Department of Molecular Biology and Biotechnology, University of Sheffield, Firth Court, Western Bank, Sheffield S10 2TN, UK1
Author for correspondence: David J. Kelly. Tel: +44 114 222 4414. Fax: +44 114 272 8697. e-mail: d.kelly{at}sheffield.ac.uk
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ABSTRACT |
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Keywords: cheY, cheW, cheV, motility, fluorescence, acetyl phosphate, phosphorylation
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INTRODUCTION |
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The paradigm of the bacterial chemotactic response is the system found in enteric bacteria such as Escherichia coli and Salmonella typhimurium, and has been extensively reviewed (Bourret et al., 1991 ; Blair, 1995
; Stock & Surette, 1996
). It has become clear, however, that many bacteria have more complex chemotaxis systems than the enterics, with additional or duplicated genes (Armitage & Schmitt, 1997
). The complete genome sequencing of two strains of H. pylori (Tomb et al., 1997
; Alm et al., 1999
) revealed the presence of six che gene homologues. These are: the response regulator gene cheY (HP1067/JHP358), a histidine kinase gene cheA (HP0392/JHP989), cheW, encoding the ternary-complex-forming protein CheW (HP0391/JHP990) and three unlinked copies of cheV. The latter form a paralagous family and have been designated cheV1 (HP0019/JHP17), cheV2 (HP0616/JHP559) and cheV3 (HP0393/JHP988) by Doig et al. (1999)
. Each cheV gene encodes a protein consisting of an N-terminal domain homologous to CheW and a C-terminal domain homologous to CheY. At least three genes are present that encode transducer-like receptor proteins, which possess several conserved glutamate residues that could be methylated as part of a stimulus-adaptation system. However, there is no evidence of potential methyltransferase (cheR) or methylesterase (cheB) genes, and so the details of adaptation are unclear. The cheA, cheW and cheV3 genes are arranged in an operon while cheY and the two further copies of cheV are located at unlinked sites on the chromosome. Beier et al. (1997)
characterized the response regulator gene cheY and showed that it was part of a larger operon containing chemotaxis-unrelated stress-induced genes. The deduced histidine kinase CheA is actually a bi-functional protein in H. pylori, as it is fused to an N-terminal CheY domain (Jackson et al., 1995
; Kelly, 1998
) and has been redesignated CheAY, encoded by the cheAY gene (Foynes et al., 2000
). Thus, H. pylori contains five CheY-like domains on separate proteins, but there is no evidence for a cheZ gene (encoding a protein which enhances the intrinsic CheY phosphatase activity) in this bacterium. Mutants in either cheY or cheAY have been shown to be non-chemotactic (Beier et al., 1997
; Foynes et al., 2000
).
One of the unusual features of the H. pylori chemotaxis machinery is the presence of the three paralogues of CheV. CheV was first identified as a novel chemotaxis protein in Bacillus subtilis (Frederick & Helmann, 1994 ) and partial redundancy with CheW was reported (Rosario et al., 1994
). However, the actual function of CheV in chemotaxis has not been elucidated. To begin to address this problem in H. pylori, we have determined, by insertional mutagenesis, the essentiality for chemotaxis of each of the three cheV paralogues in this bacterium. These genes have also been expressed in E. coli wild-type and isogenic
cheY and
cheW mutants to investigate the interaction of the H. pylori proteins with the enteric chemotaxis system. In E. coli CheY, aspartate residue D57 is the site of phosphorylation via CheA or small-molecule phosphodonors such as acetyl phosphate (Lukat et al., 1992
; McCleary & Stock, 1994
; Da Re et al., 1999
). In H. pylori, the CheY domains of each of the CheV paralogues contain this conserved aspartate, raising the possibility that in the absence of a CheZ homologue, these domains could act as phosphate sinks to control the level of phospho-CheY. In support of this, we have now shown by fluorescence studies that at least one of the H. pylori CheV proteins (CheV2) is capable of binding acetyl phosphate.
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METHODS |
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Electron microscopy.
