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Abstract：
Copper is broadly toxic to bacteria. As such, bacteria have evolved specialized copper export systems (cop operons) often consisting of a DNA-binding/copper-responsive regulator (which can be a repressor or activator), a copper chaperone, and a copper exporter. For those bacteria using DNA-binding copper repressors, few studies have examined the regulation of this operon regarding the operator DNA sequence needed for repressor binding. In Streptococcus pneumoniae (the pneumococcus), CopY is the copper repressor for the cop operon. Previously, homologs of pneumococcal CopY have been characterized to bind a 10-base consensus sequence T/GACANNTGTA known as the cop box. Using this motif, we sought to determine whether genes outside the cop operon are also regulated by the CopY repressor, which was previously shown in Lactococcus lactis We found that S. pneumoniae CopY did not bind to cop operators upstream of these candidate genes in vitro During this process, we found that the cop box sequence is necessary but not sufficient for CopY binding. Here, we propose an updated operator sequence for the S. pneumoniae cop operon to be ATTGACAAATGTAGAT binding CopY with a dissociation constant (Kd ) of ∼28 nM. We demonstrate strong cross-species interaction between some CopY proteins and CopY operators, suggesting strong evolutionary conservation. Taken together with our binding studies and bioinformatics data, we propose the consensus operator RNYKACANNYGTMRNY for the bacterial CopR-CopY copper repressor homologs.IMPORTANCE Many Gram-positive bacteria respond to copper stress by upregulating a copper export system controlled by a copper-sensitive repressor, CopR-CopY. The previous operator sequence for this family of proteins had been identified as TACANNTGTA. Here, using several recombinant proteins and mutations in various DNA fragments, we define those 10 bases as necessary but not sufficient for binding and in doing so, refine the cop operon operator to the 16-base sequence RNYKACANNTGTMRNY. Due to the sheer number of repressors that have been said to bind to the original 10 bases, including many antibiotic resistance repressors such as BlaI and MecI, we feel that this study highlights the need to reexamine many of these sites of the past and use added stringency for verifying operators in the future.
摘要：
铜对细菌具有广泛的毒性。因此，细菌已经进化出专门的铜输出系统（cop操纵子），通常由DNA结合/铜响应调节因子（可以是阻遏物或激活剂）组成，一个铜伴侣，一个铜出口商。对于那些使用DNA结合铜阻遏物的细菌，很少有研究研究了该操纵子对抑制子结合所需的操纵子DNA序列的调节。在肺炎链球菌（肺炎球菌）中，CopY是警察操纵子的铜抑制子。以前,肺炎球菌CopY同源物的特征是结合10个碱基的共有序列T/GACANNTGTA，称为copbox。使用这个主题，我们试图确定cop操纵子之外的基因是否也受CopY阻遏因子的调节，我们发现肺炎链球菌CopY在体外不与这些候选基因上游的cop算子结合。我们发现，cop框序列是必要的，但不足以进行CopY绑定。这里,我们提出肺炎链球菌cop操纵子的更新操纵子序列为ATTGACAAATGTAGAT结合CopY，解离常数（Kd）为28nM。我们证明了一些CopY蛋白和CopY算子之间的强跨物种相互作用，表明了强大的进化保守性。结合我们的结合研究和生物信息学数据，我们提出了细菌CopR-CopY铜阻遏同源物的共识算子RNYKACANNYGTMRNY。重要性许多革兰氏阳性细菌通过上调由铜敏感阻遏物控制的铜输出系统对铜胁迫的反应，CopR-CopY.该蛋白质家族的先前操纵子序列已被鉴定为TACANNTGTA。这里,使用几种重组蛋白和各种DNA片段中的突变，我们将这10个碱基定义为必要的，但不足以进行绑定，将cop操纵子运算符细化为16个碱基的序列RNYKACANNTGTMRNY。由于据说与原始10个碱基结合的阻遏物数量众多，包括许多抗生素抗性抑制物，如BlaI和MecI，我们认为，这项研究强调需要重新检查过去的许多网站，并在未来使用更严格的方式来验证运营商。
