Figure 4. Cross interactions between three pairs of M. tuberculosis relBE-like proteins.

(A) The BacterioMatch II two-hybrid system (Stratagene) was used to detect protein-protein interactions of different pairs of RelBE proteins, as described in the “Materials and Methods”. Upper right panel: an outline of the upper left panel plates (CK+: co-transformant containing pBT-LGF2 and pTRG-Gal11P as a positive control. CK-: co-transformant containing pBT and pTRG as a negative control). Each unit represents the corresponding co-transformant in the plates. Lower left panel: plate plus 10 µg/mL str and 6 mM 3AT. Lower right panel: a summarized network of protein-protein interactions between these RelBE proteins. The black circle represents toxin and the white circle represents antitoxin. (B) SPR assays. The interactions of RelB with RelK and RelG were monitored using surface plasmon resonance on a BIAcore 3000 (GE healthcare). The surface of the chip was activated by saturating the nitrilotriaceticacid sites with running buffer (100 mM Hepes-NaOH, pH 75, 50 µM EDTA, 0.1 mM dithiothreitol, 50 mM NaCl) containing 0.5 mM NiCl_2. In all graphs, time (seconds) is plotted on the X-axis; response units (RU) are plotted on the y axis. Five nmol of histidine-tagged RelB proteins were immobilized on the chip surface. Following a period of stabilization, each GST protein was passed over the chip and then allowed to dissociate for 10 min. Overlay plots depicting the interactions were produced. (C) EMSA was used to detect the cross-regulations on the binding of RelBE with Rv1247c operon promoter. Electrophoresis was performed and gels were exposed to a storage-phosphor screen overnight as described in the “Materials and Methods”. Both DNA substrate and protein/DNA complex were indicated by arrows on the left of the figure. 7.5 µM of each of RelB-like and RelE-like protein alone was used a control for detecting their respective binding with DNA. Left panel shows the cross interaction between RelB (7.5 µM) and various concentrations of RelG (2.5 µM, 5 µM, 6 µM, 7.5 µM, 11.25 µM, and 15 µM); right panel shows the interaction between RelB (7.5 µM) and various concentrations of RelK(2.5 µM, 5 µM, 6 µM, 7.5 µM, 11.25 µM, and 15 µM).