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大塚 裕一（オオツカ ユウイチ）
| 理工学研究科 生命科学部門 | 准教授 |
| 理学部 分子生物学科 |
業績情報
■ 論文- A type II toxin–antitoxin system, ECs3274-ECs3275, in enterohemorrhagic Escherichia coli O157
Yuka Sasaki; Yuna Mogi; Mizuki Yoshioka; Ke Liu; Yuichi Otsuka
Bioscience, Biotechnology, and Biochemistry, 巻：89, 号：1, 開始ページ：62, 終了ページ：71, 2025年01月, ［査読有り］, ［最終著者, 責任著者］
Abstract
The toxin–antitoxin (TA) genetic module controls various bacterial events. Novel toxins with different functions are still being discovered. This study aimed to determine whether the ECs3274-ECs3275 gene pair encoded by enterohemorrhagic Escherichia coli O157 functions as a TA system. To characterize this putative TA system, we analyzed the growth of E. coli expressing ECs3274, ECs3275, or both; the interaction between ECs3274 and ECs3275 using bacterial adenylate cyclase two-hybrid assays; and the DNA-binding ability of ECs3274 using gel-mobility shift assays. We observed that the ECs3274 antitoxin interacted with the ECs3275 toxin, was destabilized by Lon protease, and repressed its promoter activity via its helix-turn-helix (HTH) motif. These properties are consistent with those of typical type II TA antitoxins. Interestingly, ECs3275 has an HTH motif not observed in other TA toxins and is necessary for ECs3275 toxicity, suggesting that ECs3275 may exert its toxicity by regulating the expression of specific genes.
Oxford University Press (OUP), 研究論文（学術雑誌）
DOI：https://doi.org/10.1093/bbb/zbae146
DOI ID：10.1093/bbb/zbae146, eISSN：1347-6947 - Phage single-stranded DNA-binding protein or host DNA damage triggers the activation of the AbpAB phage defense system.
Takaomi Sasaki; Saya Takita; Takashi Fujishiro; Yunosuke Shintani; Satoki Nojiri; Ryota Yasui; Tetsuro Yonesaki; Yuichi Otsuka
mSphere, 巻：8, 号：6, 開始ページ：e0037223, 2023年12月, ［査読有り］, ［最終著者, 責任著者］, ［国際誌］
Although numerous phage defense systems have recently been discovered in bacteria, how these systems defend against phage propagation or sense phage infections remains unclear. The Escherichia coli AbpAB defense system targets several lytic and lysogenic phages harboring DNA genomes. A phage-encoded single-stranded DNA-binding protein, Gp32, activates this system similar to other phage defense systems such as Retron-Eco8, Hachiman, ShosTA, Nhi, and Hna. DNA replication inhibitors or defects in DNA repair factors activate the AbpAB system, even without phage infection. This is one of the few examples of activating phage defense systems without phage infection or proteins. The AbpAB defense system may be activated by sensing specific DNA-protein complexes.
英語, 研究論文（学術雑誌）
DOI：https://doi.org/10.1128/msphere.00372-23
DOI ID：10.1128/msphere.00372-23, PubMed ID：37882551, PubMed Central ID：PMC10732053 - The hokW-sokW Locus Encodes a Type I Toxin–Antitoxin System That Facilitates the Release of Lysogenic Sp5 Phage in Enterohemorrhagic Escherichia coli O157
Kosuke Takada; Kotone Hama; Takaomi Sasaki; Yuichi Otsuka
Toxins, 巻：13, 号：11, 開始ページ：796, 終了ページ：796, 2021年11月, ［査読有り］, ［最終著者, 責任著者］
The toxin-antitoxin (TA) genetic modules control various bacterial events, such as plasmid maintenance, persister cell formation, and phage defense. They also exist in mobile genetic elements, including prophages; however, their physiological roles remain poorly understood. Here, we demonstrate that hokW-sokW, a putative TA locus encoded in Sakai prophage 5 (Sp5) in enterohemorrhagic Escherichia coli O157: H7 Sakai strain, functions as a type I TA system. Bacterial growth assays showed that the antitoxic activity of sokW RNA against HokW toxin partially requires an endoribonuclease, RNase III, and an RNA chaperone, Hfq. We also demonstrated that hokW-sokW assists Sp5-mediated lysis of E. coli cells when prophage induction is promoted by the DNA-damaging agent mitomycin C (MMC). We found that MMC treatment diminished sokW RNA and increased both the expression level and inner membrane localization of HokW in a RecA-dependent manner. Remarkably, the number of released Sp5 phages decreased by half in the absence of hokW-sokW. These results suggest that hokW-sokW plays a novel role as a TA system that facilitates the release of Sp5 phage progeny through E. coli lysis.
MDPI AG, 英語, 研究論文（学術雑誌）
DOI：https://doi.org/10.3390/toxins13110796
DOI ID：10.3390/toxins13110796, eISSN：2072-6651, 共同研究・競争的資金等ID：13443555 - Manipulating Interactions between T4 Phage Long Tail Fibers and Escherichia coli Receptors
Akiyo Suga; Marina Kawaguchi; Tetsuro Yonesaki; Yuichi Otsuka
Applied and Environmental Microbiology, 巻：87, 号：13, 2021年07月, ［査読有り］, ［最終著者, 責任著者］
Understanding the host specificity of phages will lead to the development of phage therapy. The interaction between outer membrane protein C (OmpC), one of the Escherichia coli receptors, and the gp37 protein composing the digital tip (DT) region of the long tail fibers of bacteriophage T4 largely determines its host specificity.
American Society for Microbiology, 英語, 研究論文（学術雑誌）
DOI：https://doi.org/10.1128/aem.00423-21
DOI ID：10.1128/aem.00423-21, ISSN：0099-2240, eISSN：1098-5336, 共同研究・競争的資金等ID：13443555;13443556 - A short peptide derived from the ZorO toxin functions as an effective antimicrobial
Otsuka Y; Ishikawa T; Takahashi C; Masuda M
Toxins, 巻：11, 号：7, 開始ページ：DOI:10.3390/toxins11070392, 2019年07月, ［査読有り］, ［筆頭著者, 責任著者］, ［国際誌］
Antimicrobial peptides are potential molecules for the development of novel antibiotic agents. The ZorO toxin of a type I toxin-antitoxin system in Escherichia coli O157:H7 is composed of 29 amino acids and its endogenous expression inhibits E. coli growth. However, little is known about its inhibitory mechanism. In this study, we demonstrate that the ZorO localized in the inner membrane affects the plasma membrane integrity and potential when expressed in E. coli cells, which triggers the production of cytotoxic hydroxyl radicals. We further show that five internal amino acids (Ala-Leu-Leu-Arg-Leu; ALLRL) of ZorO are necessary for its toxicity. This result prompted us to address the potential of the synthetic ALLRL peptide as an antimicrobial. Exogenously-added ALLRL peptide to Gram-positive bacteria, Staphylococcus aureus and Bacillus subtilis, and a fungus, Candida albicans, trigger cell membrane damage and exhibit growth defect, while having no effect on Gram-negative bacterium, E. coli. The ALLRL peptide retains its activity under the physiological salt concentrations, which is in contrast to natural antimicrobial peptides. Importantly, this peptide has no toxicity against mammalian cells. Taken together, an effective and short peptide, ALLRL, would be an attractive antimicrobial to Gram-positive bacteria and C. albicans.
