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CHUMSAKUL Onuma大学院科学技術イノベーション研究科 科学技術イノベーション専攻助教
研究活動情報
■ 論文- 2024年06月, Experimental dermatology, 33(6) (6), e15118, 英語, 国際誌研究論文(学術雑誌)
- Partial bacterial genome reduction by genome engineering can improve the productivity of various metabolites, possibly via deletion of non-essential genome regions involved in undesirable metabolic pathways competing with pathways for the desired end products. However, such reduction may cause growth defects. Genome reduction of Bacillus subtilis MGB874 increases the productivity of cellulases and proteases but reduces their growth rate. Here, we show that this growth defect could be restored by silencing redundant or less important genes affecting exponential growth by manipulating the global transcription factor AbrB. Comparative transcriptome analysis revealed that AbrB-regulated genes were upregulated and those involved in central metabolic pathway and synthetic pathways of amino acids and purine/pyrimidine nucleotides were downregulated in MGB874 compared with the wild-type strain, which we speculated were the cause of the growth defects. By constitutively expressing high levels of AbrB, AbrB regulon genes were repressed, while glycolytic flux increased, thereby restoring the growth rate to wild-type levels. This manipulation also enhanced the productivity of metabolites including γ-polyglutamic acid. This study provides the first evidence that undesired features induced by genome reduction can be relieved, at least partly, by manipulating a global transcription regulation system. A similar strategy could be applied to other genome engineering-based challenges aiming toward efficient material production in bacteria.2022年05月, DNA research : an international journal for rapid publication of reports on genes and genomes, 29(3) (3), 英語, 国際誌研究論文(学術雑誌)
- The structure and function of bacterial chromosomes are dynamically regulated by a wide variety of nucleoid-associated proteins (NAPs) and DNA superstructures, such as DNA supercoiling. In Escherichia coli, integration host factor (IHF), a NAP, binds to specific transcription promoters and regulatory DNA elements of DNA replication such as the replication origin oriC: binding to these elements depends on the cell cycle but underlying mechanisms are unknown. In this study, we combined GeF-seq (genome footprinting with high-throughput sequencing) with synchronization of the E. coli cell cycle to determine the genome-wide, cell cycle-dependent binding of IHF with base-pair resolution. The GeF-seq results in this study were qualified enough to analyze genomic IHF binding sites (e.g., oriC and the transcriptional promoters of ilvG and osmY) except some of the known sites. Unexpectedly, we found that before replication initiation, oriC was a predominant site for stable IHF binding, whereas all other loci exhibited reduced IHF binding. To reveal the specific mechanism of stable oriC-IHF binding, we inserted a truncated oriC sequence in the terC (replication terminus) locus of the genome. Before replication initiation, stable IHF binding was detected even at this additional oriC site, dependent on the specific DnaA-binding sequence DnaA box R1 within the site. DnaA oligomers formed on oriC might protect the oriC-IHF complex from IHF dissociation. After replication initiation, IHF rapidly dissociated from oriC, and IHF binding to other sites was sustained or stimulated. In addition, we identified a novel locus associated with cell cycle-dependent IHF binding. These findings provide mechanistic insight into IHF binding and dissociation in the genome.2021年, Frontiers in microbiology, 12, 697712 - 697712, 英語, 国際誌研究論文(学術雑誌)
