Drug-efflux by pump proteins is one of the major mechanisms of antibiotic resistance in bacteria. Here, we use quantitative fluorescence microscopy to investigate the real-time dynamics of drug accumulation and efflux in live E. coli cells. We visualize simultaneously the intrinsically fluorescent protein-synthesis inhibitor tetracycline (Tc) and the fluorescently labelled Tc-specific efflux pump, TetA. We show that Tc penetrates the cells within minutes and accumulates to stable intracellular concentration after ∼20 min. The final level of drug accumulation reflects the balance between Tc-uptake by the cells and Tc-efflux by pump proteins. In wild-type Tc-sensitive cells, drug accumulation is significantly limited by the activity of the multidrug efflux pump, AcrAB-TolC. Tc-resistance wild-type cells carrying a plasmid-borne Tn10 transposon contain variable amounts of TetA protein, produced under steady-state repression by the TetR repressor. TetA content heterogeneity determines the cells’ initial ability to efflux Tc. Yet, efflux remains partial until the synthesis of additional TetA pumps allows for Tc-efflux activity to surpass Tc-uptake. Cells overproducing TetA no longer accumulate Tc and become resistant to high concentrations of the drug. This work uncovers the dynamic balance between drug entry, protein-synthesis inhibition, efflux-pump production, drug-efflux activity and drug-resistance levels.

中文翻译：

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直接可视化活大肠杆菌细胞中药物外排。

泵蛋白的药物外排是细菌对抗生素产生抗药性的主要机制之一。在这里，我们使用定量荧光显微镜技术研究活大肠杆菌中药物积累和流出的实时动态。细胞。我们同时可视化固有荧光蛋白合成抑制剂四环素（Tc）和荧光标记的Tc特异性外排泵TetA。我们显示Tc在数分钟内穿透细胞，并在约20分钟后累积至稳定的细胞内浓度。药物积累的最终水平反映了细胞摄取Tc和泵送蛋白质Tc外排之间的平衡。在野生型Tc敏感细胞中，药物积累受到多药外排泵AcrAB-TolC的活性的显着限制。携带质粒携带的Tn 10的抗Tc野生型细胞转座子含有可变数量的TetA蛋白，该蛋白在稳态抑制下由TetR阻遏物产生。TetA含量异质性决定了细胞流出Tc的初始能力。但是，外排仍然是部分的，直到合成其他TetA泵使Tc外排活动超过Tc吸收为止。过量产生TetA的细胞不再积累Tc，并且对高浓度的药物产生抗性。这项工作揭示了药物进入，蛋白质合成抑制，外排泵产生，药物外排活性和药物抗性水平之间的动态平衡。