研究者データベース

小島 久弥(コジマ ヒサヤ)
低温科学研究所 生物環境部門
助教

基本情報

所属

  • 低温科学研究所 生物環境部門

職名

  • 助教

学位

  • 博士(理学)(東京都立大学)

科研費研究者番号

  • 70400009

J-Global ID

研究キーワード

  • 硫黄循環   sulfur cycle   

研究分野

  • ライフサイエンス / 生態学、環境学

研究活動情報

論文

  • Miho Watanabe, Hisaya Kojima, Manabu Fukui
    International journal of systematic and evolutionary microbiology 70 12 6408 - 6413 2020年12月 [査読有り]
     
    A novel facultatively anaerobic, nitrate-reducing bacterium, designated MeG22T, was isolated from a freshwater lake in Japan. Cells of the strain were straight rods (0.8×2.5-10 µm), motile, and Gram-stain-negative. For growth, the optimum NaCl concentration was 0 % and the optimum temperature was 30 °C. Under anoxic conditions, strain MeG22T reduced nitrate to nitrite. Major cellular fatty acids were C15 : 1 ω6c (13.6 %), C17 : 0 (11.9 %), anteiso-C15 : 0 (10.6 %) and iso-C15 : 0 (10.6 %). The major respiratory quinone was menaquinone-7. The genome sequence of strain MeG22T consists of 5 712 279 bp with a G+C content of 40.3 mol%. Phylogenetic analysis based on the 16S rRNA gene revealed that the novel strain belonged to the family Prolixibacteraceae within the phylum Bacteroidetes. The closest relative of strain MeG22T was Sunxiuqinia faeciviva strain JAM-BA0302T with a 16S rRNA gene sequence similarity of 90.9 %. On the basis of phylogenetic and phenotypic characterization, Aquipluma nitroreducens, gen. nov., sp. nov., belonging to the family Prolixibacteraceae is proposed with the type strain MeG22T (=NBRC 112896T=DSM 106262T).
  • Jun Mochizuki, Hisaya Kojima, Manabu Fukui
    Archives of microbiology 2020年10月21日 [査読有り]
     
    Aerobic, Gram-stain-negative, obligately chemolithoautotrophic thiosulfate-oxidizing bacteria, strains AkT22T and aks77T were isolated from a brackish lake in Japan. Strains AkT22T and aks77T were isolated from samples of eelgrass and sediment, respectively. Growth on sulfide, tetrathionate, elemental sulfur, and organic substrates was not observed for both strains. Growth of the strains was observed at 5 °C or higher temperature, with optimum growth at 22 °C. Strain AkT22T grew at a pH range of 5.8-8.0, with optimum growth at pH 6.7-7.8. Strain aks77T grew at a pH range of 5.8-8.5, with optimum growth at pH 7.0-7.9. Major cellular fatty acids (> 10% of total) of strain AkT22T were C16:1, C18:1, and C16:0. The sole respiratory quinone was ubiquinone-8 in both strains. The genome of strain AkT22T consisted of a circular chromosome, with size of approximately 2.6 Mbp and G + C content of 43.2%. Those values of the genome of strain aks77T were ca. 2.7 Mbp and 45.5%, respectively. Among cultured bacteria, Thiomicrorhabdus aquaedulcis HaS4T showed the highest sequence identities of the 16S rRNA gene, to strains AkT22T (94%) and aks77T (95%). On the basis of these results, Thiosulfativibrio zosterae gen. nov., sp. nov. and Thiosulfatimonas sediminis gen. nov., sp. nov. are proposed, with type strains of AkT22T (= BCRC 81184T = NBRC 114012T = DSM 109948T) and aks77T (= BCRC 81183T = NBRC 114013T), respectively.
  • Hisaya Kojima, Mamoru Kanda, Kazuhiro Umezawa, Manabu Fukui
    Archives of microbiology 2020年09月14日 [査読有り][通常論文]
     
    A facultatively anaerobic sulfur-oxidizing bacterium, strain skT11T, was isolated from anoxic lake water of a stratified freshwater lake. As electron donor for chemolithoautotrophic growth, strain skT11T oxidized thiosulfate, tetrathionate, and elemental sulfur under nitrate-reducing conditions. Oxygen-dependent growth was observed under microoxic conditions, but not under fully oxygenated conditions. Growth was observed at a temperature range of 5-37 °C, with optimum growth at 28 °C. Strain skT11T grew at a pH range of 5.1-7.5, with optimum growth at pH 6.5-6.9. Heterotrophic growth was not observed. Major components in the cellular fatty acid profile were C16:1 and C16:0. The complete genome of strain skT11T consisted of a circular chromosome with a size of 3.8 Mbp and G + C content of 60.2 mol%. Phylogenetic analysis based on the 16S rRNA gene sequences indicated that the strain skT11T is related to sulfur-oxidizing bacteria of the genera Sulfuricella, Sulfurirhabdus, and Sulfuriferula, with sequence identities of 95.4% or lower. The analysis also indicated that these three genera should be excluded from the family Gallionellaceae, as members of another family. On the basis of its genomic and phenotypic properties, strain skT11T (= DSM 110711 T = NBRC 114323 T) is proposed as the type strain of a new species in a new genus, Sulfurimicrobium lacus gen. nov., sp. nov. In addition, emended descriptions of the families Gallionellaceae and Sulfuricellaceae are proposed to declare that Sulfuricellaceae is not a later synonym of Gallionellaceae.
  • Miho Watanabe, Yuriko Higashioka, Hisaya Kojima, Manabu Fukui
    Systematic and applied microbiology 43 5 126109 - 126109 2020年09月 [査読有り][通常論文]
     
    Strain 28bB2TT is a sulfate-reducing bacterium isolated in a previous study, obtained from a p-xylene-degrading enrichment culture. Physiological, phylogenetic and genomic characterizations of strain 28bB2TT were performed to establish the taxonomic status of the strain. Cells of strain 28bB2TT were short oval-shaped (0.8-1.2×1.2-2.7μm), motile, and Gram-negative. For growth, the optimum pH was pH 6.5-7.0 and the optimum temperature was 28-32°C. Strain 28bB2TT oxidized toluene but could not utilize p-xylene. Sulfate and thiosulfate were used as electron acceptors. The G+C content of the genomic DNA was 53.8mol%. The genome consisted of an approximately 8.3 Mb of chromosome and two extrachromosomal elements. On the basis of 16S rRNA gene analysis, strain 28bB2TT was revealed to belong to the genus Desulfosarcina, with high sequence identities to Desulfosarcina ovata oXyS1T (99.5%) and Desulfosarcina cetonica DSM 7267T (98.7%). Results of Average Nucleotide Identity (ANI) calculation and digital DNA-DNA hybridization (dDDH) analysis showed that the strain 28bB2TT should be classified as a subspecies under D. ovata. Based on physiological and phylogenetic data, strain 28bB2TT (=NBRC 106234 =DSM 23484) is proposed as the type strain of a novel species in genus Desulfosarcina, Desulfosarcina ovata subsp. sediminis subsp. nov.
  • Kazuhiro Umezawa, Hisaya Kojima, Yukako Kato, Manabu Fukui
    Systematic and applied microbiology 43 5 126110 - 126110 2020年09月 [査読有り][通常論文]
     
    The phylum Nitrospirota (previously known as Nitrospirae or Nitrospira) currently encompasses a limited number of bacterial species with validly published names, including sulfate-reducing bacteria (SRB) of the genus Thermodesulfovibrio. Some metagenome-assembled genomes (MAGs) of bacteria occur in this phylum, and genes involved in dissimilatory sulfur metabolism have been identified in them. Currently, however, there is no established way to discriminate SRB and sulfur-disproportionating bacteria (SDB), which obtain energy from the disproportionation of inorganic sulfur compounds. In this study, a thiosulfate-disproportionating enrichment culture was established from a hot spring microbial mat. The culture was dominated by a single species belonging to the phylum Nitrospirota, and growth of the novel bacterium was supported by disproportionation of thiosulfate and elemental sulfur. Its growth was not observed under sulfate-reducing conditions. Therefore, a comparative genomic analysis of SDB and SRB was performed using its draft genome sequence, in order to identify any genetic element that could be used as a marker for SDB. As a result, a characteristic gene cluster was identified as a putative genetic element that characterized the genomes of SDB. The gene cluster was found in some MAGs of the phylum Nitrospirota, and their corresponding bacteria may also be capable of the disproportionation of inorganic sulfur compounds.
  • Hisaya Kojima, Jun Mochizuki, Manabu Fukui
    International journal of systematic and evolutionary microbiology 2020年05月04日 [査読有り][通常論文]
     
    A chemolithoautotrophic sulfur-oxidizing bacterium, strain SGTMT was isolated from snow collected in Japan. As electron donors for growth, SGTMT oxidized thiosulfate, tetrathionate and elemental sulfur. Heterotrophic growth was not observed. Growth of the novel isolate was observed at a temperature range of 5-28 °C, with optimum growth at 18 °C. SGTMT grew at a pH range of 4.3-7.4, with optimum growth at pH 6.1-7.1. Major components in the cellular fatty acid profile were summed feature 3 (C16 : 1ω7c and/or C16 : 1ω6c) and C16 : 0. The complete genome of SGTMT consisted of a circular chromosome of approximately 3.4 Mbp and two plasmids. Phylogenetic analysis based on the 16S rRNA gene indicated that SGTMT represented a member of the genus Sulfuriferula, and its closest relative is Sulfuriferula thiophila mst6T with a sequence identity of 98 %. A comparative genome analysis showed dissimilarity between the genomes of SGTMT and S. thiophila mst6T, as low values of average nucleotide identity (74.9 %) and digital DNA-DNA hybridization (20.4%). On the basis of its genomic and phenotypic properties, SGTMT (=DSM 109609T=BCRC 81185T) is proposed as the type strain of a novel species, Sulfuriferula nivalis sp. nov. Some characteristics of another species in the same genus, Sulfuriferula plumbiphila, were also investigated to revise and supplement its description. The type strain of S. plumbiphila can grow on thiosulfate, tetrathionate and elemental sulfur. The strain showed optimum growth at pH 6.3-7.0 and shared major cellular fatty acids with the other species of the genus Sulfuriferula.
  • Miho Watanabe, Hisaya Kojima, Manabu Fukui
    ANTONIE VAN LEEUWENHOEK INTERNATIONAL JOURNAL OF GENERAL AND MOLECULAR MICROBIOLOGY 113 3 349 - 355 2020年03月 [査読有り][通常論文]
     
    A novel facultative anaerobic and facultative psychrophilic bacterium, designated SPP2(T), was isolated from an Antarctic marine sediment. Cells of the isolate were observed to be long rods (0.5 x 5-10 mu m), Gram-stain negative and to have gliding motility. For growth, the optimum NaCl concentration was found to be 2-3% and the optimum temperature to be 18-22 degrees C. Strain SPP2(T) cannot use sulfate and nitrate as electron acceptors in the presence of lactate. The G+C content of the genomic DNA was determined to be 36.0 mol%.. The major cellular fatty acids were identified as anteiso-C-15:0 and iso-C-15:0. MK-7 was found to be the predominant respiratory quinone. Phylogenetic analysis based on the 16S rRNA gene revealed that the novel strain belongs to the family Marinifilaceae and to be closely related to Labilibaculum manganireducens 59.10-2M(T) with 16S rRNA gene sequence identity of 98%. The OrthoANI and dDDH values between the genome sequences of strain SPP2(T) and its close relative were 84% and 27.3%, which are lower than the threshold values for species delineation. On the basis of phylogenetic and phenotypic characterisation, Labilibaculum antarcticum sp. nov. is proposed with the type strain SPP2(T) (= NBRC 111151(T) = CECT 9460(T)).
  • Miho Watanabe, Hisaya Kojima, Manabu Fukui
    Archives of microbiology 2020年02月04日 [査読有り][通常論文]
     
    An aerobic bacterium, designated strain Dysh456T, was isolated from a crude oil-contaminated soil. Cells of strain Dysh456T were rod-shaped, motile, and Gram-stain-negative. Strain Dysh456T grew at 13-48 °C and pH 4.3-7.9. Major cellular fatty acids were iso-C15:0 (42.5%), iso-C17:0 (15.3%) and summed feature 9 (iso-C17:1 ω9c/C16:0 10-methyl [13.7%]). Major respiratory quinone was ubiquinone-8. The genome of strain Dysh456T consists of a single circular chromosome of 2,874,969 bp in length with G + C content of 68.3%. Phylogenetic analysis based on the 16S rRNA gene sequences indicated that strain Dysh456T belongs to the family Rhodanobacteraceae, but none of the existing genera can accommodate this novel isolate. On the basis of physiological, chemotaxonomic, and genomic properties, strain Dysh456T (= NBRC 112897T = DSM 105662T) is proposed as the type strain representing a novel species of novel genus, for which the name Aerosticca soli gen. nov., sp. nov. is proposed.
  • Santona Khatun, Tomoya Iwata, Hisaya Kojima, Yoshiki Ikarashi, Kana Yamanami, Daichi Imazawa, Tanaka Kenta, Ryuichiro Shinohara, Hiromi Saito
    Water 12 2 402 - 402 2020年02月02日 [査読有り][通常論文]
     
    Our understanding of the source of methane (CH4) in freshwater ecosystems is being revised because CH4 production in oxic water columns, a hitherto inconceivable process of methanogenesis, has been discovered for lake ecosystems. The present study surveyed nine Japanese deep freshwater lakes to show the pattern and mechanisms of such aerobic CH4 production and subsurface methane maximum (SMM) formation. The field survey observed the development of SMM around the metalimnion in all the study lakes. Generalized linear model (GLM) analyses showed a strong negative nonlinear relationship between dissolved organic carbon (DOC) and dissolved inorganic nitrogen (DIN), as well as a similar curvilinear relationship between DIN and dissolved CH4, suggesting that the availability of organic carbon controls N accumulation in lake waters thereby influences the CH4 production process. The microbial community analyses revealed that the distribution of picocyanobacteria (i.e., Synechococcus), which produce CH4 in oxic conditions, was closely related to the vertical distribution of dissolved CH4 and SMM formation. Moreover, a cross-lake comparison showed that lakes with a more abundant Synechococcus population exhibited a greater development of the SMM, suggesting that these microorganisms are the most likely cause of methane production. Thus, we conclude that the stoichiometric balance between DOC and DIN might cause the cascading responses of biogeochemical processes, from N depletion to picocyanobacterial domination, and subsequently influence SMM formation in lake ecosystems.
  • Santona Khatun, Tomoya Iwata, Hisaya Kojima, Manabu Fukui, Takuya Aoki, Seito Mochizuki, Azusa Naito, Ai Kobayashi, Ryo Uzawa
    Science of the Total Environment 696 2019年12月15日 [査読有り][通常論文]
     
    © 2019 Elsevier B.V. Methanogenesis in freshwater lakes has classically been considered to arise from anaerobic methanogens in oxygen-depleted sediments. However, the accumulation of supersaturated methane in fully oxygenated water columns is commonly observed in many lakes, and factors responsible for the formation of the subsurface methane maximum (SMM) remain largely unknown. The present study conducted in 14 Japanese freshwater lakes showed that the SMM formation during the summer stratification period is a common feature in large and deep oligotrophic lakes. The seasonal survey of a deep oligotrophic lake revealed that SMM formation may be uncoupled with the dissolution of atmospheric methane, as well as with the transport of methane from tributary rivers, littoral sediments, and hypolimnetic anoxic sources, suggesting the contribution of in situ methane production. In fact, batch-culture experiments confirmed that bacterioplankton present in lake subsurface waters produce methane aerobically through the decomposition of methylphosphonic acid. Moreover, the development of SMM was closely associated with the seasonal dynamics of planktonic cyanobacteria such as Synechococcus, which may carry the enzyme to catabolize organophosphonate compounds. Therefore, we suggest that the predominance of Synechococcus during the thermal stratification period plays a significant role in SMM formation, and likely the methane flux from lakes to the atmosphere.
  • Hisaya Kojima, Manabu Fukui
    INTERNATIONAL JOURNAL OF SYSTEMATIC AND EVOLUTIONARY MICROBIOLOGY 69 9 2849 - 2853 2019年09月 [査読有り][通常論文]
     
