Teichoic acids in the periplasm and cell envelope of Streptococcus pneumoniae

Abstract 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. Significance The 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.