Polar Localization of PhoN2, a Periplasmic Virulence-Associated Factor of Shigella flexneri, Is Required for Proper IcsA Exposition at the Old Bacterial Pole
Figure 1
PhoN2 is a periplasmic protein.
Exponentially-growing bacteria were harvested by centrifugation and supernatant, periplasmic and membrane fractions were prepared as described in Materials and Methods. Fractions were solubilised in Laemmli buffer and analyzed by Western blot analysis using polyclonal anti-PhoN2 [21], monoclonal anti-HA, polyclonal anti-SurA and anti-OmpA antibodies, as indicated. Panel A, bacterial fractions of wild-type S. flexneri strain M90T and of its mutant derivative strain HNDHA10 carrying the phoN2::HA fusion under the control of its natural promoter (Table S1). Panel B, bacterial fractions of the ΔphoN2 mutant derivative strain HND115 complemented with plasmids pHND10, 19, 11, 23, 14, 15, 16 and 21 encoding the different HA-fused recombinant proteins under the control of an L-arabinose inducible promoter (Table S1). In these cases, 0.016% of L-arabinose was used to induce phoN2::HA expression. No specific signals of PhoN2-HA were evidenced when bacteria were grown in the absence of the inducer. Experiments were repeated at least three times and images are representative.