Language：English   Publishing type：Research paper (scientific journal)   Publisher：Oxford University Press ({OUP})  

In bacterial synthetic biology, whole genome transplantation has been achieved only in mycoplasmas that contain a small genome and are competent for foreign genome uptake. In this study, we developed Escherichia coli strains programmed by three 1-megabase (Mb) chromosomes by splitting the 3-Mb chromosome of a genome-reduced strain. The first split-chromosome retains the original replication origin (oriC) and partitioning (par) system. The second one has an oriC and the par locus from the F plasmid, while the third one has the ori and par locus of the Vibrio tubiashii secondary chromosome. The tripartite-genome cells maintained the rod-shaped form and grew only twice as slowly as their parent, allowing their further genetic engineering. A proportion of these 1-Mb chromosomes were purified as covalently closed supercoiled molecules with a conventional alkaline lysis method and anion exchange columns. Furthermore, the second and third chromosomes could be individually electroporated into competent cells. In contrast, the first split-chromosome was not able to coexist with another chromosome carrying the same origin region. However, it was exchangeable via conjugation between tripartite-genome strains by using different selection markers. We believe that this E. coli-based technology has the potential to greatly accelerate synthetic biology and synthetic genomics.

DOI： 10.1093/nar/gkab298

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Language：English   Publishing type：Research paper (scientific journal)  

Although Escherichia coli has been a popular tool for plasmid construction, this bacterium was believed to be "unsuitable" for constructing a large plasmid whose size exceeds 500 kilobases. We assumed that traditional plasmid vectors may lack some regulatory DNA elements required for the stable replication and segregation of such a large plasmid. In addition, the use of a few site-specific recombination systems may facilitate cloning of large DNA segments. Here we show two strategies for constructing 1-megabase (1-Mb) secondary chromosomes by using new bacterial artificial chromosome (BAC) vectors. First, the 3-Mb genome of a genome-reduced E. coli strain was split into two chromosomes (2-Mb and 1-Mb), of which the smaller one has the origin of replication and the partitioning locus of the Vibrio tubiashii secondary chromosome. This chromosome fission method (Flp-POP cloning) works via flippase-mediated excision, which coincides with the reassembly of a split chloramphenicol resistance gene, allowing chloramphenicol selection. Next, we developed a new cloning method (oriT-POP cloning) and a fully equipped BAC vector (pMegaBAC1H) for developing a 1-Mb plasmid. Two 0.5-Mb genomic regions were sequentially transferred from two donor strains to a recipient strain via conjugation and captured by pMegaBAC1H in the recipient strain to produce a 1-Mb plasmid. This 1-Mb plasmid was transmissible to another E. coli strain via conjugation. Furthermore, these 1-Mb secondary chromosomes were amplifiable in vitro by using the reconstituted E. coli chromosome replication cycle reaction (RCR). These strategies and technologies would make popular E. coli cells a productive factory for designer chromosome engineering.

DOI： 10.1021/acssynbio.0c00008

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Publishing type：Research paper (scientific journal)   Publisher：American Chemical Society (ACS)  

DOI： 10.1021/acssynbio.9b00055

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Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.3390/life8040043

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DOI： 10.1093/nar/gky481

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Microbiology Society  

WalRK is an essential two-component signal transduction system that plays a central role in coordinating cell wall synthesis and cell growth in Bacillus subtilis. However, the physiological role of WalRK and its essentiality for growth have not been elucidated. We investigated the behaviour of WalRK during heat stress and its essentiality for cell proliferation. We determined that the inactivation of the walHI genes which encode the negative modulator of WalK, resulted in growth defects and eventual cell lysis at high temperatures. Screening of suppressor mutations revealed that the inactivation of LytE, an dl-endopeptidase, restored the growth of the ΔwalHI mutant at high temperatures. Suppressor mutations that reduced heat induction arising from the walRK regulon were also mapped to the walK ORF. Therefore, we hypothesized that overactivation of LytE affects the phenotype of the ΔwalHI mutant. This hypothesis was corroborated by the overexpression of the negative regulator of LytE, IseA and PdaC, which rescued the growth of the ΔwalHI mutant at high temperatures. Elucidating the cause of the temperature sensitivity of the ΔwalHI mutant could explain the essentiality of WalRK. We proved that the constitutive expression of lytE or cwlO using a synthetic promoter uncouples these expressions from WalRK, and renders WalRK nonessential in the pdaC and iseA mutant backgrounds. We propose that the essentiality of WalRK is derived from the coordination of cell wall metabolism with cell growth by regulating dl-endopeptidase activity under various growth conditions.

