Skip to main content
NCBI home page
Applied and Environmental Microbiology logoLink to Applied and Environmental Microbiology
. 1996 Oct;62(10):3727–3731. doi: 10.1128/aem.62.10.3727-3731.1996

Overcoming a defect in generalized recombination in the marine fish pathogen Vibrio anguillarum 775: construction of a recA mutant by marker exchange.

J T Singer 1, C Ma 1, K J Boettcher 1
PMCID: PMC168180  PMID: 8837428

Abstract

A defect in generalized recombination has prevented the use of marker exchange for the construction of specific chromosomal mutations in the marine fish pathogen Vibrio anguillarum 775. Through the use of large segments of homologous DNA, we were successful in overcoming this defect and used marker exchange to construct a recA mutant of V. anguillarum H775-3. A recombinant cosmid carrying the recA gene of V. anguillarum 775 in the center of a 25-kb cloned DNA insert was isolated by complementation of methyl methanesulfonate (MMS) sensitivity in Escherichia coli HB101. The recA gene was inactivated by inserting a kanamycin resistance gene into recA, and the mutant gene was subsequently introduced into V. anguillarum H775-3 by conjugal mobilization. Isolation of recombinants between cosmid-borne recA::kan sequences and chromosomal DNA was facilitated by the introduction of an incompatible plasmid, and Southern hybridization was used to verify the presence of recA::kan in the chromosomal DNA of the recA mutant. V. anguillarum carrying recA::kan was considerably more sensitive to UV radiation and to MMS than was its parent, and near wild-type levels of resistance to MMS and UV light were restored by introduction of cloned recA genes from both E. coli and V. anguillarum. These results indicate that recA is required for DNA repair in V. anguillarum and demonstrate the utility of this modified marker exchange technique for the construction of mutations in this economically important fish pathogen.

Full Text

The Full Text of this article is available as a PDF (250.4 KB).

Selected References

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

  1. Balbas P., Soberon X., Bolivar F., Rodriguez R. L. The plasmid, pBR322. Biotechnology. 1988;10:5–41. doi: 10.1016/b978-0-409-90042-2.50007-6. [DOI] [PubMed] [Google Scholar]
  2. Better M., Helinski D. R. Isolation and characterization of the recA gene of Rhizobium meliloti. J Bacteriol. 1983 Jul;155(1):311–316. doi: 10.1128/jb.155.1.311-316.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Birnboim H. C., Doly J. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 1979 Nov 24;7(6):1513–1523. doi: 10.1093/nar/7.6.1513. [DOI] [PMC free article] [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. Cohen S. N., Chang A. C., Hsu L. Nonchromosomal antibiotic resistance in bacteria: genetic transformation of Escherichia coli by R-factor DNA. Proc Natl Acad Sci U S A. 1972 Aug;69(8):2110–2114. doi: 10.1073/pnas.69.8.2110. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Crosa J. H., Hodges L. L., Schiewe M. H. Curing of a plasmid is correlated with an attenuation of virulence in the marine fish pathogen Vibrio anguillarum. Infect Immun. 1980 Mar;27(3):897–902. doi: 10.1128/iai.27.3.897-902.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Crosa J. H., Schiewe M. H., Falkow S. Evidence for plasmid contribution to the virulence of fish pathogen Vibrio anguillarum. Infect Immun. 1977 Nov;18(2):509–513. doi: 10.1128/iai.18.2.509-513.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. 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]
  9. Farrand S. K., O'Morchoe S. P., McCutchan J. Construction of an Agrobacterium tumefaciens C58 recA mutant. J Bacteriol. 1989 Oct;171(10):5314–5321. doi: 10.1128/jb.171.10.5314-5321.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Feinberg A. P., Vogelstein B. A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem. 1983 Jul 1;132(1):6–13. doi: 10.1016/0003-2697(83)90418-9. [DOI] [PubMed] [Google Scholar]
  11. Figurski D. H., Helinski D. R. Replication of an origin-containing derivative of plasmid RK2 dependent on a plasmid function provided in trans. Proc Natl Acad Sci U S A. 1979 Apr;76(4):1648–1652. doi: 10.1073/pnas.76.4.1648. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. 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]
  13. Koomey J. M., Falkow S. Cloning of the recA gene of Neisseria gonorrhoeae and construction of gonococcal recA mutants. J Bacteriol. 1987 Feb;169(2):790–795. doi: 10.1128/jb.169.2.790-795.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Meade H. M., Long S. R., Ruvkun G. B., Brown S. E., Ausubel F. M. Physical and genetic characterization of symbiotic and auxotrophic mutants of Rhizobium meliloti induced by transposon Tn5 mutagenesis. J Bacteriol. 1982 Jan;149(1):114–122. doi: 10.1128/jb.149.1.114-122.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Miller R. V., Kokjohn T. A. General microbiology of recA: environmental and evolutionary significance. Annu Rev Microbiol. 1990;44:365–394. doi: 10.1146/annurev.mi.44.100190.002053. [DOI] [PubMed] [Google Scholar]
  16. 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]
  17. Ohman D. E., West M. A., Flynn J. L., Goldberg J. B. Method for gene replacement in Pseudomonas aeruginosa used in construction of recA mutants: recA-independent instability of alginate production. J Bacteriol. 1985 Jun;162(3):1068–1074. doi: 10.1128/jb.162.3.1068-1074.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Resnick D., Nelson D. R. Cloning and characterization of the Aeromonas caviae recA gene and construction of an A. caviae recA mutant. J Bacteriol. 1988 Jan;170(1):48–55. doi: 10.1128/jb.170.1.48-55.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. 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]
  20. Singer J. T., Choe W., Schmidt K. A., Makula R. A. Virulence plasmid pJM1 prevents the conjugal entry of plasmid DNA into the marine fish pathogen Vibrio anguillarum 775. J Gen Microbiol. 1992 Dec;138(12):2485–2490. doi: 10.1099/00221287-138-12-2485. [DOI] [PubMed] [Google Scholar]
  21. Singer J. T., Earley S. Identification of polypeptides encoded by cloned pJM1 iron uptake DNA isolated from Vibrio anguillarum 775. J Bacteriol. 1989 May;171(5):2293–2302. doi: 10.1128/jb.171.5.2293-2302.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Singer J. T. Molecular cloning of the recA analog from the marine fish pathogen Vibrio anguillarum 775. J Bacteriol. 1989 Nov;171(11):6367–6371. doi: 10.1128/jb.171.11.6367-6371.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Singer J. T., Schmidt K. A., Reno P. W. Polypeptides p40, pOM2, and pAngR are required for iron uptake and for virulence of the marine fish pathogen Vibrio anguillarum 775. J Bacteriol. 1991 Feb;173(3):1347–1352. doi: 10.1128/jb.173.3.1347-1352.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Tolmasky M. E., Gammie A. E., Crosa J. H. Characterization of the recA gene of Vibrio anguillarum. Gene. 1992 Jan 2;110(1):41–48. doi: 10.1016/0378-1119(92)90442-r. [DOI] [PubMed] [Google Scholar]
  25. 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]
  26. Wyman A. R., Wertman K. F. Host strains that alleviate underrepresentation of specific sequences: overview. Methods Enzymol. 1987;152:173–180. doi: 10.1016/0076-6879(87)52017-1. [DOI] [PubMed] [Google Scholar]

Articles from Applied and Environmental Microbiology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES