INTERACTIONS OF OXYR WITH THE PROMOTER REGION OF THE OXYR AND AHPC GENES FROM MYCOBACTERIUM-LEPRAE AND MYCOBACTERIUM-TUBERCULOSIS

In contrast to the intact oxyR gene (a homolog of the central regulato r of peroxide stress response in enteric bacteria) in Mycobacterium le prae, this gene is inactive in all strains of M. tuberculosis. In both species, oxyR is divergently transcribed from ahpC, which encodes a h omolog of alkyl hydroperoxide reductase. To initiate investigations of the regulation of oxidative stress in mycobacteria and consequences o f the elimination of oxyR in M. tuberculosis, in this work we tested t he hypothesis that mycobacterial OxyR acts as a DNA binding protein an d analyzed its interactions with the oxyR and ahpC promoters. M. lepra e OxyR was overproduced and purified, and its binding to the oxyR-ahpC intergenic region of M. leprae was demonstrated. By using a sequentia l series of overlapping DNA fragments, the minimal OxyR binding site w as delimited to al 30-bp DNA segment which included a palindromic sequ ence conforming with the established rules for the LysR family of regu lators. A consensus sequence for the mycobacterial OxyR recognition si te (cTTATCggc-N-3-gccGATAAg) was deduced based on its conservation in different mycobacteria. A variance in two potentially critical nucleot ides within this site was observed in M. tuberculosis, in keeping with its reduced affinity for OxyR. Transcription of plasmid-borne M. lepr ae oxyR and ahpC was investigated in M. smegmatis and M. bovis BCG by S1 nuclease protection and transcriptional fusion analyses. Two mRNA 5 ' ends were detected in each direction: (i) P(1)oxyR and P(2)oxyR and (ii) P(1)ahpC and P(2)ahpC. The binding site for OxyR overlapped P(1)o xyR, reminiscent of the autoregulatory loops controlling expression of oxyR in enteric bacteria and characteristic of the LysR superfamily i n general. This site was also centered 65 bp upstream of P(1)ahpC, mat ching the usual position of LysR-type recognition sequences in relatio nship to positively controlled promoters. Superimposed on these featur es was the less orthodox presence of multiple transcripts and their un ique arrangement, including a region of complementarity at the 5' ends of the P(2)ahpC and P(2)oxyR mRNAs, suggesting the existence of compl ex regulatory relationships controlling oxyR and ahpC expression in my cobacteria.