英語, 研究論文（学術雑誌）
DOI：https://doi.org/10.3390/toxins11070392
DOI ID：10.3390/toxins11070392, PubMed ID：31277504, PubMed Central ID：PMC6669753 - RnlB antitoxin of the Escherichia coli RnlA-RnlB toxin–antitoxin module requires RNase HI for inhibition of RnlA toxin activity
Naka K; Qi D; Yonesaki T; Otsuka Y
Toxins, 巻：9, 号：1, 開始ページ：DOI:10.3390/toxins9010029, 2017年01月, ［査読有り］, ［最終著者, 責任著者］
The Escherichia coli RnlA-RnlB toxin-antitoxin system is related to the anti-phage mechanism. Under normal growth conditions, an RnlA toxin with endoribonuclease activity is inhibited by binding of its cognate RnlB antitoxin. After bacteriophage T4 infection, RnlA is activated by the disappearance of RnlB, resulting in the rapid degradation of T4 mRNAs and consequently no T4 propagation when T4 dmd encoding a phage antitoxin against RnlA is defective. Intriguingly, E. coli RNase HI, which plays a key role in DNA replication, is required for the activation of RnlA and stimulates the RNA cleavage activity of RnlA. Here, we report an additional role of RNase HI in the regulation of RnlA-RnlB system. Both RNase HI and RnlB are associated with NRD (one of three domains of RnlA). The interaction between RnlB and NRD depends on RNase HI. Exogenous expression of RnlA in wild-type cells has no effect on cell growth because of endogenous RnlB and this inhibition of RnlA toxicity requires RNase HI and NRD. These results suggest that RNase HI recruits RnlB to RnlA through NRD for inhibiting RnlA toxicity and thus plays two contrary roles in the regulation of RnlA-RnlB system.
MDPI AG, 英語, 研究論文（学術雑誌）
DOI：https://doi.org/10.3390/toxins9010029
DOI ID：10.3390/toxins9010029, ISSN：2072-6651, Web of Science ID：WOS:000392980000029 - Characterization of the interactions between Escherichia coli receptors, LPS and OmpC, and bacteriophage T4 long tail fibers
Ayaka Washizaki; Tetsuro Yonesaki; Yuichi Otsuka
MICROBIOLOGYOPEN, 巻：5, 号：6, 開始ページ：1003, 終了ページ：1015, 2016年12月, ［査読有り］, ［最終著者, 責任著者］
Bacteriophages have strict host specificity and the step of adsorption is one of key factors for determining host specificity. Here, we systematically examined the interaction between the Escherichia coli receptors lipopolysaccharide (LPS) and outer membrane protein C (OmpC), and the long tail fibers of bacteriophage T4. Using a variety of LPS mutants, we demonstrated that T4 has no specificity for the sugar sequence of the outer core (one of three LPS regions) in the presence of OmpC but, in the absence of OmpC, can adsorb to a specific LPS which has only one or two glucose residues without a branch. These results strengthen the idea that T4 adsorbs to E. coli via two distinct modes, OmpC-dependent and OmpC--independent, suggested by previous reports (Prehm et al. 1976; Yu and Mizushima 1982). Isolation and characterization of the T4 mutants Nik (No infection to K--12 strain), Nib (No infection to B strain), and Arl (altered recognition of LPS) identified amino acids of the long tail fiber that play important roles in the interaction with OmpC or LPS, suggesting that the top surface of the distal tip head domain of T4 long tail fibers interacts with LPS and its lateral surface interacts with OmpC.
WILEY-BLACKWELL, 英語, 研究論文（学術雑誌）
DOI：https://doi.org/10.1002/mbo3.384
DOI ID：10.1002/mbo3.384, ISSN：2045-8827, Web of Science ID：WOS:000390567300008 - Structural insights into the inhibition mechanism of bacterial toxin LsoA by bacteriophage antitoxin Dmd
Hua Wan; Yuichi Otsuka; Zeng-Qiang Gao; Yong Wei; Zhen Chen; Michiaki Masuda; Tetsuro Yonesaki; Heng Zhang; Yu-Hui Dong
MOLECULAR MICROBIOLOGY, 巻：101, 号：5, 開始ページ：757, 終了ページ：769, 2016年09月, ［査読有り］, ［筆頭著者］
Bacteria have obtained a variety of resistance mechanisms including toxin-antitoxin (TA) systems against bacteriophages (phages), whereas phages have also evolved to overcome bacterial anti-phage mechanisms. Dmd from T4 phage can suppress the toxicities of homologous toxins LsoA and RnlA from Escherichia coli, representing the first example of a phage antitoxin against multiple bacterial toxins in known TA systems. Here, the crystal structure of LsoA-Dmd complex showed Dmd is inserted into the deep groove between the N-terminal repeated domain (NRD) and the Dmd-binding domain (DBD) of LsoA. The NRD shifts significantly from a 'closed' to an 'open' conformation upon Dmd binding. Site-directed mutagenesis of Dmd revealed the conserved residues (W31 and N40) are necessary for LsoA binding and the toxicity suppression as determined by pull-down and cell toxicity assays. Further mutagenesis identified the conserved Dmd-binding residues (R243, E246 and R305) of LsoA are vital for its toxicity, and suggested Dmd and LsoB may possess different inhibitory mechanisms against LsoA toxicity. Our structure-function studies demonstrate Dmd can recognize LsoA and inhibit its toxicity by occupying the active site possibly via substrate mimicry. These findings have provided unique insights into the defense and counter-defense mechanisms between bacteria and phages in their co-evolution.
WILEY-BLACKWELL, 英語, 研究論文（学術雑誌）
DOI：https://doi.org/10.1111/mmi.13420
DOI ID：10.1111/mmi.13420, ISSN：0950-382X, eISSN：1365-2958, Web of Science ID：WOS:000384410300005 - Prokaryotic toxin–antitoxin systems: novel regulations of the toxins.
