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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 3: Modulation of the uORF coding sequence alters the response to amino acid deprivation.

Figure 3:

(A-B) Dose response curves (mean ± s.e.m., n = 3 replicates) of the PwhiB7:mScarlet reporter strain for clarithromycin (translation inhibitor) and 6-FABA (tryptophan biosynthesis inhibitor), grown in the presence or absence of 1 mM tryptophan. (A) Top row = M. smegmatis; (B) bottom row = M. tuberculosis. Drug dose-response curves (percent growth) are shown in black and mScarlet fluorescence (RFU) are shown in red. The blue shaded region highlights the differential whiB7 induction of M. smegmatis and M. tuberculosis in response to tryptophan limitation by 6-FABA.

(C) Normalized fluorescence of the indicated PwhiB7:mScarlet M. smegmatis and M. tuberculosis CRISPRi strains 24 hours and 8 days after addition of ATc, respectively (mean ± s.e.m., n = 3 replicates). Statistical significance with respect to each non-targeting CRISPRi strain was calculated using a Student’s t-test; **P< 0.01, ****P< 0.0001, n.s. = non-significant.

(D) Amino acid composition (x-axis) of annotated or predicted whiB7 uORF sequences from the listed bacteria. A black X denotes lack of that amino acid within the indicated whiB7 uORF. M. tb = M. tuberculosis; M. ks = M. kansasii; M. av = M. avium; M. ul = M. ulcerans; M. mr = M. marinum; M. ft = M.fortuitum; M. ab = M. abscessus; M. sm = M. smegmatis; S. co = S. coelicolor; R. jo = R. jostii.

(E) Genetic architecture of the engineered M. smegmatis whiB7 uORF variants, transformed into the ΔwhiB7 M. smegmatis strain.

(F) whiB7 ORF mRNA fold-change of the indicated M. smegmatis CRISPRi strains 18 hours after addition of ATc (mean ± s.e.m., n = 3 biological replicates). The M. smegmatis CRISPRi strains harbor the whiB7 uORF variants as depicted in panel (E). whiB7 ORF mRNA fold change is relative to sigA and normalized to the respective non-targeting CRISPRi strain for each uORF variant. Statistical significance with respect to the WT whiB7 uORF strain was calculated for each knockdown mutant using a Student’s t-test; *P< 0.05, **P< 0.01, n.s. = non-significant. WT = wild-type.