Fig. 5

Characterization of full-length Mtb-SecB^TA/HigA1 chaperone/antitoxin complex. a SEC-MALS analysis. Continuous and dashed lines represent the variation of refractive index against elution time for Mtb-SecB^TA (red), Mtb-SecB^TA/HigA1 (blue), Mtb-HigA1^ΔC42 (violet), and Mtb-SecB^TA/HigA1/dsDNA (black) for protein and salt contributions, respectively. The experimentally measured molar mass distribution (same color code) and deduced mean molar mass (in g mol⁻¹) are indicated for each elution peak. Calculated molecular masses of proteins and protein complexes used for experiments (in daltons): Mtb-SecB^TA tetramer, 81,552; Mtb-SecB^TA/HigA1 hetero-hexamer, 115,068; Mtb-HigA1^ΔC42 dimer, 27,994; Mtb-SecB^TA/HigA1/dsDNA, 138,420. b ITC profile of 38-bp dsDNA titrated into Mtb-SecB^TA/HigA1 (upper and lower parts represent raw and integrated binding heats, respectively). c Native MS-derived relative abundance of Mtb-SecB^TA/HigA1 (blue bars) and Mtb-SecB^TA (red bars) obtained upon titration of Mtb-SecB^TA/HigA1 with 0, 1, 2, 4, 16 molar equivalents of ChAD. d Overexpression of luciferase-ChAD chimera activates Mtb-HigB1. E. coli WPtet57 strain expressing chromosomally encoded Mtb-SecB^TA under the control of an anhydrotetracycline inducible promoter was co-transformed with pC6-Mtb-HigBA1 and the pSE380-based vector (−), luciferase (Luc), or the Luc-Mtb-ChAD and Luc-Mtb-ChAD^Y114A chimeras. Transformants were grown to mid-log phase, serially diluted and spotted on LB ampicillin chloramphenicol agar plates without or with IPTG (to express Luc and Luc chimeras), arabinose (to express Mtb-HigBA1), and anhydrotetracycline (to express Mtb-SecB^TA) inducers as indicated. Plates were incubated overnight at 37 °C