FtsZ treadmilling is essential for Z-ring condensation and septal constriction initiation in Bacillus subtilis cell division

ABSTRACTDespite the central role of division in bacterial physiology, how division proteins work together as a nanoscale machine to divide the cell remains poorly understood. Cell division by cell wall synthesis proteins is guided by the cytoskeleton protein FtsZ, which assembles at mid-cell as a dense Z-ring formed of treadmilling filaments1,2. However, although FtsZ treadmilling is essential for cell division, the function of FtsZ treadmilling remains unclear2–5. Here, we systematically resolve the function of FtsZ treadmilling across each stage of division in the Gram-positive model organism Bacillus subtilis using a novel combination of nanofabrication, advanced microscopy, and microfluidics to measure the division-protein dynamics in live cells with ultrahigh sensitivity. We find that FtsZ treadmilling has two essential functions: mediating condensation of diffuse FtsZ filaments into a dense Z-ring, and initiating constriction by guiding septal cell wall synthesis. After constriction initiation, FtsZ treadmilling has a dispensable function in accelerating septal constriction rate. Our results show that FtsZ treadmilling is critical for assembling and initiating the bacterial cell division machine.

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FtsZ treadmilling is essential for Z-ring condensation and septal constriction initiation in Bacillus subtilis cell division

Nature Communications ◽

10.1038/s41467-021-22526-0 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Kevin D. Whitley ◽

Calum Jukes ◽

Nicholas Tregidgo ◽

Eleni Karinou ◽

Pedro Almada ◽

...

Keyword(s):

Cell Wall ◽

Bacillus Subtilis ◽

Cell Division ◽

Model Organism ◽

Live Cells ◽

Bacterial Cell Division ◽

Cell Wall Synthesis ◽

Bacterial Physiology ◽

Z Ring ◽

Ultrahigh Sensitivity

AbstractDespite the central role of division in bacterial physiology, how division proteins work together as a nanoscale machine to divide the cell remains poorly understood. Cell division by cell wall synthesis proteins is guided by the cytoskeleton protein FtsZ, which assembles at mid-cell as a dense Z-ring formed of treadmilling filaments. However, although FtsZ treadmilling is essential for cell division, the function of FtsZ treadmilling remains unclear. Here, we systematically resolve the function of FtsZ treadmilling across each stage of division in the Gram-positive model organism Bacillus subtilis using a combination of nanofabrication, advanced microscopy, and microfluidics to measure the division-protein dynamics in live cells with ultrahigh sensitivity. We find that FtsZ treadmilling has two essential functions: mediating condensation of diffuse FtsZ filaments into a dense Z-ring, and initiating constriction by guiding septal cell wall synthesis. After constriction initiation, FtsZ treadmilling has a dispensable function in accelerating septal constriction rate. Our results show that FtsZ treadmilling is critical for assembling and initiating the bacterial cell division machine.

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Bacteriophage SP01 Gene Product 56 Inhibits Bacillus subtilis Cell Division by Interacting with FtsL and Disrupting Pbp2B and FtsW Recruitment

Journal of Bacteriology ◽

10.1128/jb.00463-20 ◽

2020 ◽

Vol 203 (2) ◽

pp. e00463-20

Author(s):

Amit Bhambhani ◽

Isabella Iadicicco ◽

Jules Lee ◽

Syed Ahmed ◽

Max Belfatto ◽

...

Keyword(s):