    Strain HaS4(T) is an aerobic sulfur-oxidizing bacterium isolated from water of Lake Harutori in Japan. It was isolated and partially characterized in a previous study, but its taxonomic status has not been determined. The previous study revealed that the strain is an obligate chemolithoautotroph which grows at temperatures ranging from 0 to 25 degrees C (optimum, 22 degrees C) and pH from pH 6.2 to 8.8 (optimum, pH 6.6-7.4). In this study, further characterization of the strain was made to describe it as representative of a novel species. Cells of strain HaS4(T) are rod-shaped, 1.6-2.5 mu m long, 0.7-0.9 mu m wide and Gram-stain-negative. Major cellular fatty acids were summed feature 3 (C-16 : 1 omega 7c and/or C-16 : 1 omega 6c), summed feature 8 (C-18 : 1 omega 7c and/or C-18 : 1 omega 6c) and C-16 : 0. Phylogenetic analysis based on the 16S rRNA gene indicated that the strain is related to the genus Thiomicrorhabdus, but phylogenetically distinct from the type strains of existing species in the genus. On the basis of its phylogenetic and phenotypic properties, strain HaS4(T) (=NBRC 112315(T)=BCRC 81110(T)) is proposed as type strain of a new non-marine species of the genus Thiomicrorhabdus with the name Thiomicrorhabdus aquaedulcis sp. nov.
  • Melody Cabrera Ospino, Hisaya Kojima, Manabu Fukui
    FRONTIERS IN MICROBIOLOGY 10 2019年05月 [査読有り][通常論文]
     
    Microbes play essential roles in arsenic transformation in the environment. Microbial arsenite oxidation is catalyzed by either of two distantly related arsenite oxidases, referred to as AIO and ARX. The arx genes encoding ARX and its regulatory proteins were originally defined in the genomes of gammaproteobacteria isolated from an alkaline soda lake. The arx gene cluster has been identified in a limited number of bacteria, predominantly in gammaproteobacteria isolated from lakes characterized by high pH and high salinity. In the present study, a novel arsenite-oxidizing betaproteobacterium, strain M52, was isolated from a hot spring microbial mat. The strain oxidized arsenite under both microaerophilic and nitrate-reducing conditions at nearly neutral pH. Genome analysis revealed that the strain possesses the ant gene cluster in its genome and lacks genes encoding AIO. Inspection of the bacterial genomes available in the GenBank database revealed that the presence of this gene cluster is restricted to genomes of Proteobacteria, mainly in the classes Gammaproteobacteria and Betaproteobacteria. In these genomes, the structure of the gene cluster was generally well-conserved, but genes for regulatory proteins were lacking in genomes of strains belonging to a specific lineage. Phylogenetic analysis suggested that ARX encoded in the genomes can be divided into three groups, and strain M52 belongs to a group specific for organisms living in low-salt environments. The ArxA protein encoded in the genome of strain M52 was characterized by the presence of a long insertion, which was specifically observed in the same group of ARX. In clone library analyses with a newly designed primer pair, a diverse ArxA sequence with a long insertion was detected in samples of lake water and hot spring microbial mat, characterized by low salinity and a nearly neutral pH. Among the isolated bacterial strains whose arsenite oxidation has been demonstrated, strain M52 is the first betaproteobacterium that possesses the arx genes, the first strain encoding ARX of the group specific for low-salt environments, and the first organism possessing the gene encoding ArxA with a long insertion.
  • Mia Terashima, Keisuke Ohashi, Taichi E. Takasuka, Hisaya Kojima, Manabu Fukui
    ENVIRONMENTAL MICROBIOLOGY REPORTS 11 2 227 - 235 2019年04月 [査読有り][通常論文]
     
    Hymenobacter nivis P3(T) is a heterotrophic bacterium isolated from Antarctic red snow generated by algal blooms. Despite being non-photosynthetic, H. nivis was dominantly found in the red snow environment that is exposed to high light and UV irradiation, suggesting that this species can flourish under such harsh conditions. In order to further understand the adaptive strategies on the snow surface environment of Antarctica, the genome of H. nivis P3(T) was sequenced and analyzed, which identified genes putatively encoding for light-reactive proteins such as proteorhodopsin, phytochrome, photolyase and several copies of cryptochromes. Culture-based experiments revealed that H. nivis P3(T) growth was significantly enhanced under light conditions, while dark conditions had increased extracellular polymeric substances. Furthermore, the expression of several putative light-reactive proteins was determined by proteomic analysis. These results indicate that H. nivis P3(T) is able to potentially utilize light, which may explain its dominance on the red snow surface environment of Antarctica. Originality-significance statement The role of proteorhodopsin in heterotrophic bacteria is not well-characterized, as only a handful of proteorhodopsin-harbouring isolates were shown to have a light-enhanced phenotype through culture-based experiments to date. This is the first study that demonstrates light-stimulated growth and protein expression evidence of photoactive proteins for a non-marine psychrophile and for a member of the genus Hymenobacter. It is also the first study that provides comprehensive proteome information for this genus. This study presents significant results in understanding the adaptive mechanism of a heterotrophic non-photosynthetic bacterium thriving on the snow surface environment of Antarctica as well as demonstrating the role of light-utilization in promoting growth, possibly through proteorhodopsin.
  • Miho Watanabe, Hisaya Kojima, Kazuhiro Umezawa, Manabu Fukui
    FRONTIERS IN MICROBIOLOGY 10 2019年02月 [査読有り][通常論文]
     
    Desulfonema ishimotonii strain Tokyo 01(T) is a filamentous sulfate-reducing bacterium isolated from a marine sediment. In this study, the genome of this strain was sequenced and analyzed with a focus on gene transfer from phylogenetically distant organisms. While the strain belongs to the class Deltaproteobacteria, hundreds of proteins encoded in the genome showed the highest sequence similarities to those of organisms outside of the class Deltaproteobacteria, suggesting that more than 20% of the genome is putatively of foreign origins. Many of these proteins had the highest sequence identities with proteins encoded in the genomes of filamentous bacteria, including giant sulfur oxidizers of the orders Thiotrichales, cyanobacteria of various genera, and uncultured bacteria of the candidate phylum KSB3. As mobile genetic elements transferred from phylogenetically distant organisms, putative inteins were identified in the GyrB and DnaE proteins encoded in the genome of strain Tokyo 01(T). Genes involved in DNA recombination and repair were enriched in comparison to the closest relatives in the same family. Some of these genes were also related to those of organisms outside of the class Deltaproteobacteria, suggesting that they were acquired by horizontal gene transfer from diverse bacteria. The genomic data suggested significant genetic transfer among filamentous gliding bacteria in phylogenetically dispersed lineages including filamentous sulfate reducers. This study provides insights into the genomic evolution of filamentous bacteria belonging to diverse lineages, characterized by various physiological functions and different ecological roles.
  • Tomohiro Watanabe, Hisaya Kojima, Kazuhiro Umezawa, Chiaki Hori, Taichi E. Takasuka, Yukako Kato, Manabu Fukui
    FRONTIERS IN MICROBIOLOGY 10 2019年02月 [査読有り][通常論文]
     
    Even in the current era of metagenomics, the interpretation of nucleotide sequence data is primarily dependent on knowledge obtained from a limited number of microbes isolated in pure culture. Thus, it is of fundamental importance to expand the variety of strains available in pure culture, to make reliable connections between physiological characteristics and genomic information. In this study, two sulfur oxidizers that potentially represent two novel species were isolated and characterized. They were subjected to whole-genome sequencing together with 7 neutrophilic and chemolithoautotrophic sulfur-oxidizing bacteria. The genes for sulfur oxidation in the obtained genomes were identified and compared with those of isolated sulfur oxidizers in the classes Betaproteobacteria and Gammaproteobacteria. Although the combinations of these genes in the respective genomes are diverse, typical combinations corresponding to three types of core sulfur oxidation pathways were identified. Each pathway involves one of three specific sets of proteins, SoxCD, DsrABEFHCMKJOP, and HdrCBAHypHdrCB. All three core pathways contain the SoxXYZAB proteins, and a cytoplasmic sulfite oxidase encoded by soeABC is a conserved component in the core pathways lacking SoxCD. Phylogenetically close organisms share same core sulfur oxidation pathway, but a notable exception was observed in the family 'Sulfuricellaceae'. In this family, some strains have either core pathway involving DsrABEFHCMKJOP or HdrCBAHypHdrCB, while others have both pathways. A proteomics analysis showed that proteins constituting the core pathways were produced at high levels. While hypothesized function of HdrCBAHypHdrCB is similar to that of Dsr system, both sets of proteins were detected with high relative abundances in the proteome of a strain possessing genes for these proteins. In addition to the genes for sulfur oxidation, those for arsenic metabolism were searched for in the sequenced genomes. As a result, two strains belonging to the families Thiobacillaceae and Sterohbacteriaceae were observed to harbor genes encoding ArxAB, a type of arsenite oxidase that has been identified in a limited number of bacteria. These findings were made with the newly obtained genomes, including those from 6 genera from which no genome sequence of an isolated organism was previously available. These genomes will serve as valuable references to interpret nucleotide sequences.
  • Miho Watanabe, Hisaya Kojima, Manabu Fukui
    INTERNATIONAL JOURNAL OF SYSTEMATIC AND EVOLUTIONARY MICROBIOLOGY 68 9 2891 - 2899 2018年09月 [査読有り][通常論文]
     
    The genus Desulfotomaculum is a heterogeneous group of spore-forming sulfate-reducing bacteria. The type species of the genus is Desulfotomaculum nigrificans (Approved Lists 1980) emend. Visser et al. 2014. The results of phylogenetic analysis demonstrated that the genus Desulfotomaculum already has lost the clustering monophyly and was segregated into some distinct groups with low sequence similarity. Major features of the type strains in these groups were compared, and four novel genera, Desulfallas gen. nov., Desulfofundulus gen. nov., Desulfofarcimen gen. nov. and Desulfohalotomaculum gen. nov. were proposed to accommodate species transferred from the genus Desulfotomaculum.
  • Miho Watanabe, Hisaya Kojima, Manabu Fukui
    MARINE GENOMICS 39 1 - 2 2018年06月 [査読有り][通常論文]
     
    Marinifilaceae bacterium strain SPP2 is a Gram-negative facultative anaerobe, isolated from the Antarctic marine sediment. Here, we present the complete genome sequence of Marinifilaceae bacterium strain SPP2, which consists of 5,718,991 bp with a G + C content of 35.99%. The genome data provides insights of microbial evolution and adaption in the Antarctic marine ecosystem. (C) 2017 Elsevier B.V. All rights reserved.
  • Melody Cabrera Ospino, Hisaya Kojima, Tomohiro Watanabe, Tomoya Iwata, Manabu Fukui
    LIMNOLOGY 19 2 177 - 183 2018年04月 [査読有り][通常論文]
     
    Anaerobic arsenite oxidation is potentially important but the least understood process in the arsenic cycle. The catalytic subunit of the key enzyme for anaerobic arsenite oxidation is encoded by the arxA gene. In this study, a novel primer pair for the arxA gene was designed to detect diverse sequences of this notable gene. Further modification of the designed primer was made by adding extra bases to its 5'- end. This modification made it possible to analyze the PCR products with TA cloning, which provides higher throughput of investigations. With the combination of modified primer pair and TA cloning, diverse arxA gene sequences were effectively obtained from samples of lake water, spring water, and hot spring microbial mat. The sequences detected in the samples characterized by low salinity and nearly neutral pH were phylogenetically distinct from the majority of previously known arxA genes, found in the genome of alkaliphiles and halophiles.
  • Masayuki Itoh, Hisaya Kojima, Pei-Chi Ho, Chun-Wei Chang, Tzong-Yueh Chen, Silver Sung-Yun Hsiao, Yuki Kobayashi, Megumu Fujibayashi, Shuh-Ji Kao, Chih-hao Hsieh, Manabu Fukui, Noboru Okuda, Takeshi Miki, Fuh-Kwo Shiah
    Ecological Research 33 5 1 - 2 2018年03月29日 [査読有り][通常論文]
     
    The article “Integrating isotopic, microbial, and modeling approaches to understand methane dynamics in a frequently disturbed deep reservoir in Taiwan”, written by Masayuki Itoh, Hisaya Kojima, Pei-Chi Ho, Chun-Wei Chang, Tzong-Yueh Chen, Silver Sung-Yun Hsiao, Yuki Kobayashi, Megumu Fujibayashi, Shuh-Ji Kao, Chih-hao Hsieh, Manabu Fukui, Noboru Okuda, Takeshi Miki, Fuh-Kwo Shiah, was originally published electronically on the publisher’s internet portal (currently SpringerLink) on 18 September 2017 without open access.
  • Miho Watanabe, Riho Tokizawa, Hisaya Kojima, Manabu Fukui
    STANDARDS IN GENOMIC SCIENCES 12 76  2017年12月 [査読有り][通常論文]
     
    Effusibacillus lacus strain skLN1(T) is the type strain of the type species in the genus Effusibacillus which is the one of the genera in the family Alicyclobacillaceae within the phylum Firmicutes. Effusibacillus lacus strain skLN1T is a Grampositive, spore-forming thermophilic neutrophile isolated from freshwater lake sediment. Here, we present the draft genome sequence of strain skLN1(T), which consists of 3,902,380 bp with a G + C content of 50.38%.
  • Masayuki Itoh, Hisaya Kojima, Pei-Chi Ho, Chun-Wei Chang, Tzong-Yueh Chen, Silver Sung-Yun Hsiao, Yuki Kobayashi, Megumu Fujibayashi, Shuh-Ji Kao, Chih-hao Hsieh, Manabu Fukui, Noboru Okuda, Takeshi Miki, Fuh-Kwo Shiah
    ECOLOGICAL RESEARCH 32 6 861 - 871 2017年11月 [査読有り][通常論文]
     
    It has been estimated that more than 48% of global methane emissions from lakes and reservoirs occur at low latitudes (< 24 degrees). To improve this estimate, knowledge regarding underexplored ecosystems, particularly deep lakes and reservoirs in Asian monsoon regions, is needed because the magnitude of methane emissions is influenced by lake bathymetry and climatic conditions. We conducted long-term studies beginning in 2004 at Feitsui Reservoir (FTR) in Taiwan, a subtropical monomictic system with a maximum depth of 120 m to monitor seasonal and interannual variations of three key characteristics and to understand the mechanisms underlying these variations. Key characteristics investigated were as follows: (1) the balance of primary production and heterotrophic respiration as a determinant of vertical oxygen distribution, (2) methane production at the bottom of the reservoir, oxidation in the water column, and emissions from the lake surface, and (3) the contribution of methane-originated carbon to the pelagic food web through methane-oxidizing bacteria (MOB). This review highlights major achievements from FTR studies integrating isotopic, microbial, and modeling approaches. Based on our findings, we proposed two conceptual models: (1) a model of methane dynamics, which addresses the differences in methane emission mechanisms between deep and shallow lakes, and (2) a spatially explicit model linking benthic methane production to the pelagic food web, which addresses the diversity of MOB metabolisms and their dependence on oxygen availability. Finally, we address why long-term studies of subtropical lakes and reservoirs are important for better understanding the effects of climate on low- to mid-latitude ecosystems.
  • Rei Okamoto, Hisaya Kojima, Manabu Fukui
    INTERNATIONAL JOURNAL OF SYSTEMATIC AND EVOLUTIONARY MICROBIOLOGY 67 11 4773 - 4776 2017年11月 [査読有り][通常論文]
     
    cA novel acidophilic heterotrophic bacterium, strain Ok2G(T), was isolated from a freshwater lake in Japan. Cells of the isolate were Gram-stain-negative and non-motile rods (0.6-0.8 x 1.0-2.8 mu m). Growth was observed at 4-35 degrees C with an optimum growth temperature of 28 degrees C. The pH range for growth was 3.0-6.2 with an optimum pH of 4.5. The strain utilized fructose, glucose, sucrose, mannitol, sorbitol, ethanol, benzyl alcohol, pyruvate, yeast extract and tryptone as carbon and energy sources for aerobic growth. DNA G+C content was 62.6 mol%. The major cellular fatty acid and the isoprenoid quinone were summed feature 8 (C-18:1 omega 7c and/or C-18:1 omega 6c) and Q-10, respectively. Phylogenetic analysis based on the 16S rRNA gene sequence indicated that strain Ok2G(T) belongs to the genus Acidocella but is distinct from existing species with sequence similarities lower than 97 %. On the basis of these results, strain Ok2GT (= NBRC 112502(T) = DSM 104037(T)) is proposed as the type strain of a novel species, Acidocella aquatica sp. nov.
  • Tomohiro Watanabe, Aya Miura, Tomoya Iwata, Hisaya Kojima, Manabu Fukui
    ENVIRONMENTAL MICROBIOLOGY REPORTS 9 5 522 - 527 2017年10月 [査読有り][通常論文]
     
