| 1. |
Chuyuan Lu, Ryusei Yoshida, Tsutomu Katayama, Shogo Ozaki, Thermotoga maritima oriC involves a DNA unwinding element with distinct modules and a DnaA-oligomerizing region with a novel directional binding mode, Journal of Biological Chemistry, 10.1016/j.jbc.2023.104888, 104888-104888, 2023.07, Initiation of chromosomal replication requires dynamic nucleoprotein complexes. In most eubacteria, the origin oriC contains multiple DnaA box sequences to which the ubiquitous DnaA initiators bind. In Escherichia coli oriC, DnaA boxes sustain construction of higher-order complexes via DnaA-DnaA interactions, promoting the unwinding of the DNA unwinding element (DUE) within oriC and concomitantly binding the single-stranded DUE to install replication machinery. Despite the significant sequence homologies among DnaA proteins, bacterial oriC sequences are highly diverse. The present study investigated the design of oriC (tma-oriC) from Thermotoga maritima, an evolutionarily ancient eubacterium. The minimal tma-oriC sequence includes a DUE and a flanking region containing five DnaA boxes recognized by the cognate DnaA initiator (tmaDnaA). This DUE was comprised of two distinct functional modules, an unwinding module and a tmaDnaA-binding module. Three direct repeats of the trinucleotide TAG within DUE were essential for both unwinding and single-stranded DUE binding by tmaDnaA complexes constructed on the DnaA boxes. Its surrounding AT-rich sequences stimulated only duplex unwinding. Moreover, head-to-tail oligomers of ATP-bound tmaDnaA were constructed within tma-oriC, irrespective of the directions of the DnaA boxes. This binding mode was considered to be induced by flexible swiveling of DnaA domains III and IV, which were responsible for DnaA-DnaA interactions and DnaA box binding, respectively. Phasing of specific tmaDnaA boxes in tma-oriC DNA was also responsible for unwinding. These findings indicate that a single-stranded DUE recruitment mechanism was responsible for unwinding, and would enhance understanding of the fundamental molecular nature of the origin sequences present in evolutionarily divergent bacteria..  |
| 2. |
Ryusei Yoshida, Shogo Ozaki, Hironori Kawakami, Tsutomu Katayama, Single-stranded DNA recruitment mechanism in replication origin unwinding by DnaA initiator protein and HU, an evolutionary ubiquitous nucleoid protein., Nucleic acids research, 10.1093/nar/gkad389, 2023.05, The Escherichia coli replication origin oriC contains the initiator ATP-DnaA-Oligomerization Region (DOR) and its flanking duplex unwinding element (DUE). In the Left-DOR subregion, ATP-DnaA forms a pentamer by binding to R1, R5M and three other DnaA boxes. The DNA-bending protein IHF binds sequence-specifically to the interspace between R1 and R5M boxes, promoting DUE unwinding, which is sustained predominantly by binding of R1/R5M-bound DnaAs to the single-stranded DUE (ssDUE). The present study describes DUE unwinding mechanisms promoted by DnaA and IHF-structural homolog HU, a ubiquitous protein in eubacterial species that binds DNA sequence-non-specifically, preferring bent DNA. Similar to IHF, HU promoted DUE unwinding dependent on ssDUE binding of R1/R5M-bound DnaAs. Unlike IHF, HU strictly required R1/R5M-bound DnaAs and interactions between the two DnaAs. Notably, HU site-specifically bound the R1-R5M interspace in a manner stimulated by ATP-DnaA and ssDUE. These findings suggest a model that interactions between the two DnaAs trigger DNA bending within the R1/R5M-interspace and initial DUE unwinding, which promotes site-specific HU binding that stabilizes the overall complex and DUE unwinding. Moreover, HU site-specifically bound the replication origin of the ancestral bacterium Thermotoga maritima depending on the cognate ATP-DnaA. The ssDUE recruitment mechanism could be evolutionarily conserved in eubacteria.. |
| 3. |
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.. |
| 4. |
Yukari Sakiyama, Mariko Nagata, Ryusei Yoshida, Kazutoshi Kasho, Shogo Ozaki, Tsutomu Katayama, Concerted actions of DnaA complexes with DNA-unwinding sequences within and flanking replication origin oriC promote DnaB helicase loading, Journal of Biological Chemistry, 10.1016/j.jbc.2022.102051, 298, 6, 102051-102051, 2022.06. |
| 5. |
Kenya Miyoshi, Yuka Tatsumoto, Shogo Ozaki, Tsutomu Katayama, Negative feedback for DARS2–Fis complex by ATP–DnaA supports the cell cycle-coordinated regulation for chromosome replication, Nucleic Acids Research, 10.1093/nar/gkab1171, 2021.11. |
| 6. |
