FIG. 3.

Effects of linker insertions within the propeptide domain of lasA. (A) LasA proteolytic activity in 18-h culture supernatants determined as the rate of staphylolysis (OD₅₉₅/min/mg protein). The strains examined were as follows: WT, wild-type PAO1 (LasA⁺ strain); +, LasA⁺ control PDO801(pKKG08); −, LasA⁻ control PDO801(pUCP18). Numbers indicate the residues preceding a linker insertion in strain PDO801(pKKG08). (B) Western blot analysis of mature LasA (20 kDa) in 18-h culture supernatants. Note that propeptide linker mutants 87, 92, and 95 were especially reduced in both activity and protein production at 18 h. (C) Western blot analysis of 0.5-h washed-cell culture supernatants to detect secretion of proLasA (42 kDa), which showed that all propeptide mutant proteins were secreted. Note that propeptide mutant 165 displayed a double band at the position predicted for the 28-kDa intermediate form of LasA. (D) Western blot analysis of intracellular proLasA in propeptide mutants 87, 92, and 95 grown for 18 h. The proLasA was detected but did not appear to be accumulating inside the cell.