Soft Metal, Disulfide, and Cysteine Stresses in Escherichia coli

描述(申请人提供)：这项研究的目标是调查软金属、半胱氨酸输入和半胱氨酸积累可以扰乱大肠杆菌适应性的机制。这三种不同的压力通过半胱氨酸化学的中心参与而联系在一起。软金属存在于许多生境中，它们对细菌和高等生物体都构成了威胁。在整个生物群中分布着绑定和出口它们的专用解毒系统，突显了它们的毒性。其中一些金属--特别是银和汞--长期以来一直被用作抗生素。然而，我们对它们实际上是如何破坏细胞的知之甚少。在最近发表的工作中，我们证明了铜主要通过灭活依赖铁/S的脱水酶来毒害大肠杆菌。它通过结合半胱氨酸残基来实现这一点，半胱氨酸残基协调这些酶的催化铁硫簇，从而取代铁原子。初步数据表明，银、汞、镉和锌在体外也有这种作用。这项研究(目标1)将测试这些软金属是否在体内通过这种机制发挥其毒性。它还将确定软金属是否类似地取代了单核酶中的铁，单核酶使用了配位基团--通常包括半胱氨酸--这些基团更喜欢铁以外的金属。目标2集中在一个单独的硫磺问题上：当胱氨酸从有氧环境中输入时，二硫键应激的风险。二硫化物应激通常被认为是由活性氧引起的，但最近的数据并不支持这一观点。然而，当细胞快速进口二硫化物时，二硫应激是一个真正的风险，例如胱氨酸。原则上，这样的事件似乎很可能将二硫键传播到细胞质蛋白质上。我们对高通量转运蛋白的研究表明，胱氨酸的输入与还原有关，这是一种避免二硫化物释放到细胞质中的策略。在目标2中，这一模型将得到严格的测试。目标3解决快速进口半胱氨酸的后果：半胱氨酸的过度积累本身就是有毒的。我们的数据显示，大肠杆菌处理的是 这个问题是通过将半胱氨酸泵出细胞来解决的。这项调查将测试半胱氨酸毒性的三种可能机制，并确定避免半胱氨酸毒性的出口商(S)。总而言之，这项工作将阐明半胱氨酸的氧化还原活性和金属亲和力产生的化学问题，以及细胞已经获得的保护自己免受半胱氨酸影响的策略。所有这三种以硫为重点的压力--软金属暴露、二硫化物输入和半胱氨酸积累--都发生在可能存在于自然栖息地的条件下。

DESCRIPTION (provided by applicant): The goals of this study are to investigate the mechanisms by which soft metals, cystine import, and cysteine accumulation can perturb the fitness of Escherichia coli. These three distinct stresses are connected by the central involvement of cysteine chemistry. Soft metals are present in many habitats, where they comprise a threat to bacteria and higher organisms alike. Their toxicity is underscored by the distribution throughout the biota of dedicated detoxification systems that bind and export them. Some of these metals- notably silver and mercury-have a long history of being used as antibiotics. Nevertheless, we have little knowledge of how they actually damage cells. In recent published work we demonstrated that copper poisons E. coli primarily by inactivating Fe/S-dependent dehydratases. It does so by binding the cysteine residues that coordinate the catalytic iron-sulfur clusters of these enzymes, thereby displacing the iron atoms. Preliminary data demonstrate that silver, mercury, cadmium, and zinc have this effect in vitro, too. This study (Aim 1) will test whether these soft metals exert their toxicity through this mechanism in vivo. It will also determine whether soft metals similarly displace iron from mononuclear enzymes, which employ coordinating groups-often including cysteine-that prefer metals other than iron. Aim 2 focuses upon a separate sulfur problem: the risk of disulfide stress when cystine is imported from aerobic environments. Disulfide stress has conventionally been thought to arise from reactive oxygen species, but recent data do not support this idea. However, disulfide stress is a real risk when cells rapidly import disulfide compounds, such as cystine. In principle such an event would seem likely to propagate disulfide bonds to cytoplasmic proteins. Our study of the high-flux transporter suggests that cystine import is linked to reduction, a tacti that would avoid the release of this disulfide into the cytoplasm. In Aim 2 this model will be rigorously tested. Aim 3 addresses the consequence of rapid cystine import: the excessive accumulation of cysteine, which is toxic in its own right. Our data reveals that E. coli deals with this problem by pumping the cysteine back out of the cell. This investigation will test three plausible mechanisms of cysteine toxicity, and it will identify the exporter(s) that averts it. Collectively this work will illuminate chemical problems that arise from the redox activity and metal affinity of cysteine, as well as the strategies that cells have acquired to protect themselve from it. All three of these sulfur-focused stresses-soft-metal exposure, disulfide import, and cysteine accumulation-occur under conditions that are likely to exist in natural habitats.