DOI： 10.1099/mic.0.000625

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Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1093/nar/gkx822

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：CELL PRESS  

The SMC-ScpAB complex plays a crucial role in chromosome organization and segregation in many bacteria. It is composed of a V-shaped SMC dimer and an ScpAB subcomplex that bridges the two Structural Maintenance of Chromosomes (SMC) head domains. Despite its functional significance, the mechanistic details of SMC-ScpAB remain obscure. Here we provide evidence that ATP-dependent head-head engagement induces a lever movement of the SMC neck region, which might help to separate juxtaposed coiled-coil arms. Binding of the ScpA N-terminal domain (NTD) to the SMC neck region is negatively regulated by the ScpB C-terminal domain. Mutations in the ScpA NTD compromise this regulation and profoundly affect the overall shape of the complex. The SMC hinge domain is structurally relaxed when free from coiled-coil juxtaposition. Taken together, we propose that the structural parts of SMC-ScpAB are subjected to the balance between constraint and relaxation, cooperating to modulate dynamic conformational changes of the whole complex.

DOI： 10.1016/j.str.2017.02.008

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Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1111/1462-2920.12147

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：CELL PRESS  

The sliding clamp is an essential component of the replisome required for processivity of DNA synthesis and several other aspects of chromosome metabolism. However, the in vivo dynamics of the clamp are poorly understood. We have used various biochemical and cell biological methods to study the dynamics of clamp association with the replisome in Bacillus subtilis cells. We find that clamps form large assemblies on DNA, called "clamp zones." Loading depends on DnaG primase and is probably driven by Okazaki fragment initiation on the lagging strand. Unloading, which is probably regulated, only occurs after many clamps have accumulated on the DNA. On/off cycling allows chromosomal zones of about 200 accumulated clamps to follow the replisome. Since we also show that clamp zones recruit proteins bearing a clamp-binding sequence to replication foci, the results highlight the clamp as a central organizer in the structure and function of replication foci.

DOI： 10.1016/j.molcel.2011.02.024

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Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1074/jbc.m803158200

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DOI： 10.1128/jb.00044-08

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：COLD SPRING HARBOR LAB PRESS, PUBLICATIONS DEPT  

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 angstrom 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.

DOI： 10.1101/gad.1561207

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Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1111/j.1365-2958.2006.05450.x

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：BLACKWELL PUBLISHING  

The Escherichia coli hda gene codes for a DnaA-related protein that is essential for the regulatory inactivation of DnaA (RIDA), a system that controls the initiation of chromosomal replication. We have identified the ygfZ gene, which encodes a folate-binding protein, as a suppressor of hda mutations. The ygfZ null mutation suppresses an hda null mutation. The over-initiation and abortive elongation phenotypes conferred by the hda mutations are partially suppressed in an hda ygfZ background. The accumulation of the active form of DnaA, ATP-DnaA, in the hda mutant is suppressed by the disruption of the ygfZ gene, indicating that YgfZ is involved in regulating the level of ATP-DnaA. Although ygfZ is not an essential gene, the ygfZ disruptant grows slowly, especially at low temperature, demonstrating that this gene is important for cellular proliferation. We have identified mnmE (trmE) as a suppressor of the ygfZ disruption. This gene encodes a GTPase involved in tRNA modification. Examination of RNA modification in the ygfZ mutant reveals reduced levels of 2-methylthio N-6-isopentenyladenosine, indicating that YgfZ participates in the methylthio-group formation of this modified nucleoside in some tRNAs. These results suggest that YgfZ is a key factor in regulatory networks that act via tRNA modification.

DOI： 10.1111/j.1365-2958.2006.05067.x

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：BLACKWELL PUBLISHING  

The Escherichia coli hda gene codes for a DnaA-related protein that is essential for the regulatory inactivation of DnaA (RIDA), a system that controls the initiation of chromosomal replication. We have identified the ygfZ gene, which encodes a folate-binding protein, as a suppressor of hda mutations. The ygfZ null mutation suppresses an hda null mutation. The over-initiation and abortive elongation phenotypes conferred by the hda mutations are partially suppressed in an hda ygfZ background. The accumulation of the active form of DnaA, ATP-DnaA, in the hda mutant is suppressed by the disruption of the ygfZ gene, indicating that YgfZ is involved in regulating the level of ATP-DnaA. Although ygfZ is not an essential gene, the ygfZ disruptant grows slowly, especially at low temperature, demonstrating that this gene is important for cellular proliferation. We have identified mnmE (trmE) as a suppressor of the ygfZ disruption. This gene encodes a GTPase involved in tRNA modification. Examination of RNA modification in the ygfZ mutant reveals reduced levels of 2-methylthio-6-iodeadenosine, indicating that YgfZ participates in the methylthio-group formation of this modified nucleoside in some tRNAs. These results suggest that YgfZ is a key factor in regulatory networks that act via tRNA modification.

