Fig 3. SufT interacts with 4Fe-4S cluster containing proteins- SufR and Aconitase.
(A) M-PFC based demonstration of specific protein-protein interaction of SufT with Acn and SufR (4Fe-4S proteins), but not with SufTC62A. Construct 1) SufT[F3]/Acn[F1,2], 2) SufTC62A[F3]/Acn[F1,2], 3) SufT[F3]/SufR[F1,2], 4) SufTC62A[F3]/SufR[F1,2], 5) CFT10[F1,2]/ESAT6[F3] (positive control), and 6) empty vectors expressing [F3] and [F1,2] only (negative controls) were co-transformed in Msm and growth was scored on TRIM containing 7H11 plates. (B) The Mtb strain expressing pRH2502 (ATc inducible dCas9- vector control) and SufT specific gRNA in pRH2521 vectors (SufT-KD) was exposed to ATc (200 ng/mL) for 24 h and 48 h and the expression of sufT was monitored by RT-qPCR. (C) The SufT-KD strain was treated with 200 ng/mL of ATc and intracellular levels of SufT was determined at the indicated time points by immunoblotting using anti-SufT. Image density was analyzed with ImageJ software. Aconitase (Acn; 102 KDa) is used as loading control. (D) (top panel) Western blot of SufT and Acn from SufT-KD and vector control (with (+) and without (-) ATc) lysate. (lower panel) Immuno-precipitation of Acn from SufT KD (+ and − ATc) lysate with Anti-SufT IgG as well as rabbit Isotype IgG (Iso) as negative control. All M-PFC and western blot experiments were performed in triplicate, one representative image is shown here.