Herz Katia, Vimont Sophie, Padan Etana, Berche Patrick
Department of Microbial and Molecular Ecology, Institute of Life Sciences, The Hebrew University of Jerusalem, 91904 Jerusalem, Israel.
J Bacteriol. 2003 Feb;185(4):1236-44. doi: 10.1128/JB.185.4.1236-1244.2003.
Vibrio cholerae, the causative agent of cholera, is a normal inhabitant of aquatic environments, where it survives in a wide range of conditions of pH and salinity. In this work, we investigated the role of three Na+/H+ antiporters on the survival of V. cholerae in a saline environment. We have previously cloned the Vc-nhaA gene encoding the V. cholerae homolog of Escherichia coli. Here we identified two additional antiporter genes, designated Vc-nhaB and Vc-nhaD, encoding two putative proteins of 530 and 477 residues, respectively, highly homologous to the respective antiporters of Vibrio species and E. coli. We showed that both Vc-NhaA and Vc-NhaB confer Na+ resistance and that Vc-NhaA displays an antiport activity in E. coli, which is similar in magnitude, kinetic parameters, and pH regulation to that of E. coli NhaA. To determine the roles of the Na+/H+ antiporters in V. cholerae, we constructed nhaA, nhaB, and nhaD mutants (single, double, and triple mutants). In contrast to E. coli, the inactivation of the three putative antiporter genes (Vc-nhaABD) in V. cholerae did not alter the bacterial exponential growth in the presence of high Na+ concentrations and had only a slight effect in the stationary phase. In contrast, a pronounced and similar Li+-sensitive phenotype was found with all mutants lacking Vc-nhaA during the exponential phase of growth and also with the triple mutant in the stationary phase of growth. By using 2-n-nonyl-4-hydroxyquinoline N-oxide, a specific inhibitor of the electron-transport-linked Na+ pump NADH-quinone oxidoreductase (NQR), we determined that in the absence of NQR activity, the Vc-NhaA Na+/H+ antiporter activity becomes essential for the resistance of V. cholerae to Na+ at alkaline pH. Since the ion pump NQR is Na+ specific, we suggest that its activity masks the Na+/H+ but not the Li+/H+ antiporter activities. Our results indicate that the Na+ resistance of the human pathogen V. cholerae requires a complex molecular system involving multiple antiporters and the NQR pump.
霍乱弧菌是霍乱的病原体，是水生环境中的正常栖居菌，能在广泛的pH值和盐度条件下存活。在本研究中，我们调查了三种Na⁺/H⁺反向转运蛋白对霍乱弧菌在盐环境中存活的作用。我们之前已克隆了编码霍乱弧菌大肠杆菌同源物的Vc-nhaA基因。在此，我们鉴定出另外两个反向转运蛋白基因，命名为Vc-nhaB和Vc-nhaD，它们分别编码530和477个残基的两个假定蛋白，与弧菌属和大肠杆菌的相应反向转运蛋白高度同源。我们发现Vc-NhaA和Vc-NhaB都赋予Na⁺抗性，且Vc-NhaA在大肠杆菌中表现出反向转运活性，其在大小、动力学参数和pH调节方面与大肠杆菌NhaA相似。为确定Na⁺/H⁺反向转运蛋白在霍乱弧菌中的作用，我们构建了nhaA、nhaB和nhaD突变体（单突变体、双突变体和三突变体）。与大肠杆菌不同，霍乱弧菌中三个假定反向转运蛋白基因（Vc-nhaABD）的失活在高Na⁺浓度存在时并未改变细菌的指数生长，且在稳定期仅有轻微影响。相反，在生长指数期，所有缺乏Vc-nhaA的突变体以及生长稳定期的三突变体都表现出明显且相似的Li⁺敏感表型。通过使用电子传递链相关的Na⁺泵NADH-醌氧化还原酶（NQR）的特异性抑制剂2-n-壬基-4-羟基喹啉N-氧化物，我们确定在没有NQR活性的情况下，Vc-NhaA Na⁺/H⁺反向转运蛋白活性对于霍乱弧菌在碱性pH值下对Na⁺的抗性至关重要。由于离子泵NQR对Na⁺具有特异性，我们认为其活性掩盖了Na⁺/H⁺而非Li⁺/H⁺反向转运蛋白的活性。我们的结果表明，人类病原体霍乱弧菌的Na⁺抗性需要一个涉及多个反向转运蛋白和NQR泵的复杂分子系统。
