Cloning of the Vibrio cholerae recA gene and construction of a Vibrio cholerae recA mutant.
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.
D003001:Cloning, Molecular; D004262:DNA Restriction Enzymes; D015252:Deoxyribonucleases, Type II Site-Specific; D005798:Genes, Bacterial; D008242:Lysogeny; D008741:Methyl Methanesulfonate / Q000494:pharmacology; D009154:Mutation; D010957:Plasmids; D011938:Rec A Recombinases / Q000235:genetics*; D011995:Recombination, Genetic; D014161:Transduction, Genetic; D014466:Ultraviolet Rays; D014734:Vibrio cholerae / Q000187:drug effects / Q000235:genetics* / Q000502:physiology / Q000528:radiation effects; D014775:Virus Activation
D008741:Methyl Methanesulfonate; D011938:Rec A Recombinases; D004262:DNA Restriction Enzymes; C025580:endodeoxyribonuclease XBAI; D015252:Deoxyribonucleases, Type II Site-Specific