vanC cluster of vancomycin-resistant Enterococcus gallinarum BM4174

Glycopeptide-resistant enterococci of the VanC type synthesize UDP-muramyl- pentapeptide [D-Ser] for cell wall assembly and prevent synthesis of peptid oglycan precursors ending in D-Ala, The vanC cluster of Enterococcus gallin arum BM4174 consists of five genes: I vanC-1, vanXY(C), vanT, vanR(C), and vanS(C). Three genes are sufficient for resistance: vanC-1 encodes a ligase that synthesizes the dipeptide D-ALa-D-Ser for addition to UDP-MurNAc-trip eptide, vanXY(C) , encodes a D,D-dipeptidase-carboxypeptidase that hydrolyz es D-Ala-D-Ala and removes D-Ala from UDP-MurNAc-pentapeptide[D-Ala], and v anT encodes a membrane-bound serine racemase that provides D-Ser for the sy nthetic pathway. The three genes are clustered: the start codons of vanXY(C ) and vanT overlap the termination codons of vanC-1 and vanXY(C), respectiv ely, Two genes which encode proteins with homology to the VanS-VanR two-com ponent regulatory system were present downstream from the resistance genes. The predicted amino acid sequence of VanR(C) exhibited 50% identity to Van R and 33% identity to VanR(B). VanS(C) had 40% identity to VanS over a regi on of 308 amino acids and 24% identity to VanS(B) over a region of 285 amin o acids. All residues with important functions in response regulators and h istidine kinases were conserved in VanR(C) and VanS(C), respectively. Induc tion experiments based on the determination of D,D-carboxypeptidase activit y in cytoplasmic extracts confirmed that the genes were expressed constitut ively. Using a promoter-probing vector, regions upstream from the resistanc e and regulatory genes were identified that have promoter activity.