- Purpose: Photoreceptor degeneration is a major cause of blindness. Microglia are known to play key roles in the pathogenesis and progression of neural degeneration. We examined the possible use of apigenin, which is a naturally occurring flavonoid, for the treatment of photoreceptor degeneration through regulation of microglial activities. Methods: As in vitro analyses, BV2 and MG5 mouse microglia cell lines were stimulated in the presence or absence of apigenin, and their activation profile was examined. In vivo study was done using rd1 photoreceptor degeneration model, and apigenin was administered by intravitreal injection, and pathological feature was examined. Results: Cell survival was not affected by apigenin in either BV2 and MG5. Apigenin suppressed lipopolysaccharide (LPS)-induced chemokine production in both BV2 and MG5 cells, but phagocytosis was suppressed in MG5 cells but not in BV2 cells. Apigenin inhibited LPS-induced M1 activation but could not drive microglia toward the M2 phenotype. Apigenin suppressed the expression of miR-155 in a dose-dependent manner. Furthermore, the Ets protein level was suppressed by treatment of BV2 cells with apigenin. When rd1 mice were treated with apigenin by intravitreal injection, the expression of inflammatory chemokines in the retina was reduced, and activation of microglia and Müller glia was suppressed. Furthermore, the thickness of the outer nuclear layer of the retina of rd1 mice was thicker in apigenin-treated retinas. Conclusions: Taken together, local administration of apigenin to the retina is a potential therapeutic treatment for photoreceptor degeneration, which involves downregulation of microglia in the retina when photoreceptors are damaged.2020年06月, Journal of ocular pharmacology and therapeutics : the official journal of the Association for Ocular Pharmacology and Therapeutics, 36(5) (5), 311 - 319, 英語, 国際誌研究論文(学術雑誌)
- Nucleotide sequences recognized and bound by DNA-binding proteins (DBPs) are critical to control and maintain gene expression, replication, chromosome segregation, cell division, and nucleoid structure in bacterial cells. Therefore, determination of the binding sequences of DBPs is important not only to study DBP recognition mechanisms, but also to understand the fundamentals of cell homeostasis. While ChIP-seq analysis appears to be an effective way to determine DBP-binding sites on the genome, the resolution is sometimes not sufficient to identify the sites precisely. Here, we introduce a simple and effective method named Genome footprinting with high-throughput sequencing (GeF-seq) to determine binding sites of DBPs at single base-pair resolution. GeF-seq detects binding sites of DBPs as sharp peaks and thus makes it possible to identify the recognition sequence in each "binding peak" more easily and accurately than using ChIP-seq.2018年, Methods in molecular biology (Clifton, N.J.), 1837, 33 - 47, 英語, 国際誌研究論文(学術雑誌)
- Upon oxygen limitation, the Bacillus subtilis ResE sensor kinase and its cognate ResD response regulator play primary roles in the transcriptional activation of genes functioning in anaerobic respiration. The nitric oxide (NO)-sensitive NsrR repressor controls transcription to support nitrate respiration. In addition, the ferric uptake repressor (Fur) can modulate transcription under anaerobic conditions. However, whether these controls are direct or indirect has been investigated only in a gene-specific manner. To gain a genomic view of anaerobic gene regulation, we determined the genome-wide in vivo DNA binding of ResD, NsrR, and Fur transcription factors (TFs) using in situ DNase I footprinting combined with chromatin affinity precipitation sequencing (ChAP-seq; genome footprinting by high-throughput sequencing [GeF-seq]). A significant number of sites were targets of ResD and NsrR, and a majority of them were also bound by Fur. The binding of multiple TFs to overlapping targets affected each individual TF's binding, which led to combinatorial transcriptional control. ResD bound to both the promoters and the coding regions of genes under its positive control. Other genes showing enrichment of ResD at only the promoter regions are targets of direct ResD-dependent repression or antirepression. The results support previous findings of ResD as an RNA polymerase (RNAP)-binding protein and indicated that ResD can associate with the transcription elongation complex. The data set allowed us to reexamine consensus sequence motifs of Fur, ResD, and NsrR and uncovered evidence that multiple TGW (where W is A or T) sequences surrounded by an A- and T-rich sequence are often found at sites where all three TFs competitively bind.IMPORTANCE Bacteria encounter oxygen fluctuation in