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研究者情報

マスター

アカウント（マスター）

  • 氏名

    笠原　康裕(カサハラ　ヤスヒロ), カサハラ　ヤスヒロ

所属（マスター）

  • 低温科学研究所　生物環境部門

所属（マスター）

  • 低温科学研究所　生物環境部門

researchmap

プロフィール情報

学位

  • 博士（農学）（東北大学）

プロフィール情報

  • 姓

    笠原, カサハラ

  • 名

    康裕, ヤスヒロ

  • ID各種

    200901073112870143

対象リソース

業績リスト

研究キーワード

  • 微生物多様性   群集構造解析   メタオミクス   プロテオミクス   ゲノム微生物学   微生物生態学   Genomic Microbiology   Protenomics   Community structure   Soil microbiology   

研究分野

  • ライフサイエンス / 生態学、環境学
  • ライフサイエンス / システムゲノム科学
  • ライフサイエンス / ゲノム生物学
  • 環境・農学 / ランドスケープ科学
  • 環境・農学 / 環境農学

経歴

  • 2005年 - 現在 北海道大学低温科学研究所
  • 2002年 - 2005年 茨城大学農学部資源生物科学科
  • 1994年 - 2002年 奈良先端科学技術大学院大学バイオサイエンス研究科
  • 2002年 奈良先端科学技術大学院大学情報科学研究科
  • 1993年 - 1994年 大阪大学医学部

学歴

  •         - 1992年   東北大学   農学研究科
  •         - 1986年   東北大学   農学部

委員歴

  • 2013年01月 - 2015年12月   日本微生物生態学会   評議員
  • 2007年01月 - 2015年12月   日本微生物生態学会   和文誌編集幹事   日本微生物生態学会
  • 2005年01月 - 2008年12月   日本微生物生態学会   評議員   日本微生物生態学会

論文

  • Mamoru Oshiki, Yuka Toyama, Toshikazu Suenaga, Akihiko Terada, Yasuhiro Kasahara, Takashi Yamaguchi, Nobuo Araki
    Microbes and Environments 37 2 2022年 [査読有り]
  • Fumika Muramatsu, Mimori Tonomura, Mikina Yamada, Yasuhiro Kasahara, Shigeki Yamamura, Takao Iino, Seigo Amachi
    Applied and Environmental Microbiology 2020年09月25日 [査読有り][通常論文]
     
    Anaeromyxobacter sp. strain PSR-1, a dissimilatory arsenate [As(V)]-reducing bacterium, can utilize As(V) as a terminal electron acceptor for anaerobic respiration. A previous draft genome analysis revealed that strain PSR-1 lacks typical respiratory As(V) reductase genes (arrAB), which suggested the involvement of another protein in As(V) respiration. Dissimilatory As(V) reductase activity of strain PSR-1 was induced under As(V)-respiring conditions, and was localized predominantly in the periplasmic fraction. The activity was visualized on partially-denaturing gel electrophoresis, and liquid chromatography-tandem mass spectrometry analysis identified proteins involved in the active band. Among these proteins, a protein annotated as molybdopterin-dependent oxidoreductase (PSR1_00330) exhibited the highest sequence coverage of 76%. Phylogenetic analysis revealed that this protein was a homolog of tetrathionate reductase catalytic subunit TtrA. However, the crude extract of strain PSR-1 did not show significant tetrathionate reductase enzyme activity. Comparative proteomic analysis revealed that the protein PSR1_00330 and a homolog of tetrathionate reductase electron transfer subunit TtrB (PSR1_00329) were expressed abundantly and specifically under As(V)-respiring conditions, respectively. The genes encoding PSR1_00330 and PSR1_00329 formed an operon-like structure along with a c-type cytochrome gene, and their transcription was upregulated under As(V)-respiring conditions. These results suggest that the protein PSR1_00330, which lacks tetrathionate reductase activity, functions as a dissimilatory As(V) reductase in strain PSR-1. Considering the wide distribution of TtrA homologs among bacteria and archaea, they may play a hitherto unknown role along with conventional respiratory As(V) reductase (Arr) in the biogeochemical cycling of arsenic in nature. IMPORTANCE Dissimilatory As(V)-reducing prokaryotes play significant roles in arsenic release and contamination in groundwater and threaten the health of people worldwide. Generally, such prokaryotes reduce As(V) by means of a respiratory As(V) reductase designated as Arr. However, some dissimilatory As(V)-reducing prokaryotes such as Anaeromyxobacter sp. strain PSR-1 lack genes encoding Arr, suggesting the involvement of other protein in As(V) reduction. In this study, by using multiple proteomic and transcriptional analyses, it was found that the dissimilatory As(V) reductase of strain PSR-1 was a protein closely related to the tetrathionate reductase catalytic subunit (TtrA). Tetrathionate reductase is known to play a role in anaerobic respiration of Salmonella on tetrathionate, but strain PSR-1 showed neither growth on tetrathionate nor significant tetrathionate reductase enzyme activity. These results suggest the possibility that TtrA homologs encoded in a wide variety of archaeal and bacterial genomes might function as dissimilatory As(V) reductases.
  • Chihiro Yamazaki, Sumie Kashiwa, Ayaka Horiuchi, Yasuhiro Kasahara, Shigeki Yamamura, Seigo Amachi
    Environmental Microbiology 22 6 2196 - 2212 2020年06月 [査読有り][通常論文]
     
    Pseudomonas sp. strain SCT is capable of using iodate (IO3- ) as a terminal electron acceptor for anaerobic respiration. A possible key enzyme, periplasmic iodate reductase (Idr), was visualized by active staining on non-denaturing gel electrophoresis. Liquid chromatography-tandem mass spectrometry analysis revealed that at least four proteins, designated as IdrA, IdrB, IdrP1 , and IdrP2 , were involved in Idr. IdrA and IdrB were homologues of catalytic and electron transfer subunits of respiratory arsenite oxidase (Aio); however, IdrA defined a novel clade within the dimethylsulfoxide (DMSO) reductase family. IdrP1 and IdrP2 were closely related to each other and distantly related to cytochrome c peroxidase. The idr genes (idrABP 1 P 2 ) formed an operon-like structure, and their transcription was upregulated under iodate-respiring conditions. Comparative proteomic analysis also revealed that Idr proteins and high affinity terminal oxidases (Cbb3 and Cyd), various H2 O2 scavengers, and chlorite (ClO2- ) dismutase-like proteins were expressed specifically or abundantly under iodate-respiring conditions. These results suggest that Idr is a respiratory iodate reductase, and that both O2 and H2 O2 are formed as by-products of iodate respiration. We propose an electron transport chain model of strain SCT, in which iodate, H2 O2 , and O2 are used as terminal electron acceptors.
  • Tatsuya Tsuchiya, Ayaka Ehara, Yasuhiro Kasahara, Natsuko Hamamura, Seigo Amachi
    Applied and Environmental Microbiology 85 14 2019年05月17日 [査読有り][通常論文]
     
    ABSTRACT The reduction of arsenate [As(V)] to arsenite [As(III)] by dissimilatory As(V)-reducing bacteria, such as Geobacter spp., may play a significant role in arsenic release from anaerobic sediments into groundwater. The biochemical and molecular mechanisms by which these bacteria cope with this toxic element remain unclear. In this study, the expression of several genes involved in arsenic respiration (arr) and resistance (ars) was determined using Geobacter sp. strain OR-1, the only cultured Geobacter strain capable of As(V) respiration. In addition, proteins expressed differentially under As(V)-respiring conditions were identified by semiquantitative proteomic analysis. Dissimilatory As(V) reductase (Arr) of strain OR-1 was localized predominantly in the periplasmic space, and the transcription of its gene (arrA) was upregulated under As(V)-respiring conditions. The transcription of the detoxifying As(V) reductase gene (arsC) was also upregulated, but its induction required 500 times higher concentration of As(III) (500 μM) than did the arrA gene. Comparative proteomic analysis revealed that in addition to the Arr and Ars proteins, proteins involved in the following processes were upregulated under As(V)-respiring conditions: (i) protein folding and assembly for rescue of proteins with oxidative damage, (ii) DNA replication and repair for restoration of DNA breaks, (iii) anaplerosis and gluconeogenesis for sustainable energy production and biomass formation, and (iv) protein and nucleotide synthesis for the replacement of damaged proteins and nucleotides. These results suggest that strain OR-1 copes with arsenic stress by orchestrating pleiotropic processes that enable this bacterium to resist and actively metabolize arsenic. IMPORTANCE Dissimilatory As(V)-reducing bacteria, such as Geobacter spp., play significant roles in arsenic release and contamination in groundwater and threaten the health of people worldwide. However, the biochemical and molecular mechanisms by which these bacteria cope with arsenic toxicity remain unclear. In this study, it was found that both respiratory and detoxifying As(V) reductases of a dissimilatory As(V)-reducing bacterium, Geobacter sp. strain OR-1, were upregulated under As(V)-respiring conditions. In addition, various proteins expressed specifically or more abundantly in strain OR-1 under arsenic stress were identified. Strain OR-1 actively metabolizes arsenic while orchestrating various metabolic processes that repair oxidative damage caused by arsenic. Such information is useful in assessing and identifying possible countermeasures for the prevention of microbial arsenic release in nature.
  • Identification of nitrogen-fixing Bradyrhizobium associated with roots of field-grown sorghum by metagenome and proteome analyses.
    Minamisawa, K, S. Hara, T. Morikawa, S. Wasai, Y. Kasahara, T. Koshiba, K. Yamazaki, T. Fujiwara, T. Tokunaga
    Frontiers in Microbiology 10 407  2019年03月 [査読有り][通常論文]
  • Mamoru Oshiki, Toshikazu Fukushima, Shuichi Kawano, Yasuhiro Kasahara, Junichi Nakagawa
    Microbes and Environments 34 4 402 - 412 2019年 [査読有り][通常論文]
  • Bihe Kong, Lei Chen, Yasuhiro Kasahara, Akihiro Sumida, Kiyomi Ono, Jan Wild, Arata Nagatake, Ryusuke Hatano, Toshihiko Hara
    MICROBES AND ENVIRONMENTS 32 2 103 - 111 2017年06月 [査読有り][通常論文]
     
