Cloning of the Vibrio cholerae recA gene and construction of a Vibrio cholerae recA mutant

Abstract

A recombinant plasmid carrying the recA gene of Vibrio cholerae was isolated from a V. cholerae genomic library, using complementation in Escherichia coli. The plasmid complements a recA mutation in E. coli for both resistance to the DNA-damaging agent methyl methanesulfonate and recombinational activity in bacteriophage P1 transductions. After determining the approximate location of the recA gene on the cloned DNA fragment, we constructed a defined recA mutation by filling in an XbaI site located within the gene. The 4-base pair insertion resulted in a truncated RecA protein as determined by minicell analysis. The mutation was spontaneously recombined onto the chromosome of a derivative of V. cholerae strain P27459 by screening for methyl methanesulfonate-sensitive variants. Southern blot analysis confirmed the presence of the inactivated XbaI site in the chromosome of DNA isolated from one of these methyl methanesulfonate-sensitive colonies. The recA V. cholerae strain was considerably more sensitive to UV light than its parent, was impaired in homologous recombination, and was deficient in induction of a temperate vibriophage upon exposure to UV light. We conclude that the V. cholerae RecA protein has activities which are analogous to those described for the RecA protein of E. coli.