Cross-talk between the histidine protein kinase VanS and the response regulator PhoB. Characterization and identification of a VanS domain that inhibits activation of PhoB.
VanS is a two-component transmembrane sensory kinase that, together with its response regulator VanR, activates the expression of genes responsible for vancomycin resistance in Enterococcus faecium BM4147. In this report, we demonstrate that the cytoplasmic domain of VanS (including residues Met95 to Ser384) is capable of high level activation (> 500 fold) of the Escherichia coli response regulator PhoB in vivo in the absence of its signaling kinases PhoR, CreC (PhoM), or acetyl phosphate synthesis. In vitro experiments carried out on the purified proteins confirmed that the activation is due to efficient cross-talk between VanS and PhoB, since phospho-VanS catalyzed transfer of its phosphoryl group to PhoB with approximately 90% transfer in 5 min at a 1:4 VanS/PhoB stoichiometry. However, the rate of transfer was at least 100-fold slower than that observed between phospho-VanS and VanR. The in vivo activation of PhoB was used as a reporter system to identify peptide fragments of VanS capable of interfering with activation by VanS(Met95-Ser384), in order to identify an interaction domain. A library of plasmids encoding fragments of VanS(Met95-Ser384) was constructed using transposon mutagenesis, and a subpopulation of these plasmids encoded peptides that interfered with activation of PhoB by VanS(Met95-Ser384). A minimal size fragment (Met95-Ile174) was shown to be both necessary and sufficient for potent inhibition (85%) of this activation.
Fisher
S L
SL
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115, USA.
Jiang
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W
Wanner
B L
BL
Walsh
C T
CT
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D017931:DNA Primers; D004251:DNA Transposable Elements; D016984:Enterococcus faecium / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D006639:Histidine; D007700:Kinetics; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D010766:Phosphorylation; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011494:Protein Kinases / Q000737:chemistry / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D014157:Transcription Factors / Q000737:chemistry / Q000378:metabolism*
D001426:Bacterial Proteins; D017931:DNA Primers; D004251:DNA Transposable Elements; D011994:Recombinant Proteins; D014157:Transcription Factors; C061278:PhoB protein, Bacteria; C073901:VanS protein, Enterococcus; D006639:Histidine; D011494:Protein Kinases