Genetic analysis of bacteriocin 43 of vancomycin-resistant Enterococcus faecium

Abstract

A total of 636 vancomycin-resistant Enterococcus faecium (VRE) isolates obtained between 1994 and 1999 from the Medical School Hospital of the University of Michigan were tested for bacteriocin production. Of the 277 (44%) bacteriocinogenic strains, 21 were active against E. faecalis, E. faecium, E. hirae, E. durans, and Listeria monocytogenes. Of those 21 strains, a representative bacteriocin of strain VRE82, designated bacteriocin 43, was found to be encoded on mobilizable plasmid pDT1 (6.2 kbp). Nine open reading frames (ORFs), ORF1 to ORF9, were presented on pDT1 and were oriented in the same direction. The bacteriocin 43 locus (bac43) consists of the bacteriocin gene bacA (ORF1) and the immunity gene bacB (ORF2). The deduced bacA product is 74 amino acids in length with a putative signal peptide of 30 amino acids at the N terminus. The bacB gene encodes a deduced 95-amino-acid protein without a signal sequence. The predicted mature BacA protein (44 amino acids) showed sequence homology with the membrane-active class IIa bacteriocins of lactic acid bacteria and showed 86% homology with bacteriocin 31 from E. faecalis YI717 and 98% homology with bacteriocin RC714. Southern analysis with a bac43 probe of each plasmid DNA from the 21 strains showed hybridization to a specific fragment corresponding to the 6.2-kbp EcoRI fragment, suggesting that the strains harbored the pDT1-like plasmid (6.2 kb) which encoded the bacteriocin 43-type bacteriocin. The bac43 determinant was not identified among non-VRE clinical isolates.

FIG. 1.

Agarose gel electrophoresis of EcoRI-digested plasmid DNAs of bacteriocinogenic strain VRE82 and transconjugants. Lanes: 1, HindIII-digested lambda DNA; 2, E. faecium VRE82 (wild-type VRE strain); 3, nonbacteriocinogenic VRE BM4105RF transconjugant; 4, bacteriocinogenic VRE BM4105RF transconjugant. Arrow, 6.2-kb band.

FIG. 2.

Physical map of pDT1 showing deduced ORFs and transposon insertions into pDT1 of pMG501 (pAM401::pDT1). (a) Physical map of pDT1 (6.2 kbp) and deduced ORFs. Thick horizontal arrows indicate ORFs on pDT1 and the direction of transcription. (b) Map of Tn5 insertions into pDT1 of pAM401::pDT1. Open circles indicate Tn5 insertion mutants. Numbers beside symbols are mutant identification numbers. aa, amino acids.

FIG. 3.

Cloning of PCR products from the region of the bacteriocin determinant of pDT1. Thick lines represent the cloned PCR product. The numbers at the ends of the thick lines represent the 5′ and 3′ ends of the segment on the map (base pairs). The vertical bar with an arrowhead is the potential promoter. a.a., amino acids; n.d., the pasmid did not transform E. faecalis FA2-2.

FIG. 4.

Nucleotide sequence of bacA and bacB of bacteriocin 43 and deduced amino acid sequence. Potential promoters (−10 and −35) and S.D. ribosome binding sequences are underlined. The inverted repeat sequence is indicated by horizontal arrows. Primers H1 and H2, which were used for PCR analysis of the bac43 determinant in clinical isolates, are indicated by dashed lines. The accession number is AB178871.

FIG. 5.

Comparison of the amino acid sequence of the predicted BacA protein of bacteriocin 43 with the amino acid sequences of homologous bacteriocins. The sequences of the predicted BacA protein and other class IIa bacteriocins are shown. The consensus sequence Tyr-Gly-Asn-Gly-Lys (Val) (YGNGL[V]) of class IIa bacteriocins is indicated in boldfaced letters. The vertical arrow and dashed line indicate the cleavage site in the prebacteriocins. Identical amino acids (a.a.) are boxed.

FIG. 6.

EcoRI-digested plasmid DNAs isolated from 21 VRE strains that showed bacteriocin activity against E. faecalis, E. faecium, E. hirae, E. durans, and L. monocytogenes. (A) Agarose gel electrophoresis of EcoRI-digested plasmid DNAs. (B) The gel was Southern blotted and hybridized with the bacA probe. Lanes: 1 and 23, HindIII-digested lambda DNA; 2 to 22, strains 74, 78, 82, 83, 94, 252, 272, 278, 319, 330, 351, 367, 418, 419, 424, 437, 455, 477, 506, 576, and 595, respectively. Arrows, 6.2-kb bands.