vanC cluster of vancomycin-resistant Enterococcus gallinarum BM4174

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.

FIG. 1

Physical map of the vanC gene cluster. The fragments cloned in plasmids pCA10, pCA11, and pCA12 are indicated by thin solid lines. Thick arrows represent coding sequences and indicate the direction of transcription. The fragment cloned into pCA11 contains the intergenic region between orf1 and vanC-1 including the 3′ end of orf1 and the 5′ end of vanC-1. The insert of plasmid pCA12 contains the intergenic region between vanT and vanR_C (including the 3′ end of vanT and the 5′ end of vanR_C). Cloning of the inserts of pCA8 and pCA9 was performed by inverse PCR using oligonucleotides A, B, C, and D (represented by small arrows) containing SacI and XbaI sites at the 5′ and 3′ ends, respectively, after digestion and self-religation of total DNA from E. gallinarum BM4174 with HindIII and PvuI (asterisks). Numbers above each gene indicate the percentage of GC. Restriction sites: P, PvuI; S, SacI; Sl, SalI; H, HindIII; X, XbaI.

FIG. 2

Sequence of the vanR_C and vanS_C genes. The deduced amino acid sequences are shown below the nucleotide sequence. The deduced amino acid sequence of the C terminus of VanT is shown above the nucleotide sequence. Clusters of hydrophobic amino acids in VanS_C predicted to represent transmembrane regions (21) are shown in boldface. The putative RBS of vanR_C is indicated in italics.

FIG. 3

Alignment of the deduced amino acid sequence of VanRc from E. gallinarum BM4174, VanR from E. faecium BM4147 (3), and VanRb from E. faecalis V583 (16). The alignment was made by using CLUSTAL W (18). Numbers at the left correspond to the position of the first amino acid in the corresponding line. Black boxes indicate amino acid identity, and shaded boxes indicate similar amino acids. Asp10, Asp53, and Lys102 in VanR_C, which are conserved in the effector domain of other response regulators (42), are indicated above the alignment. Asp53 of VanR is phosphorylated by the associated histidine kinase VanS (40).

FIG. 4

Alignment of the deduced amino acid sequence of VanSc from E. gallinarum BM4174, VanS from E. faecium BM4147 (3), and VanSb from E. faecalis V583 (16). The alignment was made using CLUSTAL W (18). Numbers at the left correspond to the position of the first amino acid in the corresponding line. Black boxes indicate the amino acid identity, and shaded boxes indicate similar amino acids. Conserved sequence motifs H, N, G1, F, and G2 (29) are indicated above the alignment. His147 in VanS_C corresponds to the putative autophosphorylation site.

FIG. 5

Sequence of orf1 upstream from the vanC-1 gene. The deduced amino acid sequence is shown below the nucleotide sequence. The deduced amino acid sequence of the N terminus of VanC-1 is shown above the nucleotide sequence. The putative RBS of orf1 is underlined. The hexanucleotides exhibiting similarity to the −35 (TTGATC) and −10 (TAGACT) consensus promoter sequences (from P_vanH of vancomycin-resistant E. faecium) (20) (underlined above) are shown in boldface.