Department of Biology, California State University Northridge, Northridge, California, USA.
mSphere. 2022 Dec 21;7(6):e0047422. doi: 10.1128/msphere.00474-22. Epub 2022 Nov 23.
The transcriptional repressor AcrR is the main regulator of the multidrug efflux pump AcrAB-TolC, which plays a major role in antibiotic resistance and cell physiology in Escherichia coli and other . However, it remains unknown which ligands control the function of AcrR. To address this gap in knowledge, this study tested whether exogenous and/or endogenous molecules identified as potential AcrR ligands regulate the activity of AcrR. Using electrophoretic mobility shift assays (EMSAs) with purified AcrR and the promoter and gene expression experiments, we found that AcrR responds to both exogenous molecules and cellular metabolites produced by E. coli. In total, we identified four functional ligands of AcrR, ethidium bromide (EtBr), an exogenous antimicrobial known to be effluxed by the AcrAB-TolC pump and previously shown to bind to AcrR, and three polyamines produced by E. coli, namely, putrescine, cadaverine, and spermidine. We found that EtBr and polyamines bind to AcrR both and , which prevents the binding of AcrR to the promoter and, ultimately, induces the expression of . Finally, we also found that AcrR contributes to mitigating the toxicity produced by excess polyamines by directly regulating the expression of AcrAB-TolC and two previously unknown AcrR targets, the MdtJI spermidine efflux pump and the putrescine degradation enzyme PuuA. Overall, these findings significantly expand our understanding of the function of AcrR by revealing that this regulator responds to different exogenous and endogenous ligands to regulate the expression of multiple genes involved in efflux and detoxification. Multidrug efflux pumps can remove antibiotics and other toxic molecules from cells and are major contributors to antibiotic resistance and bacterial physiology. Therefore, it is essential to better understand their function and regulation. AcrAB-TolC is the main multidrug efflux pump in the family, and AcrR is its major transcriptional regulator. However, little is known about which ligands control the function of AcrR or which other genes are controlled by this regulator. This study contributes to addressing these gaps in knowledge by showing that (i) the activity of AcrR is controlled by the antimicrobial ethidium bromide and by polyamines produced by E. coli, and (ii) AcrR directly regulates the expression of AcrAB-TolC and genes involved in detoxification and efflux of excess polyamines. These findings significantly advance our understanding of the biological role of AcrR by identifying four ligands that control its function and two novel targets of this regulator.
转录抑制剂 AcrR 是多药外排泵 AcrAB-TolC 的主要调节剂，在大肠杆菌和其他 中，它在抗生素耐药性和细胞生理学中起着重要作用。然而，目前尚不清楚哪些配体控制 AcrR 的功能。为了填补这一知识空白，本研究检测了是否有鉴定为潜在 AcrR 配体的外源和/或内源性分子调节 AcrR 的活性。通过使用纯化的 AcrR 与 启动子和 基因表达实验进行电泳迁移率变动分析（EMSA），我们发现 AcrR 对外源分子和大肠杆菌产生的细胞代谢物均有反应。总的来说，我们鉴定了 AcrR 的四个功能配体，即溴化乙锭（EtBr）、一种已知被 AcrAB-TolC 泵外排的外源性抗菌剂，先前已显示与 AcrR 结合，以及由大肠杆菌产生的三种聚胺，即腐胺、尸胺和亚精胺。我们发现 EtBr 和聚胺都与 AcrR 结合 和 ，这阻止了 AcrR 与 启动子结合，最终诱导 的表达。最后，我们还发现 AcrR 通过直接调节 AcrAB-TolC 和两个先前未知的 AcrR 靶标，MdtJI 亚精胺外排泵和腐胺降解酶 PuuA 的表达，有助于减轻过量聚胺产生的毒性。总的来说，这些发现通过揭示该调节剂对外源和内源性配体的不同反应来调节参与外排和解毒的多个基因的表达，从而大大扩展了我们对 AcrR 功能的理解。多药外排泵可以将抗生素和其他有毒分子从细胞中排出，是抗生素耐药性和细菌生理学的主要贡献者。因此，了解它们的功能和调节至关重要。AcrAB-TolC 是 家族中的主要多药外排泵，AcrR 是其主要的转录调节剂。然而，目前尚不清楚哪些配体控制 AcrR 的功能，也不清楚该调节剂还控制哪些其他基因。本研究通过显示（i）AcrR 的活性受抗菌溴化乙锭和大肠杆菌产生的聚胺控制，以及（ii）AcrR 直接调节 AcrAB-TolC 和参与解毒和排出过量聚胺的外排基因的表达，为填补这些知识空白做出了贡献。这些发现通过鉴定控制其功能的四种配体和该调节剂的两个新靶标，极大地推进了我们对 AcrR 生物学作用的理解。