    Facultative autotrophs of the genus Sulfuritalea within the class Betaproteobacteria have been predicted to be an important bacterial population in stratified freshwater lakes based on previous PCR-based studies. Here, we designed a new probe specific for the genus Sulfuritalea and performed catalysed reporter deposition-fluorescence in situ hybridisation to enumerate cells of Sulfuritalea species throughout the water column in a stratified freshwater lake. The cells stained with the Sulfuritalea-specific probe were detected in all hypoxic water samples collected in different seasons and years. Their abundance ranged from 1.4 x 10(4) to 2.1 x 10(5) cells ml(-1), corresponding to 0.5-5.5% of the total DAPI-stained cells and 2.3-15% of the total bacterial cells. A high abundance of Sulfuritalea species was recorded in hypoxic water samples without nitrate, which is the only known anaerobic electron acceptor for Sulfuritalea. Nitrate-independent anaerobic respiration was further investigated using a single cultured representative of this genus, and its growth via arsenate respiration was experimentally demonstrated. In conclusion, Sulfuritalea species were found to be a major component of the planktonic bacterial community in nitrate-depleted hypoxic water, where arsenate respiration is one of the possible energy metabolisms of Sulfuritalea.
  • Hisaya Kojima, Miho Watanabe, Manabu Fukui
    INTERNATIONAL JOURNAL OF SYSTEMATIC AND EVOLUTIONARY MICROBIOLOGY 67 9 3458 - 3461 2017年09月 [査読有り][通常論文]
     
    A novel Gram-stain-negative, chemolithoautotrophic sulfur oxidizer, strain JG42(T), was isolated from a hot spring microbial mat. As an electron donor for autotrophic growth, strain JG42(T) utilized sulfide, thiosulfate, tetrathionate and elemental sulfur. Cells of strain JG42(T) were oxidase-positive and catalase-negative. The G+C content of the genomic DNA was 65 mol%. The predominant cellular fatty acid was C-16:0. Phylogenetic analysis of the 16S rRNA gene indicated that strain JG42(T) belonged to the order Chromatiales, but sequence similarities to the known species were less than 94 %. On the basis of its properties, strain JG42(T) (= DSM 104776(T) = NBRC 112696(T)) is proposed as the type strain of a novel species of a new genus, Sulfurivermis fontis gen. nov., sp. nov., which belongs to the family Thioalkalispiraceae. A new family, Thioprofundaceae fam. nov., is also proposed to accommodate the genus Thioprofundum, transferred from the family Thioalkalispiraceae.
  • Miho Watanabe, Yuriko Higashioka, Hisaya Kojima, Manabu Fukui
    INTERNATIONAL JOURNAL OF SYSTEMATIC AND EVOLUTIONARY MICROBIOLOGY 67 8 2994 - 2997 2017年08月 [査読有り][通常論文]
     
    In previous studies, two hydrocarbon-degrading sulfate-reducing bacteria, strains PP31(T) and PL12(T), were obtained from oil-polluted marine sediments of Shuaiba, Kuwait. They had been reported as organisms capable of anaerobic degradation of p-xylene and n-alkanes, respectively. The 16S rRNA gene sequence of strain PP31(T) showed 98.8% sequence similarities to that of Desulfosarcina variabilis ` Montpellier'T. Strains PL12(T) had 97.8% of sequence similarity to Desulfosarcina ovata oXys1(T). They both have been partially characterized, but not been validly published as new species of the genus Desulfosarcina. In this study, additional characterizations of these strains were made to describe them as two new species of the genus Desulfosarcina. Major cellular fatty acids of strain PP31(T) were C-15 : 0 (25.9 %) and anteiso-C-15 : 0 (22.3 %), whereas those of strain PL12(T) were C-15 : 0 (21.3 %), C-16 : 0 (17.8 %) and anteiso-15 : 0 (11.6 %). The phylogenetic tree based on 16S rRNA gene revealed that these isolates should not be classified as any of the known species in the genus Desulfosarcina. On the basis of phenotypic and phylogenetic analyses, these two sulfate reducers are proposed to form two novel species of the genus Desulfosarcina : Desulfosarcina widdelii sp. nov. (PP31(T) = JCM 31729(T) = DSM 103921(T)) and Desulfosarcina alkanivorans sp. nov. (PL12(T) = JCM 31728(T) = DSM 103901(T)). In addition, emended description of the genus Desulfosarcina is presented in this study.
  • Mia Terashima, Kazuhiro Umezawa, Shoichi Mori, Hisaya Kojima, Manabu Fukui
    FRONTIERS IN MICROBIOLOGY 8 1481  2017年08月 [査読有り][通常論文]
     
    Psychrophilic algae blooms can be observed coloring the snow during the melt season in alpine snowfields. These algae are important primary producers on the snow surface environment, supporting the microbial community that coexists with algae, which includes heterotrophic bacteria and fungi. In this study, we analyzed the microbial community of green and red-colored snow containing algae from Mount Asahi, Japan. We found that Chloromonas spp. are the dominant algae in all samples analyzed, and Chlamydomonas is the second-most abundant genus in the red snow. For the bacterial community profile, species belonging to the subphylum Betaproteobacteria were frequently detected in both green and red snow, while members of the phylum Bacteroidetes were also prominent in red snow. Furthermore, multiple independently obtained strains of Chloromonas sp. from inoculates of red snow resulted in the growth of Betaproteobacteria with the alga and the presence of bacteria appears to support growth of the xenic algal cultures under laboratory conditions. The dominance of Betaproteobacteria in algae-containing snow in combination with the detection of Chloromonas sp. with Betaproteobacteria strains suggest that these bacteria can utilize the available carbon source in algae-rich environments and may in turn promote algal growth.
  • Miho Watanabe, Hisaya Kojima, Manabu Fukui
    INTERNATIONAL JOURNAL OF SYSTEMATIC AND EVOLUTIONARY MICROBIOLOGY 67 8 2679 - 2682 2017年08月 [査読有り][通常論文]
     
    A mesophilic, endospore-forming, sulfate-reducing bacterium, designated strain NAW-5(T), was isolated from marsh soil. Cells of strain NAW-5(T) were Gram-stain-negative, curved rods that were motile. Strain NAW-5(T) grew at 18-48 degrees C (optimum 3237 degrees C) and pH 5.8-8.4 (optimum pH 6.2-7.3). Electron donors utilized were various organic acids and H-2 which support autotrophic growth. Fermentative growth occurred on carboxylic acids, but not on sugar. Sulfate, thiosulfate and elemental sulfur were used as electron acceptors. The respiratory isoprenoid quinone was MK-7. The genomic DNA G+C content of this strain was 46.6 mol%. Sequence analysis of the 16S rRNA gene showed that strain NAW-5(T) was affiliated to the family 'Desulfotomacul aceae' but the strain shared very low sequence similarity with any representatives of this family (>= 89 %). Strain NAW-5(T) belongs to Desulfotomaculum subcluster Ig which does not include any species with validly published names. On the basis of significant differences in the phylogenetic and phenotypic properties between strain NAW-5(T) and related species, strain NAW-5(T) represents a novel species of a new genus for which the name Desulfocucumis palustris gen. nov., sp. nov. is proposed. The type strain of the type species is NAW-5(T) (= DSM 102911(T) = NBRC 112242(T)).
  • Miho Watanabe, Hisaya Kojima, Manabu Fukui
    STANDARDS IN GENOMIC SCIENCES 12 34  2017年06月 [査読有り][通常論文]
     
    Desulfoplanes formicivorans strain Pf12B(T) is the type strain of the type species in the genus Desulfoplanes, which is the one of the genera in the family Desulfomicrobiaceae within the order Desulfovibrionales. This deltaproteobacterium was isolated from a blackish meromictic lake sediment. D. formicivorans strain Pf12B(T) is a Gram-negative, motile and sulfate-reducing bacterium. Cells of strain Pf12B(T) are characterized by possession of vibroid morphology and red fluorescent pigment. Here we describe the features, draft genome sequence and annotation of this organism, the sole species of the genus Desulfoplanes. The genome comprised 3,000,979 bp, 2,657 protein-coding genes and 58 RNA genes.
  • Hisaya Kojima, Miho Watanabe, Manabu Fukui
    INTERNATIONAL JOURNAL OF SYSTEMATIC AND EVOLUTIONARY MICROBIOLOGY 67 5 1355 - 1358 2017年05月 [査読有り][通常論文]
     
    A novel sulfur-oxidizing autotrophic bacterium, strain J1A(T) was isolated from a hot spring microbial mat. The cells were Gram-stain-negative, catalase-negative and oxidase-positive. As sole electron donor for chemolithoautotrophic growth, strain J1A(T) utilized sulfide, thiosulfate, elemental sulfur, and tetrathionate. The G+C content of the genomic DNA was 66 mol%. Major cellular fatty acids (>40% of total) were C-16:0 and summed feature 3 (C-16:1 omega 7c and/or C-16:1 omega 6c). The predominant quinone was Q-8. Phylogenetic analysis of the 16S rRNA gene indicated that strain J1A(T) is a relative of species of the genus Thiobacillus, but shares only 93% or lower sequence similarities with them. On the basis of its properties, strain J1A(T) represents a novel species of a new genus, for which the name Sulfuritortus calidifontis gen. nov., sp. nov. is proposed. The type strain of the type species is J1A(T) (=DSM 103923(T)=NBRC 112474(T)).
  • Tomohiro Watanabe, Hisaya Kojima, Manabu Fukui
    SCIENTIFIC REPORTS 6 2016年11月 [査読有り][通常論文]
     
    Adenylylsulfate reductase is a heterodimeric complex of two subunits, AprB and AprA, and is a key enzyme in dissimilatory sulfate reduction and sulfur oxidation. Common use of aprA as a functional marker gene has revealed the diversity of sulfur-cycle prokaryotes in diverse environments. In this study, we established a comprehensive sequence set of apr genes and employed it to reanalyze apr phylogeny, evaluate the coverage of a widely used primer set (AprA-1-FW/AprA-5-RV), and categorize environmental aprA sequences. Phylogenetic tree construction revealed new members of Apr lineage II and several previously unrecognized lateral gene transfer events. Using the established phylogenetic tree, we classified all previously reported aprA sequences amplified from freshwater lakes with the primer pair AprA-1-FW/AprA-5-RV in addition to the aprA sequences newly retrieved from freshwater lakes; the obtained results were complemented by 16S rRNA clone library analysis. Apr-based classifications of some of operational taxonomic units were supported by 16S rRNA-based analysis. This study updates our knowledge on the phylogeny of aprBA and shows the identities of several sulfur-cycle bacteria, which could not be classified to a known taxa until now. The established apr sequence set is publicly available and can be applied to assign environmental sequences to known lineages.
  • Hisaya Kojima, Miho Watanabe, Riho Tokizawa, Arisa Shinohara, Manabu Fukui
    INTERNATIONAL JOURNAL OF SYSTEMATIC AND EVOLUTIONARY MICROBIOLOGY 66 4821 - 4825 2016年11月 [査読有り][通常論文]
     
    A novel aerobic bacterial strain, P3(T), was isolated from a red snow obtained from Antarctica. Cells of strain P3(T) were rod-shaped, non-motile, catalase-negative, oxidase-positive and Gramstain-negative. Growth was observed at temperatures ranging from 0 to 25 degrees C, with optimum growth at 15 degrees C. The pH range for growth was pH 5.3-7.8. The G+C content of the genomic DNA was 55.0 mol%. The major components in the fatty acid profile were iso-C-15: 0, summed feature 4 (iso-C-17:1 I and/or anteiso-C-17:1 B), anteiso-C-15:0 and summed feature 3 (isoC(16:1)omega 7c and/or iso-C(16:1 omega)6c). The predominant isoprenoid quinone was MK-7. Phylogenetic analysis based on the 16S rRNA gene sequence indicated that the novel isolate was a member of the genus Hymenobacter, and the strain showed highest sequence similarity (94 %) with Hymenpbacter glaciei VUG-A130(T), Hymenobacter soli PB17(T) and Hymenobacter antarcticus VUG-A42aa(T). On the basis of phylogenetic and phenotypic properties strain P3(T) represents a novel species of the genus Hymenobacter (for which the name Hymenobacter nivis sp. nov. is proposed. The types strain is P3(T) (= DSM 101755(T) = NBRC 111535(T)).
  • Hisaya Kojima, Manabu Fukui
    INTERNATIONAL JOURNAL OF SYSTEMATIC AND EVOLUTIONARY MICROBIOLOGY 66 3515 - 3518 2016年09月 [査読有り][通常論文]
     
    A chemolithotrophic sulfur-oxidizing bacterium, strain aks1(T), was isolated from sediment of a brackish lake in Japan. The cells were curved rod-shaped and Gram-stain-negative. The G+C content of the genomic DNA was 53 mol%. The major components in the cellular fatty acid profile were C-16:0 and summed feature 3 (C-16 (: 1)omega 7c and/or C-16 (: 1)omega 6c). As electron donor for chemolithoautotrophic growth, strain aks1(T) oxidized thiosulfate, sulfide, and elemental sulfur. The strain could utilize oxygen and nitrate as an electron acceptor for thiosulfate oxidation. Growth was observed at a temperature range of 5-34 degrees C, with optimum growth at 30-32 degrees C. Growth of the strain was observed at a pH range of 6.4-8.7. Phylogenetic analysis based on 16S rRNA gene sequences indicated that the strain is related to members of the familyGranulosicoccaceae within the order Chromatiales, with sequence similarities around 92%. On the basis of phylogenetic and phenotypic properties, strain aks1(T) represents a novel species of a new genus, for which the name Sulfuriflexus mobilis gen. nov., sp. nov. is proposed. The type strain of the type species is aks1(T) (=DSM 102939(T) =NBRC 111889(T)).
  • Kazuhiro Umezawa, Tomohiro Watanabe, Aya Miura, Hisaya Kojima, Manabu Fukui
    STANDARDS IN GENOMIC SCIENCES 11 1 2016年09月 [査読有り][通常論文]
     
    Sulfurifustis variabilis and Sulfuricaulis limicola are autotrophic sulfur-oxidizing bacteria belonging to the family Acidiferrobacteraceae in the order Acidiferrobacterales. The type strains of these species, strain skN76(T) and strain HA5(T), were isolated from lakes in Japan. Here we describe the complete genome sequences of Sulfurifustis variabilis skN76T and Sulfuricaulis limicola HA5T. The genome of Sulfurifustis variabilis skN76T consists of one circular chromosome with size of 4.0 Mbp including 3864 protein-coding sequences. The genome of Sulfuricaulis limicola HA5T is 2.9 Mbp chromosome with 2763 protein-coding sequences. In both genomes, 46 transfer RNA-coding genes and one ribosomal RNA operon were identified. In the genomes, redundancies of the genes involved in sulfur oxidation and inorganic carbon fixation pathways were observed. This is the first report to show the complete genome sequences of bacteria belonging to the order Acidiferrobacterales in the class Gammaproteobacteria.
  • Tomohiro Watanabe, Hisaya Kojima, Manabu Fukui
    INTERNATIONAL JOURNAL OF SYSTEMATIC AND EVOLUTIONARY MICROBIOLOGY 66 2041 - 2045 2016年05月 [査読有り][通常論文]
     
    A novel sulfur-oxidizing bacterium designated strain mst6(T) was isolated from spring water of Masutomi hot spring in Japan. The cells were rod-shaped (1.2-4.0 x 0.5-0.7 mm) and Gram-stain-negative. The G+C content of genomic DNA was around 52.6 mol%. The isolate possessed summed feature 3 (C-16 : 1 omega 7c and/or C-16 : 1 omega 6c), C-16 : 0 and C-12 : 0 as major cellular fatty acids. Strain mst6(T) grew by inorganic carbon fixation and oxidation of inorganic sulfur compounds with oxygen as an electron acceptor. The isolate grew over a temperature range of 5-34 degrees C, a NaCl concentration range of 0-110 mM and a pH range of 4.6-8.1. Optimum growth occurred at 32 degrees C, in the absence of NaCl and at pH 5.9-6.2. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain mst6(T) belongs to the family Sulfuricellaceae in the class Betaproteobacteria. The closest cultured relative was Sulfuriferula multivorans TTNT with a 16S rRNA gene sequence similarity of 97.0 %. On the basis of the data obtained in this study, strain mst6(T) represents a novel species of the genus Sulfuriferula, for which the name Sulfuriferula thiophila sp. nov. is proposed. The type strain is mst6(T) (=NBRC 111150(T) =DSM 101871(T)). In addition, we propose correcting the name Sulfuriferula plumbophilus Watanabe, Kojima and Fukui 2015 to Sulfuriferula plumbiphila corrig. based on Rule 12c, Rule 61 and Appendix 9 of the International Code of Nomenclature of Prokaryotes.
  • Tomohiro Watanabe, Aya Miura, Arisa Shinohara, Hisaya Kojima, Manabu Fukui
    INTERNATIONAL JOURNAL OF SYSTEMATIC AND EVOLUTIONARY MICROBIOLOGY 66 5 1986 - 1989 2016年05月 [査読有り][通常論文]
     
