Organization of peptidoglycan synthesis in nodes and separate rings at different stages of cell division of Streptococcus pneumoniae

Abstract

Bacterial peptidoglycan (PG) synthesis requires strict spatiotemporal organization to reproduce specific cell shapes. In ovoid‐shaped Streptococcus pneumoniae (Spn), septal and peripheral (elongation) PG synthesis occur simultaneously at midcell. To uncover the organization of proteins and activities that carry out these two modes of PG synthesis, we examined Spn cells vertically oriented onto their poles to image the division plane at the high lateral resolution of 3D‐SIM (structured‐illumination microscopy). Labeling with fluorescent D‐amino acids (FDAA) showed that areas of new transpeptidase (TP) activity catalyzed by penicillin‐binding proteins (PBPs) separate into a pair of concentric rings early in division, representing peripheral PG (pPG) synthesis (outer ring) and the leading‐edge (inner ring) of septal PG (sPG) synthesis. Fluorescently tagged PBP2x or FtsZ locate primarily to the inner FDAA‐marked ring, whereas PBP2b and FtsX remain in the outer ring, suggesting roles in sPG or pPG synthesis, respectively. Pulses of FDAA labeling revealed an arrangement of separate regularly spaced “nodes” of TP activity around the division site of predivisional cells. Tagged PBP2x, PBP2b, and FtsX proteins also exhibited nodal patterns with spacing comparable to that of FDAA labeling. Together, these results reveal new aspects of spatially ordered PG synthesis in ovococcal bacteria during cell division.

The Pneumococcal Divisome: Dynamic Control of Streptococcus pneumoniae Cell Division

“…In S. pneumoniae , the initial FtsZ-ring assembly at the equators of newly divided daughter cells organises all of the components required for cell division, septal and peripheral PG synthesis and chromosome segregation ( Figure 2 ; Fleurie et al, 2014b ; Tsui et al, 2014 ; Perez et al, 2019 , 2021b ). In this regard, the pneumococcal FtsZ-ring assembly resembles the predivisional PG complexes at the septa of rod-shaped bacteria ( Buddelmeijer and Beckwith, 2002 ; den Blaauwen et al, 2008 ).…”

“…With the exception of FtsN, homologs of the E. coli divisome proteins are present in S. pneumoniae , and it seems likely that the pneumococcal divisome assembles in the same order, which will be assumed here. Notably, at a point early after the start of division, septal and peripheral PG synthesis proteins separate into concentric rings ( Perez et al, 2021b ). During this separation, some proteins remain in the closing inner-ring septal PG synthesis machine, including FtsZ and PBP2x, while other proteins partition and remain in the outer-ring peripheral PG synthesis machine, including PBP2b, FtsX, and PBP2x ( Perez et al, 2021b ).…”

“…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. Many of the key cell division proteins are highly conserved in these organisms, underlying their essentiality and universality, although their functions can vary between species.…”

“…Septal synthesis produces the cell wall separating the new daughter cells, while peripheral synthesis is responsible for cell elongation ( Berg et al, 2013 ; Tsui et al, 2014 ; Philippe et al, 2015 ; Straume et al, 2017 ). 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 ).…”

“…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 ).…”