Cell suspensions were deposited onto Formvar-coated grids, blotted off and negatively stained by the addition of 1% (w/v) phosphotungstic acid to the grids for 3 min. After blotting and air-drying, the grids were viewed and photographed using a Philips CM10 transmission electron microscope.
Preparation and sequencing of DNA.
Plasmid DNA for screening clones, sequencing and gene disruption experiments was routinely isolated using anion-exchange resin spin-columns (QIAGEN), according to the manufacturers instructions. Double-stranded sequencing of plasmid DNA was carried out using Applied Biosystems Taq DyeDeoxy terminator cycle sequencing reactions analysed on an ABI model 373A automated DNA sequencer. Synthesis of custom primers was carried out on an Applied Biosystems model 392 DNA synthesizer. H. pylori total chromosomal DNA was extracted using a modified SDS lysis procedure (Marmur, 1961 ). Restriction endonucleases, T4 DNA ligase and Taq polymerases were purchased from Promega, and Pfu polymerase from Stratagene, and were used according to the manufacturers instructions.
Construction and characterization of H. pylori isogenic insertion mutants.
A set of PCR primers was designed to amplify the entire coding regions of the cheW, cheV1, cheV2 and cheV3 genes, both for insertional activation after cloning of the products into pGEM-3Zf(-) (Promega) and for expression after subcloning into pTM30 (Morrison & Parkinson, 1994 ). The primers used were: CheW-F (5'-TCGAGGATCCAAGCAACCAATTAAAAGAT-3'), CheW-R (5'-TCGAGGTACCGAAGTCTTTTTTTAAGATTTC-3'), CheV1-F (5'-TCGAGGATCCAGCTGATAGTTTAGCGGGC-3'), CheV1-R (5'-TCGAGGTACCTGCTAATTCCAAAAATTG-3'), CheV2-F (5'-TCGAGGATCCAGTAAGAGAGATATTGACAAA-3'), CheV2-R (5'-TCGAGGTACCTTATGAAAGCGTTTTTTT-3'), CheV3-F (5'-TCGAGGATCCAGCAGAAAAAACAGCTA-3') and CheV3-R (5'-TCGAGGTACCCGCATTCTTGTCTAAAAT-3'). These primers introduced BamHI or KpnI restriction sites (shown in bold italics) for the in-frame subcloning of these genes into the pTM30 polylinker (see below). The second codon (forward primers) or final codon (reverse primers) of the gene is underlined. PCR reactions were performed with Pfu polymerase using genomic DNA from strain 26695 as template.
pGEM-3Zf(-) was linearized with HincII and blunt-end ligated with the products resulting from PCR with the above primers, generating plasmids pMSP1, pMSP4, pMSP5 and pMSP6 (Table 1). These plasmids were linearized at unique restriction sites within the H. pylori gene inserts, as detailed in Table 1
. Protruding 5' termini were in-filled using the Klenow fragment of the E. coli DNA polymerase I where necessary. For insertional inactivation, a chloramphenicol acetyltransferase (cat) cassette derived from Campylobacter coli (Wang & Taylor, 1990
) was used. The 900 bp cat cassette excised from pUCAT by HincII digestion was used to inactivate cheW and cheV2. However, it possesses rho-independent transcription terminators at the 3' terminus. A non-polar version of this cassette was used to inactivate cheV1 and cheV3. PCR primers Cat-F (5'-TCCGTCGTCGGTATCGTATGG-3') and Cat-R (5'-TTATCAGTGCGACAAACTGGG-3') were designed to amplify an 829 bp product from pUCAT containing the promoter region of the cat gene but lacking the 3' transcriptional terminators. Both cassettes were purified using a Gel Extraction Kit (QIAGEN) and blunt-end ligated into the appropriate plasmids. Recombinant clones were selected by growth on LB agar containing 30 µg chloramphenicol ml-1 and the orientation of the cassettes determined by restriction analysis. The following plasmids were generated: pMSP12, pMSP15, pMSP16 and pMSP17 (see Table 1
). To construct a plasmid suitable for generating a H. pylori cheV2/cheV3 double mutant, the C. coli-derived aphAIII cassette (Wang & Taylor 1990
) was amplified from pUKAN with Pfu polymerase, using primers Kan-F (5'-ACTGAGATCTACTCTATGAAGCGCCATATT-3') and Kan-R (5'-CAATAGATCTTTTAGACATCATCTAAATCTAGG-3'). Plasmid pMSP17 was linearized at the unique BclI site within the cheV2 gene and blunt-end ligated with the aphAIII cassette. E. coli transformants were selected by growth on LB agar containing 30 µg kanamycin sulphate ml-1. The resulting plasmid was designated pMSP18.