Alberto Reinders, Benjamin Sellner, Firas Fadel, Margo van Berkum, Andreas Kaczmarczyk, Shogo Ozaki, Johanna Rueher, Pablo Manfredi, Matteo Sangermani, Alexander Harms, Camilo Perez, Tilman Schirmer, Urs Jenal, Digital control of c-di-GMP in E. coli balances population-wide developmental transitions and phage sensitivity, bioRxiv, 10.1101/2021.10.01.462762, 2021.10, AbstractNucleotide-based signaling molecules (NSMs) are widespread in bacteria and eukaryotes, where they control important physiological and behavioral processes. In bacteria, NSM-based regulatory networks are highly complex, entailing large numbers of enzymes involved in the synthesis and degradation of active signaling molecules. How the converging input from multiple enzymes is transformed into robust and unambiguous cellular responses has remained unclear. Here we show that Escherichia coli converts dynamic changes of c-di-GMP into discrete binary signaling states, thereby generating heterogeneous populations with either high or low c-di-GMP. This is mediated by an ultrasensitive switch protein, PdeL, which senses the prevailing cellular concentration of the signaling molecule and couples this information to c-di-GMP degradation and transcription feedback boosting its own expression. We demonstrate that PdeL acts as a digital filter that facilitates precise developmental transitions, confers cellular memory, and generates functional heterogeneity in bacterial populations to evade phage predation. Based on our findings, we propose that bacteria apply ultrasensitive regulatory switches to convert dynamic changes of NSMs into binary signaling modes to allow robust decision-making and bet-hedging for improved overall population fitness.. |
| 7. |
Shogo Ozaki, Yasutaka Wakasugi, Tsutomu Katayama, Z-Ring-Associated Proteins Regulate Clustering of the Replication Terminus-Binding Protein ZapT in Caulobacter crescentus, mBio, 10.1128/mBio.02196-20, 2021.01. |
| 8. |
Chihiro Hayashi, Erika Miyazaki, Shogo Ozaki, Yoshito Abe, Tsutomu Katayama, DnaB helicase is recruited to the replication initiation complex via binding of DnaA domain I to the lateral surface of the DnaB N-terminal domain, The Journal of biological chemistry, 10.1074/jbc.RA120.014235, 295, 32, 11131-11143, 2020.08, The DNA replication protein DnaA in Escherichia coli constructs higher-order complexes on the origin, oriC, to unwind this region. DnaB helicase is loaded onto unwound oriC via interactions with the DnaC loader and the DnaA complex. The DnaB-DnaC complex is recruited to the DnaA complex via stable binding of DnaB to DnaA domain I. The DnaB-DnaC complex is then directed to unwound oriC via a weak interaction between DnaB and DnaA domain III. Previously, we showed that Phe46 in DnaA domain I binds to DnaB. Here, we searched for the DnaA domain I-binding site in DnaB. The DnaB L160A variant was impaired in binding to DnaA complex on oriC but retained its DnaC-binding and helicase activities. DnaC binding moderately stimulated DnaA binding of DnaB L160A, and loading of DnaB L160A onto oriC was consistently and moderately inhibited. In a helicase assay with partly single-stranded DNA bearing a DnaA-binding site, DnaA stimulated DnaB loading, which was strongly inhibited in DnaB L160A even in the presence of DnaC. DnaB L160A was functionally impaired in vivo On the basis of these findings, we propose that DnaB Leu160 interacts with DnaA domain I Phe46 DnaB Leu160 is exposed on the lateral surface of the N-terminal domain, which can explain unobstructed interactions of DnaA domain I-bound DnaB with DnaC, DnaG primase, and DnaA domain III. We propose a probable structure for the DnaA-DnaB-DnaC complex, which could be relevant to the process of DnaB loading onto oriC.. |
| 9. |
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.. |
| 10. |
Shogo Ozaki, Regulation of replication initiation
Lessons from Caulobacter crescentus, Genes and Genetic Systems, 10.1266/ggs.19-00011, 94, 5, 183-196, 2019.01, Chromosome replication is a fundamental process in all domains of life. To accurately transmit genetic material to offspring, the initiation of chromosome replication is tightly regulated to ensure that it occurs only once in each cell division cycle. In the model bacterium Caulobacter crescentus, the CtrA response regulator inhibits the origin of replication at the pre-replication stage. Inactivation of CtrA permits the universal DnaA initiator to form an initiation complex at the origin, leading to replication initiation. Subsequently, the initiation complex is inac-tivated to prevent extra initiation. Whereas DNA replication occurs periodically in exponentially growing cells, replication initiation is blocked under various stress conditions to halt cell cycle progression until the normal condition is restored or the cells adapt to the stress. Thus, regulating the initiation complex plays an important role in not only driving cell cycle progression, but also maintaining cell integrity under stress. Multiple regulatory signaling pathways controlling CtrA and DnaA have been identified and recent studies have advanced our knowledge of the underlying mechanistic and molecular processes. This review focuses on how bacterial cells control replication initiation, highlighting the latest findings that have emerged from studies in C. crescentus.. |
| 11. |
