Scheu Patrick D, Steinmetz Philipp A, Dempwolff Felix, Graumann Peter L, Unden Gottfried
Institute for Microbiology and Wine Research, University of Mainz, Mainz, Germany.
Microbiology, Faculty for Biology, University of Freiburg, Freiburg, Germany.
PLoS One. 2014 Dec 30;9(12):e115534. doi: 10.1371/journal.pone.0115534. eCollection 2014.
The C4-dicarboxylate responsive sensor kinase DcuS of the DcuS/DcuR two-component system of E. coli is membrane-bound and reveals a polar localization. DcuS uses the C4-dicarboxylate transporter DctA as a co-regulator forming DctA/DcuS sensor units. Here it is shown by fluorescence microscopy with fusion proteins that DcuS has a dynamic and preferential polar localization, even at very low expression levels. Single assemblies of DcuS had high mobility in fast time lapse acquisitions, and fast recovery in FRAP experiments, excluding polar accumulation due to aggregation. DctA and DcuR fused to derivatives of the YFP protein are dispersed in the membrane or in the cytosol, respectively, when expressed without DcuS, but co-localize with DcuS when co-expressed at appropriate levels. Thus, DcuS is required for location of DctA and DcuR at the poles and formation of tripartite DctA/DcuS/DcuR sensor/regulator complexes. Vice versa, DctA, DcuR and the alternative succinate transporter DauA were not essential for polar localization of DcuS, suggesting that the polar trapping occurs by DcuS. Cardiolipin, the high curvature at the cell poles, and the cytoskeletal protein MreB were not required for polar localization. In contrast, polar localization of DcuS required the presence of the cytoplasmic PAS(C) and the kinase domains of DcuS.
大肠杆菌DcuS/DcuR双组分系统中的C4 - 二羧酸响应传感器激酶DcuS是膜结合的，并呈现极性定位。DcuS利用C4 - 二羧酸转运蛋白DctA作为共调节因子，形成DctA/DcuS传感器单元。在此，通过融合蛋白的荧光显微镜观察表明，即使在非常低的表达水平下，DcuS也具有动态且优先的极性定位。在快速时间推移采集中，DcuS的单个组装体具有高迁移率，并且在荧光漂白恢复（FRAP）实验中快速恢复，排除了由于聚集导致的极性积累。当在没有DcuS的情况下表达时，与黄色荧光蛋白（YFP）衍生物融合的DctA和DcuR分别分散在膜中或细胞质中，但在适当水平共表达时与DcuS共定位。因此，DcuS是DctA和DcuR定位于细胞极以及形成三方DctA/DcuS/DcuR传感器/调节复合物所必需的。反之，DctA、DcuR和替代琥珀酸转运蛋白DauA对于DcuS的极性定位不是必需的,这表明极性捕获是由DcuS发生的。心磷脂、细胞极处的高曲率以及细胞骨架蛋白MreB对于极性定位不是必需的。相反，DcuS的极性定位需要细胞质PAS(C)和DcuS的激酶结构域的存在。
