FIG 3.

Quantitative enzymatic assay of secreted protease, PLC, endochitinase, and chitobiosidase activities. Bacterial strains were grown in LB broth for 22 h at 37°C with shaking (250 rpm), and the supernatants were filtered and assayed for enzymatic activity as described in Materials and Methods. (A) Protease assay. Values are presented as ng of protease activity per μl of culture supernatant. (B) PLC assay. p-Nitrophenylphosphorylcholine hydrolysis by PLC was monitored spectrophotometrically by the measurement of p-nitrophenol at 410 nm. (C) Endochitinase assay. One unit of activity releases 1 μmol of p-nitrophenol from 4-nitrophenyl β-d-N,N′,N″-triacetylchitotriose per min at pH 4.8 at 37°C. (D) Chitobiosidase activity. One unit of activity releases 1 μmol of p-nitrophenol from 4-nitrophenyl N,N′-diacetyl-β-d-chitobioside per min at pH 4.8 at 37°C. All numerical values are the means of three separate experiments performed in triplicate plus standard deviations (error bars). In all assays, MSHR668 was significantly different than 668 ΔgspD and 668 ΔgspE (P < 0.0001) and 668 ΔgspD/gspD was significantly different than 668 ΔgspD and 668 ΔgspE (P < 0.005).