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

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.

scholarly journals FtsA Regulates Z-Ring Morphology and Cell Wall Metabolism in an FtsZ C-Terminal Linker-Dependent Manner in Caulobacter crescentus

2020 ◽  
Vol 202 (7) ◽  
Author(s):  
Jordan M. Barrows ◽  
Kousik Sundararajan ◽  
Anant Bhargava ◽  
Erin D. Goley
Keyword(s):  
Cell Wall ◽  
Caulobacter Crescentus ◽  
Bacterial Cell Division ◽  
Cell Wall Metabolism ◽  
Gtpase Domain ◽  
Content Type ◽  
Binding Partners ◽  
Z Ring ◽  
Membrane Anchoring

ABSTRACT Bacterial cell division requires the assembly of a multiprotein division machinery, or divisome, that remodels the cell envelope to cause constriction. The cytoskeletal protein FtsZ forms a ringlike scaffold for the divisome at the incipient division site. FtsZ has three major regions: a conserved GTPase domain that polymerizes into protofilaments on binding GTP, a C-terminal conserved peptide (CTC) required for binding membrane-anchoring proteins, and a C-terminal linker (CTL) region of varied length and low sequence conservation. Recently, we demonstrated that the CTL regulates FtsZ polymerization properties in vitro and Z-ring structure and cell wall metabolism in vivo. In Caulobacter crescentus, an FtsZ variant lacking the CTL (designated ΔCTL) can recruit all known divisome members and drive local cell wall synthesis but has dominant lethal effects on cell wall metabolism. To understand the underlying mechanism of the CTL-dependent regulation of cell wall metabolism, we expressed chimeras of FtsZ domains from C. crescentus and Escherichia coli and observed that the E. coli GTPase domain fused to the C. crescentus CTC phenocopies C. crescentus ΔCTL. By investigating the contributions of FtsZ-binding partners, we identified variants of FtsA, a known membrane anchor for FtsZ, that delay or exacerbate the ΔCTL phenotype. Additionally, we observed that the ΔCTL protein forms extended helical structures in vivo upon FtsA overproduction. We propose that misregulation downstream of defective ΔCTL assembly is propagated through the interaction between the CTC and FtsA. Overall, our study provides mechanistic insights into the CTL-dependent regulation of cell wall enzymes downstream of FtsZ polymerization. IMPORTANCE Bacterial 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 assembly into the canonical Z ring and interactions with protein binding partners, all of which results in the activation of enzymes that remodel the cell wall to drive constriction. Using mutants of FtsZ, we have elaborated on the role of its C-terminal linker domain in regulating Z-ring stability and dynamics, as well as the requirement for its conserved C-terminal domain and interaction with the membrane-anchoring protein FtsA for regulating the process of cell wall remodeling for constriction.

scholarly journals The chaperonin GroESL facilitates Caulobacter crescentus cell division by supporting the function of the actin homologue FtsA

2020 ◽  
Author(s):  
Kristen Schroeder ◽  
Kristina Heinrich ◽  
Ines Neuwirth ◽  
Kristina Jonas
Keyword(s):  
Cell Cycle ◽  
Cell Wall ◽  
Cell Division ◽  
Bacterial Cell ◽  
Antimicrobial Agents ◽  
Caulobacter Crescentus ◽  
Bacterial Cell Division ◽  
Growing Cell ◽  
Bacterial Cell Cycle

AbstractThe highly conserved chaperonin GroESL performs a crucial role in protein folding, however the essential cellular pathways that rely on this chaperone are underexplored. Loss of GroESL leads to severe septation defects in diverse bacteria, suggesting the folding function of GroESL may be integrated with the bacterial cell cycle at the point of cell division. Here, we describe new connections between GroESL and the bacterial cell cycle, using the model organism Caulobacter crescentus. Using a proteomics approach, we identify candidate GroESL client proteins that become insoluble or are degraded specifically when GroESL folding is insufficient, revealing several essential proteins that participate in cell division and peptidoglycan biosynthesis. We demonstrate that other cell cycle events such as DNA replication and chromosome segregation are able to continue when GroESL folding is insufficient, and find that deficiency of the bacterial actin homologue FtsA function mediates the GroESL-dependent block in cell division. Our data suggest that a GroESL-FtsA interaction is required to maintain normal dynamics of the FtsZ scaffold and divisome functionality in C. crescentus. In addition to supporting FtsA function, we show that GroESL is required to maintain the flow of peptidoglycan precursors into the growing cell wall. Linking a chaperone to cell division may be a conserved way to coordinate environmental and internal cues that signal when it is safe to divide.ImportanceAll organisms depend on mechanisms that protect proteins from misfolding and aggregation. GroESL is a highly conserved molecular chaperone that functions to prevent protein aggregation in organisms ranging from bacteria to humans. Despite detailed biochemical understanding of GroESL function, the in vivo pathways that strictly depend on this chaperone remain poorly defined in most species. This study provides new insights into how GroESL is linked to the bacterial cell division machinery, a crucial target of current and future antimicrobial agents. We identify a functional interaction between GroESL and FtsA, a conserved bacterial actin homologue, suggesting that as in eukaryotes, some bacteria exhibit a connection between cytoskeletal actin proteins and chaperonins. Our work further defines how GroESL is integrated with cell wall synthesis, and illustrates how highly conserved folding machines ensure the functioning of fundamental cellular processes during stress.

