systems in food and water supplies and human clinical specimens. (Desmarchelier et al., J Clin Microbiol, 36:1801-1804 (1998); (Feng et al., J Clin Microbiol, 36:2339-2341 (1998); and (Paton and Paton, J Clin Microbiol, 36:598-602 (1998)). While the expression of the O157 antigen and the presence of the rfb region encoded in this antigen are necessary components of a pathogenic E. coli O0157, diverse non-toxigenic E. coli O157 exist that express H antigens 3, 16, 43, and 45 and contain sequences homologous to the E. coli O157:H7 rfb region. (Bilge et al., Infect Immun, 64:4795-4801 (1996)). Such organisms frustrate a diagnostic strategy based upon the detection of genetic differences in the rfb region. (Wang et al., Infect Immun, 66:3545-3551 (1998)).
  • The rfb cluster of genes occurs at approximately 44 minutes on the [0022] E. coli chromosome. These clusters are generally between 8 and 14 kb in length and contain approximately 8 to 12 contiguous genes that act in concert to produce the O side chain lipopolysaccharide. (Reeves, New Compr Biochem, 27:281-314 (1994); Reeves et al., Trends Microbiol, 4:495-503 (1996)). Adjacent to the rfb cluster is the gnd allele that encodes 6-phosphogluconate dehydrogenase (6-PGD) (EC 1.1.1.44), the third enzyme in the pentose-phosphate pathway. Although gnd encodes a “housekeeping” gene with critical bacterial function, this allele is highly polymorphic, when compared to other “housekeeping” genes in the E. coli chromosome. (Whittam and Ake, “Mechanisms of molecular evolution,” Sinauer, Takahata and Clark, eds., Sunderland, Mass.: 1993:223-245). It is believed by some that the polymorphisms at the gnd locus result from inter-strain or interspecies transfers and subsequent recombination with Salmonella. (Barcak and Wolf, Jr., J Bacteriol, 170:372-379 (1988); Beltran et al., Proc Natl Acad Sci USA, 85:7753-7757 (1988); Bisercic et al., J Bacteriol, 173:3894-3900 (1991); Boyd et al., J Gen Microbiol, 139:1125-1132 (1993); Dykhuizen and Green, J Bacteriol, 173:7257-7268 (1991); and Selander et al., Infect Immun, 58:2262-2275 (1990)).
  • By one model, the “hitchhiking hypothesis”, the rfb region of [0023] E. coli is believed to have been acquired via horizontal transfer from other species by virtue of sequence homology and low G+C content. That is, gnd and rfb are thought to co-transfer or “hitchhike” with rfb. (Nelson and Selander, Proc Natl Acad Sci USA, 91:10227-10231 (1994)). In support of this hypothesis are the discordant electromorphic appearances of 6-PGD of E. coli O157:H7 and its closest non-O157:H7 relative, E. coli O55:H7. Among other evolutionary events including the acquisition of bacteriophage encoding the Shiga toxin genes, the E. coli O157:H7 large plasmid, and the loss of the ability to ferment sorbitol, it has been speculated that the E. coli O55:H7 rfb region was exchanged for the E. coli O157:H7 rfb region. (Feng et al., J Clin Microbiol, 36:2339-2341 (1998)).
  • While the current paradigm explains the observed polymorphic gnd structure as being a result of selective pressures on gnd itself, the inventor set out to prove that the genetic diversity at the gnd locus resulted from the close proximity of gnd to the rfb cluster and the fact that the rfb genes encode bacterial surface molecules that are efficiently targeted by the immune system. The inventor reasoned that the gnd locus, as well as other genes within the rfb cluster, co-evolved with the immune system and, thus, the polymorphisms within these genes could be used to identify and differentiate the O157:H7 [0024] E coli from other strains of E. coli, including bacteria expressing a nonpathogenic form of the O157 antigen. Accordingly, the inventor cloned and sequenced the gnd genes of virulent strains of E. coli O157:H7, E. coli O55:H7, and E. coli that express the O157 antigen but are not as pathogenic to humans as E. coli O157:H7 and determined that, indeed, a relationship existed between polymorphisms within genes of the rfb cluster, in particular gnd and pathogenicity.
  • The gnd genes of [0025] E. coli O157:H7 and the other E. coli strains were cloned from purified bacterial DNA. To obtain genomic or plasmid DNA, bacteria were grown overnight in LB broth (Maniatis et al., Molecular cloning: a laboratory manual, Cold Spring Harbor Laboratory, (1982)) without antibiotics or with ampicillin (200 mg/mL), respectively, at 37° C. For genomic DNA, bacteria (3 ml), pelletted by centrifugation, were suspended in 50 millimolar (mM) Tris-HCl (pH8.0) and 50 mM ethylenediamine tetraacetic acid (EDTA). Ten microliters (□1) of 20% SDS were added to this mix simultaneous with the addition of 18 □1 of proteinase K (20 mg/ml). These chemicals were obtained from Sigma (St. Louis, Mo.). Bacteria were incubated at 65° C. for 2-24 hours, and were then extracted once or more times with phenyl-chloroform-isoamyl alcohol (25:24:1), and back extracted with chloroform-isoamyl alcohol (24:1). The resulting aqueous DNA was then precipitated at room temperature adding 10M ammonium acetate to a concentration of 2.5M, followed by the addition of 2.5 volumes of 100% ethanol. The precipitate was centrifuged, washed once with 100% ethanol, air dried, and solubilized in 10 mM Tris-HCl (pH8.0), containing 1 mM ETDA. Plasmids were obtained and prepared using the Qiaprep Spin Miniprep Kit (Qiagen Inc., Valencia, Calif.) and manufacture's instructions.
  • To amplify gnd from [0026] E. coli expressing the O157 antigen, the inventor initially used the primer pair (1) 5′CACGGATCCGATCACACCTGACAGGAGTA3′ (SEQ. ID. No.1) (for the rfb side) and 5′CCGGAATTCCGGGCAAAAAAAAGCCCGGTGCAA3′ (SEQ. ID. No. 2) (for the his side), which were derived from published sequences (Bisercic et al., J Bacteriol, 173:3894-3900 (1991)) and were modified to contain BamHI and EcoRI sites for cloning purposes. However, these primers failed to obtain an amplicon from E. coli O55:H7 DNA. Therefore, the consensus oligonucleotides of primer pair (2)-5′CGGAATTCCGCGCTCAACATCGANAGCCGTGG3′ (SEQ. ID. No. 3) and 5′CGGAATTCCGCCTGGATCAGGTTAGCCGG3′ (SEQ. ID. No. 4) (derived from a computerized data base of E. coli gnd sequences and having 5′ EcoRI sites) were used to prime DNA from strain TB 182A (an E. coli O55:H7 strain). (Bokete et al., J Infect Dis, 175:1382-1389 (1997)). These primers produced a PCR product of approximately 1.3 kb, consisting of the internal portion of the gnd gene. Sequence analysis of this amplicon determined that the following primer pairs would prime DNA close to the 5′ and 3′ termini, respectively, of this allele:
  • (3)-5′CGGGGTACCCCGTAAGGGACCAGTTTCTTACCTGGG3′ (SEQ. ID. No. 5) and 5′GCCCTATCTAGATAAAGG3′ (SEQ. ID. No.6); [0027]
  • (4)-5′AGTTAAAGCCTTCCGCGG3′ (SEQ. ID. No. 7) and 5′TGCCCGCTACATCTCCTC3′ (SEQ. ID. No.8); and [0028]
  • (5)-5′GTTGTACTCTTCAGACGC3′) (SEQ. ID. No. 9) and 5′TCGTCGCTTATGCGGTACAGAGCG3′ (SEQ. ID. No. 10). [0029]
  • Total genomic DNA of [0030] E. coli O55:H7 was then digested with SacII (enzyme purchased from Promega, Madison, Wis. and used according to the manufacturer's instructions). The resulting DNA fragments were then circularized by adding DNA ligase and ligase buffer (purchased from New England Biolabs, and used according to the manufacturer's instructions). Primer pairs (6)-5′CGGGGTACCCCGTAAGGGACCAGTTTCTTACCTGGG3′ (SEQ. ID. No. 5) and 5′GCCCTATCTAGATAAAGG3′ (SEQ. ID. No. 6), and (7)-5′AGTTAAAGCCTTCCGCGG3′ (SEQ. ID. No. 7) and 5′TGCCCGCTACATCTCCTC3′ (SEQ. ID. No. 8) were then used to amplify DNA beyond the 5′ and 3′ termini of the E. coli O55:H7 gnd, respectively. The resulting sequence data then prompted the design and use of the primer pair (8)-5′CCATCAGTAATAATGAAAAGGAATT3′ (SEQ. ID. No. 11) and 5′ATCATTAGCTCCTCTTAAGATCGC3′ (SEQ. ID. No.12) to amplify the E. coli O55 gnd allele. Primer pairs (9)-5′TCGTCGCTTATGCGGTACAGAGCG3′) (SEQ. ID. No. 10) or 5′GCGTTCTTAAAGAGTCCTGC3′ (SEQ. ID. No. 13) and 5′TGCCCGCTACATCTCCTC3′ (SEQ. ID. No. 8) amplified DNA spanning the 3′ ends of gnd of E. coli O157:H7, and E. coli O55:H7 and E. coli O55:H6 strains (DEC lineages 1 and 2).
  • PCR was performed using either the Expand™ Long Template PCR System (Boehringer Mannheim, Indianapolis, Ind.) (“Expand System”) or Taq DNA polymerase (Promega, Madison, Wis.). For initial pan-gnd amplifications, Taq DNA polymerase (Promega) was used. For amplifications using the Expand system, reactions were performed in 50 □1 containing [0031] BMB buffer 1 supplied by the manufacturer. DNA polymerases used were either Taq DNA polymerase supplied by Promega, catalog number M1865 (5U/□1) (A) or Taq and Pwo DNA polymerases supplied by Boehringer-Mannheim (3.5U/□1) (B). Buffers used were: Promega Taq DNA polymerase 10× reaction buffer, without MgCl2 (supplied with polymerase by manufacturer) (10× buffer is 500 mM KCl, 100 mM Tris-HCl (pH 9.0 at 25° C.), 1.0% Triton® X-100); Promega Taq DNA polymerase 10× reaction buffer, with MgCl2 (supplied by manufacturer) (10× buffer is 500 mM KCl, 15 mM MgCl2, 100 mM Tris-HCl (pH 9.0 at 25° C.), 1.0% Triton® X-100); or Boehringer-Mannheim Expand 10× Buffer 1 (supplied by manufacturer). Thermocycling conditions included: 35 cycles at 94° C. (1 min), 37° C. (1 min), and 72° C. (1 min), followed by a 7 minute incubation at 72° C.); 30 cycles at 94° C. (1 min), 37° C. (1 min), and 72° C. (1 min), followed by a 7 minute incubation at 72° C.; an initial cycle at 95° C. (3 min), 55° C. (1 min), and 74° C. (1 min), followed by 35 cycles of 95° C. (1 min), 55° C. (1 min), and 74° C. (1 min), and a final incubation at 72° C. (5 min); or an initial incubation at 92° C. (2 min), followed by 10 cycles at 92° C. (10 sec), 52° C. (30 sec), and 68° C. (1 min), and 10 more cycles at 92° C. (10 sec), 52° C. (30 sec), and 68° C. (1 min plus successive 10 second increments during each cycle). All PCR reactions were performed in a PTC™ 100 programmable thermal cycler (MI Research, Inc., Watertown, Mass.). The resulting amplicons were visualized in ethidium bromide stained agarose gels.
  • Initially, Taq-generated amplicons of the [0032] E. coli O157 gnd alleles were cloned into pSK+ (Stratagene), after digestion with BamHI and EcoRI, and an amplicon of the internal portion of the E. coli O55:H7 gnd allele was cloned into the EcoRI site of pSK+. Subsequently, the pGEM T Easy Vector (Promega, Madison, Wis.) was used for cloning and sequencing of PCR products. White colonies, which suggest that the DNA was inserted into the cloning vector, were grown in LB broth with ampicillin (200 mg/mL), and resulting plasmids were obtained and prepared using the Qiaprep Spin Miniprep Kit (Qiagen Inc., Valencia, Calif.) according to the manufacturer's instructions. Confirmation of an insert was obtained by digestion with EcoRI and agarose gel electrophoresis. Cloned inserts were sequenced using vector specific (SP6 and T7) and appropriate intervening primers, and the Perkins Elmer Applied Biosystems Dye Terminator Cycle Sequencing Ready reaction Kit (Part no 402079, Perkins Elmer, Foster City, Calif.) or the Perkins Elmer Applied Systems BigDye™ Terminator Cycle Sequencing Ready Reaction Kit (Part number 43031521, Perkins Elmer, Foster City, Calif.). Sequencing was performed at the Fred Hutchinson Cancer Research Center using a ABI 373 sequencer (Applied Biosystems) or at the University of Washington Department of Biochemistry using an ABI 377 automated sequencer (Applied Biosystems).
  • For sequences of cloned amplicons that were derived from amplification of gnd using Taq polymerase without a proofreading system, unambiguous bidirectional sequence was obtained. For each of these strains, a subsequent amplicon was prepared and cloned using the Expand System, and at least one additional confirmation of each nucleotide was obtained by sequence analysis. For amplicons obtained only by use of the Expand System, unambiguous bidirectional double stranded sequences were obtained. Sequences were aligned with the GCG program (University of Wisconsin). BLAST searches were performed using the NCBI Blast server. (Gish and States, [0033] Nat. Genet., 3:266-272 (1993)).
  • The sequence of the gnds and corresponding proteins of several toxigenic O157 [0034] E. coli strains are provided below (gnd SEQ. ID. No./6-PGD SEQ. ID. No):
  • (1) 157:H7, strain 86-24 (SEQ ID. Nos. 22 and 23); [0035]
  • (2) 157:H7, strain 2433 (from Colombia also called H8) (SEQ ID. Nos. 16 and 17); [0036]
  • (3) 157:H7, strain ADLL 1541 (a strain from Australia) (SEQ ID. Nos. 18 and 19); [0037]
  • (4) 157:H7, strain 85-07 (SEQ ID. Nos. 24 and 25); [0038]
  • (5) 157:H7, strain 87-16 (SEQ ID. Nos. 26 and 27); and [0039]
  • (6) 157:NM, strain 2755 (a non-motile, sorbitol fermentor from Germany) (SEQ ID. Nos. 20 and 21). [0040]
  • When the gnd sequences of these strains were compared, only 2 and 3 nucleotides in strains 85-07 and 87-16, respectively, differed from the sequence derived for [0041] E. coli O157:H7, strain 86-24. Further, the non-motile O157 pathogen also possessed a gnd that was almost identical to the gnd of E. coli O157:H7, its slightly greater evolutionary distance from E. coli O157:H7 notwithstanding. These findings established that the exceedingly toxigenic E. coli O157:H7 possess a gnd that has undergone only minor genetic drift and provided evidence that stable sequences associated with pathogenicity could be determined.
  • The sequence of the gnds and corresponding proteins of several non Shiga-toxigenic, nonpathogenic [0042] E. coli strains are provided below (gnd SEQ. ID. No./6-PGD SEQ. ID. No):
  • (1) 55:H7, strain TB182A (SEQ. ID. Nos. 42 and 43); [0043]
  • (2) 157:H3, strain 3004-89 (SEQ. ID. Nos. 28 and 29); [0044]
  • (3) 157:H12, strain 5933 (SEQ. ID. Nos. 30 and 31); [0045]
  • (4) 157:H16, strain13A80 (SEQ. ID. Nos. 40 and 41); [0046]
  • (5) 157:H16, strain 13A81 (SEQ. ID. Nos. 32 and 33); [0047]
  • (6) 157:H38, strain 3005-89 (SEQ. ID. Nos. 36 and 37); [0048]
  • (7) 157:H43, strain 7E (SEQ. ID. Nos. 38 and 39); and [0049]
  • (8) 157:H45, strain 13A83 (SEQ. ID. Nos. 34 and 35). [0050]
  • Upon comparison of the sequences of the highly toxigenic strains with the less toxigenic strains, the inventor discovered that several polymorphisms could be used to identify the highly toxigenic [0051] E. coli O157 strains. Table 1 lists many of the polymorphisms found, that is, the positions at which the gnds of E. coli O157:H7 strain 86-24 (the reference strain) differ from the other gnd genes that were sequenced. These polymorphisms are also depicted graphically in FIG. 1. Notably, the sites at which the gnds of the non-pathogenic strains differ from the gnds of pathogenic E. coli O157:H7 occur in a subset of positions such that distinct patterns are discernible. For example, single nucleotide polymorphisms were found in strains 13A81 and 13A83 (E. coli O157 isolates expressing H antigens 16 and 45, respectively); strains 13A80, 7E, 3005-89, 3004-89, and G5933 (E. coli O157 expressing H antigens 16, 43, 38, 3, and 12, respectively); and each of the non-H7 E. coli O157 strains. As one of skill will readily appreciate, the amino acid sequences that correspond to the polymorphisms described in Table 1 (i.e., the polymorphisms expressed in terms of the amino acid) can be rapidly determined by matching the position of the nucleotide polymorphism to the protein sequences found in the sequence listing.
  • Surprisingly, one specific polymorphism, the T218I was discovered in pathonic O157:H7 strains and the O55:H7 strain TB182A but not any of non-pathonic O157:H7 strains. The sequence data revealed that the non-pathogenic strains, except O55:H7, have a cytosine and guanine residue at nucleotide positions 653 and 654, respectively; whereas, the pathogenic strains have a thymine and cytosine at positions 653 and 654, respectively. Thus, a convenient way to distinguish pathogenic O157:H7 strains from non-pathogenic O157:H7 strains involves the identification of a “C→T” mutation at nucleotide position 653 of gnd and/or a “G→C” mutation at nucleotide position 654 or the presence of an isoleucine amino acid residue at amino acid position 218. Because the gnd of [0052] E. coli O55:H7 is only about 82% homologous to the gnd of E. coli O157:H7 (e.g., strain 86-24), these strains can be easily distinguished at several different loci, as will be described in greater detail below.
    TABLE 1
    Pos. 86-24 13A81 13A83 13A80 7E 3005 3004 5933
    24 A C C
    36 A T T
    45 G A A
    51 C T T
    54 T A A A A A
    102 T C C C C C
    103 T C
    111 A G G G G G
    114 G A A
    177 A G G G G G G G
    204 T C C
    211 T C C C C C C C
    261 T C C C C C C C
    263 A G
    267 A T T G G G G G
    291 C T
    306 T C C C C C C C
    317 A T T
    351 A C C C C C
    369 C T T
    387 T C C C C C C C
    390 T A A A A A
    393 G A A A A A A A
    395 A G
    396 G A A A A A
    399 C G G
    402 A G G G G G G G
    405 A G G G G G G G
    411 T G G
    420 T C C
    453 A G G G G G G G
    459 G T T
    466 C T T
    483 A C C
    486 G T T
    498 T C C
    501 G A A
    504 A G G
    507 C T T
    534 A G G
    547 A G
    561 C T T
    576 C T T A A A A A
    585 G A A A A A A A
    618 T C C
    621 T C C C C C C C
    627 C T T
    631 A T T
    633 C T T
    648 G A A
    653 T C C C C C C C
    654 C G G G G G
    702 T C C C C C C C
    711 C T T T T T T T
    720 T C C C C C C C
    759 C T T
    768 A G
    780 A G G
    786 G C C C C C C C
    789 C T T T T T T T
    810 A G G
    834 G A A
    861 A T T T T T T T
    864 C T T T T T T T
    888 C T T T T T
    894 T C C C C C C C
    897 G A A A A A
    910 T A A
    918 A G G G G G G G
    919 C A A
    924 G A A A A A
    933 T A A A A A A A
    939 C T T
    951 C T T
    957 A G G
    966 C T T
    972 A G G G G G G G
    1002 T C C C C C
    1008 G C C C C C C C
    1017 A G G G G G G G
    1026 G T T T T T
    1040 A T
    1098 C T T T T T
    1122 C T T T T T
    1131 T A A
    1173 T G G G G G G G
    1197 C T T T T T
    1215 C T T T T T T T
    1224 C T T T T T T T
    1233 G A A
    1266 C G G
    1284 T C C C C C
    1287 C T T T T T
    1296 C T T T T T
    1302 T A A
    1314 G A A
    1350 G A A A A A
    1392 C T T T T T T T
  • The discovered sequences were aligned with the GCG program (University of Wisconsin) and several Blast searches were performed on the NCBI Blast server using the nucleotide sequence of [0053] E. coli O157:H7, strain 86-24 as the query sequence. (Gish and States, Nat. Genet., 3:266-272 (1993)). The high scoring pairs from the E. coli strains that were used are provided in Table 2.
    TABLE 2
    High-scoring Segment Pairs: Score P(N) N
    gb|U14423|ECU14423 Eseherichia coli A8190 6-phosphogl. . . 6675 0.0 1
    gb|M63829|ECOR56 Escherichia coli 6-phosphogluconat. . . 6585 0.0 1
    gb|M63827|ECOR25 Eseherichia coli 6-phosphogluconat. . . 6549 0.0 1
    gb|M64331|ECONDGN E.coli (strain ECOR65) 6-phosphogl. . . 6540 0.0 1
    gb|M63823|ECOR18 Eseherichia coli 6-phosphogluconat. . . 6513 0.0 1
    gb|M64328|ECONDGK E.coli (strain ECOR69) 6-phosphogl. . . 6495 0.0 1
    gb|M64329|ECONDGL E.coli (strain ECOR70) 6-phosphogi. . . 6468 0.0 1
    gb|M64330|ECONDGM E.coli (strain ECOR68) 6-phosphogi. . . 6441 0.0 1
    gb|M63825|ECOR21 Escherichia coli 6-phosphogluconat. . . 6432 0.0 1
    gb|M63824|ECOR20 Escherichia coli 6-phosphogluconat. . . 6423 0.0 1
    gb|AE000294|ECAE000294 Escherichia coli K-12 MG1655 secti. . . 6414 0.0 1
    dbj|D90841|D90841 E.coli genomic DNA, Kohara clone #. . . 6414 0.0 1
    gb|M63821|ECOR10 Escherichia coli 6-phosphogluconat. . . 6405 0.0 1
    gb|K02072|ECOGND E.coli gnd gene coding for 6-phosp. . . 6405 0.0 1
    gb|M63826|ECOR23 Escherichia coli 6-phosphogluconat. . . 6369 0.0 1
    gb|M63822|ECOR11 Eseherichia coli 6-phosphogluconat. . . 6315 0.0 1
    gb|U14469|SIBU14469 Shigella boydii ATCC 8700 6-phosph. . . 6306 0.0 1
    gb|M63828|ECOR47 Escherichia coli 6-phosphogluconat. . . 6297 0.0 1
    gb|U14456|ECU14456 Eseherichia coli EC63 6-phosphoglu. . . 6288 0.0 1
    emb|X71970|SFRFBAJ S.flexneri bB, gaiF, rfbA-J, rfbX,. . . 6270 0.0 1
    gb|U14442|ECU14442 Eseherichia coli EC40 6-phosphoglu. . . 6270 0.0 1
    gb|U14436|ECU14436 Eseherichia coli EC15 6-phosphoglu. . . 6261 0.0 1
    gb|U14467|SDU14467 Shigella dysenteriac ATCC 13313 6-. . . 6252 0.0 1
    gb|U14445|ECU14445 Eseherichia coli EC43 6-phosphoglu. . . 6234 0.0 1
    gb|U14433|ECU14433 Escherichia coli E8518196-phospho. . . 6225 0.0 1
    gb|U14448|ECU14448 Eseherichia coli EC46 6-phosphoglu. . . 6216 0.0 1
    gb|U14438|ECU14438 Eseherichia coli EC25 6-phosphoglu. . . 6216 0.0 1
    gb|U14441|ECU14441 Eseherichia coli EC35 6-phosphoglu. . . 6189 0.0 1
    gb|U14455|ECU14455 Eseherichia coli EC6 6-phosphogluc. . . 6180 0.0 1
    gb|U14435|ECU14435 Eseherichia coli EC14 6-phosphoglu. . . 6180 0.0 1
    gb|U14460|ECU14460 Esoherichia coli EC69 6-phosphoglu. . . 6153 0.0 1
    gb|U14462|EFU14462 Escherichia fergusonii ATCC 35469. . . 6148 0.0 1
    gb|U14459|ECU14459 Escherichia coli EC70 6-phosphoglu. . . 6144 0.0 1
    gb|U14450|ECU14450 Escherichia coli ECS 6-phosphogluc. . . 6135 0.0 1
    gb|U14439|ECU14439 Escherichia coli EC52 6-phosphoglu. . . 6126 0.0 1
    gb|U14431|ECU14431 Escherichia coli E2666-74 6-phospli. . . 6126 0.0 1
    gb|U14458|ECU14458 Escherichia coli EC68 6-phosphogin. . . 6117 0.0 1
    gb|U14440|ECU14440 Escherichia coli EC32 6-phosphoglu. . . 6081 0.0 1
    gb|U14434|ECU14434 Escherichia coli EC10 6-phosphogln. . . 6081 0.0 1
    gb|U14470|SSU14470 Shigelia sonnei ATCC 29930 6-phosp. . . 6027 0.0 1
    gb|U14457|ECU14457 Eseherichia coli EC64 6-phosphoglu. . . 6000 0.0 1
    gb|U14446|ECU14446 Escherichia coli EC44 6-phosphoglu. . . 6000 0.0 1
    gb|U14468|SFU14468 Shigella flexneri ATCC 29903 6-pho. . . 5919 0.0 1
    gb|U14451|ECU14451 Escherichia coli EC5O 6-phosphoglu. . . 5622 0.0 1
    gb|U14449|ECU14449 Escherichia coli EC49 6-phosphoglu. . . 5613 0.0 1
    gb|M64324|ECOGNDG E.coli (strain ECOR4) 6-phosphoglu. . . 5199 0.0 1
    emb|X15651|SEGNDB S. enterica gnd gene for 6-phospho. . . 5082 0.0 1
    gb|M64332|STYGNDA S.typhimurium (strain LT2) 6-phosp. . . 5082 0.0 1
    dbj|D21242|KPNCPS Kiebsiella pneumoniae cps gene clu. . . 5001 0.0 1
    gb|M64325|ECONDGH E.coli (strain ECOR16) 6-phosphogl. . . 5001 0.0 1
    dbj|AB010150|AB010150 Escherichia coli O8 wb gene cluste. . . 4965 0.0 1
    gb|U14424|CDU14424 Citrobacter diversus CTI9 6-phosph. . . 4938 0.0 1
    gb|U14427|CDU14427 Citrobacter diversus CT4 6-phospho. . . 4929 0.0 1
    gb|U14425|CDU14425 Citrobacter diversus CT27 6-phosph. . . 4929 0.0 1
    gb|U14428|CDU14428 Citrobacter diversus CT42 6-phosph. . . 4920 0.0 1
    gb|U14429|CDU14429 Citrobacter diversus CT45 6-phosph. . . 4911 0.0 1
    gb|U14432|CDU14432 Citrobacter diversus CT9 6-phospho. . . 4893 0.0 1
    gb|L27646|ECOGNDH E. coli phosphogiuconate dehydroge. . . 4884 0.0 1
    gb|U14353|SEU14353 Salmonella enterica V serovar Broo. . . 4858 0.0 1
    gb|U14495|SEU14495 Salmonella enterica IIIa isolate S. . . 4848 0.0 1
    gb|U14481|SEU14481 Salmonella enterica V 6-phosphoglu. . . 4839 0.0 1
    gb|U14508|SEU14508 Salmonella enterica V isolate S304. . . 4830 0.0 1
    gb|U14466|CFU14466 Citrobacter freundii ATCC 8090 6-p. . . 4829 0.0 1
    gb|U14509|SEU14509 Salmonella enterica V isolate S304. . . 4821 0.0 1
    gb|U14360|SEU14360 Salmonella enterica I serovar Glos. . . 4804 0.0 1
    gb|U14500|SEU14500 Salmonella enterica H isolate S30. . . 4803 0.0 1
    gb|U14496|SEU14496 Salmonella enterica IIIa isolate S. . . 4803 0.0 1
    gb|U14485|SEU14485 Salmonella enterica I ParatyphiB 6. . . 4794 0.0 1
    gb|U14476|SEU14476 Salmonella enterica I Saintpaul 6-. . . 4794 0.0 1
    gb|U14368|SEU14368 Salmonella enterica I serovar Para. . . 4786 0.0 1
    gb|U14479|SEU14479 Salmonella enterica I Typhimurium. . . 4785 0.0 1
    gb|U14346|SEU14346 Salmonella enterica IIIa serovar A. . . 4777 0.0 1
    gb|U14340|SEU14340 Salmonella enterica II serovar Spr. . . 4777 0.0 1
    gb|U14498|SEU14498 Salmonella enterica II isolate S29. . . 4776 0.0 1
    gb|U14465|EVU14465 Escherichia vulneris ATCC 33821 6-. . . 4776 0.0 1
    gb|U14367|SEU14367 Salmonella enterica I serovar Senf. . . 4768 0.0 1
    gb|U14363|SEU14363 Salmonella enterica II serovar 1,9. . . 4768 0.0 1
    gb|U14361|SEU14361 Salmonella enterica II serovar Sof. . . 4768 0.0 1
    gb|U14338|SEU14338 Salmonella enterica II serovar 9,1. . . 4768 0.0 1
    gb|U14497|SEU14497 Salmonella enterica II isolate 529. . . 4767 0.0 1
    gb|U14493|SEU14493 Salmonella enterica IIIb isolate S. . . 4767 0.0 1
    gb|U14480|SEU14480 Salmonella enterica IIIb 6-phosph. . . 4767 0.0 1
    gb|U14477|SEU14477 Salmonella enterica I Javiana 6-ph. . . 4767 0.0 1
    gb|U14475|SEU14475 Salmonella enterica I Dublin 6-pho. . . 4767 0.0 1
    gb|U14474|SEU14474 Salmonella enterica I Choleraesuis. . . 4767 0.0 1
    gb|U14505|SEU14505 Salmonella enterica IV isolate 530. . . 4758 0.0 1
    gb|U14491|SEU14491 Salmonella enterica I Enteritidis. . . 4758 0.0 1
    gb|U14357|SEU14357 Salmonella enterica I serovar Cano. . . 4750 0.0 1
    gb|U14351|SEU14351 Salmonella enterica IV serovar 43:. . . 4750 0.0 1
    gb|U14349|SEU14349 Salmonella enterica IV serovar Ar. . . 4750 0.0 1
    gb|U14503|SEU14503 Salmonella enterica VII isolate S3. . . 4749 0.0 1
    gb|U14494|SEU14494 Salmonella enterica IIIb isolate S. . . 4749 0.0 1
    gb|U14483|SEU14483 Salmonella enterica VI isolate S30. . . 4749 0.0 1
    gb|U14478|SEU14478 Salmonella enterica I Derby 6-phos. . . 4749 0.0 1
    gb|U14437|ECU14437 Escherichia coli EC16 6-phosphoglu. . . 4749 0.0 1
    gb|U14352|SEU14352 Salmonella enterica V serovar Balb. . . 4741 0.0 1
    gb|U14350|SEU14350 Salmonella enterica IV serovar Hou. . . 4741 0.0 1
    gb|U14490|SEU14490 Salmonella enterica I ParatyphiA 6. . . 4740 0.0 1
    gb|U14487|SEU14487 Salmonella enterica I Typhi 6-phos. . . 4740 0.0 1
    gb|U14484|SEU14484 Salmonella enterica VI isolate S30. . . 4740 0.0 1
    gb|U14484|SEU14484 Salmonella enterica VI isolate S30. . . 4740 0.0 1
  • Three distinct allele groups were found in [0054] E. coli O157. (See Table 3). These alleles differed from one another at about 5% of their nucleotide residues. The “gnd allele A” is comprised of gnds of toxigenic E. coli O157:H7 and E. coli O157:NM strains. The gnd sequences of strains 85-07 and 87-16 each differed from that of strain 86-24 at only two of their 1407 nucleotides; the remaining three were identical. The “gnd allele B” is found in E. coli O157 strains expressing flagellar antigens H3, H12, H16, and H38, and in strain DEC 7E (a nonmotile O157 with an MLEE pattern identical to that of E. coli O157:H43) and differs from gnd allele A at about 4% of its nucleotides. The “gnd allele C” is found in E. coli O157:H45 and O157:H16 strains, and differs from gnd allele A at about 6% of its 1407 nucleotides.
