Regulation of expression of the vanD glycopeptide resistance gene cluster from Enterococcus faecium BM4339

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.

FIG. 1

Schematic representation of the vanD gene cluster in BM4339. (A) Map of the 8.7-kb region containing the vanR_D, vanS_D, vanY_D, vanH_D, vanD, vanX_D, and intD ORFs comprising the vanD gene cluster. Open arrows indicate sense of transcription. The PCR fragment internal to vanX_D used as a probe in hybridization experiments is indicated. Abbreviations: H, HindIII; S, Sau3AI; X, XmaI. (B) Inserts in recombinant plasmids. The inserts are represented by solid lines, and the vectors are indicated in parentheses. (C) Probes used in Northern hybridization and in RT-PCR. (D) Oligodeoxynucleotides used in RT-PCR and in primer extension. Arrowheads indicate positions and orientations of primers.

FIG. 2

Partial alignment of the deduced sequences of IntD from E. faecium BM4339, TnpI from B. thuringiensis H1.1 (accession no. P10020) (24), XerC from H. influenzae RD/KW20 (accession no. P44818) (17), and XerD from E. coli K-12 (accession no. P21891) (22). Tetrads of residues conserved in all the members of the Int family of site-specific recombinases (28) are indicated in boldface. The numbers of residues separating the different segments containing the conserved amino acids are indicated for each protein.

FIG. 3

Analysis of the vanD gene cluster transcription by Northern hybridization. Total RNA from BM4339 (lanes 1, 3, and 5) and BM4409 (lanes 2, 4, and 6) was hybridized with the vanR_D (lanes 1 and 2) and the vanS_D (lanes 3 and 4) probes (A) and the vanD (lanes 1 and 2), the vanX_D (lanes 3 and 4), and the intD (lanes 5 and 6) probes (B) (see Fig. 1C). The size of the transcripts was determined according to RNA molecular weight marker I (Boehringer) (not shown).

FIG. 4

Analysis of the transcription of the vanY_D and vanD genes. Electrophoresis of the products obtained by RT-PCR using the primers RTY and RTD (see Fig. 1D and Table 2) (A) and corresponding Southern hybridization (B and C). Incubations were carried out in the absence (lanes 1) or presence (lanes 2) of reverse transcriptase. Lanes M contained DNA from bacteriophage lambda digested with PstI as a marker. (B) Hybridization with a vanD probe (see Fig. 1C). (C) Hybridization with a vanY_D probe (see Fig. 1C).

FIG. 5

Analysis of the transcription of the vanH_D and vanD genes. Electrophoresis of the products obtained by RT-PCR using the primers RTH and RTD (see Fig. 1D and Table 2) (A) and corresponding Southern hybridization (B and C). Incubations were carried out in the absence (lanes 1) or presence (lanes 2) of reverse transcriptase. Lanes M contained DNA from bacteriophage lambda digested with PstI as a marker. (B) Hybridization with a vanD probe (see Fig. 1C). (C) Hybridization with a vanH_D probe (see Fig. 1C).

FIG. 6

Identification of the transcriptional start site for the vanR_D and vanS_D genes in BM4339 by primer extension analysis. Left panel, lane 1, primer elongation product obtained with oligodeoxynucleotide PR and 50 μg of total RNA from BM4339 (arrowhead); lane 2, control without RNA; lanes T, G, C, and A, results of sequencing reactions performed with the same primer. Right panel, sequence from nucleotide position −128 to +122 (numbering from the A of the ATG start codon of vanR_D, negative in the 3′ to 5′ direction and positive in the 5′ to 3′ direction). The +1 transcriptional start site for the vanR_D and vanS_D mRNA in BM4339 and the −35 and −10 promoter sequences located upstream are in boldface. The ATG start codon of vanR_D is indicated by an arrow, and the RBS is in boldface and underlined.

FIG. 7

Identification of the transcriptional start site for the vanY_D, vanH_D, vanD, vanX_D, and intD genes in BM4339 by primer extension analysis. Left panel, lane 1, primer elongation product obtained with oligodeoxynucleotide PY and 50 μg of total RNA from BM4339 (arrowhead); lane 2, control without RNA; lanes T, G, C, and A, results of sequencing reactions performed with the same primer. Right panel, sequence from nucleotide position −110 to +109 (numbering from the A of the ATG start codon of vanY_D, negative in the 3′-to-5′ direction and positive in the 5′-to-3′ direction). The +1 transcriptional start site for the vanY_D, vanH_D, vanD, vanX_D, and intD mRNA in BM4339 and the −35 and −10 promoter sequences located upstream are in boldface. The ATG start codon of vanY_D is indicated by an arrow, and the RBS is in boldface and underlined.

FIG. 8

CAT activity in cytoplasmic extracts from E. faecalis JH2-2 and from E. faecium BM4339 harboring plasmid pAT666 (P_YD cat). Controls were performed without addition of vancomycin to the culture medium (“not induced” bars), and induction was achieved by adding 1 μg of vancomycin/ml to cultures of JH2-2/pAT666 and 8 μg of vancomycin/ml to cultures of BM4339/pAT666 (“induced” bars). Enzymatic activity was expressed as nanomoles of product formed per minute per milligram of protein in S100 extracts. Results are means ± standard deviations obtained from three independent extracts.

FIG. 9

Analysis of derivatives of E. faecium BM4339 harboring a wild-type ddl gene on a high-copy-number plasmid (BM4409) or the same gene integrated as a single copy in the chromosome (BM4458 and BM4459). (A) Proportions of late soluble cytoplasmic peptidoglycan precursors accumulated in the presence of ramoplanin. (B) Levels of resistance to vancomycin and teicoplanin.