Yuichi Otsuka
Current Genetics, 巻：62, 号：2, 開始ページ：379, 終了ページ：382, 2016年05月, ［査読有り］, ［筆頭著者, 責任著者］
Toxin-antitoxin (TA) systems are widely conserved in prokaryotic plasmids and chromosomes and are linked to many roles in cell physiology, including plasmid maintenance, stress response, persistence and protection from phage infection. A TA system is composed of a stable toxin and a labile antitoxin that inhibits a harmful effect of the cognate toxin. When gene expression from the TA loci is repressed under certain conditions such as nutrient starvation, the toxin is freed from the rapidly degrading antitoxin and obstructs an essential cellular process, such as DNA replication, translation and peptidoglycan synthesis, which subsequently causes growth arrest. TA systems are classified into five types according to the nature and the function of antitoxins, and the activity of toxins is tightly regulated in a variety of ways. This short-review highlights several novel regulatory mechanisms for Escherichia coli toxins that we recently discovered.
SPRINGER, 英語, 研究論文（学術雑誌）
DOI：https://doi.org/10.1007/s00294-015-0557-z
DOI ID：10.1007/s00294-015-0557-z, ISSN：0172-8083, eISSN：1432-0983, Web of Science ID：WOS:000373954800022 - ADP-ribosyltransferase Alt of bacteriophage T4 negatively regulates the Escherichia coli MazF toxin of a toxin–antitoxin module.
Alawneh A. M; Qi D; Yonesaki T; Otsuka Y
Molecular Microbiology, 巻：99, 号：1, 開始ページ：188, 終了ページ：198, 2016年01月, ［査読有り］, ［最終著者, 責任著者］
Prokaryotic toxin-antitoxin (TA) systems are linked to many roles in cell physiology, such as plasmid maintenance, stress response, persistence and protection from phage infection, and the activities of toxins are tightly regulated. Here, we describe a novel regulatory mechanism for a toxin of Escherichia coli TA systems. The MazF toxin of MazE-MazF, which is one of the best characterized type II TA systems, was modified immediately after infection with bacteriophage T4. Mass spectrometry demonstrated that the molecular weight of this modification was 542 Da, corresponding to a mono-ADP-ribosylation. This modification disappeared in cells infected with T4 phage lacking Alt, which is one of three ADP-ribosyltransferases encoded by T4 phage and is injected together with phage DNA upon infection. In vivo and in vitro analyses confirmed that T4 Alt ADP-ribosylated MazF at an arginine residue at position 4. Finally, the ADPribosylation of MazF by Alt resulted in the reduction of MazF RNA cleavage activity in vitro, suggesting that it may function to inactivate MazF during T4 infection. This is the first example of the chemical modification of an E. coli toxin in TA systems to regulate activity.
WILEY-BLACKWELL, 英語, 研究論文（学術雑誌）
DOI：https://doi.org/10.1111/mmi.13225
DOI ID：10.1111/mmi.13225, ISSN：0950-382X, eISSN：1365-2958, Web of Science ID：WOS:000369157900013 - Rapid Degradation of Host mRNAs by Stimulation of RNase E Activity by Srd of Bacteriophage T4
Dan Qi; Abdulraheem M. Alawneh; Tetsuro Yonesaki; Yuichi Otsuka
GENETICS, 巻：201, 号：3, 開始ページ：977, 終了ページ：U356, 2015年11月, ［査読有り］, ［最終著者, 責任著者］
Escherichia coli messenger RNAs (mRNAs) are rapidly degraded immediately after bacteriophage T4 infection, and the host RNase E contributes to this process. Here, we found that a previously uncharacterized factor of T4 phage, Srd (Similarity with rpoD), was involved in T4-induced host mRNA degradation. The rapid decay of ompA and lpp mRNAs was partially alleviated and a decay intermediate of lpp mRNA rapidly accumulated in cells infected with T4 phage lacking srd. Exogenous expression of Srd in uninfected cells significantly accelerated the decay of these mRNAs. In addition, lpp(T) RNA, with a sequence identical to the decay intermediate of lpp mRNA and a triphosphate at 5'-end, was also destabilized by Srd. The destabilization of these RNAs by Srd was not observed in RNase E-defective cells. The initial cleavage of a primary transcript by RNase E can be either direct or dependent on the 5'-end of transcript. In the latter case, host RppH is required to convert the triphosphate at 5'-end to a monophosphate. lpp(T) RNA, but not lpp and ompA mRNAs, required RppH for Srd-stimulated degradation, indicating that Srd stimulates both 5'-end-dependent and -independent cleavage activities of RNase E. Furthermore, pull-down and immunoprecipitation analyses strongly suggested that Srd physically associates with the N-terminal half of RNase E containing the catalytic moiety and the membrane target sequence. Finally, the growth of T4 phage was significantly decreased by the disruption of srd. These results strongly suggest that the stimulation of RNase E activity by T4 Srd is required for efficient phage growth.
GENETICS SOCIETY AMERICA, 英語, 研究論文（学術雑誌）
DOI：https://doi.org/10.1534/genetics.115.180364
DOI ID：10.1534/genetics.115.180364, ISSN：0016-6731, eISSN：1943-2631, Web of Science ID：WOS:000365517200014 - AbpA and AbpB provide anti-phage activity in Escherichia coli
Ryota Yasui; Ayaka Washizaki; Yuko Furihata; Tetsuro Yonesaki; Yuichi Otsuka
Genes and Genetic Systems, 巻：89, 号：2, 開始ページ：51, 終了ページ：60, 2014年09月, ［査読有り］, ［最終著者, 責任著者］
Bacteria have a variety of resistance mechanisms for surviving bacteriophage infections. Here, we describe a novel anti-phage mechanism in Escherichia coli.Cells harboring a plasmid with the genes abpA and abpB, formerly yfjL and yfjK, blocked the propagation of bacteriophages belonging to three families: T4, T2, T7 and λ phages. Both genes were necessary for the inhibition of phage propagation, and deletion of either chromosomal gene resulted in a 20% increase of progeny compared to wild-type cells. Neither overexpression nor deficiency of AbpA and AbpB had any apparent effect on E. coli growth. We isolated seven suppressor mutants of T4 phage that grew weakly on cells overexpressing AbpA and AbpB, and found that their mutations were all located in gene 41, which encodes a replicative DNA helicase that is essential for DNA replication. Furthermore, we demonstrated that AbpA and AbpB inhibited DNA replication and late gene expression of T4 phage. Similarly, DNA replication of T7 and λ phages was also inhibited by AbpA and AbpB. These results strongly suggest that E. coli AbpA and AbpB target DNA replication of phages to block their propagation.
