Figure 5. AscarD negatively regulates carD.

(A) A schematic diagram of PUCP and PUCP_mut plasmids construction (see a detailed description in Experimental section). (B) β-Galactosidase activities of mc²155 strains transformed with PUCP or PUCP_mut plasmid. Individual data for the three biological replicates are shown in the corresponding columns. (C) CarD protein levels in different strains. Mycobacterial cells were harvested at the mid-stationary phase (MSP). The upper part shows the Western blot with CarD levels, and the histogram below it shows the quantitative statistics of Western blot results. Statistical test was done using the Student's t-test, with ** indicating p-value <0.01, *** indicating p-value <0.001, and n.s. indicating p-value >0.05. (D) The tolerance of ascarD_OE and control strains to oxidative stress, DNA damage, and antibiotic stimulation, respectively. Serially diluted bacterial suspensions were separately spotted onto normal 7H10 plate (CK) or plates containing 0.3 mM H₂O₂, 0.05% methanesulfonate (MMS), 0.2 μg/ml of ciprofloxacin (CIP), 0.1 μg/ml of streptomycin (Str), or 5 μg/ml of rifamycin (Rif), respectively.

Figure 5—figure supplement 1. The expression levels of carD and ascarD in different strains.

(A) Schematic diagram for the location of small guide RNA (sgRNA) targeting ascarD. (B) Transcript levels of ascarD, carD, and ispD in ascarD_KD and control strains. The mc²155 strain transformed with pRH2521 empty vector was used as the control. (C) Transcript levels of ascarD and carD in ascarD_OE and control strains. The mc²155 strain transformed with pMV261 empty vector was used as the control. sigA was used as the internal reference gene of qRT-PCR. Error bars indicate the standard deviation of three biological replicates. Statistical testing was done using the Student’s t-test, with *** indicating p value <0.001, n.s. indicating p value >0.05.