Dhandayuthapani S, Mudd M, Deretic V
Department of Microbiology, University of Texas Health Science Center at San Antonio, 78284-7758, USA.
J Bacteriol. 1997 Apr;179(7):2401-9. doi: 10.1128/jb.179.7.2401-2409.1997.
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.
与麻风分枝杆菌中完整的oxyR基因（肠道细菌中过氧化物应激反应中央调节因子的同源物）不同，该基因在所有结核分枝杆菌菌株中均无活性。在这两个物种中，oxyR与ahpC呈反向转录，ahpC编码烷基过氧化氢还原酶的同源物。为了启动对分枝杆菌氧化应激调节以及结核分枝杆菌中oxyR缺失后果的研究，在本研究中，我们检验了分枝杆菌OxyR作为DNA结合蛋白的假说，并分析了它与oxyR和ahpC启动子的相互作用。过量表达并纯化了麻风分枝杆菌OxyR，并证明了它与麻风分枝杆菌oxyR-ahpC基因间区域的结合。通过使用一系列连续的重叠DNA片段，将最小的OxyR结合位点限定在一个30 bp的DNA片段上，该片段包含一个符合LysR家族调节因子既定规则的回文序列。基于其在不同分枝杆菌中的保守性，推导了分枝杆菌OxyR识别位点的共有序列（cTTATCggc-N3-gccGATAAg）。在结核分枝杆菌中观察到该位点内两个潜在关键核苷酸的差异，这与其对OxyR的亲和力降低一致。通过S1核酸酶保护和转录融合分析，在耻垢分枝杆菌和牛型结核分枝杆菌卡介苗中研究了质粒携带的麻风分枝杆菌oxyR和ahpC的转录。在每个方向上检测到两个mRNA 5'末端：（i）P1oxyR和P2oxyR以及（ii）P1ahpC和P2ahpC。OxyR的结合位点与P1oxyR重叠，这让人联想到控制肠道细菌中oxyR表达的自动调节环，也是LysR超家族的一般特征。该位点也位于P1ahpC上游65 bp处，与LysR型识别序列相对于正调控启动子的通常位置相匹配。叠加在这些特征之上的是多个转录本不太正统的存在及其独特的排列，包括P2ahpC和P2oxyR mRNA 5'末端的互补区域，这表明存在控制分枝杆菌中oxyR和ahpC表达的复杂调控关系。