Ryo Sugiyama, Kazutoshi Kasho, Kenya Miyoshi, Shogo Ozaki, Wataru Kagawa, Hitoshi Kurumizaka, Tsutomu Katayama, A novel mode of DnaA-DnaA interaction promotes ADP dissociation for reactivation of replication initiation activity, Nucleic acids research, 10.1093/nar/gkz795, 47, 21, 11209-11224, 2019.12, ATP-DnaA is temporally increased to initiate replication during the cell cycle. Two chromosomal loci, DARS (DnaA-reactivating sequences) 1 and 2, promote ATP-DnaA production by nucleotide exchange of ADP-DnaA for timely initiation. ADP-DnaA complexes are constructed on DARS1 and DARS2, bearing a cluster of three DnaA-binding sequences (DnaA boxes I-III), promoting ADP dissociation. Although DnaA has an AAA+ domain, which ordinarily directs construction of oligomers in a head-to-tail manner, DnaA boxes I and II are oriented oppositely. In this study, we constructed a structural model of a head-to-head dimer of DnaA AAA+ domains, and analyzed residues residing on the interface of the model dimer. Gln208 was specifically required for DARS-dependent ADP dissociation in vitro, and in vivo analysis yielded consistent results. Additionally, ADP release from DnaA protomers bound to DnaA boxes I and II was dependent on Gln208 of the DnaA protomers, and DnaA box III-bound DnaA did not release ADP nor require Gln208 for ADP dissociation by DARS-DnaA complexes. Based on these and other findings, we propose a model for DARS-DnaA complex dynamics during ADP dissociation, and provide novel insight into the regulatory mechanisms of DnaA and the interaction modes of AAA+ domains.. |
| 12. |
Saki Taniguchi, Kazutoshi Kasho, Shogo Ozaki, Tsutomu Katayama, Escherichia coli CrfC protein, a nucleoid partition factor, localizes to nucleoid poles via the activities of specific nucleoid-associated proteins, Frontiers in Microbiology, 10.3389/fmicb.2019.00072, 10, FEB, 2019.01, The Escherichia coli CrfC protein is an important regulator of nucleoid positioning and equipartition. Previously we revealed that CrfC homo-oligomers bind the clamp, a DNA-binding subunit of the DNA polymerase III holoenzyme, promoting colocalization of the sister replication forks, which ensures the nucleoid equipartition. In addition, CrfC localizes at the cell pole-proximal loci via an unknown mechanism. Here, we demonstrate that CrfC localizes to the distinct subnucleoid structures termed nucleoid poles (the cell pole-proximal nucleoid-edges) even in elongated cells as well as in wild-type cells. Systematic analysis of the nucleoid-associated proteins (NAPs) and related proteins revealed that HU, the most abundant NAP, and SlmA, the nucleoid occlusion factor regulating the localization of cell division apparatus, promote the specific localization of CrfC foci. When the replication initiator DnaA was inactivated, SlmA and HU were required for formation of CrfC foci. In contrast, when the replication initiation was inhibited with a specific mutant of the helicase-loader DnaC, CrfC foci were sustained independently of SlmA and HU. H-NS, which forms clusters on AT-rich DNA regions, promotes formation of CrfC foci as well as transcriptional regulation of crfC. In addition, MukB, the chromosomal structure mainetanice protein, and SeqA, a hemimethylated nascent DNA region-binding protein, moderately stimulated formation of CrfC foci. However, IHF, a structural homolog of HU, MatP, the replication terminus-binding protein, Dps, a stress-response factor, and FtsZ, an SlmA-interacting factor in cell division apparatus, little or only slightly affected CrfC foci formation and localization. Taken together, these findings suggest a novel and unique mechanism that CrfC localizes to the nucleoid poles in two steps, assembly and recruitment, dependent upon HU, MukB, SeqA, and SlmA, which is stimulated directly or indirectly by H-NS and DnaA. These factors might concordantly affect specific nucleoid substructures. Also, these nucleoid dynamics might be significant in the role for CrfC in chromosome partition.. |
| 13. |
Yukari Sakiyama, Masahiro Nishimura, Chihiro Hayashi, Yusuke Akama, Shogo Ozaki, Tsutomu Katayama, The DnaA AAA+ Domain His136 residue directs DnaB replicative helicase to the unwound region of the replication origin, oriC, Frontiers in Microbiology, 10.3389/fmicb.2018.02017, 9, AUG, 2018.08, Chromosomal replication initiation requires dynamic mechanisms in higher-order nucleoprotein complexes that are constructed at the origin of replication. In Escherichia coli, DnaA molecules construct functional oligomers at the origin oriC, enabling localized unwinding of oriC and stable binding of DnaB helicases via multiple domain I molecules of oriC-bound DnaA. DnaA-bound DnaB helicases are then loaded onto the unwound region of oriC for construction of a pair of replisomes for bidirectional replication. However, mechanisms of DnaB loading to the unwound oriC remain largely elusive. In this study, we determined that His136 of DnaA domain III has an important role in loading of DnaB helicases onto the unwound oriC. DnaA H136A mutant protein was impaired in replication initiation in vivo, and in DnaB loading to the unwound oriC in vitro, whereas the protein fully sustained activities for oriC unwinding and DnaA domain I-dependent stable binding between DnaA and DnaB. Functional and structural analyses supported the idea that transient weak interactions between DnaB helicase and DnaA His136 within specific protomers of DnaA oligomers direct DnaB to a region in close proximity to single stranded DNA at unwound oriC bound to DnaA domain III of the DnaA oligomer. The aromatic moiety of His136 is basically conserved at corresponding residues of eubacterial DnaA orthologs, implying that the guidance function of DnaB is common to all eubacterial species.. |