Genetics Society of Japan, 英語, 研究論文（学術雑誌）
DOI：https://doi.org/10.1266/ggs.89.51
DOI ID：10.1266/ggs.89.51, ISSN：1880-5779, PubMed ID：25224971, SCOPUS ID：84907545882 - RNase HI stimulates the activity of RnlA toxin in Escherichia coli
Kenta Naka; Mitsunori Koga; Tetsuro Yonesaki; Yuichi Otsuka
MOLECULAR MICROBIOLOGY, 巻：91, 号：3, 開始ページ：596, 終了ページ：605, 2014年02月, ［査読有り］, ［最終著者, 責任著者］
A type II toxin-antitoxin system in Escherichia coli, rnlA-rnlB, functions as an anti-phage mechanism. RnlA is a toxin with an endoribonuclease activity and the cognate RnlB inhibits RnlA toxicity in E. coli cells. After bacteriophage T4 infection, RnlA is activated by the disappearance of RnlB, resulting in the rapid degradation of T4 mRNAs and consequently no T4 propagation, when T4 dmd is defective: Dmd is an antitoxin against RnlA for promoting own propagation. Previous studies suggested that the activation of RnlA after T4 infection was regulated by multiple components. Here, we provide the evidence that RNase HI is an essential factor for activation of RnlA. The dmd mutant phage could grow on Delta rnhA (encoding RNase HI) cells, in which RnlA-mediated mRNA cleavage activity was defective. RNase HI bound to RnlA in vivo and enhanced the RNA cleavage activity of RnlA in vitro. In addition, ectopic expression of RnlA in Delta rnlAB Delta rnhA cells has less effect on cell toxicity and RnlA-mediated mRNA degradation than in Delta rnlAB cells. This is the first example of a direct factor for activation of a toxin.
WILEY-BLACKWELL, 英語, 研究論文（学術雑誌）
DOI：https://doi.org/10.1111/mmi.12479
DOI ID：10.1111/mmi.12479, ISSN：0950-382X, eISSN：1365-2958, Web of Science ID：WOS:000345498000012 - Structure-function studies of Escherichia coli RnlA reveal a novel toxin structure involved in bacteriophage resistance
Yong Wei; Zeng-Qiang Gao; Yuichi Otsuka; Kenta Naka; Tetsuro Yonesaki; Heng Zhang; Yu-Hui Dong
MOLECULAR MICROBIOLOGY, 巻：90, 号：5, 開始ページ：956, 終了ページ：965, 2013年12月, ［査読有り］
Escherichia coli RnlA-RnlB is a newly identified toxin-antitoxin (TA) system that plays a role in bacteriophage resistance. RnlA functions as a toxin with mRNA endoribonuclease activity and the cognate antitoxin RnlB inhibits RnlA toxicity in E. coli cells. Interestingly, T4 phage encodes the antitoxin Dmd, which acts against RnlA to promote its own propagation, suggesting that RnlA-Dmd represents a novel TA system. Here, we have determined the crystal structure of RnlA refined to 2.10 angstrom. RnlA is composed of three independent domains: NTD (N-terminal domain), NRD (N repeated domain) and DBD (Dmd-binding domain), which is an organization not previously observed among known toxin structures. Small-angle X-ray scattering (SAXS) analysis revealed that RnlA forms a dimer in solution via interactions between the DBDs from both monomers. The in vitro and in vivo functional studies showed that among the three domains, only the DBD is responsible for recognition and inhibition by Dmd and subcellular location of RnlA. In particular, the helix located at the C-terminus of DBD plays a vital role in binding Dmd. Our comprehensive studies reveal the key region responsible for RnlA toxicity and provide novel insights into its structure-function relationship.
WILEY-BLACKWELL, 英語, 研究論文（学術雑誌）
DOI：https://doi.org/10.1111/mmi.12409
DOI ID：10.1111/mmi.12409, ISSN：0950-382X, eISSN：1365-2958, Web of Science ID：WOS:000327374300004 - Identification of the human PMR1 mRNA endonuclease as an alternatively processed product of the gene for peroxidasin-like protein
Shan-Qing Gu; Baskar Bakthavachalu; Joonhee Han; Deepak P. Patil; Yuichi Otsuka; Chittibabu Guda; Daniel R. Schoenberg
RNA, 巻：18, 号：6, 開始ページ：1186, 終了ページ：1196, 2012年06月, ［査読有り］
The PMR1 endonuclease was discovered in Xenopus liver and identified as a member of the large and diverse peroxidase gene family. The peroxidase genes arose from multiple duplication and rearrangement events, and their high degree of sequence similarity confounded attempts to identify human PMR1. The functioning of PMR1 in mRNA decay depends on the phosphorylation of a tyrosine in the C-terminal polysome targeting domain by c-Src. The sequences of regions that are required for c-Src binding and phosphorylation of Xenopus PMR1 were used to inform a bioinformatics search that identified two related genes as potential candidates for human PMR1: peroxidasin homolog (PXDN) and peroxidasin homolog-like (PXDNL) protein. Although each of these genes is predicted to encode a large, multidomain membrane-bound peroxidase, alternative splicing of PXDNL pre-mRNA yields a transcript whose predicted product is a 57-kDa protein with 42% sequence identity to Xenopus PMR1. Results presented here confirm the existence of the predicted 57-kDa protein, show this is the only form of PXDNL detected in any of the human cell lines examined, and confirm its identity as human PMR1. Like the Xenopus protein, human PMR1 binds to c-Src, is tyrosine phosphorylated, sediments on polysomes, and catalyzes the selective decay of a PMR1 substrate mRNA. Importantly, the expression of human PMR1 stimulates cell motility in a manner similar to that of the Xenopus PMR1 expressed in human cells, thus providing definitive evidence linking endonuclease decay to the regulation of cell motility.
COLD SPRING HARBOR LAB PRESS, PUBLICATIONS DEPT, 英語, 研究論文（学術雑誌）
DOI：https://doi.org/10.1261/rna.031369.111
DOI ID：10.1261/rna.031369.111, ISSN：1355-8382, eISSN：1469-9001, Web of Science ID：WOS:000304423000008 - Dmd of bacteriophage T4 functions as an antitoxin against Escherichia coli LsoA and RnlA toxins
Yuichi Otsuka; Tetsuro Yonesaki
MOLECULAR MICROBIOLOGY, 巻：83, 号：4, 開始ページ：669, 終了ページ：681, 2012年02月, ［査読有り］, ［筆頭著者］
Enterohaemorrhagic Escherichia coli O157:H7 harbours a cryptic plasmid, pOSAK1, that carries only three ORFs: mobA (involved in plasmid mobilization), ORF1 and ORF2. Predicted proteins encoded by these two ORFs were found to share a weak homology with RnlA and RnlB, respectively, a toxinantitoxin system encoded on the E. coli K-12 chromosome. Here, we report that lsoA (ORF1) encodes a toxin and lsoB (ORF2) an antitoxin. In spite of the homologies, RnlB and LsoB functioned as antitoxins against only their cognate toxins and not interchangeably with each other. Interestingly, T4 phage Dmd suppressed the toxicities of both RnlA and LsoA by direct interaction, the first example of a phage with an antitoxin against multiple toxins.
