ABSTRACT
A new open reading frame, encoding a putative integrase-like protein, was detected downstream from the six genes of the vanD glycopeptide resistance cluster in Enterococcus faecium BM4339 (B. Casadewall and P. Courvalin, J. Bacteriol. 181:3644-3648, 1999). In this cluster, genes coding for the VanR(D)-VanS(D) two-component regulatory system were cotranscribed from the P(R(D)) promoter, whereas transcription of the vanY(D), vanH(D), vanD, vanX(D), and intD genes was initiated from the P(Y(D)) promoter located between vanS(D) and vanY(D) (the D subscript indicates that the gene is part of the vanD operon). The VanR(D)-VanS(D) regulatory system is likely to activate transcription of the resistance genes from the promoter P(Y(D)). Glycopeptide-susceptible derivatives of BM4339 were obtained by trans complementation of the frameshift mutation in the ddl gene, restoring functional D-alanine:D-alanine ligase activity in this strain. The glycopeptide-susceptible transformant BM4409, producing only D-alanyl-D-alanine-terminating peptidoglycan precursors, did not express the resistance genes encoding the VanY(D) D,D-carboxypeptidase, the VanH(D) dehydrogenase, the VanD ligase, the VanX(D) D,D-dipeptidase, and also the IntD integrase, although the regulatory region of the vanD cluster was still transcribed. In BM4409, the absence of VanR(D)-VanS(D), apparently dependent, transcription from promoter P(Y(D)) correlated with the lack of D-alanyl-D-lactate-terminating precursors. The vanX(D) gene was transcribed in BM4339, but detectable amounts of VanX(D) D,D-dipeptidase were not synthesized. However, the gene directed synthesis of an active enzyme when cloned on a multicopy plasmid in Escherichia coli, suggesting that the enzyme was unstable in BM4339 or that it had very low activity that was detectable only under conditions of high gene dosage. This activity is not required for glycopeptide resistance in BM4339, since this strain cannot synthesize D-alanyl-D-alanine.
Subject(s)
Anti-Bacterial Agents/pharmacology , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Enterococcus faecium/drug effects , Gene Expression Regulation, Bacterial , Peptide Synthases , Vancomycin/pharmacology , Bacterial Proteins/chemistry , Base Sequence , Drug Resistance, Microbial/genetics , Enterococcus faecium/genetics , Enterococcus faecium/growth & development , Escherichia coli/genetics , Escherichia coli/metabolism , Humans , Molecular Sequence Data , Multigene Family , Plasmids/genetics , Promoter Regions, Genetic/genetics , Promoter Regions, Genetic/physiology , Transcription, GeneticABSTRACT
Glycopeptide dependence for growth in enterococci results from mutations in the ddl gene that inactivate the host D-Ala:D-Ala ligase. The strains require glycopeptides as inducers for synthesis of resistance proteins, which allows for the production of peptidoglycan precursors ending in D-Ala-D-Lac instead of D-Ala-D-Ala. The sequences of the ddl gene from nine glycopeptide-dependent Enterococcus faecium clinical isolates were determined. Each one had a mutation consisting either in a 5-bp insertion at position 41 leading to an early stop codon, an in-frame 6-bp deletion causing the loss of two residues (KDVA243-246 to KA), or single base-pair changes resulting in an amino acid substitution (E13 --> G, G99 --> R, V241 --> D, D295 --> G, P313 --> L). The potential consequences of the deletion and point mutations on the 3-D structure of the enzyme were evaluated by comparative molecular modeling of the E. faecium enzyme, using the X-ray structure of the homologous Escherichia coli D-Ala:D-Ala ligase DdlB as a template. All mutated residues were found either to interact directly with one of the substrates of the enzymatic reaction (E13 and D295) or to stabilize the position of critical residues in the active site. Maintenance of the 3-D structure in the vicinity of these mutations in the active site appears critical for D-Ala:D-Ala ligase activity.
Subject(s)
Base Sequence/genetics , Enterococcus faecium/enzymology , Models, Molecular , Mutation/genetics , Peptide Synthases/chemistry , Peptide Synthases/genetics , Amino Acid Sequence , Computational Biology/methods , Enterococcus faecium/classification , Enterococcus faecium/pathogenicity , Glycoproteins/metabolism , Imaging, Three-Dimensional , Molecular Sequence Data , Species Specificity , Structure-Activity RelationshipABSTRACT
VanD-type Enterococcus faecium BM4416 was constitutively resistant to vancomycin and to teicoplanin by synthesis of peptidoglycan precursors ending in D-alanyl-D-lactate. Like E. faecium BM4339, the only VanD-type strain described so far, BM4416 produced an impaired D-alanine:D-alanine ligase. Unlike for BM4339, which had a 5-bp insertion in ddl, inactivation of the gene in BM4416 was due to insertion of IS19.
Subject(s)
Anti-Bacterial Agents/pharmacology , Enterococcus faecium/enzymology , Peptide Synthases/genetics , Vancomycin Resistance/genetics , Amino Acid Sequence , Base Sequence , DNA, Bacterial/analysis , Enterococcus faecium/drug effects , Enterococcus faecium/genetics , Molecular Sequence Data , Mutagenesis, Insertional , Peptide Synthases/metabolism , Teicoplanin/pharmacology , Vancomycin/pharmacologyABSTRACT
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.
Subject(s)
Anti-Bacterial Agents/pharmacology , Carboxypeptidases , Drug Resistance, Microbial/genetics , Enterococcus faecium/drug effects , Enterococcus faecium/genetics , Membrane Proteins , Multigene Family , Open Reading Frames , Amino Acid Sequence , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Base Sequence , Chromosomes, Bacterial , Conserved Sequence , Enterococcus faecium/growth & development , Escherichia coli/genetics , Frameshift Mutation , Genotype , Molecular Sequence Data , Peptide Synthases/chemistry , Peptide Synthases/genetics , Plasmids , Sequence Alignment , Streptomyces/genetics , Vancomycin/pharmacologyABSTRACT
The D-alanine:D-alanine-ligase-related enzymes can have three preferential substrate specificities. Usually, these enzymes synthesize D-alanyl-D-alanine. In vancomycin-resistant Gram-positive bacteria, structurally related enzymes synthesize D-alanyl-D-lactate or d-alanyl-d-serine. The sequence of internal fragments of eight structural d-alanine:d-alanine ligase genes from enterococci has been determined. Alignment of the deduced amino acid sequences with those of other related enzymes from Gram-negative and Gram-positive bacteria revealed the presence of four distinct sequence patterns in the putative substrate-binding sites, each correlating with specificity to a particular substrate (D-alanine:D-lactate ligases exhibited two patterns). Phylogenetic analysis showed different clusters. The enterococcal subtree was largely superimposable on that derived from 16S rRNA sequences. In lactic acid bacteria, structural divergence due to differences in substrate specificity was observed. Glycopeptide resistance proteins VanA and VanB, the VanC-type ligases, and DdlA and DdlB from enteric bacteria and Haemophilus influenzae constituted separate clusters.