    A novel sulfur oxidizer, strain wk12(T), was isolated from an industrially synthesized lead (II) sulfide. The G+C content of the genomic DNA was around 58.5 mol%. The major components in the cellular fatty acid profile were summed feature 3 (C-16 : 1 omega 7c and/or C-16 : 1 omega 6c), C-16 : 0 and summed feature 8 (C-18 : 1 omega 7c and/or C-18 : 1 omega 6c). The strain oxidized lead sulfide, thiosulfate and tetrathionate as electron donors to support autotrophic growth. Cells of strain wk12(T) were motile, rod-shaped (0.5-1.060.7-2.2 mm), and Gram-stain-negative. For growth, the temperature range was 5-37 degrees C, and optimum growth was observed at 28-32 degrees C. The pH range for growth was 5.8-8.7, with optimum growth at pH 6.4-7.1. Optimum growth of the isolate was observed in medium without NaCl, and no growth was observed in the medium containing 0.5 M or more NaCl. Phylogenetic analysis based on 16S rRNA gene sequences indicated that the isolate belongs to the class Acidithiobacillia. The closest relative with a validly published name was Thermithiobacillus tepidarius DSM 3134(T), with a 16S rRNA gene sequence similarity of 96 %. On the basis of phylogenetic and phenotypic properties, strain wk12(T) represents a novel species of the genus Thermithiobacillus, for which the name Thermithiobacillus plumbiphilus sp. nov. is proposed. The type strain is wk12(T) (=NBRC 111508(T) =DSM 101799(T)).
  • Roles of Terrestrial Carbon Subsidies to Aquatic Community Metabolism in Mountain Lake Ecosystems
    Iwata,T, N. Mochizuki, T. Suzuki, Ay. Kohzu, H. Kojima, M. Fukui, J. Urabe
    In G. Kudo (ed), Structure and Function of Mountain Ecosystems in Japan: Biodiversity and Vulnerability to Climate Change 115 - 144 2016年04月01日 [査読有り][通常論文]
  • Hisaya Kojima, Kazuhiro Umezawa, Manabu Fukui
    INTERNATIONAL JOURNAL OF SYSTEMATIC AND EVOLUTIONARY MICROBIOLOGY 66 1828 - 1831 2016年04月 [査読有り][通常論文]
     
    A novel autotrophic, thermophilic bacterium, strain TF1(T), was isolated from a hot spring in Japan. Cells of strain TF1(T) were motile, Gram-stain-negative, rod-shaped, 1.0-2.0 mu m in length and 0.5-0.6 mu m in width. Major components in the cellular fatty acid profile were C-16 : 0, C-18 : 0 and anteiso-C-17 : 0. The temperature range for growth was 40-77 degrees C, and optimum temperature was 75 degrees C. The pH range for growth was 5.9-9.5, and the optimum pH was 7.5-8.8. Strain TF1(T) grew chemolithoautotrophically by disproportionation of sulfur, thiosulfate and sulfite. Phylogenetic analysis based on 16S rRNA gene sequences indicated that the strain belongs to the family Thermodesulfobacteriaceae. The closest cultivated relative was Caldimicrobium rimae DST, with highest 16S rRNA gene sequence similarity of 96 %. The genome of strain TF1(T) consists of one circular chromosome, with a size of 1.8 Mbp and G+ C content of 38.30 mol%. On the basis of its phylogenetic and phenotypic properties, strain TF1(T) (=DSM 29380(T) =NBRC 110713(T)) is proposed as the type strain of a novel species, Caldimicrobium thiodismutans sp. nov.
  • Miho Watanabe, Hisaya Kojima, Manabu Fukui
    INTERNATIONAL JOURNAL OF SYSTEMATIC AND EVOLUTIONARY MICROBIOLOGY 66 1330 - 1339 2016年03月 [査読有り][通常論文]
     
    Limnochorda pilosa is a pleomorphic facultative anaerobe and the sole species in the class Limnochordia, which has tentatively been placed in the phylum Firmicutes. In the present study, the complete genome sequence of L. pilosa HC45(T) was obtained and analysed. The genome size was 3.82 Mbp and the DNA G + C content was 69.73 %. Phylogenetic analyses based on the 30S-50S ribosomal proteins and 23S rRNA gene consistently indicated that L. pilosa is phylogenetically isolated from the other members of the phylum Firmicutes. Ultrastructural observation revealed that L. pilosa possesses a Gram-negative-type cell wall and the capacity to form endospores. Accordingly, the L. pilosa genome has characteristics that are specific to Gram-negative bacteria and contains many genes that are involved in sporulation. On the other hand, several sporulation genes were absent from the L. pilosa genome although they have been regarded as essential for the endospore-forming system of members of the phylum Firmicutes. The gyrB gene of L. pilosa possesses an intein sequence. The genome has a high percentage of GTG start codons and lacks several conserved genes related to cell division.
  • Fujii M, Hirao T, Kojima H, Fukui M
    Structure and Function of Mountain Ecosystems in Japan: Biodiversity and Vulnerability to Climate Change 145 - 169 2016年 [査読有り][通常論文]
  • Hisaya Kojima, Tomohiro Watanabe, Manabu Fukui
    INTERNATIONAL JOURNAL OF SYSTEMATIC AND EVOLUTIONARY MICROBIOLOGY 66 266 - 270 2016年01月 [査読有り][通常論文]
     
    A novel sulfur-oxidizing bacterium, strain HA5(T), was isolated from sediment of a lake in Japan. The cells were rod-shaped (0.3-0.5 x 1.2-6.0 gm) and Gram-stain-negative. The G + C content of the genomic DNA was 63 mol%. The major components in the cellular fatty acid profile were C-16 : 0 and summed feature 3 (C-16 (:) (1)omega 7c and/or C-16 : 1 omega 6c). The strain oxidized thiosulfate, tetrathionate and elemental sulfur as electron donors to support autotrophic growth. Growth was observed at a temperature range of 8-37 degrees C, with optimum growth at 28-32 degrees C. The pH range for growth was pH 6.1-9.2.Optimunn growth of the isolate was observed in medium without NaCI. Phylogenetic analysis based on 16S rRNA gene sequences indicated that the strain belongs to the family Acidiferrobacteraceae in the class Gammaproteobacteria. The closest relative was Sulfurifustis variabilis skN76(T) with the highest 16S rRNA gene sequence similarity of 93%. On the basis of phylogenetic and phenotypic properties, strain HA5(T) is proposed to represent a novel species of a new genus, Sulfuricaulis limicola gen. nov., sp. nov. The type strain of the type species is HA5(T) (=DSM 100373(T)=NBRC 110752(T)).
  • Tomohiro Watanabe, Hisaya Kojima, Arisa Shinohara, Manabu Fukui
    INTERNATIONAL JOURNAL OF SYSTEMATIC AND EVOLUTIONARY MICROBIOLOGY 66 113 - 117 2016年01月 [査読有り][通常論文]
     
    A novel sulfur-oxidizing bacterium, designated strain BiS0(T), was isolated from a sediment sample collected from a freshwater lake in Japan. The cells were rod-shaped, 1.4-4.6 x 0.4-0.7 mu m and Gram-stain-negative. The G-1-C content of the genomic DNA was around 44 mol%. The isolate possessed summed feature 3 (C-16:1 omega 7c and/or C-16:1 omega 6c), C-16:0 and C-10:0 3-OH as major cellular fatty acids. Strain BiS0(T) grew by carbon dioxide fixation and oxidation of inorganic sulfur compounds with oxygen as the electron acceptor. Growth was observed over a temperature range of 0-32 degrees C (optimum, 15-22 degrees C), an NaCI concentration range of 0-546.4 mM (optimum 0-66.7 mM) and a pH range of 5.28.1 (optimum 6.1-6.3). Phylogenetic analysis, based on 16S rRNA gene sequences, indicated that strain BiS0(T) belongs to the family Sulfuricellaceae in the class Betaproteobacteria. The closest cultured relatives were Sulfuricella denitrificans skB26(T) and Sulfuricella sp. T08, with 16S rRNA gene sequence similarities of 96.3 %. On the basis of the data obtained in this study, strain BiS0(T) represents a novel species of a novel genus, for which the name Sulfurirhabdus autotrophica gen. nov., sp. nov. is proposed. The type strain is BiS0(T) (=NBRC 110941(T)=DSM 100309(T)).
  • Yuki Kobayashi, Hisaya Kojima, Masayuki Itoh, Noboru Okuda, Manabu Fukui, Fuh-Kwo Shiah
    Plankton and Benthos Research 11 4 144 - 146 2016年 [査読有り][通常論文]
     
    Methane-oxidizing bacteria (MOB) are regarded as key players in aquatic ecosystems, which can mitigate greenhouse gas emissions to the atmosphere. Among phylogenetically diverse MOB, those of the phylum Proteobacteria have been regarded as major methane oxidizers in environments, and they are classified into two major groups, type I and type II. Another group of MOB, ‘Candidatus Methylomirabilis oxyfera’ and its close relatives have also been detected in various environments, but their predominance over proteobacterial MOB has hardly ever been reported. Feitsui Reservoir (FTR) is a subtropical reservoir situated in Taiwan, where the predominance of ‘M. oxyfera’-like phylotypes in a planktonic MOB community was first reported. In this study, planktonic MOB of three types (‘M. oxyfera’-like, type I and type II) were quantified with catalyzed reporter deposition-fluorescence in situ hybridization, to reveal seasonal variation and vertical distribution in the FTR. The MOB were enumerated for 161 samples obtained from 8 water depths on 23 sampling days over 16 months. The results obtained in this study will provide valuable basic data for a better understanding of MOB communities in the environment, giving insights into the global methane budget.
  • Hisaya Kojima, Arisa Shinohara, Manabu Fukui
    INTERNATIONAL JOURNAL OF SYSTEMATIC AND EVOLUTIONARY MICROBIOLOGY 65 3709 - 3713 2015年10月 [査読有り][通常論文]
     
    A novel autotrophic bacterium, strain skN76(T), was isolated from sediment of a lake in Japan. As sole electron donor to support chemolithoautotrophic growth, the strain oxidized thiosulfate, tetrathionate and elemental sulfur. For growth, the optimum temperature was 42-45 degrees C and the optimum pH was 6.8-8.2. The cells were Gram-stain-negative, catalase-positive and oxidase-positive. The strain exhibited changes in morphology depending on growth temperature. Cells grown at the optimum temperature were rod-shaped (0.9-3.0 mu m long and 0.3-0.5 mu m wide), whereas a filamentous form was observed when the strain was cultured at the lowest permissive growth temperatures. The G+C content of genomic DNA was 69 mol%. The major components in the fatty acid profile were C-16 : 0, summed feature 3 (C-16 : 1 omega 7c and/or C-16 : 1 omega 6c) and summed feature 9 (iso-C-17 : 1 omega 9c and/or 10-methyl C-16 : 0). Phylogenetic analysis based on 16S rRNA gene sequences indicated that the closest cultivated relative of strain skN76(T) was Acidiferrobacter thiooxydans m-1(T), with sequence similarity of 93 %. On the basis of its phylogenetic and phenotypic properties, strain skN76(T) (=DSM 100313(T)= NBRC 110942(T)) is proposed as the type strain of a novel species of a novel genus, Sulfurifustis variabilis gen. nov., sp. nov. Novel taxa, Acidiferrobacteraceae fam. nov. and Acidiferrobacterales ord. nov., are also proposed to accommodate the genera Acidiferrobacter and Sulfurifustis gen. nov.
  • Tomohiro Watanabe, Hisaya Kojima, Manabu Fukui
    MICROBIOLOGY RESOURCE ANNOUNCEMENTS 3 5 2015年09月 [査読有り][通常論文]
     
    Strain skMP5(T) is a moderately thermophilic and facultatively anaerobic bacterium, described as a representative of Mizugakiibacter sediminis. Here, we report the annotated draft genome sequence of strain skMP5(T).
  • Miho Watanabe, Hisaya Kojima, Manabu Fukui
    INTERNATIONAL JOURNAL OF SYSTEMATIC AND EVOLUTIONARY MICROBIOLOGY 65 2378 - 2384 2015年08月 [査読有り][通常論文]
     
    A novel facultatively anaerobic bacterium, strain HC45(T), was isolated from sediment of a brackish meromictic lake in Japan, Lake Harutori. Cells were pleomorphic, and filamentous bodies were 5-100 mu m in length. For growth, the optimum pH was 7.0 and the optimum temperature was 45-50 degrees C. The G+C content of the genomic DNA was 71 mol%. iso-C-15 (:) (0) and anteiso-C-15 : (0) were the major components in the cellular fatty acid profile. The predominant respiratory quinone was MK-7. Strain HC45(T) shared very low 16S rRNA gene sequence similarity with cultivated strains (<= 85 %). Phylogenetic analysis based on 16S rRNA gene sequences revealed that the isolate was distantly related to members of the family Symbiobacteriaceae and family XVII Incertae Sedis in the class Clostridia, and they formed a cluster separate from canonical species of the phylum Firmicutes. These results indicated that strain HC45(T) should not be placed in any existing class of the phylum Firmicutes. On the basis of phylogenetic and phenotypic characterization, Limnochorda pilosa gen. nov., sp. nov. is proposed with HC45(T) (=NBRC 110152(T)=DSM 28787T) as the type strain, as the first representative of novel taxa, Limnochordales ord. nov., Limnochordaceae fam. nov. in Limnochordia classis. nov.
  • Miho Watanabe, Hisaya Kojima, Manabu Fukui
    INTERNATIONAL JOURNAL OF SYSTEMATIC AND EVOLUTIONARY MICROBIOLOGY 65 1902 - 1907 2015年06月 [査読有り][通常論文]
     
    A novel sulfate-reducing bacterium, designated strain Pf12B(T), was isolated from sediment of meromictic Lake Harutori in Japan. Cells were vibroid (1.0 x 3.0-4.0 mu m), motile and Gram-stain-negative. For growth, the optimum pH was 7.0-7.5 and the optimum temperature was 42-45 degrees C. Strain Pf12B(T) used sulfate, thiosulfate and sulfite as electron acceptors. The G+C content of the genomic DNA was 55.4 mol%. Major cellular fatty acids were C-16:0 and C-18:0 The strain was desulfoviridin-positive. Phylogenetic analysis based on the 16S rRNA gene revealed that the novel strain belonged to the order Desulfovibrionales in the class Deltaproteobacteria. The closest relative was Desulfomicrobium baculatum DSM 4028(T) with which it shared 91 % 16S rRNA gene sequence similarity. On the basis of phylogenetic and phenotypic characterization, a novel species of a new genus belonging to the family Desulfomicrobiaceae is proposed, Desulfoplanes formicivorans gen. nov., sp. nov. The type strain of Desulfoplanes formicivorans is Pf12B(T) (=NBRC 110391(T)=DSM 28890(T)).
  • Tomohiro Watanabe, Hisaya Kojima, Manabu Fukui
    MICROBIOLOGY RESOURCE ANNOUNCEMENTS 3 3 2015年05月 [査読有り][通常論文]
     
    Sulfuricella sp. strain T08 is a sulfur-oxidizing autotroph newly isolated from a freshwater lake in Japan. Strain T08 is the second isolate of the genus Sulfuricella. Here, we report the annotated draft genome sequence of the isolate.
  • Hisaya Kojima, Yoshitoshi Ogura, Nozomi Yamamoto, Tomoaki Togashi, Hiroshi Mori, Tomohiro Watanabe, Fumiko Nemoto, Ken Kurokawa, Tetsuya Hayashi, Manabu Fukui
    ISME JOURNAL 9 5 1166 - 1176 2015年05月 [査読有り][通常論文]
     
    Large sulfur-oxidizing bacteria, which accumulate a high concentration of nitrate, are important constituents of aquatic sediment ecosystems. No representative of this group has been isolated in pure culture, and only fragmented draft genome sequences are available for these microorganisms. In this study, we successfully reconstituted the genome of Thioploca ingrica from metagenomic sequences, thereby generating the first complete genome sequence from this group. The Thioploca samples for the metagenomic analysis were obtained from a freshwater lake in Japan. A PCR-free paired-end library was constructed from the DNA extracted from the samples and was sequenced on the Illumina MiSeq platform. By closing gaps within and between the scaffolds, we obtained a circular chromosome and a plasmid-like element. The reconstituted chromosome was 4.8Mbp in length with a 41.2% GC content. A sulfur oxidation pathway identical to that suggested for the closest relatives of Thioploca was deduced from the reconstituted genome. A full set of genes required for respiratory nitrate reduction to dinitrogen gas was also identified. We further performed a proteomic analysis of the Thioploca sample and detected many enzymes/proteins involved in sulfur oxidation, nitrate respiration and inorganic carbon fixation as major components of the protein extracts from the sample, suggesting that these metabolic activities are strongly associated with the physiology of Thioploca ingrica in lake sediment.
  • Tomohiro Watanabe, Hisaya Kojima, Manabu Fukui
    INTERNATIONAL JOURNAL OF SYSTEMATIC AND EVOLUTIONARY MICROBIOLOGY 65 1504 - 1508 2015年05月 [査読有り][通常論文]
     