Transformation of H. pylori 26695 with pMSP12, pMSP15, pMSP16 and pMSP17, and of strain MSP300 with pMSP18, was carried out as previously described (Ferrero et al., 1992 ). Transformants were selected by addition of 30 µg chloramphenicol ml-1 or 30 µg kanamycin sulphate ml-1 to the plating medium as appropriate. Genomic DNA was extracted from putative recombinants, and correct insertion of the resistance cassettes evaluated by PCR using the above primers.
Cloning and expression of H. pylori cheW and cheV genes in pTM30 and transformation of E. coli chemotaxis mutants.
Complementation and expression experiments with the H. pylori chemotaxis genes were done by subcloning the cheW, cheV1, cheV2 and cheV3 genes in-frame into the BamHI and KpnI sites of the expression vector pTM30, and transforming the resulting constructs (Table 1) into the E. coli wild-type strain RP437, and chemotaxis mutants RP5232 (
cheY) and RP4606 (
cheW). The fidelity of the constructs was checked by DNA sequencing. Expression of proteins was induced with IPTG and monitored by SDS-PAGE on 10% (w/v) polyacrylamide gels. Swarming and motility phenotypes were determined for the complemented wild-type and mutant strains on LB plus 0·3% (w/v) Difco agar, containing IPTG in the range 01 mM.
Purification of the CheV2 protein.
pMSP11 was grown in 6x1 l batches of LB plus 100 µg ampicillin ml-1 until an OD600 of 0·5 was reached. IPTG was added to 1 mM final concentration and growth continued for 5 h, after which the cells were harvested by centrifugation (8000 g, 4 °C, 20 min), resuspended in 20 ml 10 mM HEPES buffer pH 7·5 (buffer A) and disrupted in a French press at 12000 p.s.i. (82·8 MPa). The broken cells were centrifuged (150000 g, 4 °C, 2 h) and the supernatant (cell-free extract) removed. A series of ammonium sulphate precipitations were carried out on the supernatant and the precipitates forming at 30% and 40% saturation were combined and dissolved in 3 ml buffer A. The solution was applied to a DEAE-Sepharose column connected to a BioLogic HP system equilibrated in buffer A. Proteins were eluted by applying the following salt gradient: (i) 00·2 M NaCl for 15 min, (ii) 0·2 M NaCl for 15 min, (iii) 0·20·6 M NaCl for 180 min, (iv) 0·61·0 M NaCl for 15 min. CheV2-enriched fractions eluted at 0·250·35 M NaCl. These were pooled, concentrated to about 4 ml and applied to a Superdex 75 gel-filtration column equilibrated in 10 mM HEPES pH 7·5 containing 0·2 M NaCl (buffer B) connected to a Pharmacia FPLC system. Proteins were eluted in 2 ml fractions at 2 ml min-1 in buffer B for 60 min. Purification was monitored by SDS-PAGE and fractions containing pure CheV2 were pooled and concentrated to 12 ml. N-terminal sequencing by the automated Edman degradation method (carried out by Dr A. J. G. Moir in the authors Department) was used to confirm the identity of the purified protein.
Fluorescence spectroscopy.
This was performed using a Hitachi F-2500 spectrofluorimeter with an excitation wavelength of 280 nm and an emission wavelength of 340 nm. The assay mixture consisted of 0·10·2 µM CheV2 in 1·5 ml 10 mM HEPES pH 7·5/1 mM EDTA/1 mM magnesium chloride (buffer C). The sample cuvette was maintained at 25 °C in the spectrofluorimeter housing. Fluorescence changes upon the addition of acetyl phosphate (0·550 mM final concentration) were monitored until the fluorescence change stabilized. Fluorescence values were corrected for dilution.
