scholarly journals A conserved coiled-coil protein pair focuses the cytokinetic Z-ring in Caulobacter crescentus

2017 ◽  
Vol 105 (5) ◽  
pp. 721-740 ◽  
Author(s):  
Selamawit Abi Woldemeskel ◽  
Ryan McQuillen ◽  
Alex M. Hessel ◽  
Jie Xiao ◽  
Erin D. Goley
Keyword(s):  
Protein Pair ◽  
Caulobacter Crescentus ◽  
Coiled Coil ◽  
Z Ring

scholarly journals Z-Ring-Associated Proteins Regulate Clustering of the Replication Terminus-Binding Protein ZapT in Caulobacter crescentus

mBio ◽  
2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Shogo Ozaki ◽  
Yasutaka Wakasugi ◽  
Tsutomu Katayama
Keyword(s):  
Caulobacter Crescentus ◽  
Binding Protein ◽  
Specific Interaction ◽  
Common Mechanism ◽  
Dna Complexes ◽  
Content Type ◽  
Physical Linkage ◽  
Associated Proteins ◽  
Z Ring ◽  
Replication Terminus

ABSTRACT Regulated organization of the chromosome is essential for faithful propagation of genetic information. In the model bacterium Caulobacter crescentus, the replication terminus of the chromosome is spatially arranged in close proximity to the cytokinetic Z-ring during the cell cycle. Although the Z-ring-associated proteins ZapA and ZauP interact with the terminus recognition protein ZapT, the molecular functions of the complex that physically links the terminus and the Z-ring remain obscure. In this study, we found that the physical linkage helps to organize the terminus DNA into a clustered structure. Neither ZapA nor ZauP was required for ZapT binding to the terminus DNA, but clustering of the ZapT-DNA complexes over the Z-ring was severely compromised in cells lacking ZapA or ZauP. Biochemical characterization revealed that ZapT, ZauP, and ZapA interacted directly to form a highly ordered ternary complex. Moreover, multiple ZapT molecules were sequestered by each ZauP oligomer. Investigation of the functional structure of ZapT revealed that the C terminus of ZapT specifically interacted with ZauP and was essential for timely positioning of the Z-ring in vivo. Based on these findings, we propose that ZauP-dependent oligomerization of ZapT-DNA complexes plays a distinct role in organizing the replication terminus and the Z-ring. The C termini of ZapT homologs share similar chemical properties, implying a common mechanism for the physical linkage between the terminus and the Z-ring in bacteria. IMPORTANCE Rapidly growing bacteria experience dynamic changes in chromosome architecture during chromosome replication and segregation, reflecting the importance of mechanisms that organize the chromosome globally and locally within a cell to maintain faithful transmission of genetic material across generations. During cell division in the model bacterium Caulobacter crescentus, the replication terminus of the chromosome is physically linked to the cytokinetic Z-ring at midcell. However, the functions of this physical linkage are not fully understood. We adopted biochemical and cell-biological techniques to characterize the linkage, including the terminus-binding protein ZapT and the Z-ring-associated protein ZauP. We obtained evidence that the Z-ring organizes the chromosome terminus into a compact structure at midcell via specific interaction between ZapT and ZauP oligomers. Because these proteins are conserved in diverse Gram-negative bacteria, our findings highlight a novel and conserved role for the linker complex in regulated organization of the chromosome terminus.

scholarly journals High throughput 3D super-resolution microscopy reveals Caulobacter crescentus in vivo Z-ring organization

2014 ◽  
Vol 111 (12) ◽  
pp. 4566-4571 ◽  
Author(s):  
S. J. Holden ◽  
T. Pengo ◽  
K. L. Meibom ◽  
C. Fernandez Fernandez ◽  
J. Collier ◽  
...  
Keyword(s):  
High Throughput ◽  
Caulobacter Crescentus ◽  
Super Resolution ◽  
Z Ring ◽  
Super Resolution Microscopy

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 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 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 Novel Divisome-Associated Protein Spatially Coupling the Z-Ring with the Chromosomal Replication Terminus in Caulobacter crescentus

mBio ◽  
2020 ◽  
Vol 11 (2) ◽  
Author(s):  
Shogo Ozaki ◽  
Urs Jenal ◽  
Tsutomu Katayama
Keyword(s):  
Cell Cycle ◽  
Cell Division ◽  
Caulobacter Crescentus ◽  
Enteric Bacteria ◽  
Chromosome Replication ◽  
Chromosomal Replication ◽  
Content Type ◽  
Physical Linkage ◽  
Z Ring ◽  
Replication Terminus

ABSTRACT Cell division requires proper spatial coordination with the chromosome, which undergoes dynamic changes during chromosome replication and segregation. FtsZ is a bacterial cytoskeletal protein that assembles into the Z-ring, providing a platform to build the cell division apparatus. In the model bacterium Caulobacter crescentus, the cellular localization of the Z-ring is controlled during the cell cycle in a chromosome replication-coupled manner. Although dynamic localization of the Z-ring at midcell is driven primarily by the replication origin-associated FtsZ inhibitor MipZ, the mechanism ensuring accurate positioning of the Z-ring remains unclear. In this study, we showed that the Z-ring colocalizes with the replication terminus region, located opposite the origin, throughout most of the C. crescentus cell cycle. Spatial organization of the two is mediated by ZapT, a previously uncharacterized protein that interacts with the terminus region and associates with ZapA and ZauP, both of which are part of the incipient division apparatus. While the Z-ring and the terminus region coincided with the presence of ZapT, colocalization of the two was perturbed in cells lacking zapT, which is accompanied by delayed midcellular positioning of the Z-ring. Moreover, cells overexpressing ZapT showed compromised positioning of the Z-ring and MipZ. These findings underscore the important role of ZapT in controlling cell division processes. We propose that ZapT acts as a molecular bridge that physically links the terminus region to the Z-ring, thereby ensuring accurate site selection for the Z-ring. Because ZapT is conserved in proteobacteria, these findings may define a general mechanism coordinating cell division with chromosome organization. IMPORTANCE Growing bacteria require careful tuning of cell division processes with dynamic organization of replicating chromosomes. In enteric bacteria, ZapA associates with the cytoskeletal Z-ring and establishes a physical linkage to the chromosomal replication terminus through its interaction with ZapB-MatP-DNA complexes. However, because ZapB and MatP are found only in enteric bacteria, it remains unclear how the Z-ring and the terminus are coordinated in the vast majority of bacteria. Here, we provide evidence that a novel conserved protein, termed ZapT, mediates colocalization of the Z-ring with the terminus in Caulobacter crescentus, a model organism that is phylogenetically distant from enteric bacteria. Given that ZapT facilitates cell division processes in C. crescentus, this study highlights the universal importance of the physical linkage between the Z-ring and the terminus in maintaining cell integrity.

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