(2005) identified mutations in the atpE gene leading to diarylquinone resistance in Mycobacterium tuberculosis and Mycobacterium smegmatis. By whole-genome sequencing, base substitutions suppressing relA mutations were identified (Srivatsan et al., 2008). In B. subtilis, a point mutation in the yqiD gene generated one type of l-form (Leaver et al., 2009). Makarov et al. (2009) identified the arabinan pathway as a target for benzothiazinones in M. tuberculosis. Here, we report the molecular basis for a mechanism circumventing the action of the MK-2206 clinical trial new antibiotic CmC on B. subtilis. Taq, Taq native
and Pvu DNA polymerases were purchased from Fermentas. DNase I and SuperScript™III reverse transcriptase were from Ambion and Invitrogene, respectively. Escherichia coli strains DH5α and TG1 and B. subtilis strain 168 were used and grown in Luria–Bertani (LB) medium. According to Steinfels et al. (2004), mutant
B. subtilis 8R was grown in the presence of CmC with or without the addition of 50 μM reserpine. Total RNA was prepared as described (Heidrich et al., 2006). RNA used for real-time PCR was treated with 3 μL DNase I (1 U μL−1) in 50 μL in the presence of 0.5 μL RiboLock™ RNase Inhibitor (40 U μL−1) and DNase I buffer with MgCl2 for 30 min at 37 °C, followed by 10 min at 80 °C to inactivate the enzyme. The RNA was further purified using the DNA-free RNA Kit from Zymo Research. For qRT-PCR, the Applied Biosystems StepOne real-time PCR system and the GeneAmp Fast SYBR Green Master Mix were used. The PCR conditions on the cDNA were optimized in the Applied Biosystems fast cycler ‘Verity’. Ratios were calculated using the RG-7388 clinical trial ΔΔCT method (Pfaffl, 2002). Membrane proteins were prepared using a protocol adapted from Steinfels et al. (2002). Primers pxyvcC-F and yvcC2MF_2 as well as pxyvcC-R1 and primer yvcC2MR_2 were used to generate PCR fragments. After annealing, the resulting
chimera sequences were extended and amplified using primers pxyvcC-F and pxyvcC-R1 to give rise to a long fragment of 1289 bp. Similarly, using primers yvcC1 MF_2 and yvcC1MR_2, a PCR fragment containing only the +6 mutation was generated. These fragments Chlormezanone were used to transform B. subtilis 168 and select for growth in the presence of different CmC concentrations. Preparation of B. subtilis RNAP and in vitro transcription experiments were performed as described previously (Licht et al., 2008). Gels were dried and subjected to Phosphoimaging (Fujix BAS 1000). pc bas 2.0e software was used for the quantification of the bands. Bacillus subtilis 168 grown till the late log-phase was inoculated 1 : 100 in 10 mL LB medium without an antibiotic and LB with 0.25 μM CmC [0.5 × minimal inhibitory concentration (MIC)], with 0.5 μM CmC (1 × MIC) and with 1 μM CmC (2 × MIC) and incubated for 24 h at 37 °C and 200 r.p.m., yielding turbid growth only in the 1 μM CmC culture.