Protein sequence and phylogenetic analyses.
Multiple sequence analyses were performed using the UWGCG program PILEUP (Devereux et al., 1984 ). For phylogenetic analyses, sequences were aligned in CLUSTAL X (Thompson et al., 1997
) and the output file used in PHYLIP (Felsenstein, 1993
) to produce a distance matrix tree with the confidence level of the tree topology determined by bootstrapping (100 datasets). The final trees were viewed in TREEVIEW (Page, 1996
).
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RESULTS |
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The chemotactic ability of the H. pylori mutants was determined by swarm-plate assays on semi-solid BHI medium (Fig. 2). The formation of a visible swarm surrounding the site of inoculation results from the production of a nutrient gradient during growth, to which the cells respond chemotactically by moving outwards, forming characteristic concentric rings. Non-chemotactic cells will remain near the site of inoculation. The parental strain 26695 consistently formed large swarms after 34 d growth (Fig. 2
). However swarming was abolished in the HP0391 (cheW) mutant (Fig. 2
); the cells only grew at the site of inoculation and did not move outwards to any significant extent. This gene is therefore essential for H. pylori chemotaxis. Swarming was also severely reduced in the HP0019 (cheV1) mutant in comparison to the parental strain (Fig. 2
), but was not completely abolished. In contrast, mutations in HP0616 (cheV2) or HP0393 (cheV3) did not result in any decrease in swarming ability compared to the isogenic wild-type parent (Fig. 2
). A cheV2/cheV3 double mutant was constructed to test whether these genes can replace each others function. However, this strain also clearly exhibited normal swarming behaviour (Fig. 2
). The restriction sites used for insertion of the cat cassette into cheV2 and cheV3 are located towards the 3' end of the genes, which might result in the synthesis of partially functional truncated and/or fusion proteins containing the CheW domains. In order to rule out this possibility, the expression in E. coli of CheV from the IPTG-inducible ptac promoter in pMSP10 and pMSP11 was compared to that of the same plasmids containing the cat-inactivated genes. Whereas IPTG-inducible proteins of the predicted size for CheV (33 kDa) were expressed in the case of the wild-type genes, these were clearly absent in the mutant constructs and no truncated or fusion proteins were observed (data not shown). Overall, the mutant phenotypes suggest that cheV1 plays an important role in chemotaxis and that its function cannot be substituted for by either cheV2 or cheV3.
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Evidence for phosphorylation of CheV2 by acetyl phosphate
In order to gain evidence for phosphorylation of the CheY domains of the CheV proteins, E. coli cultures harbouring pMSP8, pMSP10 or pMSP11 were induced with 1 mM IPTG, harvested, and attempts made to purify to homogeneity the overexpressed CheV proteins. Insufficient amounts of CheV1 and CheV3 were expressed from pMSP8 and pMSP10, respectively, for successful purification, but CheV2 was optimally expressed to about 20% of the soluble protein (Fig. 5a) and was successfully purified by a combination of ion-exchange and gel-filtration chromatography after an initial ammonium sulphate fractionation of crude cell-free extracts (Fig. 5b
). The N-terminal sequence of the purified protein was determined to be MLQDPVRDIDKTT. Residues 613 are identical to residues 29 of the deduced CheV2 protein (residues 15 arise from the in-frame cloning into the pTM30 vector; see Methods). In view of the presence of the appropriate conserved residues in the CheY-like domains of each of the proteins, CheV2 was considered to be a suitable representative paralogue for biochemical studies.
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Thus the Km of CheV2 for acetyl phosphate is equal to Ksk3/k2 in this scheme and can be determined from the fluorescence change assuming that the observed quenching is a direct effect of the reduced quantum yield of phospho-CheV2 relative to CheV2 (Lukat et al., 1992 ). The reciprocal of the slope of the line in Fig. 6(c)
gave a Km value of 21 mM.
