Skip to main content
Infection and Immunity logoLink to Infection and Immunity
. 1993 Jul;61(7):3032–3037. doi: 10.1128/iai.61.7.3032-3037.1993

Nucleotide sequence encoding the mannose-fucose-resistant hemagglutinin of Vibrio cholerae O1 and construction of a mutant.

V L Franzon 1, A Barker 1, P A Manning 1
PMCID: PMC280956  PMID: 8514410

Abstract

The region of DNA encoding the mannose-fucose-resistant hemagglutinin (MFRHA) of Vibrio cholerae O1 has been localized, and the nucleotide sequence has been determined. The region contains a single open reading frame encoding 230 amino acids, corresponding to a protein of 26.9 kDa. The N terminus of this protein is atypical for a protein localized in the outer membrane. A mutant lacking MFRHA activity has been constructed by allelic exchange after inactivation via the insertion of a kanamycin resistance gene cartridge. The MFRHA-negative mutant has been assessed for virulence in the infant mouse cholera model. This mutant shows a marked defect in its ability to persist in the infant mouse gut and is incapable of competing with the wild-type organism, even when given in 25-fold excess. This defect also leads to a > 100-fold increase in the 50% lethal dose. These data suggest that the MFRHA is an important colonization factor in the infant mouse model.

Full text

PDF

Selected References

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

  1. Alm R. A., Manning P. A. Characterization of the hlyB gene and its role in the production of the El Tor haemolysin of Vibrio cholerae O1. Mol Microbiol. 1990 Mar;4(3):413–425. doi: 10.1111/j.1365-2958.1990.tb00608.x. [DOI] [PubMed] [Google Scholar]
  2. Attridge S. R., Rowley D. Prophylactic significance of the nonlipopolysaccharide antigens of Vibrio cholerae. J Infect Dis. 1983 Nov;148(5):931–939. doi: 10.1093/infdis/148.5.931. [DOI] [PubMed] [Google Scholar]
  3. Bolivar F., Rodriguez R. L., Greene P. J., Betlach M. C., Heyneker H. L., Boyer H. W., Crosa J. H., Falkow S. Construction and characterization of new cloning vehicles. II. A multipurpose cloning system. Gene. 1977;2(2):95–113. [PubMed] [Google Scholar]
  4. Boyer H. W., Roulland-Dussoix D. A complementation analysis of the restriction and modification of DNA in Escherichia coli. J Mol Biol. 1969 May 14;41(3):459–472. doi: 10.1016/0022-2836(69)90288-5. [DOI] [PubMed] [Google Scholar]
  5. Clewell D. B., Helinski D. R. Supercoiled circular DNA-protein complex in Escherichia coli: purification and induced conversion to an opern circular DNA form. Proc Natl Acad Sci U S A. 1969 Apr;62(4):1159–1166. doi: 10.1073/pnas.62.4.1159. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Ditta G., Stanfield S., Corbin D., Helinski D. R. Broad host range DNA cloning system for gram-negative bacteria: construction of a gene bank of Rhizobium meliloti. Proc Natl Acad Sci U S A. 1980 Dec;77(12):7347–7351. doi: 10.1073/pnas.77.12.7347. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Finkelstein R. A., Hanne L. F. Purification and characterization of the soluble hemagglutinin (cholera lectin)( produced by Vibrio cholerae. Infect Immun. 1982 Jun;36(3):1199–1208. doi: 10.1128/iai.36.3.1199-1208.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Focareta T., Manning P. A. Distinguishing between the extracellular DNases of Vibrio cholerae and development of a transformation system. Mol Microbiol. 1991 Oct;5(10):2547–2555. doi: 10.1111/j.1365-2958.1991.tb02101.x. [DOI] [PubMed] [Google Scholar]
  9. Franzon V. L., Manning P. A. Molecular cloning and expression in Escherichia coli K-12 of the gene for a hemagglutinin from Vibrio cholerae. Infect Immun. 1986 Apr;52(1):279–284. doi: 10.1128/iai.52.1.279-284.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Freter R., Jones G. W. Adhesive properties of Vibrio cholerae: nature of the interaction with intact mucosal surfaces. Infect Immun. 1976 Jul;14(1):246–256. doi: 10.1128/iai.14.1.246-256.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Hanne L. F., Finkelstein R. A. Characterization and distribution of the hemagglutinins produced by Vibrio cholerae. Infect Immun. 1982 Apr;36(1):209–214. doi: 10.1128/iai.36.1.209-214.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Herrington D. A., Hall R. H., Losonsky G., Mekalanos J. J., Taylor R. K., Levine M. M. Toxin, toxin-coregulated pili, and the toxR regulon are essential for Vibrio cholerae pathogenesis in humans. J Exp Med. 1988 Oct 1;168(4):1487–1492. doi: 10.1084/jem.168.4.1487. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Holmes D. S., Quigley M. A rapid boiling method for the preparation of bacterial plasmids. Anal Biochem. 1981 Jun;114(1):193–197. doi: 10.1016/0003-2697(81)90473-5. [DOI] [PubMed] [Google Scholar]