WILEY-BLACKWELL, 英語, 研究論文（学術雑誌）
DOI：https://doi.org/10.1111/j.1365-2958.2012.07975.x
DOI ID：10.1111/j.1365-2958.2012.07975.x, ISSN：0950-382X, Web of Science ID：WOS:000299779200002 - Escherichia coli rnlA and rnlB compose a novel toxin–antitoxin system.
Koga M; Otsuka Y; Lemire S; Yonesaki T
Genetics, 巻：187, 号：1, 開始ページ：123, 終了ページ：130, 2011年01月, ［査読有り］
RNase LS was originally identified as a potential antagonist of bacteriophage T4 infection. When T4 dmd is defective, RNase LS activity rapidly increases after T4 infection and cleaves T4 mRNAs to antagonize T4 reproduction. Here we show that rnlA, a structural gene of RNase LS, encodes a novel toxin, and that rnlB (formally yfjO), located immediately downstream of rnlA, encodes an antitoxin against RnlA. Ectopic expression of RnlA caused inhibition of cell growth and rapid degradation of mRNAs in Delta rnlAB cells. On the other hand, RnlB neutralized these RnlA effects. Furthermore, overexpression of RnlB in wild-type cells could completely suppress the growth defect of a T4 dmd mutant, that is, excess RnlB inhibited RNase LS activity. Pull-down analysis showed a specific interaction between RnlA and RnlB. Compared to RnlA, RnlB was extremely unstable, being degraded by ClpXP and Lon proteases, and this instability may increase RNase LS activity after T4 infection. All of these results suggested that rnlA-rnlB define a new toxin-antitoxin (TA) system.
GENETICS SOC AM, 英語, 研究論文（学術雑誌）
DOI：https://doi.org/10.1534/genetics.110.121798
DOI ID：10.1534/genetics.110.121798, ISSN：0016-6731, Web of Science ID：WOS:000286100900010 - IscR Regulates RNase LS Activity by Repressing rnlA Transcription
Yuichi Otsuka; Kumiko Miki; Mitsunori Koga; Natsu Katayama; Wakako Morimoto; Yasuhiro Takahashi; Tetsuro Yonesaki
GENETICS, 巻：185, 号：3, 開始ページ：823, 終了ページ：830, 2010年07月, ［査読有り］, ［筆頭著者］
The Escherichia coli endoribonuclease LS was originally identified as a potential antagonist of bacteriophage T4. When the T4 dmd gene is defective, RNase LS cleaves T4 mRNAs and antagonizes T4 reproduction. This RNase also plays an important role in RNA metabolisms in E. coli. rnlA is an essential gene for RNase LS activity, but the transcriptional regulation of this gene remains to be elucidated. An Fe-S cluster protein, IscR, acts as a transcription factor and controls the expression of genes that are necessary for Fe-S cluster biogenesis. Here, we report that overexpression of IscR suppressed RNase LS activity, causing the loss of antagonist activity against phage T4. This suppressive effect did not require the ligation of Fe-S cluster into IscR. beta-Galactosidase reporter assays showed that transcription from an rnlA promoter increased in iscR-deleted cells compared to wild-type cells, and gel-mobility shift assays revealed specific binding of IscR to the rnlA promoter region. RT-PCR analysis demonstrated that endogenous rnlA mRNA was reduced by overexpression of IscR and increased by deletion of iscR. From these results, we conclude that IscR negatively regulates transcription of rnlA and represses RNase LS activity.
GENETICS SOC AM, 英語, 研究論文（学術雑誌）
DOI：https://doi.org/10.1534/genetics.110.114462
DOI ID：10.1534/genetics.110.114462, ISSN：0016-6731, Web of Science ID：WOS:000281906800010 - 新世代のファージ研究
米崎哲朗; 大塚裕一
生産と技術, 巻：62, 開始ページ：55, 終了ページ：58, 2010年, ［最終著者］
日本語, 研究論文（大学，研究機関等紀要） - Identification of a Cytoplasmic Complex That Adds a Cap onto 5 '-Monophosphate RNA
Yuichi Otsuka; Nancy L. Kedersha; Daniel R. Schoenberg
MOLECULAR AND CELLULAR BIOLOGY, 巻：29, 号：8, 開始ページ：2155, 終了ページ：2167, 2009年04月, ［査読有り］, ［筆頭著者］
Endonuclease decay of nonsense-containing beta-globin mRNA in erythroid cells generates 5'-truncated products that were reported previously to have a cap or caplike structure. We confirmed that this 5' modification is indistinguishable from the cap on full-length mRNA, and Western blotting, immunoprecipitation, and active-site labeling identified a population of capping enzymes in the cytoplasm of erythroid and nonerythroid cells. Cytoplasmic capping enzyme sediments in a 140-kDa complex that contains a kinase which, together with capping enzyme, converts 5'-monophosphate RNA into 5'-GpppX RNA. Capping enzyme shows diffuse and punctate staining throughout the cytoplasm, and its staining does not overlap with P bodies or stress granules. Expression of inactive capping enzyme in a form that is restricted to the cytoplasm reduced the ability of cells to recover from oxidative stress, thus supporting a role for capping in the cytoplasm and suggesting that some mRNAs may be stored in an uncapped state.
AMER SOC MICROBIOLOGY, 英語, 研究論文（学術雑誌）
DOI：https://doi.org/10.1128/MCB.01325-08
DOI ID：10.1128/MCB.01325-08, ISSN：0270-7306, Web of Science ID：WOS:000264558400015 - APPROACHES FOR STUDYING PMR1 ENDONUCLEASE-MEDIATED MRNA DECAY
Yuichi Otsuka; Daniel R. Schoenberg
RNA TURNOVER IN EUKARYOTES: NUCLEASES, PATHWAYS AND ANAYLSIS OF MRNA DECAY, 巻：448, 開始ページ：241, 終了ページ：263, 2008年, ［査読有り］, ［筆頭著者］
Although most eukaryotic mRNAs are degraded by exonucleases acting on either end of the molecule, a subset of mRNAs undergo endonuclease cleavage within the mRNA body. Endonuclease cleavage can be activated by cellular stress, extracellular signals, or by ribosome stalling, as might occur at a premature termination codon. Only a few eukaryotic mRNA endonucleases have been identified, and of these, polysomal ribonuclease 1 (PMR1) is the best characterized. A notable feature of PMR1-mediated mRNA decay is that it acts on specific mRNAs while they are engaged by translating ribosomes. This chapter begins with several procedures used to characterize in vivo endonuclease cleavage of any mRNA by any endonudease. These include approaches for identifying the 5'-end(s) downstream of an endonuclease cleavage site (S1 nuclease protection and primer extension), and a ligation-mediated RT-PCR approach developed in our laboratory for identifying the 3'-ends upstream of a cleavage site. We then describe a number of approaches used to characterize PMR1-mediated mRNA decay in cultured cells. PMR1 participates in a number of different complexes. We show several approaches for studying these complexes, and we describe techniques for isolating and characterizing PMR1-interacting proteins and its target mRNAs. Although the various techniques described here have proven their usefulness in studying PMR1, they can be generalized to studying decay by any other mRNA endonuclease.
