D-Ala-D-Ala ligases from glycopeptide antibiotic-producing organisms are highly homologous to the enterococcal vancomycin-resistance ligases VanA and VanB

Abstract

The crisis in antibiotic resistance has resulted in an increasing fear of the emergence of untreatable organisms. Resistance to the glycopeptide antibiotic vancomycin in the enterococci, and the spread of these pathogens throughout the environment, has shown that this scenario is a matter of fact rather than fiction. The basis for vancomycin resistance is the manufacture of the depsipeptide D-Ala-D-lactate, which is incorporated into the peptidoglycan cell wall in place of the vancomycin target D-Ala-D-Ala. Pivotal to the resistance mechanism is the production of a D-Ala-D-Ala ligase capable of ester formation. Two highly efficient depsipeptide ligases have been cloned from vancomycin-resistant enterococci: VanA and VanB. These ligases show high amino acid sequence similarity to each other ( approximately 75%), but less so to other D-Ala-D-X ligases (<30%). We have cloned ddls from two glycopeptide-producing organisms, the vancomycin producer Amycolatopsis orientalis and the A47934 producer Streptomyces toyocaensis. These ligases show strong predicted amino acid homology to VanA and VanB (>60%) but not to other D-Ala-D-X ligases (<35%). The D-Ala-D-Ala ligase from S. toyocaensis shows D-Ala-D-lactate synthase activity in cell-free extracts of S. lividans transformed with the ddl gene and confirms the predicted enzymatic activity. These results imply a close evolutionary relationship between resistance mechanisms in the clinics and in drug-producing bacteria.

Figure 1

Structures of glycopeptide antibiotics A47934 and vancomycin.

Figure 2

Restriction endonuclease map of the region cloned from S. toyocaensis containing ddlM and murX and the complete sequence of the ddlM gene, arrows indicate sites complementary to the PCR primers. Sa, SalI; Sm, SmaI; S1, SstI; P, PstI; N, NcoI; B, BamHI; RV, EcoRV; X, XmnI.

Figure 3

Partial sequence of the ddl gene from A. orientalis C329.2.

Figure 4

Phylogenetic relationship among Ddls. Phylogenetic tree prepared using the Clustal V method (23), the gap penalty and gap length were set to 10. The point accepted mutations (PAM250) weight table (24) was used to define the similarity between sequences (number of matching residues divided by the sum of the mismatched residues + matching residues + gapped residues) using the MegAlign software by DNAstar (Madison, WI). The horizontal axis is the percentage distance between sequences.

Figure 5

Sequence overlaps in selected regions of Ddls. (a) Sequence overlap in the ω-loop region. (b) Sequence overlap at the N-terminal d-Ala1 binding region. (c) Sequence overlap in the Mg²⁺ binding region.

Figure 6

d-Ala-d-X activity assays. The ddlM gene was cloned into plasmid pFD666 to give pFD666-Stoyddl and used to transform glycopeptide-sensitive S. lividans 1326. Assays were performed as previously described (5), and the products separated on cellulose TLC with 1-butanol:acetic acid:water (12:3:5) as solvent. d-Ala-d-Lac was identified by comigration with authentic depsipeptide produced by VanA.