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VesB is surface-associated in WT but not in the T2SS mutant.

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posted on 2018-10-23, 17:38 authored by Shilpa Gadwal, Tanya L. Johnson, Henriette Remmer, Maria Sandkvist

The ΔvesB and ΔvesBΔeps strains containing empty vector (p) or pVesB were grown for four hours in M9 media supplemented with glucose and casamino acids in the presence of 100 μg/mL carbenicillin and 50 μM IPTG. A. Supernatants (S) and whole cells (C) were isolated and analyzed for protease activity using the fluorogenic peptide Boc-Gln-Ala-Arg-AMC. Samples from three independent experiments were each analyzed in technical triplicates. Bars show mean ± S.E. B. Samples from A were analyzed by SDS-PAGE and immunoblotting with VesB antibodies. Representative blot of one of three separate experiments. C. Cells were washed and incubated with VesB antibodies followed by Alexa Fluor 488 antibodies. The cells were subjected to quantitative fluorimetry and the fluorescence units were normalized by OD600 of the original cultures and fold changes over cells with empty vector were calculated. The experiments were done in triplicates and standard error bars are shown. P values were generated by comparing ΔvesB and ΔvesBΔeps strains containing pVesB or pVesB-S221A (**p<0.01). D. Cells that were labeled with VesB antibodies followed by Alexa Fluor 488 antibodies were visualized by differential interference contrast and fluorescence microscopy. Representative images of three independent experiments are shown.

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