    In order to understand the relationships between understory bamboo and soil properties, we compared microbial community structures in the soil of a Betula ermanii boreal forest with Sasa kurilensis present and removed using high-throughput DNA sequencing. The presence of understory S. kurilensis strongly affected soil properties, including total carbon, total nitrogen, nitrate, and the C:N ratio as well as relative soil moisture. Marked differences were also noted in fungal and bacterial communities between plots. The relative abundance of the fungal phylum Ascomycota was 13.9% in the Sasa-intact plot and only 0.54% in the Sasa-removed plot. Among the Ascomycota fungi identified, the most prevalent were members of the family Pezizaceae. We found that the abundance of Pezizaceae, known to act as mycorrhizal fungi, was related to the amount of total carbon in the Sasa-intact plot. The relative abundance of Proteobacteria was significantly higher, whereas those of Planctomycetes and Actinobacteria were lower in the Sasa-intact plot than in the Sasa-removed plot. Furthermore, the results obtained suggest that some species of the phylum Planctomycetes are more likely to occur in the presence of S. kurilensis. Collectively, these results indicate that the presence of S. kurilensis affects microbial communities and soil properties in a B. ermanii boreal forest.
  • Shakhinur Islam Mondal, Md Rakibul Islam, Akira Sawaguchi, Md Asadulghani, Tadasuke Ooka, Yasuhiro Gotoh, Yasuhiro Kasahara, Yoshitoshi Ogura, Tetsuya Hayashi
    Scientific Reports 6 1 2016年12月 [査読有り][通常論文]
     
    Shiga toxin 2 (Stx2), one of the most important virulence factors of enterohaemorrhagic Escherichia coli (EHEC), is encoded by phages. These phages (Stx2 phages) are often called lambda-like. However, most Stx2 phages are short-tailed, thus belonging to the family Podoviridae, and the functions of many genes, especially those in the late region, are unknown. In this study, we performed a systematic genetic and morphological analysis of genes with unknown functions in Sp5, the Stx2 phage from EHEC O157:H7 strain Sakai. We identified nine essential genes, which, together with the terminase genes, determine Sp5 morphogenesis. Four of these genes most likely encoded portal, major capsid, scaffolding and tail fiber proteins. Although exact roles/functions of the other five genes are unknown, one was involved in head formation and four were required for tail formation. One of the four tail genes encoded an unusually large protein of 2,793 amino-acid residues. Two genes that are likely required to maintain the lysogenic state were also identified. Because the late regions of Stx2 phages from various origins are highly conserved, the present study provides an important basis for better understanding the biology of this unique and medically important group of bacteriophages.
  • Hajime Morimoto, Ryosuke Kadoya, Kazuhiro Takahashi, Yasuhiro Kasahara
    ENVIRONMENTAL MICROBIOLOGY REPORTS 8 5 825 - 832 2016年10月 [査読有り][通常論文]
     
    Knowledge of the gene expression dynamics of a single soil bacterial strain contributes to the understanding of its behaviour, physiological state and surrounding-microenvironment. Genes expressed in soil environments rather than in laboratory media are considered to particularly relevant. Here, we compared genome-wide gene expression profiles of the bacterium Pseudomonas putida F1 inoculated in three different types of nonsterile soils deduced using proteome analysis via sodium dodecyl sulfate-polyacrylamide gel electrophoresis combined with liquid chromatography-tandem mass spectrometry. Proteins commonly detected in all three samples and involved with bacterial growth and fundamental metabolism were excluded. Nine proteins were identified as specifically expressed in soil including an aldehyde dehydrogenase, a nitric oxide dioxygenase and five proteins encoded by a cluster of metabolism-associated genes. Expression factor analysis revealed that the nitric oxide dioxygenase-coding gene was induced by nitric oxide and the five clustered genes were induced under phosphate starvation. The expression of these genes can be attributed to response to soil environmental stimuli surrounding the F1 cells. These results strongly suggest that our soil metaproteome approach is useful for understanding the autecology and lifestyle of a single bacterial strain in soil environments and allows the prediction of themicroenvironment surrounding the bacterial cells.
  • 笠原 康裕
    土と微生物 70 2 41 - 44 2016年 [査読有り][通常論文]
  • Zhihua Bao, Takashi Okubo, Kengo Kubota, Yasuhiro Kasahara, Hirohito Tsurumaru, Mizue Anda, Seishi Ikeda, Kiwamu Minamisawa
    APPLIED AND ENVIRONMENTAL MICROBIOLOGY 80 16 5043 - 5052 2014年08月 [査読有り][通常論文]
     
    In a previous study by our group, CH4 oxidation and N-2 fixation were simultaneously activated in the roots of wild-type rice plants in a paddy field with no N input; both processes are likely controlled by a rice gene for microbial symbiosis. The present study examined which microorganisms in rice roots were responsible for CH4 oxidation and N-2 fixation under the field conditions. Metaproteomic analysis of root-associated bacteria from field-grown rice (Oryza sativa Nipponbare) revealed that nitrogenase complex-containing nitrogenase reductase (NifH) and the alpha subunit (NifD) and beta subunit (NifK) of dinitrogenase were mainly derived from type II methanotrophic bacteria of the family Methylocystaceae, including Methylosinus spp. Minor nitrogenase proteins such as Methylocella, Bradyrhizobium, Rhodopseudomonas, and Anaeromyxobacter were also detected. Methane monooxygenase proteins (PmoCBA and MmoXYZCBG) were detected in the same bacterial group of the Methylocystaceae. Because these results indicated that Methylocystaceae members mediate both CH4 oxidation and N-2 fixation, we examined their localization in rice tissues by using catalyzed reporter deposition-fluorescence in situ hybridization (CARD-FISH). The methanotrophs were localized around the epidermal cells and vascular cylinder in the root tissues of the field-grown rice plants. Our metaproteomics and CARD-FISH results suggest that CH4 oxidation and N-2 fixation are performed mainly by type II methanotrophs of the Methylocystaceae, including Methylosinus spp., inhabiting the vascular bundles and epidermal cells of rice roots.
  • Hiraku Takada, Sanae Fukushima-Tanaka, Masato Morita, Yasuhiro Kasahara, Satoru Watanabe, Taku Chibazakura, Hiroshi Hara, Kouji Matsumoto, Hirofumi Yoshikawa
    MOLECULAR MICROBIOLOGY 91 2 242 - 255 2014年01月 [査読有り][通常論文]
     
    The mechanism by which the membrane synthetic machinery might be co-organized with the cell-division architecture during the bacterial cell cycle remains to be investigated. We characterized a key enzyme of phospholipid and fatty acid synthesis in Bacillus subtilis, the acyl-acyl carrier protein phosphate acyltransferase (PlsX), and identified it as a component of the cell-division machinery. Comprehensive interaction analysis revealed that PlsX interacts with FtsA, the FtsZ-anchoring protein. PlsX mainly localized at the potential division site independent of FtsA and FtsZ and then colocalized with FtsA. By multidirectional approaches, we revealed that the Z-ring stabilizes the association of PlsX at the septum and pole. The localization of PlsX is also affected by the progression of DNA replication. PlsX is needed for cell division and its inactivation leads to aberrant Z-ring formation. We propose that PlsX localization is prior to Z-ring formation in the hierarchy of septum formation events and that PlsX is important for co-ordinating membrane synthesis with cell division in order to properly complete septum formation.
  • Hajime Morimoto, Masayoshi Kuwano, Yasuhiro Kasahara
    ARCHIVES OF MICROBIOLOGY 195 12 805 - 813 2013年12月 [査読有り][通常論文]
     
    Pseudomonas putida F1 can metabolize toluene, ethylbenzene, and benzene for growth. Previously, we identified proteins involved in the utilization of these compounds by P. putida F1 through culture in liquid media. However, it was unclear whether laboratory analysis of bacterial activity and catabolism accurately reflected the soil environment. We identified proteins involved in the degradation of toluene, ethylbenzene, and benzene growth in soil using two-dimensional gel electrophoresis (2-DE) or standard SDS-PAGE combined with liquid chromatography-tandem mass spectrometry (LC-MS/MS). According to 2-DE/LC-MS/MS analysis, 12 of 22 key enzymes involved in the degradation of toluene, ethylbenzene, and benzene were detected. In standard SDS-PAGE/LC-MS/MS analysis of soil with ethylbenzene, approximately 1,260 cellular proteins were identified in P. putida F1. All key enzymes and transporter and sensor proteins involved in ethylbenzene degradation were up-regulated similar to that noted in liquid cultures. In P. putida F1, aromatic hydrocarbon response in soil is the same as that observed in liquid media.
  • Yasuhiro Kasahara, Hajime Morimoto, Masayoshi Kuwano, Ryo Kadoya
    JOURNAL OF MICROBIOLOGICAL METHODS 91 3 434 - 442 2012年12月 [査読有り][通常論文]
     
    Pseudomonas putida F1 can degrade aromatic hydrocarbons to intermediate products of the tricarboxylic acid cycle. To determine key induced proteins and enzymes required for degradation of toluene, ethylbenzene, benzene, p-cymene, and p-cumate, we performed comprehensive proteome analysis using a combination of 1-D SDS-PAGE and LC-MS/MS in cells grown in the presence of each aromatic hydrocarbon. Semi-quantitative analysis using protein content calculated from the exponentially modified protein abundance index (emPAI) was performed for each proteome data set, and the resulting data were compared. Of 5250 known proteins in P. putida F1, 1733-2368 expressed proteins were identified. All of the key enzymes in the degradation pathways were identified. Additionally, the proteins induced by the aromatic hydrocarbons, regulators, and transporters were also found. Using K-means clustering analysis of the proteome data sets, substrate-specific induced proteins were characterized, ranging from 62 to 164 in number. The functions of most of these proteins were not unknown in relation to the metabolism of aromatic hydrocarbons. These results suggest that the approaches used here are ideal as a primary investigation of the various physiological characteristics of bacterial cells. (c) 2012 Elsevier B.V. All rights reserved.
  • Satoshi Shimano, Mitsuo Sambe, Yasuhiro Kasahara
    Microbes and Environments 27 2 136 - 141 2012年 [査読有り][通常論文]
     
