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Interactions of OxyR with the promoter region of the oxyR and ahpC genes from Mycobacterium leprae and Mycobacterium tuberculosis.

ABSTRACT: In contrast to the intact oxyR gene (a homolog of the central regulator of peroxide stress response in enteric bacteria) in Mycobacterium leprae, this gene is inactive in all strains of M. tuberculosis. In both species, oxyR is divergently transcribed from ahpC, which encodes a homolog of alkyl hydroperoxide reductase. To initiate investigations of the regulation of oxidative stress in mycobacteria and consequences of the elimination of oxyR in M. tuberculosis, in this work we tested the hypothesis that mycobacterial OxyR acts as a DNA binding protein and analyzed its interactions with the oxyR and ahpC promoters. M. leprae OxyR was overproduced and purified, and its binding to the oxyR-ahpC intergenic region of M. leprae was demonstrated. By using a sequential series of overlapping DNA fragments, the minimal OxyR binding site was delimited to a 30-bp DNA segment which included a palindromic sequence conforming with the established rules for the LysR family of regulators. A consensus sequence for the mycobacterial OxyR recognition site (cTTATCggc-N3-gccGATAAg) was deduced based on its conservation in different mycobacteria. A variance in two potentially critical nucleotides within this site was observed in M. tuberculosis, in keeping with its reduced affinity for OxyR. Transcription of plasmid-borne M. leprae 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) P1oxyR and P2oxyR and (ii) P1ahpC and P2ahpC. The binding site for OxyR overlapped P1oxyR, reminiscent of the autoregulatory loops controlling expression of oxyR in enteric bacteria and characteristic of the LysR superfamily in general. This site was also centered 65 bp upstream of P1ahpC, matching the usual position of LysR-type recognition sequences in relationship to positively controlled promoters. Superimposed on these features was the less orthodox presence of multiple transcripts and their unique arrangement, including a region of complementarity at the 5' ends of the P2ahpC and P2oxyR mRNAs, suggesting the existence of complex regulatory relationships controlling oxyR and ahpC expression in mycobacteria.

SUBMITTER: Dhandayuthapani S

PROVIDER: S-EPMC178979 | biostudies-other | 1997 Apr

REPOSITORIES: biostudies-other

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Interactions of OxyR with the promoter region of the oxyR and ahpC genes from Mycobacterium leprae and Mycobacterium tuberculosis.

Dhandayuthapani S S Mudd M M Deretic V V

Journal of bacteriology 19970401 7

In contrast to the intact oxyR gene (a homolog of the central regulator of peroxide stress response in enteric bacteria) in Mycobacterium leprae, this gene is inactive in all strains of M. tuberculosis. In both species, oxyR is divergently transcribed from ahpC, which encodes a homolog of alkyl hydroperoxide reductase. To initiate investigations of the regulation of oxidative stress in mycobacteria and consequences of the elimination of oxyR in M. tuberculosis, in this work we tested the hypothe ...[more]

PMID: 9079928

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Project description:Oxidative stress response in pathogenic mycobacteria is believed to be of significance for host-pathogen interactions at various stages of infection. It also plays a role in determining the intrinsic susceptibility to isoniazid in mycobacterial species. In this work, we characterized the oxyR-ahpC and furA-katG loci in the nontuberculous pathogen Mycobacterium marinum. In contrast to Mycobacterium smegmatis and like Mycobacterium tuberculosis and Mycobacterium leprae, M. marinum was shown to possess a closely linked and divergently oriented equivalents of the regulator of peroxide stress response oxyR and its subordinate gene ahpC, encoding a homolog of alkyl hydroperoxide reductase. Purified mycobacterial OxyR was found to bind to the oxyR-ahpC promoter region from M. marinum and additional mycobacterial species. Mobility shift DNA binding analyses using OxyR binding sites from several mycobacteria and a panel of in vitro-generated mutants validated the proposed consensus mycobacterial recognition sequence. M. marinum AhpC levels detected by immunoblotting, were increased upon treatment with H2O2, in keeping with the presence of a functional OxyR and its binding site within the promoter region of ahpC. In contrast, OxyR did not bind to the sequences upstream of the katG structural gene, and katG expression did not follow the pattern seen with ahpC. Instead, a new open reading frame encoding a homolog of the ferric uptake regulator Fur was identified immediately upstream of katG in M. marinum. The furA-katG linkage and arrangement are ubiquitous in mycobacteria, suggesting the presence of additional regulators of oxidative stress response and potentially explaining the observed differences in ahpC and katG expression. Collectively, these findings broaden our understanding of oxidative stress response in mycobacteria. They also suggest that M. marinum will be useful as a model system for studying the role of oxidative stress response in mycobacterial physiology, intracellular survival, and other host-pathogen interactions associated with mycobacterial diseases.

Dataset Information

Interactions of OxyR with the promoter region of the oxyR and ahpC genes from Mycobacterium leprae and Mycobacterium tuberculosis.

Publications

Interactions of OxyR with the promoter region of the oxyR and ahpC genes from Mycobacterium leprae and Mycobacterium tuberculosis.

Similar Datasets

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