    TABLE 3
    Wild type E. coli used:
    Surface
    Strain Antigens Source gnd Genbank number
    Designation O H (Reference) alleles gnd rfbE
    E. coli 86-24 157 7 WA State patients (39) A AF176356 AF163327
    O157/O55 85-07 157 7 WA State patients (39) A AF176359 AF163328
    H7 (DEC5) 87-16 157 7 WA State patients (39) A AF176360 AF163329
    lineage H8 157 7 Colombia (S. Mattar) A AF176357
    ADAL233 157 7 Australia* A AF176358
    2755 157 NM Germany (L. Beutin) A AF176361 AF163330
    TB156A 55 7 WA State (40)
    TB182A 55 7 WA State (40) AF176369
    DEC 5A-5E 55 7 Penn State (16)
    E. coli O157 3004-89 157 3 CDC (N. Strockbine) B AF176362 AF163326
    and E. coli G5933 157 12 CDC (T. Barrett) B AF176363 AF163331
    O55 in non 13A81 157 16 FDA (S. Weagant) C AF176364 AF163332
    H7 lineages 13A83 157 45** CDC (N. Strockbine) C AF176365 AF163333
    3005-89 157 38 CDC (N. Strockbine) B AF176366 AF163334
    DEC7E 157 43*** Penn State (16) B AF176367 AF163335
    13A80 157 16 CDC (N. Strockbine) B AF176368 AF163336
    DEC1A 55 6 Penn State (16) AF176370
    DEC1B 55 6 Penn State (16) AF176371
    DEC2A 55 6 Penn State (16) AF176372
    DEC2B 55 6 Penn State (16) AF176373
  • Although [0055] E. coli O55:H7 is the closest relative to E. coli O157:H7, their gnd sequences are strikingly different. The gnd sequence of E. coli O157:H7, strain 86-24 has only about 82% homology to the gnd sequence of E. coli O55:H7, strain TB182A and there appears to be no readily apparent region of conservation between these two alleles.
  • y analyzing the sequence downstream of the gnd of [0056] E. coli O55:H7, the inventor also discovered the presence of one or more mobile elements within the gnd-rfb cluster. (FIG. 2). Approximately 96% of the 1934 nucleotides beyond the 3′+3915 position relative to gnd of E. coli O55:H7 (i.e., the segment that starts 3916 nucleotides beyond the 3′ terminus of gnd of E. coli O55:H7, and extends towards his) were found to be identical to nucleotides between the 3′+52 and the 3′+1984 positions relative to gnd of E. coli O157:H7. The region common to E. coli O55:H7 and E. coli O157:H7 contained open reading frames (orfs) encoding UDP glucose-6-dehydrogenase and an O-antigen chain length determining protein. Sequences between positions 3′+1 and 3′+51, and 3′+1 and 3′+3915, relative to the respective E. coli O55:H7 and E. coli O157:H7 gnds, were not found to be homologous.
  • he DNA between [0057] positions 3′+52 and 3′+3922 relative to gnd of E. coli O55:H7 was found to have a variety of features that are pertinent to DNA mobility. Approximately, 97% of the nucleotides between positions 3′+2680 and 3′+3809 relative to the gnd allele of E. coli O55:H7 were found to be homologous to DNA encoding an E. coli Rhs-associated H-repeat (H-rpt) protein (Genbank number L02370) and eleven nucleotides (AGCTTGCCCTG) (SEQ. ID. No. 14) between positions 3′+3799 and 3′+3809, inclusive, were identical to the eleven nucleotides of an inverted repeat flanking the H-rpt unit in E. coli (Genbank number LO2370). (Zhao et al., J. Bacteriol., 175:2799-2808 (1993)). A nearly identical inversion (CAGGGAAGAT) (SEQ. ID. No. 15) of this 11-mer was also identified on the opposite end of this H-rpt gene homologous segment, between positions 3′+2655 and 3′+2665.
  • Further, the inventor discovered an orf between [0058] positions 3′+2817 and 3′+3422 that encodes a protein of 201 amino acids, which is about 98% homologous to H-repeat protein amino acids in RhsB encoded by orf-H (Genbank number LO2370). (Zhao et al., J. Bacteriol., 175:2799-2808 (1993)). Still further, the inventor found that approximately 92% of the 114 inclusive nucleotides between positions 3′+3809 and 3′+3922 relative to gnd of E. coli O55:H7, including 7 nucleotides of the sequence common to E. coli O157:H7, are identical to nucleotides adjacent to the 3′ end of tnpA of Salmonella typhimurium LT2, encoding IS200 transposase A (GenBank number AFO93749). DNA between nucleotides at the 3′+478 and 3′+1942 positions relative to gnd of E. coli O55:H7 were also found to be about 75% identical to E. coli O111 wbdJ and wbdK (Genbank number U13629). The two orfs corresponding to nucleotides between positions 3′+112 and 3′+1035, and 3′+1032 and 3′+2198 relative to gnd are 67% and 80% identical to WbdJ and Wbd K, respectively. (Bastin and Reeves, Gene, 164:17-23 (1995)). Three segments between nucleotides at positions 3′+2788 and 3′+3806 relative to the E. coli O55:H7 gnd allele are 83-96% homologous to non-coding regions of the E. coli O157:H7 rfb cluster (Genbank numbers AF061251 and AB008676).
  • Next, PCR was employed using the primers: 5′GCGTTCTTAAAGAGTCCTGC3′ (SEQ. ID. No. 13) and 5′TGCCCGCTACATCTCCTC3′ (SEQ. ID. No. 8), which correspond to the 3′ end of gnd and downstream regions, so as to obtain a 6.5 kb amplicon from the DNA of 11 [0059] E. coli O55 strains. This amplicon was not obtained when PCR was performed with these primers on DNA from E. coli O157:H7.
  • Further, the inventor has found that this amplicon can be used as a hybridization probe to efficiently detect the presence of [0060] E. coli O55 strains from diverse lineages. Genomic DNA or amplicons from E. coli HB101, E. coli O157:H7 strain 86-24, E. coli O55:H7 strains TB156A, TB182A, and 5 A-E, and E. coli O55:H6 strains 1A, 1B, 2A, and 2B were produced using the primers: 5′GCGTTCTTAAAGAGTCCTGC3′ (SEQ. ID. No. 13) and 5′TGCCCGCTACATCTCCTC3′ (SEQ. ID. No. 8). These DNAs were then digested with SacI, separated in 1% agarose in tris-borate-EDTA (Maniatis et al., Molecular cloning: a laboratory manual (Cold Spring Harbor Laboratory) (1982)), and were transferred to a nylon membrane (Micron Separations). The transferred DNA was then probed with a cloned amplicon generated by the primers: 5′GCGTTCTTAAAGAGTCCTGC3′ (SEQ. ID. No. 13) and 5′TGCCCGCTACATCTCCTC3′ (SEQ. ID. No. 8) using E. coli O55:H7 template DNA. The amplicon probe was labeled with the Megaprime DNA system (Amersham) and [−α32P]dATP (New England Nuclear Research Products). This experiment showed a strong signal in the lanes loaded with DNA from an O55 strain but not from a lane loaded with DNA from an O157 strain or the HB101 control. The study above not only provides strong evidence that the region 3′ to gnd in E. coli O55 strains contains a conserved element with sequences that are involved in DNA mobility but also teach a rapid method to differentiate E. coli O55:H7 from O157:H7.
  • The data above also shed light on the origins of gnd diversity in [0061] E. coli, and on the mobility of the rfb region. The identical structure of gnds of E. coli O55 in diverse lineages provides evidence that gnd and the O55 rfb cluster have transferred as an intact unit between E. coli strains in nature. Additionally, the nearly identical E. coli O55 gnds, regardless of clonal frame, supports the finding that the O55 gnd-rfb cluster has been recently disseminated in natural populations. The pan-allelic discordance between the gnds of E. coli expressing the O55 and O157 LPS antigens in the E. coli DEC5 lineage is also consistent with co-transfer of intact gnd-rfb region in this lineage of E. coli.
  • Sequence analysis verified that the recombination of the gnd-rfb region utilized transposition in [0062] E. coli O55 strains. A short AT-rich site of insertion into the chromosome can be identified adjacent to a 3′ remnant of tnpA (of IS200), which utilizes AT-rich target integration sites. An H-repeat protein gene, however, with an intact orf, is also significant. Not wanting to limit the scope of the invention to any particular mechanism of action and offered only for the purposes of explanation, the inventor believes that the H-rpt protein gene does indeed, encode a transposase and the intactness of this gene provides evidence that the E. coli O55 gnd-rfb cluster has only been recently acquired by E. coli O55 in the three different lineages studied. Interestingly, transposition appears to be the mechanism of insertion of the V. cholerae 0139 rfb region (Stroeher et al., Proc. Natl. Acad. Sci. USA, 92:10374-10378 (1995); Bik et al., Embo J., 14:209-216 (1995); Stroeher et al., J. Bacteriol., 179:2740-2747 (1997); Comstock et al., Mol. Microbiol., 19:815-826 (1996)), and H-rpt protein homologues have been proposed to play a role in rfb transfer in Salmonella and Vibrio. (Xiang et al., J. Bacteriol., 176:4357-4365 (1994); Hill et al., Mol. Microbiol., 12:865-871 (1994)). Moreover, two H-rpt homologues, the ISAS1element of Aeromonas salmonicida (Gustafson et al., J. Mol. Biol., 237:452-463 (1994)) and an IS1358 construct (originally found in the V. cholerae 0139 rjb region) (Dumontier et al., J. Bacteriol., 180:6101-6106 (1998)) have been demonstrated to transpose.
  • Additional components of the identified mobile element were also found. The [0063] E. coli O55 and O111 O-side chains each contain colitose (Keene et al., Carbohydr. Res., 111:289-296 (1983)), an unusual residue among known bacterial LPS sugars. The rfb regions specifying these two serogroups have genes encoding WbdK and WbdJ homologues, though on different sides of gnd. WbdK is homologous to RfbH of Yersinia pseudotuberculosis, a CDB-4-keto-6-deoxy-D-glucose-3-dehydrase in the CDP-abequose pathway. WbdK is a putative pyridoxamine 5-phosphate-dependent dehydrase at a corresponding step in the synthesis of the O111 antigen. (Bastin and Reeves, Gene, 164:17-23 (1995)). WbdJ is homologous to Orf1.9 encoded by the E. coli capsular polysaccharide gene cluster, and is believed to perform a related function in the synthesis of the E. coli O111 LPS antigen.
  • These findings have implications for understanding the evolution of this region of the [0064] E. coli chromosome. First, the near uniformity of gnd structure in E. coli O157:H7 collected during two different decades on four continents does not agree with the current paradigm that this pathogen hypermutates and evolves rapidly. (LeClerc et al., Science, 274:1208-1211 (1996)). Second, rfb genes specifying the O157 antigen associate with only a limited number of distinct gnd alleles. Third, the presence of intact gnd alleles B, and C in different lineages provides evidence that non-H7 E. coli O157 have recently acquired a putative O157 mobile element. In the disclosure below, the inventor describes several other aspects of the invention that involve software and hardware.
  • Software and Hardware Embodiments [0065]
  • It will be appreciated by those skilled in the art that a computer readable medium having the gnd sequences and/or corresponding proteins of SEQ. ID. Nos. 16-43 are useful for the determination of homologous sequences, design of probes and primers, epitope analysis, elucidation of structural and functional domains, and the construction of protein models for rational drug design. The gnd sequences and/or corresponding proteins of SEQ. ID. Nos. 16-43 can be stored, recorded, and manipulated on any medium that can be read and accessed by a computer. [0066]
  • As used herein, the words “recorded” and “stored” refer to a process for storing information on computer readable medium. A skilled artisan can readily adopt any of the presently known methods for recording information on computer readable medium to generate manufactures comprising the nucleotide or polypeptide sequence information of this embodiment of the invention. A variety of data storage structures are available to a skilled artisan for creating a computer readable medium having recorded thereon a nucleotide or polypeptide sequence. The choice of the data storage structure will generally be based on the component chosen to access the stored information. Computer readable media include magnetically readable media, optically readable media, or electronically readable media. For example, the computer readable media may be a hard disc, a floppy disc, a magnetic tape, CD-ROM, RAM, or ROM as well as other types of other media known to those skilled in the art. The computer readable media on which the sequence information is stored may be in a personal computer, a network, a server or other computer systems known to those skilled in the art. [0067]
  • Embodiments of the invention include systems, particularly computer-based systems that contain the sequence information described herein. As used herein, “a computer-based system” refers to the hardware, software, and database used to analyze the gnd sequences and/or corresponding proteins of SEQ. ID. Nos. 16-43, or fragments thereof. The computer-based system preferably includes the storage media described above, and a processor for accessing and manipulating the sequence data. The hardware of the computer-based systems of this embodiment comprise a central processing unit (CPU) and one or more databases. A skilled artisan can readily appreciate that any one of the currently available computer-based systems are suitable. [0068]
  • In one particular embodiment, the computer system includes a processor connected to a bus which is connected to a main memory (preferably implemented as RAM) and a variety of secondary storage devices, such as a hard drive and removable medium storage device. The removable medium storage device may represent, for example, a floppy disk drive, a compact disk drive, a magnetic tape drive, etc. A removable storage medium, such as a floppy disk, a compact disk, a magnetic tape, etc. containing control logic and/or data recorded therein (e.g., the gnd sequences and/or corresponding proteins of SEQ. ID. Nos. 16-43) may be inserted into the removable storage device. The computer system includes appropriate software for reading the control logic and/or the data from the removable medium storage device once inserted in the removable medium storage device. The gnd sequences and/or corresponding proteins of SEQ. ID. Nos. 16-43 may be stored in a well known manner in the main memory, any of the secondary storage devices, and/or a removable storage medium. Software for accessing and processing the gnd sequences and/or corresponding proteins of SEQ. ID. Nos. 16-43 (such as search tools, compare tools, and modeling tools etc.) reside in main memory during execution. [0069]
  • As used herein, “a database” refers to memory that can store nucleotide or polypeptide sequence information, and protein model information. Additionally, a “database” refers to a memory access component which can access manufactures having recorded thereon nucleotide or polypeptide sequence information, and/or protein model information. In other embodiments, a database stores an “[0070] E. coli pathogen profile” that comprises nucleotide and/or polypeptide sequence information, and/or protein model information on gnd genes and 6-PGD proteins and the polymorphisms therein. Advantageously, an E. coli pathogen profile has recorded or stored in a database a plurality of polymorphisms associated with highly pathogenic and/or less pathogenic E. coli strains, which would allow investigators and clinicians to rapidly identify the presence of a particular strain of E. coli in a biological sample or food or water or other biological material. Desirably, such polymorphisms are recorded in a format that facilitates the process of determining the identity of a bacterial strain, for example, the pathogen profile can be stored such that the sequences therein that correspond to specific organisms are fully searchable by sequence, organism, and/or restriction map and homology, identity and matches to queried sequences can be determined. A preferable organization of the database is as provided by NCBI, which allows BLAST-type searching, protein model searching, key word searches, and an interface with Medline. Many other types of databases and organizations are known to those of skill in the art and several will be discussed below.
  • The gnd sequences and/or corresponding proteins of SEQ. ID. Nos. 16-43 may be stored and manipulated in a variety of data processor programs in a variety of formats. For example, the sequence data may be stored as text in a word processing file, such as MicrosoftWORD or WORDPERFECT or as an ASCII file in a variety of database programs familiar to those of skill in the art, such as DB2, SYBASE, or ORACLE. A “search program” refers to one or more programs that are implemented on the computer-based system to compare a nucleotide or polypeptide sequence with other nucleotide or polypeptide sequences stored within the database. A search program also refers to one or more programs that compare one or more protein models to several protein models that exist in a database. A search program is used, for example, to compare regions of the gnd sequences and/or corresponding proteins of SEQ. ID. Nos. 16-43 that match sequences in nucleic acid and/or protein data base so as to identify homologies and structural or functional motifs. Additionally, a search program is used to compare an [0071] E. coli pathogen profile to a queried sequence so as to identify the presence of one or more polymorphisms in the queried sequence and determine the strain of the bacteria from which the queried sequence was derived.
  • A “retrieval program” refers to one or more programs that are implemented on the computer based system to identify a homologous nucleic acid sequence, a homologous protein sequence, or a homologous protein model. Further a retrieval program can be used to identify an [0072] E. coli pathogen profile that matches a queried sequence, keyword, disease characteristic, or restriction map. Preferably, the retrieval program interfaces with a display format that presents the data from the E. coli pathogen profile in a form that can be rapidly discerned. For example, the “bar code” shown in FIG. 1 is one format that can be obtained by a retrieval program that provides information on the position of polymorphisms that can be used to identify or distinguish a particular strain of E. coli.
  • In several embodiments, one of the novel sequences disclosed in (SEQ. ID. Nos. 16-43) is compared to a queried sequence and the percent sequence identity is determined. Standard methods that are commonly used to compare the similarity and position of the amino acid of two polypeptides can be used to make these comparisons. Using a computer program such as BLAST or FASTA, for example, two polypeptides can be aligned for optimal matching of their respective amino acids (either along the full length of one or both sequences, or along a predetermined portion of one or both sequences). Such programs provide “default” opening penalty and a “default” gap penalty, and a scoring matrix such as PAM 250 (a standard scoring matrix; see Dayhoff et al., in: Atlas of Protein Sequence and Structure, Vol. 5, Supp. 3 (1978)) can be used in conjunction with the computer program. The percent identity can then be calculated as:[0073]
  • total number of identical matches x 100
  • [length of the longer sequence within the matched span+number of gaps introduced into the longer sequence in order to align the two sequences]
  • Polypeptides that are at least 70% identical will typically have one or more amino acid substitutions, deletions and/or insertions. Usually, the substitutions will be conservative so as to have little or no effect on the overall net charge, polarity, or hydrophobicity of the protein but optionally may increase the activity of 6-PGD. [0074]
  • Several Blast searches (BlastP 2.0.10, see Altschul et al., [0075] Nucleic. Acids. Res. 25:3389 (1997), herein incorporated by reference) were performed on the NCBI data base (http:// www.ncbi.nlm.nih.gov/blast) to characterize the novel 6-PGD molecules, fragments of these molecules, and regions within the gnd/rfb gene cluster, in particular the region 3′ of 6-PGD. Some of the results from initial Blast searches are disclosed in Table 2. Polypeptide fragments surrounding the T218I polymorphism were searched extensively. In this particular search, the matrix was BLOSUM62, the opening gap penalty was 11, and the gap extension was 1. Additional searches included Blast 2 (BlastP 2.0.9) searches on the NCBI data base using the BLOSUM matrix with an opening penalty of 11, a gap extension of 1, and an x_-dropoff of 50. These later search parameters were used to compare 6-PGD encoded by O157:H7, strains 86-24, H8, ADAL233, and 2755 to:
  • (1) 6-PGD encoded by O157:H7, strain 87-16; [0076]
  • (2) 6-PGD encoded by O55:H7, strain TB182A; [0077]
  • (3) 6-PGD encoded by O157:H3, strain 3004-89 (an “allele B” gene product); [0078]
  • (4) 6-PGD encoded by O157:H12, strain G5933 (an “allele B” gene product); [0079]
  • (5) 6-PGD encoded by O157:H16, strain 13A81 (an “allele C” gene product); [0080]
  • (6) 6-PGD encoded by O157:H45, strain 3584-91 (an “allele C” gene product); [0081]
  • (7) 6-PGD encoded by O157:H38, strain 3005-89 (an “allele C” gene product); [0082]
  • (8) 6-PGD encoded by O157:H43, strain 7E (an “allele C” gene product); and [0083]
  • (9) 6-PGD encoded by O157:H45, strain 3260-92 (an “allele C” gene product). [0084]
  • (10) 6-PGD encoded by O157:H7, strain 8507 [0085]
  • ORFs encoded by the gnd sequences and/or corresponding proteins of SEQ. ID. Nos. 16-43 and regions within the gnd/rfb gene cluster were also compared to known amino acid sequences found in Swissprot. Many computer programs and databases may be used with embodiments of the invention. The following list is intended not to limit the invention but to provide guidance to programs and databases that are useful with the nucleic acid and protein sequence embodiments of the invention. The programs and databases that can be used include, but are not limited to: MacPattern (EMBL), DiscoveryBase (Molecular Applications Group), GeneMine (Molecular Applications Group), Look (Molecular Applications Group), MacLook (Molecular Applications Group), BLAST and BLAST2 (NCBI), BLASTN and BLASTX (Altschul et al, [0086] J. Mol. Biol. 215: 403 (1990)), FASTA (Pearson and Lipman, Proc. Natl. Acad. Sci. USA, 85: 2444 (1988)), Catalyst (Molecular Simulations Inc.), Catalyst/SHAPE (Molecular Simulations Inc.), Cerius2.DBAccess (Molecular Simulations Inc.), HypoGen (Molecular Simulations Inc.), Insight II, (Molecular Simulations Inc.), Discover (Molecular Simulations Inc.), CHARMm (Molecular Simulations Inc.), Felix (Molecular Simulations Inc.), DelPhi, (Molecular Simulations Inc.), QuanteMM, (Molecular Simulations Inc.), Homology (Molecular Simulations Inc.), Modeler (Molecular Simulations Inc.), Modeller 4 (Sali and Blundell J. Mol. Biol. 234:217-241 (1997)), ISIS (Molecular Simulations Inc.), Quanta/Protein Design (Molecular Simulations Inc.), WebLab (Molecular Simulations Inc.), WebLab Diversity Explorer (Molecular Simulations Inc.), Gene Explorer (Molecular Simulations Inc.), SeqFold (Molecular Simulations Inc.), the EMBL/Swissprotein database, the MDL Available Chemicals Directory database, the MDL Drug Data Report data base, the Comprehensive Medicinal Chemistry database, Derwents's World Drug Index database, and the BioByteMasterFile database. Many other programs and data bases would be apparent to one of skill in the art given the present disclosure.
