vanE gene cluster of vancomycin-resistant Enterococcus faecalis BM4405

Abstract

Acquired VanE-type resistance to low levels of vancomycin (MIC = 16 microg/ml) in Enterococcus faecalis BM4405 is due to the inducible synthesis of peptidoglyean precursors terminating in D-alanine-D-serine (Fines,M., B. Prichon, P. Reynolds, D. Sahm, and P. Courvalin, Antimicrob. Agents Chemother. 43:2161-2164, 1999). A chromosomal location was assigned to the vanE operon by pulsed-field gel electrophoresis and hybridization, and its sequence was determined. Three genes, encoding the VanE ligase, the VanXYE DD-peptidase, and the VanTE serine racemase, that displayed 43 to 53% identity with the corresponding genes in the vanC operon were found. In addition, two genes coding for a two-component regulatory system, VanRE-VanSE, exhibiting 60 and 44% identity with VanR,-VanS, were present downstream from vanTE. However, because of a stop codon at position 78, VanSE was probably not functional. The five genes, with the same orientation, were shown to be cotranscribed by Northern analysis and reverse transcription-PCR. The vanE, vanXYE, and vanTE genes conferred inducible low-level resistance to vancomycin after cloning in E. faecalis JH2-2, probably following cross talk with a two-component regulatory system of the host.

FIG. 1.

Schematic representation of the vanE gene cluster and of recombinant plasmids. (A) Open arrows represent coding sequences and the direction of transcription. The asterisk indicates the stop codon in vanS_E. (B) The inserts in the recombinant plasmids are represented by solid lines, and the vectors are indicated in parentheses. (C) PCR fragments used as probes in Northern hybridization. (D) Oligonucleotides used in RT-PCR and in primer extension. Arrowheads indicate positions and orientations of primers.

FIG. 2.

Comparison of the d-Ala-d-Ser gene clusters. Arrows represent coding sequences and indicate the direction of transcription. The asterisk indicates the stop codon in vanS_E. The guanosine-plus-cytosine content (% G+C) is indicated in the arrows. The percentages of amino acid (aa) identity between the deduced proteins are indicated under the arrows.

FIG. 3.

Phylogenetic tree derived from the alignment of d-Ala:Lac and d-Ala:d-Ser ligases. The tree was constructed by the neighbor-joining method, taking into account the results of maximum-parsimony and bootstrapping analysis.

FIG. 4.

Analysis of vanE gene cluster transcription by Northern hybridization. Total RNA from BM4405 was hybridized with the vanE (lane 1), vanXY_E (lane 2), vanT_E (lane 3), vanR_E (lane 4), and vanS_E (lane 5) probes. The sizes of the transcripts were determined according to RNA molecular weight marker I (Boehringer) (not shown). b, bases.

FIG. 5.

Analysis of the transcription of the vanE, vanXY_E, and vanT_E genes. Electrophoresis of the product obtained by RT-PCR with primers VDV and E15 (Fig. 1D and Table 2) (A) and corresponding Southern hybridizations with vanE (B), vanXY_E (C), and vanT_E (D) probes (Fig. 1C) are shown. Incubations were carried out in the absence (lanes 1) or presence (lanes 2) of reverse transcriptase. Lanes M, DNA from bacteriophage lambda digested by PstI as a marker.

FIG. 6.

Identification of the transcriptional start site for the vanE, vanXY_E, vanT_E, vanR_E, and vanS_E genes in BM4405 by primer extension analysis. (Left panel) Lane 1, primer elongation product obtained with oligodeoxynucleotide PE1 and 50 μg of total RNA from BM4405 (arrowhead); lanes T, G, C, and A, results of sequencing reactions performed with the same primer. Right panel, sequence from nucleotide positions −353 to +141 (numbering from the A of the ATG start codon of vanE, negative in the 3′-to-5′ direction and positive in the 5′-to-3′ direction). The +1 transcriptional start site for the vanE, vanXY_E, vanT_E, vanR_E, and vanS_E mRNA in BM4405 and the −35 and −10 promoter sequences located upstream are in boldface. The ATG start codon of vanE is indicated by an arrow, and the ribosome binding site (RBS) is in boldface and underlined.