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. The high-molecular-weight penicillin-binding proteins (PBPs) produced by Streptococcus pneumoniae are divided into two different classes. Members of class A (PBP1a, PBP1b, and PBP2a) are bifunctional, having both transpeptidase and transglycosylase activities. Class B PBPs (PBP2x and PBP2b), on the other hand, possess only transpeptidase activity (1, 2). The study of pneumococcal class A PBPs is severely hampered by their functional redundancy. Each of the genes encoding these PBPs can be deleted individually, demonstrating that none of them are essential for growth in the laboratory. It is also possible to isolate pbp1b pbp2a and pbp1a pbp1b double mutants, whereas pbp1a pbp2a double mutants are not viable (3). In contrast, both class B PBPs, PBP2x and PBP2b, are essential in S. pneumoniae (4).The peptidoglycan sacculus is a gigantic macromolecule built as a multilayered network of linear glycan chains interlinked by short peptide bridges. In S. pneumoniae, linear pentapeptides (Lalanyl-␥-D-glutaminyl-L-lysyl-D-alanyl-D-alanine) appended to N-acetylmuramic acid residues on separate glycan strands are cross-linked by formation of a direct bond involving L-lysine on one peptide strand and D-alanine on the other. In addition to these directly cross-linked peptide bridges, pneumococcal peptidoglycan also contains peptide bridges made from branched muropeptides. ...
SummaryPneumococci that are competent for natural genetic transformation express a number of proteins involved in binding, uptake, translocation and recombination of DNA. In addition, they attack and lyse noncompetent sister cells present in the same environment. This phenomenon has been termed fratricide. The key effector of pneumococcal fratricide is CbpD, a secreted protein encompassing an N-terminal CHAP domain, two SH3b domains and a C-terminal choline-binding domain (CBD). CbpD is believed to degrade the cell wall of target cells, but experimental evidence supporting this hypothesis has been lacking. Here, we show that CbpD indeed has muralytic activity, and that this activity requires functional CBD and SH3b domains. To better understand the critical role played by the non-catalytic C-terminal region of CbpD, various translational fusions were constructed between the CBD and SH3b domains and green fluorescent protein (GFP). The results showed that the SH3b domains specifically recognize and bind peptidoglycan, while the CBD domain functions as a localization signal that directs CbpD to the septal region of the pneumococcal cell. Intriguingly, transmission electron microscopy analysis revealed that target cells attacked by CbpD ruptures at the septal region, in accordance with the binding specificity displayed by the CBD domain.
The oval shape of pneumococci results from a combination of septal and lateral peptidoglycan synthesis. The septal cross-wall is synthesized by the divisome, while the elongasome drives cell elongation by inserting new peptidoglycan into the lateral cell wall. Each of these molecular machines contains penicillin-binding proteins (PBPs), which catalyze the final stages of peptidoglycan synthesis, plus a number of accessory proteins. Much effort has been made to identify these accessory proteins and determine their function. In the present paper we have used a novel approach to identify members of the pneumococcal elongasome that are functionally closely linked to PBP2b. We discovered that cells depleted in PBP2b, a key component of the elongasome, display several distinct phenotypic traits. We searched for proteins that, when depleted or deleted, display the same phenotypic changes. Four proteins, RodA, MreD, DivIVA and Spr0777, were identified by this approach. Together with PBP2b these proteins are essential for the normal function of the elongasome. Furthermore, our findings suggest that DivIVA, which was previously assigned as a divisomal protein, is required to correctly localize the elongasome at the negatively curved membrane region between the septal and lateral cell wall.
Streptococcus pneumoniae and probably most other members of the genus Streptococcus are competent for natural genetic transformation. During the competent state, S. pneumoniae produces a murein hydrolase, CbpD, that kills and lyses noncompetent pneumococci and closely related species. Previous studies have shown that CbpD is essential for efficient transfer of genomic DNA from noncompetent to competent cells in vitro. Consequently, it has been proposed that CbpD together with the cognate immunity protein ComM constitutes a DNA acquisition mechanism that enables competent pneumococci to capture homologous DNA from closely related streptococci sharing the same habitat. Although genes encoding CbpD homologs or CbpD-related proteins are present in many different streptococcal species, the genomes of a number of streptococci do not encode CbpD-type proteins. In the present study we show that the genomes of nearly all species lacking CbpD encode an unrelated competence-regulated murein hydrolase termed LytF. Using Streptococcus gordonii as a model system, we obtained evidence indicating that LytF is a functional analogue of CbpD. In sum, our results show that a murein hydrolase gene is part of the competence regulon of most or all streptococcal species, demonstrating that these muralytic enzymes constitute an essential part of the streptococcal natural transformation system.
In oval-shaped Streptococcus pneumoniae, septal and longitudinal peptidoglycan syntheses are performed by independent functional complexes: the divisome and the elongasome. Penicillin-binding proteins (PBPs) were long considered the key peptidoglycan-synthesizing enzymes in these complexes. Among these were the bifunctional class A PBPs, which are both glycosyltransferases and transpeptidases, and monofunctional class B PBPs with only transpeptidase activity. Recently, however, it was established that the monofunctional class B PBPs work together with transmembrane glycosyltransferases (FtsW and RodA) from the shape, elongation, division, and sporulation (SEDS) family to make up the core peptidoglycan-synthesizing machineries within the pneumococcal divisome (FtsW/PBP2x) and elongasome (RodA/PBP2b). The function of class A PBPs is therefore now an open question. Here we utilize the peptidoglycan hydrolase CbpD that targets the septum of S. pneumoniae cells to show that class A PBPs have an autonomous role during pneumococcal cell wall synthesis. Using assays to specifically inhibit the function of PBP2x and FtsW, we demonstrate that CbpD attacks nascent peptidoglycan synthesized by the divisome. Notably, class A PBPs could process this nascent peptidoglycan from a CbpD-sensitive to a CbpD-resistant form. The class A PBP-mediated processing was independent of divisome and elongasome activities. Class A PBPs thus constitute an autonomous functional entity which processes recently formed peptidoglycan synthesized by FtsW/PBP2×. Our results support a model in which mature pneumococcal peptidoglycan is synthesized by three functional entities, the divisome, the elongasome, and bifunctional PBPs. The latter modify existing peptidoglycan but are probably not involved in primary peptidoglycan synthesis. class A PBPs | CbpD | peptidoglycan | Streptococcus pneumoniae
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.