vanC Cluster of Vancomycin-Resistant Enterococcus gallinarum BM4174

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Abstract
Glycopeptide-resistant enterococci of the VanC type synthesize UDP-muramyl-pentapeptide[ d -Ser] for cell wall assembly and prevent synthesis of peptidoglycan precursors ending in d -Ala. The vanC cluster of Enterococcus gallinarum BM4174 consists of five genes: 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-tripeptide, vanXY C encodes a d , d -dipeptidase–carboxypeptidase that hydrolyzes d -Ala- d -Ala and removes d -Ala from UDP-MurNAc-pentapeptide[ d -Ala], and vanT encodes a membrane-bound serine racemase that provides d -Ser for the synthetic pathway. The three genes are clustered: the start codons of vanXY C and vanT overlap the termination codons of vanC-1 and vanXY C , respectively. Two genes which encode proteins with homology to the VanS-VanR two-component regulatory system were present downstream from the resistance genes. The predicted amino acid sequence of VanR C exhibited 50% identity to VanR and 33% identity to VanR B . VanS C had 40% identity to VanS over a region of 308 amino acids and 24% identity to VanS B over a region of 285 amino acids. All residues with important functions in response regulators and histidine kinases were conserved in VanR C and VanS C , respectively. Induction experiments based on the determination of d , d -carboxypeptidase activity in cytoplasmic extracts confirmed that the genes were expressed constitutively. Using a promoter-probing vector, regions upstream from the resistance and regulatory genes were identified that have promoter activity.