Isolation and characterization of nonchemotactic CheZ mutants of Escherichia coli.
The Escherichia coli CheZ protein stimulates dephosphorylation of CheY, a response regulator in the chemotaxis signal transduction pathway, by an unknown mechanism. Genetic analysis of CheZ has lagged behind biochemical and biophysical characterization. To identify putative regions of functional importance in CheZ, we subjected cheZ to random mutagenesis and isolated 107 nonchemotactic CheZ mutants. Missense mutations clustered in six regions of cheZ, whereas nonsense and frameshift mutations were scattered reasonably uniformly across the gene. Intragenic complementation experiments showed restoration of swarming activity when compatible plasmids containing genes for the truncated CheZ(1-189) peptide and either CheZA65V, CheZL90S, or CheZD143G were both present, implying the existence of at least two independent functional domains in each chain of the CheZ dimer. Six mutant CheZ proteins, one from each cluster of loss-of-function missense mutations, were purified and characterized biochemically. All of the tested mutant proteins were defective in their ability to dephosphorylate CheY-P, with activities ranging from 0.45 to 16% of that of wild-type CheZ. There was good correlation between the phosphatase activity of CheZ and the ability to form large chemically cross-linked complexes with CheY in the presence of the CheY phosphodonor acetyl phosphate. In consideration of both the genetic and biochemical data, the most severe functional impairments in this set of CheZ mutants seemed to be concentrated in regions which are located in a proposed large N-terminal domain of the CheZ protein.
Boesch
K C
KC
Department of Microbiology & Immunology, University of North Carolina, Chapel Hill 27599-7290, USA.
Silversmith
R E
RE
Bourret
R B
RB
D000595:Amino Acid Sequence; D001426:Bacterial Proteins; D002633:Chemotaxis / Q000502:physiology*; D004926:Escherichia coli / Q000235:genetics* / Q000502:physiology*; D029968:Escherichia coli Proteins; D005816:Genetic Complementation Test; D013058:Mass Spectrometry; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D000072236:Methyl-Accepting Chemotaxis Proteins; D008969:Molecular Sequence Data; D009154:Mutation; D010766:Phosphorylation; D011485:Protein Binding; D016415:Sequence Alignment
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D008565:Membrane Proteins; D000072236:Methyl-Accepting Chemotaxis Proteins; C000607606:cheY protein, E coli