PrrA modulation of Mycobacterium tuberculosis response to acidic pH and high chloride levels 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 acidic pH and high chloride levels. 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 was grown to an OD600 of ~0.6 in standing, filter-capped T-75 flasks. Bacteria were then subcultured to an OD600 = 0.3 in standing, filter-capped T-25 flasks in 7H9, pH 7 media ± 250 mM NaCl, or 7H9, pH 5.7 media ± 250 mM NaCl. Exposure to the different media conditions 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.