Skip to main content
NCBI home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1996 Mar;178(5):1310–1319. doi: 10.1128/jb.178.5.1310-1319.1996

Flagellin A is essential for the virulence of Vibrio anguillarum.

D L Milton 1, R O'Toole 1, P Horstedt 1, H Wolf-Watz 1
PMCID: PMC177804  PMID: 8631707

Abstract

A flagellin gene from the fish pathogen Vibrio anguillarum was cloned, sequenced, and mutagenized. The DNA sequence suggests that the flaA gene encodes a 40.1-kDa protein and is a single transcriptional unit. A polar mutation and four in-frame deletion mutations (180 bp deleted from the 5' end of the gene, 153 bp deleted from the 3' end of the gene, a double deletion of both the 180- and 153-bp deletions, and 942 bp deleted from the entire gene) were made. Compared with the wild type, all mutants were partially motile, and a shortening of the flagellum was seen by electron microscopy. Wild-type phenotypes were regained when the mutations were transcomplemented with the flaA gene. Protein analysis indicated that the flaA gene corresponds to a 40-kDa protein and that the flagellum consists of three additional flagellin proteins with molecular masses of 41, 42, and 45 kDa. N-terminal sequence analysis confirmed that the additional proteins were flagellins with N termini that are 82 to 88% identical to the N terminus of FlaA. Virulence studies showed that the N terminal deletion, the double deletion, and the 942-bp deletion increased the 50% lethal dose between 70- and 700-fold via immersion infection, whereas infection via intraperitoneal injection showed no loss in virulence. In contrast, the polar mutant and the carboxy-terminal deletion mutant showed approximately a 10(4)-fold increase in the 50% lethal dose by both immersion and intraperitoneal infection. In summary, FlaA is needed for crossing the fish integument and may play a role in virulence after invasion of the host.