ELSEVIER ACADEMIC PRESS INC, 英語, 論文集(書籍)内論文
DOI：https://doi.org/10.1016/S0076-6879(08)02613-X
DOI ID：10.1016/S0076-6879(08)02613-X, ISSN：0076-6879, Web of Science ID：WOS:000262252300013 - A role of Rn1A in the RNase LS activity from Escherichia coli
Yuichi Otsuka; Mitsunori Koga; Akira Iwamoto; Tetsuro Yonesaki
GENES & GENETIC SYSTEMS, 巻：82, 号：4, 開始ページ：291, 終了ページ：299, 2007年08月, ［査読有り］, ［筆頭著者］
Escherichia coli ribonuclease LS is a potential antagonist of bacteriophage T4. When the T4 dmd gene is defective, RNase LS cleaves T4 mRNAs and antagonizes T4 reproduction. Our previous work demonstrated that E. coli rnlA is essential for RNase LS activity. Here we show that His-tagged RnlA cleaves T4 soc RNA at one of the sites also cleaved by RNase LS in a cell extract. The cleavage activities of His-tagged RnlA and the RNase LS activity in a cell extract were inhibited by Dmd encoded by T4 phage. Fractionation of the RNase LS activity in a cell extract showed that it sedimented through a sucrose density gradient as a 1000-kDa complex that included RnlA. Pull-down experiments revealed more than 10 proteins associated with His-tagged RnlA. Among these, triose phosphate isomerase exhibited a remarkable affinity to RnlA. These results suggest that RnlA plays a central role in RNase LS activity and that its activity is regulated by multiple components.
GENETICS SOC JAPAN, 英語, 研究論文（学術雑誌）
ISSN：1341-7568, eISSN：1880-5779, Web of Science ID：WOS:000250609000003 - Polysome-bound endonuclease PMR1 is targeted to stress granules via stress-specific binding to TIA-1
Feng Yang; Yong Peng; Elizabeth L. Murray; Yuichi Otsuka; Nancy Kedersha; Daniel R. Schoenberg
MOLECULAR AND CELLULAR BIOLOGY, 巻：26, 号：23, 開始ページ：8803, 終了ページ：8813, 2006年12月, ［査読有り］
The generalized process of mRNA decay involves deadenylation followed by release from translating polysomes, decapping, and exonuclease decay of the mRNA body. In contrast the mRNA endonuclease PMR1 forms a selective complex with its translating substrate mRNA, where it initiates decay by cleaving within the mRNA body. In stressed cells the phosphorylation of the alpha subunit of eukaryotic initiation factor 2 causes translating mRNAs to accumulate with stalled 48S subunits in large subcellular structures termed stress granules (SGs), wherein mRNAs undergo sorting for reinitiation, storage, or decay. Given the unique relationship between translation and PMR1-mediated mRNA decay, we examined the impact of stress-induced dissociation of polysomes on this process. Arsenite stress disrupts the polysome binding of PMR1 and its substrate mRNA but has no impact on the critical tyrosine phosphorylation of PMR1, its association with substrate mRNA, or its association with the functional similar to 680-kDa mRNP complex in which it normally resides on polysomes. We show that arsenite stress drives PMR1 into an RNase-resistant complex with TIA-1, and we identify a distinct domain in the N terminus of PMR1 that facilitates its interaction with TIA-1. Finally, we show that arsenite promotes the delayed association of PMR1 with SGs under conditions which cause tristetraprolin and butyrate response factor 1, proteins that facilitate exonucleolytic mRNA, to exit SGs.
AMER SOC MICROBIOLOGY, 英語, 研究論文（学術雑誌）
DOI：https://doi.org/10.1128/MCB.00090-06
DOI ID：10.1128/MCB.00090-06, ISSN：0270-7306, Web of Science ID：WOS:000242203700012 - A novel endoribonuclease, RNase LS, in Escherichia coli.
Otsuka Y; Yonesaki T
Genetics, 巻：169, 開始ページ：13, 終了ページ：20, 2005年01月, ［査読有り］, ［筆頭著者］
英語, 研究論文（学術雑誌）
CiNii Articles ID：10024394672 - A Novel Endoribonuclease of Escherichia coli that Induces Gene Silencing in Bacteriophage T4
大塚裕一
大阪大学, 2003年12月, ［筆頭著者, 責任著者］
英語, 学位論文（博士） - 大腸菌mRNAの分解
大塚裕一; 米崎哲朗
蛋白質核酸酵素, 巻：48, 号：3, 開始ページ：240, 終了ページ：246, 2003年03月, ［査読有り］, ［筆頭著者］
共立出版, 日本語, 学位論文（その他）
ISSN：0039-9450, CiNii Articles ID：40005690889, CiNii Books ID：AN00140437 - Escherichia coli endoribonucleases involved in cleavage of bacteriophage T4 mRNAs
Y Otsuka; H Ueno; T Yonesaki
JOURNAL OF BACTERIOLOGY, 巻：185, 号：3, 開始ページ：983, 終了ページ：990, 2003年02月, ［査読有り］, ［筆頭著者］
The dmd mutant of bacteriophage T4 has a defect in growth because of rapid degradation of late-gene mRNAs, presumably caused by mutant-specific cleavages of RNA. Some such cleavages can occur in an allele-specific manner, depending on the translatability of RNA or the presence of a termination codon. Other cleavages are independent of translation. In the present study, by introducing plasmids carrying various soc alleles, we could detect cleavages of soc RNA in uninfected cells identical to those found in dmd mutant-infected cells. We isolated five Escherichia coli mutant strains in which the dmd mutant was able to grow. One of these strains completely suppressed the dmd mutant-specific cleavages of soc RNA. The loci of the E. coli mutations and the effects of mutations in known RNase-encoding genes suggested that an RNA cleavage activity causing the dmd mutant-specific mRNA degradation is attributable to a novel RNase. In addition, we present evidence that 5'-truncated soc RNA, a stable form in T4-infected cells regardless of the presence of a dmd mutation, is generated by RNase E.