their natural environment as well as in host organisms. Hence, understanding how bacteria respond to oxygen limitation will impact environmental and human health. ResD, NsrR, and Fur control transcription under anaerobic conditions. This work using in situ DNase I footprinting uncovered the genome-wide binding profile of the three transcription factors (TFs). Binding of the TFs is often competitive or cooperative depending on the promoters and the presence of other TFs, indicating that transcriptional regulation by multiple TFs is much more complex than we originally thought. The results from this study provide a more complete picture of anaerobic gene regulation governed by ResD, NsrR, and Fur and contribute to our further understanding of anaerobic physiology.2017年07月, Journal of bacteriology, 199(13) (13), 英語, 国際誌研究論文(学術雑誌)
- Although the speed of nascent DNA synthesis at individual replication forks is relatively uniform in bacterial cells, the dynamics of replication fork progression on the chromosome are hampered by a variety of natural impediments. Genome replication dynamics can be directly measured from an exponentially growing cell population by sequencing newly synthesized DNA strands that were specifically pulse-labeled with the thymidine analogue 5-bromo-2'-deoxyuridine (BrdU). However, a short pulse labeling with BrdU is impracticable for bacteria because of poor incorporation of BrdU into the cells, and thus, the genomewide dynamics of bacterial DNA replication remain undetermined. Using a new thymidine-requiring Escherichia coli strain, eCOMB, and high-throughput sequencing, we succeeded in determining the genomewide replication profile in bacterial cells. We also found that fork progression is paused in two ~200-kb chromosomal zones that flank the replication origin in the growing cells. This origin-proximal obstruction to fork progression was overcome by an increased thymidine concentration in the culture medium and enhanced by inhibition of transcription. These indicate that DNA replication near the origin is sensitive to the impediments to fork progression, namely a scarcity of the DNA precursor deoxythymidine triphosphate and probable conflicts between replication and transcription machineries.2016年08月, Genes to cells : devoted to molecular & cellular mechanisms, 21(8) (8), 907 - 14, 英語, 国際誌研究論文(学術雑誌)
- BACKGROUND: The two-component regulatory system, involving the histidine sensor kinase DegS and response regulator DegU, plays an important role to control various cell processes in the transition phase of Bacillus subtilis. The degU32 allele in strain 1A95 is characterized by the accumulation of phosphorylated form of DegU (DegU-P). RESULTS: Growing 1A95 cells elevated the pH of soytone-based medium more than the parental strain 168 after the onset of the transition phase. The rocG gene encodes a catabolic glutamate dehydrogenase that catalyzes one of the main ammonia-releasing reactions. Inactivation of rocG abolished 1A95-mediated increases in the pH of growth media. Thus, transcription of the rocG locus was examined, and a novel 3.7-kb transcript covering sivA, rocG, and rocA was found in 1A95 but not 168 cells. Increased intracellular fructose 1,6-bisphosphate (FBP) levels are known to activate the HPr kinase HPrK, and to induce formation of the P-Ser-HPr/CcpA complex, which binds to catabolite responsive elements (cre) and exerts CcpA-dependent catabolite repression. A putative cre found within the intergenic region between sivA and rocG, and inactivation of ccpA led to creation of the 3.7-kb transcript in 168 cells. Analyses of intermediates in central carbon metabolism revealed that intracellular FBP levels were lowered earlier in 1A95 than in 168 cells. A genome wide transcriptome analysis comparing 1A95 and 168 cells suggested similar events occurring in other catabolite repressive loci involving induction of lctE encoding lactate dehydrogenase. CONCLUSIONS: Under physiological conditions the 3.7-kb rocG transcript may be tightly controlled by a roadblock mechanism involving P-Ser-HPr/CcpA in 168 cells, while in 1A95 cells abolished repression of the 3.7-kb transcript. Accumulation of DegU-P in 1A95 affects central carbon metabolism involving lctE enhanced by unknown mechanisms, downregulates FBP levels earlier, and inactivates HPrK to allow the 3.7-kb transcription, and thus similar events may occur in other catabolite repressive loci.2015年02月, BMC microbiology, 15, 43 - 43, 英語, 国際誌研究論文(学術雑誌)
