School of Biosciences, University of Birmingham, Edgbaston, Birmingham, UK.
BMC Microbiol. 2012 Jun 18;12:109. doi: 10.1186/1471-2180-12-109.
The pbr resistance operon from Cupriavidus metallidurans CH34 plasmid pMOL30 confers resistance to Pb(II) salts, and is regulated by the Pb(II) responsive regulator PbrR, which is a MerR family activator. In other metal sensing MerR family regulators, such as MerR, CueR, and ZntR the cognate regulator binds to a promoter with an unusually long spacer between the -35 and -10 sequences, and activates transcription of resistance genes as a consequence of binding the appropriate metal. Cysteine residues in these regulators are essential for metal ion coordination and activation of expression from their cognate promoter. In this study we investigated the interaction of PbrR with the promoter for the structural pbr resistance genes, PpbrA, effects on transcriptional activation of altering the DNA sequence of PpbrA, and effects on Pb(II)-induced activation of PpbrA when cysteine residues in PbrR were mutated to serine.
Gel retardation and footprinting assays using purified PbrR show that it binds to, and protects from DNase I digestion, the PpbrA promoter, which has a 19 bp spacer between its -35 and -10 sites. Using β-galactosidase assays in C. metallidurans, we show that when PpbrA is changed to an 18 bp spacer, there is an increase in transcriptional activation both in the presence and absence of Pb(II) salts up to a maximum induction equivalent to that seen in the fully-induced wild-type promoter. Changes to the -10 sequence of PpbrA from TTAAAT to the consensus E. coli -10 sequence (TATAAT) increased transcriptional activation from PpbrA, whilst changing the -10 sequence to that of the Tn501 mer promoter (TAAGGT) also increased the transcriptional response, but only in the presence of Pb(II). Individual PbrR mutants C14S, C55S, C79S, C114S, C123S, C132S and C134S, and a double mutant C132S/C134S, were tested for Pb(II) response from PpbrA, using β-galactosidase assays in C. metallidurans. The PbrR C14S, C79S, C134S, and C132S/C134S mutants were defective in Pb(II)-induced activation of PpbrA.
These data show that the metal-dependent activation of PbrR occurs by a similar mechanism to that of MerR, but that metal ion coordination is through cysteines which differ from those seen in other MerR family regulators, and that the DNA sequence of the -10 promoter affects expression levels of the lead resistance genes.
铜绿假单胞菌 CH34 质粒 pMOL30 的 pbr 抗性操纵子赋予了对 Pb(II)盐的抗性，并且受到 Pb(II)响应调节剂 PbrR 的调节，PbrR 是 MerR 家族激活剂。在其他金属感应 MerR 家族调节剂中，如 MerR、CueR 和 ZntR，同源调节剂结合到启动子上，-35 和 -10 序列之间有一个异常长的间隔序列，并且由于结合了适当的金属而激活抗性基因的转录。这些调节剂中的半胱氨酸残基对于金属离子配位和从其同源启动子激活表达是必不可少的。在这项研究中，我们研究了 PbrR 与结构 pbr 抗性基因的启动子之间的相互作用，改变 PpbrA 的 DNA 序列对转录激活的影响，以及当 PbrR 中的半胱氨酸残基突变为丝氨酸时对 Pb(II)诱导的 PpbrA 激活的影响。
使用纯化的 PbrR 进行凝胶阻滞和足迹分析表明，它结合并保护 PpbrA 启动子免受 DNA 酶 I 的消化，PpbrA 启动子的-35 和 -10 位点之间有 19 个碱基对的间隔序列。我们在 C. metallidurans 中使用 β-半乳糖苷酶测定法表明，当 PpbrA 变为 18 个碱基对的间隔序列时，无论是在存在还是不存在 Pb(II)盐的情况下，转录激活都会增加，最大诱导程度相当于完全诱导的野生型启动子。将 PpbrA 的-10 序列从 TTAAAT 更改为大肠杆菌-10 序列（TATAAT）增加了 PpbrA 的转录激活，而将-10 序列更改为 Tn501 mer 启动子（TAAGGT）也增加了转录反应，但仅在存在 Pb(II)的情况下。使用 C. metallidurans 中的 β-半乳糖苷酶测定法，我们测试了 PbrR 的 C14S、C55S、C79S、C114S、C123S、C132S 和 C134S 单个突变体以及 C132S/C134S 双突变体对 PpbrA 的 Pb(II)反应。PbrR 的 C14S、C79S、C134S 和 C132S/C134S 突变体在 Pb(II)诱导的 PpbrA 激活中存在缺陷。
这些数据表明，PbrR 的金属依赖性激活是通过类似于 MerR 的机制发生的，但金属离子配位是通过与其他 MerR 家族调节剂不同的半胱氨酸发生的，并且-10 启动子的 DNA 序列会影响铅抗性基因的表达水平。