    Ciliates play important roles as prey and predators in ecosystems. Changes in the ciliate community can affect the composition and population of microfauna and microflora in ecosystems. To investigate the structure of ciliate communities, we developed a nested PCR-DGGE method, which combines a universal eukaryotic-specific primer set in the first PCR step with a ciliate-specific primer set in the second PCR step, to amplify 18S rRNA genes from ciliates. The 300 bp DGGE fragments generated more bands on the gel than the 600 bp DGGE fragments. Prior to bead beating, DNA extraction of ciliates from soil samples was optimized with a combination of freeze-thaw cycles and ultrasonication. We applied this nested PCR-DGGE method to agricultural soils amended with 0, 120, 300, and 600 t ha -1 year -1 of livestock slurry. The results from the DGGE profiles and principal component analysis (PCA) revealed that the supplement of slurry to soils influenced the ciliate communities. From phylogenetic analysis, 108 DGGE bands were assigned to six classes, which included Spirotrichea and Colpodea, of the subphylum Intramacronucleata, and one class of the subphylum Postciliodesmatophora. These results indicated that a wide variety of taxonomic groups were detected by DGGE profiling. Thus, the nested PCR-DGGE method described here could clearly differentiate between ciliate communities within soil samples and allowed for the phylogenetic identification of these ciliates at the class level.
  • Atsushi Takabayashi, Katsunori Kurihara, Masayoshi Kuwano, Yasuhiro Kasahara, Ryouichi Tanaka, Ayumi Tanaka
    PLANT AND CELL PHYSIOLOGY 52 12 2103 - 2114 2011年12月 [査読有り][通常論文]
     
    The reversible associations between the light-harvesting complexes (LHCs) and the core complexes of PSI and PSII are essential for the photoacclimation mechanisms in higher plants. Two types of Chls, Chl a and Chl b, both function in light harvesting and are required for the biogenesis of the photosystems. Chl b-less plants have been studied to determine the function of the LHCs because the Chl b deficiency has severe effects specific to the LHCs. Previous studies have shown that the amounts of the LHCs, especially the LHCII trimer, were decreased in the mutants; however, it is still unclear whether Chl b is required for the assembly of the LHCs and for the association of the LHCs with PSI and PSII. Here, to reveal the function of Chl b in the LHCs, we investigated the oligomeric states of the LHCs, PSI and PSII in the Arabidopsis Chl b-less mutant. A two-dimensional blue native-PAGE/SDS-PAGE demonstrated that the PSI-LHCI supercomplex was fully assembled in the absence of Chl b, whereas the trimeric LHCII and PSII-LHCII supercomplexes were not detected. The PSI-NAD(P)H dehydrogenase (NDH) supercomplexes were also assembled in the mutant. Furthermore, we detected two forms of monomeric LHC proteins. The faster migrating forms, which were detected primarily in the mutant, were probably apo-LHC proteins, whereas the slower migrating forms were probably the LHC proteins that contained Chl a. These findings increase our understanding of the Chl b function in the assembly of LHCs and the association of the LHCs with PSI, PSII and NDH.
  • Kana Yamamoto, Satoshi D. Ohdachi, Yasuhiro Kasahara
    MICROBES AND ENVIRONMENTS 25 3 197 - 203 2010年09月 [査読有り][通常論文]
     
    Soil bacteria play important roles as litter decomposers in most terrestrial ecosystems and microbial activity is affected by activities of soil invertebrates. In soil ecosystems of forests in Hokkaido, the long-clawed shrew is an important predator whose preying on soil invertebrates may indirectly affect soil bacterial communities. To estimate indirect top-down effects of shrews on the soil bacterial community, field experiments were conducted using enclosures in which shrews were introduced and removed, and changes in bacterial community composition, species richness, diversity, and evenness were observed using automated ribosomal intergenic spacer analysis (ARISA). Abiotic environmental conditions (ambient temperature, soil temperature, soil moisture content and soil pH) were also considered. Bacterial community structure was significantly affected by soil moisture content and soil temperature. The significant causes of the change in bacterial species richness, diversity, and evenness varied among experimental treatments; however, soil moisture tended to have significantly negative effects on these indices in all cases. In the present study, effects of shrews on the bacterial community were not detected.
  • Satoshi Shimano, Mitsuo Sanbe, Yasuhiro Kasahara
    MICROBES AND ENVIRONMENTS 23 4 356 - 359 2008年 [査読有り][通常論文]
     
    The molecular analysis of ciliates for identification and phylogeny is usually conducted through PCR amplification and DNA sequencing, with DNA extracted from a large number of cells, Therefore, the analysis of ciliates is limited to only those species that can be cultured. We propose a single-cell PCR procedure to overcome the difficulty in the analysis of unculturable species. The procedure has been tested on 6 Stichotrichia strains and uncultured Levicoleps biwae cells, targeting 18S rRNA gene sequences, after carrying out microscopic observations and obtaining photographic records, This procedure enables the systematic analysis of unculturable ciliate strains in natural environments by linking the morphology and genotype of a single cell.
  • A taxon-specific oligonucleotide primer set for PCR detection of soil ciliate.
    Tunjung Puitika, Y. Kasahara, N. Miyoshi, Y. Sato, S. Shimano
    Microbes & Environ 22 78 - 81 2007年 [査読有り][通常論文]
  • Tunjung Puitika, Yasuhiro Kasahara, Norikazu Miyoshi, Yoshinori Sato, Satoshi Shimano
    MICROBES AND ENVIRONMENTS 22 1 78 - 81 2007年 [査読有り][通常論文]
     
    A PCR primer set was designed to amplify the 18S rRNA gene from the phylum Ciliophora (ciliates). The pair amplified DNA from 5 organisms representing the major taxonomic groups of ciliates but not from 13 nonciliate organisms. To test the ability of the primers to amplify ciliate rRNA genes from environmental samples, a clone library was constructed from an agricultural soil sample and analyzed. The rRNA gene sequences of all clones were affiliated with the Ciliophora. This newly developed PCR primer set is able to amplify ciliate rRNA genes from soil, and may be useful for the detection and identification of ciliates.
  • 昭日 格図, 小松崎 将一, 佐藤 嘉則, 太田 寛行, 笠原 康裕
    土と微生物 59 2 136 - 136 日本土壌微生物学会 2005年
  • Y Ikunaga, SI Miyakawa, M Hasegawa, Y Kasahara, O Kodama, H Ohta
    SOIL SCIENCE AND PLANT NUTRITION 50 6 871 - 875 2004年12月 [査読有り][通常論文]
     
    Biodegradation activity of branched nonylphenols (BNPs) by Sphingobium amiense strain YTT and Sphingomonas cloacae strain S-3(T) was examined. High-resolution GC-MS was used to separate five isomer groups from the commercial BNP mixture. In the time course experiments of BNP degradation, the isomers with alpha-dimethyl and those with alpha-methyl, alpha-ethyl configurations were rapidly degraded by Sphingobium amiense strain YTT, while those with highly branched chains were broken down less efficiently. In contrast, Sphingomonas cloacae strain S-3(T) degraded all the isomers of the BNP groups at similar rates. The utilization and degradation of 4-n-alkylphenols (C3 to C9) and 8 phenolic acids were also tested with the strains. The results suggested that Sphingomonas cloacae strain S-3(T) displays a catabolic activity possesses the catabolism specific of branched alkylphenols, while Sphingobium amiense strain YTT is a versatile organism capable of utilizing a wide range of phenolic compounds including phenolic acids, and of degrading 4-n-alkylphenols and a limited range of BNPs.
  • S Okuda, R Igarashi, Y Kusui, Y Kasahara, H Morisaki
    JOURNAL OF BACTERIOLOGY 185 13 3711 - 3717 2003年07月 [査読有り][通常論文]
     
    Mutants of Bacillus subtilis 168 strain were obtained by inactivation of a specific gene by homologous recombination with the plasmid pMutinT3. The cell surface properties of these strains were characterized by measuring the electrophoretic mobility of the cells as a function of pH and ionic strength. The surface properties were different for the strains possessing flagella on their cells and strain FlgB, having no flagellum, due to knockout of the corresponding gene. The cell surface properties of the strains possessing flagella become similar to those of strain FlgB after acid treatment. It was confirmed that the acid treatment degraded the flagella without causing any apparent structural change on the cell surface via observations made using atomic force microscopy, transmission electron microscopy, and scanning electron microscopy. These results indicate that the flagella are a key factor influencing cell surface properties.
  • R Shingaki, Y Kasahara, T Inoue, S Kokeguchi, K Fukui
    CANADIAN JOURNAL OF MICROBIOLOGY 49 5 313 - 325 2003年05月 [査読有り][通常論文]
     
    Bacillus subtilis 168 and its major autolysin mutant, AN8, were shown to excrete two size classes of DNA when cultured in Luria-Bertani medium. Pulsed-field gel electrophoresis of DNA harvested from the cell surface demonstrated the presence of 13-kb-long and circa 50-kb-long strands. Restriction digestion of both sizes of DNA resulted in a smearing pattern, as observed by agarose gel electrophoresis. Shotgun sequencing of DNase I partial digests of 50-kb DNA fragments revealed that the strands originate from various sites on the chromosome. SDS-PAGE analysis of cell surface fractions and culture supernatants demonstrated the presence of several proteins that were thought to be associated with the DNA. Of these, three major proteins were identified, i.e., XkdG, XkdK, and XkdM, by tandem mass spectrometry, all of which were proteins of a defective prophage PBSX residing in the Bacillus subtilis chromosome. Disruption of these PBSX genes resulted in a reduction of 13-kb fragment generation and excretion and also a great reduction of 50-kb fragment excretion. Electron microscopy showed that a few mature phages and numerous membrane vesicle-like particles existed in the cell surface fractions of strain 168. The present findings suggest that the spontaneous generation and excretion of chromosome DNA fragments in Bacillus subtilis are both closely related to the expression of defective prophage genes.
  • K Kobayashi, SD Ehrlich, A Albertini, G Amati, KK Andersen, M Arnaud, K Asai, S Ashikaga, S Aymerich, P Bessieres, F Boland, SC Brignell, S Bron, K Bunai, J Chapuis, LC Christiansen, A Danchin, M Debarbouille, E Dervyn, E Deuerling, K Devine, SK Devine, O Dreesen, J Errington, S Fillinger, SJ Foster, Y Fujita, A Galizzi, R Gardan, C Eschevins, T Fukushima, K Haga, CR Harwood, M Hecker, D Hosoya, MF Hullo, H Kakeshita, D Karamata, Y Kasahara, F Kawamura, K Koga, P Koski, R Kuwana, D Imamura, M Ishimaru, S Ishikawa, Ishio, I, D Le Coq, A Masson, C Mauel, R Meima, RP Mellado, A Moir, S Moriya, E Nagakawa, H Nanamiya, S Nakai, P Nygaard, M Ogura, T Ohanan, M O'Reilly, M O'Rourke, Z Pragai, HM Pooley, G Rapoport, JP Rawlins, LA Rivas, C Rivolta, A Sadaie, Y Sadaie, M Sarvas, T Sato, HH Saxild, E Scanlan, W Schumann, JFML Seegers, J Sekiguchi, A Sekowska, SJ Seror, M Simon, P Stragier, R Studer, H Takamatsu, T Tanaka, M Takeuchi, HB Thomaides, Vagner, V, JM van Dijl, K Watabe, A Wipat, H Yamamoto, M Yamamoto, Y Yamamoto, K Yamane, K Yata, K Yoshida, H Yoshikawa, U Zuber, N Ogasawara
    PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 100 8 4678 - 4683 2003年04月 [査読有り][通常論文]
     