Full Text

The Full Text of this article is available as a PDF (631.3 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Akerley B. J., Cotter P. A., Miller J. F. Ectopic expression of the flagellar regulon alters development of the Bordetella-host interaction. Cell. 1995 Feb 24;80(4):611–620. doi: 10.1016/0092-8674(95)90515-4. [DOI] [PubMed] [Google Scholar]
  2. Attridge S. R., Rowley D. The role of the flagellum in the adherence of Vibrio cholerae. J Infect Dis. 1983 May;147(5):864–872. doi: 10.1093/infdis/147.5.864. [DOI] [PubMed] [Google Scholar]
  3. Chart H. Multiflagellate variants of Vibrio anguillarum. J Gen Microbiol. 1983 Jul;129(7):2193–2197. doi: 10.1099/00221287-129-7-2193. [DOI] [PubMed] [Google Scholar]
  4. Devereux J., Haeberli P., Smithies O. A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res. 1984 Jan 11;12(1 Pt 1):387–395. doi: 10.1093/nar/12.1part1.387. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Drake D., Montie T. C. Flagella, motility and invasive virulence of Pseudomonas aeruginosa. J Gen Microbiol. 1988 Jan;134(1):43–52. doi: 10.1099/00221287-134-1-43. [DOI] [PubMed] [Google Scholar]
  6. 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]
  7. Fuerst J. A., Perry J. W. Demonstration of lipopolysaccharide on sheathed flagella of Vibrio cholerae O:1 by protein A-gold immunoelectron microscopy. J Bacteriol. 1988 Apr;170(4):1488–1494. doi: 10.1128/jb.170.4.1488-1494.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Gay P., Le Coq D., Steinmetz M., Berkelman T., Kado C. I. Positive selection procedure for entrapment of insertion sequence elements in gram-negative bacteria. J Bacteriol. 1985 Nov;164(2):918–921. doi: 10.1128/jb.164.2.918-921.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. 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]
  10. Gerl L., Sumper M. Halobacterial flagellins are encoded by a multigene family. Characterization of five flagellin genes. J Biol Chem. 1988 Sep 15;263(26):13246–13251. [PubMed] [Google Scholar]
  11. Grant C. C., Konkel M. E., Cieplak W., Jr, Tompkins L. S. Role of flagella in adherence, internalization, and translocation of Campylobacter jejuni in nonpolarized and polarized epithelial cell cultures. Infect Immun. 1993 May;61(5):1764–1771. doi: 10.1128/iai.61.5.1764-1771.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Guentzel M. N., Berry L. J. Motility as a virulence factor for Vibrio cholerae. Infect Immun. 1975 May;11(5):890–897. doi: 10.1128/iai.11.5.890-897.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. 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]
  14. Helmann J. D. Alternative sigma factors and the regulation of flagellar gene expression. Mol Microbiol. 1991 Dec;5(12):2875–2882. doi: 10.1111/j.1365-2958.1991.tb01847.x. [DOI] [PubMed] [Google Scholar]
  15. Homma M., DeRosier D. J., Macnab R. M. Flagellar hook and hook-associated proteins of Salmonella typhimurium and their relationship to other axial components of the flagellum. J Mol Biol. 1990 Jun 20;213(4):819–832. doi: 10.1016/S0022-2836(05)80266-9. [DOI] [PubMed] [Google Scholar]
  16. Jones G. W., Freter R. Adhesive properties of Vibrio cholerae: nature of the interaction with isolated rabbit brush border membranes and human erythrocytes. Infect Immun. 1976 Jul;14(1):240–245. doi: 10.1128/iai.14.1.240-245.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Kaniga K., Delor I., Cornelis G. R. A wide-host-range suicide vector for improving reverse genetics in gram-negative bacteria: inactivation of the blaA gene of Yersinia enterocolitica. Gene. 1991 Dec 20;109(1):137–141. doi: 10.1016/0378-1119(91)90599-7. [DOI] [PubMed] [Google Scholar]
  18. Kapatral V., Minnich S. A. Co-ordinate, temperature-sensitive regulation of the three Yersinia enterocolitica flagellin genes. Mol Microbiol. 1995 Jul;17(1):49–56. doi: 10.1111/j.1365-2958.1995.mmi_17010049.x. [DOI] [PubMed] [Google Scholar]
  19. Kelly-Wintenberg K., Anderson T., Montie T. C. Phosphorylated tyrosine in the flagellum filament protein of Pseudomonas aeruginosa. J Bacteriol. 1990 Sep;172(9):5135–5139. doi: 10.1128/jb.172.9.5135-5139.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Kerridge D. Flagellar synthesis in Salmonella typhimurium: factors affecting the formation of the flagellar epsilon-N-methyllysine. J Gen Microbiol. 1966 Jan;42(1):71–82. doi: 10.1099/00221287-42-1-71. [DOI] [PubMed] [Google Scholar]
  21. Kornacker M. G., Newton A. Information essential for cell-cycle-dependent secretion of the 591-residue Caulobacter hook protein is confined to a 21-amino-acid sequence near the N-terminus. Mol Microbiol. 1994 Oct;14(1):73–85. doi: 10.1111/j.1365-2958.1994.tb01268.x. [DOI] [PubMed] [Google Scholar]
