Single-molecule detection of efflux pump machinery in Pseudomonas aeruginosa

被引:31
作者
Xu, XHN [1 ]
Brownlow, W [1 ]
Huang, S [1 ]
Chen, J [1 ]
机构
[1] Old Dominion Univ, Dept Chem & Biochem, Norfolk, VA 23529 USA
关键词
efflux pump; ethidium bromide; multi-drug resistance; membrane transport; Pseudomonas aeruginosa; single-molecule detection; fluorescence microscopy and spectroscopy; single live cell imaging;
D O I
10.1016/S0006-291X(03)00692-2
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Real-time single-molecule microscopy and spectroscopy were used to monitor single molecules moving in and out of live bacterial cells, Pseudomonas aeruginosa. Ethidium bromide (EtBr) was chosen as the fluorescence probe because it emitted a weak fluorescence in aqueous solution (outside of the cells) and became strongly fluorescent as it entered the cells and intercalated with DNA. Such changes in fluorescence intensity by individual EtBr molecules were measured to determine the influx and efflux rates of EtBr by the cells. The transport rates for EtBr through the energized extrusion pumps of these strains (WT, nalB-1, and DeltaABM) of P. aeruginoso were measured and showed stochastic behavior with the average being (2.86 +/- 0.12)7 (2.80 +/- 0.13), and (2.74 +/- 0.39) s(-1), respectively. The transport rates of the three strains were independent of substrate concentration at the single-molecule level. In contrast to bulk (many molecules) measurements, single-molecule detection allowed the influx and efflux kinetics to be observed in low substrate concentrations at the molecular level. (C) 2003 Elsevier Science (USA). All rights reserved.
引用
收藏
页码:79 / 86
页数:8
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