Table 1.
Relative competitive fitness of Streptococcus pneumoniae pbp mutants.
Figure 1.
Correlation plot between the fitness value of pbp mutants, obtained by using pbp genes from Cba-19 and Cba-28 clinical strains, and the penicillin MIC increase found (µg/ml).
A) Correlation for Cp1015 pbp19 mutants, Cp1015 pbp2b19 (diamonds), Cp1015 pbp2b19 pbp2x19 (triangles), and Cp1015 pbp2b19 pbp1a19 pbp2x19 (circles). B) Correlation for Cp1015 pbp28 mutants, Cp1015 pbp2b28 (diamonds), Cp1015 pbp2b28pbp2x28 (triangles), and Cp1015 pbp2b28 pbp2x28 pbp1a28 (circles).
Figure 2.
Effect of pbp mutations on cell morphology.
Cells were prepared for microscopy and stained with DAPI as described in Materials and Methods (bar scale, 5 µm). (A) Merged phase-contrast and DAPI fluorescence image of the pbp mutants showing a bacillar morphology, contrasting with the typical coccoid shape of the CP1015 (wt) strain. (B) Cell shape modifications of the cell populations detected by flow cytometry analysis. Exponentially growing bacterial cells were injected into a flow cytometer and the results were analyzed with the WinMDI software. To compare populations, the data for each strain were plotted on a two-dimensional graph (x-axis, forward scatter; y-axis, side scatter).
Figure 3.
Electron microscopy analysis of cell morphology of pbp mutants.
Microphotographs of the wild-type strain (Cp1015) revealed the typical morphology of S. pneumoniae, with a correct septum placement, division and symmetry of daughter cells. In the pbp2b28 mutant, two subpopulations were identified, namely rod-like and coccoid cells. The rod-like shaped cells showed multiple septa with incorrect formation and placement. The coccoid-shape cells exhibited multiple septa and intracellular accumulation of the cell wall, with the atypical septum localization resulting in irregular cell divisions and asymmetrical daughter cells (see arrows). The pbp2b28 pbp2x28 mutant partially restored the coccoid morphology, but conserved some cell alterations similar to those of the single pbp2b28 mutant. The triple pbp mutant showed a similar morphology to wt cells. Additional microphotographs are shown in Figs. S12, S13, S14 and S15 in Supporting Information S1 (bar scale, 1.6 µm).
Figure 4.
Septal localization in pbp mutants.
Exponentially grown cells were stained with DAPI and fluorescent vancomycin (Fl-Van, which labels nascent peptidoglycan synthesis and strongly marks septal localization) and analysed by using an epifluorescence microscope (see Material and Methods). The pbp2b showed a septal accumulation in rod-like shaped cells, whereas the double (pbp2b pbp2x or pbp2b pbp1a) and the triple pbp mutants restored their septal localization. Representative images are shown from experiments that were repeated independently three times (bar scale, 1 µm).
Figure 5.
Stability in vivo of wild-type and mutant PBPs.
Strains Cp1015 (Panel A), Cp1015 pbp2b28 (Panel B) and Cp1015 pbp2b28pbp2x28 pbp1a28 (Panel C) expressing HA-tagged PBPs were grown in Todd Hewitt broth with 0.5% Yeast Extract to an OD600 nm of 0.2 prior to the addition of kanamycin (500 µg/ml) to inhibit protein synthesis. At the time intervals indicated, aliquots were withdrawn and analyzed by SDS-PAGE followed by immunoblotting with an anti-HA monoclonal antibody (see Material and Methods).
Figure 6.
Protein localization in wild-type and pbp mutants by fluorescence microscopy.
A) Localization of FtsZ. Microphotographs indicate the most common patterns of FtsZ localization through the cell cycle for wt and pbp mutants. Polyclonal antibody against FtsZ was used for its detection by immunofluorescence microscopy. In the Cp1015 (wt) strain, FtsZ is localized at the equatorial ring and septum, In Cp 1015 pbp2b28, the patterns suggest FtsZ delocalization and cell cycle disruption. The triple pbp mutant showed a wild-type FtsZ placement. B) Localization of PBP2b28-GFP. Microphotographs indicate the most common patterns found for PBP2b localization through the cell cycle in the wt and pbp2b28 mutant. Representative images are shown from experiments that were repeated independently three times (bar scale, 1 µm).
Figure 7.
The percentage of interaction is defined as the ratio between the number of cells co-transformed able to grow in 3-AT selective medium and the total number of cells obtained in medium without 3-AT. The plasmid pair pBT-LGF2/pTRG-Gal11 (BacterioMatch II, Stratagene) was used as a positive control. A) Interaction of PBP2bwt-FtsZ and PBP2b28-FtsZ B) As negative controls, the following plasmid pairs were used: pBT/pTRG carrying codifying sequences of non-interacting proteins, C (-); pBT-empty/pTRG-recombinant, to evaluate the two-hybrid system auto-activation for the target proteins (-)/Autoactivation Target; pBT-PBP2b/pTRG-empty, as non-specific transcription activation control mediated by PBP2bwt or PBP2b28, C (-)/Autoactivation Bait.