OVERCOMING A DEFECT IN GENERALIZED RECOMBINATION IN THE MARINE FISH PATHOGEN VIBRIO-ANGUILLARUM-775 - CONSTRUCTION OF A RECA MUTANT BY MARKER EXCHANGE

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 def ect and used marker exchange to construct a recA mutant of V. anguilla rum H775-3, A recombinant cosmid carrying the recA gene of V. anguilla rum 775 in the center of a 25-kb cloned DNA insert was isolated by com plementation of methyl methanesulfonate (MMS) sensitivity in Escherich ia coli HB101. The recA gene was inactivated by inserting a kanamycin resistance gene into recA, and the mutant gene was subsequently introd uced 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, a nd Southern hybridization was used to verify the presence of recA::kan in the chromosomal DNA of the recA mutant, V. anguillarum carrying re cA::kan was considerably more sensitive to UV radiation and to MMS tha n was its parent, and near wild-type levels of resistance to MMS and U V light were restored by introduction of cloned recA genes from both E . coli and V. anguillarum, These results indicate that recA is require d 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.