FIG 6.
In vivo interactions of PhoP and extracytoplasmic sigma factors. To investigate stress-specific interactions of PhoP and sigma factors (SigE and SigH), the phoP::Kanr mutant, expressing a His-tagged PhoP under the control of 19-kDa mycobacterial antigen promoter in p19Kpro (49), was grown (A) under normal conditions and in the presence of 5 mM diamide or (B) at pH 7.0 and acidic pH (pH 4.5), respectively. The cell lysates with comparable amounts of total protein were incubated with Ni-NTA, and bound proteins were eluted. Panels A and B display immunoblots of eluents (lanes 1 and 2) and the corresponding cell lysates (lanes 3 and 4) detecting SigH (top panel), SigE (middle panel), and PhoP (bottom panel) in Western blots using antibodies directed against the corresponding purified proteins. (C) To examine the effect of PhoP on SigH-dependent activation of the trxB1 promoter, M. smegmatis strains harboring transcriptional fusion of the trxB1 regulatory region upstream of lacZ and the indicated constructs expressing SigH or other regulators were grown under normal conditions, and β-galactosidase activity was measured as described in Materials and Methods. In each case, average promoter activity was determined from two biological repeats, each with two technical repeats (*, P < 0.05; **, P < 0.01). Note that in the reporter assays described above, PhoP and SigH were expressed from pUAB400-phoP and pUAB300-sigH, respectively (as described in the legend to Fig. 5).