Fig 1. A rv2390c’::luciferase reporter transcription factor overexpression screen identifies PrrA as a TF that regulates Mtb response to pH and Cl^-.

(A) Mtb carrying a chromosomal rv2390c’::luciferase reporter was grown in 7H9, pH 7.0 ± 250 mM NaCl, or 7H9, pH 5.7 ± pH 250 mM NaCl for 9 days before light output (relative light units, RLU) and OD₆₀₀ were measured. Fold induction compares RLU/OD₆₀₀ in each condition to RLU/OD₆₀₀ in the control pH 7.0 condition. Data are shown as means ± SD from three experiments. (B) A library of inducible TF overexpression plasmids (P₁’::TF-FLAG-tetON) in the background of a rv2390c’::luciferase reporter Mtb strain was screened for their response to 250 mM NaCl. TF overexpression was induced with 200 ng/ml ATC 1 day prior to exposure to 7H9, pH 7.0 ± 250 mM NaCl media for 9 days, in the continued presence of ATC. RLU/OD₆₀₀ was measured and fold induction calculated as in (A) for each strain. Empty vector plasmid controls were included for comparison. (C) prrA overexpression represses Mtb response to acidic pH and high [Cl^-]. Mtb(P₁’::prrA-FLAG-tetON, rv2390c’::luciferase) was grown in 7H9, pH 7.0 ± 250 mM NaCl, or 7H9, pH 5.7 ± pH 250 mM NaCl for 9 days, with 0.1% ethanol (EtOH) as a carrier control (“control”) or 200 ng/ml ATC (“prrA OE”) added 6 days post-assay start. RLU/OD₆₀₀ was measured at the end of the assay and fold induction calculated as in (A). Data are shown as means ± SD from three experiments. p-values were obtained with an unpaired t-test. ** p<0.01, *** p<0.001, **** p<0.0001.