  • Additionally, aspects of the invention include recombinant vectors, probes, and primers comprising the gnd sequences and/or corresponding proteins of SEQ. ID. Nos. 16-43 and fragments thereof, in particular portions of the gnd gene or corresponding protein that contain a polymorphism described in Table 1. The discussion below describes these aspects of the invention. [0087]
  • Nucleic Acid Embodiments [0088]
  • Several embodiments of the invention include recombinant vectors, probes, and primers comprising the gnad sequences of SEQ. ID. Nos. 22, 16, 18, 24, 26, 20, 42, 28, 30, 40, 32, 36, 38, and 34 and fragments thereof. In addition to the full-length gnd genes described in SEQ. ID. Nos. 22, 16, 18, 24, 26, 20, 42, 28, 30, 40, 32, 36, 38, and 34, preferred nucleic acid embodiments include fragments of any gnd gene that have a polymorphism described in Table 1. The term “full-length” refers to either the entire sequence of genomic gnd or cDNA gnd depending on the context. Further embodiments include nucleic acids that complement the full-length gnd described in SEQ. ID. Nos. 22, 16, 18, 24, 26, 20, 42, 28, 30, 40, 32, 36, 38, and 34 and nucleic acids that complement fragments of gnd that have at least one polymorphism found in Table 1. Desired embodiments include nucleic acids having at least 9 consecutive bases of a gnd and at least one polymorphism found in Table 1 or a sequence complementary thereto. In this regard, the nucleic acid embodiments of the invention can have from 9 to approximately 1,406 consecutive nucleotides of SEQ. ID. Nos.: 22, 16, 18, 24, 26, 20, 42, 28, 30, 40, 32, 36, 38, and 34 or a complement to these sequences of virtually any length so long as the nucleic acid includes at least one polymorphism described in Table 1. One of skill in the art will readily appreciate that the gnd nucleic acids of the invention can be joined to an exogenous nucleic acid so as create a fusion product, which is within the scope of the invention, having virtually any length. Thus, a nucleic acid having a portion (i.e., about 9 to about 1,406 consecutive nucleotides) of SEQ. ID. Nos.: 22, 16, 18, 24, 26, 20, 42, 28, 30, 40, 32, 36, 38, and 34 or a complement to these sequences or a full-length gnd of the invention (either genomic or cDNA) are embodiments. That is, embodiments include a nucleic acid having at least one polymorphism described in Table 1 and less than or equal to 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, 700, 725, 750, 775, 800, 825, 850, 875, 900, 925, 950, 975, 1000, 1100, 1200, 1300, and 1406 nucleotides. Preferably, the nucleic acid embodiments, however, comprise at least 12, 13, 14, 15, 16, 17, 18, or 19 consecutive nucleotides from SEQ. ID. Nos.: 22, 16, 18, 24, 26, 20, 42, 28, 30, 40, 32, 36, 38, and 34 or a complement to these sequences, as conditions dictate, so long as the fragment has at least one polymorphism described in Table 1. More preferably, the nucleic acid embodiments comprise at least 20-30 consecutive nucleotides. These nucleic acid oligomers have biotechnological and diagnostic use, e.g., in nucleotide acid hybridization assays, Southern and Northern Blot analysis, etc. and the prognosis of [0089] E. coli infection. Some embodiments comprise recombinant constructs having all or part of the gnd genes disclosed in SEQ. ID. Nos. 22, 16, 18, 24, 26, 20, 42, 28, 30, 40, 32, 36, 38, and 34 or complements thereof A recombinant construct can be capable of replicating autonomously in a host cell. Alternatively, the recombinant construct can become integrated into the chromosomal DNA of the host cell. Such a recombinant polynucleotide comprises a polynucleotide of genomic or cDNA, of semi-synthetic or synthetic origin by virtue of human manipulation. Therefore, recombinant nucleic acids comprising sequences otherwise not naturally occurring are provided by embodiments of this invention.
  • The nucleic acid embodiments of this invention can also be altered by mutation such as substitutions, additions, or deletions that provide for sequences encoding functionally equivalent molecules. Due to the degeneracy of nucleotide coding sequences, other DNA sequences that encode substantially the same 6-PGD amino acid sequence as depicted in SEQ. ID. Nos.: 23, 17, 19, 25, 27, 21, 43, 29, 31, 41, 33, 37, 39, and 35 can be used in some embodiments of the invention. These include, but are not limited to, nucleic acid sequences comprising all or portions of gnd depicted in SEQ. ID. Nos.: 22, 16, 18, 24, 26, 20, 42, 28, 30, 40, 32, 36, 38, and 34 or complements thereof that have been altered by the substitution of different codons that encode a functionally equivalent amino acid residue within the sequence, thus producing a silent change. [0090]
  • In addition, recombinant gnd-encoding nucleic acid sequences and their complementary sequences can be engineered so as to modify processing or expression. For example, and not by way of limitation, the gnd genes depicted in SEQ. ID. Nos.: 22, 16, 18, 24, 26, 20, 42, 28, 30, 40, 32, 36, 38, and 34 can be combined with a promoter sequence and/or ribosome binding site, or a signal sequence may be inserted upstream of 6-PGD-encoding sequences to permit secretion of 6-PGD and thereby facilitate harvesting or bioavailability. Additionally, a given gnd nucleic acid can be mutated in vitro or in vivo, to create and/or destroy translation, initiation, and/or termination sequences, or to create variations in coding regions and/or form new restriction sites or destroy preexisting ones, or to facilitate further in vitro modification. Any technique for mutagenesis known in the art can be used, including but not limited to, in vitro site-directed mutagenesis. (Hutchinson et al., J. Biol. Chem. 253:6551 (1978)). Further, nucleic acids encoding other proteins or domains of other proteins can be joined to nucleic acids encoding 6-PGD so as to create a fusion protein. The resulting fusion proteins can be used as biotechnological tools to investigate the mobility of regions of the gnd/rfb cluster, for example, or to develop strain specific antibodies. [0091]
  • The nucleic acid embodiments can also be used as biotechnological tools for isolation procedures and diagnostic assays. By using the gnd sequences disclosed in SEQ. ID. Nos.: 22, 16, 18, 24, 26, 20, 42, 28, 30, 40, 32, 36, 38, and 34, probes that complement these sequences can be designed and manufactured by oligonucleotide synthesis. Preferred hybridization probes comprise at least one polymorphism found in Table 1. These probes can be used to screen cDNA or genomic libraries so as to isolate natural sources of the nucleic acid embodiments of the invention or can be used to identify specific strains or classes of strains of [0092] E. coli. Further, sequences from nucleic acids complementing the gnd sequences disclosed in SEQ. ID. Nos.: 22, 16, 18, 24, 26, 20, 42, 28, 30, 40, 32, 36, 38, and 34, can be used to make oligonucleotide primers by conventional oligonucleotide synthesis for use in amplification strategies, such as PCR. These oligonucleotide primers can be used, for example, to isolate the nucleic acid embodiments of this invention by amplifying the sequences resident in genomic DNA or biological samples by using PCR or other enzyme-mediated nucleic acid amplification techniques. Such diagnostic and food or water screening techniques are discussed in greater detail below.
  • Alternatively, the nucleic acids encoding the gnd sequences disclosed in SEQ. ID. Nos.: 22, 16, 18, 24, 26, 20, 42, 28, 30, 40, 32, 36, 38, and 34, or fragments thereof are manipulated using conventional techniques in molecular biology to create recombinant constructs that express 6-PGD or fragments of 6-PGD. The discussion that follows describes some of these expression constructs and protein embodiments. [0093]
  • Protein Embodiments [0094]
  • The 6-PGD polypeptide embodiments or derivatives thereof, include but are not limited to, those molecules having as a primary amino acid sequence all of the amino acid sequence substantially as depicted in SEQ. ID. Nos.: 23, 17, 19, 25, 27, 21, 43, 29, 31, 41, 33, 37, 39, and 35 and fragments of these sequences at least three amino acids in length including altered sequences in which functionally equivalent amino acid residues are substituted for residues within the sequence resulting in a silent change. Preferred fragments include at least one of the polymorphisms that can be deduced from Table 1, as described previously. It is to be understood that in the following discussion in this section, references made to 6-PGD in a general sense are intended to encompass the proteins and fragments thereof found in SEQ. ID. Nos. 23, 17, 19, 25, 27, 21, 43, 29, 31, 41, 33, 37, 39, and 35. [0095]
  • Accordingly, one or more amino acid residues within the 6-PGD polypeptide of SEQ. ID. Nos.: 23, 17, 19, 25, 27, 21, 43, 29, 31, 41, 33, 37, 39, and 35 or fragments thereof can be substituted by another amino acid of a similar polarity that acts as a functional equivalent, resulting in a silent alteration. Substitutes for an amino acid within the sequence can be selected from other members of the class to which the amino acid belongs. For example, the non-polar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine. The polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine and glutamine. The positively charged (basic) amino acids include arginine, lysine, and histidine. The negatively charged (acidic) amino acids include aspartic acid and glutamic acid. The aromatic amino acids include phenylalanine, tryptophan, and tyrosine. [0096]
  • The 6-PGD fragments of the invention can be less than or equal to 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, and 468 amino acids in length. In other aspects of the invention, the 6-PGD polypeptide of SEQ. ID. Nos.: 23, 17, 19, 25, 27, 21, 43, 29, 31, 41, 33, 37, 39, and 35 or fragments thereof or derivatives thereof are differentially modified during or after translation, e.g., by phosphorylation, glycosylation, cross-linking, acylation, proteolytic cleavage, linkage to an antibody molecule, membrane molecule, or other ligand. (Ferguson et al., Ann. Rev. Biochem. 57:285-320 (1988)). [0097]
  • In several embodiments, the 6-PGD polypeptide of SEQ. ID. Nos.: 23, 17, 19, 25, 27, 21, 43, 29, 31, 41, 33, 37, 39, and 35 or fragments thereof are expressed in a cell line. The sequences, constructs, vectors, clones, and other materials comprising the present invention can advantageously be in enriched or isolated form. As used herein, “enriched” means that the concentration of the material is at least about 2, 5, 10, 100, or 1000 times its natural concentration (for example), advantageously 0.01%, by weight, preferably at least about 0.1% by weight. Enriched preparations from about 0.5%, 1%, 5%, 10%, and 20% by weight are also contemplated. The term “isolated” requires that the material be removed from its original environment (e.g., the natural environment if it is naturally occuriang). For example, a naturally-occuring polynucleotide or polypeptide present in a living animal is not isolated, but the same polynucleotide or polypeptide, separated from some or all of the coexisting materials in the natural system, is isolated. It is also advantageous that the sequences be in purified form. The term “purified” does not require absolute purity; rather, it is intended as a relative definition. Purification of starting material or natural material to at least one order of magnitude, preferably two or three orders, and more preferably four or five orders of magnitude is expressly contemplated. [0098]
  • To express the proteins encoded by gnd or portions thereof, nucleic acids containing the coding sequence for 6-PGD or fragments of 6-PGD are obtained and cloned into a suitable expression vector such that the coding region is operably linked to a heterologous promoter. The nucleic acid encoding the protein or polypeptide to be expressed is operably linked to a promoter in an expression vector using conventional cloning technology. The expression vector can be in any of the mammalian, yeast, amphibian, insect, parasite, or bacterial expression systems known in the art. Commercially available vectors and expression systems are available from a variety of suppliers including Genetics Institute (Cambridge, Mass.), 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 can be optimized for the particular expression organism in which the expression vector is introduced, as explained by Hatfield, et al., U.S. Pat. No. 5,082,767, incorporated herein by this reference. Further, a secretory leader sequence can be incorporated so as to facilitate purification of the protein. [0099]
  • The following is provided as one exemplary method to express the proteins encoded by the nucleic acids described above. First, the methionine initiation codon for the gene and the poly A signal of the gene are identified. If the nucleic acid encoding the polypeptide to be expressed lacks a methionine to serve as the initiation site, an initiating methionine can be introduced next to the first codon of the nucleic acid using conventional techniques. Similarly, if the nucleic acid lacks a poly A signal, this sequence can be added to the construct by, for example, splicing out the Poly A signal from pSG5 (Stratagene) using BglI and SalI restriction endonuclease enzymes and incorporating it into the mammalian expression vector pXT1 (Stratagene). The vector 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. [0100]
  • The nucleic acid encoding the polypeptide to be expressed can be obtained by PCR from the bacterial vector using oligonucleotide primers complementary to the nucleic acid and containing restriction endonuclease sequences for Pst I incorporated into the 5′ primer and BglII at the 5′ end of the [0101] corresponding cDNA 3′ primer, taking care to ensure that the nucleic acid is positioned in frame with the poly A signal. The purified fragment obtained from the resulting PCR reaction is digested with PstI, blunt ended with an exonuclease, digested with Bgl II, purified and ligated to pXT1, now containing a poly A signal and digested with BglII. The ligated product is transfected into a suitable cell line, e.g., 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 μg/ml G418 (Sigma, St. Louis, Mo.). Preferably the expressed protein is released into the culture medium, thereby facilitating purification.
  • Another embodiment utilizes the “Xpress system for expression and purification” (Invitrogen, San Diego, Calif.). The Xpress system is designed for high-level production and purification of recombinant proteins from bacterial, mammalian, and insect cells. The Xpress vectors produce recombinant proteins fused to a short N-terminal leader peptide that has a high affinity for divalent cations. Using a nickel-chelating resin (Invitrogen), the recombinant protein can be purified in one step and the leader can be subsequently removed by cleavage with enterokinase. [0102]
  • One preferred vector for the expression of 6-PGD and fragments of 6-PGD is the pBlueBacHis2 Xpress. The pBlueBacHis2 Xpress vector is a Baculovirus expression vector containing a multiple cloning site, an ampicillin resistance gene, and a lac z gene. By one approach, the gnd nucleic acid, or portion thereof is cloned into the pBlueBacHis2 Xpress vector and SF9 cells are infected. The expression protein is then isolated or purified according to the maufacturer's instructions. Several other cultured cell lines having recombinant constructs or vectors comprising gnd or portions thereof are embodiments of the present invention and their manufacture would be routine given the present disclosure. [0103]
  • Proteins in the culture medium can also be separated by gel electrophoresis. The separated proteins are then detected using techniques such as Coomassie or silver staining or by using antibodies against the protein. Coomassie, silver staining, and immunolabeling of proteins are techniques familiar to those skilled in the art. If desired, the proteins can also be ammonium sulfate precipitated or separated based on size or charge prior to electrophoresis. [0104]
  • The protein encoded by gnd or portion thereof can also be purified using standard immunochromatography techniques. In such procedures, a solution containing the protein, such as the culture medium or a cell extract, is applied to a column having antibodies against the protein attached to the chromatography matrix. The protein is allowed to bind the immunochromatography column. Thereafter, the column is washed to remove non-specifically bound proteins. The specifically bound protein is then released from the column and recovered using standard techniques. [0105]
  • Further, gnd or portion therof can be incorporated into expression vectors designed for use in purification schemes employing chimeric polypeptides. In such strategies, the coding sequence of gnd or portion therof is inserted in frame with the gene encoding the other half of the chimera. The other half of the chimera may be β-globin or a nickel binding polypeptide encoding sequence. A chromatography matrix having antibody to β-globin or nickel attached thereto is then used to purify the chimeric protein. Protease cleavage sites can be engineered between the β-globin gene or the nickel binding polypeptide and the gnd cDNA such as enterokinase. Thus, the two polypeptides of the chimera can be separated from one another by protease digestion. [0106]
  • One useful expression vector for generating β-globin chimerics is pSG5 (Stratagene), which 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 published in methods texts such as Davis et al., ([0107] Basic Methods in Molecular Biology, L. G. Davis, M. D. Dibner, and J. F. Battey, ed., Elsevier Press, NY, 1986) and many of the methods are available from Stratagene, Life Technologies, Inc., or Promega. Polypeptide may additionally be produced from the construct using in vitro translation systems, such as the In vitro Express™ Translation Kit (Stratagene).
  • In addition to isolating or purifying 6-PGD and fragments of 6-PGD by using recombinant DNA techniques, these molecules can be prepared by chemical synthesis methods (such as solid phase peptide synthesis) using methods known in the art such as those set forth by Merrifield et al., J. Am. Chem. Soc. 85:2149 (1964), Houghten et al., Proc. Natl. Acad. Sci. USA, 82:51:32 (1985), and Stewart and Young (solid phase peptide synthesis, Pierce Chem Co., Rockford, Ill. (1984). Such polypeptides can be synthesized with or without a methionine on the amino terminus. Chemically synthesized 6-PGD and fragments of 6-PGD can be oxidized using methods set forth in these references to form disulfide bridges. 6-PGD and fragments of 6-PGD can be employed as biologically active or immunological substitutes for natural, purified 6-PGD and fragments of 6-PGD. Analogs of 6-PGD or fragments of 6-PGD include small molecules modeled on the peptides. These small molecules are also known as peptidomimetics. A peptidomimetic is a molecule that has the same effect as a peptide, usually because it has the same critical ‘shape’, but is not itself a peptide and hence is not broken down by proteases and is cheaper to produce. Thus, peptidomimetics that structurally and/or functionally resemble 6-PGD or fragments of 6-PGD can be made and evaluated for their ability to interact with 6-PGD in a 6-PGD characterization assay (e.g., inhibit the function of natural 6-PGD or fragment thereof) or induce an immune response in a subject. Several approaches to make peptidomimetics that resemble polypeptides are described in the art. A vast number of methods, for example, can be found in U.S. Pat. Nos. 5,288,707; 5,552,534; 5,811,515; 5,817,626; 5,817,879; 5,821,231; and 5, 874,529, herein incorporated by reference in their entirety. [0108]
  • Following synthesis or expression and isolation or purification of the proteins encoded by gnd or a portion thereof, the isolated or purified proteins can be used to generate antibodies and tools for identifying agents that interact with 6-PGD and fragments of 6-PGD. Antibodies that recognize 6-PGD and fragments of 6-PGD have many uses including, but not limited to, biotechnological applications, therapeutic/prophylactic applications, and diagnostic applications. Such antibodies include, but are not limited to, polyclonal, monoclonal, chimeric, single chain, Fab fragments and fragments produced by a Fab expression library. [0109]
  • For the production of antibodies, various hosts including goats, rabbits, rats, mice, etc can be immunized by injection with 6-PGD or any portion, fragment or oligopeptide that retains immunogenic properties. Depending on the host species, various adjuvants can be used to increase immunological response. Such adjuvants include but are not limited to Freund's, mineral gels such as aluminum hydroxide, and surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, and dinitrophenol. BCG (Bacillus Calmette-Guerin) and [0110] Corynebacterium parvum are potentially useful adjuvants.
  • Peptides used to induce specific antibodies can have an amino acid sequence consisting of at least three amino acids, preferably at least 10 or 15 amino acids that include a polymorphism as can be deduced from Table 1. Preferred antibodies, for example, include ones that specifically bind to a polypeptide having the T218I polymorphism or a nucleic acid having either the C653T or G654C but not 6-PGD or gnd that has the Thr218 or thymine or cytosine polymorphisms at nucleic acid positions 653 and 654, respectively. That is, preferred antibodies recognize an epitope that uniquely identifies the Iso218 polymorphism but not the Thr218 polymorphism or vice versa or the antibodies recognize an epitope that uniquely identifies a cytosine at nucleic acid position 653 and/or a guanine at nucleic acid position 654 or a thymine at position 653 and/or a cytosine at nucleic acid position 654. Desirably, short stretches of amino acids encoding fragments of 6-PGD are fused with those of another protein such as keyhole limpet hemocyanin and antibody is produced against the chimeric molecule. While antibodies capable of specifically recognizing 6-PGD can be generated by injecting into mice synthetic 3-mer, 10-mer, and 15-mer peptides that correspond to a protein sequence of 6-PGD, a more diverse set of antibodies can be generated by using recombinant or purified 6-PGD and fragments of 6-PGD. [0111]
  • To generate antibodies to 6-PGD and fragments of 6-PGD, substantially pure 6-PGD or a fragment of 6-PGD is isolated from a transfected or transformed cell. The concentration of the polypeptide 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 polypeptide of interest can then be prepared as follows: [0112]
  • Monoclonal antibodies to 6-PGD or a fragment of 6-PGD can be prepared using any technique that provides for the production of antibody molecules by continuous cell lines in culture. These include but are not limited to the hybridoma technique originally described by Koehler and Milstein (Nature 256:495-497 (1975), the human B-cell hybridoma technique (Kosbor et al. Immunol Today 4:72 (1983); Cote et al Proc Natl Acad Sci 80:2026-2030 (1983), and the EBV-hybridoma technique Cole et al. Monoclonal Antibodies and Cancer Therapy, Alan R. Liss Inc, New York N.Y., pp 77-96 (1985). In addition, techniques developed for the production of “chimeric antibodies”, the splicing of mouse antibody genes to human antibody genes to obtain a molecule with appropriate antigen specificity and biological activity can be used. (Morrison et al. Proc Natl Acad Sci 81:6851-6855 (1984); Neuberger et al. Nature 312:604-608(1984); Takeda et al. Nature 314:452-454(1985). Alternatively, techniques described for the production of single chain antibodies (U.S. Pat. No. 4,946,778) can be adapted to produce 6-PGD -specific single chain antibodies. Antibodies can also be produced by inducing in vivo production in the lymphocyte population or by screening recombinant immunoglobulin libraries or panels of highly specific binding reagents as disclosed in Orlandi et al., Proc Natl Acad Sci 86: 3833-3837 (1989), and Winter G. and Milstein C; Nature 349:293-299 (1991). [0113]
  • Antibody fragments that contain specific binding sites for 6-PGD can also be generated. For example, such fragments include, but are not limited to, the F(ab′)[0114] 2 fragments that can be produced by pepsin digestion of the antibody molecule and the Fab fragments that can be generated by reducing the disulfide bridges of the F(ab′)2 fragments. Alternatively, Fab expression libraries can be constructed to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity. (Huse W. D. et al. Science 256:1275-1281 (1989)).
  • By one approach, monoclonal antibodies to 6-PGD of fragments thereof are made as follows. Briefly, a mouse is repetitively inoculated with a few micrograms of the selected protein or peptides derived therefrom 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 in the presence 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., [0115] Meth. Enzymol. 70:419 (1980), and derivative 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.
  • Polyclonal antiserum containing antibodies to heterogenous epitopes of a single protein can be prepared by immunizing suitable animals with the expressed protein or peptides derived therefrom 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 others 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 intraderinal sites appears to be most reliable. An effective immunization protocol for rabbits can be found in Vaitukaitis, J. et al. [0116] J. Clin. Endocrinol. Metab. 33:988-991 (1971).
  • 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, for example, Ouchterlony, O. et al., Chap. 19 in: [0117] Handbook of Experimental Immunology D. Wier (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, 2d Ed. (Rose and Friedman, Eds.) Amer. Soc. For Microbiol., Washington, D.C. (1980). Antibody preparations prepared according to either protocol are useful in quantitative immunoassays that determine concentrations of antigen-bearing substances in biological samples; they are also used semi-quantitatively or qualitatively (e.g., in diagnostic embodiments that identify the presence of 6-PGD in biological samples).
  • Diagnostic and Screening Embodiments [0118]
  • Generally, the diagnostics and screening methods of the invention can be classified according to whether the embodiment is a nucleic acid or protein based assay. These assays preferably identify and distinguish the strain and extent of pathogenicity of an [0119] E. coli present in a biological sample (e.g., a sample from a patient, food source, or liquid source) by detecting the presence of one or more polymorphisms at the gnd locus. That is, several of the diagnostic and screening embodiments focus on the detection of one or more polymorphisms provided in Table 1 or that can be deduced from Table 1 in a nucleic acid or protein sample. Additionally, the manufacture of kits that incorporate the reagents and methods described in the following embodiments so as to allow for the rapid detection and identification of highly pathogenic O157:H7 E. coli are contemplated. The diagnostic kits can include a nucleic acid probe or an antibody or combinations thereof, which specifically detect the one or more polymorphisms described in Table 1 or that can be deduced from Table 1. The detection component of these kits will typically be supplied in combination with one or more of the following reagents. A support capable of absorbing or otherwise binding DNA, RNA, or protein will often be supplied. Available supports include membranes of nitrocellulose, nylon or derivatized nylon that can be characterized by bearing an array of positively charged substituents. One or more restriction enzymes, control reagents, buffers, amplification enzymes, and non-human polynucleotides like calf-thymus or salmon-sperm DNA can be supplied in these kits.
  • Useful nucleic acid-based diagnostic techniques include, but are not limited to, direct DNA sequencing, Southern Blot analysis, single-stranded confirmation analysis (SSCA), RNase protection assay, dot blot analysis, nucleic acid amplification, and combinations of these approaches. The starting point for these analysis is isolated or purified DNA from a biological sample. Most simply, fecal material is obtained from a subject to be tested or a food or water sample is provided. While the bacterial can be cultures to obtain a sufficient amount of DNA to test, in some embodiments, the bactrerial DNA is extracted from the sample and amplified by a DNA amplification technique such as PCR using primers that correspond to regions of the gnd locus and/or the gnd/rfb cluster, preferably regions having a polymorphism listed in Table 1. [0120]
  • Several methods can be used to detect a polymorphism in a biological sample. Direct DNA sequencing, either manual sequencing or automated fluorescent sequencing can detect such sequence variations. Another approach is the single-stranded confirmation polymorphism assay (SSCA) (Orita et al., [0121] Proc. Natl. Acad. Sci. USA 86:2776-2770 (1989), herein incorporated by reference). This method, however, does not detect all sequence changes, especially if the DNA fragment size is greater than 200 base pairs, but can be optimized to detect most DNA sequence variation. The reduced detection sensitivity is a disadvantage, but the increased throughput possible with SSCA makes it an attractive, viable alternative to direct sequencing for mutation detection. The fragments which have shifted mobility on SSCA gels are then sequenced to determine the exact nature of the DNA sequence variation. Other approaches based on the detection of mismatches between the two complimentary DNA strands include clamped denaturing gel electrophoresis (CDGE) (Sheffield et al., Am. J. Hum. Genet. 49:699-706 (1991)), heteroduplex analysis (HA) (White et al., Genomics 12:301-306 (1992)), and chemical mismatch cleavage (CMC) (Grompe et al., Proc. Natl. Acad. Sci. USA 86:5855-5892 (1989)). A review of currently available methods of detecting DNA sequence variation can be found in Grompe, Nature Genetics 5:111-117 (1993).
