Localization of components of the chemotaxis machinery of Escherichia coli using fluorescent protein fusions

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Abstract
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.