Holtmann Gudrun, Bakker Evert P, Uozumi Nobuyuki, Bremer Erhard
Laboratory for Microbiology, Department of Biology, Philipps University Marburg, D-35032 Marburg, Germany.
J Bacteriol. 2003 Feb;185(4):1289-98. doi: 10.1128/JB.185.4.1289-1298.2003.
Recently, a new type of K+ transporter, Ktr, has been identified in the bacterium Vibrio alginolyticus (T. Nakamura, R. Yuda, T. Unemoto, and E. P. Bakker, J. Bacteriol. 180:3491-3494, 1998). The Ktr transport system consists of KtrB, an integral membrane subunit, and KtrA, a subunit peripherally bound to the cytoplasmic membrane. The genome sequence of Bacillus subtilis contains two genes for each of these subunits: yuaA (ktrA) and ykqB (ktrC) encode homologues to the V. alginolyticus KtrA protein, and yubG (ktrB) and ykrM (ktrD) encode homologues to the V. alginolyticus KtrB protein. We constructed gene disruption mutations in each of the four B. subtilis ktr genes and used this isogenic set of mutants for K+ uptake experiments. Preliminary K+ transport assays revealed that the KtrAB system has a moderate affinity with a Km value of approximately 1 mM for K+, while KtrCD has a low affinity with a Km value of approximately 10 mM for this ion. A strain defective in both KtrAB and KtrCD exhibited only a residual K+ uptake activity, demonstrating that KtrAB and KtrCD systems are the major K+ transporters of B. subtilis. Northern blot analyses revealed that ktrA and ktrB are cotranscribed as an operon, whereas ktrC and ktrD, which occupy different locations on the B. subtilis chromosome, are expressed as single transcriptional units. The amount of K+ in the environment or the salinity of the growth medium did not influence the amounts of the various ktr transcripts. A strain with a defect in KtrAB is unable to cope with a sudden osmotic upshock, and it exhibits a growth defect at elevated osmolalities which is particularly pronounced when KtrCD is also defective. In the ktrAB strain, the osmotically mediated growth defect was associated with a rapid loss of K+ ions from the cells. Under these conditions, the cells stopped synthesizing proteins but the transcription of the osmotically induced proHJ, opuA, and gsiB genes was not impaired, demonstrating that a high cytoplasmic K+ concentration is not essential for the transcriptional activation of these genes at high osmolarity. Taken together, our data suggest that K+ uptake via KtrAB and KtrCD is an important facet in the cellular defense of B. subtilis against both suddenly imposed and prolonged osmotic stress.
最近，在溶藻弧菌中发现了一种新型钾离子转运蛋白Ktr（T. Nakamura、R. Yuda、T. Unemoto和E. P. Bakker，《细菌学杂志》180:3491 - 3494，1998年）。Ktr转运系统由一个整合膜亚基KtrB和一个与细胞质膜外周结合的亚基KtrA组成。枯草芽孢杆菌的基因组序列中这两个亚基各有两个基因：yuaA（ktrA）和ykqB（ktrC）编码与溶藻弧菌KtrA蛋白同源的蛋白，yubG（ktrB）和ykrM（ktrD）编码与溶藻弧菌KtrB蛋白同源的蛋白。我们构建了枯草芽孢杆菌四个ktr基因的基因敲除突变体，并使用这组同基因突变体进行钾离子摄取实验。初步的钾离子转运分析表明，KtrAB系统对钾离子具有中等亲和力，Km值约为1 mM，而KtrCD对该离子的亲和力较低，Km值约为10 mM。在KtrAB和KtrCD均有缺陷的菌株中，仅表现出残余的钾离子摄取活性，这表明KtrAB和KtrCD系统是枯草芽孢杆菌主要的钾离子转运体。Northern印迹分析显示，ktrA和ktrB作为一个操纵子共转录，而位于枯草芽孢杆菌染色体不同位置的ktrC和ktrD则作为单个转录单元表达。环境中的钾离子含量或生长培养基的盐度并不影响各种ktr转录本的量。KtrAB有缺陷的菌株无法应对突然的渗透压升高，并且在渗透压升高时表现出生长缺陷，当KtrCD也有缺陷时这种缺陷尤为明显。在ktrAB菌株中，渗透压介导的生长缺陷与细胞中钾离子的快速流失有关。在这些条件下，细胞停止合成蛋白质，但渗透压诱导的proHJ、opuA和gsiB基因的转录并未受损，这表明高细胞质钾离子浓度对于这些基因在高渗透压下的转录激活并非必不可少。综上所述，我们的数据表明，通过KtrAB和KtrCD摄取钾离子是枯草芽孢杆菌细胞抵御突然施加和长期存在的渗透压胁迫的一个重要方面。
