Localization of Components of the Chemotaxis Machinery of Escherichia Coli Using Fluorescent Protein Fusions

Mol Microbiol. 2000 Aug;37(4):740-51. doi: 10.1046/j.1365-2958.2000.02044.x.

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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Bacterial Proteins*
  • Base Sequence
  • Chemotaxis*
  • DNA Primers
  • DNA, Bacterial
  • Escherichia coli / metabolism*
  • Escherichia coli / physiology
  • Escherichia coli Proteins
  • Green Fluorescent Proteins
  • Histidine Kinase
  • Luminescent Proteins / genetics
  • Membrane Proteins / genetics
  • Membrane Proteins / metabolism*
  • Methyl-Accepting Chemotaxis Proteins
  • Microscopy, Fluorescence

Substances

  • Bacterial Proteins
  • DNA Primers
  • DNA, Bacterial
  • Escherichia coli Proteins
  • Luminescent Proteins
  • Membrane Proteins
  • Methyl-Accepting Chemotaxis Proteins
  • cheY protein, E coli
  • Green Fluorescent Proteins
  • Histidine Kinase
  • cheA protein, E coli