Molecular cloning and characterization of the recA gene of Rhizobium phaseoli and construction of recA mutants

Abstract

The Rhizobium phaseoli recA gene has been cloned by interspecific complementation of the Fec phenotype of bacteriophage lambda. The cloned gene restored the recombination proficiency and conferred resistance to DNA-damaging agents (methyl methanesulfonate and nitrofurantoin) to an Escherichia coli recA mutant. The direction of transcription and the localization of the recA gene were determined by mutagenesis with phage MudIIPR13 and heterologous hybridization with an E. coli recA probe. An R. phaseoli recA::Spcr mutation was introduced in two R. phaseoli strains by homogenotization. The R. phaseoli recA mutants were more sensitive to DNA-damaging agents and exhibited a 100-fold reduction in recombination frequency as compared with their parental strains. A deletion of the symbiotic plasmid abolishing nodulation was found at high frequency (10(-2)) in R. phaseoli CNF42. This event was recA dependent. In R. phaseoli CFN285, two events of symbiotic instability were found at high frequency (10(-3]: one was a deletion in the symbiotic plasmid, and the other was the loss of whole symbiotic plasmid. In the CFN285 recA::Spcr mutant, only the loss of the symbiotic plasmid was observed.