    A sulfur-oxidizing bacterium, strain TINT, was isolated from a Thioploca sample obtained from a freshwater lake in Japan. The isolate shared 97.1% 16S rRNA gene sequence similarity with an obligately aerobic chemolithoautotroph, 'Thiobacillus plumbophilus' Gro7(T). Cells were rods, motile, and Gram-stain-negative. The G+C content of the genomic DNA was approximately 66 mol%. Strain TTNT grew over a temperature range of 8-32 degrees C (optimum 22-25 degrees C), an NaCl concentration range of 0-133.3 mM (optimum 0-3.3 mM) and a pH range of 5.3-8.6 (optimum pH 6.4-7.0). Strain TTNT was facultatively anaerobic and could utilize nitrate as an electron acceptor. The isolate oxidized tetrathionate, thiosulfate and elemental sulfur as the sole energy sources for autotrophic growth, and could also grow heterotrophically on a number of organic substrates. Based on its phylogenetic and phenotypic properties, strain TTNT represents a novel species of a novel genus, for which the name Sulfuriferula multivorans gen. nov., sp. nov. is proposed. The type strain is TTNT (=NBRC 110683(T)=DSM 29343(T)). Along with this, the reclassification of 'Thiobacillus plumbophilus' as Sulfuriferula plumbophilus sp. nov. (type strain Gro7(T)=NBRC 107929(T)=DSM 6690(T)) is proposed. Based on the data obtained in this study, we describe the designations Sulfuricellaceae fam. nov. and Sulfuricellales ord. nov.
  • Yoshinori Takano, Hisaya Kojima, Eriko Takeda, Yusuke Yokoyama, Manabu Fukui
    PROGRESS IN EARTH AND PLANETARY SCIENCE 2 2015年04月 [査読有り][通常論文]
     
    We report a 6,000 years record of subglacial weathering and biogeochemical processes in two perennially ice-covered glacial lakes at Rundvagshetta, on the Soya Coast of Lutzow-Holm Bay, East Antarctica. The two lakes, Lake Maruwan Oike and Lake Maruwan-minami, are located in a channel that drains subglacial water from the base of the East Antarctic ice sheet. Greenish-grayish organic-rich laminations in sediment cores from the lakes indicate continuous primary production affected by the inflow of subglacial meltwater containing relict carbon, nitrogen, sulfur, and other essential nutrients. Biogenic silica, amorphous hydrated silica, and DNA-based molecular signatures of sedimentary facies indicate that diatom assemblages are the dominant primary producers, supported by the input of inorganic silicon (Si) from the subglacial inflow. This study highlights the significance of subglacial water-rock interactions during physical and chemical weathering processes and the importance of such interactions for the supply of bioavailable nutrients.
  • Yuriko Higashioka, Hisaya Kojima, Tomohiro Watanabe, Manabu Fukui
    Genome Announcements 3 6 2015年 [査読有り][通常論文]
     
    Desulfatitalea tepidiphila S28bFT is a sulfate-reducing bacterium closely related to Desulfosarcina species. Here, the draft genome sequence of strain S28bFT is reported.
  • Masanori Fujii, Toshihide Hirao, Hisaya Kojima, Manabu Fukui
    AQUATIC MICROBIAL ECOLOGY 75 3 251 - 257 2015年 [査読有り][通常論文]
     
    We examined bacterioplankton community composition (BCC) in 27 oligotrophic freshwater lakes with similar environments, located in the mountainous regions of eastern Japan. We determined BCC by the relative sequence abundance of each taxon using polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) targeting the 16S rRNA gene region. Using multiple regression analyses, we investigated the relative importance of environmental and geospatial processes on community assembly of the general bacterioplankton, as well as of the taxonomic groups Betaproteobacteria and Bacteroidetes. Both processes had significant relationships with community assembly. In particular, environmental processes were relevant, however small the variations among study sites. Community composition of Betaproteobacteria was mainly correlated with environmental factors, whereas that of Bacteroidetes was primarily associated with geospatial variables, such as the presence of Tsugaru Strait as a geographic barrier. Thus, inter-taxonomic differences are significant in assessing relationships of environmental and geospatial processes with freshwater BCC. Finally, our study provides evidence that biogeographic events are potentially significant for the distribution and diversity of microorganisms, as is the case with macroorganisms. These perspectives should be taken into consideration in order to fully understand bacterial community assembly.
  • Itoh, Masayuki, Kobayashi, Yuki, Chen, Tzong-Yueh, Tokida, Takeshi, Fukui, Manabu, Kojima, Hisaya, Miki, Takeshi, Tayasu, Ichiro, Shiah, Fuh-Kwo, Okuda, Noboru
    Journal of Geophysical Research-Biogeosciences 120 7 1246 - 1261 2015年 [査読有り][通常論文]
  • Hisaya Kojima, Riho Tokizawa, Manabu Fukui
    INTERNATIONAL JOURNAL OF SYSTEMATIC AND EVOLUTIONARY MICROBIOLOGY 64 3983 - 3987 2014年12月 [査読有り][通常論文]
     
    A novel, moderately thermophilic, bacterial strain (skMP5(T)) was isolated from sediment of a freshwater lake in Japan. The cells were rod-shaped, motile and Gram-stain-negative. Growth was observed at temperatures ranging from 25 to 52 degrees C, with optimum growth observed at 48-50 degrees C. The pH range for growth was pH 5.0-8.2, with optimum growth at pH 6.0-7.0. The G+C content of genomic DNA was 72 mol%. The major components in the fatty acid profile were iso-C-17:0 and iso-C-17:1 omega 9c. The predominant isoprenoid quinone of the strain was ubiquinone Q-8. The strain was facultatively anaerobic, and reduced nitrate to nitrite under anoxic conditions. Phylogenetic analysis based on 16S rRNA gene sequences indicated that the isolate was a member of the family Xanthomonadaceae within the class Gammaproteobacteria, showing highest sequence similarity with Tahibacter aquaticus RaM5-2 (93.6%) and Metallibacterium scheffleri DKE6(T) (93.3%). On the basis of phylogenetic and phenotypic properties, strain 5kMP5(T) represents a novel species of a new genus, Mizugakiibacter sediminis gen. nov., sp. nov. The type strain of the type species is skMP5(T) (=DSM 27098(T)=NBRC 109608(T)).
  • Hisaya Kojima, Johanna Moll, Joerg Kahnt, Manabu Fukui, Seigo Shima
    ENVIRONMENTAL MICROBIOLOGY 16 11 3431 - 3442 2014年11月 [査読有り][通常論文]
     
    Consortia of anaerobic methanotrophic (ANME) archaea and delta-proteobacteria anaerobically oxidize methane coupled to sulfate reduction to sulfide. The metagenome of ANME-1 archaea contains genes homologous to genes otherwise only found in methanogenic archaea, and transcription of some of these genes in ANME-1 cells has been shown. We now have heterologously expressed three of these genes in Escherichia coli, namely those homologous to genes for formylmethanofuran:tetrahydromethanopterin formyltransferase, methenyltetrahydromethanopterin cyclohydrolase (Mch) and coenzyme F-420-dependent methylenetetrahydromethanopterin dehydrogenase (Mtd), and have characterized the overproduced enzymes with respect to their coenzyme specificity and other catalytic properties. The three enzymes from ANME-1 were found to catalyse the same reactions and with similar specific activities using identical coenzymes as the respective enzymes in methanogenic archaea, the apparent K-m for their substrates being in the same concentration range. The results support the proposal that anaerobic oxidation of methane to CO2 in ANME involves the same enzymes and coenzymes as CO2 reduction to methane in methanogenic archaea. Interestingly, the activity of Mch and the stability of Mtd from ANME-1 were found to be dependent on the presence of 0.5-1.0M potassium phosphate, which suggested that ANME-1 archaea contain high concentrations of lyotropic salts, presumably as compatible solutes.
  • Kyoko Kubo, Hisaya Kojima, Manabu Fukui
    SYSTEMATIC AND APPLIED MICROBIOLOGY 37 7 510 - 519 2014年10月 [査読有り][通常論文]
     
    The vertical distribution of sulfate-reducing bacteria was investigated in a shallow, eutrophic, meromictic lake, Lake Harutori, located in a residential area of Kushiro, Japan. A steep chemocline, characterized by gradients of oxygen, sulfide and salinity, was found at a depth of 3.5-4.0 m. The sulfide concentration at the bottom of the lake was high (up to a concentration of 10.7 mM). Clone libraries were constructed using the aprA gene, which encodes adenosine-5'-phosphosulfate reductase subunit A, in order to monitor sulfate-reducing bacteria. In the aprA clone libraries, the most abundant sequences were those from the Desulfosarcina-Desulfococcus (DSS) group. A primer set for a DSS group-specific 16S rRNA gene was used to construct another clone library, analysis of which revealed that the uncultured group of sulfate-reducing bacteria, SEEP SRB-1, accounted for nearly half of the obtained sequences. Quantification of the major bacterial groups by catalyzed reporter deposition-fluorescence in situ hybridization demonstrated that the DSS group accounted for 3.2-4.8% of the total bacterial community below the chemocline. The results suggested that the DSS group was one of the major groups of sulfate-reducing bacteria and that these presumably metabolically versatile bacteria might play an important role in sulfur cycling in Lake Harutori. (C) 2014 Elsevier GmbH. All rights reserved.
  • Tomohiro Watanabe, Hisaya Kojima, Manabu Fukui
    SYSTEMATIC AND APPLIED MICROBIOLOGY 37 6 387 - 395 2014年09月 [査読有り][通常論文]
     
    Despite detailed studies of marine sulfur-oxidizing bacteria, our knowledge concerning their counterparts in freshwater lake ecosystems is limited. Genome sequencing of the freshwater sulfur-oxidizing betaproteobacteria Sulfuricella denitrificans skB26 and Sulfuritalea hydrogenivorans sk43H have been completed. Strain skB26 possessed a circular plasmid of 86.6-kbp in addition to its chromosome, and an approximate 18-kbp region of the plasmid was occupied by an arxA-like operon, encoding a new clade of anaerobic arsenite oxidase. Multilocus sequence analysis showed that strain skB26 could not be assigned to any existing order; thus a novel order, Sulfuricellales, is proposed. The genomes of strains skB26 and sk43H were examined, focusing on the composition and the phylogeny of genes involved in the oxidation of inorganic sulfur compounds. Strains skB26 and sk43H shared a common pathway, which consisted of Sqr, SoxEF, SoxXYZAB, Dsr proteins, AprBA, Sat, and SoeABC. Comparative genomics of betaproteobacterial sulfur oxidizers showed that this pathway was also shared by the freshwater sulfur oxidizers Thiobacillus denitrificans and Sideroxydans lithotrophicus. It also revealed the presence of a conserved gene cluster, which was located immediately upstream of the betaproteobacterial dsr operon. (C) 2014 Elsevier GmbH. All rights reserved.
  • Miho Watanabe, Hisaya Kojima, Manabu Fukui
    INTERNATIONAL JOURNAL OF SYSTEMATIC AND EVOLUTIONARY MICROBIOLOGY 64 2770 - 2774 2014年08月 [査読有り][通常論文]
     
    A novel thermophilic, facultatively anaerobic bacterium, strain skLN1(T), was isolated from the sediment of a freshwater lake in Japan. Cells of strain skLN1(T) were rod-shaped and Gram-stain-variable. A KOH lysis test suggested that the cell wall of the isolate has a Gram-positive structure. For aerobic growth, the optimum pH was pH 7.25-7.50 and the optimum temperature was 50 52 degrees C. The G+C content of the genomic DNA was 50.8 mol%. Nitrate was reduced to nitrite. Alicyclic fatty acids were not detected, and branched-chain fatty acids were major components in the cellular fatty acid profile. MK-7 was the predominant respiratory quinone. Phylogenetic analysis based on 16S rRNA gene sequences revealed that the isolated strain was related most closely to Alicyclobacillus consociatus CCUG 53762(T) (95 % similarity). This analysis also showed that the monophyly of the genus Alicyclobacillus had been lost. On the basis of phylogenetic and phenotypic characterization, Effusibacillus lacus gen. nov., sp. nov. is proposed. The type strain of Effusibacillus lacus is skLN1(T) (=NBRC 109614(T)=DSM 27172(T)). It is also proposed that Alicyclobacillus pohliae and Alicyclobacillus consociatus should be reclassified to the genus Effusibacillus as Effusibacillus pohliae comb. nov. and Effusibacillus consociatus comb. nov., respectively.
  • Hisaya Kojima, Riho Tokizawa, Kouhei Kogure, Yuki Kobayashi, Masayuki Itoh, Fuh-Kwo Shiah, Noboru Okuda, Manabu Fukui
    SCIENTIFIC REPORTS 4 2014年07月 [査読有り][通常論文]
     
    Methane-oxidizing bacteria (MOB) gain energy from the oxidation of methane and may play important roles in freshwater ecosystems. In this study, the community structure of planktonic MOB was investigated in a subtropical reservoir. Bacterial community structure was investigated through the analysis of the 16S rRNA gene. Three groups of phylogenetically distinct MOB were detected in the clone libraries of polymerase chain reaction products obtained with universal primers. The groups belonged to the class Gammaproteobacteria, the class Alphaproteobacteria, and the candidate phylum NC10. The last group, which consists of close relatives of the nitrite reducer 'Candidatus Methylomirabilis oxyfera', was frequently detected in the clone libraries of deep-water environments. The presence of 3 groups of MOB in deep water was also shown by a cloning analysis of the pmoA gene encoding particulate methane monooxygenase. The dominance of 'M. oxyfera'-like organisms in deep water was confirmed by catalyzed reporter depositionfluorescence in situ hybridization, in which cells stained with a specific probe accounted for 16% of total microbial cells. This is the first study to demonstrate that close relatives of the nitrite reducer can be major component of planktonic MOB community which may affect carbon flow in aquatic ecosystems.
  • Hisaya Kojima, Manabu Fukui
    INTERNATIONAL JOURNAL OF SYSTEMATIC AND EVOLUTIONARY MICROBIOLOGY 64 1587 - 1592 2014年05月 [査読有り][通常論文]
     
    A novel facultatively autotrophic bacterium, strain BSN1(T) was isolated from sediment of a freshwater lake in Japan. The cells were rod-shaped, motile and Gram-stain-negative. As sole energy sources for autotrophic growth, the strain oxidized thiosulfate, elemental sulfur and hydrogen. Strain BSN1T was a facultative anaerobe utilizing nitrate as an electron acceptor. Growth was observed at temperatures lower than 34 C, and the optimum growth was observed at 30-32 degrees C. The range of pH for growth was pH 6.8-8.8, and the optimum pH was pH 7.8-8.1. The optimum growth of the isolate occurred at concentrations of NaCl less than 50 mM. The G+C content of genomic DNA was 67 mol%. The major component in the fatty acid profile of strain BSN1(T) grown on fumarate was summed feature 3 (C-16:1 omega 7c and/or iso-C-15:0 2-OH). Phylogenetic analysis based on 165 rRNA gene sequences indicated that the strain was a member of the class Betaproteobacteria, and it showed the highest sequence similarity with Georgfuchsia toluolica G5G6(T) (96.2%). Phylogenetic analyses were also performed on genes involved in sulfur oxidation. On the basis of its phylogenetic and phenotypic properties, strain BSN1(T) (= DSM 26916(T)=NBRC 109412(T)) is proposed as the type strain of a novel species of a novel genus, Sulfurisoma sediminicola gen. nov., sp. nov.
  • Hisaya Kojima, Tomohiro Watanabe, Tomoya Iwata, Manabu Fukui
    PLOS ONE 9 4 2014年04月 [査読有り][通常論文]
     
    Planktonic sulfur oxidizers are important constituents of ecosystems in stratified water bodies, and contribute to sulfide detoxification. In contrast to marine environments, taxonomic identities of major planktonic sulfur oxidizers in freshwater lakes still remain largely unknown. Bacterioplankton community structure was analyzed in a stratified freshwater lake, Lake Mizugaki in Japan. In the clone libraries of 16S rRNA gene, clones very closely related to a sulfur oxidizer isolated from this lake, Sulfuritalea hydrogenivorans, were detected in deep anoxic water, and occupied up to 12.5% in each library of different water depth. Assemblages of planktonic sulfur oxidizers were specifically analyzed by constructing clone libraries of genes involved in sulfur oxidation, aprA, dsrA, soxB and sqr. In the libraries, clones related to betaproteobacteria were detected with high frequencies, including the close relatives of Sulfuritalea hydrogenivorans.
  • Miho Watanabe, Hisaya Kojima, Manabu Fukui
    INTERNATIONAL JOURNAL OF SYSTEMATIC AND EVOLUTIONARY MICROBIOLOGY 63 3574 - 3578 2013年10月 [査読有り][通常論文]
     
