Freton, C. | bioRxiv Science

bioRxiv2024

New assessment of teichoic acids in the cell envelope of Streptococcus pneumoniae

Teichoic acids (TA) are linear phospho-saccharidic polymers and important constituents of the cell envelope of Gram-positive bacteria, either bound to the peptidoglycan as wall teichoic acids (WTA) or to the membrane as lipoteichoic acids (LTA). The chemical composition of TA varies greatly but the presence of both WTA and LTA is highly conserved, hinting at an underlying fundamental function that is distinct from their numerous specific roles in diverse organisms. We report here the observation of a periplasmic space in the Gram-positive Streptococcus pneumoniae by cryo-electron microscopy of vitreous sections. The thickness and appearance of this region change upon deletion of genes involved in the attachment of teichoic acids, supporting the role of TA in the maintenance of a periplasmic space in Gram-positive bacteria as a possible universal function. Consequences of these mutations were further examined by super-resolved microscopy (dSTORM), following metabolic and fluorophore coupling by click-chemistry in pulse and pulse-chase experiments. This novel labeling method also enabled in-gel analysis of cell fractions, revealing that LTA-containing membranes sediment at low centrifugal forces. Owing to this easy separation approach, we were able to titrate the actual amount of TA per cell and to determine the ratio of WTA to LTA. In addition, we followed the change of TA length during growth phases, and discovered that a mutant devoid of LTA accumulates the membrane-bound polymerized TA precursor. SignificanceThe existence of a periplasmic space in Gram-positive bacteria has long been debated. The finding that compromising the attachment of teichoic acids changes the appearance and thickness of the periplasm in the pneumococcus indicates a role of these polymers in the maintenance of this space between the membrane and the cell wall. Metabolic labeling and electrophoresis showed that LTA-containing membranes are easily sedimented. This finding indicates that the LTA/WTA ratios reported in previous studies were likely underestimated, since most LTA were probably unknowingly discarded in these studies. Our method of TA analysis opens a new era in the investigation of these important and poorly known bacterial polymers and their role in the periplasmic space of Gram-positive organisms.

microbiology↗

bioRxiv2024

DivIVA controls the dynamics of septum splitting and cell elongation in Streptococcus pneumoniae

Bacterial shape and division rely on the dynamics of cell wall assembly, which involves regulated synthesis and cleavage of the peptidoglycan. In ovococci, these processes are coordinated in an annular mid-cell region with nanometric dimensions. More precisely, the cross-wall that is synthesized by the divisome is split to generate lateral wall, whose expansion is insured by insertion of so-called peripheral peptidoglycan by the elongasome. Septum cleavage and peripheral peptidoglycan synthesis are thus crucial remodeling events for ovococcal cell division and elongation. The structural DivIVA protein has long been known as a major regulator of these processes but its mode of action remains unknown. Here, we integrate click chemistry-based peptidoglycan labeling, direct stochastic optical reconstruction microscopy and in silico modeling, as well as epifluorescence and stimulated emission depletion microscopy to investigate the role of DivIVA in Streptococcus pneumoniae cell morphogenesis. Our work reveals two distinct phases of peptidoglycan remodeling along the cell cycle, that are differentially controlled by DivIVA. In particular, we show that DivIVA ensures homogeneous septum cleavage and peripheral peptidoglycan synthesis around the division site, and their maintenance throughout the cell cycle. Our data additionally suggest that DivIVA impacts the contribution of the elongasome and class A PBPs to cell elongation. We also report the position of DivIVA on either side of the septum, consistent with its known affinity for negatively curved membranes. Finally, we take the opportunity provided by these new observations to propose hypotheses for the mechanism of action of this key morphogenetic protein. IO_SCPLOWMPORTANCEC_SCPLOWThis study sheds light on fundamental processes governing bacterial cell growth and division, using integrated click chemistry, advanced microscopy and computational modeling approaches. More precisely, it addresses mechanisms involved in the regulation of cell wall synthesis and remodeling in Streptococcus pneumoniae. This bacterium belongs to the morphological group of ovococci, which includes many human pathogens, such as streptococci and enterococci. In this study, we have dissected the function of DivIVA, which is a structural protein involved in cell division, cell morphogenesis and chromosome partitioning in Gram-positive bacteria. This work unveils the role of DivIVA in the orchestration of cell division and elongation along the pneumococcal cell cycle. It not only helps understanding how ovoid bacteria proliferate, but also offers an opportunity to consider how DivIVA might serve as a scaffold and sensor for particular membrane regions, and thus be involved in various processes associated with the cell cycle.

microbiology↗