Fig. 1: Functional characterization of MenT3 and MenT4 toxins belonging to MenAT subfamily of TA systems from M. tuberculosis.

a–f Overexpression of MenT3 and MenT4 inhibits the growth of E. coli and M. tuberculosis. a, b These panels show growth patterns of E. coli Bl-21 (pLysS, λDE3) strains harboring pET28b derivatives expressing either wild type or mutant MenT3 (a) or wild type or mutant MenT4 (b) proteins in the absence or presence of inducer. c, d These panels depict growth patterns of E. coli BL21 (pLysS, λDE3) strains harboring pET-Duet constructs overexpressing MenT3 (c) or MenT4 (d) either alone or along with their cognate or non-cognate antitoxins. The growth of various strains was determined by measuring OD_600nm. The data shown in these panels are representative of two independent experiments. e, f The growth patterns of M. tuberculosis H₃₇Rv harboring pTetR derivatives expressing either MenT3 or MenT4 are shown in these panels. The growth of recombinant strains was determined by measuring either OD_600nm (e) or bacterial counts (f). The data shown in (e) is representative of two independent experiments. The data shown in (f) is mean ± SD of log₁₀ CFU obtained from two independent experiments, each performed with duplicate cultures. p values depicted on the graphs were assessed using one-way ANOVA. g, h Construction of ΔmenT4ΔT3 strain of M. tuberculosis. g Schematic representation of menT3 and menT4 locus in parental, ΔmenT4 and ΔmenT4ΔT3 strain of M. tuberculosis Erdman is shown. The open reading frame of menT4 was replaced with the hygromycin resistance gene in ΔmenT4 strain of M. tuberculosis. In the double mutant strain, ΔmenT4ΔT3, the open reading frame of menT3 and menT4 were replaced with kanamycin and hygromycin resistance gene, respectively. h The replacement of menT3 and menT4 with kanamycin and hygromycin resistance gene, respectively, in their respective single and double mutant strain was confirmed by PCR using gene-specific primers. Source Data are provided as a Source Data file.