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DISCUSSION |
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The results from insertional inactivation of each of the three cheV genes in H. pylori revealed that CheV1 is critical for chemotaxis, as a cheV1 null mutant was severely affected in its ability to swarm on soft agar, although swarming was not completely abolished. The expression of the two other cheV genes may be responsible for this residual level of swarming. However, the single cheV2 and cheV3 mutants were fully chemotactic and formed swarms on semi-solid agar indistinguishable from those of the parental strain. These genes cannot simply substitute for each others function, as a cheV2/cheV3 double mutant strain was also fully chemotactic. In the double mutant it seems likely that CheV1 can subsitute for the roles of both CheV2 and CheV3. The results suggest a dominant and specialized function for CheV1 in H. pylori chemotaxis. The expression of H. pylori cheV2 or cheV3 in the E. coli cheW or
cheY mutants did not restore a swarming phenotype. However, as with cheW, when expressed in the isogenic E. coli wild-type strain these genes caused a clear inhibition of swarming. Therefore, CheV2 and CheV3 can interact with the E. coli chemotaxis system but as they consist of both a CheW and a CheY domain, the mechanism of interaction may be complex. The CheW domains could bind to the chemoreceptors and inhibit chemotaxis in the same manner as that described for the H. pylori CheW. However, the CheY domains could also compete with the native E. coli CheY for phosphate from CheA. The overall effect of this would be to sequester phosphoryl groups from the native E. coli CheY, thus causing a build-up of unphosphorylated E. coli CheY which would be unable to bind to the motor switch. The net effect would be that the cells would tumble rarely, giving a smooth-swimming phenotype and an inability to swarm. The ability of CheV2 and CheV3 to perturb chemotaxis in E. coli demonstrates that these proteins do have a role in chemotaxis, despite the fact that normal swarming behaviour was still observed for H. pylori cheV2 and cheV3 mutants. However, in this study we have not examined the swimming patterns of the H. pylori mutants in detail, and it would be informative to study their behaviour by video tracking, to reveal any motility defects not apparent in simple swarm assays.
Thus, the most likely roles for the CheV proteins in H. pylori could involve the ability of their CheY domains to be phosphorylated. All of the proteins contain the necessary active-site residues, and we have shown using fluorescence spectroscopy that CheV2, as a representative of the CheV family, is able to bind the small-molecule phosphodonor acetyl phosphate in a similar manner to that which is characteristic of CheY and other response-regulators (Lukat et al., 1992 ; McCleary, 1996
). This is the first evidence for phosphorylation of any CheV protein. The low affinity observed indicates that acetyl phosphate itself is not physiologically important as a phosphodonor to CheV in vivo; the current view of E. coli CheY from stopped-flow kinetic studies of acetyl phosphate binding is that CheA provides a high local concentration of phosphate groups (Da Re et al., 1999
). Phosphorylation of CheV proteins may function in controlling the flow of phosphate to CheY itself, by the CheV proteins acting as phosphate sinks. The N-terminal CheW domain of CheV may interact with a site on the cytoplasmic signalling domain of the receptors and catalyse phosphoryl transfer from CheA onto the C-terminal CheY domain of the protein. The receptor-binding site may be the same as used by the CheW protein, thus inhibiting the flow of phosphate to the CheY protein itself by a process of competitive binding. The need for a phosphate sink may be related to the absence of a cheZ homologue in H. pylori, as is the case with other non-enteric bacteria. For example, there are no cheZ homologues in Rb. sphaeroides and Sinorhizobium meliloti but these organisms do possess several copies of cheY (Armitage & Schmitt, 1997
; Sourjik & Schmitt, 1996
). A phosphate sink function to control phospho-CheY levels has been suggested for these additional CheY proteins (Armitage & Schmitt, 1997
) and for the CheY domain of CheAY in H. pylori (Foynes et al., 2000
). Our results suggest that the CheY domains of the CheV proteins also have an important role as phospho-acceptors.
The purification of all of the H. pylori chemotaxis proteins, and the reconstitution of the signal-transduction cascade in vitro to demonstrate how intramolecular and intermolecular phosphotransfer from CheAY to the other CheY domains occurs, will be needed to fully explain the role and mechanism of the individual CheV proteins, and to further our understanding of this novel chemotaxis system.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Alm, R. A., Ling, L.-S. L., Moir, D. T. & 20 other authors (1999). Genomic sequencing comparison of two unrelated isolates of the human gastric pathogen Helicobacter pylori. Nature 397, 176180.[Medline]
Armitage, J. P. & Schmitt, R. (1997). Bacterial chemotaxis: Rhodobacter sphaeroides and Sinorhizobium meliloti variations on a theme? Microbiology 143, 3671-3682.[Abstract]
Beier, D., Spohn, G., Rappuoli, R. & Scarlato, V. (1997). Identification and characterisation of an operon of Helicobacter pylori that is involved in motility and stress adaptation. J Bacteriol 179, 4676-4683.[Abstract]
Blair, D. F. (1995). How bacteria sense and swim. Annu Rev Microbiol 49, 489-522.[Medline]
Bourret, R. B., Borkovich, K. A. & Simon, M. I. (1991). Signal transduction involving phosphorylation in prokaryotes. Annu Rev Biochem 60, 401-441.[Medline]
Chalker, A. F., Mineheart, H. W., Hughes, N. J. & 8 other authors (2001). Systematic identification of selective essential genes in Helicobacter pylori by genome prioritization and allelic replacement mutagenesis. J Bacteriol 183, 12591268.
Da Re, S. S., Deville-Bonne, D., Tolstyk, T., Veron, M. & Stock, J. B. (1999). Kinetics of CheY phosphorylation by small molecule phosphodonors. FEBS Lett 457, 323-326.[Medline]
Devereux, J., Haeberli, P. & Smithies, O. (1984). A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res 12, 387-395.[Abstract]
Doig, P., de Jonge, B. L., Alm, R. A. & 10 other authors (1999). Helicobacter pylori physiology predicted from genomic comparison of two strains. Microbiol Mol Biol Rev 63, 675707.
Eaton, K. A., Morgan, D. R. & Krakowa, S. (1992). Motility as a factor in the colonisation of gnotobiotic piglets by Helicobacter pylori. J Med Microbiol 37, 123-127.[Abstract]
Eaton, K. A., Suerbaum, S., Josenhans, C. & Krakowa, S. (1996). Colonisation of gnotobiotic piglets by Helicobacter pylori deficient in two flagellin genes. Infect Immun 64, 2445-2448.[Abstract]
Felsenstein, J. (1993). PHYLIP: Phylogeny Inference Package. Version 3.5. Seattle, WA: University of Washington.
Ferrero, R. L., Cussac, V., Courcoux, P. & Labigne-Roussel (1992). Construction of isogenic urease-negative mutants of Helicobacter pylori by allelic exchange. J Bacteriol 174, 4212-4217.[Abstract]
Forman, D., Newell, D. G., Fullerton, F., Yarnell, J. W. G., Stacey, A. R., Wald, N. & Sitas, F. (1991). Association between infection with Helicobacter pylori and risk of gastric cancer: evidence from a perspective investigation. Br Med J 302, 1302-1305.[Medline]
Foynes, S., Dorrell, N., Ward, S. J., Stabler, R. A., McColm, A. A., Rycroft, A. N. & Wren, B. W. (2000). Helicobacter pylori possesses two CheY response regulators and a histidine kinase sensor, CheA, which are essential for chemotaxis and colonization of the gastric mucosa. Infect Immun 68, 2016-2023.