    A novel spore-forming, sulfate-reducing bacterium, strain SR45(T), was isolated from sediment of a freshwater lake, Lake Mizugaki, in Japan. Cells of strain SR45 were rod-shaped (1.0-1.5x2.0-5.0 mu m) and weakly motile; Gram staining and the KOH lysis test were negative. For growth, the optimum pH was 6.4-6.8 and the optimum temperature was 42-45 degrees C. Strain SR45(T) used sulfate, thiosulfate, sulfite and elemental sulfur as electron acceptors but not Fe(III). The G+C content of the genomic DNA was 41.1 mol%. Phylogenetic analyses based on genes for the 16S rRNA and DNA gyrase (gyrB) revealed that the isolated strain belonged to the family Peptococcaceae in the class Clostridia. The closest relative is Desulfotomaculum acetoxidans 5575(T), with 16S rRNA gene sequence similarity of 92-94 %. It is suggested that the strain is the second isolated member of Desulfotomaculum subcluster le. The isolate had multiple 16S rRNA gene copies, with 13 different sequences. On the basis of phylogenetic and phenotypic characterization, the name Desulfotomaculum intricatum sp. nov. is proposed, with the type strain SR45(T) (=NBRC 109411(T)=DSM 26801(T)).
  • Tomohiro Watanabe, Hisaya Kojima, Yoshinori Takano, Manabu Fukui
    SYSTEMATIC AND APPLIED MICROBIOLOGY 36 6 436 - 443 2013年09月 [査読有り][通常論文]
     
    The diversity of sulfate-reducing prokaryotes (SRPs) and sulfur-oxidizing prokaryotes (SOPs) in freshwater lake ecosystems was investigated by cloning and sequencing of the aprA gene, which encodes for a key enzyme in dissimilatory sulfate reduction and sulfur oxidation. To understand their diversity better, the spatial distribution of aprA genes was investigated in sediments collected from six geographically distant lakes in Antarctica and Japan, including a hypersaline lake for comparison. The microbial community compositions of freshwater sediments and a hypersaline sediment showed notable differences. The clones affiliated with Desulfobacteraceae and Desulfobulbaceae were frequently detected in all freshwater lake sediments. The SOP community was mainly composed of four major phylogenetic groups. One of them formed a monophyletic cluster with a sulfur-oxidizing betaproteobacterium, Sulfuricella denitrificans, but the others were not assigned to specific genera. In addition, the AprA sequences, which were not clearly affiliated to either SRP or SOP lineages, dominated the libraries from four freshwater lake sediments. The results showed the wide distribution of some sulfur-cycle prokaryotes across geographical distances and supported the idea that metabolic flexibility is an important feature for SRP survival in low-sulfate environments. (C) 2013 Elsevier GmbH. All rights reserved.
  • Takato Matsui, Hisaya Kojima, Manabu Fukui
    ESTUARINE COASTAL AND SHELF SCIENCE 119 139 - 144 2013年03月 [査読有り][通常論文]
     
    Most sedimentary mineralization occurs along coasts under anaerobic conditions. In the absence of oxygen, high-molecular weight organic matter in marine sediments is gradually decomposed by hydrolysis, fermentation and sulfate reduction. Because of the different responses of the respective steps to temperature, degradation may be specifically slowed or stopped in certain step. To evaluate the effect of temperature on cellobiose degradation, culture experiments were performed at six different temperatures (3 degrees C, 8 degrees C, 13 degrees C, 18 degrees C, 23 degrees C, and 28 degrees C) under sulfate-reducing conditions. This study measured the concentrations of sulfide, dissolved organic carbon (DOC), and organic acids during that degradation. Degradation patterns were divided into three temperature groups: 3 degrees C, 8/13 degrees C, and 18/23/28 degrees C. The decrease in DOC proceeded in two steps, except at 3 degrees C. The length of the stagnant phase separating these two steps differed greatly between temperatures of 8/13 degrees C and 18/23/28 degrees C. In the first step, organic carbon was consumed by hydrolysis, fermentation and sulfate reduction. In the second step, acetate accumulated during the first step was oxidized by sulfate reduction. Bacterial communities in the cultures were analyzed by denaturing gradient gel electrophoresis (DGGE); the major differences among the three temperature groups were attributed to shifts in acetate-using sulfate reducers of the genus Desulfobacter. This suggests that temperature characteristics of dominant acetate oxidizers are important factors in determining the response of carbon flow in coastal marine sediments in relation to the changes in temperature. (C) 2013 Elsevier Ltd. All rights reserved.
  • Yuriko Higashioka, Hisaya Kojima, Miho Watanabe, Manabu Fukui
    INTERNATIONAL JOURNAL OF SYSTEMATIC AND EVOLUTIONARY MICROBIOLOGY 63 761 - 765 2013年02月 [査読有り][通常論文]
     
    A novel sulfate-reducing bacterium, strain S28bF(T), was isolated from tidal flat sediment from Tokyo Bay, Japan. Cells of strain S28bF(T) were rod-shaped (0.5-0.6x1.7-3.8 mu m), motile and Gram-stain-negative. For growth, the optimum pH was pH 6.8-7.3 and the optimum temperature was 34-42 degrees C. Strain S28bF(T) used sulfate and thiosulfate as electron acceptors, but not nitrate. The G+C content of the genomic DNA was 56.6 mol%. The fatty acid profile of strain S28bF(T) was characterized by the presence of anteiso-C-15:0 and C-16:0 as the major components. Phylogenetic analyses based on genes for 16S rRNA, the alpha subunit of dissimilatory sulfite reductase (dsrA) and adenosine-5'-phosphosulfate reductase (aprA) revealed that the isolated strain belonged to the class Deltaproteobacteria. Its closest relative was Desulfosarcina cetonica DSM 7267(T) with a 16S rRNA gene sequence similarity of 93.3%. Two other strains, S28OL1 and S28OL2 were also isolated from the same sediment. These strains were closely related to S28bF(T) with 16S rRNA gene sequence similarities of 99%, and the same physiological characteristics were shared with strain S28bF(T). On the basis of phylogenetic and phenotypic characterization, a novel species in a new genus, Desulfatitalea tepidiphila gen. nov., sp. nov., is proposed to accommodate the strains obtained in this study. The type strain is S28bF(T) (=NBRC 107166(T)=DSM 23472(T)).
  • Yoshinori Takano, Jonathan James Tyler, Hisaya Kojima, Yusuke Yokoyama, Yukiko Tanabe, Takaharu Sato, Nanako O. Ogawa, Naohiko Ohkouchi, Manabu Fukui
    APPLIED GEOCHEMISTRY 27 12 2546 - 2559 2012年12月 [査読有り][通常論文]
     
    The paleolimnology of two lakes which were isolated as a result of the crustal uplift during the late Holocene along the Soya Coast, Lutzow-Holm Bay, East Antarctica were studied. The focus was on temporal variations in the biogeochemical composition of sediment cores recovered from Lake Skallen at Skallen and Lake Oyako at Skarvsnes. Both sets of lake sediments record environmental changes associated with a transition from marine to lacustrine settings, as indicated by analyses of C and N contents, nitrogen isotopic compositions (delta N-15), and major element concentrations. Changes in the dominant primary producers during the marine-lacustrine transition (marine diatom to cyanobacteria) at L. Skallen was clearly revealed by biogenic opal-A, diatom assemblages, and molecular signature from denaturing gradient gel electrophoresis (DGGE) with 16S ribosomal RNA (rRNA) gene analysis. Radiocarbon dating of acid-insoluble organic C suggested that the environmental transition from marine to fresh water occurred at 2940 +/- 100 cal yr BP at L. Skallen and 1060 +/- 90 cal yr BP at L. Oyako. Based on these data, a mean crustal uplift rate of 3.2 mm yr (1) is inferred for the history of marine-lacustrine transition via brackish conditions. The geological setting causing glacio-isostatic uplift was the primary factor in controlling the transition event in sedimentary and biological facies. (C) 2012 Elsevier Ltd. All rights reserved.
  • Tomohiro Watanabe, Hisaya Kojima, Manabu Fukui
    APPLIED AND ENVIRONMENTAL MICROBIOLOGY 78 18 6545 - 6549 2012年09月 [査読有り][通常論文]
     
    Except for several conspicuous cases, very little is known about sulfur oxidizers living in natural freshwater environments. Sulfuricella denitrificans skB26 is a psychrotolerant sulfur oxidizer recently isolated from a freshwater lake as a representative of a new genus in the class Betaproteobacteria. In this study, an approximately 3.2-Mb draft genome sequence of strain skB26 was obtained. In the draft genome, consisting of 23 contigs, a single rRNA operon, 43 tRNA genes, and 3,133 coding sequences were identified. The identified genes include those required for sulfur oxidation, denitrification, and carbon fixation. Comparative proteomic analysis was conducted to assess cold adaptation mechanisms of this organism. From cells grown at 22 degrees C and 5 degrees C, proteins were extracted for analysis by nano-liquid chromatography electrospray ionization tandem miss spectrometry. In the cells cultured at 5 degrees C, relative abundances of ribosomal proteins, cold shock proteins, and DEAD/DEAH box RNA helicases were increased in comparison to those at 22 degrees C. These results suggest that maintenance of proper translation is critical for growth under low-temperature conditions, similar to the case for other cold-adapted prokaryotes.
  • Yuriko Higashioka, Hisaya Kojima, Manabu Fukui
    MICROBES AND ENVIRONMENTS 27 3 273 - 277 2012年09月 [査読有り][通常論文]
     
    A novel strain of p-xylene-degrading sulfate reducer was isolated in pure culture. Strain PP31 was obtained from a p-xylene-degrading enrichment culture established from polluted marine sediment. Analyses of the 16S rRNA gene and two functional genes involved in sulfate respiration and anaerobic degradation of aromatic compounds revealed that the isolate was closely related to members of the genus Desulfosarcina. Strain PP31 was capable of growing on p-xylene under sulfate-reducing conditions, and the ratio of generated sulfide and consumed p-xylene suggested complete oxidation by the novel isolate. The strain could not grow on benzene, toluene, ethylbenzene, m-xylene oxylene, or n-hexane as an electron donor. Strain PP31 is the first isolated bacterium that degrades p-xylene anaerobically, and will be useful to understanding the mechanism of anaerobic degradation of p-xylene.
  • Hisaya Kojima, Masazumi Tsutsumi, Kanako Ishikawa, Tomoya Iwata, Marc Mussmann, Manabu Fukui
    SYSTEMATIC AND APPLIED MICROBIOLOGY 35 4 233 - 238 2012年06月 [査読有り][通常論文]
     
    Methane oxidation coupled to denitrification is mediated by 'Candidatus methylomirabilis oxyfera', which belongs to the candidate phylum NC10. The distribution of putative denitrifying methane-oxidizing bacteria related to "M. oxyfera" was investigated in a freshwater lake, Lake Biwa, Japan. In the surface layer of the sediment from a profundal site, a phylotype closely related to "M. oxyfera" was most frequently detected among NC10 bacteria in PCR analysis of the 16S rRNA gene. In the sediment, sequences related to "M. oxyfera" were also detected in a pmoA gene library. The presence of NC10 bacteria was also confirmed by catalyzed reporter deposition fluorescence in situ hybridization (CARD-FISH). Denaturing gradient gel electrophoresis (DGGE) and quantitative real-time PCR indicated that the abundance of the "M. oxyfera"-related phylotype was higher in the upper layers of the profundal sediment. The horizontal distribution of the putative methanotrophs in lake sediment was also analyzed by DGGE, which revealed that their occurrence was restricted to deep water areas. These results agreed with those in a previous study of another freshwater lake, and suggested that the upper layer of the profundal sediments is the main habitat for denitrifying methanotrophs. (c) 2012 Elsevier GmbH. All rights reserved.
  • Masanori Fujii, Hisaya Kojima, Tomoya Iwata, Jotaro Urabe, Manabu Fukui
    MICROBIAL ECOLOGY 63 3 496 - 508 2012年04月 [査読有り][通常論文]
     
    Relationships between environmental factors and bacterial communities were investigated in 41 freshwater lakes located in mountainous regions of eastern Japan. Bacterioplankton community composition (BCC) was determined by polymerase chain reaction-denaturing gradient gel electrophoresis of the 16S rRNA gene and then evaluated on the basis of physicochemical and biological variables of the lakes. Canonical correspondence analysis revealed that BCC of oligotrophic lakes was significantly influenced by dissolved organic carbon (DOC) content, but its effect was not apparent in the analysis covering all lakes including mesotrophic and eutrophic ones. The generalized linear model showed the negative association of DOC on the taxon richness of bacterioplankton communities. DOC was positively correlated with the catchment area per lake volume, suggesting that a large fraction of DOC supplied to the lake was derived from terrestrial sources. These results suggest that allochthonous DOC has a significant effect on bacterioplankton communities especially in oligotrophic lakes. The genus Polynucleobacter was detected most frequently. The occurrence of Polynucleobacter species was positively associated with DOC and negatively associated with total phosphorus (TP) levels. In addition, TP had a stronger effect than DOC, suggesting that oligotrophy is the most important factor on the occurrence of this genus.
  • Masazumi Tsutsumi, Hisaya Kojima, Manabu Fukui
    MICROBES AND ENVIRONMENTS 27 1 67 - 71 2012年03月 [査読有り][通常論文]
     
    Vertical profiles of the abundance, community composition, and potential activity of methane-oxidizing bacteria (MOB) were investigated in the sediment of Lake Biwa. Sediment samples were obtained from two sites at different water depths. The abundance of MOB was assessed as the copy number of the pmoA gene (encoding the alpha subunit of particulate methane monooxygenase), measured with quantitative real-time PCR. Abundance of the pmoA gene peaked in the 5-8 cm layer of the sediment from both sites. MOB community composition was investigated by denaturing gradient gel electrophoresis (DGGE) analysis of pmoA and 16S rRNA genes. The band patterns observed in DGGE did not significantly differ with sediment depths or sampling sites. Sequence analysis of the DGGE bands indicated the dominance of the genus Methylobacter. Potential activity, which was measured in the presence of sufficient amounts of methane and oxygen, decreased linearly from the sediment surface to deeper layers. These results suggest that the pmoA gene copy number cannot be regarded as an indicator of aerobic MOB that retain potential activity in sediments.
  • Fumiko Nemoto, Hisaya Kojima, Akifumi Ohtaka, Manabu Fukui
    AQUATIC MICROBIAL ECOLOGY 66 3 295 - 300 2012年 [査読有り][通常論文]
     
    Members of the genus Thioploca are uncultured filamentous sulfur-oxidizing bacteria that live in freshwater/brackish sediments and have the ability to store nitrate in high concentrations in their cells. Their close relatives that inhabit marine sediments, such as Thiomargarita and 'Candidatus Marithioploca', are thought to greatly influence cycles of sulfur, nitrogen, and phosphorus. To date, the genus Thioploca has been reported only from temperate and subarctic areas. Our demonstration of Thioploca in Lake Tonle Sap, Cambodia, is the first report of this genus in a tropical lake. The filaments obtained from Lake Tonle Sap were morphologically similar to those of other lakes. Phylogenetic analysis based on genes for 16S rRNA, 23S rRNA, and the 16S-23S rRNA internal transcribed spacer (ITS) region revealed that 3 distinct lineages coexist in this lake. These results indicate that the geographical distribution and phylogenetic diversity of the genus Thioploca is greater than previously thought.
  • Fumiko Nemoto, Hisaya Kojima, Manabu Fukui
    MICROBIAL ECOLOGY 62 4 753 - 764 2011年11月 [査読有り][通常論文]
     
