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. 1989 Apr;171(4):1870–1878. doi: 10.1128/jb.171.4.1870-1878.1989

Broad-host-range vectors for delivery of TnphoA: use in genetic analysis of secreted virulence determinants of Vibrio cholerae.

R K Taylor 1, C Manoil 1, J J Mekalanos 1
PMCID: PMC209834  PMID: 2539354

Abstract

Gene fusions between the cholera toxin structural genes and phoA, which encodes bacterial alkaline phosphatase, were identified after TnphoA mutagenesis of the cloned genes in Escherichia coli and were then mobilized into Vibrio cholerae. The activities of the hybrid proteins were detectable in V. cholerae and suggested that, like cholera toxin, they were secreted beyond the cytoplasm. To extend the utility of TnphoA to identify additional genetic export signals in V. cholerae and other gram-negative bacteria, TnphoA delivery vectors utilizing broad-host-range plasmids were developed. By using V. cholerae as a model system, insertion mutants carrying active phoA gene fusions were identified as colonies expressing alkaline phosphatase, which appeared blue on agar containing the indicator 5-bromo-4-chloro-3-indolyl phosphate. Since alkaline phosphatase is active only upon export from the cytoplasm, PhoA+ colonies resulting from the mutagenesis procedure were enriched for insertions in genes that encode secreted proteins. Insertion mutations were identified in the gene encoding a major outer membrane protein, OmpV, and in tcpA, which encodes a pilus (fimbrial) subunit. Mutant strains harboring chromosomal insertions isolated in this manner can be used to assess the role of the corresponding inactivated gene products on survival of V. cholerae in vivo. The expression of the hybrid proteins as determined by measuring alkaline phosphatase activity also allowed the convenient study of virulence gene expression.