DOI： 10.1111/j.1365-2958.2005.04932.x

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Language：English   Publishing type：Research paper (international conference proceedings)   Publisher：IEEE  

The Escherichia coli initiator protein, DnaA binds to the replicational origin of the chromosome (oriC) and initiates the replication in a manner depending on ATP ' binding. Replication of mini-chromosome (a plasmid bearing oriC) has been reconstituted in vitro using crude extracts or purified proteins. In these in vitro systems, only ATP-bound form of DnaA is active to initiate a series of initiation reactions which leads to loading of the DNA polymerase III holoenzyme (pol III HE) onto DNA for DNA synthesis. In a manner dependent on Hda protein and DNA loading of pol III HE, ATP bound on DnaA is hydrolyzed. The resultant ADP-bound form of DnaA is inactive for the initiation. This inactivation system called RIDA (regulatory inactivation of DnaA) ensures the replication initiation only once per cell cycle. We here describe recent progress on our system to reconstitute the inactivation of DnaA by RIDA and a novel system to regenerate active DnaA. The reconstitution of the Hda-dependent DnaA-ATP hydrolysis requires the DNA-loaded form of the sliding clamp. This complex is formed by loading of the beta subunit of pol III HE onto DNA during replication process. Hda protein forms a stable complex with the sliding clamp, and most likely has a direct contact to ATP bound on DnaA. The cellular ATP-DnaA level fluctuates during the cell cycle. Regeneration from ADP-DnaA to ATP-DnaA is observed in vivo before the next round of the replication. Recently we have found a specific DNA segment DARS (DnaA-reactivating sequences) that has an activity to release ADP bound on DnaA. ATP-DnaA is regenerated by nucleotide-exchange in the presence of DARS and the elevated concentrations of ATP. The resultant ATP-DnaA was active in the mini-chromosome replication reaction in vitro. Thus, DARS can functionally reactivate ADP-DnaA. The inactivation-reactivation cycle of DnaA might be reconstituted in vitro in a manner controled by RIDA and DARS.

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DOI： 10.1046/j.1365-2443.2003.00671.x

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Language：English   Publishing type：Research paper (scientific journal)  

DnaA protein binds specifically to a group of binding sites collectively called as DnaA boxes within the bacterial replication origin to induce local unwinding of duplex DNA. The DNA-binding domain of DnaA, domain IV, comprises the C-terminal 94 amino acid residues of the protein. We overproduced and purified a protein containing only this domain plus a methionine residue. This protein was stable as a monomer and maintained DnaA box-specific binding activity. We then analyzed its solution structure by CD spectrum and heteronuclear multi-dimensional NMR experiments. We established extensive assignments of the 1H, 13C, and 15N nuclei, and revealed by obtaining combined analyses of chemical shift index and NOE connectivities that DnaA domain IV contains six α-helices and no β-sheets, consistent with results of CD analysis. Mutations known to reduce DnaA box-binding activity were specifically located in or near two of the α-helices. These findings indicate that the DNA-binding fold of DnaA domain IV is unique among origin-binding proteins. © 2002 Elsevier Science (USA). All rights reserved.

DOI： 10.1016/S0006-291X(02)02590-1

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Language：English   Publishing type：Research paper (scientific journal)  

The DnaA protein, which initiates chromosomal replication in Escherichia coli, is negatively regulated by both the sliding clamp of DNA polymerase III holoenzyme and the IdaB protein. We have found that, when the amount of minichromosome is limited in an in vitro replication system, minichromosomal replication-stimulated hydrolysis of DnaA-bound ATP yields the ADP-bound inactive form. The number of sliding clamps formed during replication was at least five per minichromosome, which is 2.7-fold higher than the number formed during incubation without replication. These results support the notion that coupling of DnaA-ATP hydrolysis to DNA replication is the outcome of enhanced clamp formation. We have also found that the amino acid substitution R334H in DnaA severely inhibits the hydrolysis of bound ATP in vitro. Whereas ATP bound to wild-type DnaA is hydrolysed in a DNA-dependent intrinsic manner or in a sliding clamp-dependent manner, ATP bound to DnaA R334H protein was resistant to hydrolysis under the same conditions. This arginine residue may be located in the vicinity where ATP binds, and therefore may play an essential role in ATP hydrolysis. This residue is highly conserved among DnaA homologues and also in the Box VIII motif of the AAA+ protein family.

DOI： 10.1046/j.1365-2958.2001.02378.x

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Authorship：Last author,　Corresponding author   Language：Japanese   Publisher：(株)羊土社  

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：GENETICS SOC JAPAN  

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Applicant：オリシロジェノミクス株式会社

Application no：特願2019-527961  Date applied：5 7 2018

Patent/Registration no：特許第6701450号  Date registered：8 5 2020 

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Application no：JP2019029793  Date applied：30 7 2019

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Application no：JP2018025528  Date applied：5 7 2018

Publication no：WO2019-009361  Date published：10 1 2019

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Application no：JP2018007485  Date applied：28 2 2018

Publication no：WO2018-159669  Date published：7 9 2018

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Application no：特願2016-560254  Date applied：18 11 2015

Patent/Registration no：特許第6262877号  Date registered：22 12 2017 

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Applicant：国立研究開発法人科学技術振興機構

Application no：JP2017018472  Date applied：17 5 2017

Publication no：WO2017-199991  Date published：23 11 2017

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