Immunochemical analysis of Psp in P. fluorescens SBW25 and derived mutants. (A) Double immunodiffusion of cell supernatants and lysates. The central well in a 1.5% agarose plate contained rabbit anti-DING (Psp from SBW25) antiserum. The peripheral wells contained (clockwise, from the top) PBR826 (Δpsp) supernatant (Δpsp-sup), PBR828 (ΔhxcR) supernatant (ΔhxcR-sup), Psp standard (DING), SBW25 lysate (wt-lysate), PBR826 (Δpsp) lysate (Δpsp-lysate), PBR828 (ΔhxcR) lysate (ΔhxcR-lysate), SBW25 supernatant (wt-sup). (B) Western blotting of supernatants for SBW25 grown in different media. Concentrated cell supernatants were separated on a 12% SDS-PAGE gel, and Psp was detected with anti-human DING antiserum. Lane A, protein ladder; B, PBR826 (Δpsp) in LB broth (negative control); C, SBW25 in PL broth; D, E, SBW25 in PR broth; F, SBW25 in LB broth; G, recombinant Psp (positive control). (C) Western blotting of cell supernatants for SBW25 mutants grown on PL medium. Concentrated cell fractions were separated on a 12% SDS-PAGE gel, and Psp was detected with anti-Psp antiserum. Lane A and D, wild-type; B and E, PBR826 (Δpsp); C and F, PBR828 (ΔhxcR); G, Psp standard.