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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., (© 2020 John Wiley & Sons Ltd.)

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 mid-cell 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 centres 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 centres 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 remodelling., (© 2014 John Wiley & Sons Ltd.)

Bacterial cell division and peptidoglycan (PG) synthesis are orchestrated by the coordinated dynamic movement of essential protein complexes. Recent studies show that bidirectional treadmilling of FtsZ filaments/bundles is tightly coupled to and limiting for both septal PG synthesis and septum closure in some bacteria, but not in others. Here we report the dynamics of FtsZ movement leading to septal and equatorial ring formation in the ovoid-shaped pathogen, Streptococcus pneumoniae. Conventional and singlemolecule total internal reflection fluorescence microscopy (TIRFm) showed that nascent rings of FtsZ and its anchoring and stabilizing proteins FtsA and EzrA move out from mature septal rings coincident with MapZ rings early in cell division. This mode of continuous nascent ring movement contrasts with a failsafe streaming mechanism of FtsZ/FtsA/EzrA observed in a ΔmapZ mutant and another Streptococcus species. This analysis also provides several parameters of FtsZ treadmilling in nascent and mature rings, including treadmilling velocity in wild-type cells and ftsZ(GTPase) mutants, lifetimes of FtsZ subunits in filaments and of entire FtsZ filaments/bundles, and the processivity length of treadmilling of FtsZ filament/bundles. In addition, we delineated the motion of the septal PBP2x transpeptidase and its FtsW glycosyl transferasebinding partner relative to FtsZ treadmilling in S. pneumoniae cells. Five lines of evidence support the conclusion that movement of the bPBP2x:FtsWcomplex in septa depends on PG synthesis and not on FtsZ treadmilling. Together, these results support a model in which FtsZ dynamics and associations organize and distribute septal PG synthesis, but do not control its rate in S. pneumoniae. [ABSTRACT FROM AUTHOR]

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 mid-cell regions of S treptococcus 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 centres of constricting septa in mid-to-late divisional cells resolved by high-resolution 3 D- SIM microscopy. Dual-protein and protein-fluorescent vancomycin 2 D and 3 D- SIM immunofluorescence microscopy ( IFM) of cells at different division stages corroborate that Pbp2x separates to the centres 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 remodelling. [ABSTRACT FROM AUTHOR]

Summary Construction of the cell plate during plant cell division requires the precise insertion of materials around the circumferentially growing phragmoplast. New work shows that two kinesin-4 motor proteins act to shorten the domain of overlapping microtubules at the phragmoplast perimeter, limiting the site of material deposition. [ABSTRACT FROM AUTHOR]