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首页> 外文期刊>Proceedings of the National Academy of Sciences of the United States of America >Recruitment of genes and enzymes conferring resistance to the nonnatural toxin bromoacetate
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Recruitment of genes and enzymes conferring resistance to the nonnatural toxin bromoacetate

机译：招募对非天然毒素溴乙酸盐有抗性的基因和酶

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摘要

Microbial niches contain toxic chemicals capable of forcing organ-isms into periods of intense natural selection to afford survival. Elucidating the mechanisms by which microbes evade environmen-tal threats has direct relevance for understanding and combating the rise of antibiotic resistance. In this study we used a toxic small-molecule, bromoacetate, to model the selective pressures imposed by antibiotics and anthropogenic toxins. We report the results of genetic selection experiments that identify nine genes from Escherichia coli whose overexpression affords survival in the presence of a normally lethal concentration of bromoacetate. Eight of these genes encode putative transporters or transmembrane proteins, while one encodes the essential peptidoglycan biosyn-thetic enzyme, UDP-W-acetylglucosamine enolpyruvoyl transferase (MurA). Biochemical studies demonstrate that the primary physio-logical target of bromoacetate is MurA, which becomes irreversibly inactivated via alkylation of a critical active-site cysteine. We also screened a comprehensive library of E. coli single-gene deletion mutants and identified 63 strains displaying increased susceptibil-ity to bromoacetate. One hypersensitive bacterium lacks yliJ, a gene encoding a predicted glutathione transferase. Herein, YliJ is shown to catalyze the glutathione-dependent dehalogenation of bromoacetate with a k_(cat)/K_m value of 5.4 × 10~3 M~(-1)s~(-1). YliJ displays exceptional substrate specificity and produces a rate enhancement exceeding 5 orders of magnitude, remarkable char-acteristics for reactivity with a nonnatural molecule. This study illustrates the wealth of intrinsic survival mechanisms that can be exploited by bacteria when they are challenged with toxins.
机译：微生物生态位含有能够迫使生物进入激烈自然选择期以维持生存的有毒化学物质。阐明微生物逃避环境威胁的机制与理解和抵抗抗生素耐药性的上升有着直接的关系。在这项研究中，我们使用了有毒的小分子溴乙酸盐来模拟抗生素和人为毒素所施加的选择性压力。我们报告了遗传选择实验的结果，该实验从大肠杆菌中鉴定了9个基因，这些基因的过量表达可在正常致死浓度的溴乙酸盐存在下存活。这些基因中的八个编码假定的转运蛋白或跨膜蛋白，而一个则编码必需的肽聚糖生物合成酶UDP-W-乙酰氨基葡糖烯醇式丙酮酸转移酶（MurA）。生化研究表明，溴乙酸盐的主要生理学靶标是MurA，它通过关键活性位点半胱氨酸的烷基化作用不可逆地失活。我们还筛选了一个完整的大肠杆菌单基因缺失突变体文库，并鉴定了63个菌株，这些菌株对溴乙酸盐的敏感性增加。一种超敏细菌缺乏yliJ，yliJ是编码预测的谷胱甘肽转移酶的基因。在此，YliJ被证明以5.4×10〜3 M〜（-1）s〜（-1）的k_（cat）/ K_m值催化溴乙酸盐的谷胱甘肽依赖性脱卤。 YliJ显示出卓越的底物特异性，并产生超过5个数量级的速率增强，具有与非天然分子反应的显着特征。这项研究说明了细菌受到毒素攻击时可以利用的丰富的内在生存机制。

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