Pbp2x localizes separately from Pbp2b and other peptidoglycan synthesis proteins during later stages of cell division of Streptococcus pneumoniae D39

Abstract

The relative localization patterns of class B penicillin-binding proteins Pbp2x and Pbp2b were used as positional indicators of septal and peripheral (side-wall-like) peptidoglycan (PG) synthesis, respectively, in the midcell regions of Streptococcus pneumoniae cells at different stages of division. We confirm that Pbp2x and Pbp2b are essential in the strain D39 genetic background, which differs from that of laboratory strains. We show that Pbp2b, like Pbp2x and class A Pbp1a, follows a different localization pattern than FtsZ and remains at division septa after FtsZ reappears at the equators of daughter cells. Pulse-experiments with fluorescent D-amino acids (FDAAs) were performed in wild-type cells and in cells in which Pbp2x activity was preferentially inhibited by methicillin or Pbp2x amount was depleted. These experiments show that Pbp2x activity separates from that of other PBPs to the centers of constricting septa in mid-to-late divisional cells resolved by high-resolution 3D-SIM microscopy. Dual-protein and protein-fluorescent vancomycin 2D and 3D-SIM immunofluorescence microscopy (IFM) of cells at different division stages corroborate that Pbp2x separates to the centers of septa surrounded by an adjacent constricting ring containing Pbp2b, Pbp1a, and regulators, StkP and MreC. The separate localization of Pbp2x suggests distinctive roles in completing septal PG synthesis and remodeling.

Lyme disease and relapsing feverBorreliaelongate through zones of peptidoglycan synthesis that mark division sites of daughter cells

“…Perhaps more importantly, the spatial and temporal regulation of PG synthesis contributing to either cell elongation or septum formation are vastly different between B. burgdorferi and S. pneumoniae, ultimately shaping their cell morphology. In S. pneumoniae, cell elongation occurs at the same time and place as septum formation, such that cells adopt a prolate spheroid shape (6,(25)(26)(27)(28)(29). This is in contrast to B. burgdorferi, in which cell elongation and septum formation are largely uncoupled, resulting in long, rod-shaped cells.…”

“…This suggests that cells are able to "sense" the cellular 1/4 and 3/4 positions to establish zones of PG synthesis. B. burgdorferi is, in some aspects, similar to the firmicute Streptococcus pneumoniae, because S. pneumoniae is born with a ring of PG synthesis at midcell and establishes new rings of PG synthesis at 1/4 and 3/4 positions before division and cell separation are complete (25,26). However, there are important differences between B. burgdorferi and S. pneumoniae.…”

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

“…These aliquots were centrifuged at 16,000 ϫ g for 2.5 min at 4°C, supernatants were discarded, and pellets were resuspended in 50 l of cold 1ϫ PBS. An amount of 1.5 l of labeled live cells (no fixation) was placed on a slide, covered with a glass coverslip, and imaged and processed as described previously using a Nikon E-400 epifluorescence phase-contrast microscope and NIS-Elements AR imaging software (Nikon) (33,46).…”

“…Moreover, cell chaining, which is especially sensitive to the function of PG hydrolases, favors colonization in the nasopharynx but makes cells vulnerable to phagocytosis in the lung (30,31). In this paper, we used fluorescent D-amino acid (FDAA) probes (32,33) to visualize PG dynamics in capsulated and unencapsulated mutants of S. pneumoniae growing in culture and in host-relevant biofilms in vitro. We also used FDAA probes to visualize defects in PG morphology caused by PG hydrolases and key cell division mutants.…”

The bacterial divisome: ready for its close-up

“…In addition, 3D-SIM was used to localize divisome components of Streptococcus pneumoniae, which grow and divide as small ovococci. Using 3D-SIM for IFM, Malcolm Winkler's group found that penicillin-binding proteins Pbp1A and Pbp2x localize to division septa like FtsZ, but form rings clearly outside the Z ring and remain at maturing division septa even after FtsZ has migrated to future division sites [70]. Moreover, at a certain point in the S. pneumoniae division cycle, the Pbp1a ring is detectably outside the Pbp2x ring, suggesting that these proteins form concentric layers.…”