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Fig. 5 | BMC Microbiology

Fig. 5

From: Characterization of pneumococcal Ser/Thr protein phosphatase phpP mutant and identification of a novel PhpP substrate, putative RNA binding protein Jag

Fig. 5

D192 and D231 are essential for PhpP catalytic activity in vivo. a Phosphorylation pattern after induction of expression of GFP-PhpP-WT (Sp140), GFP-PhpP-D192A (Sp292) and GFP-PhpP-D231A (Sp293) in ΔphpP genetic background. Total cell lysates from cultures grown in C + Y medium in the presence or absence of ZnSO4 were separated by SDS-PAGE and immunoblotted with α-pThr antibody to document protein phosphorylation. α-GFP antibody was used to show expression level of GFP-PhpP and immunodetection of RpoA was used as a loading control. Position of StkP and its substrates is indicated by arrows. b Morphology of strains expressing GFP-PhpP-WT (Sp140), GFP-PhpP-D192A (Sp292) and GFP-PhpP-D231A (Sp293) in ΔphpP genetic background. Pneumococcal strains were cultivated in C + Y medium supplemented with ZnSO4. Phase contrast images show cell morphology in the presence of 0.2 and 0.3 mM ZnSO4 and median cell lengths ± MAD (n = 300) corresponding to each image are shown below. Bar, 5 μm. c Cell length analysis. Cell length parameters were analyzed and plotted in box-and-whiskers graph. Mann-Whitney U test: * cell length in the presence of inducer is significantly different from uninduced conditions (0 mM ZnSO4) P < 0.0001. 300 cells were scored per sample. d Localization of PhpP. Strains expressing GFP-PhpP-WT (Sp140), GFP-PhpP-D192A (Sp292) and GFP-PhpP-D231A (Sp293) were cultivated in C + Y medium supplemented with 0.2 mM ZnSO4. GFP signal and overlay of phase contrast and GFP signal are shown. Enrichment of PhpP at midcell of cells at first stage of cell division (predivisional cells) is indicated by full arrow; cells showing cytoplasmic localization of PhpP are indicated by open arrow. Bar, 1 μm. Predivisional cells (n = 20) showing either midcell enrichment of PhpP (WT and D192A) or cytoplasmic localization (D231A) were selected to quantify distribution of GFP-PhpP along the cell axis. Fluorescence intensity (arbitrary units) versus cell length is plotted in corresponding graphs (error bars show SD)

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