Characterization of the recA gene from Pseudomonas fluorescens OE 28.3 and construction of a recA mutant

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Abstract
The recA gene of Pseudomonas fluorescens OE 28.3 was isolated by complementation of the Fec- phenotype of recombinant lambda EMBL3 phages in a RecA- Escherichia coli strain. The subcloned recA restored resistance to UV and methyl methanesulphonate (MMS) exposure in recA mutants of E. coli. DNA sequence analysis showed that the coding region of the P. fluorescens gene, specifying a protein of 352 amino acid residues, was preceded by an SOS box highly similar to those of Pseudomonas aeruginosa and Azotobacter vinelandii. The deduced amino acid sequence displayed highest homology to the RecA proteins from P. aeruginosa (87.8% identity) and A. vinelandii (84.3% identity). In both the regulatory region and the structural gene, a relatively high degree of sequence divergence from the Pseudomonas cepacia gene was observed. A mutant of P. fluorescens was constructed by inserting a kanamycin resistance cassette into its recA gene. This mutant exhibited an increased sensitivity to UV irradiation and MMS, and was strongly impaired in homologous recombinational activity.