    To estimate the minimal gene set required to sustain bacterial life in nutritious conditions, we carried out a systematic inactivation of Bacillus subtilis genes. Among approximate to4,100 genes of the organism, only 192 were shown to be indispensable by this or previous work. Another 79 genes were predicted to be essential. The vast majority of essential genes were categorized in relatively few domains of cell metabolism, with about half involved in information processing, one-fifth involved in the synthesis of cell envelope and the determination of cell shape and division, and one-tenth related to cell energetics. Only 4% of essential genes encode unknown functions. Most essential genes are present throughout a wide range of Bacteria, and almost 70% can also be found in Archaea and Eucarya. However, essential genes related to cell envelope, shape, division, and respiration tend to be lost from bacteria with small genomes. Unexpectedly, most genes involved in the Embden-Meyerhof-Parnas pathway are essential. Identification of unknown and unexpected essential genes opens research avenues to better understanding of processes that sustain bacterial life.
  • Daisuke Miyake, Yasuhiro Kasahara, Hisao Morisaki
    Microbes and Environments 18 1 24 - 31 2003年 [査読有り][通常論文]
     
    The distribution of antibiotic-resistant bacteria in Lake Biwa sediment with depth from a relatively shallow (0-1 cm) to a relatively deep (9-10 cm) layer was investigated. More colonies formed on 100-fold diluted nutrient broth (DNB) agar medium than nutrient broth (NB) medium. The colony number and its difference between the two media decreased with depth. The resistance to antibiotics, ampicillin, chloramphenicol, erythromycin, rifampicin, streptomycin, sulfamethoxazole and tetracycline, was examined. The ratio of antibiotic-resistant strains increased with the depth of sediment. The isolates obtained from the deepest layer using antibiotic-free DNB medium included significantly more strains which were resistant to higher concentrations and many more kinds of antibiotics. In contrast, the isolates from the antibiotic-containing medium showed no such features. According to the 16S rDNA sequence, the isolates from the shallowest layer belonged to the Actinobacteria phylum and the Alpha-, Beta- and Gamma-proteobacteria. The isolates from the deepest layer, however, belonged to the Firmicutes phylum and the Alphaproteobacteria with some exceptions. Many of the multi-resistant isolates were similar to the genus Ralstonia, Afipia, or Bacillus cereus, known for the biodegradation of xenobiotics or pathogenicities mediated via plasmids. © 2003, Japanese Society of Microbial Ecology & The Japanese Society of Soil Microbiology. All rights reserved.
  • Shingaki, R, Y. Kasahara, M. Iwano, M. Kuwano, T. Takatsuka, T. Inoue, S. Kokeguchi, K. Fukui
    Microbiology 149 2501 - 2511 2003年 [査読有り][通常論文]
  • The electrophoretic mobility of Bacillus subtilis knockout mutants with and without flagella.
    Okuda, S, R. Igarashi, Y. Kusui, Y. Kasahara, H. Morisaki
    J. Bacteriology 149 2501 - 2511 2003年 [査読有り][通常論文]
  • R Kuwana, Y Kasahara, M Fujibayashi, H Takamatsu, N Ogasawara, K Watabe
    MICROBIOLOGY-SGM 148 3971 - 3982 2002年12月 [査読有り][通常論文]
     
    The spores of Bacillus subtilis have characteristic properties and consist of complex structures including various types of proteins. To perform comprehensive analysis of the protein composition of the spores, the proteins extracted from the spore were analysed by a combination of one-dimensional PAGE and liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) using Turboquest SEQUEST software interfaced with the DNA sequence database of B. subtilis. A total of 154 proteins were identified, and 69 of them were novel. The remaining 85 proteins have been previously reported as sporulation-specific proteins or as proteins that are synthesized in vegetative cells. The expression pattern of each gene deduced to encode novel spore proteins was analysed using a series of strains carrying a lacZ reporter gene. The results revealed that the expression of 26 genes was dependent on sporulation-specific sigma factors, namely sigma(F), sigma(E), sigma(G) and sigma(k). In this study, it is demonstrated that the combination of the techniques of SDS-PAGE and LC-MS/MS, with the mutant library of B. subtilis, is an effective tool for the analysis of complicated cellular structures.
  • R Kadoya, AKM Hassan, Y Kasahara, N Ogasawara, S Moriya
    MOLECULAR MICROBIOLOGY 45 1 73 - 87 2002年07月 [査読有り][通常論文]
     
    Current views of bacterial chromosome segregation vary in respect of the likely presence or absence of an active segregation mechanism involving a mitotic-like apparatus. Furthermore, little is known about cis-acting elements for chromosome segregation in bacteria. In this report, we show that two separate DNA regions, a 3' coding region of dnaA and the AT-rich sequence between dnaA and dnaN (the initial opening site of duplex DNA during replication), are necessary for efficient segregation of the chromosome in Bacillus subtilis. When a plasmid replicon was integrated into argG, far from oriC, on the chromosome and then the oriC function was disrupted, the oriC-deleted mutant formed anucleate cells at 5% possibly because of defects in chromosome segregation. However, when the two DNA sequences were added near oriN, frequency of anucleate cells decreased to 1%. In these cells, the origin (argG) regions were localized near cell poles, whereas they were randomly distributed in cells without the two DNA sequences. These results suggest that the two DNA sequences in and downstream of the dnaA gene participate in correct positioning of the replication origin region within the cell and that this function is associated with accurate chromosome segregation in B. subtilis .
  • In-Gi Kim, Yasuhiro Kasahara, Kyung-Sook Whang
    Microbes and Environments 16 3 169 - 176 2001年 [査読有り][通常論文]
     
    A phylogenetic analysis of heterotrophic bacterial populations inhabiting streams and groundwater contaminated by acid mine drainage (AMD) was conducted. The samples were collected from sites around an inactive underground Dae-Sung coal mine at Keumsan, Korea. The investigation showed pigment-forming bacteria to be the major strains inhabiting the pollutant, accounting for up to approximately 50% of all isolates. Twenty-six pigment-forming bacteria were isolated and their taxonomic characteristics determined by phenotypic, chemotaxonomic and phylogenetic analyses. Based on a phylogenetic analysis using 16S ribosomal RNA gene nucleotide sequences, these isolates were found to fall within four major phylogenetic groups: α, β, and γ subdivisions of Proteobacteria and low-G+C gram-positive bacteria. The α-Proteobacteria were further separated into α-1, α-2 and α-4 subclasses. Many isolates from the polluted stream (site P5) and groundwater (site G) were identified as Sphingomonas of the Proteobacteria α-4 subclass. Because strains P5-21 and P5-11 appeared to be novel species within the genus Sphingomonas, the discussion was focused on their taxonomy as well as abundance in the polluted regions. © 2001, Japanese Society of Microbial Ecology & The Japanese Society of Soil Microbiology. All rights reserved.
  • Ynte P. de Vries, Y. Takahara, Y. Ikunaga, Y. Ushiba, M. Hasegawa, Y. Kasahara, H. Shimomura, S. Hayashi, Y. Hirai, H. Ohta
    Microbes & Environ. 45 4 73 - 87 日本微生物生態学会 2001年 [査読有り][通常論文]
     
    A conventional enrichment culture on branched nonylphenol (NP) with diluted nutrient broth as an additional source of organic nutrients yielded a bacterial strain able to degrade branched NP. The isolate (designated YT) was identified as Sphingomonas sp. based on an analysis of its 16S ribosomal RNA genes and cellular lipids. The degradation of NP by strain YT occurred primarily during the exponential phase of cell growth in cultures on a yeast extract-mineral salts medium. The degree of degradation was directly proportional to the amount of yeast extract present in the medium and no significant growth occurred when NP was the sole source of carbon and energy. Gas chromatography-mass spectrometry (GC-MS) of resting cell suspensions incubated with branched NP revealed that the degradation did not yield any metabolites containing aromatic residues but only branched alcohols. When a linear NP was used as the target substrate, GS-MS of the suspensions indicated the appearance of a hydroxylated linear NP as an intermediate during the degradation. Strain YT is expected to attack NP by an initial oxidative cleavage of the phenol ring.
  • K Fukuchi, Y Kasahara, K Asai, K Kobayashi, S Moriya, N Ogasawara
    MICROBIOLOGY-UK 146 1573 - 1583 2000年07月 [査読有り][通常論文]
     
    Essential two-component systems are now being identified in bacteria. The Bacillus subtilis yycF gene encoding a response regulator, and its orthologue in Staphylococcus aureus, were reported recently to be essential for cell growth, although genes under their control have yet to be identified. The essential nature of the yycF regulator gene and its cognate kinase gene, yycG, in B. subtilis was also noted during the course of construction of a knockout mutant bank of newly identified genes in the genome sequence project. It was found that yycG could be deleted in the presence of an active form of the YycF protein, thereby suggesting direct interaction between YycG and YycF. Production of mini-cells and reduction in cell length occurred when the YycF regulator was overproduced in B. subtilis. These observations led to the finding that YycF overproduction up-regulated the expression from the P1 promoter of the cell division operon, ftsAZ. In addition, the YycF protein binds to the Pt promoter region in vitro. These results clearly indicate that the essential two-component regulatory system encoded by yycF and yycG genes has the potential to modulate expression of the ftsAZ operon in B. subtilis.
  • K Asai, SH Baik, Y Kasahara, S Moriya, N Ogasawara
    MICROBIOLOGY-UK 146 263 - 271 2000年02月 [査読有り][通常論文]
     
