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

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.

SUBMITTER: Schroeder K

PROVIDER: S-EPMC8262945 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description: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.

Project description:Cell polarity development is the prerequisite for cell differentiation and generating biodiversity. In the model bacterium Caulobacter crescentus, the polarization of the scaffold protein PopZ during the predivisional cell stage plays a central role in asymmetric cell division. However, our understanding of the spatiotemporal regulation of PopZ localization remains incomplete. In the current study, a direct interaction between PopZ and the new pole scaffold PodJ is revealed, which plays a primary role in triggering the new pole accumulation of PopZ. The coiled-coil 4-6 domain in PodJ is responsible for interacting with PopZ in vitro and promoting PopZ transition from monopolar to bipolar in vivo. Elimination of the PodJ-PopZ interaction impairs the PopZ-mediated chromosome segregation by affecting both the positioning and partitioning of the ParB-parS centromere. Further analyses of PodJ and PopZ from other bacterial species indicate this scaffold-scaffold interaction may represent a widespread strategy for spatiotemporal regulation of cell polarity in bacteria. IMPORTANCE Caulobacter crescentus is a well-established bacterial model to study asymmetric cell division for decades. During cell development, the polarization of scaffold protein PopZ from monopolar to bipolar plays a central role in C. crescentus asymmetric cell division. Nevertheless, the spatiotemporal regulation of PopZ has remained unclear. Here, we demonstrate that the new pole scaffold PodJ functions as a regulator in triggering PopZ bipolarization. The primary regulatory role of PodJ was demonstrated in parallel by comparing it with other known PopZ regulators, such as ZitP and TipN. Physical interaction between PopZ and PodJ ensures the timely accumulation of PopZ at the new cell pole and the inheritance of the polarity axis. Disruption of the PodJ-PopZ interaction impaired PopZ-mediated chromosome segregation and may lead to a decoupling of DNA replication from cell division during the cell cycle. Together, the scaffold-scaffold interaction may provide an underlying infrastructure for cell polarity development and asymmetric cell division.

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

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