Minimal Peptidoglycan (PG) Turnover in Wild-Type and PG Hydrolase and Cell Division Mutants of Streptococcus pneumoniae D39 Growing Planktonically and in Host-Relevant Biofilms

Abstract

We determined whether there is turnover of the peptidoglycan (PG) cell wall of the ovococcus bacterial pathogen Streptococcus pneumoniae (pneumococcus). Pulse-chase experiments on serotype 2 strain D39 radiolabeled with N-acetylglucosamine revealed little turnover and release of PG breakdown products during growth compared to published reports of PG turnover in Bacillus subtilis. PG dynamics were visualized directly by long-pulse-chase-new-labeling experiments using two colors of fluorescent D-amino acid (FDAA) probes to microscopically detect regions of new PG synthesis. Consistent with minimal PG turnover, hemispherical regions of stable "old" PG persisted in D39 and TIGR4 (serotype 4) cells grown in rich brain heart infusion broth, in D39 cells grown in chemically defined medium containing glucose or galactose as the carbon source, and in D39 cells grown as biofilms on a layer of fixed human epithelial cells. In contrast, B. subtilis exhibited rapid sidewall PG turnover in similar FDAA-labeling experiments. High-performance liquid chromatography (HPLC) analysis of biochemically released peptides from S. pneumoniae PG validated that FDAAs incorporated at low levels into pentamer PG peptides and did not change the overall composition of PG peptides. PG dynamics were also visualized in mutants lacking PG hydrolases that mediate PG remodeling, cell separation, or autolysis and in cells lacking the MapZ and DivIVA division regulators. In all cases, hemispheres of stable old PG were maintained. In PG hydrolase mutants exhibiting aberrant division plane placement, FDAA labeling revealed patches of inert PG at turns and bulge points. We conclude that growing S. pneumoniae cells exhibit minimal PG turnover compared to the PG turnover in rod-shaped cells. IMPORTANCEPG cell walls are unique to eubacteria, and many bacterial species turn over and recycle their PG during growth, stress, colonization, and virulence. Consequently, PG breakdown products serve as signals for bacteria to induce antibiotic resistance and as activators of innate immune responses. S. pneumoniae is a commensal bacterium that colonizes the human nasopharynx and opportunistically causes serious respiratory and invasive diseases. The results presented here demonstrate a distinct demarcation between regions of old PG and regions of new PG synthesis and minimal turnover of PG in S. pneumoniae cells growing in culture or in host-relevant biofilms. These findings suggest that S. pneumoniae minimizes the release of PG breakdown products by turnover, which may contribute to evasion of the innate immune system. P eptidoglycan (PG) biosynthesis and placement are dynamic processes that determine the shapes, sizes, chaining, and resistance to turgor of bacterial cells (1-6). In Gram-positive bacteria, PG also serves as the scaffolding for covalent attachment of surface wall teichoic acid, capsule, and sortase-attached proteins (7-9). The seminal work of Park and Uehara demonstrated that PG is rapidly turned over and the breakdown components recycled in...

Chromosome segregation drives division site selection in Streptococcus pneumoniae

“…Although the FtsZ-mCherry strain showed a normal cell size distribution in a wild-type background, when combined with the ΔmapZ mutant, a clear synthetic phenotype arose and cells were misformed (SI Appendix, Fig. S2 A and B), suggesting that the previously described mapZ phenotype in the presence of FtsZ fusions should be interpreted with caution (30). Therefore, we replaced FtsZmCherry by a more functional FtsZ-CFP or FtsZ-mKate2 (FtsZ-RFP) fusion (SI Appendix, Fig.…”

“…MapZ has been proposed as the division site selector in S. pneumoniae, and Z-ring positioning and angle were shown to be perturbed in mapZ-null mutants (22,23). In contrast to what can be expected for a protein involved in division site selection, ΔmapZ mutants are not elongated but are on average shorter than wild-type cells with relatively minor distortions in cell morphology (22,23), raising questions on what the actual biological function of MapZ is (30). To reassess the ΔmapZ phenotype, we fused MapZ at its N terminus to a monomeric superfolder green fluorescent protein (GFP).…”

“…Because MapZ has a large extracellular PG binding domain and is controlled by the Ser/Thr kinase StkP (22,36), which is proposed to be a key player in tuning peripheral and septal PG synthesis (24,37), it is tempting to speculate that MapZ has a function in cell wall remodeling and subsequently maintaining the perpendicular Z-ring plane. Although this remains to be verified experimentally, it has been shown that mutations in PG hydrolases as well as depletion of Pbp2b involved in cell elongation can lead to nonparallel division and cell-shape alterations in S. pneumoniae (30,38,39), similar to the phenotype of ΔmapZ cells.…”

Novel Electrophilic Scaffold for Imaging of Essential Penicillin-Binding Proteins in Streptococcus pneumoniae

“…However, their nonspecificity for individual PBPs can only indicate regions of the cell where there is TP activity. 21 …”

Roles of the Essential Protein FtsA in Cell Growth and Division in Streptococcus pneumoniae

“…Given the possibility that PG synthesis is dispersed upon depletion of FtsA, we compared the sites of active PG synthesis in Rx1 ΔftsA//P Zn gfp-ftsA ϩ cells by staining with HADA (HCC-amino-Dalanine), a fluorescent hydroxy-coumarin derivative of D-alanine (56). Short pulses of HADA incorporate into PG in regions of active penicillin-binding protein transpeptidase activity (56,57). Rx1 ΔftsA//P Zn gfp-ftsA ϩ cells were grown in the absence or presence of different ZnCl 2 concentrations and observed by fluorescence microscopy (Fig.…”