    Transport systems for C-4-dicarboxylates, such as malate, fumarate and succinate, are poorly understood in Cram-positive bacteria, The whole genome sequence of Bacillus subtilis revealed two genes, ydbE and ydbH, whose deduced products are highly homologous to binding proteins and transporters for C-4-dicarboxylates in Gram-negative bacteria. Between ydbE and ydbH, genes ydbF and ydbG encoding a sensor-regulator pair, were located. Inactivation of each one of the ydbEFGH genes caused a deficiency in utilization of fumarate or succinate but not of malate. Expression of ydbH, encoding a putative transporter, was stimulated in a minimal salt medium containing 0.05% yeast extract but repressed by the addition of malate to the medium. inactivation of the putative sensor-regulator pair or solute-binding protein, ydbFG or ydbE, caused complete loss of ydbH expression. The utilization of fumarate and stimulation of ydbH expression resumed in a ydbE null mutant in which ydbFGH were overproduced. Based on these observations, together with analysis of the sequence similarities of the deduced product, we conclude that YdbH is a C-4-dicarboxylate-transport protein and its expression is regulated by a C-4-dicarboxylate sensor kinase-regulator pair, YdbF and YdbG. Furthermore, it is suggested that YdbE does not directly participate in transport of C-4-dicarboxylates, but plays a sensory role in the ydbF-ydbG two-component system, giving rise to specificity or increased efficiency to the system. Deletion analysis of the promoter region of ydbH revealed that a direct repeat sequence was required for the activation of ydbH expression. A catabolite-responsive element (CRE) was also found in the -10 region of the promoter, suggesting negative regulation by a CRE-binding protein.
  • Y Fujita, KI Yoshida, Y Miwa, N Yanai, E Nagakawa, Y Kasahara
    JOURNAL OF BACTERIOLOGY 180 16 4309 - 4313 1998年08月 [査読有り][通常論文]
     
    The Bacillus subtilis fbp gene encoding fructose-1,6-bisphosphatase (FBPase) was originally identified as yydE. The fbp gene was expressed at a fairly constant level in cells undergoing glycolysis or gluconeogenesis,fbp transcription was initiated 94 bp upstream of the translation initiation codon, resulting in a 2.4-kb monocistronic transcript. Interestingly, B. subtilis FBPase exhibited no significant similarity to other FBPases in protein sequence databases.
  • F Kunst, N Ogasawara, Moszer, I, AM Albertini, G Alloni, Azevedo, V, MG Bertero, P Bessieres, A Bolotin, S Borchert, R Borriss, L Boursier, A Brans, M Braun, SC Brignell, S Bron, S Brouillet, CV Bruschi, B Caldwell, Capuano, V, NM Carter, SK Choi, JJ Codani, IF Connerton, NJ Cummings, RA Daniel, F Denizot, KM Devine, A Dusterhoft, SD Ehrlich, PT Emmerson, KD Entian, J Errington, C Fabret, E Ferrari, D Foulger, C Fritz, M Fujita, Y Fujita, S Fuma, A Galizzi, N Galleron, SY Ghim, P Glaser, A Goffeau, EJ Golightly, G Grandi, G Guiseppi, BJ Guy, K Haga, J Haiech, CR Harwood, A Henaut, H Hilbert, S Holsappel, S Hosono, MF Hullo, M Itaya, L Jones, B Joris, D Karamata, Y Kasahara, M KlaerrBlanchard, C Klein, Y Kobayashi, P Koetter, G Koningstein, S Krogh, M Kumano, K Kurita, A Lapidus, S Lardinois, J Lauber, Lazarevic, V, SM Lee, A Levine, H Liu, S Masuda, C Mauel, C Medigue, N Medina, RP Mellado, M Mizuno, D Moestl, S Nakai, M Noback, D Noone, M OReilly, K Ogawa, A Ogiwara, B Oudega, SH Park, Parro, V, TM Pohl, D Portetelle, S Porwollik, AM Prescott, E Presecan, P Pujic, B Purnelle, G Rapoport, M Rey, S Reynolds, M Rieger, C Rivolta, E Rocha, B Roche, M Rose, Y Sadaie, T Sato, E Scanlan, S Schleich, R Schroeter, F Scoffone, J Sekiguchi, A Sekowska, SJ Seror, P Serror, BS Shin, B Soldo, A Sorokin, E Tacconi, T Takagi, H Takahashi, K Takemaru, M Takeuchi, A Tamakoshi, T Tanaka, P Terpstra, A Tognoni, Tosato, V, S Uchiyama, M Vandenbol, F Vannier, A Vassarotti, A Viari, R Wambutt, E Wedler, H Wedler, T Weitzenegger, P Winters, A Wipat, H Yamamoto, K Yamane, K Yasumoto, K Yata, K Yoshida, HF Yoshikawa, E Zumstein, H Yoshikawa, A Danchin
    NATURE 390 6657 249 - 256 1997年11月 [査読有り][通常論文]
     
    Bacillus subtilis Is the best-characterized member of the Gram-positive bacteria. Its genome of 4,214,810 base pairs comprises 4,100 protein-coding genes. Of these protein-coding genes, 53% are represented once, while a quarter of the genome corresponds to several gene families that have been greatly expanded by gene duplication, the largest family containing 77 putative ATP-binding transport proteins. In addition, a large proportion of the genetic capacity is devoted to the utilization of a variety of carbon sources, including many plant-derived molecules. The identification of five signal peptidase genes, as well as several genes for components of the secretion apparatus, is important given the capacity of Bacillus strains to secrete large amounts of industrially important enzymes. Many of the genes are involved in the synthesis of secondary metabolites, Including antibiotics, that are more typically associated with Streptomyces species. The genome contains at least ten prophages or remnants of prophages, indicating that bacteriophage Infection has played an important evolutionary role in horizontal gene transfer, in particular In the propagation of bacterial pathogenesis.
  • C Beloin, S Ayora, R Exley, L Hirschbein, N Ogasawara, Y Kasahara, JC Alonso, F LeHegarat
    MOLECULAR & GENERAL GENETICS 256 1 63 - 71 1997年09月 [査読有り][通常論文]
     
    In the course of the Bacillus subtilis genome sequencing project, we identified an open reading frame encoding a putative 16.4 kDa protein. This protein shows, respectively, 34% and 25% identity with the Escherichia coli regulatory proteins Lrp and AsnC. Phylogenetic analysis suggests that it represents a new group in the AsnC-Lrp family. Sequence comparisons, as well as immunodetection experiments, lead to the conclusion that the product of this B. subtilis lrp-like-gene is a bona fide Lrp protein - the first one to be detected in gram-positive bacteria. When expressed in E. coli, the B. subtilis Lrp-like protein is able to repress, by about two-fold, the expression of the ilvIH operon which is normally regulated by E. coli Lrp, indicating functional similarity in their regulatory targets. Vegetative growth of a B. subtilis lrp-like mutant is not affected in rich medium. However, the lrp-like mutation causes a transitory inhibition of growth in minimal medium in the presence of valine and isoleucine, which is relieved by leucine. This points to a possible role in regulation of amino acid metabolism. In addition, sporogenesis occurs earlier in the lrp-like mutant than in the reference strain, implying that the B. subtilis Lrp-like protein plays a role in the growth phase transition.
  • T Hattori, H Mitsui, H Haga, N Wakao, S Shikano, K Gorlach, Y Kasahara, A ElBeltagy, R Hattori
    ANTONIE VAN LEEUWENHOEK INTERNATIONAL JOURNAL OF GENERAL AND MOLECULAR MICROBIOLOGY 72 1 21 - 28 1997年07月 [査読有り][通常論文]
     
    Recent studies on the colony formation of soil bacteria opened the way to categorize soil bacteria into colony forming curve (CFC) groups of different growth rates. A bacterial culture collection comprising organisms from every CFC group is called an ecocollection (EC). Outlines of ECs of paddy soil 1992 and grassland soil 1987 and 1992 were described. Phylogenetic studies by 16S rDNA sequencing showed a great diversity of culture strains of the ecocollections. A set of alternative concepts was proposed; the active and the quiescent forms of bacterial cells in soil. The former is able to be cultivated and thus counted by the plate method, while the latter is not unless it transforms into the former. Based on the results several points required for extensive cataloguing of soil bacteria were noted.
  • Y Sadaie, K Yata, M Fujita, H Sagai, M Itaya, Y Kasahara, N Ogasawara
    MICROBIOLOGY-UK 143 1861 - 1866 1997年06月 [査読有り][通常論文]
     
    A 36 kb sequence of the phoB-rrnE-groESL region of the Bacillus subtilis chromosome at around 55 degrees has been determined. The sequenced region contains 36 ORFs including the phoB and groESL genes, and the whole rrnE operon. The phoB gene is transcribed in the direction opposite to that of chromosome replication, while most ORFs, including groESL and the rrnE operon, are transcribed in the same direction. Two newly identified tRNA genes upstream of the rrnE operon were those for Arg-tRNA and Gly-tRNA. The sequenced region contains an operon consisting of genes for degradation and uptake of mannan. The rrnE operon and its downstream ORFs are well conserved among Mycoplasma genitalium, Haemophilus influenzae, Synechocystis so. and Methanococcus jannaschii. sigma(H) consensus sequences are present in the promoter regions of three ORFs, including groESL.
  • Yasuhiro Kasahara, Sumiko Nakai, Naotake Ogasawara, Katsunori Yata, Yoshito Sadaie
    DNA Research 4 5 335 - 339 1997年 [査読有り][通常論文]
     
