An ortholog of OxyR in Legionella pneumophila is expressed postexponentially and negatively regulates the alkyl hydroperoxide reductase (ahpC2D) operon
An ortholog of OxyR in Legionella pneumophila is expressed postexponentially and negatively regulates the alkyl hydroperoxide reductase (ahpC2D) operon
复制标题
DOI：
10.1128/jb.00141-08
复制
发表时间：
2008-05-01
影响因子：
3.2
通讯作者：
Hoffman, Paul S.
中科院分区：
文献类型：
--
作者：
LeBlanc, Jason J.;Brassinga, Ann Karen C.;Hoffman, Paul S.
嗜肺军团菌表达两种过氧化物清除烷基氢过氧化物还原酶系统（AhpC 1和AhpC 2D），在细菌生长周期中表达差异。指数后表达的AhpC 1系统的功能损失通过指数表达的AhpC 2D系统的表达增加来补偿。在这项研究中，我们使用了丙烯酰胺捕获的DNA结合复合物（ACDC）技术和质谱鉴定的蛋白质结合的ahpC 2D操纵子的启动子区域。捕获的主要蛋白质是OxyR的直系同源物（OxyR（Lp））。遗传学研究表明，oxyR（Lp）是一个必需的基因表达后，只有部分补充大肠杆菌oxyR突变体（GS 077）。凝胶迁移试验证实OxyRLp与ahpC 2D启动子序列特异性结合，但不与ahpC 1或oxyR（Lp）启动子结合;然而，OxyRLp与E. coliOxyR调节的启动子（katG、oxyR和ahpCf）。DNA酶I保护研究表明，OxyR（Lp）结合基序跨越启动子和转录起始序列的ahpC 2和保护区是不变的处理与还原剂或过氧化氢（H2 O2）。此外，OxyR（Lp）（pBADLpoxyR）介导的ahpC 2-gfp报告基因构建体在E. coli GS 077（oxyR突变株）不能被H_（102）攻击逆转。与其他OxyR蛋白的比对显示了几个氨基酸取代预测消融硫醇氧化或激活所需的构象变化。我们建议这些突变锁定OxyR（Lp）在一个活跃的DNA结合构象，这使得从氧化应激调节剂的细胞周期调节剂的功能分歧，也许控制基因表达在postexponential分化。
Legionella pneumophila expresses two peroxide-scavenging alkyl hydroperoxide reductase systems (AhpC1 and AhpC2D) that are expressed differentially during the bacterial growth cycle. Functional loss of the postexponentially expressed AhpC1 system is compensated for by increased expression of the exponentially expressed AhpC2D system. In this study, we used an acrylamide capture of DNA-bound complexes (ACDC) technique and mass spectrometry to identify proteins that bind to the promoter region of the ahpC2D operon. The major protein captured was an ortholog of OxyR (OxyR(Lp)). Genetic studies indicated that oxyR(Lp) was an essential gene expressed postexponentially and only partially complemented an Escherichia coli oxyR mutant (GS077). Gel shift assays confirmed specific binding of OxyRLp to ahpC2D promoter sequences, but not to promoters of ahpC1 or oxyR(Lp); however, OxyRLp weakly bound to E. coli OxyR-regulated promoters (katG, oxyR, and ahpCf). DNase I protection studies showed that the OxyR(Lp) binding motif spanned the promoter and transcriptional start sequences of ahpC2 and that the protected region was unchanged by treatments with reducing agents or hydrogen peroxide (H2O2). Moreover, the OxyR(Lp) (pBADLpoxyR)-mediated repression of an ahpC2-gfp reporter construct in E. coli GS077 (the oxyR mutant) was not reversed by H,02 challenge. Alignments with other OxyR proteins revealed several amino acid substitutions predicted to ablate thiol oxidation or conformational changes required for activation. We suggest these mutations have locked OxyR(Lp) in an active DNA-binding conformation, which has permitted a divergence of function from a regulator of oxidative stress to a cell cycle regulator, perhaps controlling gene expression during postexponential differentiation.