1 M sodium acetate, pH 3.0, 5 mM MgSO4, and 0.3 U/μl DNase I (Roche Diagnostics, Mannheim, Germany) for 30 min at 37°C. After heat inactivation for 5 min at 75°C, the RNA was precipitated with LiCl as described by . After denaturation for 5 min at 65°C, reverse transcription of 500 ng RNA was performed with Omniscript Reverse Transcriptase (QIAGEN, Hilden, Germany) according to the manufacturer’s instructions by using random hexamer primers (Invitrogen, Karlsruhe, Germany). Subsequently, the cDNA was amplified using combinations of the primers A (bioY-RBS_fw, bioY_rev), B (bioY-int_fw, bioM-int_rev) Dabrafenib nmr and C (bioMN-RBS_fw, bioYMN_rev). As a control, cDNA of dnaE was amplified using primers RT-dnaE-fw and RT-dnaE-rev.
To determine transcriptional starts by RACE-PCR RNA was prepared and purified as described above. Primers binding downstream of the annotated translational starts of bioY and bioM (bioY_rev, bioM_rev) along with 2.0 μg total RNA were used for cDNA synthesis reverse transcription
with Superscript II (Invitrogen, Karlsruhe, Germany) according to the supplier’s protocol. After RNA digestion with RNase H (Fermentas, St. Leon-Roth, Germany) and purification the cDNA was then selleckchem modified by terminal deoxynucleotidyl transferase (Fermentas, St. Leon-Roth, Germany) and dATP respectively dCTP to determinate the transcriptional start accurately. Subsequently, the cDNA was amplified using combinations of oligo(dT) or oligo(dG) primer and either bioY-int_rev or bioM-int_rev. The obtained PCR products were cloned into the pGEM-T Easy vector (Promega, Mannheim, Germany) and transferred into E. coli DH5α cells. At least two different clones per gene were selected for plasmid preparation and DNA sequencing (BigDye Terminator v3.1 Cycle Sequencing Kit and ABI Prism Capillary Sequencer Model 3730, Applied Biosystems, Forster-City, USA). Transport assays Biotin-limited (2.5 μg/l) precultures of C. glutamicum WT(pEKEx3) and biotin-sufficient (200 μg/l) precultures of WT(pEKEx3) and WT(pEKEx3-bioYMN) were used to inoculate glucose minimal medium cultures
with either 1 μg/l or 200 μg/l biotin and allowed to grow to mid-exponential phase in minimal medium CGXII supplied with glucose as the sole carbon source. 1 mM Smoothened IPTG was used in this culture for 17 h for the induction of pEKEx3-bioYMN expression. Subsequently, cells were washed two times with the assay buffer (0.1 M sodium chloride, 25 mM potassium phosphate, pH 7.5) and incubated on ice until the measurement. The cells were energized by incubation for 3 min at 30°C with 20 mM glucose at an optical density (600 nm) of 5 in an assay volume of 2 ml before biotin was added. Finally, 7 kBq of 3H-labeled biotin (1.11-2.22 TBq/mmol, PerkinElmer, Rodgau, Germany) was applied in an 2 ml assay at concentrations indicated in the respective experiments, and 200 μl samples were taken at 15, 30, 45, 60, 90 s in order to determine initial uptake rates.