Molecular characterization of the vanD gene cluster and a novel insertion element in a vancomycin-resistant enterococcus isolated in Canada

Abstract

A single vanD-containing Enterococcus faecium strain (N97-330) was isolated in Canada. The vanD-containing region was cloned and sequenced. Although the proteins have more than 96% identity to a previously described vanD region in BM4339, the vanS(D) gene contains a frameshift mutation that leads to a predicted truncated protein. Furthermore, sequence analysis of the ddl gene revealed the presence of an IS982-like element (ISEfm1) which interrupted the D-Ala-D-Ala ligase. This suggested the constitutive expression of the vanD operon, which was confirmed. Pulsed-field gel electrophoresis fingerprinting demonstrated that BM4339 was not related to N97-330 (>15 band differences). Both strains contained multiple copies of the IS982-like element.

FIG. 1

Schematic representation of the vanD gene cluster and sequence of a putative stem-loop structure. (A) Predicted ORFs from a 6,793-bp region sequenced from two λ clones and a PCR product. Open arrows represent the coding sequences for the vanR_D, vanS_D, vanY_D, vanH_D, vanD, vanX_D, and orfR1 genes. The vertical line in the vanS_D ORF represents the position of the frameshift mutation leading to a predicted truncated protein. The vanD probe used in hybridization experiments to isolate the λ clones is shown below the corresponding region. (B) Sequence of the putative stem-loop structure downstream of vanX_D.

FIG. 2

Sequence of the ISEfm1 element found in the ddl gene and prevalence of the insertion element in selected strains. (A) Complete nucleotide sequence and predicted amino acid sequence of the ISEfm1 element and partial ddl gene. Capital letters indicate nucleotide differences in ddl compared to the published sequence of E. faecium BM4147-1. Open arrows indicate the coding sequence for the ddl and tnp genes. ddl′ denotes the sequence of the ddl gene downstream of the ISEfm1 element. DR, IR-L, and IR-R indicate direct repeats, the left inverted repeat, and the right inverted repeat, respectively. The open box indicates the translational frameshift region. (B) Southern blot of ClaI-digested DNA probed with the ISEfm1 PCR product. Lane 1, 1-kb extension ladder (Life Technologies; sizes, in kilobases, of the fragments are indicated at the left); lane 2, E. faecium N97-330 (vanD); lane 3, E. faecium BM4339 (vanD); lane 4, E. faecium N98-638 (vanA); lane 5, E. faecium ATCC 19434; lane 6, E. faecalis ATCC 29212.