Fig. 3.

The DivIVA kinase is AfsK. The phosphorylation state of DivIVA, before and after the inhibition of cell wall synthesis, was analyzed in (A) single, double, and triple mutants corresponding to three PASTA domain-containing STKs of S. coelicolor, SCO2110, SCO3821, and SCO3848, (B) 13 other constructed STK mutants, and (C) a constructed afsK mutant. Growing cultures of WT S. coelicolor and of the STK mutants, each expressing FLAG-divIVA, were incubated with 50 μg/mL bacitracin for 30 min before harvest, preparation of cell extracts, and immunoprecipitation of FLAG-DivIVA/DivIVA. (D) Complementation of the afsK-null mutant restores DivIVA phosphorylation. afsK was cloned into the integrative vector pMS82 to create pKF256, which was introduced into the afsK null mutant and into WT S. coelicolor. The phosphorylation state of DivIVA, before and after the inhibition of cell wall synthesis, was analyzed in each strain by Western blot analysis of crude cell extracts. Closed arrowheads indicate phosphorylated DivIVA and open arrowheads indicate nonphosphorylated DivIVA.