| 14. |
Badri N. Dubey, Christian Lori, Shogo Ozaki, Geoffrey Fucile, Ivan Plaza-Menacho, Urs Jenal, Tilman Schirmer, Cyclic di-GMP mediates a histidine kinase/phosphatase switch by noncovalent domain cross-linking, Science advances, 10.1126/sciadv.1600823, 2, 9, 2016.09, Histidine kinases are key components of regulatory networks in bacteria. Although many of these enzymes are bifunctional, mediating both phosphorylation and dephosphorylation of downstream targets, the molecular details of this central regulatory switch are unclear. We showed recently that the universal second messenger cyclic di–guanosine monophosphate (c-di-GMP) drives Caulobacter crescentus cell cycle progression by forcing the cell cycle kinase CckA from its default kinase into phosphatase mode. We use a combination of structure determination, modeling, and functional analysis to demonstrate that c-di-GMP reciprocally regulates the two antagonistic CckA activities through noncovalent cross-linking of the catalytic domain with the dimerization histidine phosphotransfer (DHp) domain. We demonstrate that both c-di-GMP and ADP (adenosine diphosphate) promote phosphatase activity and propose that c-di-GMP stabilizes the ADP-bound quaternary structure, which allows the receiver domain to access the dimeric DHp stem for dephosphorylation. In silico analyses predict that c-di-GMP control is widespread among bacterial histidine kinases, arguing that it can replace or modulate canonical transmembrane signaling.. |
| 15. |
C. Lori, Shogo Ozaki, S. Steiner, R. Böhm, S. Abel, B. N. Dubey, T. Schirmer, S. Hiller, U. Jenal, Cyclic di-GMP acts as a cell cycle oscillator to drive chromosome replication, Nature, 10.1038/nature14473, 523, 7559, 236-239, 2015.07, Fundamental to all living organisms is the capacity to coordinate cell division and cell differentiation to generate appropriate numbers of specialized cells. Whereas eukaryotes use cyclins and cyclin-dependent kinases to balance division with cell fate decisions, equivalent regulatory systems have not been described in bacteria. Moreover, the mechanisms used by bacteria to tune division in line with developmental programs are poorly understood. Here we show that Caulobacter crescentus, a bacterium with an asymmetric division cycle, uses oscillating levels of the second messenger cyclic diguanylate (c-di-GMP) to drive its cell cycle. We demonstrate that c-di-GMP directly binds to the essential cell cycle kinase CckA to inhibit kinase activity and stimulate phosphatase activity. An upshift of c-di-GMP during the G1-S transition switches CckA from the kinase to the phosphatase mode, thereby allowing replication initiation and cell cycle progression. Finally, we show that during division, c-di-GMP imposes spatial control on CckA to install the replication asymmetry of future daughter cells. These studies reveal c-di-GMP to be a cyclin-like molecule in bacteria that coordinates chromosome replication with cell morphogenesis in Caulobacter. The observation that c-di-GMP-mediated control is conserved in the plant pathogen Agrobacterium tumefaciens suggests a general mechanism through which this global regulator of bacterial virulence and persistence coordinates behaviour and cell proliferation.. |
| 16. |
Alberto Reinders, Chee Seng Hee, Shogo Ozaki, Adam Mazur, Alex Boehm, Tilman Schirmer, Urs Jenal, Expression and genetic activation of cyclic di-GMP-specific phosphodiesterases in Escherichia coli, Journal of Bacteriology, 10.1128/JB.00604-15, 198, 3, 448-462, 2015.01, Intracellular levels of the bacterial second messenger cyclic di-GMP (c-di-GMP) are controlled by antagonistic activities of diguanylate cyclases and phosphodiesterases. The phosphodiesterase PdeH was identified as a key regulator of motility in Escherichia coli, while deletions of any of the other 12 genes encoding potential phosphodiesterases did not interfere with motility. To analyze the roles of E. coli phosphodiesterases, we demonstrated that most of these proteins are expressed under laboratory conditions. We next isolated suppressor mutations in six phosphodiesterase genes, which reinstate motility in the absence of PdeH by reducing cellular levels of c-di-GMP. Expression of all mutant alleles also led to a reduction of biofilm formation. Thus, all of these proteins are bona fide phosphodiesterases that are capable of interfering with different c-di-GMP-responsive output systems by affecting the global c-di-GMP pool. This argues that E. coli possesses several phosphodiesterases that are inactive under laboratory conditions because they lack appropriate input signals. Finally, one of these phosphodiesterases, PdeL, was studied in more detail. We demonstrated that this protein acts as a transcription factor to control its own expression. Motile suppressor alleles led to a strong increase of PdeL activity and elevated pdeL transcription, suggesting that enzymatic activity and transcriptional control are coupled. In agreement with this, we showed that overall cellular levels of c-di-GMP control pdeL transcription and that this control depends on PdeL itself. We thus propose that PdeL acts both as an enzyme and as a c-di-GMP sensor to couple transcriptional activity to the c-di-GMP status of the cell.. |
| 17. |
Shogo Ozaki, Annina Schalch-Moser, Ludwig Zumthor, Pablo Manfredi, Anna Ebbensgaard, Tilman Schirmer, Urs Jenal, Activation and polar sequestration of PopA, a c-di-GMP effector protein involved in Caulobacter crescentus cell cycle control, Molecular Microbiology, 10.1111/mmi.12777, 94, 3, 580-594, 2014.01, Summary: When Caulobacter crescentus enters S-phase the replication initiation inhibitor CtrA dynamically positions to the old cell pole to be degraded by the polar ClpXP protease. Polar delivery of CtrA requires PopA and the diguanylate cyclase PleD that positions to the same pole. Here we present evidence that PopA originated through gene duplication from its paralogue response regulator PleD and subsequent co-option as c-di-GMP effector protein. While the C-terminal catalytic domain (GGDEF) of PleD is activated by phosphorylation of the N-terminal receiver domain, functional adaptation has reversed signal transduction in PopA with the GGDEF domain adopting input function and the receiver domain serving as regulatory output. We show that the N-terminal receiver domain of PopA specifically interacts with RcdA, a component required for CtrA degradation. In contrast, the GGDEF domain serves to target PopA to the cell pole in response to c-di-GMP binding. In agreement with the divergent activation and targeting mechanisms, distinct markers sequester PleD and PopA to the old cell pole upon S-phase entry. Together these data indicate that PopA adopted a novel role as topology specificity factor to help recruit components of the CtrA degradation pathway to the protease specific old cell pole of C. crescentus.. |
| 18. |
Nicole J. Davis, Yaniv Cohen, Stefano Sanselicio, Coralie Fumeaux, Shogo Ozaki, Jennifer Luciano, Ricardo C. Guerrero-Ferreira, Elizabeth R. Wright, Urs Jenal, Patrick H. Viollier, De- and repolarization mechanism of flagellar morphogenesis during a bacterial cell cycle, Genes and Development, 10.1101/gad.222679.113, 27, 18, 2049-2062, 2013.09, Eukaryotic morphogenesis is seeded with the establishment and subsequent amplification of polarity cues at key times during the cell cycle, often using (cyclic) nucleotide signals. We discovered that flagellum de- and repolarization in the model prokaryote Caulobacter crescentus is precisely orchestrated through at least three spatiotemporal mechanisms integrated at TipF. We show that TipF is a cell cycle-regulated receptor for the second messenger-bis-(3′-5′)-cyclic dimeric guanosine monophosphate (c-di-GMP)-that perceives and transduces this signal through the degenerate c-di-GMP phosphodiesterase (EAL) domain to nucleate polar flagellum biogenesis. Once c-di-GMP levels rise at the G1 → S transition, TipF is activated, stabilized, and polarized, enabling the recruitment of downstream effectors, including flagellar switch proteins and the PflI positioning factor, at a preselected pole harboring the TipN landmark. These c-di-GMP-dependent events are coordinated with the onset of tipF transcription in early S phase and together enable the correct establishment and robust amplification of TipF-dependent polarization early in the cell cycle. Importantly, these mechanisms also govern the timely removal of TipF at cell division coincident with the drop in c-di-GMP levels, thereby resetting the flagellar polarization state in the next cell cycle after a preprogrammed period during which motility must be suspended.. |
| 19. |
Shogo Ozaki, Yusaku Matsuda, Kenji Keyamura, Hironori Kawakami, Yasunori Noguchi, Kazutoshi Kasho, Komomo Nagata, Tamami Masuda, Yukari Sakiyama, Tsutomu Katayama, A replicase clamp-binding dynamin-like protein promotes colocalization of nascent DNA strands and equipartitioning of chromosomes in E.coli, Cell Reports, 10.1016/j.celrep.2013.07.040, 4, 5, 985-995, 2013.05, In Escherichia coli, bidirectional chromosomal replication is accompanied by the colocalization of sister replication forks. However, the biological significance of this mechanism and the key factors involved are still largely unknown. In this study, we found that a protein, termed CrfC, helps sustain the colocalization of nascent DNA regions of sister replisomes and promote chromosome equipartitioning. CrfC formed homomultimers that bound to multiple molecules of the clamp, a replisome subunit that encircles DNA, and colocalized with nascent DNA regions in a clamp-binding-dependent manner in living cells. CrfC is a dynamin homolog; however, it lacks the typical membrane-binding moiety and instead possesses a clamp-binding motif. Given that clamps remain bound to DNA after Okazaki fragment synthesis, we suggest that CrfC sustains the colocalization of sister replication forks in a unique manner by linking together the clamp-loaded nascent DNA strands, thereby laying the basis for subsequent chromosome equipartitioning. |
| 20. |
Shogo Ozaki, Yasunori Noguchi, Yasuhisa Hayashi, Erika Miyazaki, Tsutomu Katayama, Differentiation of the DnaA-oriC subcomplex for DNA unwinding in a replication initiation complex, Journal of Biological Chemistry, 10.1074/jbc.M112.372052, 287, 44, 37458-37471, 2012.10, Background: Multiple DnaA molecules form highly ordered complexes on the origin DNA to initiate chromosomal replication. Results: Novel structural motifs of DnaA are specifically required for the formation of the DNA unwinding-specific DnaA subcomplex. Conclusion: Distinct inter-DnaA interactions are required for the unwinding-specific subcomplex. Significance: Differentiation of the unwinding-specific subcomplex and a key mechanism underlying it are revealed.. |
| 21. |
Shogo Ozaki, Yasunori Noguchi, Masahiro Nishimura, Tsutomu Katayama, Stable nucleotide binding to DnaA requires a specific glutamic acid residue within the AAA+ box II motif, Journal of Structural Biology, 10.1016/j.jsb.2012.05.001, 179, 2, 242-250, 2012.08, In complex with ATP, but not ADP, DnaA protein multimers unwind a specific region of duplex DNA within the chromosomal replication origin, oriC, triggering a series of reactions that result in initiation of DNA replication. Following replication initiation, ATP hydrolysis, which is coupled to DNA replication, results in the generation of initiation-incompetent ADP-DnaA. Suppression of overinitiation of replication requires that ADP-DnaA complexes be stably maintained until the next round of replication. Thus, the functional and structural requirements that ensure stable nucleotide binding to DnaA are crucial for proper regulation of replication. Here, we demonstrate that Glu143 of DnaA, located within the AAA+ box II N-linker motif, is a key residue involved in stable nucleotide binding. A Glu143 substitution variant of DnaA (DnaA E143A) bound to ADP on ice with an affinity similar to wild-type DnaA, but the resultant ADP-DnaA E143A complex was more labile at 37. °C than wild-type ADP-DnaA complexes. Consistent with this, conversion of ADP-DnaA E143A to ATP-DnaA E143A was stimulated at 37. °C in the presence of ATP, which also stimulated replication of a minichromosome in an in vitro reconstitution reaction. Expression of DnaA E143A in vivo inhibited cell growth in an oriC-dependent manner, suggesting that DnaA E143A caused over-initiation of replication, consistent with the in vitro results. Glu is a highly conserved residue at the corresponding position of γ-proteobacterial DnaA orthologs. Our finding of the novel role for the DnaA N-linker region may represent a conserved function of this motif among those DnaA orthologs.. |
| 22. |
Shogo Ozaki, Tsutomu Katayama, Highly organized DnaA-oriC complexes recruit the single-stranded DNA for replication initiation, Nucleic Acids Research, 10.1093/nar/gkr832, 40, 4, 1648-1665, 2012.02, In Escherichia coli, the replication origin oriC consists of two functional regions: the duplex unwinding element (DUE) and its flanking DnaA-assembly region (DAR). ATP-DnaA molecules multimerize on DAR, unwinding DUE for DnaB helicase loading. However, DUE-unwinding mechanisms and functional structures in DnaA-oriC complexes supporting those remain unclear. Here, using various in vitro reconstituted systems, we identify functionally distinct DnaA sub-complexes formed on DAR and reveal novel mechanisms in DUE unwinding. The DUE-flanking left-half DAR carrying high-affinity DnaA box R1 and the ATP-DnaA-preferential DnaA box R5, τ1-2 and I1-2 sites formed a DnaA sub-complex competent in DUE unwinding and ssDUE binding, thereby supporting basal DnaB loading activity. This sub-complex is further subdivided into two; the DUE-distal DnaA sub-complex formed on the ATP-DnaA-preferential sites binds ssDUE. Notably, the DUE-flanking, DnaA box R1-DnaA sub-complex recruits DUE to the DUE-distal DnaA sub-complex in concert with a DNA-bending nucleoid protein IHF, thereby promoting DUE unwinding and binding of ssDUE. The right-half DAR-DnaA sub-complex stimulated DnaB loading, consistent with in vivo analyses. Similar features are seen in DUE unwinding of the hyperthermophile, Thermotoga maritima, indicating evolutional conservation of those mechanisms.. |
| 23. |
Tsutomu Katayama, Shogo Ozaki, Kenji Keyamura, Kazuyuki Fujimitsu, Regulation of the replication cycle
Conserved and diverse regulatory systems for DnaA and oriC, Nature Reviews Microbiology, 10.1038/nrmicro2314, 8, 3, 163-170, 2010.03, Chromosomal replication must be limited to once and only once per cell cycle. This is accomplished by multiple regulatory pathways that govern initiator proteins and replication origins. A principal feature of DNA replication is the coupling of the replication reaction to negative-feedback regulation. Some of the factors that are important in this process have been discovered, including the clamp (DNA polymerase III subunit-Β (DnaN)), the datA locus, SeqA, DnaA homologue protein (Hda) and YabA, as well as factors that are involved at other stages of the regulatory mechanism, such as DnaA initiator-associating protein (DiaA), the DnaA-reactivating sequence (DARS) loci and Soj. Here, we describe the regulation of DnaA, one of the central proteins involved in bacterial DNA replication, by these factors in Escherichia coli, Bacillus subtilis and Caulobacter crescentus.. |
