Fig 2. Kinetics of in vivo phosphorylation of wild type and Δ127–129 BvgA.
A. B. pertussis strain QC3216 harboring plasmids conferring expression of wild-type BvgA (pSS4893, lanes 3–8) and BvgAΔ127–129 (pQC1894, lane 9–14), respectively, were grown in PLB liquid media, induced with 1 mM IPTG and sampled at various time points post induction. The collected samples were analyzed by Phos-tag gel electrophoresis, followed by Western blot with anti-BvgA detection, as previously described [9]. Control lanes contained 1 pmol of purified BvgA incubated in the presence (+, lane 1) or absence (-, lane 2) of acetyl phosphate as described previously [9]. B. The intensities of BvgA (black bar) and BvgA~P (grey bar) for lanes 5–8 and 11–14 were quantified and reported as integrated density using ImageJ software. C. The quantitative intensities derived from four IPTG-induction time points (30 min, 60 min, 180 min and 360 min) in panel B were used to calculate the ratios of BvgA~P to BvgA for the wild type and the mutant BvgA, respectively, and to obtain the means, standard deviations, as indicated by error bars, and statistical analysis by one-way ANOVA. Outcome of the latter analysis is presented using the symbol: ns, P > 0.05.