In vivo cross-linking experiments.

The ⁴³PPPP⁴⁶ motif of PhoN2 is not required for the PhoN2-OmpA interaction. Cross-linking of HND115 (pHND10) was achieved by treating bacteria with formaldehyde to a final concentration of 1%, as described in Materials and Methods. Samples were suspended in Laemmli buffer and either heated at 37°C for 10 min to maintain cross-links or at 95°C for 20 min to break cross-links. Equal amounts of proteins were analyzed by Western blot. A protein molecular weight marker (Pierce) was used to determine the molecular weight of proteins. Immunoblotting was carried out with monoclonal anti-HA (Panels A and B) or polyclonal anti-OmpA antibodies (Panels C and D). Expression of PhoN2-HA was achieved by growing bacteria in the presence of 0.016% of L-arabinose. Panels A and C, bacteria not induced with L-arabinose; Panels B and D, L-arabinose induced bacteria. OmpA (U), unfolded OmpA; OmpA (F), folded OmpA [48]. Experiments were repeated at least three times and typical results are shown.