Construction and characterization of recA mutant strains of Corynebacterium glutamicum and Brevibacterium lactofermentum

Abstract

An internal fragment of the Corynebacterium glutamicum recA gene was amplified by the polymerase chain reaction (PCR) using degenerate primers corresponding to two short sequences that are well conserved in procaryotic RecA proteins. The deduced amino acid sequence of the amplified 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 identity (91%) was shared with the gram-positive Mycobacterium tuberculosis RecA sequence. The amplified fragment was cloned into a conditional suicide vector, pBGS, and used to generate recA deficient strains of C. glutamicum and Brevibacterium lactofermentum by insertional inactivation. These strains exhibited classical RecA phenotypes including reduced recombinational activity and increased sensitivity to DNA-damaging agents such as UV irradiation, mitomycin C and methyl-methanesulphonate.