Cell Wall ◽

Bacillus Subtilis ◽

Cell Division ◽

Gene Product ◽

Fluorescent Protein ◽

Lytic Bacteriophage ◽

Cell Wall Synthesis ◽

Content Type ◽

Ftsz Ring ◽

Green Fluorescent

ABSTRACTPrevious work identified gene product 56 (gp56), encoded by the lytic bacteriophage SP01, as being responsible for inhibition of Bacillus subtilis cell division during its infection. Assembly of the essential tubulin-like protein FtsZ into a ring-shaped structure at the nascent site of cytokinesis determines the timing and position of division in most bacteria. This FtsZ ring serves as a scaffold for recruitment of other proteins into a mature division-competent structure permitting membrane constriction and septal cell wall synthesis. Here, we show that expression of the predicted 9.3-kDa gp56 of SP01 inhibits later stages of B. subtilis cell division without altering FtsZ ring assembly. Green fluorescent protein-tagged gp56 localizes to the membrane at the site of division. While its localization does not interfere with recruitment of early division proteins, gp56 interferes with the recruitment of late division proteins, including Pbp2b and FtsW. Imaging of cells with specific division components deleted or depleted and two-hybrid analyses suggest that gp56 localization and activity depend on its interaction with FtsL. Together, these data support a model in which gp56 interacts with a central part of the division machinery to disrupt late recruitment of the division proteins involved in septal cell wall synthesis.IMPORTANCE Studies over the past decades have identified bacteriophage-encoded factors that interfere with host cell shape or cytokinesis during viral infection. The phage factors causing cell filamentation that have been investigated to date all act by targeting FtsZ, the conserved prokaryotic tubulin homolog that composes the cytokinetic ring in most bacteria and some groups of archaea. However, the mechanisms of several phage factors that inhibit cytokinesis, including gp56 of bacteriophage SP01 of Bacillus subtilis, remain unexplored. Here, we show that, unlike other published examples of phage inhibition of cytokinesis, gp56 blocks B. subtilis cell division without targeting FtsZ. Rather, it utilizes the assembled FtsZ cytokinetic ring to localize to the division machinery and to block recruitment of proteins needed for septal cell wall synthesis.

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Modification of cell wall polysaccharide spatially controls cell division in Streptococcus mutans

10.1101/2020.06.26.173716 ◽

2020 ◽

Author(s):

Svetlana Zamakhaeva ◽

Catherine T. Chaton ◽

Jeffrey S. Rush ◽

Sowmya Ajay Castro ◽

Alexander E. Yarawsky ◽

...

Keyword(s):

Cell Wall ◽

Cell Division ◽

Streptococcus Mutans ◽

Cell Separation ◽

Ring Structure ◽

Cell Wall Polysaccharide ◽

Bacterial Cell Division ◽

Glycerol Phosphate ◽

Immature Form ◽

Z Ring

AbstractBacterial cell division is driven by a tubulin homolog FtsZ, which assembles into the Z-ring structure leading to the recruitment of the cell division machinery. In ovoid-shaped Gram-positive bacteria, such as streptococci, MapZ guides Z-ring positioning at cell equators through an, as yet, unknown mechanism. The cell wall of the important dental pathogen Streptococcus mutans is composed of peptidoglycan decorated with Serotype c Carbohydrates (SCCs). Here, we show that an immature form of SCC, lacking the recently identified glycerol phosphate (GroP) modification, coordinates Z-ring positioning. Pulldown assays using S. mutans cell wall combined with binding affinity analysis identified the major cell separation autolysin, AtlA, as an SCC binding protein. Importantly, AtlA binding to mature SCC is attenuated due to GroP modification. Using fluorescently-labeled AtlA, we mapped SCC distribution on the streptococcal surface to reveal that GroP-deficient immature SCCs are exclusively present at the cell poles and equators. Moreover, the equatorial GroP-deficient SCCs co-localize with MapZ throughout the S. mutans cell cycle. Consequently, in GroP-deficient mutant bacteria, proper AtlA localization is abrogated resulting in dysregulated cellular autolysis. In addition, these mutants display morphological abnormalities associated with MapZ mislocalization leading to Z-ring misplacement. Altogether, our data support a model in which GroP-deficient immature SCCs spatially coordinate the localization of AtlA and MapZ. This mechanism ensures cell separation by AtlA at poles and Z-ring alignment with the cell equator.Graphical abstract

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DivIVA Is Required for Polar Growth in the MreB-Lacking Rod-Shaped Actinomycete Corynebacterium glutamicum

Journal of Bacteriology ◽

10.1128/jb.01934-07 ◽

2008 ◽

Vol 190 (9) ◽

pp. 3283-3292 ◽

Cited By ~ 94

Author(s):

Michal Letek ◽

Efrén Ordóñez ◽

José Vaquera ◽

William Margolin ◽

Klas Flärdh ◽

...

