摘要
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质粒携带的抗生素抗性基因（ARGs）在废水中的传播正成为一个紧迫的问题。先前的研究主要集中在共存污染物对质粒结合的影响上，但忽略了废水处理过程中不可避免地释放的一些副产物的潜在贡献。在此，我们首次证明了废水氮循环的中间体一氧化氮（NO）可以显著促进质粒RP4从大肠杆菌K12向不同受体（大肠杆菌HB101、鼠伤寒沙门氏菌和废水微生物群）的偶联转移。表型和基因型测试证实，NO诱导的促进不是由SOS反应引起的，SOS反应是公认的水平基因转移的驱动因素。相反，NO暴露通过抑制参与脂多糖生物合成的关键基因（如waaJ）的表达，增加了供体和受体的外膜通透性，从而降低了结合的膜屏障。另一方面，NO暴露不仅导致细胞内色氨酸的积累，还引发细胞内甲硫氨酸的缺乏，这两种物质都被证实在调节质粒RP4的全局调节基因（korA、korB和trbA）、激活其编码转移装置（以trfAp和trbBp为代表）方面发挥关键作用。总的来说，我们的研究结果强调了NO在废水微生物群中传播ARGs的风险，并更新了质粒结合的调节机制。
Abstract
Abstract Image
The dissemination of plasmid-borne antibiotic resistance genes (ARGs) in wastewater is becoming an urgent concern. Previous studies mainly focused on the effects of coexisting contaminants on plasmid conjugation, but ignored the potential contribution of some byproducts inevitably released from wastewater treatment processes. Herein, we demonstrate for the first time that nitric oxide (NO), an intermediate of the wastewater nitrogen cycle, can significantly boost the conjugative transfer of plasmid RP4 from Escherichia coli K12 to different recipients (E. coli HB101, Salmonella typhimurium, and wastewater microbiota). Phenotypic and genotypic tests confirmed that NO-induced promotion was not attributed to the SOS response, a well-recognized driver for horizontal gene transfer. Instead, NO exposure increased the outer membrane permeability of both the donor and recipient by inhibiting the expression of key genes involved in lipopolysaccharide biosynthesis (such as waaJ), thereby lowering the membrane barrier for conjugation. On the other hand, NO exposure not only resulted in the accumulation of intracellular tryptophan but also triggered the deficiency of intracellular methionine, both of which were validated to play key roles in regulating the global regulatory genes (korA, korB, and trbA) of plasmid RP4, activating its encoding transfer apparatus (represented by trfAp and trbBp). Overall, our findings highlighted the risks of NO in spreading ARGs among wastewater microbiota and updated the regulation mechanism of plasmid conjugation.

https://pubs.acs.org/doi/abs/10.1021/acs.est.2c01889