FIG 4.

Recruitment of PhoP and DosR within hypoxia-inducible promoters. (A) In vivo experiments compared the recruitment of DosR in WT and ΔphoP strains grown under hypoxia. ChIP-qPCR experiments utilized anti-FLAG antibodies (Thermo Scientific) to determine the fold enrichments with respect to mock IP (without adding antibody) sample, as described in Materials and Methods, and the inset compares DosR expression in 10-μg crude cell lysates; RpoB was used as the loading control. **, P < 0.01; *, P < 0.05. (B) Crude cell lysates of ΔphoP strains expressing His₆-tagged PhoP (p19kpro-phoP) (Table S3) were incubated with Ni-NTA and eluted with 200 mM imidazole. Lane 1, input sample; lane 2, elution from the crude lysate of cells lacking phoP expression; lane 3, detection of DosR coelution with PhoP. The upper and lower panels identify DosR and PhoP by using anti-DosR and anti-His antibodies, respectively, and Western blotting as described in Materials and Methods.