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 Met to Ser) 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 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(Met-Ser), in order to identify an interaction domain. A library of plasmids encoding fragments of VanS(Met-Ser) was constructed using transposon mutagenesis, and a subpopulation of these plasmids encoded peptides that interfered with activation of PhoB by VanS(Met-Ser). A minimal size fragment (Met-Ile) was shown to be both necessary and sufficient for potent inhibition (85%) of this activation.

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