Figure 5: whiB7 coordinates a feedback loop with aspC.

(A) ChIP RT-qPCR of the aspC promoter in the indicated M. smegmatis WhiB7 N-terminal 3X-FLAG strains. Fold-enrichment of aspC promoter qPCR signal relative to the control trpC promoter (WhiB7-independent) is indicated. whiB7 was induced either by CRISPRi knockdown of ettA or aspC (18 hours +ATc), or treatment with clarithromycin for 12 hours. Statistical significance with respect to the non-targeting CRISPRi strain or DMSO control was calculated using a Student’s t-test, **P< 0.01, ***P<0.001 n.s. = non-significant.

(B) Relative mRNA levels of the indicated genes in the indicated M. smegmatis CRISPRi strains 15 hours after addition of ATc (mean ± s.e.m., n = 3 biological replicates). mRNA fold-change for the indicated gene at the bottom of each pair of bar graphs was calculated relative to sigA and normalized to the respective non-targeting CRISPRi strain for WT M. smegmatis or the $\Delta$whiB7 strain. Grey = WT, blue = $\Delta$whiB7. Statistical significance between the WT and $\Delta$whiB7 strain was calculated using a Student’s t-test, **P< 0.01, ***P<0.001.

(C) Relative mRNA levels of the indicated genes in the indicated M. tuberculosis CRISPRi strains 5 days after addition of ATc (mean ± s.e.m., n = 3 biological replicates). ettA dual CRISPRi knockdown strains are shown for whiB7 and the downstream whiB7 regulon gene, tap, which serves as a negative control. mRNA fold-change for the gene indicated on the y-axis was calculated relative to sigA and normalized to the respective non-targeting CRISPRi dual non-targeting CRISPRi strain. Statistical significance for each strain was calculated with respect to the dual non-targeting (NT) strain for each of the indicated genes using a Student’s t-test, *P<0.05, **P< 0.01, ***P<0.001, ****P<0.0001.

(D) Relative mRNA levels of the indicated genes in ettA knockdown CRISPRi strains (M. smegmatis) 18 hours after addition of ATc (mean ± s.e.m., n = 3 biological replicates). mRNA fold-change for the gene indicated on the y-axis was calculated relative to sigA and normalized to the respective non-targeting CRISPRi strain for WT M. smegmatis or the $\Delta$whiB7 strain. Grey = WT, blue = $\Delta$whiB7. Statistical significance between the WT and $\Delta$whiB7 strain was calculated using a Student’s t-test, **P< 0.01.

(E,F) Proposed model (E) and mechanism (F) for the whiB7-aspC feedback loop.