Fig 4.

Regulation exerted by the PhoP-PhoQ system in nongrowing dormant intracellular bacteria matches the regulatory pattern observed in extracellular bacteria incubated in acidified growth medium. Western assays showing the relative levels of three distinct 3× FLAG-tagged proteins regulated by the PhoP-PhoQ system (VirK, PagN, and PagP) in extracellular and intracellular bacteria. Induction in intracellular bacteria was monitored by analysis of protein levels in extracellular bacteria used to infect the NRK-49F fibroblasts (inoculum, nonshaking growth conditions) and intracellular bacteria collected at 24 h postinfection. These samples are marked as extra and intra, respectively. The positive regulation of these three proteins by PhoP-PhoQ was tested in low Mg²⁺ concentrations and acid pH using the N and PCN media, respectively (see Materials and Methods). Shown are the levels of VirK, PagN, and PagP detected in extracellular bacteria grown in inducing (either 8 μM Mg²⁺ or pH 5.8) or repressing (either 10 mM Mg²⁺ or pH 7.4) conditions. Note that the response observed in acidified PCN medium matches, to a large extent, that observed in intracellular bacteria. However, the marked increase of PagP levels observed in 8 μM Mg²⁺ is not observed in nonproliferating intracellular bacteria. Loading controls based on DnaK are shown for the pagN::3×FLAG-tagged strains with equivalent results obtained for the other sets of strains shown.