Crosstalk between the serine/threonine kinase StkP and the response regulator ComE controls the stress response and intracellular survival of Streptococcus pneumoniae
(A) ComE is phosphorylated at a threonine residue by StkP. Top: nitrocellulose membrane stained with Ponceau S as a loading control. Bottom: Immunodetection of phosphorylated proteins. Phosphorylation reactions were carried out with purified GST-StkP and substrate proteins (0.5 μg each) mixed in kinase buffer and incubated at 37°C for 1 hour. Phosphorylated proteins were detected with an anti-phosphothreonine polyclonal antibody. Lane 1: Hisx6-ComE. Lane 2: Hisx6-ComE + GST-StkP. Lane 3: Hisx6-GFP. Lane 4: Hisx6-GFP + GST-StkP. Lane 5: LytA(N)-Hisx6. Lane 6: LytA(N)-Hisx6 + GST-StkP. Lane 7: Hisx6-DivIVA. Lane 8: Hisx6-DivIVA + GST-StkP. (B) ComE phosphorylation assays with different StkP:ComE molar ratios. GST-StkP and Hisx6-ComE were mixed at different molar ratios in kinase buffer and incubated at 37°C for 1 hour. Detection of phosphorylated proteins was performed as described above. (C) In vivo StkP-dependent and acid-induced ComE phosphorylation. C-terminal His-tagged ComE was purified from wt and ΔstkP strains grown in ABM (pH 7.8), and exposed to acidic stress in medium MD5, pH 6.0. Protein samples were separated by SDS-PAGE and phosphorylated or total ComE-His was detected with Pro-Q Diamond and SYPRO Ruby staining, respectively.