    Phylogenetic diversity among filamentous sulfur-oxidizing bacteria of the genus Thioploca inhabiting freshwater/brackish environments was analyzed in detail. The 16S rRNA gene sequence of Thioploca found in a freshwater lake in Japan, Lake Okotanpe, was identical to that of Thioploca from Lake Ogawara, a brackish lake. The samples of the two lakes could be differentiated by the sequences of their 23S rRNA genes and 16S-23S rRNA internal transcribed spacer (ITS) regions. The 23S rRNA-based phylogenetic relationships between Thioploca samples from four lakes (Lake Okotanpe, Lake Ogawara, Lake Biwa, and Lake Constance) were similar to those based on the 16S rRNA gene sequences. In addition, multiple types of the ITS sequences were obtained from Thioploca inhabiting Lake Okotanpe and Lake Constance. Variations within respective Thioploca populations were also observed in the analysis of the soxB gene, involved in sulfur oxidation. As major members of the sheath-associated microbial community, bacteria of the phylum Chloroflexi were consistently detected in the samples from different lakes. Fluorescence in situ hybridization revealed that they were filamentous and abundantly distributed within the sheaths of Thioploca.
  • Hisaya Kojima, Manabu Fukui
    INTERNATIONAL JOURNAL OF SYSTEMATIC AND EVOLUTIONARY MICROBIOLOGY 61 1651 - 1655 2011年07月 [査読有り][通常論文]
     
    A novel facultatively autotrophic bacterium, designated strain sk43H(T), was isolated from water of a freshwater lake in Japan. Cells of the isolate were curved rods, motile and Gram-reaction-negative. Strain sk43HT was facultatively anaerobic and autotrophic growth was observed only under anaerobic conditions. The isolate oxidized thiosulfate, elemental sulfur and hydrogen as sole energy sources for autotrophic growth and could utilize nitrate as an electron acceptor. Growth was observed at 8-32 degrees C (optimum 25 degrees C) and 6.4-7.6 (optimum pH 6.7-6.9). Optimum growth of the isolate occurred at NaCl concentrations of less than 50 mM. The G +C content of genomic DNA was around 67 mol%. The fatty acid profile of strain sk43HT when grown on acetate under aerobic conditions was characterized by the presence of C(16:0) and summed feature 3 (C(16:1)omega 7c and/or iso-C(15:0) 2-OH) as the major components. Phylogenetic analysis based on 16S rRNA gene sequences indicated that the strain was a member of the class Beta proteobacteria showing highest sequence similarity with Georgfuchsia toluolica G5G6(T) (94.7%) and Denitratisoma oestradiolicum AcBE2-1(T) (94.3%). Phylogenetic analyses were also performed using genes involved in sulfur oxidation. On the basis of its phylogenetic and phenotypic properties, strain sk43H(T) (=DSM 22779(T) =NBRC 105852(T)) represents a novel species of a new genus, for which the name Sulfuritalea hydrogenivorans gen. nov., sp. nov. is proposed.
  • Masazumi Tsutsumi, Tomoya Iwata, Hisaya Kojima, Manabu Fukui
    FRESHWATER BIOLOGY 56 2 342 - 351 2011年02月 [査読有り][通常論文]
     
    1. The assemblage of aerobic methane-oxidising bacteria (MOB) was investigated in different seasons in the water column of a stratified freshwater lake. Species composition was analysed by performing denaturing gradient gel electrophoresis (DGGE) of the 16S rRNA genes and cloning analysis of the pmoA gene, which encodes the alpha-subunit of particulate methane monooxygenase. The relative abundance of MOB to total bacteria was deduced from the copy number of the pmoA gene and 16S rRNA gene using real-time polymerase chain reaction.2. The profiles of the DGGE banding patterns changed with water depth, and these changes correlated with oxygen concentration and water temperature. The sequences of the DGGE bands obtained were all associated with the genus Methylobacter. During the analysis of pmoA gene, all clones sequenced were that of the Methylobacter/Methylosarcina group. The relative abundances of pmoA gene peaked around the oxycline, and small peaks of pmoA gene were also observed near the surface when peaks of methane were observed at the corresponding depth.3. Profiles of the DGGE banding patterns suggested that ecophysiological characteristics differ among members of the genus Methylobacter; this indicates the importance of investigating the MOB assemblage at the species level or lower. Planktonic MOB seemed to be abundant around oxycline.
  • Y. Higashioka, H. Kojima, M. Fukui
    JOURNAL OF APPLIED MICROBIOLOGY 110 1 314 - 322 2011年01月 [査読有り][通常論文]
     
    Aim: The aim of this study was to characterize the microbial community involved in anaerobic degradation of petroleum hydrocarbon under low- and moderate-temperature conditions. Methods and Results: Sulfate-reducing enrichment cultures growing on crude oil and p-xylene were established at low and moderate temperatures. Bacterial community structures of the cultures were characterized by 16S rRNA gene-based analysis and organisms responsible for degradation of p-xylene were investigated by analysis of the bamA gene, involved in anaerobic degradation of aromatic compounds. The PCR-denaturing gradient gel electrophoresis analysis indicated significant differences in microbial community structures among the cultures, depending on the temperatures of incubation. Difference depending on the temperatures was also observed in the cloning analysis of the bamA gene performed on the p-xylene-degrading enrichment cultures. Majority of clones detected in the culture of moderate temperature were related to Desulfosarcina ovata, whereas more diverse bamA gene sequences were obtained from the culture incubated at low temperature. Conclusions: Temperature-dependent differences in microbial community were demonstrated by the analyses of two genes. It was suggested that sulfate-reducing bacteria of phylogenetically different groups might be involved in the degradation of petroleum hydrocarbons in different temperature environments. Significance and Impact of the Study: This study is the first report of p-xylene-degrading sulfate-reducing enrichment culture at low temperature. The results of the experiments at low temperature were distinctly different from those reported in previous studies performed at moderate temperatures.
  • Hisaya Kojima, Manabu Fukui
    INTERNATIONAL JOURNAL OF SYSTEMATIC AND EVOLUTIONARY MICROBIOLOGY 60 2862 - 2866 2010年12月 [査読有り][通常論文]
     
    A novel facultatively anaerobic, sulfur-oxidizing bacterium, strain skB26(T), was isolated from anoxic water of a freshwater lake in Japan. The cells were rod-shaped, motile and Gram-negative. Strain skB26(T) oxidized elemental sulfur and thiosulfate to sulfate as sole energy sources. Strain skB26(T) was microaerobic and could also utilize nitrate as an electron acceptor, reducing it to nitrogen. Growth was observed at temperatures below 28 degrees C; optimum growth was observed at 22 degrees C. The pH range for growth was 6.0-9.0, and the optimum pH was 7.5-8.0. Optimum growth of the isolate was observed in medium without NaCl, and no growth was observed in medium containing more than 220 mM NaCl. The G + C content of genomic DNA was around 59 mol%. Phylogenetic analysis based on the 16S rRNA gene sequence indicated that the strain was a member of the class Betaproteobacteria, and the closest cultivated relative was Thiobacillus plumbophilus' DSM 6690, with 93% sequence similarity. Phylogenetic analyses were also performed using sequences of genes involved in sulfur oxidation, inorganic carbon fixation and nitrate respiration. On the basis of its phylogenetic and phenotypic properties, strain skB26(T) (=NBRC 105220(T) =DSM 22764(T)) is proposed as the type strain of a novel species of a new genus, Sulfuricella denitrificans gen. nov., sp. nov.
  • Keiichi Tabuchi, Hisaya Kojima, Manabu Fukui
    MICROBIAL ECOLOGY 60 3 551 - 560 2010年10月 [査読有り][通常論文]
     
    Seasonal changes in the mineralization of organic compounds in sediments were investigated in temperate, sublittoral zone sediments (Tokyo Bay, Japan). The total mineralization rate and sulfate reduction rate showed large seasonal variations over the year, and although the fluctuations in both rates correlated with temperature, the latter was irregularly high in May. The concentration of organic carbon dissolved in interstitial water was specifically high in April. A culture-based experiment was also conducted under temperatures corresponding to the seasonal changes. In the culture incubated at a temperature corresponding to April (13 A degrees C), hydrolysis and fermentation proceeded, but terminal oxidation was hindered, thereby resulting in acetate accumulation. At a temperature corresponding to May (22 A degrees C), acetate oxidation coupled with sulfate reduction was observed. The temperature-related differences were also reflected in the bacterial community structure in the cultures analyzed by DGGE. In the culture incubated at the lower temperature, sulfate-reducing bacterium of incomplete oxidizer was detected, while sequence found in the culture incubated at the higher temperature was related to complete oxidizers. These results suggest that complete and incomplete-oxidizing sulfate-reducing bacteria act as distinct functional groups, responding to temperature in different ways, particularly in environments characterized by large temperature fluctuations.
  • Masanori Fujii, Yoshinori Takano, Hisaya Kojima, Tamotsu Hoshino, Ryouichi Tanaka, Manabu Fukui
    MICROBIAL ECOLOGY 59 3 466 - 475 2010年04月 [査読有り][通常論文]
     
    "Red snow" refers to red-colored snow, caused by bloom of cold-adapted phototrophs, so-called snow algae. The red snow found in Langhovde, Antarctica, was investigated from several viewpoints. Various sizes of rounded red cells were observed in the red snow samples under microscopy. Pigment analysis demonstrated accumulation of astaxanthin in the red snow. Community structure of microorganisms was analyzed by culture-independent methods. In the analyses of small subunit rRNA genes, several species of green algae, fungus, and various phylotypes of bacteria were detected. The detected bacteria were closely related to psychrophilic or psychrotolerant heterotrophic strains, or sequences detected from low-temperature environments. As predominant lineage of bacteria, members of the genus Hymenobacter were consistently detected from samples obtained in two different years. Nitrogen isotopic compositions analysis indicated that the red snow was significantly N-15-enriched. Based on an estimation of trophic level, it was suggested that primary nitrogen sources of the red snow were supplied from fecal pellet of seabirds including a marine top predator of Antarctica.
  • Takano, Y, Yokoyama, Y, Tyler, J.J, Kojima, H, Fukui, M, Sato, T, Ogawa, N.O, Suzuki, N, Kitazato, H, Ohkouch, N
    Biogeosciences Discuss 7 4341 - 4384 2010年 [査読無し][通常論文]
  • Hisaya Kojima, Haruo Fukuhara, Manabu Fukui
    SYSTEMATIC AND APPLIED MICROBIOLOGY 32 6 429 - 437 2009年09月 [査読有り][通常論文]
     
    Reddish-brown colored snow, containing spherical brown particles, has been observed in several mires in Japan. In order to characterize this remarkable phenomenon, the microbial community and chemical species in snow were analyzed. A core sample of snow which had a colored region was investigated and it revealed vertical shifts in physicochemical characteristics and the microbial community structure. The abundance of particles peaked within the colored layer, and correlated with the amount of reducible Fe(III). The interstitial water of the colored layer was enriched with Fe(11), and characterized by reduced concentration of dissolved methane. The bacterial community in the colored region was characterized by higher relative abundance of iron-reducing bacteria and methanotrophs. Aggregates of the brown particles were found as precipitates in snow melt pools, and were subjected to cloning analyses targeting several different genes. The majority of bacterial 16S rRNA gene clones belonged to the class Beta proteo bacteria or the phylum Bacteroidetes. No snow algae were detected in the eukaryotic small subunit rRNA gene clone library. As a possible carbon source to sustain the community in the snow, involvements of carbon dioxide and methane were investigated by analyzing the genes involved in their assimilation. In the analyses of genes for ribulose-1,5-biphosphate carboxylase/oxygenase, clones related to sulfur oxidizers were obtained. The analysis or particulate methane monooxygenase genes indicated dominance of Methylobacter species. These results emphasized the uniqueness of this phenomenon, and iron reducers of the genus Geobacter are suggested to be the key organisms that could be investigated in order to understand the mechanism of this phenomenon. (C) 2009 Elsevier GmbH. All rights reserved.
  • Y. Higashioka, H. Kojima, S. Sato, M. Fukui
    JOURNAL OF APPLIED MICROBIOLOGY 107 1 126 - 135 2009年07月 [査読有り][通常論文]
     
    The analyses targeting multiple functional genes were performed on the samples of crude oil-contaminated soil, to investigate community structures of organisms involved in monoaromatic hydrocarbon degradation. Environmental samples were obtained from two sites that were contaminated with different components of crude oil. The analysis on 16S rRNA gene revealed that bacterial community structures were clearly different between the two sites. The cloning analyses were performed by using primers specific for the catabolic genes involved in the aerobic or anaerobic degradation of monoaromatic hydrocarbons, i.e. xylene monooxygenase (xylM), catechol 2,3-dioxygenase (C23O), and benzoyl-CoA reductase (bcr) genes. From the result of xylM gene, it was suggested that there are lineages specific to the respective sites, reflecting the differences of sampling sites. In the analysis of the C23O gene, the results obtained with two primer sets were distinct from each other. A comparison of these suggested that catabolic types of major bacteria carrying this gene were different between the two sites. As for the bcr gene, no amplicon was obtained from one sample. Phylogenetic analysis revealed that the sequences obtained from the other sample were distinct from the known sequences. The differences between the two sites were demonstrated in the analyses of all tested genes. As for aerobic cleavage of the aromatic ring, it was also suggested that analysis using two primer sets provide more detailed information about microbial communities in the contaminated site. The present study demonstrated that analysis targeting multiple functional genes as molecular markers is practical to examine microbial community in crude oil-contaminated environments.
  • Hisaya Kojima, Tomoya Iwata, Manabu Fukui
    FRESHWATER BIOLOGY 54 7 1501 - 1509 2009年07月 [査読有り][通常論文]
     
    1. The assemblage of planktonic methanotrophs in a stratified freshwater lake was investigated. Vertical patterns were analysed by denaturing gradient gel electrophoresis, using the primer pair specific for 16S rRNA genes of type I methanotrophs. 2. The resulting banding patterns could be divided into three distinct groups, and sequenced bands were all related to the Methylobacter species. No amplicon was obtained with the primer pair specific for type II methanotrophs. 3. Cloning analysis of the pmoA gene was performed using samples from three water depths (epilimnion, metalimnion and hypolimnion). The compositions of the clone libraries from the three depths were distinct from each other but all three libraries were dominated by clones related to Methylobacter species.
  • Akiko Kamono, Jun Matsumoto, Hisaya Kojima, Manabu Fukui
    SOIL BIOLOGY & BIOCHEMISTRY 41 6 1324 - 1330 2009年06月 [査読有り][通常論文]
     
    Myxomycetes are amoeboid organisms, and their life cycle includes a stage in which they produce fruiting bodies. Extensive field collections of their fruiting bodies have been conducted in various terrestrial ecosystems. However. little is known about the ecology of myxomycetes in their natural habitat, especially during their trophic stages. To study the presence and distribution of myxomycetes such as those belonging to Didymiaceae and Physaraceae in soil, we used a polymerase chain reaction (PCR)-based method. First, optimization of the reverse transcription (RT)-PCR conditions was performed with the RNA extracted from soil samples. A semi-nested RT-PCR approach was used to improve the specificity of detection. We used newly and previously designed primers in the first round of amplification, which was followed by a second round with the previously described primer pair for PCR-denaturing gradient gel electrophoresis. Next, this method and nucleoticle sequence analyses were applied to study several soil samples collected from five study sites. While no marked dynamic seasonal change was detected in the community composition of myxomycetes belonging to Didymiaceae and Physaraceae in the soil samples collected from the single site, their composition significantly differed between different sites. This is the first study that demonstrates the presence of myxomycetes in the soil and characterizes their community composition by RNA-based molecular analysis. (C) 2009 Elsevier Ltd. All rights reserved.
  • Yuriko Higashioka, Hisaya Kojima, Tatsunori Nakagawa, Shinya Sato, Manabu Fukui
    BIODEGRADATION 20 3 383 - 390 2009年06月 [査読有り][通常論文]
     
    A p-xylene-degrading, sulfate-reducing enrichment culture was characterized by analyzing the response of its members to changes in the available substrate. The culture was inoculated into media containing other substrates, resulting in the establishment of benzoate-, acetate-, and lactate-utilizing enrichment cultures. PCR-denaturing gradient gel electrophoresis (DGGE) analysis of the enriched cultures targeting 16S rRNA genes showed quite simple band patterns. The predominant band from the benzoate-utilizing enrichment culture was identical to that from the original enrichment culture utilizing p-xylene. A single, dominant DGGE band was observed in common from the acetate- and lactate-utilizing enrichment cultures. A novel sulfate-reducing bacterium, strain PL12, was isolated from the lactate-utilizing enrichment culture. The 16S rRNA gene sequence of strain PL12 was identical to that of the dominant DGGE band in the acetate- and lactate-utilizing enrichment cultures and distinct from the dominant sequences in the original p-xylene-degrading and benzoate-utilizing enrichment cultures. Phylogenetic analysis of the 16S rRNA gene sequences showed that the isolate belonged to the family Desulfobacteraceae in the class Deltaproteobacteria. The isolated strain PL12 could utilize n-hexane and n-decane as substrates, but could not utilize benzoate, p-xylene and other aromatic hydrocarbons. These results suggest that the p-xylene degradation observed in the original enrichment culture was performed by the dominant bacterium corresponding to DGGE band pXy-K-13 (Nakagawa et al. 2008). The novel strain PL12 might have been utilizing metabolites of p-xylene.
  • Masazumi Tsutsumi, Hisaya Kojima, Shigeru Uemura, Kiyomi Ono, Akihiro Sumida, Toshihiko Hara, Manabu Fukui
    SOIL BIOLOGY & BIOCHEMISTRY 41 2 403 - 408 2009年02月 [査読有り][通常論文]
     
