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. 1997 Feb;179(4):987–997. doi: 10.1128/jb.179.4.987-997.1997

Cloning and characterization of the Helicobacter pylori flbA gene, which codes for a membrane protein involved in coordinated expression of flagellar genes.

A Schmitz 1, C Josenhans 1, S Suerbaum 1
PMCID: PMC178789  PMID: 9023175

Abstract

Flagellar motility has been shown to be an essential requirement for the ability of Helicobacter pylori to colonize the gastric mucosa. While some flagellar structural components have been studied in molecular detail, nothing was known about factors that play a role in the regulation of flagellar biogenesis. We have cloned and characterized an H. pylori homolog (named flbA) of the lcrD/flbF family of genes. Many proteins encoded by these genes are known to be involved in flagellar biogenesis or secretion of virulence-associated proteins via type III secretion systems. The H. pylori flbA gene (2,196 bp) is capable of coding for a predicted 732-amino-acid, 80.9-kDa protein that has marked sequence similarity with other known members of the LcrD/FlbF protein family. An isogenic strain with a mutation in the flbA gene was constructed by disruption of the gene with a kanamycin resistance cassette and electroporation-mediated allelic exchange mutagenesis. The mutant strain expressed neither the FlaA nor the FlaB flagellin protein. The expression of the FlgE hook protein was reduced in comparison with the wild-type strain, and the extent of this reduction was growth phase dependent. The flbA gene disruption was shown to downregulate the expression of these flagellar genes on the transcriptional level. The flbA mutants were aflagellate and completely nonmotile. Occasionally, assembled hook structures could be observed, indicating that export of axial flagellar filament components was still possible in the absence of the flbA gene product. The hydrophilic part of the FlbA protein was expressed in Escherichia coli, purified, and used to raise a polyclonal rabbit antiserum against the FlbA protein. Western blot experiments with this antiserum indicated that the FlbA protein is predominantly associated with the cytoplasmic membrane in H. pylori. The antiserum cross-reacted with two other proteins (97 and 43 kDa) whose expression was not affected by the flbA gene disruption and which might represent further H. pylori homologs of the LcrD/FlbF protein family.