  22. 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]
  23. Kuwajima G., Kawagishi I., Homma M., Asaka J., Kondo E., Macnab R. M. Export of an N-terminal fragment of Escherichia coli flagellin by a flagellum-specific pathway. Proc Natl Acad Sci U S A. 1989 Jul;86(13):4953–4957. doi: 10.1073/pnas.86.13.4953. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Logan S. M., Trust T. J., Guerry P. Evidence for posttranslational modification and gene duplication of Campylobacter flagellin. J Bacteriol. 1989 Jun;171(6):3031–3038. doi: 10.1128/jb.171.6.3031-3038.1989. [DOI] [PMC free article] [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. Lövgren A., Zhang M. Y., Engström A., Landén R. Identification of two expressed flagellin genes in the insect pathogen Bacillus thuringiensis subsp. alesti. J Gen Microbiol. 1993 Jan;139(1):21–30. doi: 10.1099/00221287-139-1-21. [DOI] [PubMed] [Google Scholar]
  27. McCarter L. L. Genetic and molecular characterization of the polar flagellum of Vibrio parahaemolyticus. J Bacteriol. 1995 Mar;177(6):1595–1609. doi: 10.1128/jb.177.6.1595-1609.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. McSweegan E., Walker R. I. Identification and characterization of two Campylobacter jejuni adhesins for cellular and mucous substrates. Infect Immun. 1986 Jul;53(1):141–148. doi: 10.1128/iai.53.1.141-148.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Miller V. L., Mekalanos J. J. A novel suicide vector and its use in construction of insertion mutations: osmoregulation of outer membrane proteins and virulence determinants in Vibrio cholerae requires toxR. J Bacteriol. 1988 Jun;170(6):2575–2583. doi: 10.1128/jb.170.6.2575-2583.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Milton D. L., Norqvist A., Wolf-Watz H. Cloning of a metalloprotease gene involved in the virulence mechanism of Vibrio anguillarum. J Bacteriol. 1992 Nov;174(22):7235–7244. doi: 10.1128/jb.174.22.7235-7244.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Minnich S. A., Ohta N., Taylor N., Newton A. Role of the 25-, 27-, and 29-kilodalton flagellins in Caulobacter crescentus cell motility: method for construction of deletion and Tn5 insertion mutants by gene replacement. J Bacteriol. 1988 Sep;170(9):3953–3960. doi: 10.1128/jb.170.9.3953-3960.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Norqvist A., Hagström A., Wolf-Watz H. Protection of rainbow trout against vibriosis and furunculosis by the use of attenuated strains of Vibrio anguillarum. Appl Environ Microbiol. 1989 Jun;55(6):1400–1405. doi: 10.1128/aem.55.6.1400-1405.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Norqvist A., Wolf-Watz H. Characterization of a novel chromosomal virulence locus involved in expression of a major surface flagellar sheath antigen of the fish pathogen Vibrio anguillarum. Infect Immun. 1993 Jun;61(6):2434–2444. doi: 10.1128/iai.61.6.2434-2444.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. 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]
  35. Pleier E., Schmitt R. Identification and sequence analysis of two related flagellin genes in Rhizobium meliloti. J Bacteriol. 1989 Mar;171(3):1467–1475. doi: 10.1128/jb.171.3.1467-1475.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Richardson K. Roles of motility and flagellar structure in pathogenicity of Vibrio cholerae: analysis of motility mutants in three animal models. Infect Immun. 1991 Aug;59(8):2727–2736. doi: 10.1128/iai.59.8.2727-2736.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Thomashow L. S., Rittenberg S. C. Isolation and composition of sheathed flagella from Bdellovibrio bacteriovorus 109J. J Bacteriol. 1985 Sep;163(3):1047–1054. doi: 10.1128/jb.163.3.1047-1054.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Totten P. A., Lory S. Characterization of the type a flagellin gene from Pseudomonas aeruginosa PAK. J Bacteriol. 1990 Dec;172(12):7188–7199. doi: 10.1128/jb.172.12.7188-7199.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Wieland F., Paul G., Sumper M. Halobacterial flagellins are sulfated glycoproteins. J Biol Chem. 1985 Dec 5;260(28):15180–15185. [PubMed] [Google Scholar]
  40. Yao R., Burr D. H., Doig P., Trust T. J., Niu H., Guerry P. Isolation of motile and non-motile insertional mutants of Campylobacter jejuni: the role of motility in adherence and invasion of eukaryotic cells. Mol Microbiol. 1994 Dec;14(5):883–893. doi: 10.1111/j.1365-2958.1994.tb01324.x. [DOI] [PubMed] [Google Scholar]

Articles from Journal of Bacteriology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES