Fig. 1
PhpP regulates phosphorylation of StkP and its substrates. a Kinetics of transfer of phpP mutation. WT strain was transformed by Sp100 genomic DNA carrying Janus cassette inserted into phpP gene (phpP::kan rpsL). Number of kanamycin resistant transformants was plotted as a function of genomic DNA concentration in logarithmic scale. Transfer of tested marker follows linear kinetics with slope of line equal to 1 (y = ax + b, a = slope). b Phosphorylation profile of ΔphpP mutant. Membrane fraction from S. pneumoniae WT (Sp1), ΔphpP (Sp113) and ΔstkP (Sp10) was isolated and 30 μg of proteins was subjected to SDS-PAGE and immunoblotted with anti-phospho-threonine antibody (α-pThr) to detect phosphorylated proteins. The level of PhpP and StkP proteins was detected with anti-PhpP (α-PhpP) and anti-StkP antibody (α-StkP). Immunodetection of membrane protein LocZ was used as loading control. Arrows indicate position of StkP and its known (LocZ, DivIVA) and unknown (P15, P28, P35, P40, P55) substrates. Relative phosphorylation values represent mean ± SD. c Complementation of phpP mutation. Comparison of phosphoprotein pattern of wild type strain WT (Sp1), reverted wild type strain WTR (Sp222), ΔphpP strain (Sp113) and complementation strain ΔphpP PZn-phpP (Sp120) cultivated in C + Y medium in the presence or absence of inducer (ZnSO4). Total protein lysates (30 μg) were subjected to SDS-PAGE and immunoblotted with α-pThr antibody to detect phosphorylated proteins. The total amount of PhpP was detected using α-PhpP antibody. Immunodetection of α-subunit of RNA polymerase (α-RpoA) was used as loading control of total cell lysate. Arrows indicate position of StkP substrates. d PhpP dephosphorylates DivIVA. e PhpP dephosphorylates LocZ. Purified DivIVA-Flag and Flag-LocZ were incubated with His-PhpP in vitro and reaction was stopped at indicated times. Samples were subjected to SDS-PAGE and immunoblotted with α-pThr antibody to visualize dephosphorylation in time. PhpP, LocZ and DivIVA were detected with specific antibodies as described above. To exclude the spontaneous decay, phosphorylated form of both proteins, DivIVA-Flag and Flag-LocZ, was incubated for 30 and 60 min, respectively, in phosphatase reaction buffer without addition of His-PhpP