scholarly journals Species- and C-terminal linker-dependent variations in the dynamic behavior of FtsZ on membranesin vitro

2018 ◽  
Author(s):  
Kousik Sundararajan ◽  
Anthony Vecchiarelli ◽  
Kiyoshi Mizuuchi ◽  
Erin D. Goley
Keyword(s):  
Cell Wall ◽  
Lipid Bilayers ◽  
Caulobacter Crescentus ◽  
Dynamic Structure ◽  
Bacterial Cell Division ◽  
Filament Bundles ◽  
Z Ring ◽  
Local Cell

SummaryBacterial cell division requires the assembly of FtsZ protofilaments into a dynamic structure called the ‘Z-ring’. The Z-ring recruits the division machinery and directs local cell wall remodeling for constriction. The organization and dynamics of protofilaments within the Z-ring coordinate local cell wall synthesis during cell constriction, but their regulation is largely unknown. The disordered C-terminal linker (CTL) region ofCaulobacter crescentusFtsZ (CcFtsZ) regulates polymer structure and turnover in solutionin vitro, and regulates Z-ring structure and activity of cell wall enzymesin vivo. To investigate the contributions of the CTL to the polymerization properties of FtsZ on its physiological platform, the cell membrane, we reconstitutedCcFtsZ polymerization on supported lipid bilayers (SLB) and visualized polymer dynamics and structure using total internal reflection fluorescence microscopy. UnlikeE. coliFtsZ protofilaments that organized into large, bundled patterns,CcFtsZ protofilaments assembled into small, dynamic clusters on SLBs. Moreover,CcFtsZ lacking its CTL formed large networks of straight filament bundles that underwent slower turnover than the dynamic clusters of wildtype FtsZ. Ourin vitrocharacterization provides novel insights into species- and CTL-dependent differences between FtsZ assembly properties that are relevant to Z-ring assembly and function on membranesin vivo.

scholarly journals Lateral interactions between protofilaments of the bacterial tubulin homolog FtsZ are essential for cell division

eLife ◽  
2018 ◽  
Vol 7 ◽  
Author(s):  
Fenghui Guan ◽  
Jiayu Yu ◽  
Jie Yu ◽  
Yang Liu ◽  
Ying Li ◽  
...  
Keyword(s):  
Cell Division ◽  
Bacterial Cell ◽  
Dynamic Structure ◽  
Living Cells ◽  
Lateral Interactions ◽  
Bacterial Cell Division ◽  
Z Ring ◽  
Crystallographic Structures ◽  
Order Structures

The prokaryotic tubulin homolog FtsZ polymerizes into protofilaments, which further assemble into higher-order structures at future division sites to form the Z-ring, a dynamic structure essential for bacterial cell division. The precise nature of interactions between FtsZ protofilaments that organize the Z-ring and their physiological significance remain enigmatic. In this study, we solved two crystallographic structures of a pair of FtsZ protofilaments, and demonstrated that they assemble in an antiparallel manner through the formation of two different inter-protofilament lateral interfaces. Our in vivo photocrosslinking studies confirmed that such lateral interactions occur in living cells, and disruption of the lateral interactions rendered cells unable to divide. The inherently weak lateral interactions enable FtsZ protofilaments to self-organize into a dynamic Z-ring. These results have fundamental implications for our understanding of bacterial cell division and for developing antibiotics that target this key process.

