Fig 2. Perturbation of PrrA globally alters Mtb response to pH and Cl-.
(A) prrA overexpression does not significantly alter Mtb transcriptional profile in standard pH 7.0 growth conditions. Mtb(P1’::prrA-FLAG-tetON, rv2390c’::luciferase) was grown in 7H9, pH 7.0 media and treated with 0.1% EtOH or 200 ng/ml ATC for 6 hours before RNA was extracted for RNA sequencing analysis. Log2-fold change compares gene expression in the ATC (“prrA OE”) versus EtOH (“cont”) treatment sets. (B and C) prrA overexpression alters Mtb response to acidic pH and high [Cl-]. Mtb(P1’::prrA-FLAG-tetON, rv2390c’::luciferase) was grown in 7H9, pH 7.0 media and treated with 0.1% EtOH or 200 ng/ml ATC for 2 hours, before exposure to 7H9, pH 7.0 or 7H9, pH 5.7 + 250 mM NaCl for 4 hours in the continued presence of EtOH or ATC as appropriate. RNA was extracted for RNA sequencing analysis (B) or qRT-PCR (C). In (B), log2-fold change compares gene expression in the 7H9, pH 5.7 + 250 mM NaCl condition versus the 7H9, pH 7 control condition for each of the EtOH (“cont”) or ATC (“prrA OE”) treatment sets. Genes marked in red had a log2-fold change difference ≥0.25 between the ATC and EtOH treatment sets (p<0.05, FDR<0.01 in both sets, with log2-fold change ≥1 in the EtOH set). In (C), fold change compares gene expression in the pH 5.7/250 mM NaCl versus the control pH 7.0 condition for each of the EtOH (“control”) or ATC (“prrA OE”) treatment sets. sigA was used as the control gene, and data are shown as means ± SD from 3 technical replicates. p-values were obtained with an unpaired t-test. N.S. not significant, ** p<0.01, *** p<0.001, **** p<0.0001.