CONSTRUCTION AND CHARACTERIZATION OF RECA MUTANT STRAINS OF CORYNEBACTERIUM-GLUTAMICUM AND BREVIBACTERIUM-LACTOFERMENTUM

An internal fragment of the Corynebacterium glutamicum recA gene was a mplified by the polymerase chain reaction (PCR) using degenerate prime rs corresponding to two short sequences that are well conserved in pro caryotic RecA proteins. The deduced amino acid sequence of the amplifi ed fragment shared significant homology with RecA sequences from other bacteria including the ''invariant'' and functionally conserved amino acids Leu-126, Asp-144, Gly-157, Arg-169 and Asn-193. Highest identit y (91%) was shared with the gram-positive Mycobacterium tuberculosis R ecA sequence. The amplified fragment was cloned into a conditional sui cide vector, pBGS8, and used to generate recA deficient strains of C. glutamicum and Brevibacterium lactofermentum by insertional inactivati on. These strains exhibited classical RecA phenotypes including reduce d recombinational activity and increased sensitivity to DNA-damaging a gents such as UV irradiation, mitomycin C and methyl-methanesulphonate .