CONSTRUCTION OF A RECA MUTANT OF BURKHOLDERIA (FORMERLY PSEUDOMONAS),CEPACIA

A recA mutant was constructed of a soil isolate of Burkholderia cepaci a, strain ATCC 17616. Prior to mutagenesis, the recA gene was cloned f rom this strain by its ability to complement the methyl methanesulfona te sensitivity of an Escherichia coli recA mutant. Sequence analysis o f the strain showed high sequence similarity (94% nucleic acid and 99% amino acid identity) with the recA gene previously cloned from a clin ical isolate of B. cepacia, strain JN25. The subcloned recA gene from B. cepacia ATCC 17616 restored UV resistance and recombination profici ency to recA mutants of E. coli and Pseudomonas aeruginosa, as well as restoring the ability of D3 prophages to be induced to lytic growth f rom a RecA(-) strain of P. aeruginosa. The recA mutant of B. cepacia A TCC 17616 was constructed by lambda-mediated Tn5 mutagenesis of the cl oned recA gene in E. coli, followed by replacement of the Tn5-interrup ted gene for the wild-type allele in the chromosome of B. cepacia by m arker exchange. The RecA(-) phenotype of the mutant was demonstrated b y the loss of UV resistance as compared to the parental strain. Southe rn hybridization analysis of chromosomal DNA from the mutant indicated the presence of Tn5 in the recA gene, and the location of the Tn5 ins ertion in the recA allele was identified by nucleotide sequence analys is. A test using the recA mutant to see if acquired resistance to D-se rine toxicity in B. cepacia might be a result of RecA-mediated activit ies proved negative; nevertheless, RecA activity potentially contribut es to the overall genomic plasticity of B. cepacia and a recA mutant w ill be useful in bioengineering of this species.