  • A rapid preliminary analysis to detect polymorphisms and DNA sequences can be performed by looking at a series of Southern Blots of DNA cut with one or more restriction enzymes preferably with a large number of restriction enzymes. Each block contains lanes of DNA from uninfected individuals and the DNA to be tested. Southern Blots displaying hybridizing fragments when probed with sequences corresponding to one or more polymorphisms described in Table 1 indicate the presence of the specific [0122] E. coli strain. The detection of point mutations can also be accomplished by amplifying the DNA directly from the sample using primers corresponding to the regions flanking one or more polymorphisms described in Table 1 by standard PCR techniques and sequencing the amplicons, as will be discussed in greater detail below.
  • Seven well-known nucleic acid-based methods for confirming the presence of one or more polymorphisms described in Table 1 are provided below. Provided for exemplary purposes only and not intended to limit any aspect of the invention, these methods include: [0123]
  • (1) single-stranded confirmation analysis (SSCA) (Orita et al.); [0124]
  • (2) denaturing gradient gel electrophoresis (DGGE) (Wartell et al., [0125] Nucl. Acids Res. 18:2699-2705 (1990) and Sheffield et al., Proc. Natl. Acad. Sci. USA 86:232-236 (1989)), both references herein incorporated by reference;
  • (3) RNase protection assays (Finkelstein et al., [0126] Genomics 7:167-172 (1990) and Kinszler et al., Science 251:1366-1370 (1991)) both references herein incorporated by reference;
  • (4) the use of proteins which recognize nucleotide mismatches, such as the [0127] E. Coli mutS protein (Modrich, Ann. Rev. Genet. 25:229-253 (1991), herein incorporated by reference;
  • (5) allele-specific PCR (Rano and Kidd, Nucl. Acids Res. 17:8392 (1989), herein incorporated by reference), which involves the use of primers that hybridize at their 3′ ends to a polymorphism and, if the polymorphism is not present, an amplification product is not observed; and [0128]
  • (6) Amplification Refractory Mutation System (ARMS), as disclosed in European Patent Application Publication No. 0332435 and in Newton et al., [0129] Nucl. Acids Res. 17:2503-2516 (1989), both references herein incorporated by reference; and
  • (7) temporal temperature gradient gel electrophoresis (TTGE), as described by Bio-Rad in U.S./E.G. Bulletin 2103, herein incorporated by reference. [0130]
  • In SSCA, DGGE, TTGE, and RNase protection assay, a new electrophoretic band appears when the polymorphism is present. SSCA and TTGE detect a band that migrates differentially because the sequence change causes a difference in single-strand, intramolecular base pairing, which is detectable electrophoretically. RNase protection involves cleavage of the mutant polynucleotide into two or more smaller fragments. DGGE detects differences in migration rates of sequences compared to less pathogenic strain gnd sequences, using a denaturing gradient gel. In an allele-specific oligonucleotide assay (ASOs) (Conner et al., [0131] Proc. Natl. Acad. Sci. USA 80:278-282 (1983)), an oligonucleotide is designed that detects a specific sequence, and an assay is performed by detecting the presence or absence of a hybridization signal. In the mutS assay, the protein binds only to sequences that contain a nucleotide mismatch in a heteroduplex between polymorphic and non-polymorphic sequences. Mismatches, in this sense of the word refers to hybridized nucleic acid duplexes in which the two strands are not 100% complementary. The lack of total homology results from the presence of one or more polymorphisms in an amplicon obtained from a biological sample, for example, that has been hybridized to a non-polymorphic strand. Mismatched detection can be used to detect point mutations in the gnd gene or in its mRNA product. While these techniques are less sensitive than sequencing, they are easily performed on a large number of biological samples and are amenable to array technology.
  • In preferred embodiments, the nucleic acid embodiments of the present invention are attached to a support in an ordered array wherein a plurality of nucleic acid probes are attached to distinct regions of the support that do not overlap with each other. Preferably, such an ordered array is designed to be “addressable” where the distinct locations of the probe are recorded and can be accessed as part of an assay procedure. In some embodiments, addressable nucleic acid arrays comprise a plurality of nucleic acid probes that complement a plurality of polymorphisms listed in Table 1. These probes are joined to a support in different known locations. The knowledge of the precise location of each nucleic acid probe makes these “addressable” arrays particularly useful in binding assays. The nucleic acids from a preparation of several biological samples are then labeled by conventional approaches (e.g., radioactivity or fluorescence) and the labeled samples are applied to the array under conditions that permit hybridization. If a nucleic acid in the samples hybridizes to a probe on the array, then a signal will be detected at a position on the support that corresponds to the location of the hybrid. Since the identity of each labeled sample is known and the region of the support on which the labeled sample was applied is known, an identification of the presence and polymorphic variant (i.e., the strain of [0132] E. coli) can be rapidly determined. Conventional methods in DNA amplification, as will be discussed below, can also be incorporated so as to detect the presence of less than 10 bacterial cells. These approaches are easily automated using technology known to those of skill in the art of high throughput diagnostic or detection analysis.
  • Additionally, an opposite approach to that presented above can be employed. Nucleic acids present in biological samples can be disposed on a support so as to create an addressable array. Preferably, the samples are disposed on the support at known positions that do not overlap. The presence of nucleic acids having a desired polymorphism in each sample is determined by applying labeled nucleic acid probes that complement nucleic acids that encode the polymorphism and detecting the presence of a signal at locations on the array that correspond to the positions at which the biological samples were disposed. Because the identity of the biological sample and its position on the array is known, the identification of the polymorphic variant can be rapidly determined. As above, conventional methods in DNA amplification can be incorporated so as to detect the presence of very few bacterial cells. These approaches are also easily automated using technology known to those of skill in the art of high throughput diagnostic analysis. [0133]
  • Any addressable array technology known in the art can be employed with this aspect of the invention. One particular embodiment of polynucleotide arrays is known as Genechips™, and has been generally described in U.S. Pat. No. 5,143,854; PCT publications WO 90/15070 and 92/10092. These arrays are generally produced using mechanical synthesis methods or light directed synthesis methods, which incorporate a combination of photolithographic methods and solid phase oligonucleotide synthesis. (Fodor et al., [0134] Science, 251:767-777, (1991)). The immobilization of arrays of oligonucleotides on solid supports has been rendered possible by the development of a technology generally identified as “Very Large Scale Immobilized Polymer Synthesis” (VLSIPS™) in which, typically, probes are immobilized in a high density array on a solid surface of a chip. Examples of VLSIPS™ technologies are provided in U.S. Pat. Nos. 5,143,854 and 5,412,087 and in PCT Publications WO 90/15070, WO 92/10092 and WO 95/11995, which describe methods for forming oligonucleotide arrays through techniques such as light-directed synthesis techniques. In designing strategies aimed at providing arrays of nucleotides immobilized on solid supports, further presentation strategies were developed to order and display the oligonucleotide arrays on the chips in an attempt to maximize hybridization patterns and diagnostic information. Examples of such presentation strategies are disclosed in PCT Publications WO 94/12305, WO 94/11530, WO 97/29212, and WO 97/31256.
  • A wide variety of labels and conjugation techniques are known by those skilled in the art and can be used in various nucleic acid assays. There are several ways to produce labeled nucleic acids for hybridization or PCR including, but not limited to, oligolabeling, nick translation, end-labeling, or PCR amplification using a labeled nucleotide. Alternatively, a nucleic acid encoding 6-PGD, or any portion of it, can be cloned into a vector for the production of an mRNA probe. Such vectors are known in the art, are commercially available, and can be used to synthesize RNA probes in vitro by addition of an appropriate RNA polymerase such as T7, T3 or SP6 and labeled nucleotides. A number of companies such as Pharmacia Biotech (Piscataway N.J.), Promega (Madison Wis.), and U.S. Biochemical Corp (Cleveland Ohio) supply commercial kits and protocols for these procedures. Suitable reporter molecules or labels include those radionuclides, enzymes, fluorescent, chemiluminescent, or chromogenic agents, as well as, substrates, cofactors, inhibitors, magnetic particles and the like. [0135]
  • An example of a mismatch cleavage technique that is amenable to array technology is the RNase protection method. In practice, the method involves the use of a labeled riboprobe which is complementary to a gnd sequence having a polymorphism (e.g., the C653T and G654C polymorphism that distinguishes highly pathogenic O157:H7 and O55:H7 from less pathogenic [0136] E. coli strains). The riboprobe and either mRNA or DNA isolated and amplified from a biological sample are annealed (hybridized) and subsequently digested with the enzyme RNase A, which is able to detect mismatches in a duplex RNase structure. If a mismatch is detected by RNase A, the polymorphic variant is not present in the sample and the enzyme cleaves at the site of the mismatch and destroys the riboprobe. Thus, when the annealed RNA is separated on a electrophoretic gel matrix, if a mismatch has been detected and cleaved by RNase A, an RNA product will be seen which is much smaller than the full length duplex RNA for the riboprobe and the mRNA or DNA. Alternatively, complements to the riboprobe can be dispersed on an array and stringently probed with the products from the Rnase A digestion after denaturing any remaining hybrids. In this case, if a mismatch is detected and probe destroyed by Rnase A, the complements on the array will not anneal with the degraded RNA under stringent conditions. A plurality of riboprobes can be employed to screen for multiple polymorphisms in this manner so long as care is taken that the probes and complements do not cross hybridize. Panels having such arrays that screen several loci are particularly useful for the development of E. coli pathogen profiles, as described above. In a similar fashion, DNA probes can be used to detect mismatches, through enzymatic or chemical cleavage. See, e.g., Cotton, et al., Proc. Natl. Acad. Sci. USA 85:4397 (1988); Shenk et al., Proc. Natl. Acad. Sci. USA 72:989 (1975); and Novack et al., Proc. Natl. Acad. Sci. USA 83:586 (1986).
  • Alternatively, mismatches can be detected by shifts in the electrophoretic ability of mismatched duplexes relative to matched duplexes. (See, e.g., Cariello, Human [0137] Genetics 42:726 (1988), herein incorporated by reference). With either riboprobes or DNA probes, the cellular mRNA or DNA that corresponds to regions of gnd containing polymorphisms can be amplified by PCR before hybridization. DNA sequences isolated from biological samples which have been amplified by use of PCR can then be screened using allele-specific probes. These probes are nucleic acid oligomers, each of which contains a region including one or more polymorphisms present in Table 1. For example, one oligomer may be about 30 nucleotides in length and corresponds to the C653T and G654C polymorphism. By use of a battery of such allele-specific probes, PCR amplification products can be screened to identify the presence of specific polymorphisms. Of course, the most definitive test for the presence of a highly pathogenic E. coli in a sample is to directly compare nucleotide or protein sequences isolated from a biological sample with one or more of the polymorphisms present in Table 1.
  • A variety of PCR techniques are familiar to those skilled in the art. For a review of PCR technology, see Molecular Cloning to Genetic Engineering White, B. A. Ed. in [0138] Methods in Molecular Biology 67: Humana Press, Totowa (1997), the disclosure of which is incorporated herein by reference in its entirety and the publication entitled “PCR Methods and Applications” (1991, Cold Spring Harbor Laboratory Press), the disclosure of which is incorporated herein by reference in its entirety. For amplification of mRNAs, it is within the scope of the present invention to reverse transcribe mRNA into cDNA followed by PCR (RT-PCR); or, to use a single enzyme for both steps as described in U.S. Pat. No. 5,322,770, the disclosure of which is incorporated herein by reference in its entirety, or, to use Reverse Transcriptase Asymmetric Gap Ligase Chain Reaction (RT-AGLCR), as described by Marshall R. L. et al. (PCR Methods and Applications 4:80-84, 1994), the disclosure of which is incorporated herein by reference in its entirety.
  • In each of these amplification procedures, primers on either side of the sequence to be amplified are added to a suitably prepared nucleic acid sample along with dNTPs and a thermostable polymerase such as Taq polymerase, Pfu polymerase, or Vent polymerase. The nucleic acid in the sample is denatured and the primers are specifically hybridized to complementary nucleic acid sequences in the sample. The hybridized primers are extended. Thereafter, another cycle of denaturation, hybridization, and extension is initiated. The cycles are repeated multiple times to produce an amplified fragment containing the nucleic acid sequence between the primer sites. PCR has further been described in several patents including U.S. Pat. Nos. 4,683,195, 4,683,202 and 4,965,188, the disclosure of which is incorporated herein by reference in its entirety. [0139]
  • The primers are selected to be substantially complementary to a portion of the sequence of gnd DNA or mRNA and a portion of the sequence that complements the sequence of gnd DNA or mRNA, thereby allowing the sequences between the primers to be amplified. The length of the primers for use with this aspect of the invention is identical to most of the lengths of the nucleic acid embodiments provided previously. That is, primer length can be less than or equal to 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, 700, 725, 750, 775, 800, 825, 850, 875, 900, 925, 950, 975, 1000, 1100, 1200, 1300, and 1406 nucleotides. Preferably, however primers are 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 and 30 nucleotides in length. Shorter primers tend to lack specificity for a target nucleic acid sequence and generally require cooler temperatures to form sufficiently stable hybrid complexes with the template. Longer primers are expensive to produce and can sometimes self-hybridize to form hairpin structures. The formation of stable hybrids depends on the melting temperature (Tm) of the DNA. The Tm depends on the length of the primer, the ionic strength of the solution and the G+C content. The higher the G+C content of the primer, the higher is the melting temperature because G:C pairs are held by three H bonds whereas A:T pairs have only two. The G+C content of the amplification primers of the present invention preferably ranges between 10 and 75%, more preferably between 35 and 60%, and most preferably between 40 and 55%. The appropriate length for primers under a particular set of assay conditions may be empirically determined by one of skill in the art. [0140]
  • The spacing of the primers determines the length of the segment to be amplified. In the context of the present invention amplified segments carrying nucleic acid sequence encoding fragments of 6-PGD can range in size from at least about 25 bp to 35 kb. Amplification fragments from 25-1407 bp are typical, fragments from 50-1000 bp are preferred and fragments from 100-600 bp are highly preferred. It will be appreciated that amplification primers for the gnd genes of the invention can be of any sequence that allows for specific amplification of a region of the gnd genes disclosed in SEQ. ID. Nos. 22, 16, 18, 24, 26, 20, 42, 28, 30, 40, 32, 36, 38, and 34 and can, for example, include modifications such as restriction sites to facilitate cloning. [0141]
  • In a preferred embodiment, highly pathogenic O157:H7 [0142] E. coli are identified and differentiated from less pathogenic E. coli by employing PCR amplification with two sets of primers. A first set of primers is designed to produce an amplicon containing at least the C653T and G654C polymorphisms, which distinguish the highly pathogenic O157:H7 and O55:H7 E. coli from less pathogenic strains. A second set of primers is designed to produce an amplicon that is unique to the O55:H7 parasite, e.g., the primer pair: 5′GCGTTCTTAAAGAGTCCTGC3′ (SEQ. ID. No. 13) and 5′TGCCCGCTACATCTCCTC3′ (SEQ. ID. No. 8), which correspond to the 3′ end of gnd and downstream regions yield a 6.5 kb amplicon from the DNA of 11 E. coli O55 strains but not E. coli O157:H7. By using SSCP or TTGE and simple gel electrophoresis, one of skill can rapidly identify the presence of the C653T and G654C polymorphism and determine whether or not the polymorphic variant detected is O157:H7 or O55:H7. In a similar fashion, primers and combinations of primers that uniquely identify other polymorphisms, as described in Table 1, can be employed to identify and differentiate other E. coli strains.
  • The presence of a 6-PGD protein of the invention can also be detected by using conventional assays. For example, monoclonal antibodies immunoreactive with a polymorphism found on a specific 6-PGD sequence can be used to screen biological samples for the presence of a particular strain of [0143] E. coli and can be used to distinguish one strain from another. Because the T218I polymorphism can distinguish highly pathogenic O157:H7 and O55:H7 from less pathogenic O157:H7, diagnostic and screening assays that comprise reagents and methods that involve the detection of the presence or absence of the T218I polymorphism are preferred embodiments. These diagnostic assays can also include a reagent that specifically differentiates the O55:H7 and O157:H7 parasites, for example, an antibody directed to an epitope found in a region of the O55:H7 6-PGD protein that is not homologous to the 6-PGD protein from an O157:H7 parasite. Such immunological assays can be done in many convenient formats.
  • In one embodiment, antibodies are used to immunoprecipitate the 6-PGD of the invention from solution and, in another embodiment, antibodies are used to react with 6-PGD on Western or Immuneblots of a polyacrylamide gel. Favored embodiments for detecting 6-PGD include enzyme-linked immunosorbant assays (ELISA), radioimmunoassays (RIA), immunoradiometric assays (IRMA) and immunoenzymatic assays (IEMA), including sandwich assays using monoclonal and/or polyclonal antibodies. Exemplary sandwich assays are described by David et al., in U.S. Pat. Nos. 4,376,110 and 4,486,530, hereby incorporated by reference. Other embodiments employ aspects of the immune-strip technology disclosed in U.S. Pat. Nos. 5,290,678; 5,604,105; 5,710,008; 5,744,358; and 5,747,274, herein incorporated by reference, which allow for the rapid, visual identification of the presence of multiple analytes in a sample. These teachings can be readily adapted to allow for the rapid detection of the 6-PGD polymorphisms that can be deduced from Table 1. [0144]
  • In preferred protein-based diagnostic and/or detection embodiments, antibodies of the present invention are attached to a support in an ordered array wherein a plurality of antibodies are attached to distinct regions of the support that do not overlap with each other. As with the nucleic acid-based arrays, the protein-based arrays are ordered arrays that are designed to be “addressable” such that the distinct locations are recorded and can be accessed as part of an assay procedure. [0145]
  • In some embodiments, addressable antibody arrays comprise a plurality of antibodies that recognize the 6-PGD polymorphisms that can be deduced from Table 1. These probes are joined to a support in different known locations. The knowledge of the precise location of each probe makes these “addressable” arrays particularly useful in binding assays. For example, an addressable array can comprise a support having several regions to which are joined a plurality of antibody probes that recognize the 6-PGD polymorphisms that can be deduced from Table 1. Proteins obtained from biological samples are labeled by conventional approaches (e.g., radioactivity, calorimetrically, or fluorescently) and the labeled samples are applied to the array under conditions that permit binding. If a protein in the sample binds to an antibody probe on the array, then a signal will be detected at a position on the support that corresponds to the location of the antibody-protein complex. Since the identity of each labeled sample is known and the region of the support on which the labeled sample was applied is known, an identification of the presence, concentration, and/or expression level is rapidly determined. That is, by employing labeled standards of a known concentration of 6-PGD, an investigator can accurately determine the protein concentration of 6-PGD in a sample and from this information can assess the expression level of 6-PGD. Conventional methods in densitometry can also be used to more accurately determine the concentration or expression level of 6-PGD. These approaches are easily automated using technology known to those of skill in the art of high throughput diagnostic analysis. [0146]
  • In another embodiment, an opposite approach to that presented above can be employed. Proteins present in biological samples can be disposed on a support so as to create an addressable array. Preferably, the protein samples are disposed on the support at known positions that do not overlap. The presence of a protein encoding a specific form of 6-PGD in each sample is then determined by applying labeled antibody probes that recognize epitopes of 6-PGD that correspond to the polymorphisms that can be deduced from Table 1 and detecting a signal at locations on the array that correspond to the positions at which the biological samples were disposed. Because the identity of the biological sample and its position on the array is known, an identification of the presence, concentration, and/or expression level of a particular 6-PGD can be rapidly determined. That is, by employing labeled standards of a known concentration of 6-PGD, an investigator can accurately determine the concentration of 6-PGD in a sample and from this information can assess the expression level of 6-PGD. Conventional methods in densitometry can also be used to more accurately determine the concentration or expression level of 6-PGD. These approaches are also easily automated using technology known to those of skill in the art of high throughput diagnostic analysis. As detailed above, any addressable array technology known in the art can be employed with this aspect of the invention and display the protein arrays on the chips in an attempt to maximize antibody binding patterns and diagnostic information. [0147]
  • As discussed above, the presence or detection of one or more polymorphisms in 6-PGD can provide a diagnosis of a subject's disease or indicate the contamination of a food or water supply. Additional embodiments include the preparation of diagnostic kits comprising detection components such as antibodies specific for one or more polymorphisms of 6-PGD. The detection component will typically be supplied in combination with one or more of the following reagents. A support capable of absorbing or otherwise binding RNA or protein will often be supplied. Available supports for this purpose include, but are not limited to, membranes of nitrocellulose, nylon or derivatized nylon that can be characterized by bearing an array of positively charged substituents, and Genechips™ or their equivalents. One or more enzymes, such as Reverse Transcriptase and/or Taq polymerase, can be furnished in the kit, as can dNTPs, buffers, or non-human polynucleotides like calf-thymus or salmon-sperm DNA. Results from the kit assays can be interpreted by a healthcare provider or a diagnostic laboratory. Alternatively, diagnostic kits are manufactured and sold to private individuals for self-diagnosis. [0148]
  • Example 1 below describes an approach that can be used to identify other regions in the rfb/gnd gene cluster that have polymorphisms useful to identify and distinguish [0149] E. Coli strains.
    • EXAMPLE 1
  • With reference to FIG. 3, discriminating sequences flanking the gnd locus can be found by using restriction mapping and PCR cloning techniques. As shown in FIG. 3, restriction site “A” is present in fragment “B-G” (i.e., “A[0150] 1, A2, and A3”), defined below. “B” corresponds to the left-hand border of a pathogenicity or antigenicity island and “G” corresponds to the right hand border of this element. “A1” is the first restriction site A site to the left of B, and “A2” is the first restriction site A site to the right of G. If the sequence of fragment B-G is known e.g., gnd, the BG island flanking this sequence can be determined by using inverse PCR. Primers “C”, “D”, “E”, and “F” are derived from the sequence of the unique pathogenicity/antigenicity island. Actual sequence is derived from the raw data, depicted in the 5′ to 3′ direction, as indicated under the line shown in FIG. 3. The primers are in the same (primer D, primer F) or opposite orientation (primer C and primer E).
  • Next, [0151] E. coli DNA is digested to completion with enzyme A. Ligase is then added and the resulting fragments are re-circularized. Primers are added in separate tubes with a heat stable polymerase and PCR is conducted to obtain amplicons. The amplicons are cloned and sequenced. This approach identifies sequences beyond the 5′ and 3′ ends of the known pathogenicity/antigenicity islands and primers derived from these sequences are used to amplify this region in a variety of pathogens and non-pathogens, as was performed for the gnd allele. The resulting amplicons are then sequenced to identify differentiating polymorphisms.
