Casadewall B, Reynolds P E, Courvalin P
Unité des Agents Antibactériens, Institut Pasteur, 28 rue du Docteur Roux, 75724 Paris Cedex 15, France.
J Bacteriol. 2001 Jun;183(11):3436-46. doi: 10.1128/JB.183.11.3436-3446.2001.
A new open reading frame, encoding a putative integrase-like protein, was detected downstream from the six genes of the vanD glycopeptide resistance cluster in Enterococcus faecium BM4339 (B. Casadewall and P. Courvalin, J. Bacteriol. 181:3644-3648, 1999). In this cluster, genes coding for the VanR(D)-VanS(D) two-component regulatory system were cotranscribed from the P(R(D)) promoter, whereas transcription of the vanY(D), vanH(D), vanD, vanX(D), and intD genes was initiated from the P(Y(D)) promoter located between vanS(D) and vanY(D) (the D subscript indicates that the gene is part of the vanD operon). The VanR(D)-VanS(D) regulatory system is likely to activate transcription of the resistance genes from the promoter P(Y(D)). Glycopeptide-susceptible derivatives of BM4339 were obtained by trans complementation of the frameshift mutation in the ddl gene, restoring functional D-alanine:D-alanine ligase activity in this strain. The glycopeptide-susceptible transformant BM4409, producing only D-alanyl-D-alanine-terminating peptidoglycan precursors, did not express the resistance genes encoding the VanY(D) D,D-carboxypeptidase, the VanH(D) dehydrogenase, the VanD ligase, the VanX(D) D,D-dipeptidase, and also the IntD integrase, although the regulatory region of the vanD cluster was still transcribed. In BM4409, the absence of VanR(D)-VanS(D), apparently dependent, transcription from promoter P(Y(D)) correlated with the lack of D-alanyl-D-lactate-terminating precursors. The vanX(D) gene was transcribed in BM4339, but detectable amounts of VanX(D) D,D-dipeptidase were not synthesized. However, the gene directed synthesis of an active enzyme when cloned on a multicopy plasmid in Escherichia coli, suggesting that the enzyme was unstable in BM4339 or that it had very low activity that was detectable only under conditions of high gene dosage. This activity is not required for glycopeptide resistance in BM4339, since this strain cannot synthesize D-alanyl-D-alanine.
在屎肠球菌BM4339的vanD糖肽抗性簇的六个基因下游检测到一个新的开放阅读框，其编码一种假定的整合酶样蛋白（B.卡萨德瓦尔和P.库尔瓦林，《细菌学杂志》181:3644 - 3648，1999年）。在这个簇中，编码VanR(D)-VanS(D)双组分调节系统的基因从P(R(D))启动子共转录，而vanY(D)、vanH(D)、vanD、vanX(D)和intD基因的转录从位于vanS(D)和vanY(D)之间的P(Y(D))启动子起始（D下标表示该基因是vanD操纵子的一部分）。VanR(D)-VanS(D)调节系统可能从启动子P(Y(D))激活抗性基因的转录。通过对ddl基因移码突变进行反式互补，获得了BM4339的糖肽敏感衍生物，恢复了该菌株中功能性D-丙氨酸:D-丙氨酸连接酶活性。糖肽敏感转化体BM4409仅产生以D-丙氨酰-D-丙氨酸结尾的肽聚糖前体，不表达编码VanY(D) D,D-羧肽酶、VanH(D)脱氢酶、VanD连接酶、VanX(D) D,D-二肽酶以及IntD整合酶的抗性基因，尽管vanD簇的调节区域仍在转录。在BM4409中，从启动子P(Y(D))明显依赖的VanR(D)-VanS(D)转录缺失与缺乏以D-丙氨酰-D-乳酸结尾的前体相关。vanX(D)基因在BM4339中被转录，但未合成可检测量的VanX(D) D,D-二肽酶。然而，当该基因克隆到大肠杆菌的多拷贝质粒上时，指导合成了一种活性酶，这表明该酶在BM4339中不稳定，或者其活性非常低，仅在高基因剂量条件下才可检测到。这种活性对于BM4339中的糖肽抗性不是必需的，因为该菌株不能合成D-丙氨酰-D-丙氨酸。
