Abstract
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.
Highlights
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
Analogous signal transduction pathways have recently been identified in eukaryotic organisms including yeast [15, 16], plants [17], and neurospora [18]. These systems are characterized by a sensor kinase, which undergoes autophosphorylation on a histidine residue, and this phosphoryl group is transferred to an aspartate residue on a response regulator protein, which usually acts as a transcriptional activator
Activation of PhoB in vivo and subsequent phoA expression (Scheme 1) may result from phosphorylation by the sensor kinases PhoR or CreC or in response to acetyl phosphate synthesis in E. coli; none of these mechanisms occur in BW20992 due to their loss by mutation [25]
Summary
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. Vancomycin causes cell death by disrupting cell wall biosynthesis; the drug binds to the terminal D-Ala-DAla moieties of the growing peptidoglycan cell wall, thereby inhibiting the transpeptidation and transglycosylation steps necessary for peptidoglycan cell wall maturation [2] This unique mode of action has made the drug relatively immune to resistance problems; over the past decade, an increasing number of highly resistant strains of Enterococcus have been identified in clinical isolates and waste water systems [3]. Analogous signal transduction pathways have recently been identified in eukaryotic organisms including yeast [15, 16], plants [17], and neurospora [18] These systems are characterized by a sensor kinase (often a transmembrane signaling kinase such as VanS), which undergoes autophosphorylation on a histidine residue, and this phosphoryl group is transferred to an aspartate residue on a response regulator protein (in this case, VanR), which usually acts as a transcriptional activator. Sensor kinases and response regulators of two-component regulatory systems share extensive sequence similarities to other family members, even of phylogenetically distant species
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