  • Although the invention has been described with reference to embodiments and examples, it should be understood that various modifications can be made without departing from the spirit of the invention. Accordingly, the invention is limited only by the following claims. All references cited herein are hereby expressly incorporated by reference. [0152]
  • 1 43 1 29 DNA Artificial Sequence primer 1 cacggatccg atcacacctg acaggagta 29 2 33 DNA Artificial Sequence primer 2 ccggaattcc gggcaaaaaa aagcccggtg caa 33 3 32 DNA Artificial Sequence primer 3 cggaattccg cgctcaacat cganagccgt gg 32 4 29 DNA Artificial Sequence primer 4 cggaattccg cctggatcag gttagccgg 29 5 36 DNA Artificial Sequence primer 5 cggggtaccc cgtaagggac cagtttctta cctggg 36 6 18 DNA Artificial Sequence primer 6 gccctatcta gataaagg 18 7 18 DNA Artificial Sequence primer 7 agttaaagcc ttccgcgg 18 8 18 DNA Artificial Sequence primer 8 tgcccgctac atctcctc 18 9 18 DNA Artificial Sequence primer 9 gttgtactct tcagacgc 18 10 24 DNA Artificial Sequence primer 10 tcgtcgctta tgcggtacag agcg 24 11 25 DNA Artificial Sequence primer 11 ccatcagtaa taatgaaaag gaatt 25 12 24 DNA Artificial Sequence primer 12 atcattagct cctcttaaga tcgc 24 13 20 DNA Artificial Sequence primer 13 gcgttcttaa agagtcctgc 20 14 11 DNA Artificial Sequence primer 14 agcttgccct g 11 15 10 DNA Artificial Sequence primer 15 cagggaagat 10 16 1407 DNA Escherichia coli CDS (1)...(1407) 16 atg tca aag caa cag atc ggc gta gtc ggt atg gca gtg atg ggg cgc 48 Met Ser Lys Gln Gln Ile Gly Val Val Gly Met Ala Val Met Gly Arg 1 5 10 15 aac ctt gcg ctc aac atc gaa agc cgt ggt tat acc gtc tct att ttc 96 Asn Leu Ala Leu Asn Ile Glu Ser Arg Gly Tyr Thr Val Ser Ile Phe 20 25 30 aac cgt tcc cgt gaa aag acg gaa gaa gtg att gcc gaa aat cca ggc 144 Asn Arg Ser Arg Glu Lys Thr Glu Glu Val Ile Ala Glu Asn Pro Gly 35 40 45 aag aaa ctg gtt cct tac tat acg gtg aaa gaa ttt gtt gaa tct ctg 192 Lys Lys Leu Val Pro Tyr Tyr Thr Val Lys Glu Phe Val Glu Ser Leu 50 55 60 gaa acg cct cgt cgc atc ttg tta atg gtg aaa gca ggt gca ggc acg 240 Glu Thr Pro Arg Arg Ile Leu Leu Met Val Lys Ala Gly Ala Gly Thr 65 70 75 80 gat gct gct att gat tcc ctt aag cca tac ctc gat aaa ggt gac atc 288 Asp Ala Ala Ile Asp Ser Leu Lys Pro Tyr Leu Asp Lys Gly Asp Ile 85 90 95 atc att gat ggt ggt aat acc ttc ttc cag gac acc att cgt cgt aac 336 Ile Ile Asp Gly Gly Asn Thr Phe Phe Gln Asp Thr Ile Arg Arg Asn 100 105 110 cgt gag ctt tct gca gaa ggc ttt aac ttc atc ggt acc ggt gtt tcc 384 Arg Glu Leu Ser Ala Glu Gly Phe Asn Phe Ile Gly Thr Gly Val Ser 115 120 125 ggt ggt gag gag ggc gca cta aaa ggt cct tcc att atg cct ggt ggg 432 Gly Gly Glu Glu Gly Ala Leu Lys Gly Pro Ser Ile Met Pro Gly Gly 130 135 140 cag aaa gaa gcc tat gaa cta gtt gcg ccg atc ctg acc aaa atc gcc 480 Gln Lys Glu Ala Tyr Glu Leu Val Ala Pro Ile Leu Thr Lys Ile Ala 145 150 155 160 gca gtg gct gaa gac ggt gag cca tgc gtt acc tat att ggt gcc gat 528 Ala Val Ala Glu Asp Gly Glu Pro Cys Val Thr Tyr Ile Gly Ala Asp 165 170 175 ggc gca ggt cac tat gtg aag atg gtt cac aac ggt att gaa tac ggc 576 Gly Ala Gly His Tyr Val Lys Met Val His Asn Gly Ile Glu Tyr Gly 180 185 190 gat atg cag ctg att gct gaa gcc tat tct ctg ctt aaa ggt ggt ctg 624 Asp Met Gln Leu Ile Ala Glu Ala Tyr Ser Leu Leu Lys Gly Gly Leu 195 200 205 aac ctc acc aac gaa gaa ctg gcg cag atc ttt acc gag tgg aat aac 672 Asn Leu Thr Asn Glu Glu Leu Ala Gln Ile Phe Thr Glu Trp Asn Asn 210 215 220 ggt gaa ctg agc agc tac ctg atc gac att acc aaa gac atc ttc act 720 Gly Glu Leu Ser Ser Tyr Leu Ile Asp Ile Thr Lys Asp Ile Phe Thr 225 230 235 240 aaa aaa gat gaa gac ggt aac tac ctg gtt gat gtg atc ctg gat gaa 768 Lys Lys Asp Glu Asp Gly Asn Tyr Leu Val Asp Val Ile Leu Asp Glu 245 250 255 gcg gca aac aaa ggt acg ggc aaa tgg acc agc cag agc gca ctg gat 816 Ala Ala Asn Lys Gly Thr Gly Lys Trp Thr Ser Gln Ser Ala Leu Asp 260 265 270 ctc ggc gaa ccg ctg tcg ctg att acc gag tct gtg ttt gca cga tac 864 Leu Gly Glu Pro Leu Ser Leu Ile Thr Glu Ser Val Phe Ala Arg Tyr 275 280 285 atc tct tct ctg aaa gat cag cgc gtt gct gcg tct aaa gtt ctc tct 912 Ile Ser Ser Leu Lys Asp Gln Arg Val Ala Ala Ser Lys Val Leu Ser 290 295 300 ggc cca caa gcg cag cca gct ggc gac aag gct gag ttc atc gaa aaa 960 Gly Pro Gln Ala Gln Pro Ala Gly Asp Lys Ala Glu Phe Ile Glu Lys 305 310 315 320 gtt cgc cgt gca ctg tat ctg ggc aaa atc gtt tct tac gct cag ggg 1008 Val Arg Arg Ala Leu Tyr Leu Gly Lys Ile Val Ser Tyr Ala Gln Gly 325 330 335 ttc tct caa ctg cgt gcg gcg tct gaa gag tac aac tgg gat ctg aac 1056 Phe Ser Gln Leu Arg Ala Ala Ser Glu Glu Tyr Asn Trp Asp Leu Asn 340 345 350 tac ggc gaa atc gcg aag att ttc cgt gct ggc tgc atc atc cgt gcg 1104 Tyr Gly Glu Ile Ala Lys Ile Phe Arg Ala Gly Cys Ile Ile Arg Ala 355 360 365 cag ttc ctg cag aaa atc acc gat gct tat gcc gaa aat ccg cag atc 1152 Gln Phe Leu Gln Lys Ile Thr Asp Ala Tyr Ala Glu Asn Pro Gln Ile 370 375 380 gct aac ctg ctg ctg gct cct tac ttc aag caa att gcc gat gac tac 1200 Ala Asn Leu Leu Leu Ala Pro Tyr Phe Lys Gln Ile Ala Asp Asp Tyr 385 390 395 400 cag cag gcg ctg cgc gat gtc gtc gct tat gcg gta cag aac ggt atc 1248 Gln Gln Ala Leu Arg Asp Val Val Ala Tyr Ala Val Gln Asn Gly Ile 405 410 415 ccg gtt ccg acc ttc gcc gct gcg gtt gcc tat tat gac agc tac cgc 1296 Pro Val Pro Thr Phe Ala Ala Ala Val Ala Tyr Tyr Asp Ser Tyr Arg 420 425 430 gcc gct gtt ctg cct gcg aac ctg atc cag gca cag cgt gac tat ttc 1344 Ala Ala Val Leu Pro Ala Asn Leu Ile Gln Ala Gln Arg Asp Tyr Phe 435 440 445 ggt gcg cat act tat aag cgc att gat aaa gaa ggt gtg ttc cat acc 1392 Gly Ala His Thr Tyr Lys Arg Ile Asp Lys Glu Gly Val Phe His Thr 450 455 460 gaa tgg ctg gat taa 1407 Glu Trp Leu Asp * 465 17 467 PRT Escherichia coli 17 Ser Lys Gln Gln Ile Gly Val Val Gly Met Ala Val Met Gly Arg Asn 1 5 10 15 Leu Ala Leu Asn Ile Glu Ser Arg Gly Tyr Thr Val Ser Ile Phe Asn 20 25 30 Arg Ser Arg Glu Lys Thr Glu Glu Val Ile Ala Glu Asn Pro Gly Lys 35 40 45 Lys Leu Val Pro Tyr Tyr Thr Val Lys Glu Phe Val Glu Ser Leu Glu 50 55 60 Thr Pro Arg Arg Ile Leu Leu Met Val Lys Ala Gly Ala Gly Thr Asp 65 70 75 80 Ala Ala Ile Asp Ser Leu Lys Pro Tyr Leu Asp Lys Gly Asp Ile Ile 85 90 95 Ile Asp Gly Gly Asn Thr Phe Phe Gln Asp Thr Ile Arg Arg Asn Arg 100 105 110 Glu Leu Ser Ala Glu Gly Phe Asn Phe Ile Gly Thr Gly Val Ser Gly 115 120 125 Gly Glu Glu Gly Ala Leu Lys Gly Pro Ser Ile Met Pro Gly Gly Gln 130 135 140 Lys Glu Ala Tyr Glu Leu Val Ala Pro Ile Leu Thr Lys Ile Ala Ala 145 150 155 160 Val Ala Glu Asp Gly Glu Pro Cys Val Thr Tyr Ile Gly Ala Asp Gly 165 170 175 Ala Gly His Tyr Val Lys Met Val His Asn Gly Ile Glu Tyr Gly Asp 180 185 190 Met Gln Leu Ile Ala Glu Ala Tyr Ser Leu Leu Lys Gly Gly Leu Asn 195 200 205 Leu Thr Asn Glu Glu Leu Ala Gln Ile Phe Thr Glu Trp Asn Asn Gly 210 215 220 Glu Leu Ser Ser Tyr Leu Ile Asp Ile Thr Lys Asp Ile Phe Thr Lys 225 230 235 240 Lys Asp Glu Asp Gly Asn Tyr Leu Val Asp Val Ile Leu Asp Glu Ala 245 250 255 Ala Asn Lys Gly Thr Gly Lys Trp Thr Ser Gln Ser Ala Leu Asp Leu 260 265 270 Gly Glu Pro Leu Ser Leu Ile Thr Glu Ser Val Phe Ala Arg Tyr Ile 275 280 285 Ser Ser Leu Lys Asp Gln Arg Val Ala Ala Ser Lys Val Leu Ser Gly 290 295 300 Pro Gln Ala Gln Pro Ala Gly Asp Lys Ala Glu Phe Ile Glu Lys Val 305 310 315 320 Arg Arg Ala Leu Tyr Leu Gly Lys Ile Val Ser Tyr Ala Gln Gly Phe 325 330 335 Ser Gln Leu Arg Ala Ala Ser Glu Glu Tyr Asn Trp Asp Leu Asn Tyr 340 345 350 Gly Glu Ile Ala Lys Ile Phe Arg Ala Gly Cys Ile Ile Arg Ala Gln 355 360 365 Phe Leu Gln Lys Ile Thr Asp Ala Tyr Ala Glu Asn Pro Gln Ile Ala 370 375 380 Asn Leu Leu Leu Ala Pro Tyr Phe Lys Gln Ile Ala Asp Asp Tyr Gln 385 390 395 400 Gln Ala Leu Arg Asp Val Val Ala Tyr Ala Val Gln Asn Gly Ile Pro 405 410 415 Val Pro Thr Phe Ala Ala Ala Val Ala Tyr Tyr Asp Ser Tyr Arg Ala 420 425 430 Ala Val Leu Pro Ala Asn Leu Ile Gln Ala Gln Arg Asp Tyr Phe Gly 435 440 445 Ala His Thr Tyr Lys Arg Ile Asp Lys Glu Gly Val Phe His Thr Glu 450 455 460 Trp Leu Asp 465 18 1407 DNA Escherichia coli CDS (1)...(1407) 18 atg tca aag caa cag atc ggc gta gtc ggt atg gca gtg atg ggg cgc 48 Met Ser Lys Gln Gln Ile Gly Val Val Gly Met Ala Val Met Gly Arg 1 5 10 15 aac ctt gcg ctc aac atc gaa agc cgt ggt tat acc gtc tct att ttc 96 Asn Leu Ala Leu Asn Ile Glu Ser Arg Gly Tyr Thr Val Ser Ile Phe 20 25 30 aac cgt tcc cgt gaa aag acg gaa gaa gtg att gcc gaa aat cca ggc 144 Asn Arg Ser Arg Glu Lys Thr Glu Glu Val Ile Ala Glu Asn Pro Gly 35 40 45 aag aaa ctg gtt cct tac tat acg gtg aaa gaa ttt gtt gaa tct ctg 192 Lys Lys Leu Val Pro Tyr Tyr Thr Val Lys Glu Phe Val Glu Ser Leu 50 55 60 gaa acg cct cgt cgc atc ttg tta atg gtg aaa gca ggt gca ggc acg 240 Glu Thr Pro Arg Arg Ile Leu Leu Met Val Lys Ala Gly Ala Gly Thr 65 70 75 80 gat gct gct att gat tcc ctt aag cca tac ctc gat aaa ggt gac atc 288 Asp Ala Ala Ile Asp Ser Leu Lys Pro Tyr Leu Asp Lys Gly Asp Ile 85 90 95 atc att gat ggt ggt aat acc ttc ttc cag gac acc att cgt cgt aac 336 Ile Ile Asp Gly Gly Asn Thr Phe Phe Gln Asp Thr Ile Arg Arg Asn 100 105 110 cgt gag ctt tct gca gaa ggc ttt aac ttc atc ggt acc ggt gtt tcc 384 Arg Glu Leu Ser Ala Glu Gly Phe Asn Phe Ile Gly Thr Gly Val Ser 115 120 125 ggt ggt gag gag ggc gca cta aaa ggt cct tcc att atg cct ggt ggg 432 Gly Gly Glu Glu Gly Ala Leu Lys Gly Pro Ser Ile Met Pro Gly Gly 130 135 140 cag aaa gaa gcc tat gaa cta gtt gcg ccg atc ctg acc aaa atc gcc 480 Gln Lys Glu Ala Tyr Glu Leu Val Ala Pro Ile Leu Thr Lys Ile Ala 145 150 155 160 gca gtg gct gaa gac ggt gag cca tgc gtt acc tat att ggt gcc gat 528 Ala Val Ala Glu Asp Gly Glu Pro Cys Val Thr Tyr Ile Gly Ala Asp 165 170 175 ggc gca ggt cac tat gtg aag atg gtt cac aac ggt att gaa tac ggc 576 Gly Ala Gly His Tyr Val Lys Met Val His Asn Gly Ile Glu Tyr Gly 180 185 190 gat atg cag ctg att gct gaa gcc tat tct ctg ctt aaa ggt ggt ctg 624 Asp Met Gln Leu Ile Ala Glu Ala Tyr Ser Leu Leu Lys Gly Gly Leu 195 200 205 aac ctc acc aac gaa gaa ctg gcg cag atc ttt acc gag tgg aat aac 672 Asn Leu Thr Asn Glu Glu Leu Ala Gln Ile Phe Thr Glu Trp Asn Asn 210 215 220 ggt gaa ctg agc agc tac ctg atc gac att acc aaa gac atc ttc act 720 Gly Glu Leu Ser Ser Tyr Leu Ile Asp Ile Thr Lys Asp Ile Phe Thr 225 230 235 240 aaa aaa gat gaa gac ggt aac tac ctg gtt gat gtg atc ctg gat gaa 768 Lys Lys Asp Glu Asp Gly Asn Tyr Leu Val Asp Val Ile Leu Asp Glu 245 250 255 gcg gca aac aaa ggt acg ggc aaa tgg acc agc cag agc gca ctg gat 816 Ala Ala Asn Lys Gly Thr Gly Lys Trp Thr Ser Gln Ser Ala Leu Asp 260 265 270 ctc ggc gaa ccg ctg tcg ctg att acc gag tct gtg ttt gca cga tac 864 Leu Gly Glu Pro Leu Ser Leu Ile Thr Glu Ser Val Phe Ala Arg Tyr 275 280 285 atc tct tct ctg aaa gat cag cgc gtt gct gcg tct aaa gtt ctc tct 912 Ile Ser Ser Leu Lys Asp Gln Arg Val Ala Ala Ser Lys Val Leu Ser 290 295 300 ggc cca caa gcg cag cca gct ggc gac aag gct gag ttc atc gaa aaa 960 Gly Pro Gln Ala Gln Pro Ala Gly Asp Lys Ala Glu Phe Ile Glu Lys 305 310 315 320 gtt cgc cgt gca ctg tat ctg ggc aaa atc gtt tct tac gct cag ggg 1008 Val Arg Arg Ala Leu Tyr Leu Gly Lys Ile Val Ser Tyr Ala Gln Gly 325 330 335 ttc tct caa ctg cgt gcg gcg tct gaa gag tac aac tgg gat ctg aac 1056 Phe Ser Gln Leu Arg Ala Ala Ser Glu Glu Tyr Asn Trp Asp Leu Asn 340 345 350 tac ggc gaa atc gcg aag att ttc cgt gct ggc tgc atc atc cgt gcg 1104 Tyr Gly Glu Ile Ala Lys Ile Phe Arg Ala Gly Cys Ile Ile Arg Ala 355 360 365 cag ttc ctg cag aaa atc acc gat gct tat gcc gaa aat ccg cag atc 1152 Gln Phe Leu Gln Lys Ile Thr Asp Ala Tyr Ala Glu Asn Pro Gln Ile 370 375 380 gct aac ctg ctg ctg gct cct tac ttc aag caa att gcc gat gac tac 1200 Ala Asn Leu Leu Leu Ala Pro Tyr Phe Lys Gln Ile Ala Asp Asp Tyr 385 390 395 400 cag cag gcg ctg cgc gat gtc gtc gct tat gcg gta cag aac ggt atc 1248 Gln Gln Ala Leu Arg Asp Val Val Ala Tyr Ala Val Gln Asn Gly Ile 405 410 415 ccg gtt ccg acc ttc gcc gct gcg gtt gcc tat tat gac agc tac cgc 1296 Pro Val Pro Thr Phe Ala Ala Ala Val Ala Tyr Tyr Asp Ser Tyr Arg 420 425 430 gcc gct gtt ctg cct gcg aac ctg atc cag gca cag cgt gac tat ttc 1344 Ala Ala Val Leu Pro Ala Asn Leu Ile Gln Ala Gln Arg Asp Tyr Phe 435 440 445 ggt gcg cat act tat aag cgc att gat aaa gaa ggt gtg ttc cat acc 1392 Gly Ala His Thr Tyr Lys Arg Ile Asp Lys Glu Gly Val Phe His Thr 450 455 460 gaa tgg ctg gat taa 1407 Glu Trp Leu Asp * 465 19 467 PRT Escherichia coli 19 Ser Lys Gln Gln Ile Gly Val Val Gly Met Ala Val Met Gly Arg Asn 1 5 10 15 Leu Ala Leu Asn Ile Glu Ser Arg Gly Tyr Thr Val Ser Ile Phe Asn 20 25 30 Arg Ser Arg Glu Lys Thr Glu Glu Val Ile Ala Glu Asn Pro Gly Lys 35 40 45 Lys Leu Val Pro Tyr Tyr Thr Val Lys Glu Phe Val Glu Ser Leu Glu 50 55 60 Thr Pro Arg Arg Ile Leu Leu Met Val Lys Ala Gly Ala Gly Thr Asp 65 70 75 80 Ala Ala Ile Asp Ser Leu Lys Pro Tyr Leu Asp Lys Gly Asp Ile Ile 85 90 95 Ile Asp Gly Gly Asn Thr Phe Phe Gln Asp Thr Ile Arg Arg Asn Arg 100 105 110 Glu Leu Ser Ala Glu Gly Phe Asn Phe Ile Gly Thr Gly Val Ser Gly 115 120 125 Gly Glu Glu Gly Ala Leu Lys Gly Pro Ser Ile Met Pro Gly Gly Gln 130 135 140 Lys Glu Ala Tyr Glu Leu Val Ala Pro Ile Leu Thr Lys Ile Ala Ala 145 150 155 160 Val Ala Glu Asp Gly Glu Pro Cys Val Thr Tyr Ile Gly Ala Asp Gly 165 170 175 Ala Gly His Tyr Val Lys Met Val His Asn Gly Ile Glu Tyr Gly Asp 180 185 190 Met Gln Leu Ile Ala Glu Ala Tyr Ser Leu Leu Lys Gly Gly Leu Asn 195 200 205 Leu Thr Asn Glu Glu Leu Ala Gln Ile Phe Thr Glu Trp Asn Asn Gly 210 215 220 Glu Leu Ser Ser Tyr Leu Ile Asp Ile Thr Lys Asp Ile Phe Thr Lys 225 230 235 240 Lys Asp Glu Asp Gly Asn Tyr Leu Val Asp Val Ile Leu Asp Glu Ala 245 250 255 Ala Asn Lys Gly Thr Gly Lys Trp Thr Ser Gln Ser Ala Leu Asp Leu 260 265 270 Gly Glu Pro Leu Ser Leu Ile Thr Glu Ser Val Phe Ala Arg Tyr Ile 275 280 285 Ser Ser Leu Lys Asp Gln Arg Val Ala Ala Ser Lys Val Leu Ser Gly 290 295 300 Pro Gln Ala Gln Pro Ala Gly Asp Lys Ala Glu Phe Ile Glu Lys Val 305 310 315 320 Arg Arg Ala Leu Tyr Leu Gly Lys Ile Val Ser Tyr Ala Gln Gly Phe 325 330 335 Ser Gln Leu Arg Ala Ala Ser Glu Glu Tyr Asn Trp Asp Leu Asn Tyr 340 345 350 Gly Glu Ile Ala Lys Ile Phe Arg Ala Gly Cys Ile Ile Arg Ala Gln 355 360 365 Phe Leu Gln Lys Ile Thr Asp Ala Tyr Ala Glu Asn Pro Gln Ile Ala 370 375 380 Asn Leu Leu Leu Ala Pro Tyr Phe Lys Gln Ile Ala Asp Asp Tyr Gln 385 390 395 400 Gln Ala Leu Arg Asp Val Val Ala Tyr Ala Val Gln Asn Gly Ile Pro 405 410 415 Val Pro Thr Phe Ala Ala Ala Val Ala Tyr Tyr Asp Ser Tyr Arg Ala 420 425 430 Ala Val Leu Pro Ala Asn Leu Ile Gln Ala Gln Arg Asp Tyr Phe Gly 435 440 445 Ala His Thr Tyr Lys Arg Ile Asp Lys Glu Gly Val Phe His Thr Glu 450 455 460 Trp Leu Asp 465 20 1407 DNA Escherichia coli CDS (1)...(1407) 20 atg tca aag caa cag atc ggc gta gtc ggt atg gca gtg atg ggg cgc 48 Met Ser Lys Gln Gln Ile Gly Val Val Gly Met Ala Val Met Gly Arg 1 5 10 15 aac ctt gcg ctc aac atc gaa agc cgt ggt tat acc gtc tct att ttc 96 Asn Leu Ala Leu Asn Ile Glu Ser Arg Gly Tyr Thr Val Ser Ile Phe 20 25 30 aac cgt tcc cgt gaa aag acg gaa gaa gtg att gcc gaa aat cca ggc 144 Asn Arg Ser Arg Glu Lys Thr Glu Glu Val Ile Ala Glu Asn Pro Gly 35 40 45 aag aaa ctg gtt cct tac tat acg gtg aaa gaa ttt gtt gaa tct ctg 192 Lys Lys Leu Val Pro Tyr Tyr Thr Val Lys Glu Phe Val Glu Ser Leu 50 55 60 gaa acg cct cgt cgc atc ttg tta atg gtg aaa gca ggt gca ggc acg 240 Glu Thr Pro Arg Arg Ile Leu Leu Met Val Lys Ala Gly Ala Gly Thr 65 70 75 80 gat gct gct att gat tcc ctt aag cca tac ctc gat aaa ggt gac atc 288 Asp Ala Ala Ile Asp Ser Leu Lys Pro Tyr Leu Asp Lys Gly Asp Ile 85 90 95 atc att gat ggt ggt aat acc ttc ttc cag gac acc att cgt cgt aac 336 Ile Ile Asp Gly Gly Asn Thr Phe Phe Gln Asp Thr Ile Arg Arg Asn 100 105 110 cgt gag ctt tct gca gaa ggc ttt aac ttc atc ggt acc ggt gtt tcc 384 Arg Glu Leu Ser Ala Glu Gly Phe Asn Phe Ile Gly Thr Gly Val Ser 115 120 125 ggt ggt gag gag ggc gca cta aaa ggt cct tcc att atg cct ggt ggg 432 Gly Gly Glu Glu Gly Ala Leu Lys Gly Pro Ser Ile Met Pro Gly Gly 130 135 140 cag aaa gaa gcc tat gaa cta gtt gcg ccg atc ctg acc aaa atc gcc 480 Gln Lys Glu Ala Tyr Glu Leu Val Ala Pro Ile Leu Thr Lys Ile Ala 145 150 155 160 gca gtg gct gaa gac ggt gag cca tgc gtt acc tat att ggt gcc gat 528 Ala Val Ala Glu Asp Gly Glu Pro Cys Val Thr Tyr Ile Gly Ala Asp 165 170 175 ggc gca ggt cac tat gtg aag atg gtt cac aac ggt att gaa tac ggc 576 Gly Ala Gly His Tyr Val Lys Met Val His Asn Gly Ile Glu Tyr Gly 180 185 190 gat atg cag ctg att gct gaa gcc tat tct ctg ctt aaa ggt ggt ctg 624 Asp Met Gln Leu Ile Ala Glu Ala Tyr Ser Leu Leu Lys Gly Gly Leu 195 200 205 aac ctc acc aac gaa gaa ctg gcg cag atc ttt acc gag tgg aat aac 672 Asn Leu Thr Asn Glu Glu Leu Ala Gln Ile Phe Thr Glu Trp Asn Asn 210 215 220 ggt gaa ctg agc agc tac ctg atc gac att acc aaa gac atc ttc act 720 Gly Glu Leu Ser Ser Tyr Leu Ile Asp Ile Thr Lys Asp Ile Phe Thr 225 230 235 240 aaa aaa gat gaa gac ggt aac tac ctg gtt gat gtg atc ctg gat gaa 768 Lys Lys Asp Glu Asp Gly Asn Tyr Leu Val Asp Val Ile Leu Asp Glu 245 250 255 gcg gca aac aaa ggt acg ggc aaa tgg acc agc cag agc gca ctg gat 816 Ala Ala Asn Lys Gly Thr Gly Lys Trp Thr Ser Gln Ser Ala Leu Asp 260 265 270 ctc ggc gaa ccg ctg tcg ctg att acc gag tct gtg ttt gca cga tac 864 Leu Gly Glu Pro Leu Ser Leu Ile Thr Glu Ser Val Phe Ala Arg Tyr 275 280 285 atc tct tct ctg aaa gat cag cgc gtt gct gcg tct aaa gtt ctc tct 912 Ile Ser Ser Leu Lys Asp Gln Arg Val Ala Ala Ser Lys Val Leu Ser 290 295 300 ggc cca caa gcg cag cca gct ggc gac aag gct gag ttc atc gaa aaa 960 Gly Pro Gln Ala Gln Pro Ala Gly Asp Lys Ala Glu Phe Ile Glu Lys 305 310 315 320 gtt cgc cgt gca ctg tat ctg ggc aaa atc gtt tct tac gct cag ggg 1008 Val Arg Arg Ala Leu Tyr Leu Gly Lys Ile Val Ser Tyr Ala Gln Gly 325 330 335 ttc tct caa ctg cgt gcg