Département Génomes et Génétique, Unité Plasticité du Génome Bactérien, Institut Pasteur, 75015 Paris, France and Centre National de la Recherche Scientifique, CNRS, UMR3525 Paris, France.
Nucleic Acids Res. 2014 Feb;42(4):2366-79. doi: 10.1093/nar/gkt1259. Epub 2013 Dec 6.
Sub-inhibitory concentrations (sub-MIC) of antibiotics play a very important role in selection and development of resistances. Unlike Escherichia coli, Vibrio cholerae induces its SOS response in presence of sub-MIC aminoglycosides. A role for oxidized guanine residues was observed, but the mechanisms of this induction remained unclear. To select for V. cholerae mutants that do not induce low aminoglycoside-mediated SOS induction, we developed a genetic screen that renders induction of SOS lethal. We identified genes involved in this pathway using two strategies, inactivation by transposition and gene overexpression. Interestingly, we obtained mutants inactivated for the expression of proteins known to destabilize the RNA polymerase complex. Reconstruction of the corresponding mutants confirmed their specific involvement in induction of SOS by low aminoglycoside concentrations. We propose that DNA lesions formed on aminoglycoside treatment are repaired through the formation of single-stranded DNA intermediates, inducing SOS. Inactivation of functions that dislodge RNA polymerase leads to prolonged stalling on these lesions, which hampers SOS induction and repair and reduces viability under antibiotic stress. The importance of these mechanisms is illustrated by a reduction of aminoglycoside sub-MIC. Our results point to a central role for transcription blocking at DNA lesions in SOS induction, so far underestimated.
亚抑菌浓度（sub-MIC）的抗生素在选择和耐药性发展中起着非常重要的作用。与大肠杆菌不同，霍乱弧菌在亚抑菌浓度的氨基糖苷类药物存在下诱导其 SOS 反应。观察到氧化鸟嘌呤残基的作用，但这种诱导的机制尚不清楚。为了筛选不会诱导低浓度氨基糖苷类药物介导的 SOS 诱导的霍乱弧菌突变体，我们开发了一种遗传筛选方法，使 SOS 的诱导致死。我们使用两种策略（转座失活和基因过表达）确定了该途径中涉及的基因。有趣的是，我们获得了表达已知破坏 RNA 聚合酶复合物的蛋白质的失活突变体。相应突变体的重建证实了它们在低浓度氨基糖苷类药物诱导 SOS 中的特异性参与。我们提出，氨基糖苷类药物处理形成的 DNA 损伤通过形成单链 DNA 中间体进行修复，从而诱导 SOS。RNA 聚合酶脱落功能的失活导致这些损伤上的延长停顿，这阻碍了 SOS 的诱导和修复，并在抗生素应激下降低了存活率。亚抑菌浓度的氨基糖苷类药物的减少说明了这些机制的重要性。我们的结果表明，转录在 SOS 诱导中的 DNA 损伤阻断起着核心作用，迄今为止这一作用被低估了。