AMER SOC MICROBIOLOGY, 英語, 研究論文（学術雑誌）
DOI：https://doi.org/10.1128/JB.185.3.983-990.2003
DOI ID：10.1128/JB.185.3.983-990.2003, ISSN：0021-9193, Web of Science ID：WOS:000180834300031 - Gene 61.3 of bacteriophage t4 is the spackle gene
T Kai; H Ueno; Y Otsuka; W Morimoto; T Yonesaki
VIROLOGY, 巻：260, 号：2, 開始ページ：254, 終了ページ：259, 1999年08月, ［査読有り］
The bacteriophage T4 e gene encodes lysozyme (e-lysozyme), which releases progeny phage after normal infection of Escherichia coli cells. A mutation in the spackle gene suppresses the defect in e-lysozyme (Emrich, 1968). The spackle gene was mapped between genes 41 and 61, but its precise location has not previously been determined. In the current study, we constructed an amber mutant of gene 61.3, amST14, by site-directed mutagenesis. The gene 61.3 mutant shares phenotypes with spackle mutants: The amST14 mutant forms large plaques with sharp edges and exhibits truncated lysis inhibition, and furthermore, the mutation can suppress the defect in e-lysozyme activity. In addition, cloned gene 61.3 can rescue (by homologous recombination) as well as complement the S12 mutation in the spackle gene. These results strongly suggest that gene 61.3 is the spackle gene. Indeed, the S12 mutant has one base deletion of five in a consecutive A tract in the gene 61.3 coding region, substituting an unrelated 6-amino acid sequence for the 9 C-terminal amino acids in the gene 61.3 protein. The gene 61.3 protein is predicted to localize in the periplasmic space after cleavage of a signal sequence. (C) 1999 Academic Press.
ACADEMIC PRESS INC, 英語, 研究論文（学術雑誌）
ISSN：0042-6822, Web of Science ID：WOS:000082027100005
- RNA Damage and Repair
Yuichi Otsuka, ［共著］
Kotta-Loizou Ioly・Springer, 2021年07月
ISBN：9783030765712, 共同研究・競争的資金等ID：36529828;13443556
- 細菌とファージの生存競争
大塚裕一
第96回日本細菌学会総会, 2023年03月, ［招待有り］
口頭発表（招待・特別） - 細菌とファージの攻防 〜トキシン－アンチトキシン系〜
大塚裕一
第17回微生物研究会, 2018年11月, ［招待有り］, ［国内会議］
日本語, 口頭発表（招待・特別） - この世はウイルスだらけ
大塚裕一
日本微生物学連盟フォーラム「微生物：変わり者たちの素顔」, 2017年12月, ［招待有り］, ［国内会議］
日本語, 口頭発表（招待・特別） - 細菌とファージの生存戦略：トキシン－アンチトキシン系の制御
大塚裕一
第39回日本分子生物学会年会, 2016年12月, ［招待有り］, ［国内会議］
日本語, 口頭発表（招待・特別） - 細菌のトキシン－アンチトキシン系とファージ感染との関係
大塚裕一
第89回日本細菌学会総会, 2016年03月, ［招待有り］, ［国内会議］
日本語, 口頭発表（招待・特別） - ファージ療法・T4ファージを用いた新しいチャレンジ
大塚裕一
日本農芸化学会西日本支部大会シンポジウム, 2014年09月, ［招待有り］, ［国内会議］
日本語, 口頭発表（招待・特別） - Toxin–Antitoxin Systems in Escherichia coli O157 and Bacteriophage T4 Infection
大塚裕一
BIT’s 2nd Annual World Congress of Microbes–2012, 2012年07月, ［招待有り］, ［国際会議］
英語, 口頭発表（一般） - トキシン－アンチトキシンとT4ファージ
大塚裕一
第3回ファージ研究会「バクテリオファージ研究の可能性と課題」, 2010年09月, ［招待有り］, ［国内会議］
日本語, 口頭発表（招待・特別） - 5' capping of endonuclease-generated decay intermediates by a cytoplasmic capping enzyme complex
大塚裕一
Keystone Symposia 2008: Translational Regulatory Mechanisms, 2010年01月, ［招待有り］, ［国際会議］
英語, 口頭発表（招待・特別）
■ 共同研究・競争的資金等の研究課題
- 新規の抗ファージ機構が薬剤耐性菌の拡大を抑える
日本学術振興会, 科学研究費助成事業, 基盤研究(C), 2023年04月01日 - 2026年03月31日
大塚 裕一, 埼玉大学
配分額（総額）：4810000, 配分額（直接経費）：3700000, 配分額（間接経費）：1110000
本課題の目的は、大腸菌の新規因子群 AbpAとAbpB （以下、AbpAB）による抗ファージ機構が新規であることを明確にし、AbpABが形質導入や形質転換、接合を抑える因子として機能するかを検証するものである。その成果は、薬剤耐性遺伝子の水平伝播により拡大する薬剤耐性菌の蔓延を抑える対策に繋がることが期待される。本年度は4つの研究計画のうち、以下の3つの計画に取り組んだ。
計画①「ファージ感染によるAbpABの活性化機構を明らかにする」では、AbpABがファージの一本鎖DNA結合タンパク質の発現により活性化すること明らかにした。また、ファージ感染なしの条件において、DNA複製やDNA修復の阻害によりAbpABが活性化することも明らかにした。AbpABはある特定のDNA－タンパク質複合体を感知して活性化することが示唆される。
