Fig 4.

Effects of mutating the catalytic triad upon the export of GspB736flag and native GspB. Western blot analysis of GspB736flag (A) and native full-length GspB (B) export by S. gordonii strains carrying the designated serine-alanine substitutions within Asp2. Residues constituting the catalytic triad of Asp2 are highlighted in red. Culture media (M) and protoplasts (P) were collected from exponentially growing strains, while cell wall (CW) proteins were released by mutanolysin digestion, as described in the Materials and Methods. Proteins were separated by SDS-PAGE (3 to 8%) and analyzed by Western blotting, using anti-Flag antibody to detect GspB736flag and anti-GspB serum to detect native GspB. GspB*, the excessively glycosylated form of GspB due to Asp2 mutation; Asp2◆, an overexposure of native GspB detected with anti-GspB serum.