Keyword(s):

Cell Wall ◽

Bacillus Subtilis ◽

Cell Division ◽

Streptococcus Pneumoniae ◽

Corynebacterium Glutamicum ◽

Chromosome Segregation ◽

Polar Growth ◽

Cell Wall Synthesis ◽

Peptidoglycan Synthesis ◽

Polarized Cell Growth

ABSTRACT The actinomycete Corynebacterium glutamicum grows as rod-shaped cells by zonal peptidoglycan synthesis at the cell poles. In this bacterium, experimental depletion of the polar DivIVA protein (DivIVACg) resulted in the inhibition of polar growth; consequently, these cells exhibited a coccoid morphology. This result demonstrated that DivIVA is required for cell elongation and the acquisition of a rod shape. DivIVA from Streptomyces or Mycobacterium localized to the cell poles of DivIVACg-depleted C. glutamicum and restored polar peptidoglycan synthesis, in contrast to DivIVA proteins from Bacillus subtilis or Streptococcus pneumoniae, which localized at the septum of C. glutamicum. This confirmed that DivIVAs from actinomycetes are involved in polarized cell growth. DivIVACg localized at the septum after cell wall synthesis had started and the nucleoids had already segregated, suggesting that in C. glutamicum DivIVA is not involved in cell division or chromosome segregation.

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A conserved subcomplex within the bacterial cytokinetic ring activates cell wall synthesis by the FtsW-FtsI synthase

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2004598117 ◽

2020 ◽

Vol 117 (38) ◽

pp. 23879-23885 ◽

Cited By ~ 1

Author(s):

Lindsey S. Marmont ◽

Thomas G. Bernhardt

Keyword(s):

Pseudomonas Aeruginosa ◽

Cell Wall ◽

Cell Division ◽

Polymerase Activity ◽

Bacterial Cell Division ◽

Cell Wall Synthesis ◽

Negative Regulators ◽

Major Function ◽

Osmotic Lysis ◽

Direct Activator

Cell division in bacteria is mediated by a multiprotein assembly called the divisome. A major function of this machinery is the synthesis of the peptidoglycan (PG) cell wall that caps the daughter poles and prevents osmotic lysis of the newborn cells. Recent studies have implicated a complex of FtsW and FtsI (FtsWI) as the essential PG synthase within the divisome; however, how PG polymerization by this synthase is regulated and coordinated with other activities within the machinery is not well understood. Previous results have implicated a conserved subcomplex of division proteins composed of FtsQ, FtsL, and FtsB (FtsQLB) in the regulation of FtsWI, but whether these proteins act directly as positive or negative regulators of the synthase has been unclear. To address this question, we purified a five-memberPseudomonas aeruginosadivision complex consisting of FtsQLB-FtsWI. The PG polymerase activity of this complex was found to be greatly stimulated relative to FtsWI alone. Purification of complexes lacking individual components indicated that FtsL and FtsB are sufficient for FtsW activation. Furthermore, support for this activity being important for the cellular function of FtsQLB was provided by the identification of two division-defective variants of FtsL that still form normal FtsQLB-FtsWI complexes but fail to activate PG synthesis. Thus, our results indicate that the conserved FtsQLB complex is a direct activator of PG polymerization by the FtsWI synthase and thereby define an essential regulatory step in the process of bacterial cell division.

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Bacteriophage SP01 Gene Product 56 (gp56) Inhibits Bacillus subtilis Cell Division by Interacting with DivIC/FtsL to Prevent Pbp2B/FtsW Recruitment

10.1101/2020.02.06.938217 ◽

2020 ◽

Author(s):

Amit Bhambhani ◽

Isabella Iadicicco ◽

Jules Lee ◽

Syed Ahmed ◽

Max Belfatto ◽

...

Keyword(s):