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Selected References

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  1. Achtman M., Mercer A., Kusecek B., Pohl A., Heuzenroeder M., Aaronson W., Sutton A., Silver R. P. Six widespread bacterial clones among Escherichia coli K1 isolates. Infect Immun. 1983 Jan;39(1):315–335. doi: 10.1128/iai.39.1.315-335.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Blair D. F. How bacteria sense and swim. Annu Rev Microbiol. 1995;49:489–522. doi: 10.1146/annurev.mi.49.100195.002421. [DOI] [PubMed] [Google Scholar]
  3. Bukanov N. O., Berg D. E. Ordered cosmid library and high-resolution physical-genetic map of Helicobacter pylori strain NCTC11638. Mol Microbiol. 1994 Feb;11(3):509–523. doi: 10.1111/j.1365-2958.1994.tb00332.x. [DOI] [PubMed] [Google Scholar]
  4. Casadaban M. J., Cohen S. N. Analysis of gene control signals by DNA fusion and cloning in Escherichia coli. J Mol Biol. 1980 Apr;138(2):179–207. doi: 10.1016/0022-2836(80)90283-1. [DOI] [PubMed] [Google Scholar]
  5. Cover T. L., Blaser M. J. Helicobacter pylori infection, a paradigm for chronic mucosal inflammation: pathogenesis and implications for eradication and prevention. Adv Intern Med. 1996;41:85–117. [PubMed] [Google Scholar]
  6. Eaton K. A., Brooks C. L., Morgan D. R., Krakowka S. Essential role of urease in pathogenesis of gastritis induced by Helicobacter pylori in gnotobiotic piglets. Infect Immun. 1991 Jul;59(7):2470–2475. doi: 10.1128/iai.59.7.2470-2475.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Eaton K. A., Morgan D. R., Krakowka S. Motility as a factor in the colonisation of gnotobiotic piglets by Helicobacter pylori. J Med Microbiol. 1992 Aug;37(2):123–127. doi: 10.1099/00222615-37-2-123. [DOI] [PubMed] [Google Scholar]
  8. Eaton K. A., Suerbaum S., Josenhans C., Krakowka S. Colonization of gnotobiotic piglets by Helicobacter pylori deficient in two flagellin genes. Infect Immun. 1996 Jul;64(7):2445–2448. doi: 10.1128/iai.64.7.2445-2448.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Ferrero R. L., Cussac V., Courcoux P., Labigne A. Construction of isogenic urease-negative mutants of Helicobacter pylori by allelic exchange. J Bacteriol. 1992 Jul;174(13):4212–4217. doi: 10.1128/jb.174.13.4212-4217.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Galán J. E., Ginocchio C., Costeas P. Molecular and functional characterization of the Salmonella invasion gene invA: homology of InvA to members of a new protein family. J Bacteriol. 1992 Jul;174(13):4338–4349. doi: 10.1128/jb.174.13.4338-4349.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Geis G., Leying H., Suerbaum S., Mai U., Opferkuch W. Ultrastructure and chemical analysis of Campylobacter pylori flagella. J Clin Microbiol. 1989 Mar;27(3):436–441. doi: 10.1128/jcm.27.3.436-441.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Ginocchio C. C., Olmsted S. B., Wells C. L., Galán J. E. Contact with epithelial cells induces the formation of surface appendages on Salmonella typhimurium. Cell. 1994 Feb 25;76(4):717–724. doi: 10.1016/0092-8674(94)90510-x. [DOI] [PubMed] [Google Scholar]
  13. Goodwin C. S., McCulloch R. K., Armstrong J. A., Wee S. H. Unusual cellular fatty acids and distinctive ultrastructure in a new spiral bacterium (Campylobacter pyloridis) from the human gastric mucosa. J Med Microbiol. 1985 Apr;19(2):257–267. doi: 10.1099/00222615-19-2-257. [DOI] [PubMed] [Google Scholar]
  14. Guerry P., Logan S. M., Thornton S., Trust T. J. Genomic organization and expression of Campylobacter flagellin genes. J Bacteriol. 1990 Apr;172(4):1853–1860. doi: 10.1128/jb.172.4.1853-1860.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Gygi D., Bailey M. J., Allison C., Hughes C. Requirement for FlhA in flagella assembly and swarm-cell differentiation by Proteus mirabilis. Mol Microbiol. 1995 Feb;15(4):761–769. doi: 10.1111/j.1365-2958.1995.tb02383.x. [DOI] [PubMed] [Google Scholar]
  16. Harshey R. M., Toguchi A. Spinning tails: homologies among bacterial flagellar systems. Trends Microbiol. 1996 Jun;4(6):226–231. doi: 10.1016/0966-842X(96)10037-8. [DOI] [PubMed] [Google Scholar]