| 24. |
Shogo Ozaki, Tsutomu Katayama, DnaA structure, function, and dynamics in the initiation at the chromosomal origin, Plasmid, 10.1016/j.plasmid.2009.06.003, 62, 2, 71-82, 2009.09, Escherichia coli DnaA is the initiator of chromosomal replication. Multiple ATP-DnaA molecules assemble at the oriC replication origin in a highly regulated manner, and the resultant initiation complexes promote local duplex unwinding within oriC, resulting in open complexes. DnaB helicase is loaded onto the unwound single-stranded region within oriC via interaction with the DnaA multimers. The tertiary structure of the functional domains of DnaA has been determined and several crucial residues in the initiation process, as well as their unique functions, have been identified. These include specific DNA binding, inter-DnaA interaction, specific and regulatory interactions with ATP and with the unwound single-stranded oriC DNA, and functional interaction with DnaB helicase. An overall structure of the initiation complex is also proposed. These are important for deepening our understanding of the molecular mechanisms that underlie DnaA assembly, oriC duplex unwinding, regulation of the initiation reaction, and DnaB helicase loading. In this review, we summarize recent progress on the molecular mechanisms of the functions of DnaA on oriC. In addition, some members of the AAA+ protein family related to the initiation of replication and its regulation (e.g., DnaA) are briefly discussed.. |
| 25. |
Shogo Ozaki, Hironori Kawakami, Kenta Nakamura, Norie Fujikawa, Wataru Kagawa, Sam Yong Park, Shigeyuki Yokoyama, Hitoshi Kurumizaka, Tsutomu Katayama, A common mechanism for the ATP-DnaA-dependent formation of open complexes at the replication origin, Journal of Biological Chemistry, 10.1074/jbc.M708684200, 283, 13, 8351-8362, 2008.03, Initiation of chromosomal replication and its cell cycle-coordinated regulation bear crucial and fundamental mechanisms in most cellular organisms. Escherichia coli DnaA protein forms a homomultimeric complex with the replication origin (oriC). ATP-DnaA multimers unwind the duplex within the oriC unwinding element (DUE). In this study, structural analyses suggested that several residues exposed in the central pore of the putative structure of DnaA multimers could be important for unwinding. Using mutation analyses, we found that, of these candidate residues, DnaA Val-211 and Arg-245 are prerequisites for initiation in vivo and in vitro. Whereas DnaA V211A and R245A proteins retained normal affinities for ATP/ADP and DNA and activity for the ATP-specific conformational change of the initiation complex in vitro, oriC complexes of these mutant proteins were inactive in DUE unwinding and in binding to the single-stranded DUE. Unlike oriC complexes including ADP-DnaA or the mutant DnaA, ATP-DnaA-oriC complexes specifically bound the upper strand of single-stranded DUE. Specific T-rich sequences within the strand were required for binding. The corresponding conserved residues of the DnaA ortholog in Thermotoga maritima, an ancient eubacterium, were also required for DUE unwinding, consistent with the idea that the mechanism and regulation for DUE unwinding can be evolutionarily conserved. These findings provide novel insights into mechanisms for pore-mediated origin unwinding, ATP/ADP-dependent regulation, and helicase loading of the initiation complex.. |
| 26. |
Kenji Keyamura, Norie Fujikawa, Takuma Ishida, Shogo Ozaki, Masayuki Su'etsugu, Kazuyuki Fujimitsu, Wataru Kagawa, Shigeyuki Yokoyama, Hitoshi Kurumizaka, Tsutomu Katayama, The interaction of DiaA and DnaA regulates the replication cycle in E. coli by directly promoting ATP-DnaA-specific initiation complexes, Genes and Development, 10.1101/gad.1561207, 21, 16, 2083-2099, 2007.08, Escherichia coli DiaA is a DnaA-binding protein that is required for the timely initiation of chromosomal replication during the cell cycle. In this study, we determined the crystal structure of DiaA at 1.8 Å resolution. DiaA forms a homotetramer consisting of a symmetrical pair of homodimers. Mutational analysis revealed that the DnaA-binding activity and formation of homotetramers are required for the stimulation of initiation by DiaA. DiaA tetramers can bind multiple DnaA molecules simultaneously. DiaA stimulated the assembly of multiple DnaA molecules on oriC, conformational changes in ATP-DnaA-specific initiation complexes, and unwinding of oriC duplex DNA. The mutant DiaA proteins are defective in these stimulations. DiaA associated also with ADP-DnaA, and stimulated the assembly of inactive ADP-DnaA-oriC complexes. Specific residues in the putative phosphosugar-binding motif of DiaA were required for the stimulation of initiation and formation of ATP-DnaA-specific- oriC complexes. Our data indicate that DiaA regulates initiation by a novel mechanism, in which DiaA tetramers most likely bind to multiple DnaA molecules and stimulate the assembly of specific ATP-DnaA-oriC complexes. These results suggest an essential role for DiaA in the promotion of replication initiation in a cell cycle coordinated manner.. |
