Identification of a phosphorylation site and functional analysis of conserved aspartic acid residues of OmpR, a transcriptional activator for ompF and ompC in Escherichia coli.
In Escherichia coli the OmpR and EnvZ proteins regulate the expression of the outer membrane porin proteins OmpC and OmpF. EnvZ and OmpR belong to a family of sensor/effector protein pairs that control adaptation to a variety of environmental conditions. EnvZ acts as the sensor protein that phosphorylates OmpR, which in turn regulates porin gene expression. The level of phosphorylated OmpR appears to be a determining factor for ompC and ompF regulation. Phosphorylation of OmpR is considered to occur at one or more aspartic acid residues (Asp-11, Asp-12 and/or Asp-55) that are highly conserved among the effector proteins. In this report we biochemically characterized the aspartic acid residue(s) in OmpR that were phosphorylated by EnvZ. Reduction of aspartyl phosphate residues in the amino-terminal domain of OmpR with [3H]-NaBH4 indicated that Asp-55 was a primary site of modification. We further studied the role of the highly conserved aspartate residues by creating OmpR mutants having aspartate to alanine substitutions at positions 11 (D11A), 12 (D12A) and 55 (D55A). Studies of ompF and ompC expression as well as in vivo and in vitro phosphorylation experiments also demonstrated that while Asp-55 is the primary phosphate acceptor site in OmpR, Asp-11 may also serve as a phosphorylation site, particularly in the absence of Asp-55.
Delgado
J
J
Department of Biochemistry, Robert Wood Johnson Medical School, University of Medicine and Dentistry of New Jersey, Piscataway 08854.
Forst
S
S
Harlocker
S
S
Inouye
M
M
D000595:Amino Acid Sequence; D001224:Aspartic Acid / Q000235:genetics; D001425:Bacterial Outer Membrane Proteins / Q000235:genetics* / Q000378:metabolism*; D001665:Binding Sites; D017124:Conserved Sequence; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism*; D029968:Escherichia coli Proteins; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D015534:Trans-Activators / Q000235:genetics* / Q000378:metabolism*
D001425:Bacterial Outer Membrane Proteins; D029968:Escherichia coli Proteins; D009097:Multienzyme Complexes; D015534:Trans-Activators; D001224:Aspartic Acid; C057843:envZ protein, E coli