PrrA modulates Mycobacterium tuberculosis response to nitric oxide

The purpose of this study was to understand the impact of prrA overexpression on global M. tuberculosis transcriptional response to nitric oxide. Overall design: Mtb strain CDC1551 harboring a tetracycline inducible prrA-FLAG construct was grown in aerated conditions to mid-log phase (OD600 ~0.6), and treated with either ethanol (EtOH) as a carrier control or 200 ng/ml anhydrotetracycline (ATC) for 2 hours. Bacteria were subcultured to an OD600 = 0.3 in filter-capped T-75 flasks laid flat, in 12 ml 7H9, pH 7 media ± 100 µM DETA NONOate (an NO donor), with continued presence of EtOH or ATC as appropriate. Exposure to DETA NONOate was for 4 hours in a humidified, 37°C, 5% CO2 incubator before RNA was extracted. RNA was prepared by removing the rRNA and library prepping with a TruSeq Stranded kit (Illumina) before high-throughput sequencing with an Illumina HiSeq 2500 (High Output v4) (100 bp single end reads, one lane). Two biological samples per condition were sequenced.