| 27. |
Hironori Kawakami, Shogo Ozaki, Shigeo Suzuki, Kenta Nakamura, Takayuki Senriuchi, Masayuki Su'etsugu, Kazuyuki Fujimitsu, Tsutomu Katayama, The exceptionally tight affinity of DnaA for ATP/ADP requires a unique aspartic acid residue in the AAA+ sensor 1 motif, Molecular Microbiology, 10.1111/j.1365-2958.2006.05450.x, 62, 5, 1310-1324, 2006.12, Escherichia coli DnaA, an AAA+ superfamily protein, initiates chromosomal replication in an ATP-binding-dependent manner. Although DnaA has conserved Walker A/B motifs, it binds adenine nucleotides 10- to 100-fold more tightly than do many other AAA+ proteins. This study shows that the DnaA Asp-269 residue, located in the sensor 1 motif, plays a specific role in supporting high-affinity ATP/ADP binding. The affinity of the DnaA D269A mutant for ATP/ADP is at least 10- to 100-fold reduced compared with that of the wild-type and DnaA R270A proteins. In contrast, the abilities of DnaA D269A to bind a typical DnaA box, unwind oriC duplex in the presence of elevated concentrations of ATP, load DnaB onto DNA and support minichromosomal replication in a reconstituted system are retained. Whereas the acidic Asp residue is highly conserved among eubacterial DnaA homologues, the corresponding residue in many other AAA+ proteins is Asn/Thr and in some AAA+ proteins these neutral residues are essential for ATP hydrolysis but not ATP binding. As the intrinsic ATPase activity of DnaA is extremely weak, this study reveals a novel and specific function for the sensor 1 motif in tight ATP/ADP binding, one that depends on the alternate key residue Asp.. |
| 28. |
Shogo Ozaki, Kazuyuki Fujimitsu, Hitoshi Kurumizaka, Tsutomu Katayama, The DnaA homolog of the hyperthermophilic eubacterium Thermotoga maritima an open complex with a minimal 149-bp origin region in an ATP-dependent manner, Genes to Cells, 10.1111/j.1365-2443.2006.00950.x, 11, 4, 425-438, 2006.04, In Escherichia coli, ATP-DnaA, but not ADP-DnaA, forms an initiation complex that undergoes site-specific duplex DNA unwinding, open complex formation. However, it remains unclear how highly the ATP-dependent activation of the initiation factor is conserved in evolution. The hyperthermophile Thermotoga maritima is one of the most ancient eubacteria in evolution. Here, we show that the DnaA homolog (tmaDnaA) of this bacterium forms open complexes with the predicted origin region (tma-oriC) in vitro. Tma DnaA has a strong and specific affinity for ATP/ADP as well as for 12-mer repeating sequences within the tma-oriC. Unlike ADPtma DnaA, ATPtma DnaA ishighly cooperative in DNA binding and forms open complexes in a manner that depends on temperature and the superhelical tension of the tma-oriC-bearing plasmid. The minimal tma-oriC required for unwinding is a 149-bp region containing five repeats of the 12-mer sequence and two AT-rich 9-mer repeats. Tma DnaA-binding to the 12-mer motif provokes DNA bending. The 9-mer region is the duplex-unwinding site. The tma DnaA-binding and unwinding motifs of tma-oriC share sequence homology with corresponding archaeal and eukaryotic sequences. These findings suggest that the ATP-dependent molecular switch of the initiator and the mechanisms in the replication initiation complex are highly conserved in eubacterial evolution.. |
| 29. |
Toh Ru Shimuta, Kiyotaka Nakano, Yoko Yamaguchi, Shogo Ozaki, Kazuyuki Fujimitsu, Chika Matsunaga, Kenji Noguchi, Akiko Emoto, Tsutomu Katayama, Novel heat shock protein HspQ stimulates the degradation of mutant DnaA protein in Escherichia coli, Genes to Cells, 10.1111/j.1365-2443.2004.00800.x, 9, 12, 1151-1166, 2004.12, Escherichia coli DnaA protein initiates chromosomal replication and is an important regulatory target during the replication cycle. In this study, a suppressor mutation isolated by transposon mutagenesis was found to allow growth of the temperature-sensitive dnaA508 and dnaA167 mutants at 40 °C. The suppressor consists of a transposon insertion in a previously annotated ORF, here termed hspQ, a novel heat shock gene whose promoter is recognized by the major heat shock sigma factor σ 32. Expression of hspQ on a pBR322 derivative inhibits growth of the dnaA508 and dnaA167 mutants at 30 °C, whereas growth of dnaA46 and other dnaA mutants is insensitive to changes in the level of hspQ. Cellular DnaA308 protein is degraded rapidly at elevated temperature, but hspQ disruption impedes this process. In contrast, DnaA46 protein is rapidly degraded in an hspQ-independent manner. Gel-filtration and chemical cross-linking experiments suggest that HspQ forms a stable homodimer in solution and can form homomultimers consisting of about four monomers. Heat-shock induced proteases such as Clp contain homomultimers of subunit proteins. We propose that HspQ is a new factor involved in the quality control of proteins and that it functions by excluding denatured proteins.. |
