Analysis of SmaI-digested genomic DNAs of the VanB-type clinical isolates by pulsed-field gel electrophoresis. Bacteriophage λ concatemers (lane M; New England BioLabs) were used as molecular size markers, and the sizes are indicated in kilobases at the left. The single band shift in revertant strain BM4661 in comparison with resistant strain BM4659 and dependent strain BM4660 is indicated by the arrowhead.

Schematic representation of the vanB gene cluster of strain BM4659 and the sequence of the vanS_B-vanY_B intergenic region, including the P_YB resistance promoter. (A) Open arrows represent coding sequences and indicate the direction of transcription. The sizes, in base pairs, of the PCR products and the EcoRI fragments are indicated in bold. The probes used in Southern or Northern hybridizations and the oligodeoxynucleotides used for RT-PCR analysis are also shown. Arrowheads indicate the positions and orientations of the primers. The sizes of the PCR products are in bold. (B) The transcriptional start site (+1) is in bold, and the −35 and −10 sequences of P_YB are boxed. The translational start site (ATG) of vanY_B and the RBS are in boldface italics. Regions protected from DNase I cleavage by VanR_B and phosphorylated VanR_B are delineated by brackets I and II, respectively, and the conserved heptanucleotide direct repeats are underlined. The 21-bp consensus binding site of VanR_B and phosphorylated VanR_B is in boldface. The deduced amino acid sequences of the C-terminal portion of VanS_B and the N-terminal portion of VanY_B are shown above the nucleotide sequence and aligned with the first nucleotide of each codon. Horizontal arrows indicate regions of dyad symmetry. The G-to-C substitution in the inverted repetition of revertant strain BM4661 is in bold italics and underlined. The numbers indicate the bases in the transcription terminator. (C) Most stable conformation of the predicted RNA secondary structure of the transcription terminator in resistant strain BM4659, dependent strain BM4660 (top), and revertant strain BM4661 (bottom). The circle indicates the mutation in the transcription terminator of revertant strain BM4461. ΔG values were calculated with RNAfold (21).

Analysis by agarose gel electrophoresis of plasmid DNA from the VanB-type clinical isolates digested (A) with EcoRI and HindIII or from resistant strain BM4659 digested (B) with EcoRI or HindIII, followed by hybridization with vanB and vanS_B probes. The sizes, in kilobases, of molecular size markers (lane M, 100-bp DNA ladder; Invitrogen) are indicated on the left. The sizes, in bases (b), of the fragments that hybridized are indicated on the right.

Transcription analysis of the vanB gene cluster by (A) Northern hybridization and (B) RT. (A) Total RNAs from resistant strain BM4659 and revertant strain BM4661 left uninduced (NI) or induced (I) with vancomycin were hybridized successively with vanR_B, vanS_B, and vanY_B probes. The size of the transcript indicated on the side was determined according to RNA molecular weight marker I (Boehringer) (not shown). (B) Transcription analysis of the vanR_B-vanS_B, vanS_B-vanY_B, and vanY_BWH_B genes of resistant strain BM4659, dependent strain BM4660, and revertant strain BM4661. Agarose gel electrophoresis of RT-PCR products from noninduced (NI) and induced (I) strains with primer pairs VB78-VB77, VB26-VB80, and VB82-VB23, respectively (Fig. 3B and Table 2). Lanes M, bacteriophage λ DNA digested with PstI and used as size markers. The sizes of the PCR products are in base pairs.