Regulation of expression of the vanD glycopeptide resistance gene cluster from Enterococcus faecium BM4339

A new open reading frame, encoding a putative integrase-like protein, was d etected downstream from the six genes of the vanD glycopeptide resistance c luster in Enterococcus faecium BM4339 (B. Casadewall and P. Courvalin, J. B acteriol. 181:3644-3648, 1999). In this cluster, genes coding for the VanR( D)-VanS(D) two-component regulatory system were cotranscribed from the P-RD promoter, whereas transcription of the vartY(D), vanH(D), vanD, vanX(D), a nd into genes was initiated from the P-YD promoter located between vanS(D) and vanY(D) (the D subscript indicates that the gene is part of the vanD op eron). The VanR(D)-VanS(D) regulatory system is likely to activate transcri ption of the resistance genes from the promoter P-YD. Glycopeptide-suscepti ble derivatives of BM14339 were obtained by trans complementation of the fr ameshift 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 precurs ors, did not express the resistance genes encoding the VanY(D) D,D-carboxyp eptidase, the VanH(D) dehydrogenase, the VanD ligase, the VanX(D) D,D-dipep tidase, 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-YD 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 w ere not synthesized. However, the gene directed synthesis of an active enzy me 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 s ynthesize D-alanyl-D-alanine.