Arthur M, Depardieu F, Gerbaud G, Galimand M, Leclercq R, Courvalin P
Unité des Agents Antibactériens, Centre National de la Recherche Scientifique EP J0058, Institut Pasteur, Paris, France.
J Bacteriol. 1997 Jan;179(1):97-106. doi: 10.1128/jb.179.1.97-106.1997.
Transposon Tn1546 from Enterococcus faecium BM4147 encodes a histidine protein kinase (VanS) and a response regulator (VanR) that regulate transcription of the vanHAX operon encoding a dehydrogenase (VanH), a ligase (VanA), and a D,D-dipeptidase (VanX). These last three enzymes confer resistance to glycopeptide antibiotics by production of peptidoglycan precursors ending in the depsipeptide D-alanyl-D-lactate. Transcription of vanS and the role of VanS in the regulation of the vanHAX operon were analyzed by inserting a cat reporter gene into vanS. Transcription of cat and vanX was inducible by glycopeptides in partial diploids harboring vanS and vanS(omega)cat but was constitutive in strains containing only vanS(omega)cat. Promoters P(R) and P(H), located upstream from vanR and vanH, respectively, were cloned into a promoter probing vector to study transactivation by chromosomally encoded VanR and VanS. The promoters were inactive in the absence of vanR and vanS, inducible by glycopeptides in the presence of both genes, and constitutively activated by VanR in the absence of VanS. Thus, induction of the vanHAX operon involves an amplification loop resulting from binding of phospho-VanR to the P(R) promoter and increased transcription of the vanR 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 phosphorylation by a heterologous histidine protein kinase.
来自粪肠球菌BM4147的转座子Tn1546编码一种组氨酸蛋白激酶（VanS）和一种应答调节因子（VanR），它们调节编码脱氢酶（VanH）、连接酶（VanA）和D,D-二肽酶（VanX）的vanHAX操纵子的转录。后三种酶通过产生以depsipeptide D-丙氨酰-D-乳酸结尾的肽聚糖前体赋予对糖肽抗生素的抗性。通过将cat报告基因插入vanS中，分析了vanS的转录以及VanS在vanHAX操纵子调控中的作用。在含有vanS和vanS(ω)cat的部分二倍体中，cat和vanX的转录可被糖肽诱导，但在仅含有vanS(ω)cat的菌株中是组成型的。分别位于vanR和vanH上游的启动子P(R)和P(H)被克隆到一个启动子探测载体中，以研究染色体编码的VanR和VanS的反式激活作用。在没有vanR和vanS的情况下，这些启动子是无活性的，在两个基因都存在时可被糖肽诱导，并且在没有VanS的情况下被VanR组成型激活。因此，vanHAX操纵子的诱导涉及一个放大环，该放大环由磷酸化的VanR与P(R)启动子结合以及vanR和vanS基因转录增加所导致。在没有VanS的情况下观察到VanR对P(R)和P(H)的完全激活，这表明该传感器在没有糖肽的情况下通过去磷酸化对VanR进行负调控。在没有VanS的情况下VanR应答调节因子的激活可能涉及VanR与乙酰磷酸的自磷酸化或由异源组氨酸蛋白激酶进行的磷酸化。