  14. Häse C. C., Finkelstein R. A. Cloning and nucleotide sequence of the Vibrio cholerae hemagglutinin/protease (HA/protease) gene and construction of an HA/protease-negative strain. J Bacteriol. 1991 Jun;173(11):3311–3317. doi: 10.1128/jb.173.11.3311-3317.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Kaufman M. R., Seyer J. M., Taylor R. K. Processing of TCP pilin by TcpJ typifies a common step intrinsic to a newly recognized pathway of extracellular protein secretion by gram-negative bacteria. Genes Dev. 1991 Oct;5(10):1834–1846. doi: 10.1101/gad.5.10.1834. [DOI] [PubMed] [Google Scholar]
  16. Koski P., Hirvas L., Vaara M. Complete sequence of the ompH gene encoding the 16-kDa cationic outer membrane protein of Salmonella typhimurium. Gene. 1990 Mar 30;88(1):117–120. doi: 10.1016/0378-1119(90)90068-3. [DOI] [PubMed] [Google Scholar]
  17. Kyte J., Doolittle R. F. A simple method for displaying the hydropathic character of a protein. J Mol Biol. 1982 May 5;157(1):105–132. doi: 10.1016/0022-2836(82)90515-0. [DOI] [PubMed] [Google Scholar]
  18. Manning P. A., Heuzenroeder M. W., Yeadon J., Leavesley D. I., Reeves P. R., Rowley D. Molecular cloning and expression in Escherichia coli K-12 of the O antigens of the Inaba and Ogawa serotypes of the Vibrio cholerae O1 lipopolysaccharides and their potential for vaccine development. Infect Immun. 1986 Aug;53(2):272–277. doi: 10.1128/iai.53.2.272-277.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Pierce N. F., Cray W. C., Jr, Kaper J. B., Mekalanos J. J. Determinants of immunogenicity and mechanisms of protection by virulent and mutant Vibrio cholerae O1 in rabbits. Infect Immun. 1988 Jan;56(1):142–148. doi: 10.1128/iai.56.1.142-148.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Rella M., Mercenier A., Haas D. Transposon insertion mutagenesis of Pseudomonas aeruginosa with a Tn5 derivative: application to physical mapping of the arc gene cluster. Gene. 1985;33(3):293–303. doi: 10.1016/0378-1119(85)90237-9. [DOI] [PubMed] [Google Scholar]
  21. Sanger F., Coulson A. R., Barrell B. G., Smith A. J., Roe B. A. Cloning in single-stranded bacteriophage as an aid to rapid DNA sequencing. J Mol Biol. 1980 Oct 25;143(2):161–178. doi: 10.1016/0022-2836(80)90196-5. [DOI] [PubMed] [Google Scholar]
  22. 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]
  23. Sharma D. P., Stroeher U. H., Thomas C. J., Manning P. A., Attridge S. R. The toxin-coregulated pilus (TCP) of Vibrio cholerae: molecular cloning of genes involved in pilus biosynthesis and evaluation of TCP as a protective antigen in the infant mouse model. Microb Pathog. 1989 Dec;7(6):437–448. doi: 10.1016/0882-4010(89)90024-7. [DOI] [PubMed] [Google Scholar]
  24. Southern E. M. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol. 1975 Nov 5;98(3):503–517. doi: 10.1016/s0022-2836(75)80083-0. [DOI] [PubMed] [Google Scholar]
  25. Stoebner J. A., Payne S. M. Iron-regulated hemolysin production and utilization of heme and hemoglobin by Vibrio cholerae. Infect Immun. 1988 Nov;56(11):2891–2895. doi: 10.1128/iai.56.11.2891-2895.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Stroeher U. H., Karageorgos L. E., Morona R., Manning P. A. Serotype conversion in Vibrio cholerae O1. Proc Natl Acad Sci U S A. 1992 Apr 1;89(7):2566–2570. doi: 10.1073/pnas.89.7.2566. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Taylor R. K., Miller V. L., Furlong D. B., Mekalanos J. J. Use of phoA gene fusions to identify a pilus colonization factor coordinately regulated with cholera toxin. Proc Natl Acad Sci U S A. 1987 May;84(9):2833–2837. doi: 10.1073/pnas.84.9.2833. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Van Dongen W. M., De Graaf F. K. Molecular cloning of a gene coding for a Vibrio cholerae haemagglutinin. J Gen Microbiol. 1986 Aug;132(8):2225–2234. doi: 10.1099/00221287-132-8-2225. [DOI] [PubMed] [Google Scholar]
  29. Venkatesan M. M., Buysse J. M., Kopecko D. J. Characterization of invasion plasmid antigen genes (ipaBCD) from Shigella flexneri. Proc Natl Acad Sci U S A. 1988 Dec;85(23):9317–9321. doi: 10.1073/pnas.85.23.9317. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Vieira J., Messing J. The pUC plasmids, an M13mp7-derived system for insertion mutagenesis and sequencing with synthetic universal primers. Gene. 1982 Oct;19(3):259–268. doi: 10.1016/0378-1119(82)90015-4. [DOI] [PubMed] [Google Scholar]
  31. Whitchurch C. B., Hobbs M., Livingston S. P., Krishnapillai V., Mattick J. S. Characterisation of a Pseudomonas aeruginosa twitching motility gene and evidence for a specialised protein export system widespread in eubacteria. Gene. 1991 May 15;101(1):33–44. doi: 10.1016/0378-1119(91)90221-v. [DOI] [PubMed] [Google Scholar]
  32. 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]
  33. von Heijne G. How signal sequences maintain cleavage specificity. J Mol Biol. 1984 Feb 25;173(2):243–251. doi: 10.1016/0022-2836(84)90192-x. [DOI] [PubMed] [Google Scholar]
  34. von Heijne G. Signal sequences. The limits of variation. J Mol Biol. 1985 Jul 5;184(1):99–105. doi: 10.1016/0022-2836(85)90046-4. [DOI] [PubMed] [Google Scholar]

Articles from Infection and Immunity are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES