Cleavage of VesB by rhombosortase.
A. The indicated mutant strains with pVesB-S221A were grown for four hours in M9 media supplemented with casamino acids, glucose, 100 μg/mL carbenicillin and 50 μM IPTG. Culture supernatant (S) and cells (C) were isolated, matched by equivalent OD600 units (except for the ΔvesABC culture supernatant and the cell samples of the ΔvesABC, ΔglpGΔeps, and ΔvesBΔeps strains which were diluted 1:10) and analyzed by SDS-PAGE and western blotting with VesB antibodies. Representative blot is shown. B. WT and the indicated mutants containing empty vector (p), pRssP, pRssP-S102A or pRssP-H160A were grown for 4 hours in LB broth with 100 μg/mL carbenicillin and 10 μM IPTG. Culture supernatants and whole cells were isolated and analyzed for protease activity using the fluorogenic peptide Boc-Gln-Ala-Arg-AMC. The assays were performed on samples from three separate experiments in technical triplicates. The means with corresponding standard errors are shown. The difference in activity of VesB in the WT strain was statistically significant (p<0.01) when compared to the activity of VesB in the other samples except for ΔglpGp and rssp::kan pRssP supernatants and cells (NS).
