This Article Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Singer, J. T. Articles by Boettcher, K. J. Search for Related Content PubMed PubMed Citation Articles by Singer, J. T. Articles by Boettcher, K. J. Agricola Articles by Singer, J. T. Articles by Boettcher, K. J.

 Previous Article  |  Next Article 

Appl. Environ. Microbiol., Oct 1996, 3727-3731, Vol 62, No. 10
Copyright © 1996, American Society for Microbiology

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

JT Singer, C Ma and KJ Boettcher
Department of Biochemistry, Microbiology and Molecular Biology, University of Maine, Orono 04469-5735, USA. jsinger@maine.maine.edu

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.

This article has been cited by other articles:

• Okuda, J., Nakai, T., Chang, P. S., Oh, T., Nishino, T., Koitabashi, T., Nishibuchi, M. (2001). The toxR Gene of Vibrio (Listonella) anguillarum Controls Expression of the Major Outer Membrane Proteins but Not Virulence in a Natural Host Model. Infect. Immun. 69: 6091-6101 [Abstract] [Full Text]