  17. Jones C. J., Aizawa S. Genetic control of the bacterial flagellar regulon. Curr Opin Genet Dev. 1991 Oct;1(3):319–323. doi: 10.1016/s0959-437x(05)80294-1. [DOI] [PubMed] [Google Scholar]
  18. Jones C. J., Aizawa S. The bacterial flagellum and flagellar motor: structure, assembly and function. Adv Microb Physiol. 1991;32:109–172. doi: 10.1016/s0065-2911(08)60007-7. [DOI] [PubMed] [Google Scholar]
  19. Josenhans C., Labigne A., Suerbaum S. Comparative ultrastructural and functional studies of Helicobacter pylori and Helicobacter mustelae flagellin mutants: both flagellin subunits, FlaA and FlaB, are necessary for full motility in Helicobacter species. J Bacteriol. 1995 Jun;177(11):3010–3020. doi: 10.1128/jb.177.11.3010-3020.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Kostrzynska M., Betts J. D., Austin J. W., Trust T. J. Identification, characterization, and spatial localization of two flagellin species in Helicobacter pylori flagella. J Bacteriol. 1991 Feb;173(3):937–946. doi: 10.1128/jb.173.3.937-946.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Kutsukake K. Excretion of the anti-sigma factor through a flagellar substructure couples flagellar gene expression with flagellar assembly in Salmonella typhimurium. Mol Gen Genet. 1994 Jun 15;243(6):605–612. doi: 10.1007/BF00279569. [DOI] [PubMed] [Google Scholar]
  22. Labigne-Roussel A., Courcoux P., Tompkins L. Gene disruption and replacement as a feasible approach for mutagenesis of Campylobacter jejuni. J Bacteriol. 1988 Apr;170(4):1704–1708. doi: 10.1128/jb.170.4.1704-1708.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Labigne A., Cussac V., Courcoux P. Shuttle cloning and nucleotide sequences of Helicobacter pylori genes responsible for urease activity. J Bacteriol. 1991 Mar;173(6):1920–1931. doi: 10.1128/jb.173.6.1920-1931.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Leying H., Suerbaum S., Geis G., Haas R. Cloning and genetic characterization of a Helicobacter pylori flagellin gene. Mol Microbiol. 1992 Oct;6(19):2863–2874. doi: 10.1111/j.1365-2958.1992.tb01466.x. [DOI] [PubMed] [Google Scholar]
  25. Lugtenberg B., Meijers J., Peters R., van der Hoek P., van Alphen L. Electrophoretic resolution of the "major outer membrane protein" of Escherichia coli K12 into four bands. FEBS Lett. 1975 Oct 15;58(1):254–258. doi: 10.1016/0014-5793(75)80272-9. [DOI] [PubMed] [Google Scholar]
  26. Majewski S. I., Goodwin C. S. Restriction endonuclease analysis of the genome of Campylobacter pylori with a rapid extraction method: evidence for considerable genomic variation. J Infect Dis. 1988 Mar;157(3):465–471. doi: 10.1093/infdis/157.3.465. [DOI] [PubMed] [Google Scholar]
  27. Messing J., Vieira J. A new pair of M13 vectors for selecting either DNA strand of double-digest restriction fragments. Gene. 1982 Oct;19(3):269–276. doi: 10.1016/0378-1119(82)90016-6. [DOI] [PubMed] [Google Scholar]
  28. Miller S., Pesci E. C., Pickett C. L. A Campylobacter jejuni homolog of the LcrD/FlbF family of proteins is necessary for flagellar biogenesis. Infect Immun. 1993 Jul;61(7):2930–2936. doi: 10.1128/iai.61.7.2930-2936.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Minamino T., Iino T., Kutuskake K. Molecular characterization of the Salmonella typhimurium flhB operon and its protein products. J Bacteriol. 1994 Dec;176(24):7630–7637. doi: 10.1128/jb.176.24.7630-7637.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Mobley H. L., Island M. D., Hausinger R. P. Molecular biology of microbial ureases. Microbiol Rev. 1995 Sep;59(3):451–480. doi: 10.1128/mr.59.3.451-480.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Nuijten P. J., van Asten F. J., Gaastra W., van der Zeijst B. A. Structural and functional analysis of two Campylobacter jejuni flagellin genes. J Biol Chem. 1990 Oct 15;265(29):17798–17804. [PubMed] [Google Scholar]
