Bolhuis H, Poelarends G, van Veen H W, Poolman B, Driessen A J, Konings W N
Department of Microbiology, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Haren, The Netherlands.
J Biol Chem. 1995 Nov 3;270(44):26092-8. doi: 10.1074/jbc.270.44.26092.
To genetically dissect the drug extrusion systems of Lactococcus lactis, a chromosomal DNA library was made in Escherichia coli and recombinant strains were selected for resistance to high concentrations of ethidium bromide. Recombinant strains were found to be resistant not only to ethidium bromide but also to daunomycin and tetraphenylphosphonium. The drug resistance is conferred by the lmrP gene, which encodes a hydrophobic polypeptide of 408 amino acid residues with 12 putative membrane-spanning segments. Some sequence elements in this novel membrane protein share similarity to regions in the transposon Tn10-encoded tetracycline resistance determinant TetA, the multidrug transporter Bmr from Bacillus subtilis, and the bicyclomycin resistance determinant Bcr from E. coli. Drug resistance associated with lmrP expression correlated with energy-dependent extrusion of the molecules. Drug extrusion was inhibited by ionophores that dissipate the proton motive force but not by the ATPase inhibitor ortho-vanadate. These observations are indicative for a drug-proton antiport system. A lmrP deletion mutant was constructed via homologous recombinant using DNA fragments of the flanking region of the gene. The L. lactis (delta lmrP) strain exhibited residual ethidium extrusion activity, which in contrast to the parent strain was inhibited by ortho-vanadate. The results indicate that in the absence of the functional drug-proton anti-porter LmrP, L. lactis is able to overexpress another, ATP-dependent, drug extrusion system. These findings substantiate earlier studies on the isolation and characterization of drug-resistant mutants of L. lactis (Bolhuis, H., Molenaar, D., Poelarends, G., van Veen, H. W., Poolman, B., Driessen, A. J. M., and Konings, W. N. (1994) J. Bacteriol. 176, 6957-6964).
为了从基因层面剖析乳酸乳球菌的药物外排系统，构建了一个大肠杆菌染色体DNA文库，并筛选出对高浓度溴化乙锭具有抗性的重组菌株。结果发现，重组菌株不仅对溴化乙锭具有抗性，还对柔红霉素和四苯基鏻具有抗性。这种耐药性由lmrP基因赋予，该基因编码一个由408个氨基酸残基组成的疏水多肽，具有12个假定的跨膜区段。这种新型膜蛋白中的一些序列元件与转座子Tn10编码的四环素抗性决定簇TetA、枯草芽孢杆菌的多药转运蛋白Bmr以及大肠杆菌的双环霉素抗性决定簇Bcr中的区域具有相似性。与lmrP表达相关的耐药性与分子的能量依赖性外排相关。药物外排受到耗散质子动力的离子载体的抑制，但不受ATP酶抑制剂原钒酸盐的抑制。这些观察结果表明存在一种药物-质子反向转运系统。通过使用该基因侧翼区域的DNA片段进行同源重组构建了一个lmrP缺失突变体。乳酸乳球菌（ΔlmrP）菌株表现出残留的溴化乙锭外排活性，与亲本菌株不同的是，该活性受到原钒酸盐的抑制。结果表明，在缺乏功能性药物-质子反向转运蛋白LmrP的情况下，乳酸乳球菌能够过表达另一种依赖ATP的药物外排系统。这些发现证实了早期关于乳酸乳球菌耐药突变体的分离和表征的研究（Bolhuis, H., Molenaar, D., Poelarends, G., van Veen, H. W., Poolman, B., Driessen, A. J. M., and Konings, W. N. (1994) J. Bacteriol. 176, 6957 - 6964）。
