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Effects of low PBP2b levels on cell morphology and peptidoglycan composition in Streptococcus pneumoniae R6
Kari Helene Berg1, Gro Anita Stamsås, Daniel Straume
1Department of Chemistry, Biotechnology and Food Science, Norwegian University of Life Sciences, Ås, Norway.
Abstract:
Streptococcus pneumoniae produces two class B penicillin-binding proteins, PBP2x and PBP2b, both of which are essential. It is generally assumed that PBP2x is specifically involved in septum formation, while PBP2b is dedicated to peripheral cell wall synthesis. However, little experimental evidence exists to substantiate this belief. In the present study, we obtained evidence that strongly supports the view that PBP2b is essential for peripheral peptidoglycan synthesis. Depletion of PBP2b expression gave rise to long chains of cells in which individual cells were compressed in the direction of the long axis and looked lentil shaped. This morphological change is consistent with a role for pneumococcal PBP2b in the synthesis of the lateral cell wall. Depletion of PBP2x, on the other hand, resulted in lemon-shaped and some elongated cells with a thickened midcell region. Low PBP2b levels gave rise to changes in the peptidoglycan layer that made pneumococci sensitive to exogenously added LytA during logarithmic growth and refractory to chain dispersion upon addition of LytB. Interestingly, analysis of the cell wall composition of PBP2b-depleted pneumococci revealed that they had a larger proportion of branched stem peptides in their peptidoglycan than the corresponding undepleted cells. Furthermore, MurM-deficient mutants, i.e., mutants lacking the ability to synthesize branched muropeptides, were found to require much higher levels of PBP2b to sustain growth than those required by MurM-proficient strains. These findings might help to explain why increased incorporation of branched muropeptides is required for high-level beta-lactam resistance in S. pneumoniae.
Insights
Penicillin-binding protein 2b (PBP2b) is crucial for Streptococcus pneumoniae peripheral cell wall synthesis. Its depletion causes distinct cell shape changes and alters peptidoglycan composition, impacting bacterial growth and beta-lactam resistance.
Area of Science:
- Microbiology
- Cell Biology
- Biochemistry
Background:
- Streptococcus pneumoniae utilizes two essential class B penicillin-binding proteins (PBPs): PBP2x and PBP2b.
- PBP2x is typically associated with septum formation, while PBP2b is presumed to be involved in peripheral cell wall synthesis.
- Experimental evidence for PBP2b's specific role in peripheral synthesis is limited.
Purpose of the Study:
- To investigate the precise function of PBP2b in Streptococcus pneumoniae cell wall synthesis.
- To elucidate the morphological and compositional consequences of PBP2b depletion.
- To explore the relationship between PBP2b, peptidoglycan structure, and beta-lactam resistance.
Main Methods:
- Conditional depletion of PBP2b expression in Streptococcus pneumoniae.
- Microscopic analysis of cell morphology following PBP2b depletion.
- Analysis of peptidoglycan composition, including stem peptide branching.
- Assessment of bacterial sensitivity to autolysins (LytA, LytB).
- Growth studies of MurM-deficient mutants with varying PBP2b levels.
Main Results:
- PBP2b depletion resulted in lentil-shaped cells, indicating a role in lateral cell wall synthesis.
- PBP2x depletion led to lemon-shaped and elongated cells with thickened midcells.
- Low PBP2b levels increased sensitivity to LytA and impaired chain dispersion by LytB.
- PBP2b-depleted cells showed a higher proportion of branched stem peptides in peptidoglycan.
- MurM-deficient mutants required significantly higher PBP2b levels for growth.
Conclusions:
- PBP2b is essential for peripheral peptidoglycan synthesis in Streptococcus pneumoniae.
- PBP2b function influences cell shape, autolysin sensitivity, and peptidoglycan stem peptide composition.
- The findings provide a potential explanation for the role of branched muropeptides in high-level beta-lactam resistance.
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