  32. O'Toole P. W., Kostrzynska M., Trust T. J. Non-motile mutants of Helicobacter pylori and Helicobacter mustelae defective in flagellar hook production. Mol Microbiol. 1994 Nov;14(4):691–703. doi: 10.1111/j.1365-2958.1994.tb01307.x. [DOI] [PubMed] [Google Scholar]
  33. Peek R. M., Jr, Miller G. G., Tham K. T., Pérez-Pérez G. I., Cover T. L., Atherton J. C., Dunn G. D., Blaser M. J. Detection of Helicobacter pylori gene expression in human gastric mucosa. J Clin Microbiol. 1995 Jan;33(1):28–32. doi: 10.1128/jcm.33.1.28-32.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Plano G. V., Barve S. S., Straley S. C. LcrD, a membrane-bound regulator of the Yersinia pestis low-calcium response. J Bacteriol. 1991 Nov;173(22):7293–7303. doi: 10.1128/jb.173.22.7293-7303.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Plano G. V., Straley S. C. Multiple effects of lcrD mutations in Yersinia pestis. J Bacteriol. 1993 Jun;175(11):3536–3545. doi: 10.1128/jb.175.11.3536-3545.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Ramakrishnan G., Zhao J. L., Newton A. The cell cycle-regulated flagellar gene flbF of Caulobacter crescentus is homologous to a virulence locus (lcrD) of Yersinia pestis. J Bacteriol. 1991 Nov;173(22):7283–7292. doi: 10.1128/jb.173.22.7283-7292.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Rosqvist R., Håkansson S., Forsberg A., Wolf-Watz H. Functional conservation of the secretion and translocation machinery for virulence proteins of yersiniae, salmonellae and shigellae. EMBO J. 1995 Sep 1;14(17):4187–4195. doi: 10.1002/j.1460-2075.1995.tb00092.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Sanders L. A., Van Way S., Mullin D. A. Characterization of the Caulobacter crescentus flbF promoter and identification of the inferred FlbF product as a homolog of the LcrD protein from a Yersinia enterocolitica virulence plasmid. J Bacteriol. 1992 Feb;174(3):857–866. doi: 10.1128/jb.174.3.857-866.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Shapiro L. The bacterial flagellum: from genetic network to complex architecture. Cell. 1995 Feb 24;80(4):525–527. doi: 10.1016/0092-8674(95)90505-7. [DOI] [PubMed] [Google Scholar]
  41. Straley S. C., Plano G. V., Skrzypek E., Haddix P. L., Fields K. A. Regulation by Ca2+ in the Yersinia low-Ca2+ response. Mol Microbiol. 1993 Jun;8(6):1005–1010. doi: 10.1111/j.1365-2958.1993.tb01644.x. [DOI] [PubMed] [Google Scholar]
  42. Suerbaum S., Geis G., Josenhans C., Opferkuch W. Biochemical studies of Helicobacter mustelae fatty acid composition and flagella. Infect Immun. 1992 Apr;60(4):1695–1698. doi: 10.1128/iai.60.4.1695-1698.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Suerbaum S., Josenhans C., Labigne A. Cloning and genetic characterization of the Helicobacter pylori and Helicobacter mustelae flaB flagellin genes and construction of H. pylori flaA- and flaB-negative mutants by electroporation-mediated allelic exchange. J Bacteriol. 1993 Jun;175(11):3278–3288. doi: 10.1128/jb.175.11.3278-3288.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Suerbaum S. The complex flagella of gastric Helicobacter species. Trends Microbiol. 1995 May;3(5):168–171. doi: 10.1016/s0966-842x(00)88913-1. [DOI] [PubMed] [Google Scholar]
  45. Suerbaum S., Thiberge J. M., Kansau I., Ferrero R. L., Labigne A. Helicobacter pylori hspA-hspB heat-shock gene cluster: nucleotide sequence, expression, putative function and immunogenicity. Mol Microbiol. 1994 Dec;14(5):959–974. doi: 10.1111/j.1365-2958.1994.tb01331.x. [DOI] [PubMed] [Google Scholar]
  46. Telford J. L., Ghiara P., Dell'Orco M., Comanducci M., Burroni D., Bugnoli M., Tecce M. F., Censini S., Covacci A., Xiang Z. Gene structure of the Helicobacter pylori cytotoxin and evidence of its key role in gastric disease. J Exp Med. 1994 May 1;179(5):1653–1658. doi: 10.1084/jem.179.5.1653. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Yanisch-Perron C., Vieira J., Messing J. Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene. 1985;33(1):103–119. doi: 10.1016/0378-1119(85)90120-9. [DOI] [PubMed] [Google Scholar]

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