VanD-type vancomycin-resistant Enterococcus faecium 10/96A

Abstract

VanD type Enterococcus faecium 10/96A is constitutively resistant to vancomycin and to low levels of teicoplanin by nearly exclusive synthesis of peptidoglycan precursors terminating in D-alanyl-D-lactate (L. M. Dalla Costa, P. E. Reynolds, H. A. Souza, D. C. Souza, M. F. Palepou, and N. Woodford, Antimicrob. Agents Chemother. 44:3444-3446, 2000). A G(184)S mutation adjacent to the serine involved in the binding of D-Ala1 in the D-alanine:D-alanine ligase (Ddl) led to production of an impaired Ddl and accounts for the lack of D-alanyl-D-alanine-containing peptidoglycan precursors. The sequence of the vanD gene cluster revealed eight open reading frames. The organization of this operon, assigned to a chromosomal location, was similar to those in other VanD type strains. The distal part encoded the VanH(D) dehydrogenase, the VanD ligase, and the VanX(D) dipeptidase, which were homologous to the corresponding proteins in VanD-type strains. Upstream from the structural genes for these proteins was the vanY(D) gene; a frameshift mutation in this gene resulted in premature termination of the encoded protein and accounted for the lack of penicillin-susceptible D,D-carboxypeptidase activity. Analysis of the translated sequence downstream from the stop codon, but in a different reading frame because of the frameshift mutation, indicated homology with penicillin binding proteins (PBPs) with a high degree of identity with VanY(D) from VanD-type strains. The 5' end of the gene cluster contained the vanR(D)-vanS(D) genes for a putative two-component regulatory system. Insertion of ISEfa4 in the vanS(D) gene led to constitutive expression of vancomycin resistance. This new insertion belonged to the IS605 family and was composed of two open reading frames encoding putative transposases of two unrelated insertion sequence elements, IS200 and IS1341.

FIG. 1.

Schematic representation of vanD gene clusters and of recombinant plasmids. Open arrows represent coding sequences and indicate the direction of transcription. The double-headed arrow indicates the 2,368-bp PCR product previously sequenced (23). The PCR fragment internal to the vanD gene of strain 10/96A used as a probe in hybridization experiments is indicated above the corresponding region. Vertical dashed lines indicate separation of the vanS_D gene of 10/96A into two parts S_D′ and vanS_D′. The vertical bar in the vanY_D gene of 10/96A indicates the position of the frameshift mutation leading to a predicted truncated protein. The inserts in recombinant plasmids are represented by solid lines, and the vectors are indicated in parentheses. Arrowheads represent binding sites and orientation of oligodeoxynucleotides used for amplification.

FIG. 2.

Schematic representation of d-Ala:d-Ala ligases from E. faecium BM4147 and from VanD-type enterococci. The positions of the amino acids implicated in the binding of d-Ala1, d-Ala2, and ATP are indicated by dotted, hatched, and black bars, respectively (25, 51). In strain BM4339, a 5-bp insertion (italics) at the position corresponding to amino acid 13 is responsible for a frameshift mutation leading to the synthesis of a 26-amino-acid peptide instead of the putative 358-amino-acid Ddl (21). In BM4416, a copy of IS19 is inserted at position 762 of the ddl gene. Checkerboard boxes, 9-bp duplications of target DNA; black arrowheads, 21-bp perfect inverted repeat sequences; horizontally striped box, putative transposase (17, 43). In 10/96A, the single base difference with ddl from E. faecium BM4147 leading to a Gly-to-Ser substitution at position 184 is indicated in italics.

FIG. 3.

Comparison of vanD gene clusters. Arrows represent coding sequences and indicate direction of transcription. The two-component regulatory systems are represented by dotted arrows, the d,d-carboxypeptidases are represented by hatched arrows, and the genes necessary for resistance are represented by open arrows. The guanosine-plus-cytosine content (% GC) is indicated in the arrows. The percentages of identity between the deduced proteins relative to those of BM4339 are indicated under the arrows. Insertion sequence ISEfa4 in 10/96A is indicated by a double-headed arrow, and horizontally dashed arrows correspond to ORFA and ORFB. The vertical bars in vanS_D of BM4416 and vanY_D of 10/96A indicate the positions of the frameshift mutations leading to predicted truncated proteins.

FIG. 4.

Partial alignment of the deduced sequences of d,d-carboxypeptidases in VanD-type E. faecium strains 10/96A, BM4339, and BM4416 and PBPs from Streptomyces sp. strain K15 (40), Bacillus subtilis MB24 (DacF) (56), and E. coli (PBP6) (18). Conserved motifs involved in the scaffolding of the active site are indicated in boldface type. 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. 5.

Alignment of deduced amino acid sequences of VanS_D sensors. Numbers at the left refer to the first amino acid in the corresponding sequence. Numbers at the right refer to the last amino acid in the corresponding line. Identical amino acids are indicated by asterisks, and the isofunctional amino acids are indicated by dots below the alignment. Conserved motifs H, N, G1, F, and G2 are indicated above the alignment by dashes lines (41). The histidine residue in boldface lettering is the putative autophosphorylation site. The proline (P) at position 173 putatively responsible for constitutive expression of resistance in BM4339 is indicated in boldface italic lettering. The amino acid sequence of VanS_D from 10/96A starts after the insertion of ISEfa4.

FIG. 6.

Binding of benzyl[¹⁴C]penicillin to membrane proteins of VanD-type E. faecium. Membrane preparations of the strains indicated at the top were incubated with benzyl[¹⁴C]penicillin (1 μg/ml) for 5 min at 37°C; the proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis on a 12% polyacrylamide gel; the gel was stained with Coomassie blue, destained, and dried; and the PBPs were revealed by autoradiography.