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Selected References

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

  1. Benson S. A., Silhavy T. J. Information within the mature LamB protein necessary for localization to the outer membrane of E coli K12. Cell. 1983 Apr;32(4):1325–1335. doi: 10.1016/0092-8674(83)90313-6. [DOI] [PubMed] [Google Scholar]
  2. 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]
  3. Brockman R. W., Heppel L. A. On the localization of alkaline phosphatase and cyclic phosphodiesterase in Escherichia coli. Biochemistry. 1968 Jul;7(7):2554–2562. doi: 10.1021/bi00847a016. [DOI] [PubMed] [Google Scholar]
  4. Casadaban M. J., Martinez-Arias A., Shapira S. K., Chou J. Beta-galactosidase gene fusions for analyzing gene expression in escherichia coli and yeast. Methods Enzymol. 1983;100:293–308. doi: 10.1016/0076-6879(83)00063-4. [DOI] [PubMed] [Google Scholar]
  5. Datta N., Hedges R. W., Shaw E. J., Sykes R. B., Richmond M. H. Properties of an R factor from Pseudomonas aeruginosa. J Bacteriol. 1971 Dec;108(3):1244–1249. doi: 10.1128/jb.108.3.1244-1249.1971. [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. Gutierrez C., Barondess J., Manoil C., Beckwith J. The use of transposon TnphoA to detect genes for cell envelope proteins subject to a common regulatory stimulus. Analysis of osmotically regulated genes in Escherichia coli. J Mol Biol. 1987 May 20;195(2):289–297. doi: 10.1016/0022-2836(87)90650-4. [DOI] [PubMed] [Google Scholar]
  8. Hoffman C. S., Wright A. Fusions of secreted proteins to alkaline phosphatase: an approach for studying protein secretion. Proc Natl Acad Sci U S A. 1985 Aug;82(15):5107–5111. doi: 10.1073/pnas.82.15.5107. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Holmgren J. Comparison of the tissue receptors for Vibrio cholerae and Escherichia coli enterotoxins by means of gangliosides and natural cholera toxoid. Infect Immun. 1973 Dec;8(6):851–859. doi: 10.1128/iai.8.6.851-859.1973. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Knapp S., Mekalanos J. J. Two trans-acting regulatory genes (vir and mod) control antigenic modulation in Bordetella pertussis. J Bacteriol. 1988 Nov;170(11):5059–5066. doi: 10.1128/jb.170.11.5059-5066.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Kolter R., Inuzuka M., Helinski D. R. Trans-complementation-dependent replication of a low molecular weight origin fragment from plasmid R6K. Cell. 1978 Dec;15(4):1199–1208. doi: 10.1016/0092-8674(78)90046-6. [DOI] [PubMed] [Google Scholar]
  12. 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]
  13. Liu L., Whalen W., Das A., Berg C. M. Rapid sequencing of cloned DNA using a transposon for bidirectional priming: sequence of the Escherichia coli K-12 avtA gene. Nucleic Acids Res. 1987 Nov 25;15(22):9461–9469. doi: 10.1093/nar/15.22.9461. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Manoil C., Beckwith J. A genetic approach to analyzing membrane protein topology. Science. 1986 Sep 26;233(4771):1403–1408. doi: 10.1126/science.3529391. [DOI] [PubMed] [Google Scholar]
  15. Manoil C., Beckwith J. TnphoA: a transposon probe for protein export signals. Proc Natl Acad Sci U S A. 1985 Dec;82(23):8129–8133. doi: 10.1073/pnas.82.23.8129. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Mekalanos J. J., Swartz D. J., Pearson G. D., Harford N., Groyne F., de Wilde M. Cholera toxin genes: nucleotide sequence, deletion analysis and vaccine development. Nature. 1983 Dec 8;306(5943):551–557. doi: 10.1038/306551a0. [DOI] [PubMed] [Google Scholar]
  17. Messing J. New M13 vectors for cloning. Methods Enzymol. 1983;101:20–78. doi: 10.1016/0076-6879(83)01005-8. [DOI] [PubMed] [Google Scholar]
  18. Michaelis S., Inouye H., Oliver D., Beckwith J. Mutations that alter the signal sequence of alkaline phosphatase in Escherichia coli. J Bacteriol. 1983 Apr;154(1):366–374. doi: 10.1128/jb.154.1.366-374.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. 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]
  20. Miller V. L., Mekalanos J. J. Synthesis of cholera toxin is positively regulated at the transcriptional level by toxR. Proc Natl Acad Sci U S A. 1984 Jun;81(11):3471–3475. doi: 10.1073/pnas.81.11.3471. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Olsen R. H., Shipley P. Host range and properties of the Pseudomonas aeruginosa R factor R1822. J Bacteriol. 1973 Feb;113(2):772–780. doi: 10.1128/jb.113.2.772-780.1973. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Palva E. T. Major outer membrane protein in Salmonella typhimurium induced by maltose. J Bacteriol. 1978 Oct;136(1):286–294. doi: 10.1128/jb.136.1.286-294.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Pearson G. D., Mekalanos J. J. Molecular cloning of Vibrio cholerae enterotoxin genes in Escherichia coli K-12. Proc Natl Acad Sci U S A. 1982 May;79(9):2976–2980. doi: 10.1073/pnas.79.9.2976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Pohlner J., Meyer T. F., Jalajakumari M. B., Manning P. A. Nucleotide sequence of ompV, the gene for a major Vibrio cholerae outer membrane protein. Mol Gen Genet. 1986 Dec;205(3):494–500. doi: 10.1007/BF00338088. [DOI] [PubMed] [Google Scholar]
  25. Ruvkun G. B., Ausubel F. M. A general method for site-directed mutagenesis in prokaryotes. Nature. 1981 Jan 1;289(5793):85–88. doi: 10.1038/289085a0. [DOI] [PubMed] [Google Scholar]
  26. 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]
  27. Stevenson G., Leavesley D. I., Lagnado C. A., Heuzenroeder M. W., Manning P. A. Purification of the 25-kDa Vibrio cholerae major outer-membrane protein and the molecular cloning of its gene: ompV. Eur J Biochem. 1985 Apr 15;148(2):385–390. doi: 10.1111/j.1432-1033.1985.tb08850.x. [DOI] [PubMed] [Google Scholar]
  28. 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]
  29. 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]

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