    We have determined a 35-kb sequence of the groESL-gutR-cotA (45°-52°) region of the Bacillus subtilis genome. In addition to the groESL, gutRB and cotA genes reported previously, we have newly identified 24 ORFs including gutA and fruC genes, encoding glucitol permease and fructokinase, respectively. The inherent restriction/modification system genes, hsdMR and hsdMM, were mapped between groESL and gutRB, and we have identified two open reading frames (ORFs) encoding 5-methylcytosine forming DNA methyl transferase and an operon probably encoding a restriction enzyme complex. The unusual genome structure of few ORFs and lower GC content around the restriction/modification genes strongly suggests that the region originated from a bacteriophage integrated during evolution.
  • Yasuhiro Kasahara, Sumiko Nakai, Naotake Ogasawara
    DNA Research 4 2 155 - 159 1997年 [査読有り][通常論文]
     
    Within the framework of an international Bacillus subtilis genome sequencing project, we have determined a 36-kb sequence covering the region between the gntZ and trnY genes. In addition to five genes sequenced and characterized previously, 27 putative protein coding sequences (open reading frame ORF) were identified. A homology search for the newly identified ORFs revealed that six of them had similarities to known proteins. It is notable that new ORFs belonging to response-regulator aspartate phosphatase (Rap) and its regulator (Phr) families, and response regulator and sensory kinase families of two-component signal transduction systems have been identified. Furthermore, we found that some 180-bp non-coding sequence, that might be an remnant of an ancient IS element, is preserved in at least five loci of the B. subtilis genome.
  • Liu H, K. Haga, Y. Kasahara, N. Ogasawara, H. Takahashi, H. Yoshikawa
    DNA research 4 5 325 - 328 1997年 [査読有り][通常論文]
     
    As a part of the Bacillus subtilis genome sequencing project, we have determined a 25-kb sequence covering the 17°-19° region. This region contains 26 complete open reading frames (ORFs) including the alkA and adaA/B operon, which encode genes for adaptive response to DNA alkylation. A homology search for the newly identified 21 ORFs revealed that 4 of them exhibit a significant similarity to known proteins, e.g., methicillin-resistant Staphylococcus aureus (MRSA) protein homolog, proteins involved in chloramphenicol resistance, glucosamine synthase and an ABC transporter protein. The remaining 17 ORFs did not show any significant sequence similarities to known gene products in the database.
  • Akagawa E, K. Kurita, T. Sugawara, K. Nakamura, Y. Kasahara, N. Ogasawara, K. Yamane
    Microbiology 141 3241 - 3245 1995年12月 [査読有り][通常論文]
     
    We have determined a 17484 bp nucleotide sequence around the 39 degrees region, located about 480 kb downstream from the zero position of the Bacillus subtilis chromosome physical map. Among the 17 putative ORFs identified, orf1 and orf2 seem to correspond to mtlA and mtlB, encoding mannitol-specific phosphotransferase enzyme II and mannitol-1-phosphate dehydrogenase. respectively. orf4 seems to be another signal peptidase I gene (sipS) of B. subtilis. The putative products of six ORFs were similar to known proteins in data banks, namely a hypothetical 29.7 kDa protein of Escherichia coli (Orf7), a lactam utilization protein (Orf8), the urea amidolyase of yeast (Orf12), the IclR regulatory protein for aceAB of Salmonella typhimurium (Orf13), penicillin-binding protein 2 (Orf16) and aryl-alcohol dehydrogenase (Orf17). The amino acid sequence of Qrf3 showed 34% identity with that of LeuC of B. subtilis, though they seem to be functionally different. The remaining seven ORFs did not show similarity to any known proteins.
  • Y KASAHARA, H MORISAKI, T HATTORI
    JOURNAL OF GENERAL AND APPLIED MICROBIOLOGY 39 4 381 - 388 1993年08月 [査読有り][通常論文]
     
    The cell surface tension of 250 strains of bacteria isolated from grassland soil was examined by measuring the contact angle of a liquid droplet on the cell layer. The values of the contact angle of aqueous 0.1M NaCl solution and alpha-bromonaphthalene on the cell layer were used to calculate the polar and non-polar components of surface tension for the isolates. The polar component of the ''slow-growing'' isolates was smaller than that of the ''fast-growing'' isolates. On the other hand, no difference could be observed in the non-polar component between the fast- and slow-growing isolates. These results indicate that slow-growing isolates have more hydrophobic cell surface than fast-growing isolates.
  • H MORISAKI, Y KASAHARA, T HATTORI
    JOURNAL OF GENERAL AND APPLIED MICROBIOLOGY 39 1 65 - 74 1993年02月 [査読有り][通常論文]
     
    The cell surface charge of 101 strains of bacteria isolated from grassland soil was examined by determining the electrophoretic mobility (EPM) of the cells. The isolates were cultured in diluted nutrient broth medium at 27-degrees-C and the EPM of the cells was measured by micro-electrophoresis in 10 mm phosphate buffer solution at 25-degrees-C as a function of pH varying from 3.1 to 9.0. All the isolates showed negative EPM values at pH 7. These values varied characteristically among strains, i.e., slow-growing isolates showed a lower level of negative EPM values compared with fast-growing isolates. The EPM of each strain did not greatly differ from pH 9 to 6, whereas it showed a lower level of negative values under more acidic conditions. This change in the EPM value was smaller for the slow-growing isolates than that for the fast-growing ones. Under the most acidic condition (pH 3.1), some isolates, mostly the fastest-growing, Gram-positive bacteria, showed positive EPM values. The above detailed changes in EPM suggest that the fast-growing isolates have a greater number of acidic groups at the cell surface, compared with the slow-growing isolates. Furthermore, the finding of positive EPM values for the fastest-growing bacteria indicates the existence of some basic groups at their cell surface.
  • H MORISAKI, Y KASAHARA, S TANIGAWA, T HATTORI
    JOURNAL OF GENERAL AND APPLIED MICROBIOLOGY 38 2 165 - 177 1992年04月 [査読有り][通常論文]
     
    The changes in the sedimentation, the surface properties, and the attachment of Pseudomonas syringae pv. atropurpurea NIAES 1309 induced by introduction of the plasmid pBPW1=Tn7 were studied by comparing wild-type (WT) cells and cells containing the plasmid (pBPW1 cells). The cell sedimentation rate, which was markedly enhanced by the introduction of the plasmid, was investigated quantitatively by using an image analyzer. The element causing the sedimentation was assumed to exist at the cell surface. The pBPW1 cells showed a greater negative surface charge than the WT cells. Infrared spectra together with the data of the cell surface charge suggested that the pBPW1 cells contained more protein but less sugar residues at the cell surface compared with WT cells. Little difference could be found in the surface free energy, based on dispersion and polar components, between WT and pBPW1 cells. Microbial cell attachment to the surface of glass was observed continuously in a parallel plate flow system. The number of pBPW1 cells attached to the glass surface was greater than that of the WT cells: about 5 times greater after 6h from the beginning of the experiment. A factor other than the surface charge was assumed to be responsible for the cell sedimentation and/or the cell attachment to the glass surface.
  • Y KASAHARA, T HATTORI
    FEMS MICROBIOLOGY ECOLOGY 86 2 95 - 101 1991年12月 [査読有り][通常論文]
     
    The process of colony formation by bacteria from grassland soil sampled in April, July and September was simulated by a colony-forming curve (CFC). The CFC was a super-imposition of several component curves (cCFC) given theoretically by the first order reaction (FOR) model [3,6]. The pattern of FOR model curves was not influenced by the time of sampling and four cCFCs were always recognized during an incubation period of 160 h. It was considered that the CFC describes an inherent property of the bacterial population of the field. Bacterial isolates were obtained from colonies produced in each of four cCFCs on agar plates. Isolates corresponding to one cCFC were classified as one group. The bacterial isolates were characterized by morphological and physiological tests and subsequently clustered. Few oligotrophic bacteria were obtained among bacteria which produced visible colonies within 63 h of incubation time. On the other hand, approx. 50% of bacteria which produced colonies after 63 h were oligotrophic bacteria. The time required for the appearance of the first colony, t(r) of the FOR model, was very similar in the isolates belonging to one group. A close linear relationship was observed between t(r) value and doubling time of isolates.

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書籍等出版物

  • 環境変動による土壌微生物生態系の影響
    笠原 康裕 (担当:分担執筆範囲:低温科学便覧（北海道大学低温科学研究所編）)
    丸善出版 2015年
  • 微生物ってなに？
    日科技連 2006年
  • 微生物生態学入門
    日科技連 2004年
  • PCR Tips
    秀潤社 2000年
  • 基礎生化学実験法、第4巻核酸・遺伝子実験
    東京化学同人 2000年

