Nanoscale dynamics of peptidoglycan assembly during the cell cycle of Streptococcus pneumoniae

Abstract

Highlights d Metabolic labeling allows dSTORM analysis of pneumococcal peptidoglycan synthesis d Septal and peripheral peptidoglycan syntheses progress separately d Septal peptidoglycan is synthesized and remodeled from the start of the cell cycle d Peripheral synthesis persists after septal synthesis is completed

The Pneumococcal Divisome: Dynamic Control of Streptococcus pneumoniae Cell Division

“…In pneumococcus the septal and peripheral machineries both remain at midcell throughout division, but form spatially distinct concentric rings as division proceeds, with the septal machine moving with FtsZ to the inner edge of the constricting septal annulus, whilst the peripheral machine remains in the outer ring ( Figures 1B,C , 3 , top; Land et al, 2013 ; Tsui et al, 2014 , 2016 ; Rued et al, 2017 ; Sharifzadeh et al, 2017 , 2020 ; Perez et al, 2021b ). Concentric rings of newly synthesised PG consistent with this model were recently visualised by both 3D-SIM ( Perez et al, 2021b ) and by dSTORM ( Trouve et al, 2021 ). As division begins, a portion of FtsZ, EzrA, and FtsA begin to migrate to the equatorial sites of the developing daughter cells, guided by MapZ (also called LocZ) ( Figure 1 ; Fleurie et al, 2014a ; Holečková et al, 2014 ; Perez et al, 2019 ).…”

“…Studies have been carried out in a number of model organisms, including rod-shaped Escherichia coli and Bacillus subtilis , as well as those with particular morphological and biomedical interest ( Eswara and Ramamurthi, 2017 ; Hsu et al, 2019 ). In addition, metabolic labelling has been achieved using azide-bound D-Ala-D-Ala incorporation to allow Direct Stochastic Optical Reconstruction Microscopy (dSTORM) analysis of PG synthesis with very high resolution ( Trouve et al, 2021 ). Besides dSTORM, other microscopy advances such as the use of 3D-Structured Illumination Microscopy (3D-SIM; Tsui et al, 2014 ; Perez et al, 2021b ) and Total Internal Reflection Fluorescence Microscopy (TIRFm; Zhao et al, 2018 ; Perez et al, 2019 ; Squyres et al, 2021 ) have contributed studies of division and motion in relatively small bacterial cells.…”

“…Two concentric rings of newly synthesised PG surrounding the existing sacculus form as a result, whilst a portion of MapZ, FtsZ, EzrA, and FtsA (MapZ Ring) begin to migrate to the new equators. For simplicity, newly synthesised peripheral PG is drawn as a single orange colour, but likely consists of some mixture of newly synthesised peripheral PG attached to remodelled septal PG (see Trouve et al, 2021 ) (B) . Septal PG synthesis continues to close the central septum whilst peripheral PG synthesis continues to elongate the cell from midcell (C) .…”

High-Resolution Imaging of Bacterial Spatial Organization with Vertical Cell Imaging by Nanostructured Immobilisation (VerCINI)

“…VerCINI provides a complementary approach by orienting bacteria vertically and imaging along their short axes, thereby vastly improving the imaging of many biologically important structures and dynamic processes in non-spherical bacteria that were previously difficult to observe. VerCINI has already found multiple applications in high resolution imaging of bacterial spatial organization in hands of a small number of early-adopter labs 5,[12][13][14][15][16] . We hope that the methods and protocols presented here will allow many other labs to use VerCINI to look at diverse questions in bacterial cell biology from a different angle.…”

“…With μVerCINI we were also able to demonstrate that the FtsZ-targeting antibiotic PC190723 totally arrests FtsZ filament motion within seconds (Figure 3b-c). Beyond this, VerCINI has been applied to image division in several different bacterial species using a variety of imaging methods: The dynamics of FtsZ treadmilling in the ovococcoid bacterial pathogen Streptococcus pneumoniae 13 , the organization of FtsZ and FtsN in Escherichia coli using STED super-resolution imaging 14 , the division-associated cell wall synthase FtsI in E. coli using single-particle tracking 15 , and peptidoglycan synthesis during division and elongation in S. pneumoniae using dSTORM with fluorescently-labelled 'clickable' D-amino acids 16 .…”