Interactions of OxyR with the promoter region of the oxyR and ahpC genes from Mycobacterium leprae and Mycobacterium tuberculosis
Interactions of OxyR with the promoter region of the oxyR and ahpC genes from Mycobacterium leprae and Mycobacterium tuberculosis
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DOI：
10.1128/jb.179.7.2401-2409.1997
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发表时间：
1997-04-01
影响因子：
3.2
通讯作者：
Deretic, V
中科院分区：
文献类型：
--
作者：
Dhandayuthapani, S;Mudd, M;Deretic, V
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的中央调节因子的同源物）相反，该基因在所有结核分枝杆菌菌株中均失活。在这两个物种中，oxyR 的转录方式与 ahpC 不同，ahpC 编码烷基氢过氧化物还原酶的同源物。为了开始研究分枝杆菌氧化应激的调节以及结核分枝杆菌中 oxyR 消除的后果，在这项工作中，我们测试了分枝杆菌 OxyR 作为 DNA 结合蛋白的假设，并分析了其与 oxyR 和 ahpC 启动子的相互作用。麻风分枝杆菌 OxyR 被过量生产和纯化，并且证明了它与麻风分枝杆菌的 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，最小的 OxyR 结合位点被限定为所有 30 bp DNA 片段，其中包括符合 LysR 调节因子家族既定规则的回文序列。根据分枝杆菌 OxyR 识别位点 (cTTATCggc-N-3-gccGATAAg) 在不同分枝杆菌中的保守性，推导出分枝杆菌 OxyR 识别位点的共有序列。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变化，与其对 OxyR 亲和力降低的情况一致。通过 S1 核酸酶保护和转录融合分析，研究了质粒携带的麻风分枝杆菌 oxyR 和 ahpC 在耻垢分枝杆菌和牛分枝杆菌 BCG 中的转录。在每个方向检测到两个 mRNA 5' 末端：(i) P(1)oxyR 和 P(2)oxyR 和 (ii) P(1)ahpC 和 P(2)ahpC。 OxyR 的结合位点与 P(1)oxyR 重叠，让人想起控制肠道细菌中 oxyR 表达的自动调节环以及 LysR 超家族的一般特征。该位点也位于 P(1)ahpC 上游 65 bp 处，与正控制启动子相关的 LysR 型识别序列的通常位置相匹配。叠加在这些特征上的是多个转录本的不那么正统的存在及其独特的排列，包括 P(2)ahpC 和 P(2)oxyR mRNA 5' 端的互补区域，表明分枝杆菌中存在控制 oxyR 和 ahpC 表达的复杂调控关系。
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 al 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-N-3-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) P(1)oxyR and P(2)oxyR and (ii) P(1)ahpC and P(2)ahpC. The binding site for OxyR overlapped P(1)oxyR, 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 P(1)ahpC, 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 P(2)ahpC and P(2)oxyR mRNAs, suggesting the existence of complex regulatory relationships controlling oxyR and ahpC expression in mycobacteria.