FIG. 5.

Secretion of a truncated recombinant Srr1 protein in NEM316 and isogenic mutant derivatives. (A) The structure of the cell wall-anchored LPXTG protein Srr1 is shown, and the relevant features encoded by the fragment cloned in pAT18P are indicated (Sp, signal peptide; Srr, serine-rich repeat). (B) Western blot analysis of the recombinant Srr1 protein in GBS strains. The 5′ half of the srr1 gene was amplified and cloned in pAT18P and the resulting plasmid introduced in NEM316 and the ΔgtfAB, ΔsecA2, and ΔgtfCDEFGH (ΔgtfC-H) mutant derivatives. NEM316 harboring pAT18P was used as a control strain. Culture supernatants of these strains were concentrated 50-fold and the proteins, separated on 3-to-8%-gradient Tris-acetate Criterion XT SDS-PAGE gels, were silver stained or revealed in-gel by immunoblotting with rabbit anti-Srr1 pAb (α-Srr1) or with the biotinylated lectin sWGA (succinylated wheat germ lectin). Ten μg of protein was loaded in each well.