VesB is not localized to the cell surface when produced without the GlyGly-CTERM domain.

Cultures of indicated strains expressing VesB or C-terminally deleted VesBΔ5 and VesBΔ30, were grown in LB broth with 100 μg/mL carbenicillin and 40 μM IPTG. Samples were collected at time points indicated, separated into culture supernatant and cells and analyzed for VesB activity and localization. The protease activity assays were performed on samples from three independent experiments and each sample was analyzed in technical triplicates. Bars represent mean ± S.E. p values were generated by comparing culture supernatants and cells containing VesB to those having VesBΔ5 and VesBΔ30. These were all statistically significant at the 4–8.5 hour time points in both supernatants and cells, except for VesB and VesBΔ5 at the 7 hour supernatant time point. A. Using the fluorogenic peptide, Boc-Gln-Ala-Arg-AMC, the activity of VesB, VesBΔ5 and VesBΔ30 was measured in culture supernatants. B. The activity of cell-associated VesB, VesBΔ5 and VesBΔ30 was measured in intact cell suspensions as in A. C. The culture supernatants and cells were subjected to SDS-PAGE and immunoblot analysis using VesB antibodies.