OXIDATIVE STRESS-RESPONSE AND CHARACTERIZATION OF THE OXYR-AHPC AND FURA-KATG LOCI IN MYCOBACTERIUM-MARINUM

Oxidative stress response in pathogenic mycobacteria is believed to be of significance for host-pathogen interactions at various stages of i nfection. It also plays a role in determining the intrinsic susceptibi lity to isoniazid in mycobacterial species. In this work, we character ized the oxyR-ahpC and furA-katC loci in the nontuberculous pathogen M ycobacterium marinum. In contrast to Mycobacterium smegmatis and like Mycobacterium tuberculosis and Mycobacterium leprae, M. marinum was sh own to possess a closely linked and divergently oriented equivalents o f the regulator of peroxide stress response oxyR and its subordinate g ene ahpC, encoding a homolog of alkyl hydroperoxide reductase. Purifie d mycobacterial OxyR was found to bind to the oxyR-ahpC promoter regio n from M. marinum and additional mycobacterial species. Mobility shift DNA binding analyses using OxyR binding sites from several mycobacter ia and a panel of in vitro-generated mutants validated the proposed co nsensus mycobacterial recognition sequence. M. marinum AhpC levels det ected by immunoblotting, were increased upon treatment with H2O2, in k eeping with the presence of a functional OxyR and its binding site wit hin the promoter region of ahpC. In contrast, OxyR did not bind to the sequences upstream of the katG structural gene, and katG expression d id not follow the pattern seen with ahpC. Instead, a new open reading frame encoding a homolog of the ferric uptake regulator Fur was identi fied immediately upstream of katG in M. marinum. The furA-katG linkage and arrangement are ubiquitous in mycobacteria, suggesting the presen ce of additional regulators of oxidative stress response and potential ly explaining the observed differences in ahpC and katG expression. Co llectively, these findings broaden our understanding of oxidative stre ss response in mycobacteria. They also suggest that M. marinum will be useful as a model system for studying the role of oxidative stress re sponse in mycobacterial physiology, intracellular survival, and other host-pathogen inter actions associated,vith mycobacterial diseases.