FIG. 1.
Schematic representation of the vanD gene cluster from E. raffinosus strain GV5 and Northern blot analysis of the vanD cluster. (A) Open arrows represent coding sequences and indicate the direction of transcription. The PCR fragments internal to the vanSD, vanYD, and vanD genes used in the hybridization experiments are indicated below the corresponding regions. Amino acids with arrows within parentheses indicate substitutions compared with the reported sequence of the vanD4 operon of E. faecium 10/96A (8). (B) Northern hybridization was performed according to a protocol described previously (13). RNAs were prepared from strains cultured with 6 μg/ml of vancomycin (+VCM) or without vancomycin (−VCM) for 2 h. Thirty micrograms of RNA was used in each lane. The sizes of the RNAs were determined by using the sizes of RNA molecular weight markers (Invitrogen, Inc.), and the arrows and the numbers on the left indicate the positions and sizes of the largest bands in each experiment.