Kyushu University [Ozaki Shogo (Associate Professor) Faculty of Pharmaceutical Sciences, Department of Pharmaceutical Health Care and Sciences]

Growing bacteria require careful tuning of cell division processes with dynamic organization of replicating chromosomes. The objective of our project is to understand molecular mechanisms for coordination of chromosome replication with division. To achieve this we make use of different model bacteria including Escherichia coli, Caulobacter crescentus, and Thermotoga maritima.

Research

Research Interests

Mechanism and regulation for initiation complex that promotes local duplex DNA unwinding at replication origin during the initiation of chromosomal replication
keyword : DNA replication, cell cycle, DnaA, AAA+, conformational change
2005.10.

Academic Activities

Reports

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1.	Katayama, T, Ozaki, S, Keyamura, K, Fujimitsu, K., Regulation of the replication cycle: conserved and diverse regulatory systems for DnaA and oriC, Nat Rev Microbiol, 2010, 2010.03.
2.	Ozaki S., Katayama T., DnaA structure, function, and dynamics in the initiation at the chromosomal origin., Plasmid, 2009.07.

Papers

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Shogo Ozaki, Dengyu Wang, Yasutaka Wakasugi, Naoto Itani, Tsutomu Katayama, The Caulobacter crescentus DciA promotes chromosome replication through topological loading of the DnaB replicative helicase at replication forks, Nucleic Acids Research, 10.1093/nar/gkac1146, 2022.12, Abstract

The replicative DNA helicase translocates on single-stranded DNA to drive replication forks during chromosome replication. In most bacteria the ubiquitous replicative helicase, DnaB, co-evolved with the accessory subunit DciA, but how they function remains incompletely understood. Here, using the model bacterium Caulobacter crescentus, we demonstrate that DciA plays a prominent role in DNA replication fork maintenance. Cell cycle analyses using a synchronized Caulobacter cell population showed that cells devoid of DciA exhibit a severe delay in fork progression. Biochemical characterization revealed that the DnaB helicase in its default state forms a hexamer that inhibits self-loading onto single-stranded DNA. We found that upon binding to DciA, the DnaB hexamer undergoes conformational changes required for encircling single-stranded DNA, thereby establishing the replication fork. Further investigation of the functional structure of DciA revealed that the C-terminus of DciA includes conserved leucine residues responsible for DnaB binding and is essential for DciA in vivo functions. We propose that DciA stimulates loading of DnaB onto single strands through topological isomerization of the DnaB structure, thereby ensuring fork progression. Given that the DnaB-DciA modules are widespread among eubacterial species, our findings suggest that a common mechanism underlies chromosome replication..

Shogo Ozaki, Urs Jenal, Tsutomu Katayama, Novel divisome-associated protein spatially coupling the z-ring with the chromosomal replication terminus in caulobacter crescentus, mBio, 10.1128/mBio.00487-20, 11, 2, 2020.03, 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 inter-acts 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 coin-cided 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 pro-cesses. 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 phylo-genetically 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..

Presentations

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1.	Shogo Ozaki, Dengyu Wang, Yasutaka Wakasugi, Naoto Itani, and Tsutomu Katayama, Analysis on the loading mechanism of the bacterial replicative DnaB helicase in the alpha-proteobacterium Caulobacter crescentus., NIG International Symposium 2022, 2022.10.
2.	Shogo Ozaki,Tsutomu Katayama, Analysis of the chromosomal replication mechanism in the eubacterium Caulobacter crescentus, 日本分子生物学会第44回大会, 2021.12.
3.	Shogo Ozaki、Yasutaka Wakasugi、Urs Jenal, Tsutomu Katayama, A novel divisome-associated protein spatially couples the Z-ring with the chromosomal replication terminus in Caulobacter crescentus, CAULOCONFERENCE 2020, 2020.04.
4.	尾崎省吾, 若杉泰敬, 片山勉, Identification and characterization of a novel DNA binding protein that colocalizes the chromosome replication terminus with cell division apparatus in Caulobacter crescentus, 第４２回日本分子生物学会年会, 2019.12, The bacterial chromosomes are spatially and timely organized to execute chromoso me replication and segregation in a manner coupled to cell division. In the gram -negative, alphaproteobacterium Caulobacter crescentus, subcellular posit ioning of the individual chromosomal loci alters dynamically during chromosome r eplication. In a pre-replication stage, the origin of chromosome replication is sequestered to one pole of the cell while the terminus region is localized at th e other cell pole. Upon initiation of chromosome replication, one of the newly-r eplicated origin DNAs is driven toward the other cell pole to establish bipolar localization of the origin DNAs. Concomitantly, the terminus region is recruited to the midcell position where the bacterial tubulin homolog FtsZ colocalizes to form cytokinetic Z-rings. Although this colocalization likely coordinates cell division with dynamic chromosome architecture, the molecular basis for a physica l link between the Z-rings and the terminus region remains unknown. In this stud y, we identify the terminus-interactive DNA binding protein ZapT and show that i t directly binds to a component associated with the Z-rings. Moreover, the za pT gene is crucial to maintain normal cell division control in C. crescentus . Together, we propose that ZapT acts as a molecular bridge that physically link s the terminus region to the Z-rings, thereby ensuring precise processes in chro mosome segregation and cell division. Because ZapT orthologs are conserved among diverse proteobacterial species, our findings may represent a general mechanism to coordinate cell division with chromosome organization in time and space..
5.	Shogo Ozaki, Lori Christian, Urs Jenal, The second messenger signaling drives chromosome replication in the asymmetrically dividing bacterium Caulobacter crescentus, 第５６回日本生物物理学会年会, 2018.09.
6.	Analysis on the minimal functional structure of the DnaA complex for the regulation of duplex DNA unwinding.
7.	Analysis on the minimal functional elements of the replication origin in duplex DNA unwinding by DnaA.

Membership in Academic Society

American Society for Microbiology
The Japanese Biochemical Society
The Genetics Society of Japan
The Molecular Biology Society of Japan

Awards

Best Papers Awards in the 80th Annual Meeting of the Genetic Society of Japan
Best Papers Awards in the 79th Annual Meeting of the Genetic Society of Japan

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