講演・口頭発表等

  • 動物の死骸は誰のもの？: 死肉食性昆虫と雑食性食肉目の消費型競争  [通常講演]
    橋詰茜, 松島義治, 幸田良介, 笠原康裕, 大舘智志, 中島啓裕
    日本生態学会第68回全国大会 2021年03月 ポスター発表
  • 火熱撹乱による森林土壌の細菌と真菌群集の応答と回復
    笠原康裕, 高橋一弘, 小椋義俊, 三木 健, 新井大輔, 林哲也, 佐藤雅志
    第14回日本ゲノム微生物学会年会 2020年03月 ポスター発表
  • 火熱撹乱による森林土壌微生物群集の応答と回復  [通常講演]
    笠原康裕, 高橋一弘, 小椋義俊, 森本一, 三木 健, 新井大輔, Larry Lopez, 林哲也, 佐藤雅志
    日本土壌微生物学会2019年度大会（札幌） 2019年06月 ポスター発表
  • 肉の腐敗にどう抗うか？－微生物への対抗ともう１つの戦略－  [通常講演]
    橋詰 茜, 山中康如, 笠原康裕, 大舘智志, 幸田良介, 中島啓裕
    第66回日本生態学会大会 2019年03月 ポスター発表
  • ソルガム根の窒素固定活性とその原因窒素固定細菌のOmic解析による同定  [通常講演]
    南澤 究, 原新太郎, 森川峻志, 笠原康裕, 小柴太一, 山崎清志, 藤原 徹, 徳永 毅
    第27回植物微生物研究会 2017年09月 口頭発表（一般）
  • メタゲノム解析に基づくソルガム根の窒素固定細菌の分離  [通常講演]
    森川峻志、原新太郎、新井沙和、笠原康裕、小柴太一、山崎清志、藤原 徹、徳永 毅、南澤 究
    第27回植物微生物研究会 2017年09月 ポスター発表
  • メソルガム根の窒素固定活性とその原因窒素固定細菌の探索  [通常講演]
    森川峻志、原新太郎、笠原康裕、小柴太一、山崎清志、藤原 徹、徳永 毅、南澤 究
    環境微生物系学会合同大会2017 2017年08月 ポスター発表
  • ゲノム科学から見えてくる微生物によるヒ素循環  [通常講演]
    天知誠吾、土屋達哉、笠原康裕、濱村奈津子
    JpGU-AGU Joint Meeting 2017 2017年05月 シンポジウム・ワークショップパネル（指名）
  • Microbial community structure and soil properties in the rhizosphere of understory dwarf bamboo in Betula ermanii forest, northern Japan.
    Bihe Kong, Lei Chen, Yasuhiro Kasahara, Akihiro Sumida, Kiyomi Ono, Arata Nagatake, Ryusuke Hatano, Jan Wild, Toshihiko Hara
    第31回日本微生物生態学会 2016年10月 ポスター発表
  • 異化的ヒ酸還元細菌によるヒ素ストレス応答機構の発現解析  [通常講演]
    土屋達哉、笠原康裕、濱村奈津子、天知誠吾
    第31回日本微生物生態学会 2016年10月 ポスター発表
  • 土壌プロテオミクスから考える微生物生態系機能  [招待講演]
    笠原康裕
    日本土壌微生物学会2016年度大会 2016年06月 口頭発表（招待・特別）
  • Understory dwarf bamboo affects microbial structure and soil properties in a Betula ermanii forest in northern Japan
    Bihe Kong, Lei Chen, Yasuhiro Kasahara, Akihiro Sumida, Kiyomi Ono, Jan Wild, Arata Nagatake, Ryusuke Hatano, Toshihiko Hara
    日本生態学会第64回大会 2016年03月 口頭発表（一般）
  • 異化的ヒ酸還元細菌によるヒ素ストレス応答の網羅的解析
    土屋達哉, 笠原康裕, 天知誠吾
    日本農芸化学会2016年度大会 2016年03月 口頭発表（一般）
  • プロテオーム解析を用いたPseudomonas putida F1株の土壌特異的発現遺伝子の同定と特性解析  [通常講演]
    森本 一、門屋亨介、高橋一弘、笠原康裕
    第10回日本ゲノム微生物学会年会 2016年03月 ポスター発表
  • Proteome analysis of the response of Pseudomonas putida F1 to aromatic hydrocarbon in soil  [通常講演]
    第26回日本微生物生態学会 2010年 ポスター発表
  • Micro-eukaryote diversity in soil environments of a grass field.  [通常講演]
    ISEP 2010年 ポスター発表
  • ゲノム未解析株のプロテオーム解析の評価  [通常講演]
    第4回日本ゲノム微生物学会年会 2010年 ポスター発表
  • 間接タンパク質抽出法を用いた土壌メタプロテオミクスの基盤構築  [通常講演]
    第4回日本ゲノム微生物学会年会 2010年 ポスター発表
  • 半定量的発現プロテオミクスによる芳香族分解の代謝経路解析  [通常講演]
    第4回日本ゲノム微生物学会年会 2010年
  • 環境変動と土壌微生物群集変動  [通常講演]
    第56回日本生態学会大会 2009年
  • 大腸菌のVBNC状態関与遺伝子のゲノムワイドスクリーニング  [通常講演]
    第3回ゲノム微生物学会年会 2009年 ポスター発表
  • Biodiversity of micro-eukaryotes in soil environments of a grass field.  [通常講演]
    Canadian Society for Ecology and Evolution (CSEE) 2009 meeting 2009年 ポスター発表
  • シングルセルPCR法による繊毛虫類の塩基配列解析  [通常講演]
    日本土壌動物学会第31回大会 2008年 ポスター発表
  • 土壌微生物群集に対する高次捕食者トガリネズミの効果（予報）  [通常講演]
    第24回日本微生物生態学会 2008年 ポスター発表
  • Ciliate community analysis of agricultural soil based on SSU rDNA with new taxon-specific primer  [通常講演]
    59th Annual Meeting, and the International Society for Evolutionary Protistology 2008年 ポスター発表
  • Soil ciliate diversity in upland soil studied by molecular and culturing approaches  [通常講演]
    日本微生物生態学会 2007年
  • Effect of phylogenetic diversity using multiple displacement amplification for bacterial community structure.  [通常講演]
    日本微生物生態学会 2007年
  • 難培養性細菌のゲノム解析を目指して－１細胞微量ゲノム増幅法の検討－  [通常講演]
    日本土壌微生物学会 2006年 ポスター発表
  • 環境由来DNA配列に基づいた土壌原生生物の群集解析法に関する検討  [通常講演]
    日本土壌動物学会 2006年
  • 細菌1細胞からのゲノム解析を目指して －微量DNA増幅法の検討  [通常講演]
    ワークショップ微生物ゲノム研究のフロンティア 2006年 ポスター発表
  • Analysis of soil ciliates community using 18S rDNA  [通常講演]
    日本微生物生態学会 2005年 ポスター発表
  • 大腸菌の生きているが培養不能（VBNC）状態のプロテオーム解析  [通常講演]
    日本微生物生態学会 2005年 ポスター発表
  • 枯草菌ペリクルの形成因子の同定  [通常講演]
    日本微生物生態学会 2005年 ポスター発表
  • 枯草菌のペリクル形成過程の顕微鏡学的およびプロテオーム解析  [通常講演]
    日本微生物生態学会 2005年 ポスター発表
  • Degradation profiles of alkylphenols by two strains belong to the Sphingomonas group.  [通常講演]
    International Symposium on Microbial Ecology 2004年 ポスター発表
  • Community Analysis of Ciliates in Soil Based on 18S rDNA  [通常講演]
    日本微生物生態学会 2004年 ポスター発表
  • 枯草菌ペリクル構造体の形成過程と発現蛋白質プロファイル  [通常講演]
    日本微生物生態学会 2004年 ポスター発表
  • ポストゲノム微生物研究における展開－枯草菌のプロテオミクス－  [通常講演]
    日本土壌肥料学会 2004年

所属学協会

  • 国際微生物生態学会（International Society for Microbial Ecology）   日本ゲノム微生物学会   日本土壌微生物学会   日本農芸化学会   日本微生物生態学会   

共同研究・競争的資金等の研究課題

  • 日本学術振興会：科学研究費助成事業 基盤研究(C)
    研究期間 : 2021年04月 -2024年03月 
    代表者 : 笠原 康裕, 小椋 義俊
     
    山火事は生態系にとって重大な撹乱であり、土壌微生物にも多大な影響を与える。火災後の土壌生態系の回復に微生物が重要な役割を果たしているが、微生物群集の回復メカニズムは群集の構造・多様性・遷移に関する経時的な解析データの不足により未解明である。さらに生態系機能レベルの群集集合の研究不足も回復メカニズムの理解を阻んでいる。本研究は、火入れ後の回復過程における細菌群集と細菌機能群の構造変化を経時的に解析し、一連の解析結果から、構造と機能の関連性を見いだし、細菌群集の回復の変化とメカニズムを明らかにすることである。 細菌群集代謝プロファイル解析として、基質31種類が入った96穴プレートEcoPlateを用いて、土壌の基質分解能の有無を発色から判断し、代謝プロファイル解析を行う。さらに、発色ウェルから、DNAとRNAを同時に抽出する。DNAについて16SrDNA遺伝子を増幅、解読し、同定と組成解析を行う。抽出RNAについて、RNA-seq解析を行い、ネットワーク分析や群集機能解析を行う。 本年度は、EcoPlateの発色ウェルからDNAとRNAの同時抽出の検討を行った。プレートでの試料について、土壌そのままと土壌懸濁液のどちらかの適正を判断するため、接種、培養、判定、回収を行い、解析能やウェルから扱いや簡便性を評価した。これより、土壌懸濁液の使用に決定した。核酸抽出では、DNAの次世代シークエンス解析やRNAのRNA-seq解析が可能な質量を得るための、ウェル数（１か3）の比較を行った。これより１ウェルからでも解析が可能である。本研究では、試料間やウェル間のばらつきをできるだけ避けるために、３ウェルをまとめて処理することにした。これより、次年度の機能解析の方法を確立することができ、解析を進めていく。
  • 日本学術振興会：科学研究費助成事業
    研究期間 : 2018年04月 -2021年03月 
    代表者 : 中島 啓裕, 笠原 康裕, 大舘 智志
     
    本研究では，脊椎動物の死体をモデルとして，非近縁分類群間の相互作用が死体の消失にどのような影響を与えているのかを明らかにした．具体的には，北海道八雲町の落葉広葉樹林において，有害駆除されたアライグマの死体を設置し，直接観察および自動撮影カメラによる観察を行い，死肉食性昆虫（とくにホホグロオビキンバエ）と微生物による死体の消費が，脊椎動物による死体発見までの時間や利用頻度に与える影響を定量的に評価した．この結果，ハエ幼虫による消費によって哺乳綱食肉目（アカギツネとタヌキ）による消費可能量が大きく制限されること，一方で，キンバエ幼虫の活動によって食肉目の死体発見が迅速化することが明らかとなった．
  • 熱撹乱による森林土壌微生物生態系の維持機構の包括的解析
    日本学術振興会：科学研究費補助金
    研究期間 : 2014年04月 -2018年03月 
    代表者 : 笠原 康裕
  • 土壌プロテオミクスによる自然環境下の細菌個生態学的研究
    日本学術振興会：科学研究費補助金
    研究期間 : 2013年04月 -2015年03月 
    代表者 : 笠原 康裕
  • 北方森林土壌において温暖化が及ぼす微生物と原生生物の群集構造変化と連鎖関係
    日本学術振興会：科学研究費補助金
    研究期間 : 2008年04月 -2012年03月 
    代表者 : 笠原 康裕
  • Post-genomics of microbial community in natural environment.
    Basic Science Research Program
    研究期間 : 2005年 -2010年
  • 寒冷圏生態系機能モニタリングシステムの構築
    北海道大学低温科学研究所：先導的研究助成
    研究期間 : 2008年04月 -2009年03月 
    代表者 : 笠原 康裕
  • 環境プロテオミクスに基づく汚染土壌修復法の開発
    科学技術振興機構：シーズ発掘試験（発掘型）研究
    研究期間 : 2008年04月 -2009年03月 
    代表者 : 笠原 康裕
  • 電熱線大規模埋設実験による落葉広葉樹林生態系の温暖化に対する応答の解明
    日本学術振興会：科学研究費補助金
    研究期間 : 2007年04月 -2009年03月
  • 低温生態系微生物の相互作用ネットワークの解明
    北海道大学低温科学研究所：リーダーシップ研究助成
    研究期間 : 2007年04月 -2008年03月 
    代表者 : 笠原 康裕
  • 日本学術振興会：科学研究費助成事業
    研究期間 : 2007年 -2008年 
    代表者 : 新垣 隆資, 笠原 康裕, 井上 哲圭
     
