THE VANS SENSOR NEGATIVELY CONTROLS VANR-MEDIATED TRANSCRIPTIONAL ACTIVATION OF GLYCOPEPTIDE RESISTANCE GENES OF TN1546 AND RELATED ELEMENTS IN THE ABSENCE OF INDUCTION

Transposon Tn1546 from Enterococcus faecium BM4147 encodes a histidine protein kinase (VanS) and a response regulator (VanR) that regulate t ranscription of the vanHAX operon encoding a dehydrogenase (VanH), a l igase (VanA), and a D,D-dipeptidase (VanX). These last three enzymes c onfer resistance to glycopeptide antibiotics by production of peptidog lycan precursors ending in the depsipeptide D-alanyl-D-lactate. Transc ription of vanS and the role of VanS in the regulation of the vanHAX o peron were analyzed by inserting a cat reporter gene into vanS. Transc ription of cat and vanX was inducible by glycopeptides in partial dipl oids harboring vanS and vanS Omega cat but was constitutive in strains containing only vanS Omega cat. Promoters P-R and P-H, located upstre am from vanR and vanH, respectively, were cloned into a promoter probi ng vector to study transactivation by chromosomally encoded VanR and V anS, The promoters were inactive in the absence of vanR and vanS, indu cible by glycopeptides in the presence of both genes, and constitutive ly activated by VanR in the absence of VanS, Thus, induction of the va nHAX operon involves an amplification loop resulting from binding of p hospho-VanR to the P-R promoter and increased transcription of the van R and vanS genes, Full activation of P-R and P-H by VanR was observed in the absence of VanS, indicating that the sensor negatively controls VanR in the absence of glycopeptides, presumably by dephosphorylation , Activation of the VanR response regulator in the absence of VanS may involve autophosphorylation of VanR with acetyl phosphate or phosphor ylation by a heterologous histidine protein kinase.