<i>In vitro</i> phosphorylation of ParB and mutant derivatives.

<p><b>(A)</b><i>in vitro</i> phosphorylation of <i>M</i>. <i>tb</i> ParB by PknB. The soluble domains of seven recombinant <i>M</i>. <i>tb</i> STPKs (PknA to PknL) were expressed and purified as GST-tagged fusions and incubated with purified His-tagged ParB and [γ-³³P]ATP. The amount of the STPKs used varied from 0,3 to 2 μg to obtain the optimal autophosphorylation activity for each kinase. Samples were separated by SDS-PAGE, stained with Coomassie Blue (upper panel), and visualized by autoradiography after overnight exposure to a film (lower panel). Upper bands reflect the phosphorylation signal of ParB, and the lower bands correspond to the autophosphorylation activity of each kinase. <i>M</i>, molecular mass markers. <b>(B)</b><i>in vitro</i> phosphorylation of the ParB_Ala mutant. Purified ParB and phosphoablative ParB (ParB_Ala) were incubated with PknB and [γ³³-P]ATP. Samples were separated by SDS-PAGE, stained with Coomassie Blue (upper panel), and visualized by autoradiography (lower panel) after overnight exposure to a film.