Fraimow H S, Greenman J B, Leviton I M, Dougherty T J, Miller M H
Department of Medicine, Montefiore Hospital and Medical Center, Bronx, New York.
J Bacteriol. 1991 May;173(9):2800-8. doi: 10.1128/jb.173.9.2800-2808.1991.
Aminoglycoside antibiotics such as streptomycin and tobramycin must traverse the bacterial cytoplasmic membrane prior to initiating lethal effects. Previous data on Escherichia coli, Staphylococcus aureus, and Bacillus subtilis have demonstrated that transport of aminoglycosides is regulated by delta psi, the electrical component of the proton motive force. However, several laboratories have observed that growth of bacterial cells can occur in the apparent absence of delta psi, and we wished to confirm these studies with E. coli and further investigate whether transport of aminoglycosides could occur in the absence of a membrane potential. Treatment of acrA strain CL2 with the protonophore carbonyl cyanide m-chlorophenylhydrazone (CCCP) dissipated delta psi, decreased intracellular ATP levels, and resulted in cessation of growth; after a variable period of time (3 to 7 h), growth resumed, ultimately achieving growth rates comparable to those of untreated cells. Absence of delta psi in these cells was confirmed by absence of [3H]tetraphenyl phosphonium+ uptake as measured by membrane filtration, lack of flagellar motion, and inability of these cells to transport proline (but not methionine). Regrowth was associated with restoration of normal intracellular ATP as measured by luciferin-luciferase bioluminescence assay. Unlike unacclimatized CL2 cells treated with CCCP, these cells transported [3H]tobramycin similarly to untreated cells; aminoglycoside-induced killing was seen in association with transport. These studies suggest that under certain circumstances aminoglycoside transport can be driven by ATP (or other high-energy activated phosphate donors) alone, in the absence of a measurable delta psi. delta uncBC mutants of CL2 incapable of interconverting delta psi and ATP were treated with CCCP, resulting in dissipation of delta psi but no alteration in ATP content. Despite maintenance of normal ATP, there was no transport of [3H] bramycin, confirming that under normal growth conditions ATP has no role in the transport of aminoglycosides.
链霉素和妥布霉素等氨基糖苷类抗生素在产生致死效应之前必须穿过细菌细胞质膜。先前关于大肠杆菌、金黄色葡萄球菌和枯草芽孢杆菌的数据表明，氨基糖苷类的转运受质子动力势的电成分Δψ调节。然而，几个实验室观察到，细菌细胞在明显没有Δψ的情况下也能生长，我们希望用大肠杆菌来证实这些研究，并进一步研究在没有膜电位的情况下氨基糖苷类的转运是否会发生。用质子载体羰基氰化物间氯苯腙（CCCP）处理acrA菌株CL2会使Δψ消散，降低细胞内ATP水平，并导致生长停止；在一段可变的时间（3至7小时）后，生长恢复，最终达到与未处理细胞相当的生长速率。通过膜过滤测量[3H]四苯基鏻离子的摄取缺失、鞭毛运动的缺乏以及这些细胞无法转运脯氨酸（但能转运蛋氨酸），证实了这些细胞中不存在Δψ。通过荧光素 - 荧光素酶生物发光测定法测量，再生长与正常细胞内ATP的恢复有关。与用CCCP处理的未适应CL2细胞不同，这些细胞与未处理细胞一样转运[3H]妥布霉素；氨基糖苷类诱导的杀伤与转运相关。这些研究表明，在某些情况下，氨基糖苷类的转运可以仅由ATP（或其他高能活化磷酸盐供体）驱动，而不存在可测量的Δψ。用CCCP处理不能将Δψ和ATP相互转换的CL2的ΔuncBC突变体，导致Δψ消散但ATP含量没有改变。尽管维持了正常的ATP水平，但[3H]布拉霉素没有转运，这证实了在正常生长条件下ATP在氨基糖苷类的转运中不起作用。
