Department of Chemistry, University of Kentucky, Lexington, KY 40506, USA.
Saha Cardiovascular Research Center, College of Medicine, University of Kentucky, Lexington, KY 40536, USA.
J Mol Biol. 2022 Jan 30;434(2):167376. doi: 10.1016/j.jmb.2021.167376. Epub 2021 Nov 24.
AcrAB-TolC and CusBAC are two of the most well-studied Resistance-Nodulation-Division (RND) family tripartite efflux pumps in E. coli. AcrAB-TolC is a multidrug efflux system, while CusBAC transports Cu(I), Cu(II) and Ag(I). The RND pump complexes span both the inner membrane (IM) and the outer membrane (OM). The long axis dimension of the fully assembled AcrAB-TolC is ∼3 nm longer than that of CusBAC. To probe if these two efflux systems with different dimensions affect each other when they need to work simultaneously in the same cell, two real-time assays were used to monitor the efflux activities of these two pumps and their impact on each other. The results showed that the presence of AcrAB-TolC substrates accelerated the accumulation of Cu(I) in BW25113 but not in BW25113ΔcusBA or BW25113ΔtolC strains. Similarly, the presence of Ag(I) slowed down the Nile red efflux in the parent strain more significantly than in the CusBA deficient mutant. To further investigate the impact of the OM/IM distance on the function of these tripartite complexes, we experimented with strains lacking the lipoprotein Lpp or containing Lpp mutant of different lengths. Data from efflux/accumulation assays and susceptibility tests revealed that mutation of Lpp resulted in functional deficiency of both AcrAB-TolC and CusBAC. In conclusion, this study demonstrated that when AcrAB-TolC and CusBAC functioned simultaneously, it took the cell a few minutes to adjust. Furthermore, the presence of Lpp of proper length is important to support full efflux activity of transporters spanning both membrane layers in E. coli.
AcrAB-TolC 和 CusBAC 是大肠杆菌中研究最为深入的两种 Resistance-Nodulation-Division（RND）家族三联外排泵。AcrAB-TolC 是一种多药外排系统，而 CusBAC 则转运 Cu(I)、Cu(II)和 Ag(I)。RND 泵复合物横跨内膜（IM）和外膜（OM）。完全组装的 AcrAB-TolC 的长轴尺寸比 CusBAC 长约 3nm。为了探究这两种具有不同尺寸的外排系统在同一细胞中同时工作时是否会相互影响，我们使用了两种实时测定法来监测这两种泵的外排活性及其相互影响。结果表明，AcrAB-TolC 底物的存在加速了 BW25113 中 Cu(I)的积累，但在 BW25113ΔcusBA 或 BW25113ΔtolC 菌株中则没有。同样，Ag(I)的存在使亲株中的尼罗红外排明显减缓，而在 CusBA 缺失突变株中则减缓得更为显著。为了进一步研究 OM/IM 距离对这些三联复合物功能的影响，我们实验了缺乏脂蛋白 Lpp 或含有不同长度 Lpp 突变体的菌株。外排/积累测定和药敏试验的数据表明，Lpp 的突变导致 AcrAB-TolC 和 CusBAC 的功能缺失。总之，本研究表明，当 AcrAB-TolC 和 CusBAC 同时发挥作用时，细胞需要几分钟的时间来调整。此外，适当长度的 Lpp 的存在对于支持横跨大肠杆菌两层膜的转运蛋白的充分外排活性是重要的。