scholarly journals Architecture of the ring formed by the tubulin homologue FtsZ in bacterial cell division

eLife ◽  
2014 ◽  
Vol 3 ◽  
Author(s):  
Piotr Szwedziak ◽  
Qing Wang ◽  
Tanmay A M Bharat ◽  
Matthew Tsim ◽  
Jan Löwe
Keyword(s):  
Cell Division ◽  
Molecular Model ◽  
Three Dimensional ◽  
Bacterial Cell Division ◽  
Electron Cryomicroscopy ◽  
E Coli ◽  
Ftsz Ring ◽  
Z Ring

Membrane constriction is a prerequisite for cell division. The most common membrane constriction system in prokaryotes is based on the tubulin homologue FtsZ, whose filaments in E. coli are anchored to the membrane by FtsA and enable the formation of the Z-ring and divisome. The precise architecture of the FtsZ ring has remained enigmatic. In this study, we report three-dimensional arrangements of FtsZ and FtsA filaments in C. crescentus and E. coli cells and inside constricting liposomes by means of electron cryomicroscopy and cryotomography. In vivo and in vitro, the Z-ring is composed of a small, single-layered band of filaments parallel to the membrane, creating a continuous ring through lateral filament contacts. Visualisation of the in vitro reconstituted constrictions as well as a complete tracing of the helical paths of the filaments with a molecular model favour a mechanism of FtsZ-based membrane constriction that is likely to be accompanied by filament sliding.

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

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

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

2018 ◽  
Vol 9 (1) ◽  
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

scholarly journals Publisher Correction: Z-ring membrane anchors associate with cell wall synthases to initiate bacterial cell division

2019 ◽  
Vol 10 (1) ◽  
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

scholarly journals The Chaperonin GroESL Facilitates Caulobacter crescentus Cell Division by Supporting the Functions of the Z-Ring Regulators FtsA and FzlA

mBio ◽  
2021 ◽  
Vol 12 (3) ◽  
Author(s):  
Kristen Schroeder ◽  
Kristina Heinrich ◽  
Ines Neuwirth ◽  
Kristina Jonas
Keyword(s):  
Cell Cycle ◽  
Cell Wall ◽  
Cell Division ◽  
Bacterial Cell ◽  
Antimicrobial Agents ◽  
Caulobacter Crescentus ◽  
Content Type ◽  
Growing Cell ◽  
Z Ring ◽  
Bacterial Cell Cycle

ABSTRACT The highly conserved chaperonin GroESL performs a crucial role in protein folding; however, the essential cellular pathways that rely on this chaperone are underexplored. Loss of GroESL leads to severe septation defects in diverse bacteria, suggesting the folding function of GroESL may be integrated with the bacterial cell cycle at the point of cell division. Here, we describe new connections between GroESL and the bacterial cell cycle using the model organism Caulobacter crescentus. Using a proteomics approach, we identify candidate GroESL client proteins that become insoluble or are degraded specifically when GroESL folding is insufficient, revealing several essential proteins that participate in cell division and peptidoglycan biosynthesis. We demonstrate that other cell cycle events, such as DNA replication and chromosome segregation, are able to continue when GroESL folding is insufficient. We further find that deficiency of two FtsZ-interacting proteins, the bacterial actin homologue FtsA and the constriction regulator FzlA, mediate the GroESL-dependent block in cell division. Our data show that sufficient GroESL is required to maintain normal dynamics of the FtsZ scaffold and divisome functionality in C. crescentus. In addition to supporting divisome function, we show that GroESL is required to maintain the flow of peptidoglycan precursors into the growing cell wall. Linking a chaperone to cell division may be a conserved way to coordinate environmental and internal cues that signal when it is safe to divide. IMPORTANCE All organisms depend on mechanisms that protect proteins from misfolding and aggregation. GroESL is a highly conserved molecular chaperone that functions to prevent protein aggregation in organisms ranging from bacteria to humans. Despite detailed biochemical understanding of GroESL function, the in vivo pathways that strictly depend on this chaperone remain poorly defined in most species. This study provides new insights into how GroESL is linked to the bacterial cell division machinery, a crucial target of current and future antimicrobial agents. We identify a functional interaction between GroESL and the cell division proteins FzlA and FtsA, which modulate Z-ring function. FtsA is a conserved bacterial actin homologue, suggesting that as in eukaryotes, some bacteria exhibit a connection between cytoskeletal actin proteins and chaperonins. Our work further defines how GroESL is integrated with cell wall synthesis and illustrates how highly conserved folding machines ensure the functioning of fundamental cellular processes during stress.

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

2020 ◽  
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 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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