- Bacterial bio-production during the stationary phase is expected to lead to a high target yield because the cells do not consume the substrate for growth. Bacillus subtilis is widely used for bio-production, but little is known about the metabolism during the stationary phase. In this study, we focused on the dipicolinic acid (DPA) production by B. subtilis and investigated the metabolism. We found that DPA production competes with acetoin synthesis and that acetoin synthesis genes (alsSD) deletion increases DPA productivity by 1.4-fold. The mutant showed interesting features where the glucose uptake was inhibited, whereas the cell density increased by approximately 50%, resulting in similar volumetric glucose consumption to that of the parental strain. The metabolic profiles revealed accumulation of pyruvate, acetyl-CoA, and the TCA cycle intermediates in the alsSD mutant. Our results indicate that alsSD-deleted B. subtilis has potential as an effective host for stationary-phase production of compounds synthesized from these intermediates.2015年, Bioscience, biotechnology, and biochemistry, 79(12) (12), 2073 - 80, 英語, 国際誌研究論文(学術雑誌)
- The amino acid sequence of the RNA polymerase (RNAP) α-subunit is well conserved throughout the Eubacteria. Its C-terminal domain (α-CTD) is important for the transcriptional regulation of specific promoters in both Escherichia coli and Bacillus subtilis, through interactions with transcription factors and/or a DNA element called the "UP element". However, there is only limited information regarding the α-CTD regulated genes in B. subtilis and the importance of this subunit in the transcriptional regulation of B. subtilis. Here, we established strains and the growth conditions in which the α-subunit of RNAP was replaced with a C-terminally truncated version. Transcriptomic and ChAP-chip analyses revealed that α-CTD deficiency reduced the transcription and RNAP binding of genes related to the utilization of secondary carbon sources, transition state responses, and ribosome synthesis. In E. coli, it is known that α-CTD also contributes to the expression of genes related to the utilization of secondary carbon sources and ribosome synthesis. Our results suggest that the biological importance of α-CTD is conserved in B. subtilis and E. coli, but that its specific roles have diversified between these two bacteria.2015年, PloS one, 10(7) (7), e0131588, 英語, 国際誌研究論文(学術雑誌)
- The ResD response regulator activates transcription of diverse genes in Bacillus subtilis in response to oxygen limitation. ResD regulon genes that are the most highly induced during nitrate respiration include the nitrite reductase operon (nasDEF) and the flavohemoglobin gene (hmp), whose products function in nitric oxide (NO) metabolism. Transcription of these genes is also under the negative control of the NO-sensitive NsrR repressor. Recent studies showed that the NsrR regulon contains genes with no apparent relevance to NO metabolism and that the ResD response regulator and NsrR coordinately regulate transcription. To determine whether these genes are direct targets of NsrR and ResD, we used chromatin affinity precipitation coupled with tiling chip (ChAP-chip) and ChAP followed by quantitative PCR (ChAP-qPCR) analyses. The study showed that ResD and NsrR directly control transcription of the ykuNOP operon in the Fur regulon. ResD functions as an activator at the nasD and hmp promoters, whereas it functions at the ykuN promoter as an antirepressor of Fur and a corepressor for NsrR. This mechanism likely participates in fine-tuning of transcript levels in response to different sources of stress, such as oxygen limitation, iron limitation, and exposure to NO.2014年01月, Journal of bacteriology, 196(2) (2), 493 - 503, 英語, 国際誌研究論文(学術雑誌)