gcg tct gaa gag tac aac tgg gat ctg aac 1056 Phe Ser Gln Leu Arg Ala Ala Ser Glu Glu Tyr Asn Trp Asp Leu Asn 340 345 350 tac ggc gaa atc gcg aag att ttc cgt gct ggc tgc atc atc cgt gcg 1104 Tyr Gly Glu Ile Ala Lys Ile Phe Arg Ala Gly Cys Ile Ile Arg Ala 355 360 365 cag ttc ctg cag aaa atc acc gat gct tat gcc gaa aat ccg cag atc 1152 Gln Phe Leu Gln Lys Ile Thr Asp Ala Tyr Ala Glu Asn Pro Gln Ile 370 375 380 gct aac ctg ctg ctg gct cct tac ttc aag caa att gcc gat gac tac 1200 Ala Asn Leu Leu Leu Ala Pro Tyr Phe Lys Gln Ile Ala Asp Asp Tyr 385 390 395 400 cag cag gcg ctg cgc gat gtc gtc gct tat gcg gta cag aac ggt atc 1248 Gln Gln Ala Leu Arg Asp Val Val Ala Tyr Ala Val Gln Asn Gly Ile 405 410 415 ccg gtt ccg acc ttc gcc gct gcg gtt gcc tat tat gac agc tac cgc 1296 Pro Val Pro Thr Phe Ala Ala Ala Val Ala Tyr Tyr Asp Ser Tyr Arg 420 425 430 gcc gct gtt ctg cct gcg aac ctg atc cag gca cag cgt gac tat ttc 1344 Ala Ala Val Leu Pro Ala Asn Leu Ile Gln Ala Gln Arg Asp Tyr Phe 435 440 445 ggt gcg cat act tat aag cgc att gat aaa gaa ggt gtg ttc cat acc 1392 Gly Ala His Thr Tyr Lys Arg Ile Asp Lys Glu Gly Val Phe His Thr 450 455 460 gaa tgg ctg gat taa 1407 Glu Trp Leu Asp * 465 21 467 PRT Escherichia coli 21 Ser Lys Gln Gln Ile Gly Val Val Gly Met Ala Val Met Gly Arg Asn 1 5 10 15 Leu Ala Leu Asn Ile Glu Ser Arg Gly Tyr Thr Val Ser Ile Phe Asn 20 25 30 Arg Ser Arg Glu Lys Thr Glu Glu Val Ile Ala Glu Asn Pro Gly Lys 35 40 45 Lys Leu Val Pro Tyr Tyr Thr Val Lys Glu Phe Val Glu Ser Leu Glu 50 55 60 Thr Pro Arg Arg Ile Leu Leu Met Val Lys Ala Gly Ala Gly Thr Asp 65 70 75 80 Ala Ala Ile Asp Ser Leu Lys Pro Tyr Leu Asp Lys Gly Asp Ile Ile 85 90 95 Ile Asp Gly Gly Asn Thr Phe Phe Gln Asp Thr Ile Arg Arg Asn Arg 100 105 110 Glu Leu Ser Ala Glu Gly Phe Asn Phe Ile Gly Thr Gly Val Ser Gly 115 120 125 Gly Glu Glu Gly Ala Leu Lys Gly Pro Ser Ile Met Pro Gly Gly Gln 130 135 140 Lys Glu Ala Tyr Glu Leu Val Ala Pro Ile Leu Thr Lys Ile Ala Ala 145 150 155 160 Val Ala Glu Asp Gly Glu Pro Cys Val Thr Tyr Ile Gly Ala Asp Gly 165 170 175 Ala Gly His Tyr Val Lys Met Val His Asn Gly Ile Glu Tyr Gly Asp 180 185 190 Met Gln Leu Ile Ala Glu Ala Tyr Ser Leu Leu Lys Gly Gly Leu Asn 195 200 205 Leu Thr Asn Glu Glu Leu Ala Gln Ile Phe Thr Glu Trp Asn Asn Gly 210 215 220 Glu Leu Ser Ser Tyr Leu Ile Asp Ile Thr Lys Asp Ile Phe Thr Lys 225 230 235 240 Lys Asp Glu Asp Gly Asn Tyr Leu Val Asp Val Ile Leu Asp Glu Ala 245 250 255 Ala Asn Lys Gly Thr Gly Lys Trp Thr Ser Gln Ser Ala Leu Asp Leu 260 265 270 Gly Glu Pro Leu Ser Leu Ile Thr Glu Ser Val Phe Ala Arg Tyr Ile 275 280 285 Ser Ser Leu Lys Asp Gln Arg Val Ala Ala Ser Lys Val Leu Ser Gly 290 295 300 Pro Gln Ala Gln Pro Ala Gly Asp Lys Ala Glu Phe Ile Glu Lys Val 305 310 315 320 Arg Arg Ala Leu Tyr Leu Gly Lys Ile Val Ser Tyr Ala Gln Gly Phe 325 330 335 Ser Gln Leu Arg Ala Ala Ser Glu Glu Tyr Asn Trp Asp Leu Asn Tyr 340 345 350 Gly Glu Ile Ala Lys Ile Phe Arg Ala Gly Cys Ile Ile Arg Ala Gln 355 360 365 Phe Leu Gln Lys Ile Thr Asp Ala Tyr Ala Glu Asn Pro Gln Ile Ala 370 375 380 Asn Leu Leu Leu Ala Pro Tyr Phe Lys Gln Ile Ala Asp Asp Tyr Gln 385 390 395 400 Gln Ala Leu Arg Asp Val Val Ala Tyr Ala Val Gln Asn Gly Ile Pro 405 410 415 Val Pro Thr Phe Ala Ala Ala Val Ala Tyr Tyr Asp Ser Tyr Arg Ala 420 425 430 Ala Val Leu Pro Ala Asn Leu Ile Gln Ala Gln Arg Asp Tyr Phe Gly 435 440 445 Ala His Thr Tyr Lys Arg Ile Asp Lys Glu Gly Val Phe His Thr Glu 450 455 460 Trp Leu Asp 465 22 1407 DNA Escherichia coli 22 atgtcaaagc aacagatcgg cgtagtcggt atggcagtga tggggcgcaa ccttgcgctc 60 aacatcgaaa gccgtggtta taccgtctct attttcaacc gttcccgtga aaagacggaa 120 gaagtgattg ccgaaaatcc aggcaagaaa ctggttcctt actatacggt gaaagaattt 180 gttgaatctc tggaaacgcc tcgtcgcatc ttgttaatgg tgaaagcagg tgcaggcacg 240 gatgctgcta ttgattccct taagccatac ctcgataaag gtgacatcat cattgatggt 300 ggtaatacct tcttccagga caccattcgt cgtaaccgtg agctttctgc agaaggcttt 360 aacttcatcg gtaccggtgt ttccggtggt gaggagggcg cactaaaagg tccttccatt 420 atgcctggtg ggcagaaaga agcctatgaa ctagttgcgc cgatcctgac caaaatcgcc 480 gcagtggctg aagacggtga gccatgcgtt acctatattg gtgccgatgg cgcaggtcac 540 tatgtgaaga tggttcacaa cggtattgaa tacggcgata tgcagctgat tgctgaagcc 600 tattctctgc ttaaaggtgg tctgaacctc accaacgaag aactggcgca gatctttacc 660 gagtggaata acggtgaact gagcagctac ctgatcgaca ttaccaaaga catcttcact 720 aaaaaagatg aagacggtaa ctacctggtt gatgtgatcc tggatgaagc ggcaaacaaa 780 ggtacgggca aatggaccag ccagagcgca ctggatctcg gcgaaccgct gtcgctgatt 840 accgagtctg tgtttgcacg atacatctct tctctgaaag atcagcgcgt tgctgcgtct 900 aaagttctct ctggcccaca agcgcagcca gctggcgaca aggctgagtt catcgaaaaa 960 gttcgccgtg cactgtatct gggcaaaatc gtttcttacg ctcaggggtt ctctcaactg 1020 cgtgcggcgt ctgaagagta caactgggat ctgaactacg gcgaaatcgc gaagattttc 1080 cgtgctggct gcatcatccg tgcgcagttc ctgcagaaaa tcaccgatgc ttatgccgaa 1140 aatccgcaga tcgctaacct gctgctggct ccttacttca agcaaattgc cgatgactac 1200 cagcaggcgc tgcgcgatgt cgtcgcttat gcggtacaga acggtatccc ggttccgacc 1260 ttcgccgctg cggttgccta ttatgacagc taccgcgccg ctgttctgcc tgcgaacctg 1320 atccaggcac agcgtgacta tttcggtgcg catacttata agcgcattga taaagaaggt 1380 gtgttccata ccgaatggct ggattaa 1407 23 468 PRT Escherichia coli 23 Met Ser Lys Gln Gln Ile Gly Val Val Gly Met Ala Val Met Gly Arg 1 5 10 15 Asn Leu Ala Leu Asn Ile Glu Ser Arg Gly Tyr Thr Val Ser Ile Phe 20 25 30 Asn Arg Ser Arg Glu Lys Thr Glu Glu Val Ile Ala Glu Asn Pro Gly 35 40 45 Lys Lys Leu Val Pro Tyr Tyr Thr Val Lys Glu Phe Val Glu Ser Leu 50 55 60 Glu Thr Pro Arg Arg Ile Leu Leu Met Val Lys Ala Gly Ala Gly Thr 65 70 75 80 Asp Ala Ala Ile Asp Ser Leu Lys Pro Tyr Leu Asp Lys Gly Asp Ile 85 90 95 Ile Ile Asp Gly Gly Asn Thr Phe Phe Gln Asp Thr Ile Arg Arg Asn 100 105 110 Arg Glu Leu Ser Ala Glu Gly Phe Asn Phe Ile Gly Thr Gly Val Ser 115 120 125 Gly Gly Glu Glu Gly Ala Leu Lys Gly Pro Ser Ile Met Pro Gly Gly 130 135 140 Gln Lys Glu Ala Tyr Glu Leu Val Ala Pro Ile Leu Thr Lys Ile Ala 145 150 155 160 Ala Val Ala Glu Asp Gly Glu Pro Cys Val Thr Tyr Ile Gly Ala Asp 165 170 175 Gly Ala Gly His Tyr Val Lys Met Val His Asn Gly Ile Glu Tyr Gly 180 185 190 Asp Met Gln Leu Ile Ala Glu Ala Tyr Ser Leu Leu Lys Gly Gly Leu 195 200 205 Asn Leu Thr Asn Glu Glu Leu Ala Gln Ile Phe Thr Glu Trp Asn Asn 210 215 220 Gly Glu Leu Ser Ser Tyr Leu Ile Asp Ile Thr Lys Asp Ile Phe Thr 225 230 235 240 Lys Lys Asp Glu Asp Gly Asn Tyr Leu Val Asp Val Ile Leu Asp Glu 245 250 255 Ala Ala Asn Lys Gly Thr Gly Lys Trp Thr Ser Gln Ser Ala Leu Asp 260 265 270 Leu Gly Glu Pro Leu Ser Leu Ile Thr Glu Ser Val Phe Ala Arg Tyr 275 280 285 Ile Ser Ser Leu Lys Asp Gln Arg Val Ala Ala Ser Lys Val Leu Ser 290 295 300 Gly Pro Gln Ala Gln Pro Ala Gly Asp Lys Ala Glu Phe Ile Glu Lys 305 310 315 320 Val Arg Arg Ala Leu Tyr Leu Gly Lys Ile Val Ser Tyr Ala Gln Gly 325 330 335 Phe Ser Gln Leu Arg Ala Ala Ser Glu Glu Tyr Asn Trp Asp Leu Asn 340 345 350 Tyr Gly Glu Ile Ala Lys Ile Phe Arg Ala Gly Cys Ile Ile Arg Ala 355 360 365 Gln Phe Leu Gln Lys Ile Thr Asp Ala Tyr Ala Glu Asn Pro Gln Ile 370 375 380 Ala Asn Leu Leu Leu Ala Pro Tyr Phe Lys Gln Ile Ala Asp Asp Tyr 385 390 395 400 Gln Gln Ala Leu Arg Asp Val Val Ala Tyr Ala Val Gln Asn Gly Ile 405 410 415 Pro Val Pro Thr Phe Ala Ala Ala Val Ala Tyr Tyr Asp Ser Tyr Arg 420 425 430 Ala Ala Val Leu Pro Ala Asn Leu Ile Gln Ala Gln Arg Asp Tyr Phe 435 440 445 Gly Ala His Thr Tyr Lys Arg Ile Asp Lys Glu Gly Val Phe His Thr 450 455 460 Glu Trp Leu Asp 465 24 1407 DNA Escherichia coli 24 atgtcaaagc aacagatcgg cgtagtcggt atggcagtga tggggcgcaa ccttgcgccc 60 aacatcgaaa gccgtggtta taccgtctct attttcaacc gttcccgtga aaagacggaa 120 gaagtgattg ccgaaaatcc aggcaagaaa ctggttcctt actatacggt gaaagaattt 180 gttgaatctc tggaaacgcc tcgtcgcatc ttgttaatgg tgaaagcagg tgcaggcacg 240 gatgctgcta ttgattccct taagccatac ctcgataaag gtgacatcat cattgatagt 300 ggtaatacct tcttccagga caccattcgt cgtaaccgtg agctttctgc agaaggcttt 360 aacttcatcg gtaccggtgt ttccggtggt gaggagggcg cactaaaagg tccttccatt 420 atgcctggtg ggcagaaaga agcctatgaa ctagttgcgc cgatcctgac caaaatcgcc 480 gcagtggctg aagacggtga gccatgcgtt acctatattg gtgccgatgg cgcaggtcac 540 tatgtgaaga tggttcacaa cggtattgaa tacggcgata tgcagctgat tgctgaagcc 600 tattctctgc ttaaaggtgg tctgaacctc accaacgaag aactggcgca gatctttacc 660 gagtggaata acggtgaact gagcagctac ctgatcgaca ttaccaaaga catcttcact 720 aaaaaagatg aagacggtaa ctacctggtt gatgtgatcc tggatgaagc ggcaaacaaa 780 ggtacgggca aatggaccag ccagagcgca ctggatctcg gcgaaccgct gtcgctgatt 840 accgagtctg tgtttgcacg atacatctct tctctgaaag atcagcgcgt tgctgcgtct 900 aaagttctct ctggcccaca agcgcagcca gctggcgaca aggctgagtt catcgaaaaa 960 gttcgccgtg cactgtatct gggcaaaatc gtttcttacg ctcaggggtt ctctcaactg 1020 cgtgcggcgt ctgaagagta caactgggat ctgaactacg gcgaaatcgc gaagattttc 1080 cgtgctggct gcatcatccg tgcgcagttc ctgcagaaaa tcaccgatgc ttatgccgaa 1140 aatccgcaga tcgctaacct gctgctggct ccttacttca agcaaattgc cgatgactac 1200 cagcaggcgc tgcgcgatgt cgtcgcttat gcggtacaga acggtatccc ggttccgacc 1260 ttcgccgctg cggttgccta ttatgacagc taccgcgccg ctgttctgcc tgcgaacctg 1320 atccaggcac agcgtgacta tttcggtgcg catacttata agcgcattga taaagaaggt 1380 gtgttccata ccgaatggct ggattaa 1407 25 468 PRT Escherichia coli 25 Met Ser Lys Gln Gln Ile Gly Val Val Gly Met Ala Val Met Gly Arg 1 5 10 15 Asn Leu Ala Pro Asn Ile Glu Ser Arg Gly Tyr Thr Val Ser Ile Phe 20 25 30 Asn Arg Ser Arg Glu Lys Thr Glu Glu Val Ile Ala Glu Asn Pro Gly 35 40 45 Lys Lys Leu Val Pro Tyr Tyr Thr Val Lys Glu Phe Val Glu Ser Leu 50 55 60 Glu Thr Pro Arg Arg Ile Leu Leu Met Val Lys Ala Gly Ala Gly Thr 65 70 75 80 Asp Ala Ala Ile Asp Ser Leu Lys Pro Tyr Leu Asp Lys Gly Asp Ile 85 90 95 Ile Ile Asp Ser Gly Asn Thr Phe Phe Gln Asp Thr Ile Arg Arg Asn 100 105 110 Arg Glu Leu Ser Ala Glu Gly Phe Asn Phe Ile Gly Thr Gly Val Ser 115 120 125 Gly Gly Glu Glu Gly Ala Leu Lys Gly Pro Ser Ile Met Pro Gly Gly 130 135 140 Gln Lys Glu Ala Tyr Glu Leu Val Ala Pro Ile Leu Thr Lys Ile Ala 145 150 155 160 Ala Val Ala Glu Asp Gly Glu Pro Cys Val Thr Tyr Ile Gly Ala Asp 165 170 175 Gly Ala Gly His Tyr Val Lys Met Val His Asn Gly Ile Glu Tyr Gly 180 185 190 Asp Met Gln Leu Ile Ala Glu Ala Tyr Ser Leu Leu Lys Gly Gly Leu 195 200 205 Asn Leu Thr Asn Glu Glu Leu Ala Gln Ile Phe Thr Glu Trp Asn Asn 210 215 220 Gly Glu Leu Ser Ser Tyr Leu Ile Asp Ile Thr Lys Asp Ile Phe Thr 225 230 235 240 Lys Lys Asp Glu Asp Gly Asn Tyr Leu Val Asp Val Ile Leu Asp Glu 245 250 255 Ala Ala Asn Lys Gly Thr Gly Lys Trp Thr Ser Gln Ser Ala Leu Asp 260 265 270 Leu Gly Glu Pro Leu Ser Leu Ile Thr Glu Ser Val Phe Ala Arg Tyr 275 280 285 Ile Ser Ser Leu Lys Asp Gln Arg Val Ala Ala Ser Lys Val Leu Ser 290 295 300 Gly Pro Gln Ala Gln Pro Ala Gly Asp Lys Ala Glu Phe Ile Glu Lys 305 310 315 320 Val Arg Arg Ala Leu Tyr Leu Gly Lys Ile Val Ser Tyr Ala Gln Gly 325 330 335 Phe Ser Gln Leu Arg Ala Ala Ser Glu Glu Tyr Asn Trp Asp Leu Asn 340 345 350 Tyr Gly Glu Ile Ala Lys Ile Phe Arg Ala Gly Cys Ile Ile Arg Ala 355 360 365 Gln Phe Leu Gln Lys Ile Thr Asp Ala Tyr Ala Glu Asn Pro Gln Ile 370 375 380 Ala Asn Leu Leu Leu Ala Pro Tyr Phe Lys Gln Ile Ala Asp Asp Tyr 385 390 395 400 Gln Gln Ala Leu Arg Asp Val Val Ala Tyr Ala Val Gln Asn Gly Ile 405 410 415 Pro Val Pro Thr Phe Ala Ala Ala Val Ala Tyr Tyr Asp Ser Tyr Arg 420 425 430 Ala Ala Val Leu Pro Ala Asn Leu Ile Gln Ala Gln Arg Asp Tyr Phe 435 440 445 Gly Ala His Thr Tyr Lys Arg Ile Asp Lys Glu Gly Val Phe His Thr 450 455 460 Glu Trp Leu Asp 465 26 1407 DNA Escherichia coli 26 atgtcaaagc aacagatcgg cgtagtcggt atggcagtga tggggcgcaa ccttgcgctc 60 aacatcgaaa gccgtggtta taccgtctct attttcaacc gttcccgtga aaagacggaa 120 gaagtgattg ccgaaaatcc aggcaagaaa ctggttcctt actatacggt gaaagaattt 180 gttgaatctc tggaaacgcc tcgtcgcatc ttgttaatgg tgaaagcagg tgcaggcacg 240 gatgctgcta ttgattccct taagccatac ctcgataaag gtgacatcat cattgatggt 300 ggtaatacct tcttccagga caccattcgt cgtaaccgtg agctttctgc agaaggcttt 360 aacttcatcg gtaccggtgt ttccggtggt gaggagggcg cactaaaagg tccttccatt 420 atgcctggtg ggcagaaaga agcctatgaa ctagttgcgc cgatcctgac caaaatcgcc 480 gcagtggctg aagacggtga gccatgcgtt acctatattg gtgccgatgg cgcaggtcac 540 tatgtgaaga tggttcacaa cggtattgaa tacggcgata tgcagctgat tgctgaagcc 600 tattctctgc ttaaaggtgg tctgaacctc accaacgaag aactggcgca gatctttacc 660 gagtggaata acggtgaact gagcagctac ctgatcgaca ttaccaaaga catcttcact 720 aaaaaagatg aagacggtaa ctacctggtt gatgtgatcc tggatgaggc ggcaaacaaa 780 ggtacgggca aatggaccag ccagagcgca ctggatctcg gcgaaccgct gtcgctgatt 840 accgagtctg tgtttgcacg atacatctct tctctgaaag atcagcgcgt tgctgcgtct 900 aaagttctct ctggcccaca agcgcagcca gctggcgaca aggctgagtt catcgaaaaa 960 gttcgccgtg cactgtatct gggcaaaatc gtttcttacg ctcaggggtt ctctcaactg 1020 cgtgcggcgt ctgaagagta caactgggat ctgaactacg gcgaaatcgc gaagattttc 1080 cgtgctggct gcatcatccg tgcgcagttc ctgcagaaaa tcaccgatgc ttatgccgaa 1140 aatccgcaga tcgctaacct gctgctggct ccttacttca agcaaattgc cgatgactac 1200 cagcaggcgc tgcgcgatgt cgtcgcttat gcggtacaga acggtatccc ggttccgacc 1260 ttcgccgctg cggttgccta ttatgacagc taccgcgccg ctgttctgcc tgcgaacctg 1320 atccaggcac agcgtgacta tttcggtgcg catacttata agcgcgttga taaagaaggt 1380 gtgttccata ccgaatggct ggattaa 1407 27 468 PRT Escherichia coli 27 Met Ser Lys Gln Gln Ile Gly Val Val Gly Met Ala Val Met Gly Arg 1 5 10 15 Asn Leu Ala Leu Asn Ile Glu Ser Arg Gly Tyr Thr Val Ser Ile Phe 20 25 30 Asn Arg Ser Arg Glu Lys Thr Glu Glu Val Ile Ala Glu Asn Pro Gly 35 40 45 Lys Lys Leu Val Pro Tyr Tyr Thr Val Lys Glu Phe Val Glu Ser Leu 50 55 60 Glu Thr Pro Arg Arg Ile Leu Leu Met Val Lys Ala Gly Ala Gly Thr 65 70 75 80 Asp Ala Ala Ile Asp Ser Leu Lys Pro Tyr Leu Asp Lys Gly Asp Ile 85 90 95 Ile Ile Asp Gly Gly Asn Thr Phe Phe Gln Asp Thr Ile Arg Arg Asn 100 105 110 Arg Glu Leu Ser Ala Glu Gly Phe Asn Phe Ile Gly Thr Gly Val Ser 115 120 125 Gly Gly Glu Glu Gly Ala Leu Lys Gly Pro Ser Ile Met Pro Gly Gly 130 135 140 Gln Lys Glu Ala Tyr Glu Leu Val Ala Pro Ile Leu Thr Lys Ile Ala 145 150 155 160 Ala Val Ala Glu Asp Gly Glu Pro Cys Val Thr Tyr Ile Gly Ala Asp 165 170 175 Gly Ala Gly His Tyr Val Lys Met Val His Asn Gly Ile Glu Tyr Gly 180 185 190 Asp Met Gln Leu Ile Ala Glu Ala Tyr Ser Leu Leu Lys Gly Gly Leu 195 200 205 Asn Leu Thr Asn Glu Glu Leu Ala Gln Ile Phe Thr Glu Trp Asn Asn 210 215 220 Gly Glu Leu Ser Ser Tyr Leu Ile Asp Ile Thr Lys Asp Ile Phe Thr 225 230 235 240 Lys Lys Asp Glu Asp Gly Asn Tyr Leu Val Asp Val Ile Leu Asp Glu 245 250 255 Ala Ala Asn Lys Gly Thr Gly Lys Trp Thr Ser Gln Ser Ala Leu Asp 260 265 270 Leu Gly Glu Pro Leu Ser Leu Ile Thr Glu Ser Val Phe Ala Arg Tyr 275 280 285 Ile Ser Ser Leu Lys Asp Gln Arg Val Ala Ala Ser Lys Val Leu Ser 290 295 300 Gly Pro Gln Ala Gln Pro Ala Gly Asp Lys Ala Glu Phe Ile Glu Lys 305 310 315 320 Val Arg Arg Ala Leu Tyr Leu Gly Lys Ile Val Ser Tyr Ala Gln Gly 325 330 335 Phe Ser Gln Leu Arg Ala Ala Ser Glu Glu Tyr Asn Trp Asp Leu Asn 340 345 350 Tyr Gly Glu Ile Ala Lys Ile Phe Arg Ala Gly Cys Ile Ile Arg Ala 355 360 365 Gln Phe Leu Gln Lys Ile Thr Asp Ala Tyr Ala Glu Asn Pro Gln Ile 370 375 380 Ala Asn Leu Leu Leu Ala Pro Tyr Phe Lys Gln Ile Ala Asp Asp Tyr 385 390 395 400 Gln Gln Ala Leu Arg Asp Val Val Ala Tyr Ala Val Gln Asn Gly Ile 405 410 415 Pro Val Pro Thr Phe Ala Ala Ala Val Ala Tyr Tyr Asp Ser Tyr Arg 420 425 430 Ala Ala Val Leu Pro Ala Asn Leu Ile Gln Ala Gln Arg Asp Tyr Phe 435 440 445 Gly Ala His Thr Tyr Lys Arg Val Asp Lys Glu Gly Val Phe His Thr 450 455 460 Glu Trp Leu Asp 465 28 1407 DNA Escherichia coli 28 atgtcaaagc aacagatcgg cgtagtcggt atggcagtga tggggcgcaa cctagcgctc 60 aacatcgaaa gccgtggtta taccgtctct attttcaacc gcccccgtga gaagacggaa 120 gaagtgattg ccgaaaatcc aggcaagaaa ctggttcctt actatacggt gaaagagttt 180 gttgaatctc tggaaacgcc tcgccgcatc ctgttaatgg tgaaagcagg tgcaggcacg 240 gatgctgcta ttgattccct caggccgtac ctcgataaag gtgacatcat cattgatggt 300 ggtaacacct tcttccagga caccattcgt cgtaaccgtg agctttctgc cgaaggcttt 360 aacttcatcg gtaccggtgt ttccggcgga gaagaaggcg cgctgaaagg tccttccatt 420 atgcctggtg ggcagaaaga agcctatgaa ctggttgcgc cgatcctgac caaaatcgcc 480 gcagtggctg aagacggtga gccatgcgtt acctatattg gtgccgatgg cgcaggtcac 540 tatgtgaaga tggttcacaa cggtattgaa tacggagata tgcaactgat tgctgaagcc 600 tattctctgc ttaaaggtgg cctgaacctc accaacgaag aactggcgca gacgtttacc 660 gagtggaata acggtgaact gagcagctac ctgatcgaca tcaccaaaga tatcttcacc 720 aaaaaagatg aagacggtaa ctacctggtt gatgtgatcc tggatgaagc ggctaacaaa 780 ggtaccggta aatggaccag ccagagcgca ctggatctcg gcgaaccgct gtcgctgatt 840 accgagtctg tgtttgcacg ttatatctct tctctgaaag atcagcgtgt tgccgcatct 900 aaagttctct ctggcccgca agcacagcca gcaggcgaca aggctgagtt catcgaaaaa 960 gttcgccgtg cgctgtatct gggcaaaatc gtttcttacg cccagggctt ctctcagctg 1020 cgtgctgcgt ctgaagagtt caactgggat ctgaactacg gcgaaatcgc gaagattttc 1080 cgtgctggct gcatcattcg tgcgcagttc ctgcagaaaa ttaccgatgc ttatgccgaa 1140 aatccgcaga tcgctaacct gctgctggct ccgtacttca agcaaattgc cgatgattac 1200 cagcaggcgc tgcgtgatgt cgttgcttat gcggtacaga acggtatccc ggttccgacc 1260 ttcgccgctg cggttgccta ttacgatagc taccgtgccg ctgttctgcc tgcgaacctg 1320 atccaggcac agcgtgacta tttcggtgca catacttata agcgcattga taaagaaggt 1380 gtgttccata ctgaatggct ggattaa 1407 29 468 PRT Escherichia coli 29 Met Ser Lys Gln Gln Ile Gly Val Val Gly Met Ala Val Met Gly Arg 1 5 10 15 Asn Leu Ala Leu Asn Ile Glu Ser Arg Gly Tyr Thr Val Ser Ile Phe 20 25 30 Asn Arg Pro Arg Glu Lys Thr Glu Glu Val Ile Ala Glu Asn Pro Gly 35 40 45 Lys Lys Leu Val Pro Tyr Tyr Thr Val Lys Glu Phe Val Glu Ser Leu 50 55 60 Glu Thr Pro Arg Arg Ile Leu Leu Met Val Lys Ala Gly Ala Gly Thr 65 70 75 80 Asp Ala Ala Ile Asp Ser Leu Arg Pro Tyr Leu Asp Lys Gly Asp Ile 85 90 95 Ile Ile Asp Gly Gly Asn Thr Phe Phe Gln Asp Thr Ile Arg Arg Asn 100 105 110 Arg Glu Leu Ser Ala Glu Gly Phe Asn Phe Ile Gly