Localization of components of the chemotaxis machinery of Escherichia coli using fluorescent protein fusions
Localization of components of the chemotaxis machinery of Escherichia coli using fluorescent protein fusions
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DOI：
10.1046/j.1365-2958.2000.02044.x
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发表时间：
2000-08-01
影响因子：
3.6
通讯作者：
Berg, HC
中科院分区：
文献类型：
--
作者：
Sourjik, V;Berg, HC
我们制备了黄色荧光蛋白[绿色荧光蛋白（GFP）的YFP变体]与细胞质趋化蛋白CheY、CheZ和CheA以及鞭毛运动蛋白FliM的融合体，并研究了它们在野生型和突变型大肠杆菌细胞中的定位。除 CheA 融合外，所有融合均具有功能。细胞质蛋白 CheY、CheZ 和 CheA 倾向于以类似于之前观察到的甲基接受趋化蛋白 (MCP) 的方式聚集在细胞两极，但前提是存在 MCP。正如预期的那样，CheY 和 CheZ 与 MCP 的共定位是 CheA 依赖性的，而 CheA 与 MCP 的共定位是 CheW 依赖性的。使用抗 MCP 一抗通过免疫荧光证实与 MCP 的共定位。运动蛋白 FliM 在细胞侧面以离散点的形式出现。这些在野生型细胞和 fliN 突变体中可见，但在 flhC 或 fliG 突变体中未见。使用抗钩一抗通过免疫荧光证实了与鞭毛结构的共定位。令人惊讶的是，即使在细胞翻滚的条件下，我们也没有观察到 CheY 与电机的共定位。
We prepared fusions of yellow fluorescent protein [the YFP variant of green fluorescent protein (GFP)] with the cytoplasmic chemotaxis proteins CheY, CheZ and CheA and the flagellar motor protein FliM, and studied their localization in wild-type and mutant cells of Escherichia coli. All but the CheA fusions were functional. The cytoplasmic proteins CheY, CheZ and CheA tended to cluster at the cell poles in a manner similar to that observed earlier for methyl-accepting chemotaxis proteins (MCPs), but only if MCPs were present. Co-localization of CheY and CheZ with MCPs was CheA dependent, and co-localization of CheA with MCPs was CheW dependent, as expected. Co-localization with MCPs was confirmed by immunofluorescence using an anti-MCP primary antibody. The motor protein FliM appeared as discrete spots on the sides of the cell. These were seen in wild-type cells and in a fliN mutant, but not in flhC or fliG mutants. Co-localization with flagellar structures was confirmed by immunofluorescence using an antihook primary antibody. Surprisingly, we did not observe co-localization of CheY with motors, even under conditions in which cells tumbled.