Department of Molecular Biology and Genetics, Gebze Technical University, Kocaeli, Turkey.
Centro Nacional de Biotecnología-CSIC, Campus de Cantoblanco, Madrid 28049, Spain.
Environ Microbiol. 2019 Jan;21(1):314-326. doi: 10.1111/1462-2920.14459. Epub 2018 Dec 3.
The molecular mechanisms behind the mutagenic effect of reactive oxygen species (ROS) released by defective metabolization of xenobiotic 2,4-dinitrotoluene (DNT) by a still-evolving degradation pathway were studied. To this end, the genes required for biodegradation of DNT from Burkholderia cepacia R34 were implanted in Escherichia coli and the effect of catabolizing the nitroaromatic compound monitored with stress-related markers and reporters. Such a proxy of the naturally-occurring scenario faithfully recreated the known accumulation of ROS caused by faulty metabolism of DNT and the ensuing onset of an intense mutagenesis regime. While ROS triggered an oxidative stress response, neither homologous recombination was stimulated nor the recA promoter activity increased during DNT catabolism. Analysis of single-nucleotide changes occurring in rpoB during DNT degradation suggested a relaxation of DNA replication fidelity rather than direct damage to DNA. Mutants frequencies were determined in strains defective in either converting DNA damage into mutagenesis or mediating inhibition of mismatch repair through a general stress response. The results revealed that the mutagenic effect of ROS was largely SOS-independent and stemmed instead from stress-induced changes of rpoS functionality. Evolution of novel metabolic properties thus resembles the way sublethal antibiotic concentrations stimulate the appearance of novel resistance genes.
本研究旨在探讨外来物质 2,4-二硝基甲苯（DNT）代谢缺陷产生的活性氧（ROS）致突变作用的分子机制，该物质的代谢途径仍在不断进化中。为此，将降解 DNT 所需的基因植入大肠杆菌中，并通过应激相关标志物和报告基因监测硝基芳烃化合物的代谢情况。这种自然发生情况的模拟忠实再现了已知的 DNT 代谢缺陷导致的 ROS 积累，以及随后发生的强烈突变机制。虽然 ROS 引发了氧化应激反应，但在 DNT 代谢过程中既没有刺激同源重组，也没有增加 recA 启动子的活性。在 DNT 降解过程中 rpoB 中发生的单核苷酸变化分析表明，DNA 复制保真度降低，而不是 DNA 直接受损。在将 DNA 损伤转化为突变或通过一般应激反应介导错配修复抑制的缺陷菌株中，确定了突变体频率。结果表明，ROS 的致突变作用在很大程度上与 SOS 无关，而是源于 rpoS 功能的应激诱导变化。因此，新代谢特性的进化类似于亚致死抗生素浓度刺激新型抗性基因出现的方式。