Thr Gly Val Ser 115 120 125 Gly Gly Glu Glu Gly Ala Leu Lys Gly Pro Ser Ile Met Pro Gly Gly 130 135 140 Gln Lys Glu Ala Tyr Glu Leu Val Ala Pro Ile Leu Thr Lys Ile Ala 145 150 155 160 Ala Val Ala Glu Asp Gly Glu Pro Cys Val Thr Tyr Ile Gly Ala Asp 165 170 175 Gly Ala Gly His Tyr Val Lys Met Val His Asn Gly Ile Glu Tyr Gly 180 185 190 Asp Met Gln Leu Ile Ala Glu Ala Tyr Ser Leu Leu Lys Gly Gly Leu 195 200 205 Asn Leu Thr Asn Glu Glu Leu Ala Gln Thr Phe Thr Glu Trp Asn Asn 210 215 220 Gly Glu Leu Ser Ser Tyr Leu Ile Asp Ile Thr Lys Asp Ile Phe Thr 225 230 235 240 Lys Lys Asp Glu Asp Gly Asn Tyr Leu Val Asp Val Ile Leu Asp Glu 245 250 255 Ala Ala Asn Lys Gly Thr Gly Lys Trp Thr Ser Gln Ser Ala Leu Asp 260 265 270 Leu Gly Glu Pro Leu Ser Leu Ile Thr Glu Ser Val Phe Ala Arg Tyr 275 280 285 Ile Ser Ser Leu Lys Asp Gln Arg Val Ala Ala Ser Lys Val Leu Ser 290 295 300 Gly Pro Gln Ala Gln Pro Ala Gly Asp Lys Ala Glu Phe Ile Glu Lys 305 310 315 320 Val Arg Arg Ala Leu Tyr Leu Gly Lys Ile Val Ser Tyr Ala Gln Gly 325 330 335 Phe Ser Gln Leu Arg Ala Ala Ser Glu Glu Phe Asn Trp Asp Leu Asn 340 345 350 Tyr Gly Glu Ile Ala Lys Ile Phe Arg Ala Gly Cys Ile Ile Arg Ala 355 360 365 Gln Phe Leu Gln Lys Ile Thr Asp Ala Tyr Ala Glu Asn Pro Gln Ile 370 375 380 Ala Asn Leu Leu Leu Ala Pro Tyr Phe Lys Gln Ile Ala Asp Asp Tyr 385 390 395 400 Gln Gln Ala Leu Arg Asp Val Val Ala Tyr Ala Val Gln Asn Gly Ile 405 410 415 Pro Val Pro Thr Phe Ala Ala Ala Val Ala Tyr Tyr Asp Ser Tyr Arg 420 425 430 Ala Ala Val Leu Pro Ala Asn Leu Ile Gln Ala Gln Arg Asp Tyr Phe 435 440 445 Gly Ala His Thr Tyr Lys Arg Ile Asp Lys Glu Gly Val Phe His Thr 450 455 460 Glu Trp Leu Asp 465 30 1407 DNA Escherichia coli 30 atgtcaaagc aacagatcgg cgtagtcggt atggcagtga tggggcgcaa cctagcgctc 60 aacatcgaaa gccgtggtta taccgtctct attttcaacc gctcccgtga gaagacggaa 120 gaagtgattg ccgaaaatcc aggcaagaaa ctggttcctt actatacggt gaaagagttt 180 gttgaatctc tggaaacgcc tcgccgcatc ctgttaatgg tgaaagcagg tgcaggcacg 240 gatgctgcta ttgattccct caagccgtac ctcgataaag gtgacatcat tattgatggt 300 ggtaacacct tcttccagga caccattcgt cgtaaccgtg agctttctgc cgaaggcttt 360 aacttcatcg gtaccggtgt ttccggcgga gaagaaggcg cgctgaaagg tccttccatt 420 atgcctggtg ggcagaaaga agcctatgaa ctggttgcgc cgatcctgac caaaatcgcc 480 gcagtggctg aagacggtga gccatgcgtt acctatattg gtgccgatgg cgcaggtcac 540 tatgtgaaga tggttcacaa cggtattgaa tacggagata tgcaactgat tgctgaagcc 600 tattctctgc ttaaaggtgg cctgaacctc accaacgaag aactggcgca gacgtttacc 660 gagtggaata acggtgaact gagcagctac ctgatcgaca tcaccaaaga tatcttcacc 720 aaaaaagatg aagacggtaa ctacctggtt gatgtgatcc tggatgaagc ggctaacaaa 780 ggtaccggta aatggaccag ccagagcgca ctggatctcg gcgaaccgct gtcgctgatt 840 accgagtctg tgtttgcacg ttatatctct tctctgaaag atcagcgtgt tgccgcatct 900 aaagttctct ctggcccgca agcacagcca gcaggcgaca aggctgagtt catcgaaaaa 960 gttcgccgtg cgctgtatct gggcaaaatc gtttcttacg cccagggctt ctctcagctg 1020 cgtgctgcgt ctgaagagta caactgggat ctgaactacg gcgaaatcgc gaagattttc 1080 cgtgctggct gcatcattcg tgcgcagttc ctgcagaaaa ttaccgatgc ttatgccgaa 1140 aatccgcaga tcgctaacct gctgctggct ccgtacttca agcaaattgc cgatgattac 1200 cagcaggcgc tgcgtgatgt cgttgcttat gcggtacaga acggtatccc ggttccgacc 1260 ttcgccgctg cggttgccta ttacgatagc taccgtgccg ctgttctgcc tgcgaacctg 1320 atccaggcac agcgtgacta tttcggtgca catacttata agcgcattga taaagaaggt 1380 gtgttccata ctgaatggct ggattaa 1407 31 468 PRT Escherichia coli 31 Met Ser Lys Gln Gln Ile Gly Val Val Gly Met Ala Val Met Gly Arg 1 5 10 15 Asn Leu Ala Leu Asn Ile Glu Ser Arg Gly Tyr Thr Val Ser Ile Phe 20 25 30 Asn Arg Ser Arg Glu Lys Thr Glu Glu Val Ile Ala Glu Asn Pro Gly 35 40 45 Lys Lys Leu Val Pro Tyr Tyr Thr Val Lys Glu Phe Val Glu Ser Leu 50 55 60 Glu Thr Pro Arg Arg Ile Leu Leu Met Val Lys Ala Gly Ala Gly Thr 65 70 75 80 Asp Ala Ala Ile Asp Ser Leu Lys Pro Tyr Leu Asp Lys Gly Asp Ile 85 90 95 Ile Ile Asp Gly Gly Asn Thr Phe Phe Gln Asp Thr Ile Arg Arg Asn 100 105 110 Arg Glu Leu Ser Ala Glu Gly Phe Asn Phe Ile Gly Thr Gly Val Ser 115 120 125 Gly Gly Glu Glu Gly Ala Leu Lys Gly Pro Ser Ile Met Pro Gly Gly 130 135 140 Gln Lys Glu Ala Tyr Glu Leu Val Ala Pro Ile Leu Thr Lys Ile Ala 145 150 155 160 Ala Val Ala Glu Asp Gly Glu Pro Cys Val Thr Tyr Ile Gly Ala Asp 165 170 175 Gly Ala Gly His Tyr Val Lys Met Val His Asn Gly Ile Glu Tyr Gly 180 185 190 Asp Met Gln Leu Ile Ala Glu Ala Tyr Ser Leu Leu Lys Gly Gly Leu 195 200 205 Asn Leu Thr Asn Glu Glu Leu Ala Gln Thr Phe Thr Glu Trp Asn Asn 210 215 220 Gly Glu Leu Ser Ser Tyr Leu Ile Asp Ile Thr Lys Asp Ile Phe Thr 225 230 235 240 Lys Lys Asp Glu Asp Gly Asn Tyr Leu Val Asp Val Ile Leu Asp Glu 245 250 255 Ala Ala Asn Lys Gly Thr Gly Lys Trp Thr Ser Gln Ser Ala Leu Asp 260 265 270 Leu Gly Glu Pro Leu Ser Leu Ile Thr Glu Ser Val Phe Ala Arg Tyr 275 280 285 Ile Ser Ser Leu Lys Asp Gln Arg Val Ala Ala Ser Lys Val Leu Ser 290 295 300 Gly Pro Gln Ala Gln Pro Ala Gly Asp Lys Ala Glu Phe Ile Glu Lys 305 310 315 320 Val Arg Arg Ala Leu Tyr Leu Gly Lys Ile Val Ser Tyr Ala Gln Gly 325 330 335 Phe Ser Gln Leu Arg Ala Ala Ser Glu Glu Tyr Asn Trp Asp Leu Asn 340 345 350 Tyr Gly Glu Ile Ala Lys Ile Phe Arg Ala Gly Cys Ile Ile Arg Ala 355 360 365 Gln Phe Leu Gln Lys Ile Thr Asp Ala Tyr Ala Glu Asn Pro Gln Ile 370 375 380 Ala Asn Leu Leu Leu Ala Pro Tyr Phe Lys Gln Ile Ala Asp Asp Tyr 385 390 395 400 Gln Gln Ala Leu Arg Asp Val Val Ala Tyr Ala Val Gln Asn Gly Ile 405 410 415 Pro Val Pro Thr Phe Ala Ala Ala Val Ala Tyr Tyr Asp Ser Tyr Arg 420 425 430 Ala Ala Val Leu Pro Ala Asn Leu Ile Gln Ala Gln Arg Asp Tyr Phe 435 440 445 Gly Ala His Thr Tyr Lys Arg Ile Asp Lys Glu Gly Val Phe His Thr 450 455 460 Glu Trp Leu Asp 465 32 1407 DNA Escherichia coli 32 atgtcaaagc aacagatcgg cgtcgtcggt atggctgtga tgggacgcaa tcttgcgctc 60 aacatcgaaa gccgtggtta taccgtctct attttcaacc gttcccgtga aaaaacggaa 120 gaagtgattg ccgaaaatcc aggcaagaaa ctggttcctt actatacggt gaaagagttt 180 gttgaatctc tggaaacgcc tcgtcgcatc ctgttaatgg tgaaagcagg tgcaggcacg 240 gatgctgcta ttgattccct caagccttac ctcgataaag gtgacatcat cattgatggt 300 ggtaacacct tcttcctgga caccattcgt cgtaaccgtg agctttctgc agaaggcttt 360 aacttcattg gtaccggtgt ttccggcggt gaaggggggg cgctgaaagg gccttccatc 420 atgcctggtg ggcagaaaga agcctatgaa ctggttgctc cgatcttgac caaaatcgcc 480 gccgttgctg aagacggcga accgtgtgtt acctatattg gtgccgatgg cgcgggtcac 540 tatgtggaga tggttcacaa tggtattgaa tacggtgata tgcaactgat tgctgaagcc 600 tattctctgc ttaaaggcgg cctgaatctc tctaacgaag aactggcaca gacctttacc 660 gagtggaata acggtgaact gagcagctac ctgatcgaca tcaccaaaga tatcttcacc 720 aaaaaagatg aagacggtaa ctacctggtt gatgtgattc tggatgaagc ggctaacaag 780 ggtaccggta aatggaccag ccagagcgcg ctggatctcg gcgaaccgct gtcactgatt 840 accgagtctg tgtttgcacg ttatatctct tctctgaaag atcagcgcgt tgccgcgtct 900 aaagttctca ctggcccgaa agcgcagcca gcaggcgata aggctgagtt tatcgagaaa 960 gttcgtcgtg cgctgtatct gggcaaaatc gtttcttacg ctcagggctt ctctcagctg 1020 cgtgcggcgt ctgaagagta caactgggat ctgaactacg gcgaaatcgc gaagattttc 1080 cgtgctggct gcatcatccg tgcgcagttc ctgcagaaaa tcaccgatgc atatgccgaa 1140 aatccgcaga tcgctaacct gctgctggct ccgtacttca agcaaattgc cgatgactac 1200 cagcaggcgc tgcgtgatgt cgttgcttat gcagtacaga acggtatccc ggttccgacc 1260 ttcgcggctg cggttgccta ttatgacagc taccgcgccg cagttctgcc tgcaaacctg 1320 atccaggcac agcgtgacta tttcggtgcg catacttata agcgcattga taaagaaggt 1380 gtgttccata ctgaatggct ggattaa 1407 33 468 PRT Escherichia coli 33 Met Ser Lys Gln Gln Ile Gly Val Val Gly Met Ala Val Met Gly Arg 1 5 10 15 Asn Leu Ala Leu Asn Ile Glu Ser Arg Gly Tyr Thr Val Ser Ile Phe 20 25 30 Asn Arg Ser Arg Glu Lys Thr Glu Glu Val Ile Ala Glu Asn Pro Gly 35 40 45 Lys Lys Leu Val Pro Tyr Tyr Thr Val Lys Glu Phe Val Glu Ser Leu 50 55 60 Glu Thr Pro Arg Arg Ile Leu Leu Met Val Lys Ala Gly Ala Gly Thr 65 70 75 80 Asp Ala Ala Ile Asp Ser Leu Lys Pro Tyr Leu Asp Lys Gly Asp Ile 85 90 95 Ile Ile Asp Gly Gly Asn Thr Phe Phe Leu Asp Thr Ile Arg Arg Asn 100 105 110 Arg Glu Leu Ser Ala Glu Gly Phe Asn Phe Ile Gly Thr Gly Val Ser 115 120 125 Gly Gly Glu Gly Gly Ala Leu Lys Gly Pro Ser Ile Met Pro Gly Gly 130 135 140 Gln Lys Glu Ala Tyr Glu Leu Val Ala Pro Ile Leu Thr Lys Ile Ala 145 150 155 160 Ala Val Ala Glu Asp Gly Glu Pro Cys Val Thr Tyr Ile Gly Ala Asp 165 170 175 Gly Ala Gly His Tyr Val Glu Met Val His Asn Gly Ile Glu Tyr Gly 180 185 190 Asp Met Gln Leu Ile Ala Glu Ala Tyr Ser Leu Leu Lys Gly Gly Leu 195 200 205 Asn Leu Ser Asn Glu Glu Leu Ala Gln Thr Phe Thr Glu Trp Asn Asn 210 215 220 Gly Glu Leu Ser Ser Tyr Leu Ile Asp Ile Thr Lys Asp Ile Phe Thr 225 230 235 240 Lys Lys Asp Glu Asp Gly Asn Tyr Leu Val Asp Val Ile Leu Asp Glu 245 250 255 Ala Ala Asn Lys Gly Thr Gly Lys Trp Thr Ser Gln Ser Ala Leu Asp 260 265 270 Leu Gly Glu Pro Leu Ser Leu Ile Thr Glu Ser Val Phe Ala Arg Tyr 275 280 285 Ile Ser Ser Leu Lys Asp Gln Arg Val Ala Ala Ser Lys Val Leu Thr 290 295 300 Gly Pro Lys Ala Gln Pro Ala Gly Asp Lys Ala Glu Phe Ile Glu Lys 305 310 315 320 Val Arg Arg Ala Leu Tyr Leu Gly Lys Ile Val Ser Tyr Ala Gln Gly 325 330 335 Phe Ser Gln Leu Arg Ala Ala Ser Glu Glu Tyr Asn Trp Asp Leu Asn 340 345 350 Tyr Gly Glu Ile Ala Lys Ile Phe Arg Ala Gly Cys Ile Ile Arg Ala 355 360 365 Gln Phe Leu Gln Lys Ile Thr Asp Ala Tyr Ala Glu Asn Pro Gln Ile 370 375 380 Ala Asn Leu Leu Leu Ala Pro Tyr Phe Lys Gln Ile Ala Asp Asp Tyr 385 390 395 400 Gln Gln Ala Leu Arg Asp Val Val Ala Tyr Ala Val Gln Asn Gly Ile 405 410 415 Pro Val Pro Thr Phe Ala Ala Ala Val Ala Tyr Tyr Asp Ser Tyr Arg 420 425 430 Ala Ala Val Leu Pro Ala Asn Leu Ile Gln Ala Gln Arg Asp Tyr Phe 435 440 445 Gly Ala His Thr Tyr Lys Arg Ile Asp Lys Glu Gly Val Phe His Thr 450 455 460 Glu Trp Leu Asp 465 34 1407 DNA Escherichia coli 34 atgtcaaagc aacagatcgg cgtcgtcggt atggctgtga tgggacgcaa tcttgcgctc 60 aacatcgaaa gccgtggtta taccgtctct attttcaacc gttcccgtga aaaaacggaa 120 gaagtgattg ccgaaaatcc aggcaagaaa ctggttcctt actatacggt gaaagagttt 180 gttgaatctc tggaaacgcc tcgtcgcatc ctgttaatgg tgaaagcagg tgcaggcacg 240 gatgctgcta ttgattccct caagccttac ctcgataaag gtgacatcat cattgatggt 300 ggtaacacct tcttcctgga caccattcgt cgtaaccgtg agctttctgc agaaggcttt 360 aacttcattg gtaccggtgt ttccggcggt gaagaggggg cgctgaaagg gccttccatc 420 atgcctggtg ggcagaaaga agcctatgaa ctggttgctc cgatcttgac caaaatcgcc 480 gccgttgctg aagacggcga accgtgtgtt acctatattg gtgccgatgg cgcgggtcac 540 tatgtgaaga tggttcacaa tggtattgaa tacggtgata tgcaactgat tgctgaagcc 600 tattctctgc ttaaaggcgg cctgaatctc tctaacgaag aactggcaca gacctttacc 660 gagtggaata acggtgaact gagcagctac ctgatcgaca tcaccaaaga tatcttcacc 720 aaaaaagatg aagacggtaa ctacctggtt gatgtgattc tggatgaggc ggctaacaag 780 ggtaccggta aatggaccag ccagagcgcg ctggatctcg gcgaaccgct gtcactgatt 840 accgagtctg tgtttgcacg ttatatctct tctctgaaag atcagcgcgt tgccgcgtct 900 aaagttctca ctggcccgaa agcgcagcca gcaggcgata aggctgagtt tatcgagaaa 960 gttcgtcgtg cgctgtatct gggcaaaatc gtttcttacg ctcagggctt ctctcagctg 1020 cgtgcggcgt ctgaagagta caactgggat ctgaactacg gcgaaatcgc gaagattttc 1080 cgtgctggct gcatcatccg tgcgcagttc ctgcagaaaa tcaccgatgc atatgccgaa 1140 aatccgcaga tcgctaacct gctgctggct ccgtacttca agcaaattgc cgatgactac 1200 cagcaggcgc tgcgtgatgt cgttgcttat gcagtacaga acggtatccc ggttccgacc 1260 ttcgcggctg cggttgccta ttatgacagc taccgcgccg cagttctgcc tgcaaacctg 1320 atccaggcac agcgtgacta tttcggtgcg catacttata agcgcattga taaagaaggt 1380 gtgttccata ctgaatggct ggattaa 1407 35 468 PRT Escherichia coli 35 Met Ser Lys Gln Gln Ile Gly Val Val Gly Met Ala Val Met Gly Arg 1 5 10 15 Asn Leu Ala Leu Asn Ile Glu Ser Arg Gly Tyr Thr Val Ser Ile Phe 20 25 30 Asn Arg Ser Arg Glu Lys Thr Glu Glu Val Ile Ala Glu Asn Pro Gly 35 40 45 Lys Lys Leu Val Pro Tyr Tyr Thr Val Lys Glu Phe Val Glu Ser Leu 50 55 60 Glu Thr Pro Arg Arg Ile Leu Leu Met Val Lys Ala Gly Ala Gly Thr 65 70 75 80 Asp Ala Ala Ile Asp Ser Leu Lys Pro Tyr Leu Asp Lys Gly Asp Ile 85 90 95 Ile Ile Asp Gly Gly Asn Thr Phe Phe Leu Asp Thr Ile Arg Arg Asn 100 105 110 Arg Glu Leu Ser Ala Glu Gly Phe Asn Phe Ile Gly Thr Gly Val Ser 115 120 125 Gly Gly Glu Glu Gly Ala Leu Lys Gly Pro Ser Ile Met Pro Gly Gly 130 135 140 Gln Lys Glu Ala Tyr Glu Leu Val Ala Pro Ile Leu Thr Lys Ile Ala 145 150 155 160 Ala Val Ala Glu Asp Gly Glu Pro Cys Val Thr Tyr Ile Gly Ala Asp 165 170 175 Gly Ala Gly His Tyr Val Lys Met Val His Asn Gly Ile Glu Tyr Gly 180 185 190 Asp Met Gln Leu Ile Ala Glu Ala Tyr Ser Leu Leu Lys Gly Gly Leu 195 200 205 Asn Leu Ser Asn Glu Glu Leu Ala Gln Thr Phe Thr Glu Trp Asn Asn 210 215 220 Gly Glu Leu Ser Ser Tyr Leu Ile Asp Ile Thr Lys Asp Ile Phe Thr 225 230 235 240 Lys Lys Asp Glu Asp Gly Asn Tyr Leu Val Asp Val Ile Leu Asp Glu 245 250 255 Ala Ala Asn Lys Gly Thr Gly Lys Trp Thr Ser Gln Ser Ala Leu Asp 260 265 270 Leu Gly Glu Pro Leu Ser Leu Ile Thr Glu Ser Val Phe Ala Arg Tyr 275 280 285 Ile Ser Ser Leu Lys Asp Gln Arg Val Ala Ala Ser Lys Val Leu Thr 290 295 300 Gly Pro Lys Ala Gln Pro Ala Gly Asp Lys Ala Glu Phe Ile Glu Lys 305 310 315 320 Val Arg Arg Ala Leu Tyr Leu Gly Lys Ile Val Ser Tyr Ala Gln Gly 325 330 335 Phe Ser Gln Leu Arg Ala Ala Ser Glu Glu Tyr Asn Trp Asp Leu Asn 340 345 350 Tyr Gly Glu Ile Ala Lys Ile Phe Arg Ala Gly Cys Ile Ile Arg Ala 355 360 365 Gln Phe Leu Gln Lys Ile Thr Asp Ala Tyr Ala Glu Asn Pro Gln Ile 370 375 380 Ala Asn Leu Leu Leu Ala Pro Tyr Phe Lys Gln Ile Ala Asp Asp Tyr 385 390 395 400 Gln Gln Ala Leu Arg Asp Val Val Ala Tyr Ala Val Gln Asn Gly Ile 405 410 415 Pro Val Pro Thr Phe Ala Ala Ala Val Ala Tyr Tyr Asp Ser Tyr Arg 420 425 430 Ala Ala Val Leu Pro Ala Asn Leu Ile Gln Ala Gln Arg Asp Tyr Phe 435 440 445 Gly Ala His Thr Tyr Lys Arg Ile Asp Lys Glu Gly Val Phe His Thr 450 455 460 Glu Trp Leu Asp 465 36 1407 DNA Escherichia coli 36 atgtcaaagc aacagatcgg cgtagtcggt atggcagtga tggggcgcaa cctagcgctc 60 aacatcgaaa gccgtggtta taccgtctct attttcaacc gctcccgtga gaagacggaa 120 gaagtgattg ccgaaaatcc aggcaagaaa ctggttcctt actatacggt gaaagagttt 180 gttgaatctc tggaaacgcc tcgtcgcatc ctgttaatgg tgaaagcagg tgcaggcacg 240 gatgctgcta ttgattccct caagccgtac ctcgataaag gtgacatcat cattgatggt 300 ggtaacacct tcttccagga caccattcgt cgtaaccgtg agctttctgc cgaaggcttt 360 aacttcatcg gtaccggtgt ttccggcgga gaagaaggcg cgctgaaagg tccttccatt 420 atgcctggtg ggcagaaaga agcctatgaa ctggttgcgc cgatcctgac caaaatcgcc 480 gcagtggctg aagacggtga gccatgcgtt acctatattg gtgccgatgg cgcaggtcac 540 tatgtgaaga tggttcacaa cggtattgaa tacggagata tgcaactgat tgctgaagcc 600 tattctctgc ttaaaggtgg cctgaacctc accaacgaag aactggcgca gacgtttacc 660 gagtggaata acggtgaact gagcagctac ctgatcgaca tcaccaaaga tatcttcacc 720 aaaaaagatg aagacggtaa ctacctggtt gatgtgatcc tggatgaagc ggctaacaaa 780 ggtaccggta aatggaccag ccagagcgca ctggatctcg gcgaaccgct gtcgctgatt 840 accgagtctg tgtttgcacg ttatatctct tctctgaaag atcagcgtgt tgccgcatct 900 aaagttctct ctggcccgca agcacagcca gcaggcgaca aggctgagtt catcgaaaaa 960 gttcgccgtg cgctgtatct gggcaaaatc gtttcttacg cccagggctt ctctcagctg 1020 cgtgctgcgt ctgaagagta caactgggat ctgaactacg gcgaaatcgc gaagattttc 1080 cgtgctggct gcatcattcg tgcgcagttc ctgcagaaaa ttaccgatgc ttatgccgaa 1140 aatccgcaga tcgctaacct gctgctggct ccgtacttca agcaaattgc cgatgattac 1200 cagcaggcgc tgcgtgatgt cgttgcttat gcggtacaga acggtatccc ggttccgacc 1260 ttcgccgctg cggttgccta ttacgatagc taccgtgccg ctgttctgcc tgcgaacctg 1320 atccaggcac agcgtgacta tttcggtgca catacttata agcgcattga taaagaaggt 1380 gtgttccata ctgaatggct ggattaa 1407 37 468 PRT Escherichia coli 37 Met Ser Lys Gln Gln Ile Gly Val Val Gly Met Ala Val Met Gly Arg 1 5 10 15 Asn Leu Ala Leu Asn Ile Glu Ser Arg Gly Tyr Thr Val Ser Ile Phe 20 25 30 Asn Arg Ser Arg Glu Lys Thr Glu Glu Val Ile Ala Glu Asn Pro Gly 35 40 45 Lys Lys Leu Val Pro Tyr Tyr Thr Val Lys Glu Phe Val Glu Ser Leu 50 55 60 Glu Thr Pro Arg Arg Ile Leu Leu Met Val Lys Ala Gly Ala Gly Thr 65 70 75 80 Asp Ala Ala Ile Asp Ser Leu Lys Pro Tyr Leu Asp Lys Gly Asp Ile 85 90 95 Ile Ile Asp Gly Gly Asn Thr Phe Phe Gln Asp Thr Ile Arg Arg Asn 100 105 110 Arg Glu Leu Ser Ala Glu Gly Phe Asn Phe Ile Gly Thr Gly Val Ser 115 120 125 Gly Gly Glu Glu Gly Ala Leu Lys Gly Pro Ser Ile Met Pro Gly Gly 130 135 140 Gln Lys Glu Ala Tyr Glu Leu Val Ala Pro Ile Leu Thr Lys Ile Ala 145 150 155 160 Ala Val Ala Glu Asp Gly Glu Pro Cys Val Thr Tyr Ile Gly Ala Asp 165 170 175 Gly Ala Gly His Tyr Val Lys Met Val His Asn Gly Ile Glu Tyr Gly 180 185 190 Asp Met Gln Leu Ile Ala Glu Ala Tyr Ser Leu Leu Lys Gly Gly Leu 195 200 205 Asn Leu Thr Asn Glu Glu Leu Ala Gln Thr Phe Thr Glu Trp Asn Asn 210 215 220 Gly Glu Leu Ser Ser Tyr Leu Ile Asp Ile Thr Lys Asp Ile Phe Thr 225 230 235 240 Lys Lys Asp Glu Asp Gly Asn Tyr Leu Val Asp Val Ile Leu Asp Glu 245 250 255 Ala Ala Asn Lys Gly Thr Gly Lys Trp Thr Ser Gln Ser Ala Leu Asp 260 265 270 Leu Gly Glu Pro Leu Ser Leu Ile Thr Glu Ser Val Phe Ala Arg Tyr 275 280 285 Ile Ser Ser Leu Lys Asp Gln Arg Val Ala Ala Ser Lys Val Leu Ser 290 295 300 Gly Pro Gln Ala Gln Pro Ala Gly Asp Lys Ala Glu Phe Ile Glu Lys 305 310 315 320 Val Arg Arg Ala Leu Tyr Leu Gly Lys Ile Val Ser Tyr Ala Gln Gly 325 330 335 Phe Ser Gln Leu Arg Ala Ala Ser Glu Glu Tyr Asn Trp Asp Leu Asn 340 345 350 Tyr Gly Glu Ile Ala Lys Ile Phe Arg Ala Gly Cys Ile Ile Arg Ala 355 360 365 Gln Phe Leu Gln Lys Ile Thr Asp Ala Tyr Ala Glu Asn Pro Gln Ile 370 375 380 Ala Asn Leu Leu Leu Ala Pro Tyr Phe Lys Gln Ile Ala Asp Asp Tyr 385 390 395 400 Gln Gln Ala Leu Arg Asp Val Val Ala Tyr Ala Val Gln Asn Gly Ile 405 410 415 Pro Val Pro Thr Phe Ala Ala Ala Val Ala Tyr Tyr Asp Ser Tyr Arg 420 425 430 Ala Ala Val Leu Pro Ala Asn Leu Ile Gln Ala Gln Arg Asp Tyr Phe 435 440 445 Gly Ala His Thr Tyr Lys Arg Ile Asp Lys Glu Gly Val Phe His Thr 450 455 460 Glu Trp Leu Asp 465 38 1407 DNA Escherichia coli 38 atgtcaaagc aacagatcgg cgtagtcggt atggcagtga tggggcgcaa cctagcgctc 60 aacatcgaaa gccgtggtta