Cell Wall ◽

Bacillus Subtilis ◽

Cell Division ◽

Gene Product ◽

Lytic Bacteriophage ◽

Cell Wall Synthesis ◽

Ftsz Ring ◽

Ring Assembly ◽

Ring Shaped Structure ◽

Two Hybrid

ABSTRACTPrevious work identified gp56, encoded by the lytic bacteriophage SP01, as responsible for inhibition of Bacillus subtilis cell division during its infection. Assembly of the essential tubulin-like protein FtsZ into a ring-shaped structure at the nascent site of cytokinesis determines the timing and position of division in most bacteria. This FtsZ ring serves as a scaffold for recruitment of other proteins into a mature division-competent structure permitting membrane constriction and septal cell wall synthesis. Here we show that expression of the predicted 9.3-kDa gene product 56 (gp56) of SP01 inhibits latter stages of B. subtilis cell division without altering FtsZ ring assembly. GFP-tagged gp56 localizes to the membrane at the site of division. While its localization permits recruitment of early division proteins, gp56 interferes with the recruitment of late division proteins, including Pbp2b and FtsW. Imaging of cells with specific division components deleted or depleted and two-hybrid analysis suggest that gp56 localization and activity depends on its interaction with mid-recruited proteins DivIC and/or FtsL. Together these data support a model where gp56 interacts with a central part of the division machinery to disrupt late recruitment of the division proteins involved in septal cell wall synthesis.IMPORTANCEResearch over the past decades has uncovered bacteriophage-encoded factors that interfere with host cell shape or cytokinesis during viral infection. Phage factors that cause cell filamentation that have been investigated to date all act by targeting FtsZ, the conserved prokaryotic tubulin homolog that composes the cytokinetic ring in most bacteria and some groups of archaea. However, the mechanism of several identified phage factors that inhibit cytokinesis remain unexplored, including gp56 of bacteriophage SP01 of Bacillus subtilis. Here, we show that unlike related published examples of phage inhibition of cyotkinesis, gp56 blocks B. subtilis cell division without targeting FtsZ. Rather, it utilizes the assembled FtsZ cytokinetic ring to localize to the division machinery and block recruitment of proteins needed for the septal cell wall synthesis.

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Z-ring membrane anchors associate with cell wall synthases to initiate bacterial cell division

Nature Communications ◽

10.1038/s41467-018-07559-2 ◽

2018 ◽

Vol 9 (1) ◽

Cited By ~ 27

Author(s):

Manuel Pazos ◽

Katharina Peters ◽

Mercedes Casanova ◽

Pilar Palacios ◽

Michael VanNieuwenhze ◽

...

Keyword(s):

Cell Wall ◽

Cell Division ◽

Bacterial Cell ◽

Bacterial Cell Division ◽

Z Ring

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Determinants of FtsZ C-terminal linker-dependent regulation of cell wall metabolism in Caulobacter crescentus

10.1101/632075 ◽

2019 ◽

Author(s):

Kousik Sundararajan ◽

Jordan M. Barrows ◽

Erin D. Goley

Keyword(s):

Cell Wall ◽

Cell Division ◽

Caulobacter Crescentus ◽

Bacterial Cell Division ◽

Cell Wall Metabolism ◽

Gtpase Domain ◽

Binding Partners ◽

Z Ring ◽

Membrane Anchoring

AbstractBacterial cell division requires assembly of a multi-protein machinery or “divisome” that remodels the cell envelope to cause constriction. The cytoskeletal protein FtsZ forms a ring-like scaffold for the divisome at the incipient division site. FtsZ has three major regions – a conserved, polymerizing GTPase domain; a C-terminal conserved (CTC) peptide required for binding membrane-anchoring proteins; and a C-terminal linker (CTL) of poor length and sequence conservation. We previously demonstrated that, in Caulobacter crescentus, the CTL regulates FtsZ polymerization in vitro and cell wall metabolism in vivo. To understand the mechanism of CTL-dependent regulation of cell wall metabolism, here we investigated the impact of the CTL on Z-ring structure in cells and employed genetics to identify molecular determinants of the dominant lethal effects of ΔCTL. Deleting the CTL specifically resulted in formation of dense, asymmetric, non-ring FtsZ assemblies in vivo. Moreover, we observed that production of an FtsZ variant with the GTPase domain of Escherichia coli FtsZ fused to the CTC of C. crescentus FtsZ phenocopied the effects of C. crescentus ΔCTL, suggesting the CTC mediates signaling to cell wall metabolism. Finally, whereas overproduction of ZapA, FzlC, or FtsEX had slight protective effects against ΔCTL, depletion of FtsA partially suppressed the effects of ΔCTL. From these results, we propose that the cell wall misregulation downstream of ΔCTL results from its aberrant assembly properties and is propagated through the interaction between the CTC of FtsZ and FtsA. Our study provides mechanistic insights into CTL-dependent regulation of cell wall enzymes downstream of FtsZ.ImportanceBacterial cell division is essential and requires the recruitment and regulation of a complex network of proteins needed to initiate and guide constriction and cytokinesis. FtsZ serves as a master regulator for this process, and its function is highly dependent on both its self-assembly into a canonical “Z-ring” and interaction with protein binding partners, which results in the activation of enzymes that remodel the cell wall to drive constriction. Using mutants of FtsZ and its binding partners, we have established the role of its C-terminal linker domain in regulating Z-ring organization, as well as the requirement for its C-terminal conserved peptide and interaction with the membrane-anchoring protein FtsA for regulating cell wall remodeling for constriction.

