Fig. 6

CapM and CapB1 are protein targets of the ESTK PknB. a CapM is phosphorylated by PknB. Putative target proteins (3 µg) were incubated with PknB in the presence of [³³P]ATP and reactions were analyzed by SDS-PAGE and phosphoimaging. b Impact of PknB kinase activity on CapM GT activity. CapM was incubated in presence of either active PknB (light grey) or heat-inactivated PknB (dark grey) with UDP-d-[¹⁴C]FucNAc and C₅₅P. After extraction with BuOH/PyrAc, reactions were analyzed by TLC and phosphoimaging. Experiments were performed in triplicate. The error bars represent the ± SD from three biological replicates. Statistical significance was analyzed by an unpaired t-test (**p < 0.05). c CapB1 is phosphorylated by PknB. Putative target proteins (3 µg) were incubated with PknB in the presence of [³³P]ATP, and reactions were analyzed by SDS-PAGE and phosphoimaging. d Quantification of CapB autophosphorylation in the absence and presence of PknB. Experiments were performed in triplicate. The error bars represent the ± SD from three biological replicates. Statistical significance was analyzed by an unpaired t-test (***p < 0.005). e ELISA-based quantification of CP production in a ∆pknB deletion strain, the corresponding parental strain S. aureus Newman (WT) and complementation of the ∆pknB mutant in trans. Experiments were performed in triplicate. The error bars represent the ± standard error of the mean (SEM) from three biological replicates. Statistical significance was analyzed by an unpaired t-test (***p < 0.005)