PrrA modulation of Mycobacterium tuberculosis response to nitric oxide is critically regulated by serine/threonine protein kinases

The purpose of this study was to understand how prevention of serine/threonine protein kinase (STPK) phosphorylation of PrrA impacts PrrA modulation of M. tuberculosis transcriptional response to nitric oxide. Overall design: Mtb strain CDC1551 with a deletion of the native copy of prrA, and harboring a prrA-FLAG-DAS4 (wild type prrA allele - prrA-DUC/?prrA) or prrA-T6A-FLAG-DAS4 (T6A STPK-phosphoablative prrA allele - prrA-T6A-DUC/?prrA) construct were grown in aerated conditions to mid-log phase (OD600 ~0.6), before the 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). 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.