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. Author manuscript; available in PMC: 2025 Apr 18.
Published in final edited form as: Cell Chem Biol. 2024 Jan 23;31(4):669–682.e7. doi: 10.1016/j.chembiol.2023.12.020

Figure 1. Development and validation of whiB7 reporters.

Figure 1.

(A) Genetic architecture of PwhiB7 reporter constructs. Both reporters were cloned into single-copy, integrating plasmids.

(B) Zeocin resistance profiles of M. smegmatis strains (~1,000 colony forming units (CFU)/well) harboring the zeocin resistance gene driven by the indicated promoters. EM7 is a strong, constitutive promoter derived from the T7 promoter. Plates contain either no drug (left) or 100 ng/ml = 0.25X minimum inhibitory concentration (MIC) of the known whiB7 inducing drug, clarithromycin (right). The red rectangle marks clarithromycin-dependent growth of the M. smegmatis PwhiB7:zeoR strain in the presence of 10–20 μg/ml zeocin.

(C) Growth of the indicated M. smegmatis CRISPRi strains on agar plates containing zeocin at 0, 10, or 20 ug/mL. NT = non-targeting; KD = knockdown.

(D) Dose response curves (mean ± s.e.m., n = 3 replicates) of the PwhiB7:mScarlet reporter M. smegmatis strain for clarithromycin (top graph) and isoniazid (bottom graph). Drug dose-response curves (percent growth) are shown in black and mScarlet fluorescence (RFU) are shown in red.

(E) Normalized fluorescence values of the indicated M. smegmatis CRISPRi strains at 0, 12, and 24 hours after addition of ATc to activate CRISPRi (mean ± s.e.m., n = 3 replicates). Both ettA and argD are targeted with two different sgRNAs each, denoted #1 and #2. P300 is a strong, constitutive promoter. EV = empty vector.

(F) Normalized fluorescence values for the PwhiB7:mScarlet reporter strain (top row: M. smegmatis, bottom row: M. tuberculosis) in response to the listed panel of drugs. Fluorescence values are indicative of the highest value obtained at a sub-MIC concentration of each drug. Normalized RFU: M. smegmatis = −18.2 to 2,000; M. tuberculosis = 13.2 to ≥6,000. RIF = rifampicin; LVX = levofloxacin; EMB = ethambutol; INH = isoniazid; BDQ = bedaquiline; STR = streptomycin; AMK = amikacin; CAP = capreomycin; CAM = chloramphenicol; LZD = linezolid; TGC = tigecycline; FUS = fusidic acid; CLR = clarithromycin; AZT = azithromycin; LNC = lincomycin; CND = clindamycin; SPT = spectinomycin; BRT = bortezomib; ECU = ecumicin50.