Localization of components of the chemotaxis machinery of Escherichia coli using fluorescent protein fusions.
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.
Sourjik
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Department of Molecular and Cellular Biology, The Biological Laboratories, Harvard University, Cambridge, MA 02138, USA.
Berg
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D001426:Bacterial Proteins; D001483:Base Sequence; D002633:Chemotaxis; D017931:DNA Primers; D004269:DNA, Bacterial; D004926:Escherichia coli / Q000378:metabolism* / Q000502:physiology; D029968:Escherichia coli Proteins; D049452:Green Fluorescent Proteins; D000071677:Histidine Kinase; D008164:Luminescent Proteins / Q000235:genetics; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D000072236:Methyl-Accepting Chemotaxis Proteins; D008856:Microscopy, Fluorescence
D001426:Bacterial Proteins; D017931:DNA Primers; D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; D008164:Luminescent Proteins; D008565:Membrane Proteins; D000072236:Methyl-Accepting Chemotaxis Proteins; C000607606:cheY protein, E coli; D049452:Green Fluorescent Proteins; D000071677:Histidine Kinase; C000607608:cheA protein, E coli