Characterization of the vanD glycopeptide resistance gene cluster from Enterococcus faecium BM4339

Abstract

VanD-type resistance to glycopeptides in Enterococcus faecium BM4339 is due to constitutive synthesis of D-alanyl-D-lactate-terminating peptidoglycan precursors (B. Périchon, P. Reynolds, and P. Courvalin, Antimicrob. Agents Chemother. 41:2016-2018, 1997). The sequence of a 5,780-bp fragment was determined and revealed six open reading frames. The 3' distal part encoded the VanHD dehydrogenase, the VanD ligase, and the VanXD DD-dipeptidase, which were highly similar to the corresponding proteins in VanA and VanB types of resistance. The deduced VanYD protein was homologous to penicillin-binding proteins that display DD-carboxypeptidase activity. The 5' end coded for the putative VanRD-VanSD two-component regulatory system. Due to a frameshift mutation in the chromosomal ddl gene, BM4339 produced an impaired D-alanine:D-alanine ligase. However, since expression of the resistance genes is constitutive, growth of E. faecium BM4339 was not dependent on the presence of glycopeptides in the culture medium.

FIG. 1

Schematic representation of the vanD gene cluster and of recombinant plasmids. (A) Map of the 7.2-kb HindIII-Sau3AI fragment containing the vanR_D, vanS_D, vanY_D, vanH_D, vanD, and vanX_D genes. Open arrows represent coding sequences. The PCR fragments internal to the vanR_D and vanD genes used as probes in hybridization experiments are indicated above the corresponding regions. Abbreviations for restriction sites used for cloning are as follows: C, ClaI; H, HindIII; and S, Sau3AI. (B) Inserts in recombinant plasmids. The inserts are represented by solid lines, and the vectors are indicated in parentheses. Arrowheads indicate binding sites and orientations of the VR and SD oligodeoxynucleotides used for amplification.

FIG. 2

Partial alignment of the deduced amino acid sequences of VanY_D from E. faecium BM4339, PBP from Streptomyces sp. strain K15 (20), the putative DacF dd-carboxypeptidase from B. subtilis MB24 (34), and PBP 6 from E. coli (9). Conserved motifs involved in the scaffolding of the active site are indicated in boldface. The numbers of amino acids between the NH₂ terminus and motif I, motifs I and II, motifs II and III, and motif III and the COOH terminus are indicated.

FIG. 3

Partial alignment of the nucleotide sequence of the ddl genes from E. faecium BM4339 and E. faecium BM4147 (14a). Numbers at the left refer to the position of the first nucleotide in the corresponding line. Identical bases are indicated by dashes in the BM4147 sequence. The putative RBS is indicated in boldface lettering. The 5-bp insertion in the BM4339 sequence is underlined and corresponds to a gap represented by dots in the BM4147 sequence. The deduced amino acid sequence of the E. faecium BM4339 ddl gene is indicated below the alignment. Numbers at the right refer to the position of the last amino acid of the corresponding line. The putative translation stop codon is indicated by an asterisk.