計画②「AbpAとAbpBの作用機序を明らかにする」では、AlphaFold2とDali-serverを用いて、AbpAとAbpBの立体構造を予測した。AbpAはCap4ヌクレアーゼドメインと高い類似性を示した。変異解析によりAbpAのヌクレアーゼ活性が抗ファージ作用に必要であることが示唆された。AbpBはDEAD-box RNAヘリカーゼファミリーに属するヒトRNAヘリカーゼDbp5と高い類似性を示した。また、AbpABの抗ファージ作用にはAbpBのRNAヘリカーゼ活性が必要であることが示唆された。現在、両タンパク質の活性を生化学的に調べるために発現と精製に取り組んでいる。
計画③「形質導入に対するAbpABの抑制作用を明らかにする」では、AbpABの発現により、溶原ファージSp5の誘発と溶原化が抑制されることを明らかにした。過去の結果と合わせて、AbpABは溶菌ファージだけでなく溶原ファージの増殖も抑制することがわかった。よって、ファージによる形質導入を抑制することを明らかにした。
課題番号：23K06526 - トキシン－アンチトキシン系を利用して薬剤耐性菌の拡大を抑える
日本学術振興会, 科学研究費助成事業 基盤研究(C), 基盤研究(C), 2020年04月 - 2024年03月
大塚 裕一, 埼玉大学, 研究代表者
配分額（総額）：4290000, 配分額（直接経費）：3300000, 配分額（間接経費）：990000
本課題は３つの研究項目からなるが、そのうちの1つ「形質導入に対するトキシン－アンチトキシン（TA）系の抑制作用を明らかにする」について本年度実験を実施した。本項目の目的は、TA系がファージを介しておこる形質導入を抑える可能性を検証し、その抑制メカニズムを明らかにするものであり、その成果は形質導入による薬剤耐性菌の蔓延を抑える対策に繋がることが期待される。形質導入は、外界ストレスにより溶原ファージが誘発される過程（誘発）と溶原ファージ感染後にファージゲノムが細菌ゲノムに組み込まれる過程（溶原化）からなる。
大腸菌K12株のTA系が形質導入に与える影響を調べるために、K12株が持つ36種類のTA系のうち、トキシンがRNaseである11種類のTA系を全て欠失した株（11TA欠失株）とトキシンが細胞膜損傷を引き起こす16種類のTA系を全て欠失した株（16TA欠失株）の作製を試みた。現在11TA欠失株は作製済みであり、16TA欠失株については6種類のTA系の欠失まで終了している。11TA欠失株を用いて志賀毒素をコードするSp5溶原ファージの誘発を調べたところ、RNaseをコードするTA系の複数もしくはどれかが誘発を抑制することが示唆された。現在どのTA系が誘発を抑制するのか同定中である。同定後はトキシンの作用機序を参考にして抑制メカニズムの解明に取り組む予定である。Sp5ファージの溶原化についても調べたが、RNaseをコードする11種類のTA系は溶原化には関与しないことが示唆された。今後は16TA欠失株を作製して、細胞膜損傷を引き起こすTA系が溶原ファージの誘発や溶原化に与える影響について調べる予定である。
課題番号：20K07493
書籍等出版物ID：36829728 - 薬剤耐性菌や病原菌の蔓延の抑制：トキシン－アンチトキシン系を利用して
日本学術振興会, 科学研究費助成事業 基盤研究(C), 基盤研究(C), 2017年04月 - 2021年03月
大塚 裕一, 研究代表者
配分額（総額）：4810000, 配分額（直接経費）：3700000, 配分額（間接経費）：1110000
細菌のトキシン－アンチトキシン系（TA）は、ストレスを乗り越える戦略の一つとして自身の増殖を制御するしくみである。本研究では、まず腸管出血性大腸菌O157株がもつTAのZorOトキシンの作用機序を明らかにした。次に、ZorOの毒性に必要な5アミノ酸（Ala-Leu-Leu-Arg-Leu; ALLRL）が黄色ブドウ球菌（MRSAを含む）や枯草菌、カンジダ属の菌の細胞膜に作用して増殖を阻害することを明らかにした。一方、哺乳類細胞に対しては細胞毒性を示さなかった。最後に、TAが遺伝子水平伝播の一因となるファージの形質導入を抑制することを明確にした。
課題番号：17K08837
論文ID：36529263 - 細菌が持つトキシン-アンチトキシン系の機能解析とその応用
日本学術振興会, 科学研究費助成事業 若手研究(B), 若手研究(B), 2013年04月 - 2017年03月
大塚 裕一, 研究代表者
配分額（総額）：4420000, 配分額（直接経費）：3400000, 配分額（間接経費）：1020000
細菌に広く保存されるトキシン－アンチトキシン系（TAS）は、ストレス時に発現するトキシンが自身の増殖を停止させる仕組みである。本研究では、腸管出血性大腸菌O157株が持つTAS、z3289-sRNA1を研究対象として、z3289トキシンが持つ毒性の分子機構とsRNA1アンチトキシンによるz3289の翻訳抑制機構を明らかにした。またTASの生物学的役割として抗ファージ作用を明確にした。さらに、改変したz3289トキシンが抗菌ペプチドとして作用することも示した。
課題番号：25870386
論文ID：36529314, 書籍等出版物ID：36829728 - ファージによる宿主認識機構の検証
日本学術振興会, 科学研究費助成事業 基盤研究(C), 基盤研究(C), 2011年04月 - 2015年03月
米崎 哲朗; 大塚 裕一, 大阪大学, 研究分担者
配分額（総額）：5200000, 配分額（直接経費）：4000000, 配分額（間接経費）：1200000
K12株のLPS合成に関わる種々遺伝子の変異体を用いて様々なLPS構造とT4吸着能との関係およびOmpC依存性の有無を系統的に調べた。さらに、OmpCに依存しないB株には吸着できるがOmpCに依存するK12株には吸着できない、あるいはその逆の性質を示すT4ファージ変異体の単離に成功したことから、T４はOmpCに依存した吸着とOmpCに依存しない吸着という異なる機構を駆使することが明らかとなった。また、本研究からT４が認識するOmpCの構造、その認識に必要なT４尾部繊維先端部位のアミノ酸、について初めて具体的な情報が得られた。
課題番号：23570211 - 細胞質におけるRNAキャップ付加反応と細胞ストレスや翻訳の関係
日本学術振興会, 科学研究費助成事業 研究活動スタート支援, 研究活動スタート支援, 2009年04月 - 2011年03月
大塚 裕一, 大阪大学, 研究代表者
配分額（総額）：2574000, 配分額（直接経費）：1980000, 配分額（間接経費）：594000
核のみで起こると考えられるmRNAキャップ付加反応が細胞質でも起こる可能性を検証することが本研究の目的である。期間内に、細胞質でキャップ付加されるmRNAを複数同定し、さらにそれらRNAの3'末端が修飾(ポリU化)されている可能性を示唆した。また異なるプロジェクトとして、大腸菌に感染するT4ファージが大腸菌の毒素に対して抗毒素作用を持つ因子を備え、自らの増殖を可能にしていることも明らかにした。
課題番号：21870020 - Relationship of cytoplasmic capping to translation and cell stress
米国心臓協会, ポスドク助成金, 研究活動スタート支援, 2008年07月 - 2009年03月
大塚裕一, 大阪大学, 研究代表者
核のみで起こると考えられるmRNAキャップ付加反応が細胞質でも起こる可能性を検証することが本研究の目的である。期間内に、細胞質でキャップ付加されるmRNAを複数同定し、さらにそれらRNAの3'末端が修飾(ポリU化)されている可能性を示唆した。また異なるプロジェクトとして、大腸菌に感染するT4ファージが大腸菌の毒素に対して抗毒素作用を持つ因子を備え、自らの増殖を可能にしていることも明らかにした。
競争的資金, 課題番号：21870020 - Relationship of cytoplasmic capping to translation and mRNA decay
米国心臓協会, ポスドク助成金, 2006年07月 - 2008年06月
大塚裕一, 研究代表者
競争的資金