Frederick, K. L. & Helmann, J. D. (1994). Dual chemotaxis signalling pathways in Bacillus subtilis: a D-dependent gene encodes a novel protein with both CheW and CheY homologous domains. J Bacteriol 176, 2727-2735.[Abstract]
Hamblin, P. A., Bourne, N. A. & Armitage, J. P. (1997). Characterisation of the chemotaxis protein CheW from Rhodobacter sphaeroides and its effect on the behaviour of Escherichia coli. Mol Microbiol 24, 41-51.[Medline]
Jackson, C. J., Kelly, D. J. & Clayton, C. L. (1995). The cloning and characterisation of chemotaxis genes in Helicobacter pylori. Gut 37(S1), A18.[Medline]
Kelly, D. J. (1998). The physiology and metabolism of the human gastric pathogen Helicobacter pylori. Adv Microb Physiol 40, 137-189.[Medline]
Lukat, G. S., McCleary, W. R., Stock, A. M. & Stock, J. B. (1992). Phosphorylation of bacterial response regulator proteins by low-molecular weight phosphodonors. Proc Natl Acad Sci USA 89, 718-722.[Abstract]
McCleary, W. R. (1996). The activation of PhoB by acetylphosphate. Mol Microbiol 20, 1155-1163.[Medline]
McCleary, W. R. & Stock, J. B. (1994). Acetyl-phosphate and the activation of two-component response-regulators. J Biol Chem 269, 31567-31572.
Marmur, J. (1961). A procedure for the isolation of deoxyribonucleic acid from microorganisms. J Mol Biol 3, 208-218.
Marshall, B. J., Armstrong, J. A., McGechie, D. B. & Glancy, R. J. (1985). Attempt to fulfil Kochs postulates for pyloric campylobacter. Med J Aust 142, 436-439.[Medline]
Morrison, T. B. & Parkinson, J. S. (1994). Liberation of an interaction domain from the phosphotransfer region of CheA, a signaling kinase of Escherichia coli. Proc Natl Acad Sci USA 91, 5485-5489.[Abstract]
Moss, S. & Calam, J. (1992). Helicobacter pylori and peptic ulcers: the present position. Gut 33, 289-292.[Medline]
Page, R. D. M. (1996). Treeview: an application to display phylogenetic trees on personal computers. Comput Appl Biosci 12, 357-358.[Medline]
Rosario, M. M. L., Frederick, K. L., Ordal, G. W. & Helmann, J. D. (1994). Chemotaxis in Bacillus subtilis requires either of two functionally redundant CheW homologues. J Bacteriol 176, 2736-2739.[Abstract]
Sanders, D. A., Gillece-Castro, B. L., Stock, A. M., Burlingame, A. L. & Koshland, D. E.Jr (1989a). Identification of the site of phosphorylation of the chemotaxis protein response regulator, CheY. J Biol Chem 264, 21770-21778.
Sanders, D. A., Mendez, B. & Koshland, D. E.Jr (1989b). Role of the CheW protein in bacterial chemotaxis: overexpression is equivalent to absence. J Bacteriol 171, 6271-6278.[Medline]
Sourjik, V. & Schmitt, R. (1996). Different roles of CheY1 and CheY2 in the chemotaxis of Rhizobium meliloti. Mol Microbiol 22, 427-436.[Medline]
Stock, J. B. & Surette, M. G. (1996). Chemotaxis. In Escherichia coli and Salmonella typhimurium, pp. 11031129. Edited by F. C. Neidhardt and others. Washington, DC: American Society for Microbiology.
Studier, F. W. & Moffat, B. A. (1986). Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes. J Mol Biol 189, 113-130.[Medline]
Thompson, J. D., Gibson, T. J., Plewiniak, F., Jeanmougin, F. & Higgins, D. A. (1997). The CLUSTAL_X Windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res 25, 4876-4882.
Tomb, J.-F., White, O., Kerlavage, A. R. & 39 other authors (1997). The complete genome sequence of the gastric pathogen Helicobacter pylori. Nature 388, 539547.[Medline]
Volz, K. (1993). Structural conservation in the CheY superfamily. Biochemistry 32, 11741-11753.[Medline]
Wang, Y. & Taylor, D. E. (1990). Chloramphenicol resistance in Campylobacter coli nucleotide-sequence, expression, and cloning vector construction. Gene 94, 23-28.[Medline]
Wyatt, J. I., Rathbone, B. J., Dixon, M. F. & Heatley, R. V. (1987). Campylobacter pyloridis and acid-induced metaplasia in the pathogenesis of duodenitis. J Clin Pathol 40, 841-848.[Abstract]
Received 2 March 2001;
revised 4 May 2001;
accepted 10 May 2001.