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Treadmilling by FtsZ filaments drives peptidoglycan synthesis and bacterial cell division

10.1101/077560 ◽

2016 ◽

Cited By ~ 3

Author(s):

Alexandre W. Bisson Filho ◽

Yen-Pang Hsu ◽

Georgia R. Squyres ◽

Erkin Kuru ◽

Fabai Wu ◽

...

Keyword(s):

Cell Wall ◽

Cell Division ◽

Division Ring ◽

Bacterial Cell ◽

Cytoplasmic Membrane ◽

Bacterial Cell Division ◽

Cell Wall Synthesis ◽

Division Plane ◽

Peptidoglycan Synthesis

AbstractHow bacteria produce a septum to divide in two is not well understood. This process is mediated by periplasmic cell-wall producing enzymes that are positioned by filaments of the cytoplasmic membrane-associated actin FtsA and the tubulin FtsZ (FtsAZ). To understand how these components act in concert to divide cells, we visualized their movements relative to the dynamics of cell wall synthesis during cytokinesis. We find that the division septum is built at discrete sites that move around the division plane. Furthermore, FtsAZ filaments treadmill in circumferential paths around the division ring, pulling along the associated cell-wall-synthesizing enzymes. We show that the rate of FtsZ treadmilling controls both the rate of cell wall synthesis and cell division. The coupling of both the position and activity of the cell wall synthases to FtsAZ treadmilling guides the progressive insertion of new cell wall, synthesizing increasingly small concentric rings to divide the cell.One-sentence summaryBacterial cytokinesis is controlled by circumferential treadmilling of FtsAZ filaments that drives the insertion of new cell wall.

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FtsEX acts on FtsA to regulate divisome assembly and activity

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1606656113 ◽

2016 ◽

Vol 113 (34) ◽

pp. E5052-E5061 ◽

Cited By ~ 60

Author(s):

Shishen Du ◽

Sebastien Pichoff ◽

Joe Lutkenhaus

Keyword(s):

Escherichia Coli ◽

Cell Wall ◽

Cell Division ◽

Atpase Activity ◽

Critical Role ◽

Bacterial Cell Division ◽

Cell Wall Synthesis ◽

Atp Binding Cassette Transporter ◽

Inactive Form ◽

Cell Wall Hydrolysis

Bacterial cell division is driven by the divisome, a ring-shaped protein complex organized by the bacterial tubulin homolog FtsZ. Although most of the division proteins inEscherichia colihave been identified, how they assemble into the divisome and synthesize the septum remains poorly understood. Recent studies suggest that the bacterial actin homolog FtsA plays a critical role in divisome assembly and acts synergistically with the FtsQLB complex to regulate the activity of the divisome. FtsEX, an ATP-binding cassette transporter-like complex, is also necessary for divisome assembly and inhibits division when its ATPase activity is inactivated. However, its role in division is not clear. Here, we find that FtsEX acts on FtsA to regulate both divisome assembly and activity. FtsX interacts with FtsA and this interaction is required for divisome assembly and inhibition of divisome function by ATPase mutants of FtsEX. Our results suggest that FtsEX antagonizes FtsA polymerization to promote divisome assembly and the ATPase mutants of FtsEX block divisome activity by locking FtsA in the inactive form or preventing FtsA from communicating with other divisome proteins. Because FtsEX is known to govern cell wall hydrolysis at the septum, our findings indicate that FtsEX acts on FtsA to promote divisome assembly and to coordinate cell wall synthesis and hydrolysis at the septum. Furthermore, our study provides evidence that FtsA mutants impaired for self-interaction are favored for division, and FtsW plays a critical role in divisome activation in addition to the FtsQLB complex.

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