taccgtctct attttcaacc gctcccgtga gaagacggaa 120 gaagtgattg ccgaaaatcc aggcaagaaa ctggttcctt actatacggt gaaagagttt 180 gttgaatctc tggaaacgcc tcgtcgcatc ctgttaatgg tgaaagcagg tgcaggcacg 240 gatgctgcta ttgattccct caagccgtac ctcgataaag gtgacatcat cattgatggt 300 ggtaacacct tcttccagga caccattcgt cgtaaccgtg agctttctgc cgaaggcttt 360 aacttcatcg gtaccggtgt ttccggcgga gaagaaggcg cgctgaaagg tccttccatt 420 atgcctggtg ggcagaaaga agcctatgaa ctggttgcgc cgatcctgac caaaatcgcc 480 gcagtggctg aagacggtga gccatgcgtt acctatattg gtgccgatgg cgcaggtcac 540 tatgtgaaga tggttcacaa cggtattgaa tacggagata tgcaactgat tgctgaagcc 600 tattctctgc ttaaaggtgg cctgaacctc accaacgaag aactggcgca gacgtttacc 660 gagtggaata acggtgaact gagcagctac ctgatcgaca tcaccaaaga tatcttcacc 720 aaaaaagatg aagacggtaa ctacctggtt gatgtgatcc tggatgaagc ggctaacaaa 780 ggtaccggta aatggaccag ccagagcgca ctggatctcg gcgaaccgct gtcgctgatt 840 accgagtctg tgtttgcacg ttatatctct tctctgaaag atcagcgtgt tgccgcatct 900 aaagttctct ctggcccgca agcacagcca gcaggcgaca aggctgagtt catcgaaaaa 960 gttcgccgtg cgctgtatct gggcaaaatc gtttcttacg cccagggctt ctctcagctg 1020 cgtgctgcgt ctgaagagta caactgggat ctgaactacg gcgaaatcgc gaagattttc 1080 cgtgctggct gcatcattcg tgcgcagttc ctgcagaaaa ttaccgatgc ttatgccgaa 1140 aatccgcaga tcgctaacct gctgctggct ccgtacttca agcaaattgc cgatgattac 1200 cagcaggcgc tgcgtgatgt cgttgcttat gcggtacaga acggtatccc ggttccgacc 1260 ttcgccgctg cggttgccta ttacgatagc taccgtgccg ctgttctgcc tgcgaacctg 1320 atccaggcac agcgtgacta tttcggtgca catacttata agcgcattga taaagaaggt 1380 gtgttccata ctgaatggct ggattaa 1407 39 468 PRT Escherichia coli 39 Met Ser Lys Gln Gln Ile Gly Val Val Gly Met Ala Val Met Gly Arg 1 5 10 15 Asn Leu Ala Leu Asn Ile Glu Ser Arg Gly Tyr Thr Val Ser Ile Phe 20 25 30 Asn Arg Ser Arg Glu Lys Thr Glu Glu Val Ile Ala Glu Asn Pro Gly 35 40 45 Lys Lys Leu Val Pro Tyr Tyr Thr Val Lys Glu Phe Val Glu Ser Leu 50 55 60 Glu Thr Pro Arg Arg Ile Leu Leu Met Val Lys Ala Gly Ala Gly Thr 65 70 75 80 Asp Ala Ala Ile Asp Ser Leu Lys Pro Tyr Leu Asp Lys Gly Asp Ile 85 90 95 Ile Ile Asp Gly Gly Asn Thr Phe Phe Gln Asp Thr Ile Arg Arg Asn 100 105 110 Arg Glu Leu Ser Ala Glu Gly Phe Asn Phe Ile Gly Thr Gly Val Ser 115 120 125 Gly Gly Glu Glu Gly Ala Leu Lys Gly Pro Ser Ile Met Pro Gly Gly 130 135 140 Gln Lys Glu Ala Tyr Glu Leu Val Ala Pro Ile Leu Thr Lys Ile Ala 145 150 155 160 Ala Val Ala Glu Asp Gly Glu Pro Cys Val Thr Tyr Ile Gly Ala Asp 165 170 175 Gly Ala Gly His Tyr Val Lys Met Val His Asn Gly Ile Glu Tyr Gly 180 185 190 Asp Met Gln Leu Ile Ala Glu Ala Tyr Ser Leu Leu Lys Gly Gly Leu 195 200 205 Asn Leu Thr Asn Glu Glu Leu Ala Gln Thr Phe Thr Glu Trp Asn Asn 210 215 220 Gly Glu Leu Ser Ser Tyr Leu Ile Asp Ile Thr Lys Asp Ile Phe Thr 225 230 235 240 Lys Lys Asp Glu Asp Gly Asn Tyr Leu Val Asp Val Ile Leu Asp Glu 245 250 255 Ala Ala Asn Lys Gly Thr Gly Lys Trp Thr Ser Gln Ser Ala Leu Asp 260 265 270 Leu Gly Glu Pro Leu Ser Leu Ile Thr Glu Ser Val Phe Ala Arg Tyr 275 280 285 Ile Ser Ser Leu Lys Asp Gln Arg Val Ala Ala Ser Lys Val Leu Ser 290 295 300 Gly Pro Gln Ala Gln Pro Ala Gly Asp Lys Ala Glu Phe Ile Glu Lys 305 310 315 320 Val Arg Arg Ala Leu Tyr Leu Gly Lys Ile Val Ser Tyr Ala Gln Gly 325 330 335 Phe Ser Gln Leu Arg Ala Ala Ser Glu Glu Tyr Asn Trp Asp Leu Asn 340 345 350 Tyr Gly Glu Ile Ala Lys Ile Phe Arg Ala Gly Cys Ile Ile Arg Ala 355 360 365 Gln Phe Leu Gln Lys Ile Thr Asp Ala Tyr Ala Glu Asn Pro Gln Ile 370 375 380 Ala Asn Leu Leu Leu Ala Pro Tyr Phe Lys Gln Ile Ala Asp Asp Tyr 385 390 395 400 Gln Gln Ala Leu Arg Asp Val Val Ala Tyr Ala Val Gln Asn Gly Ile 405 410 415 Pro Val Pro Thr Phe Ala Ala Ala Val Ala Tyr Tyr Asp Ser Tyr Arg 420 425 430 Ala Ala Val Leu Pro Ala Asn Leu Ile Gln Ala Gln Arg Asp Tyr Phe 435 440 445 Gly Ala His Thr Tyr Lys Arg Ile Asp Lys Glu Gly Val Phe His Thr 450 455 460 Glu Trp Leu Asp 465 40 1407 DNA Escherichia coli 40 atgtcaaagc aacagatcgg cgtagtcggt atggcagtga tggggcgcaa cctagcgctc 60 aacatcgaaa gccgtggtta taccgtctct attttcaacc gctcccgtga gaagacggaa 120 gaagtgattg ccgaaaatcc aggcaagaaa ctggttcctt actatacggt gaaagagttt 180 gttgaatctc tggaaacgcc tcgtcgcatc ctgttaatgg tgaaagcagg tgcaggcacg 240 gatgctgcta ttgattccct caagccgtac ctcgataaag gtgacatcat cattgatggt 300 ggtaacacct tcttccagga caccattcgt cgtaaccgtg agctttctgc cgaaggcttt 360 aacttcatcg gtaccggtgt ttccggcgga gaagaaggcg cgctgaaagg tccttccatt 420 atgcctggtg ggcagaaaga agcctatgaa ctggttgcgc cgatcctgac caaaatcgcc 480 gcagtggctg aagacggtga gccatgcgtt acctatattg gtgccgatgg cgcaggtcac 540 tatgtgaaga tggttcacaa cggtattgaa tacggagata tgcaactgat tgctgaagcc 600 tattctctgc ttaaaggtgg cctgaacctc accaacgaag aactggcgca gacgtttacc 660 gagtggaata acggtgaact gagcagctac ctgatcgaca tcaccaaaga tatcttcacc 720 aaaaaagatg aagacggtaa ctacctggtt gatgtgatcc tggatgaagc ggctaacaaa 780 ggtaccggta aatggaccag ccagagcgca ctggatctcg gcgaaccgct gtcgctgatt 840 accgagtctg tgtttgcacg ttatatctct tctctgaaag atcagcgtgt tgccgcatct 900 aaagttctct ctggcccgca agcacagcca gcaggcgaca aggctgagtt catcgaaaaa 960 gttcgccgtg cgctgtatct gggcaaaatc gtttcttacg cccagggctt ctctcagctg 1020 cgtgctgcgt ctgaagagta caactgggat ctgaactacg gcgaaatcgc gaagattttc 1080 cgtgctggct gcatcattcg tgcgcagttc ctgcagaaaa ttaccgatgc ttatgccgaa 1140 aatccgcaga tcgctaacct gctgctggct ccgtacttca agcaaattgc cgatgattac 1200 cagcaggcgc tgcgtgatgt cgttgcttat gcggtacaga acggtatccc ggttccgacc 1260 ttcgccgctg cggttgccta ttacgatagc taccgtgccg ctgttctgcc tgcgaacctg 1320 atccaggcac agcgtgacta tttcggtgca catacttata agcgcattga taaagaaggt 1380 gtgttccata ctgaatggct ggattaa 1407 41 468 PRT Escherichia coli 41 Met Ser Lys Gln Gln Ile Gly Val Val Gly Met Ala Val Met Gly Arg 1 5 10 15 Asn Leu Ala Leu Asn Ile Glu Ser Arg Gly Tyr Thr Val Ser Ile Phe 20 25 30 Asn Arg Ser Arg Glu Lys Thr Glu Glu Val Ile Ala Glu Asn Pro Gly 35 40 45 Lys Lys Leu Val Pro Tyr Tyr Thr Val Lys Glu Phe Val Glu Ser Leu 50 55 60 Glu Thr Pro Arg Arg Ile Leu Leu Met Val Lys Ala Gly Ala Gly Thr 65 70 75 80 Asp Ala Ala Ile Asp Ser Leu Lys Pro Tyr Leu Asp Lys Gly Asp Ile 85 90 95 Ile Ile Asp Gly Gly Asn Thr Phe Phe Gln Asp Thr Ile Arg Arg Asn 100 105 110 Arg Glu Leu Ser Ala Glu Gly Phe Asn Phe Ile Gly Thr Gly Val Ser 115 120 125 Gly Gly Glu Glu Gly Ala Leu Lys Gly Pro Ser Ile Met Pro Gly Gly 130 135 140 Gln Lys Glu Ala Tyr Glu Leu Val Ala Pro Ile Leu Thr Lys Ile Ala 145 150 155 160 Ala Val Ala Glu Asp Gly Glu Pro Cys Val Thr Tyr Ile Gly Ala Asp 165 170 175 Gly Ala Gly His Tyr Val Lys Met Val His Asn Gly Ile Glu Tyr Gly 180 185 190 Asp Met Gln Leu Ile Ala Glu Ala Tyr Ser Leu Leu Lys Gly Gly Leu 195 200 205 Asn Leu Thr Asn Glu Glu Leu Ala Gln Thr Phe Thr Glu Trp Asn Asn 210 215 220 Gly Glu Leu Ser Ser Tyr Leu Ile Asp Ile Thr Lys Asp Ile Phe Thr 225 230 235 240 Lys Lys Asp Glu Asp Gly Asn Tyr Leu Val Asp Val Ile Leu Asp Glu 245 250 255 Ala Ala Asn Lys Gly Thr Gly Lys Trp Thr Ser Gln Ser Ala Leu Asp 260 265 270 Leu Gly Glu Pro Leu Ser Leu Ile Thr Glu Ser Val Phe Ala Arg Tyr 275 280 285 Ile Ser Ser Leu Lys Asp Gln Arg Val Ala Ala Ser Lys Val Leu Ser 290 295 300 Gly Pro Gln Ala Gln Pro Ala Gly Asp Lys Ala Glu Phe Ile Glu Lys 305 310 315 320 Val Arg Arg Ala Leu Tyr Leu Gly Lys Ile Val Ser Tyr Ala Gln Gly 325 330 335 Phe Ser Gln Leu Arg Ala Ala Ser Glu Glu Tyr Asn Trp Asp Leu Asn 340 345 350 Tyr Gly Glu Ile Ala Lys Ile Phe Arg Ala Gly Cys Ile Ile Arg Ala 355 360 365 Gln Phe Leu Gln Lys Ile Thr Asp Ala Tyr Ala Glu Asn Pro Gln Ile 370 375 380 Ala Asn Leu Leu Leu Ala Pro Tyr Phe Lys Gln Ile Ala Asp Asp Tyr 385 390 395 400 Gln Gln Ala Leu Arg Asp Val Val Ala Tyr Ala Val Gln Asn Gly Ile 405 410 415 Pro Val Pro Thr Phe Ala Ala Ala Val Ala Tyr Tyr Asp Ser Tyr Arg 420 425 430 Ala Ala Val Leu Pro Ala Asn Leu Ile Gln Ala Gln Arg Asp Tyr Phe 435 440 445 Gly Ala His Thr Tyr Lys Arg Ile Asp Lys Glu Gly Val Phe His Thr 450 455 460 Glu Trp Leu Asp 465 42 1407 DNA Escherichia coli 42 atgtccaagc aacagatcgg cgtcatcggt atggctgtga tggggcgcaa cttggctcta 60 aacatcgaga gccgtggtta taccgtatcc gtctataatc gctcgcgtga aaaaactgaa 120 gaggttgttg ccgaaaaccc aggtaagaaa ctggtccctt attacacggt taaagagttc 180 gtcgagtctc ttgaaactcc acgccgtatc ctgttaatgg tcaaagcggg tgctggcact 240 gatgctgcga ttaattccct gaagccctat ctagataaag gcgacatcat cattgatggc 300 ggtaatacct tctttcagga cacaattcgt cgtaaccgtg aactttccgc ggaaggcttt 360 aactttatcg gggccggggt ttcaggtggt gaagagggcg cgctgaaagg cccatctatc 420 atgcctggtg gccagaaaga tgcgtatgaa atggttgtgc caatcctgac caagattgcc 480 gcgatagctg aagatggtga accgtgcgtg acgtatattg gtgcggatgg tgcaggtcat 540 tacgtgaaga tggtgcacaa cggtatcgaa tatggcgata tgcaattgat agctgaagcc 600 tattctctgc tgaaaggtgc cctaaatctg tctaatgaag agttagcctc tatctttaat 660 gaatggaatg aaggcgagct gagcagctat ctgattgaca tcactaagga tatcttcaac 720 aaaaaagatg aagagggtaa atacttggtt gatgtgattt tggacgaagc tgcgaacaaa 780 ggtacaggca aatggaccag ccagagctct cttgatctag gcgaaccgct gtcgttgatc 840 accgaatccg tatttgcccg ctacatctcc tctctgaaag accagcgtgt tgcggcctct 900 aaagtgctgt ctggcccgca ggctaaactg gctagtgata aagctgagtt tgttgagaaa 960 gtacgccgtg ccttgtacct aggcaaaatt gtctcttatg cccaaggctt ttctcaactt 1020 cgtgccgcat cagagcaata caactgggat ttgaactacg gtgaaatcgc gaaaattttc 1080 cgcgcaggct gcattattcg tgcacagttc ctccagaaaa tcaccgacgc ttatgctgaa 1140 aacaaagata ttgcaaacct gctgcttgct ccgtatttca aacatatcgc tgatgaatat 1200 caacaagccc tccgtgatgt agtgtcttat gctgtgcaga acggtattcc ggtaccgact 1260 ttctccgccg ctgtagctta ctacgacagc taccgttctg cggttctgcc ggctaacttg 1320 atccaagcac agcgtgatta tttcggtgcg cacacgtata aacgcattga taaagaaggt 1380 gtttttcata cagaatggct agaataa 1407 43 468 PRT Escherichia coli 43 Met Ser Lys Gln Gln Ile Gly Val Ile Gly Met Ala Val Met Gly Arg 1 5 10 15 Asn Leu Ala Leu Asn Ile Glu Ser Arg Gly Tyr Thr Val Ser Val Tyr 20 25 30 Asn Arg Ser Arg Glu Lys Thr Glu Glu Val Val Ala Glu Asn Pro Gly 35 40 45 Lys Lys Leu Val Pro Tyr Tyr Thr Val Lys Glu Phe Val Glu Ser Leu 50 55 60 Glu Thr Pro Arg Arg Ile Leu Leu Met Val Lys Ala Gly Ala Gly Thr 65 70 75 80 Asp Ala Ala Ile Asn Ser Leu Lys Pro Tyr Leu Asp Lys Gly Asp Ile 85 90 95 Ile Ile Asp Gly Gly Asn Thr Phe Phe Gln Asp Thr Ile Arg Arg Asn 100 105 110 Arg Glu Leu Ser Ala Glu Gly Phe Asn Phe Ile Gly Ala Gly Val Ser 115 120 125 Gly Gly Glu Glu Gly Ala Leu Lys Gly Pro Ser Ile Met Pro Gly Gly 130 135 140 Gln Lys Asp Ala Tyr Glu Met Val Val Pro Ile Leu Thr Lys Ile Ala 145 150 155 160 Ala Ile Ala Glu Asp Gly Glu Pro Cys Val Thr Tyr Ile Gly Ala Asp 165 170 175 Gly Ala Gly His Tyr Val Lys Met Val His Asn Gly Ile Glu Tyr Gly 180 185 190 Asp Met Gln Leu Ile Ala Glu Ala Tyr Ser Leu Leu Lys Gly Ala Leu 195 200 205 Asn Leu Ser Asn Glu Glu Leu Ala Ser Ile Phe Asn Glu Trp Asn Glu 210 215 220 Gly Glu Leu Ser Ser Tyr Leu Ile Asp Ile Thr Lys Asp Ile Phe Asn 225 230 235 240 Lys Lys Asp Glu Glu Gly Lys Tyr Leu Val Asp Val Ile Leu Asp Glu 245 250 255 Ala Ala Asn Lys Gly Thr Gly Lys Trp Thr Ser Gln Ser Ser Leu Asp 260 265 270 Leu Gly Glu Pro Leu Ser Leu Ile Thr Glu Ser Val Phe Ala Arg Tyr 275 280 285 Ile Ser Ser Leu Lys Asp Gln Arg Val Ala Ala Ser Lys Val Leu Ser 290 295 300 Gly Pro Gln Ala Lys Leu Ala Ser Asp Lys Ala Glu Phe Val Glu Lys 305 310 315 320 Val Arg Arg Ala Leu Tyr Leu Gly Lys Ile Val Ser Tyr Ala Gln Gly 325 330 335 Phe Ser Gln Leu Arg Ala Ala Ser Glu Gln Tyr Asn Trp Asp Leu Asn 340 345 350 Tyr Gly Glu Ile Ala Lys Ile Phe Arg Ala Gly Cys Ile Ile Arg Ala 355 360 365 Gln Phe Leu Gln Lys Ile Thr Asp Ala Tyr Ala Glu Asn Lys Asp Ile 370 375 380 Ala Asn Leu Leu Leu Ala Pro Tyr Phe Lys His Ile Ala Asp Glu Tyr 385 390 395 400 Gln Gln Ala Leu Arg Asp Val Val Ser Tyr Ala Val Gln Asn Gly Ile 405 410 415 Pro Val Pro Thr Phe Ser Ala Ala Val Ala Tyr Tyr Asp Ser Tyr Arg 420 425 430 Ser Ala Val Leu Pro Ala Asn Leu Ile Gln Ala Gln Arg Asp Tyr Phe 435 440 445 Gly Ala His Thr Tyr Lys Arg Ile Asp Lys Glu Gly Val Phe His Thr 450 455 460 Glu Trp Leu Glu 465

Claims (28)

What is claimed is:
1. An isolated polynucleotide encoding gnd, wherein the polynucleotide comprises one of the sequences of SEQ ID Nos: 22, 16, 18, 24, 26, 20, 42, 28, 30, 40, 32, 36, 38, and 34.
2. The isolated polynucleotide of claim 1, wherein the polynucleotide comprises at least 9 consecutive bases of one of the sequences of SEQ ID Nos: 22, 16, 18, 24, 26, 20, 42, 28, 30, 40, 32, 36, 38, and 34 and contains a polymorphism described in Table 1.
3. The isolated polynucleotide of claim 1, wherein the polynucleotide encodes a polypeptide deduced from one of the sequences of SEQ ID Nos: 22, 16, 18, 24, 26, 20, 42, 28, 30, 40, 32, 36, 38, and 34.
4. The isolated polynucleotide of claim 1, wherein the polynucleotide comprises at least 9 bases that hybridize to the nucleotide sequence of one of the sequences of SEQ ID Nos: 22, 16, 18, 24, 26, 20, 42, 28, 30, 40, 32, 36, 38, and 34 or a sequence complementary thereto under the following conditions: 7% sodium dodecyl sulfate (SDS), 0.5M NaPO4 pH 7.0, 1 mM EDTA at 50° C.; and washing with 1% SDS at 42° C.
5. A recombinant construct comprising one of the sequences of SEQ ID Nos: 22, 16, 18, 24, 26, 20, 42, 28, 30, 40, 32, 36, 38, and 34 operably linked to a heterologous promoter.
6. A vector comprising the isolated DNA of claim 1.
7. A vector comprising the isolated DNA of claim 2.
8. A method of detecting a polymorphism in a gene encoding 6-PGD comprising:
obtaining a biological sample containing polynucleotides; and analyzing the biological sample for the presence of a diagnostic polynucleotide having at least one polymorphism described in Table 1.
9. The method of claim 8, wherein the polymorphism is C653T or G653C.
10. The method of claim 8, wherein the analysis of the biological sample further comprises a DNA amplification step.
11. A method of identifying a pathogenic or non-pathogenic E. coli comprising:
obtaining a biological sample containing polynucleotides;
analyzing the biological sample for the presence of a diagnostic polynucleotide having at least one polymorphism described in Table 1; and
identifying the E. coli as a pathogenic or non-pathogenic strain based on the presence or absence of at least one polymorphism described in Table 1.
12. The method of claim 11, wherein the polymorphism is C653T or G653C.
13. The method of claim 11, wherein the analysis of the biological sample further comprises a DNA amplification step.
14. An isolated protein comprising the sequence of SEQ ID Nos: 23, 17, 19, 25, 27, 21, 43, 29, 31, 41, 33, 37, 39, and 35.
15. An isolated polypeptide comprising at least 3 consecutive amino acids of one of the sequences of SEQ ID Nos: 23, 17, 19, 25, 27, 21, 43, 29, 31, 41, 33, 37, 39, and 35, wherein the polypeptide contains at least one polymorphism that can be deduced from Table 1.
16. A method of making a 6-PGD protein comprising:
obtaining a cDNA comprising one of the sequences of SEQ ID Nos: 22, 16, 18, 24, 26, 20, 42, 28, 30, 40, 32, 36, 38, and 34;
inserting the cDNA in an expression vector such that the cDNA is operably linked to a promoter; and
introducing the expression vector into a host cell whereby the host cell produces the protein encoded by the cDNA.
17. The method of claim 16, further comprising isolating the protein.
18. A method for constructing a transformed host cell that expresses one of the sequences of SEQ ID Nos: 23, 17, 19, 25, 27, 21, 43, 29, 31, 41, 33, 37, 39, and 35 comprising transforming a host cell with a recombinant DNA vector suitable for gene expression.
19. A cultured cell line comprising the vector of claim 6.
20. A cultured cell line comprising the vector of claim 7.
21. An isolated antibody capable of specifically binding to a protein having one of the sequences of SEQ ID Nos: 23, 17, 19, 25, 27, 21, 43, 29, 31, 41, 33, 37, 39, and 35, wherein the epitope corresponds to at least one polymorphism that can be deduced from Table 1.
22. An isolated antibody capable of binding to a polypeptide comprising at least 9 consecutive amino acids of the sequences of SEQ ID Nos: 23, 17, 19, 25, 27, 21, 43, 29, 31, 41, 33, 37, 39, and 35, wherein the epitope corresponds to at least one polymorphism that can be deduced from Table 1.
23. The antibody of claim 21 or 22, wherein the antibody is a monoclonal antibody.
24. A nucleic acid probe for detecting the presence of E. coli O157:H7 consisting of an isolated nucleic acid molecule at least 7 nucleotides in length, said isolated nucleic acid molecule hybridizing to DNA of gnd of E. coli O157:H7 and not to DNA of gnd of non-H7 E. coli O157 strains.
25. A nucleic acid primer for detecting the presence of E. coli O157:H7 consisting of an isolated nucleic acid molecule at least 7 nucleotides in length, said isolated nucleic acid molecule priming DNA of gnd of E. coli O157:H7 and not DNA of gnd of non-H7 E. coli O157 strains.
26. A method for detecting the presence of E. coli O157:H7 in a sample comprising the steps of:
(a) contacting said sample, under hybridization conditions, with a nucleic acid probe that selectively hybridizes to a nucleic acid sequence from gnd of E. coli O157:H7 and not to nucleic acid sequence from gnd of non-H7 E. coli O157 strains, to form a hybridization complex; and
(b) detecting formation of said hybridization complex as an indication of the presence of E. coli O157:H7 in the sample.
27. A plurality of the nucleic acid probes of claim 24 on a substrate.
28. A plurality of the nucleic acid probes of claim 24 in a microarray on a chip.
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KR20010090879A (en) 2001-10-19
NZ512088A (en) 2004-01-30
US8597877B2 (en) 2013-12-03
JP2002531130A (en) 2002-09-24
US20080102458A1 (en) 2008-05-01
EP1147196A4 (en) 2002-10-16
EP1147196A1 (en) 2001-10-24

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