Genomic DNA of the drrA–drrB null mutant was cut with BamHI and ligated to the dephosphorylated BamHI ends of pBluescript SK−. Escherichia coli cells transformed with ligated DNA were selected on ampicillin and apramycin (positive selection) plates. This recombinant clone pRESAB was sequenced with appropriate primers (Genetic Analyzer ABI310) to confirm the presence of the

chromosome–marker junction sequence on the drrB side of integration. check details Digestion of pRESAB with XbaI–BamHI released a 2.1-kb fragment comprising a drrB carboxy end and the adjacent drrD/dnrW gene. This was cloned in pOK12 (Vieira & Messing, 1991) and sequenced with M13 forward and reverse primers. The medium used for the study was prepared

as described previously (Dekleva et al., 1985). Single-colony S. peucetius was inoculated into 25 mL nitrate defined medium with 0.5% yeast extract and grown for 36 h at 30 °C, 180 r.p.m. Mycelia were collected OSI906 by centrifugation at 2000 g for 20 min at 4 °C. One gram wet weight of mycelia was inoculated in 100 mL of nitrate defined medium (NDM) with 5% maltose as the carbon source and grown for 120 h at 30 °C. Anthracylines were extracted and analyzed by HPLC (Shimadzu, Japan) as described earlier (Bartel et al., 1990). A C18 reverse-phase octadecyl column (Shimadzu) was used. The mobile phase was 65% methanol and 35% phosphorylated water, pH 2.0. DNR (Sigma Aldrich, Bangalore, India) was used as the standard. HPLC was set at a flow rate of 1 mL min−1 and A254 nm was measured. A series of dilutions were analyzed by HPLC to construct the standard graph. DNR levels were estimated based on the peak area of the DNR standard. The drrA–drrB null mutant and WT cells were tested for levels of resistance to DNR in the culture medium. R2YE plates with 0, 1, 2, 4, 6, 8 and 10 μg mL−1 DNR were prepared. The cells were grown in NDM liquid for 120 h and 10 μL of the ADAMTS5 culture was placed on an agar surface. Plates were incubated

for 90 h and photographed to record growth inhibition. Total RNA was prepared using the RNeasy Plant Mini Kit (Qiagen) according to the instructions of the manufacturer. The RNA was treated with Turbo DNAse (Ambion) according to the manufacturer’s instructions. RNA was quantified using a Nanodrop ND-1000 spectrophotometer, and the quality of RNA was analyzed on an agarose gel as described by Kieser et al. (1998). In a 10-μL reaction, 1 μg RNA, 1 mM dNTP mix and 250 ng of random hexamer (Promega) were heated to 80 °C for 5 min and rapidly chilled on ice. Two hundred units of M-MLV reverse transcriptase and 20 U Rnasin (Sigma Aldrich) were added and the volume was made up to 20 μL. The mixture was incubated at 37 °C for 60 min; the reaction was stopped by heating at 90 °C for 5 min. Control reactions were carried out without reverse transcriptase.