- Accurate identification of the DNA-binding sites of transcription factors and other DNA-binding proteins on the genome is crucial to understanding their molecular interactions with DNA. Here, we describe a new method: Genome Footprinting by high-throughput sequencing (GeF-seq), which combines in vivo DNase I digestion of genomic DNA with ChIP coupled with high-throughput sequencing. We have determined the in vivo binding sites of a Bacillus subtilis global regulator, AbrB, using GeF-seq. This method shows that exact DNA-binding sequences, which were protected from in vivo DNase I digestion, were resolved at a comparable resolution to that achieved by in vitro DNase I footprinting, and this was simply attained without the necessity of prediction by peak-calling programs. Moreover, DNase I digestion of the bacterial nucleoid resolved the closely positioned AbrB-binding sites, which had previously appeared as one peak in ChAP-chip and ChAP-seq experiments. The high-resolution determination of AbrB-binding sites using GeF-seq enabled us to identify bipartite TGGNA motifs in 96% of the AbrB-binding sites. Interestingly, in a thousand binding sites with very low-binding intensities, single TGGNA motifs were also identified. Thus, GeF-seq is a powerful method to elucidate the molecular mechanism of target protein binding to its cognate DNA sequences.2013年08月, DNA research : an international journal for rapid publication of reports on genes and genomes, 20(4) (4), 325 - 38, 英語, 国際誌研究論文(学術雑誌)
- AbrB is a global transcriptional regulator of Bacillus subtilis that represses the expression of many genes during exponential growth. Here, we demonstrate that AbrB and its homolog Abh bind to hundreds of sites throughout the entire B. subtilis genome during exponential growth. Comparison of regional binding of AbrB and Abh in wild-type, ΔabrB and Δabh backgrounds revealed that they bind as homomer and/or heteromer forms with different specificities and affinities. We found four AbrB and Abh binding patterns were major. Three of these contain pairs of TGGNA motifs connected by A/T-rich sequences, differing in arrangement and spacing. We also assessed the direct involvement of these complexes in the control of gene expression. Our data indicate that AbrB usually acts as a repressor, and that the ability of Abh to act as a transcriptional regulator was limited. We found that changes to AbrB/Abh levels affect their binding at several promoters and consequently transcriptional regulation. Surprisingly, most AbrB/Abh binding events had no impact on transcription, suggesting an interesting possibility that AbrB/Abh binding is analogous to nucleoid-associated protein binding in Escherichia coli.2011年01月, Nucleic acids research, 39(2) (2), 414 - 28, 英語, 国際誌研究論文(学術雑誌)
- 共著, Human press, 2018年, ISBN: 9781493986750GeF-seq: A Simple Procedure for Base Pair Resolution ChIP-seq
- 共著, 羊土社, 2014年実験医学別冊,次世代シーケンス解析スタンダード : 高精度で結合領域を決定するGeF-seq
- The Association for Research in Vision and Ophthalmology Conference (ARVO), 2019年, 英語Apigenin Inhibits LPS-induced Microglia Activation by Modulating MicroRNA Expression.ポスター発表
- Retina Research Meeting (RRM), 2019年, 英語Apigenin regulates activation of microglia and counteracts retinal degeneration口頭発表(一般)
- International Conference on Gram-positive Microorganism, 2015年, 英語pH adaptation mechanism in Leuconostoc mesenteroidesポスター発表
- International Conference on Gram-positive Microorganism, 2013年, 英語High-resolution mapping of in vivo genomic transcription factor binding sites using in situ DNase I footprinting and ChIP-seq.ポスター発表
- Congress of European Microbiologists (FEMS), 2011年, 英語Genome-wide binding profiles of the Bacillus subtilis transition state regulator AbrB and its homolog Abhポスター発表
- 超高分子ガンマ-ポリグルタミン酸、該ポリグルタミン酸を産生するバチルス属細菌変異株及び該バチルス属細菌変異株のスクリーニング方法特願2022-159018, 2022年09月30日, 国立大学法人神戸大学, 特開2024-052354, 2024年04月11日特許権
- 後眼部疾患の予防、改善、又は治療用組成物特願2018-090656, 2018年05月09日, ロート製薬株式会社, 特開2018-188438, 2018年11月29日, 特許第7105532号, 2022年07月14日特許権
- 組換え微生物及びその利用特願2015-211548, 2015年10月28日, 花王株式会社, 国立大学法人 奈良先端科学技術大学院大学, 特開2017-079640, 2017年05月18日, 特許第6791623号, 2020年11月09日特許権
- 枯草菌変異株及びその利用特願2015-211547, 2015年10月28日, 花王株式会社, 国立大学法人 奈良先端科学技術大学院大学, 特開2017-079639, 2017年05月18日, 特許第6693723号, 2020年04月20日特許権
- 枯草菌変異株及びその利用特願2015-226957, 2015年11月19日, 花王株式会社, 国立大学法人 奈良先端科学技術大学院大学, 特開2017-093321, 2017年06月01日, 特許第6660718号, 2020年02月13日特許権