The biogenesis of invasin is BamA- and SurA- dependent.
(A) Levels of invasin, BamA and DegP in whole-cell lysates. Bacteria were grown for 2 h under BamA depletion (–) or rescue (+) conditions, and then expression of invasin was induced. Whole-cell lysates were prepared 0, 60 and 120 min after the induction of invasin expression and analyzed by SDS-PAGE and western blotting using antibodies directed against invasin, BamA and DegP. (B) Effect of periplasmic chaperones on invasin levels in the outer membrane. Invasin protein levels in OM fractions of skp, degP and surA knockout strains and the corresponding wild-type E. coli were assessed 2 h after induction of invasin expression. Invasin, GyrA (loading control for whole-cell lysates), Skp, DegP and SurA protein were detected by Western blot analysis. The presence of invasin protein was tested in whole-cell lysates as well as in membrane fractions to demonstrate how the absence of specific chaperones influences membrane insertion of invasin. (C) Complementation of SurA. E. coli BW25113 ΔsurA expressing invasin (+ AHTC) was grown in the presence (+) or absence (–) of arabinose for induction of surA transcription from pBAD28_surA. Whole-cell lysates were analyzed in SDS gels 2 h after induction of invasin expression and Western blots were immunodecorated with anti-SurA and anti-InvA as well as anti-GroEL as loading control.