Fig 4.

Deletion of lcrQ but not yopN can partially rescue the secretion defect of the ΔrfaL mutant. (A) Secretion assays performed with wild-type, ΔrfaL, ΔyopN, and ΔrfaL ΔyopN strains. (B) Secretion assays performed with wild type, ΔrfaL, ΔlcrQ, and ΔrfaL ΔlcrQ strains. Assays were done by growing bacteria in DMEM at 37°C with or without EGTA (−Ca or +Ca, respectively). Following incubation, cultures were centrifuged to separate secreted proteins (S) from cell pellet (P). The proteins in both fractions were trichloroacetic acid precipitated, and immunoblotting was performed using the indicated antibodies. (C) Needle cross-linking by the wild type or ΔlcrQ, ΔrfaL, ΔrfaL ΔlcrQ, ΔyscF, or ΔrfaL ΔyscF mutants. Bacteria were incubated in TMH with or without CaCl₂ (+Ca or −Ca, respectively) at 37°C. Cultures were centrifuged, and the cell pellets were gently resuspended in HEPES with or without CaCl₂ and then incubated with BS³ (+) or with water (−). Proteins were trichloroacetic acid precipitated and immunoblotted using anti-YscF and anti-RpoA antibodies. mon., monomer.