    細胞膜を構成する主要脂肪酸として一部の細菌が持つメチル分岐を有した脂肪酸のある種の分子(12-メチルテトラデカン酸ならびに13-メチルテトラデカン酸)がグラム陽性菌に対して示す抗菌作用と、一部のグラム陰性菌が示すswarming運動性阻害活性について解析を行い、C-55キャリアーリピド依存的に行われる細胞壁物質や菌体外多糖様物質の合成・輸送を阻害する機構を解析した。
  • Phylogenetic analysis of unculturable bacterial cells
    0104 (Japanese Only)
    研究期間 : 2005年 -2008年
  • Genetic analysis of pellicle formation of Bacillus subtills
    Basic Science Research Program
    研究期間 : 2003年 -2008年
  • 北方森林土壌の微生物および原生生物の群集構造解析
    科学研究費補助金
    研究期間 : 2008年
  • 日本学術振興会：科学研究費助成事業
    研究期間 : 2005年 -2007年 
    代表者 : 村上 敏文, 小柳 敦史, 中元 朋実, 島野 智之, 笠原 康裕
     
    根は土壌中にあって、生育環境や土壌生物の影響を受けながら、養水分の吸収を行っている。しかし、研究手法が不十分なため、その実態はほとんどわかっていない。そこで、手法の開発を中心に研究を行った。 染色液及び樹脂を用いた根の分布解明手法の開発では、根の染色システムを耐高圧仕様に改良し、野外で隣接するトマトの根の相互の分布を明らかにした。また、単子葉植物の茎を樹脂で固めて根を染色できるようにした。残された課題としては、樹脂を使った根の立体標本の作製法の開発がある。 土壌環境及び品種特性と根分布の関係の解明手法の開発では、過湿条件でのコムギの根の遺伝特性を明らかにするため、実際の水田条件に近い湿害再現系(代掻き法)を考案し、ミズタカモジ及びライコムギがコムギよりも耐湿性が強いことを明らかにした。 土壌生物と根のマクロ的相互作用の解明手法の開発では、土壌生物の水平分布と垂直分布を同時に調査する方法を開発し、耕起土壌では不耕起土壌よりも根が土壌生物の機能強化に果たす役割が大きいことを示した。また、土壌生物と根の養分吸収を明らかにするため、リターバッグ法を用いたミクロコズム実験系を開発した。 土壌原生動物と根・微生物のミクロ的相互作用の解明手法の開発では、根圏環境の原生生物繊毛虫の群集動態を解析するためPCR-DGGE法を確立した。この方法により、顕微鏡的手法に較べてよく多くの種、かつ広範囲な分類群を検出できるようになった。 土壌微生物と根のミクロ的相互作用の解明手法の開発では、バクテリアの群集構造を解析するため、捕食者である繊毛虫の動態解析と合わせて環境DNAに基づく分子生物学的研究で比較した。その結果、原生生物の存在がバクテリアの群集構造に影響を与えていることが示唆された。 以上、開発した方法は、根-土壌生物相互作用の解明に大きく貢献すると思われる。
  • 日本学術振興会：科学研究費助成事業
    研究期間 : 2005年 -2007年 
    代表者 : 太田 寛行, 久保田 正亜, 笠原 康裕, 東 照雄, 上條 隆志, 難波 謙二
     
    本研究では、三宅島火山噴火に曝された土壌-植物生態系において、土壌微生物生態糸の動態を解析して、生態系再生のプロセスとポテンシャルを探ろうとしたものである。本研究によって、微量有機炭素しか検出されない初成土壌と位置づけられる火山灰堆積層(堆積後5年)において、独立栄養微生物による炭酸ガス固定だけでなく、大気窒素の固定も活発に起こっていることを明らかにした。また、その炭酸ガス固定と窒素固定に係わる微生物が、鉄酸化細菌のLeptospirillum属とAcidithiobacillus属であることを同定した。これらの結果は、初成土壌生成における有機物集積の実態とパイオニア微生物の群集構造初めて明らかにした点で大きな意義があると言える。すなわち、火山噴火によって破壊された土壌-植物生態系において、土壌微生物生態系の再生は極めて早く起こっていることを初めて示した。本研究でば土壌の微生物活性測定、微生物の分離と同定、土壌DNAの解析による微生物群集構造の推定、安定同位体プロービング法による機能微生物の特定など、多種の方法を用いて、上記の結論を導いた。さらに、堆積後22年を経た火山噴火物堆積物地帯で、植生回復がない地点から分離したLimnobacter属とHerbaspirillum属の細菌株について、様々な性状を分析し、新種であることを明らかにして、Limnobacter litoralis、Herbaspirllum scorialisとして記載した。Herbaspirillum属には植物に内部共生する菌種が多く記載されており、植生のない地点にHerbaspirillum scorialisが多く存在することは、新たな知見であり、植生回復のパイオニア生物として機能していることが推察された。
  • 土壌中における大腸菌O157の培養不可能（VBNC）状態のモニタリング解析
    日本学術振興会：科学研究費補助金
    研究期間 : 2004年04月 -2006年03月 
    代表者 : 笠原 康裕
  • 低温生態系微生物の相互作用ネットワークの解明
    研究期間 : 2005年
  • 日本学術振興会：科学研究費助成事業
    研究期間 : 2000年 -2004年 
    代表者 : 小笠原 直毅, 金谷 重彦, 河村 富士夫, 吉川 博文, 渡部 一仁, 佐藤 勉, 小林 和夫, 笠原 康裕, 守家 成紀
     
    1)新たに約300の変異株を作製することにより、枯草菌の全機能未知遺伝子を含む変異株バンクが作製された。その結果、富栄養培地・37度での増殖には271遺伝子が必須であることが明らかとなり、細胞増殖の基本システムが明らかとなった。 2)機能未知の必須遺伝子の一つがtRNA中の塩基修飾にかかわること、必須膜タンパク質YneSが、PlsXとの共に、リン脂質合成の第一ステップに関与することを発見した。必須GTP結合タンパク質YlqFについて、50Sリボゾーム粒子の形成の最終段階に関与することを明らかにした。 3)57種のリボソームタンパク質遺伝子の破壊株の構築を試み、既知の破壊可能遺伝子を含め、合計22種のリボソームタンパク質遺伝子が単独破壊可能であることが明らかとなった。 4)枯草菌のパラログ遺伝子の解析により、polAと-ypcPの2重破壊は、岡崎プライマーRNAの除去機能が失われ致死となるという、新しい知見を得た。 5)プロテオーム、トランスクリプトーム、遺伝学的解析により、既知119遺伝子に加えて、新たに175遺伝子がスポア形成期に特異的に発現することを明らかにし、その発現を制御するシグマ因子を同定した。さらに、約90種類の胞子タンパク質についてGFPを用いた局在解析を行い、少なくとも10種類の局在パターンを見出し、胞子中でのタンパク質の配置という、今後の重要な研究テーマを明らかにした。 6)酵母2ハイブリッド法、細胞内複合体の質量分析計による解析による、相互作用解析も試み、ECFシグマ因子の活性を制御するアンチシグマ因子、Comタンパク質の活性を制御するRapタンパク質、胞子形成開始キナーゼの活性制御にかかわるYheIHタンパク質、FtsZと結合し、細胞分裂に関与するYlqFタンパク質、DnaA, Nと相互作用し、複製開始の制御に関わるYabAタンパク質を見出した。
  • 日本学術振興会：科学研究費助成事業
    研究期間 : 1999年 -2002年 
    代表者 : 小笠原 直毅, 小林 和夫, 笠原 康裕, 守家 成紀, 朝井 計
     
    本研究は、枯草菌の細胞周期を構成する染色体複製、染色休分配や細胞分裂に関与する因子の細胞内動態を細胞生物学的手法により調べると共にこれらの因子間の相互作用も解析し、細胞周期の制御機構を解明することを目的とし、以下の新知見を得た。 (1)枯草菌の染色体複製は、開始蛋白質DnaAと複製開始点oriCの複合体形成に始まり、DnaB, D, I蛋白質により、DnaCヘリカーゼが導入される。DnaIはDnaCと、DnaDはDnaAと相互作用することを明らかにした。 (2)DnaAは細胞質に分布するのに対し、DnaB, IはoriCと同様に核様体の周辺部に局在すること、DNAポリメラーゼが複製開始時にその位置に移動することを明らかにし、複製開始は核様体周辺部で起こる事が示唆された。 (3)複製開始が起こる時期をDnaA蛋白質量が規定する可能性を、人為的にその発現量を制御することにより示した。また、spo0J破壊株において、開始点の分離に異常が生じ、過剰な複製開始が起こることが観察された。その影響はsoj遺伝子の欠失により失われ、一方、Sojの過剰発現は過剰複製を誘導した。Spo0Jは複製開始点の移動に働いており、Sojは複製開始に関与する遺伝子発現を制御していると考えられる。さらに、新たな複製開始制御蛋白質としてGTP結合蛋白質BexとYqeHを同定した。 (4)染色体分配において、セントロメアとして機能していると考えられるcis配列を細菌で始めて同定した。 (5)染色体の高次構造形成に関与しているSmc蛋白質と複合体を形成する新規蛋白質、ScpA, Bを同定した。それらの細胞内動態を解析し、Smcはレプリゾーム近傍に結合している割合が高く、Smcの染色体への結合はSpcABに依存することが示された。染色体DNAの複製直後にSmc複合体により染色体の高次構造が形成されると考えられる。
  • Metaproteomcs

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  • 繊毛虫検出用プライマー及びそれを用いる繊毛虫の検出方法

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