MYCOBACTERIAL RECA IS COTRANSCRIBED WITH A POTENTIAL REGULATORY GENE CALLED RECX

The recA gene of Mycobacterium smegmatis has been cloned and sequenced . The amino acid sequence of the RecA protein is highly homologous to other RecA proteins. Three other potential open reading frames were id entified. One, of these showed extensive homology to a protein, HypB, involved in the incorporation of nickel into hydrogenases. Another, fo und downstream of and overlapping recA, was similar to a gene, recX, w hich has been proposed to play a regulatory role related to recA funct ion. The homology between the M. smesmatis sequence and that of Mycoba cterium tuberculosis extended upstream of the recA coding region for 1 40bp including a motif identical to the Cheo-box consensus sequence wh ich has been shown to bind LexA. In addition, the transcriptional star t sites were found to be identical to those identified previously for IM. tuberculosis. Transcriptional fusions to the reporter gene chloram phenicol acetyltransferase (CAT) revealed that recA was DNA-damage ind ucible and that expression required sequences at some distance from th e mapped transcriptional start sites. Although a motif with only one m ismatch to the Cheo box was found in the intergenic region between orf l and orf2 these open reading frames were not DNA-damage inducible, no r was this motif required for regulation of recA expression. Gel retar dation assays revealed that the reason for this was that LexA did not bind to this sequence containing a mismatch. Reverse transcription/pol ymerase chain reaction analysis of M. smegmatis RNA demonstrated that recA and orf3 (recX) are within the same trancriptional unit.