    In a Japanese forest, CH4 uptake rate and methanotrophic community structure in the soil were investigated at four sites of different vegetation. At two of these Sites, undergrowth was dominated by Sasa senanensis, and that of another was dominated by Sasa kurilensis. At the rest site, undergrowth had been removed artificially. The tree-layer composition differed between the two sites with S. senanensis but, tree layer of the other two sites were dominated by the same species. At the site lacking undergrowth, observed CH4 uptake rate was twice as high as that at the other sites. Under laboratory conditions, soil sample from the site lacking undergrowth exhibited CH4 consumption rate higher than that of the adjacent site with the same dominating tree species. The community structures of methanotrophs were investigated with denaturing gradient gel electrophoresis (DGGE) of the gene encoding particulate methane monooxygenase (pmoA). The banding patterns observed were different depending on the type of undergrowth vegetation. The sequences of the DGGE bands were closely related to each other and belonged to the "upland soil cluster alpha" (USC alpha). These results imply possible close relationship between the undergrowth vegetation and methanotrophic communities in forest soils. (c) 2008 Elsevier Ltd. All rights reserved.
  • Akiko Kamono, Hisaya Kojima, Jun Matsumoto, Kimitaka Kawamura, Manabu Fukui
    NATURWISSENSCHAFTEN 96 1 147 - 151 2009年01月 [査読有り][通常論文]
     
    Myxomycetes are organisms characterized by a life cycle that includes a fruiting body stage. Myxomycete fruiting bodies contain spores, and wind dispersal of the spores is considered important for this organism to colonize new areas. In this study, the presence of airborne myxomycetes and the temporal changes in the myxomycete composition of atmospheric particles (aerosols) were investigated with a polymerase chain reaction (PCR)-based method for Didymiaceae and Physaraceae. Twenty-one aerosol samples were collected on the roof of a three-story building located in Sapporo, Hokkaido Island, northern Japan. PCR analysis of DNA extracts from the aerosol samples indicated the presence of airborne myxomycetes in all the samples, except for the one collected during the snowfall season. Denaturing gradient gel electrophoresis (DGGE) analysis of the PCR products showed seasonally varying banding patterns. The detected DGGE bands were subjected to sequence analyses, and four out of nine obtained sequences were identical to those of fruiting body samples collected in Hokkaido Island. It appears that the difference in the fruiting period of each species was correlated with the seasonal changes in the myxomycete composition of the aerosols. Molecular evidence shows that newly formed spores are released and dispersed in the air, suggesting that wind-driven dispersal of spores is an important process in the life history of myxomycetes. This study is the first to detect airborne myxomycetes with the use of molecular ecological analyses and to characterize their seasonal distribution.
  • Hisaya Kojima, Takuo Nakajima, Manabu Fukui
    FEMS MICROBIOLOGY ECOLOGY 59 1 23 - 31 2007年01月 [査読有り][通常論文]
     
    Carbon source utilization of Thioploca species from freshwater and brackish lakes in Japan was investigated. Microautoradiography demonstrated that freshwater and brackish Thioploca samples assimilate acetate. In addition, vertical nitrate transportation by freshwater Thioploca was examined by measuring substances accumulated in Thioploca filaments. The filaments of Thioploca sp. from Lake Biwa, a Japanese mesotrophic lake, contained nitrate at concentrations higher than ambient by two to three orders of magnitude. They also accumulated high concentrations of sulfate and abundant elemental sulfur. The results suggest that the Thioploca-specific strategy for sulfur oxidation, migration with accumulated nitrate, is effective even in freshwater habitats of lower sulfide supply.
  • Hisaya Kojima, Yoshikazu Koizumi, Manabu Fukui
    MICROBIAL ECOLOGY 52 4 765 - 773 2006年11月 [査読有り][通常論文]
     
    Bacterial communities associated with sheaths of Thioploca spp. from two freshwater lakes (Lake Biwa, Japan, and Lake Constance, Germany) and one brackish lake (Lake Ogawara, Japan) were analyzed with denaturing gradient gel electrophoresis (DGGE) of 16S rRNA gene fragments. The comparison between the DGGE band patterns of bulk sediment and Thioploca filaments of Lake Biwa suggested the presence of specific bacterial communities associated with Thioploca sheaths. As members of sheath-associated communities, bacteria belonging to Bacteroidetes were detected from the samples of both freshwater lakes. A DGGE band from Thioploca of Lake Biwa, belonging to candidate division OP8, was quite closely related to another DGGE band detected from that of Lake Constance. In contrast to the case of freshwater lakes, no bacterium of Bacteroidetes or OP8 was detected from Thioploca of Lake Ogawara. However, two DGGE bands from Lake Ogawara, belonging to Chloroflexi, were quite closely related to a DGGE band from Lake Constance. Two DGGE bands obtained from Lake Biwa were closely related to phylogenetically distant dissimilatory Fe(III)-reducing bacteria. Cloning analyses for a dissimilatory sulfite reductase gene were performed on the same samples used for DGGE analysis. The results of the analyses suggest that sheaths of freshwater/brackish Thioploca have little ecological significance for the majority of sulfate reducers.
  • Y Koizumi, H Kojima, M Fukui
    FEMS MICROBIOLOGY ECOLOGY 52 3 297 - 305 2005年05月 [査読有り][通常論文]
     
    The effects of light and of added electron donors and sulfur compounds on sulfur metabolisms in the microbial mat dilutions from the saline meromictic Lake Kaiike were investigated. Sulfide concentrations in the mat dilution without any electron donor gradually increased by approximate to 0.6-1 mM in the dark. Additions of lactate, acetate, H-2/CO2, propionate and iso-butylate stimulated sulfide production, whereas benzoate did not, indicating the limitation of sulfate reduction by available electron donor concentrations. More sulfide was produced, without a decrease of sulfate, in an elemental sulfur-amended dilution than in a non-amended control. In contrast, the addition of a high concentration of sulfide slowed down sulfide production. After enrichment under various conditions, microbial communities in the dilutions were characterized by a denaturing gradient gel electrophoresis of PCR-amplified 16S rRNA gene and sequencing. As a result, microorganisms affiliated with mesophilic sulfate-reducing bacteria group within the Deltaproteobacteria and the Epsilonproteobacteria were mainly enriched by the addition of electrons used in this study, suggesting that these microorganisms might play an important role in sulfur metabolisms within the surficial sediment of Lake Kaiike. (c) 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.
  • Y Koizumi, H Kojima, M Fukui
    APPLIED AND ENVIRONMENTAL MICROBIOLOGY 70 8 4930 - 4940 2004年08月 [査読有り][通常論文]
     
    Vertical distributions of dominant bacterial populations in saline meromictic Lake Kaiike were investigated throughout the water column and sediment by quantitative oligonucleotide probe membrane hybridization. Three oligonucleotide probes specific for the small-subunit (SSU) rRNA of three groups of Chlorobiaceae were newly designed. In addition, three general domain (Bacteria, Archaea, and Eukarya)-specific probes, two delta-Proteobacteria -specific probes, a Chlorobiaceae-specific probe, and a Chloroflexi-specific probe were used after optimization of their washing conditions. The abundance of the sum of SSU rRNAs hybridizing with probes specific for three groups of Chlorobiaceae relative to total SSU rRNA peaked in the chemocline, accounting for up to 68%. The abundance of the delta-proteobacterial SSU rRNA relative to total SSU rRNA rapidly increased just below the chemocline up to 29% in anoxic water and peaked at the 2- to 3-cm sediment depth at ca. 34%. The abundance of SSU rRNAs hybridizing with the probe specific for the phylum Chloroflexi relative to total SSU rRNA was highest (31 to 54%) in the top of the sediment but then steeply declined with depth and became stable at 11 to 19%, indicating the robust coexistence of sulfate-reducing bacteria and Chloroflexi in the top of the sediment. Any SSU rRNA of Chloroflexi in the water column was under the detection limit. The summation of the signals of group-specific probes used in this study accounted for up to 89% of total SSU rRNA, suggesting that the DGGE-oligonucleotide probe hybridization approach, in contrast to conventional culture-dependent approaches, was very effective in covering dominant populations.
  • Y Koizumi, H Kojima, K Oguri, H Kitazato, M Fukui
    ENVIRONMENTAL MICROBIOLOGY 6 6 622 - 637 2004年06月 [査読有り][通常論文]
     
    The vertical and temporal changes in microbial communities were investigated throughout the water column and sediment of the saline meromictic Lake Kaiike by PCR-denaturing gradient gel electrophoresis (DGGE) of 16S rDNA. Marked depth-related changes in microbial communities were observed at the chemocline and the sediment-water interface. However, no major temporal changes in the microbial community below the chemocline were observed during the sampling period, suggesting that the ecosystem in the anoxic zone of Lake Kaiike was nearly stable. Although the sequence of the most conspicuous DGGE band throughout the anoxic water and in the top of the microbial mat was most similar to that of an anoxic, photosynthetic, green sulphur bacterium, Pelodyction luteolum DSM273 (97% similarity), it represented a new phylotype. A comparison of DGGE banding patterns of the water column and sediment samples demonstrated that specific bacteria accumulated on the bottom from the anoxic water layers, and that indigenous microbial populations were present in the sediment. The measurements of bicarbonate assimilation rates showed significant phototrophic assimilation in the chemocline and lithoautotrophic assimilation throughout the anoxic water, but were not clearly linked with net sulphide turnover rates, indicating that sulphur and carbon metabolisms were not directly correlated.
  • Y Koizumi, H Kojima, M Fukui
    FEMS MICROBIOLOGY ECOLOGY 46 2 147 - 157 2003年11月 [査読有り][通常論文]
     
    Vertical changes of bacterial community structure in a mesophilic lake sediment were investigated by denaturing gradient gel electrophoresis (DGGE) of amplified 16S rDNA and reversely transcribed 16S rRNA fragments. Comparison of community structure analyses based on 16S rDNA and rRNA revealed that the diversity indices from the 16S rDNA-based DGGE profiles were greater than those from the 16S rRNA-based DGGE profiles. In addition, the diversity indices based on 16S rDNA did not change drastically throughout the layers, but the diversity indices based on 16S rRNA decreased with sediment depth. This result indicates that the diversity of active bacteria decreases and inactive bacteria accumulate in association with sedimentation. Dendrograms deduced from DGGE profiles of either 16S rDNA or rRNA were different, and the rRNA-based dendrogram showed a significant difference between the upper layers (0-2, 2-5, and 5-8 cm) and lower layers (8-11, 11-14, 14-17, and 17-20 cm). The sequences of 13 DGGE bands were determined. Phylogenetic analysis of these rDNA fragments revealed that the most conspicuous band in both rDNA- and rRNA-based DGGE was closely related to the genus Nitrospira (95% sequence similarity), and the relative signal intensity was strong especially in the deep layers. Membrane hybridization with a probe targeting the genus Nitrospira also supported the observation that 16S rRNAs derived from Nitrospira-like microorganisms were abundant in this sediment (8.6-16.8% of bacterial 16S rRNA) and that the relative abundance increased with depth. Overall, our results demonstrated that parallel characterization of community structure based on both 16S rDNA and rRNA is important for assessing whole microbial populations and active microbial populations in sediments. (C) 2003 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.
  • A Haraguchi, C Hasegawa, A Hirayama, H Kojima
    EUROPEAN JOURNAL OF SOIL BIOLOGY 39 4 191 - 196 2003年10月 [査読有り][通常論文]
     
    Decomposition rates of peat and cellulose, and oxygen consumption rates were studied in three minerotrophic peat mires in Sasakami, central Japan. These mires had differences in topography, pedology and hydrology. Two dominant vegetation types in each mire, a Sphagnum palustre-S. cuspidatum community and a Rhynchospora fauriei community, were selected as the decomposition study sites. The objective of this study was to examine how the environmental and vegetational differences in mires correlate with the activity of decomposition. Decomposition rates of peat and the rates of cellulolysis were studied in the field for 6 months. Oxygen consumption rates were measured in the field using a closed chamber equipped with an oxygen electrode. In situ peat decomposition rates showed significant differences among the three mires, whereas in situ cellulolysis rates showed significant differences between communities. Peat mass loss rates positively correlated with the nitrogen and carbon concentration of the peat. Cellulolysis rates positively correlated with the range of water table fluctuation. Oxygen consumption rates showed significant negative correlation with the averaged and minimum water table depth, and positive correlation with the range of water table fluctuation. There was a significant positive correlation between cellulolysis rates and oxygen consumption rates. These are useful parameters for evaluating how the decomposition activity in soil depends on the vegetation types and water conditions. (C) 2003 Editions scientifiques et medicales Elsevier SAS. All rights reserved.
  • H Kojima, M Fukui
    WATER RESEARCH 37 13 3216 - 3223 2003年07月 [査読有り][通常論文]
     
    Nitrate-accumulating filamentous bacteria from organic rich sediment of Tokyo Bay, morphologically similar to Beggiatoa species, were phylogenctically analyzed. From several sites in Tokyo Bay, Beggiatoa-like bacteria were collected. Each sample contained narrower or wider type (10 and 30 mum, respectively) of trichomes. With the nested PCR using specific primers for Beggiatoa, fragments of 16S rRNA gene were amplified and then subjected to denaturing gradient gel electrophoresis (DGGE) analysis. Sequencing and the following phylogenetic analysis indicated that they are related to large Beggiatoa species. The wider type was related to uncultured Beggiatoa clones of other geographical localities and distinct from the narrower type in Tokyo Bay. Among the narrower types, a sample from a tidal flat was genetically distinct from the sample from sites of 10 and 20 m water depth. These narrower types form a new branch in Beggiatoa/Thioploca cluster. The result of phylogenetic analysis was in accordance with the previous studies that indicate possession of nitrate-accumulation capability is congruent with phylogeny based on 16S rRNA sequences. (C) 2003 Elsevier Science Ltd. All rights reserved.
  • H Kojima, A Teske, M Fukui
    APPLIED AND ENVIRONMENTAL MICROBIOLOGY 69 1 390 - 398 2003年01月 [査読有り][通常論文]
     
    Filamentous, gliding, sulfide-oxidizing bacteria of the genus Thioploca were found on sediments in profundal areas of Lake Biwa, a Japanese freshwater mesotrophic lake, and were characterized morphologically and phylogenetically. The Lake Biwa Thioploca resembled morphologically Thioploca ingrica, a brackish water species from a Danish fjord. The diameters of individual trichomes were 3 to 5.6 mum; the diameters of complete Thioploca filaments ranged from 18 to 75 mum. The cell lengths ranged from 1.2 to 3.8 mum. In transmission electron microscope specimens stained with uranyl acetate, dense intracellular particles were found, which did not show any positive signals for phosphorus and sulfur in an X-ray analysis. The 16S rRNA gene of the Thioploca from Lake Biwa was amplified by using newly designed Thioploca-specific primers (706-Thioploca, Biwa160F, and Biwa829R) in combination with general bacterial primers in order to avoid nonspecific amplification of contaminating bacterial DNA. Denaturing gradient gel electrophoresis (DGGE) analysis of the three overlapping PCR products resulted in single DGGE bands, indicating that a single 16S rRNA gene had been amplified. With the same method, the Thioploca from Lake Constance was examined. The 16S rRNA sequence was verified by performing fluorescence in situ hybridization targeted at specific motifs of the Lake Biwa Thioploca. Positive signals were obtained with the bacterial probe EUB-338, the gamma-proteobacterial probe GAM42a, and probe Biwa829 targeting the Lake Biwa Thioploca. Based on the nearly complete 16S rRNA sequence and on morphological similarities, the Thioploca from Lake Biwa and the Thioploca from Lake Constance are closely related to T. ingrica and to each other.
  • A Haraguchi, H Kojima, C Hasegawa, Y Takahashi, T Iyobe
    EUROPEAN JOURNAL OF SOIL BIOLOGY 38 1 89 - 95 2002年02月 [査読有り][通常論文]
     
    Decomposition of organic materials, oxygen consumption, and carbon dioxide emission were investigated in Masukata raire, a small minerotrophic mire in central Japan. We selected three dominant community types in the mire, a Sphagnum palustre community, a Phragmites australis community, and an Alnus japonica community, for the decomposition study sites. Decomposition rates were measured in the field by examining mass loss of peat and cellulose for 6 months. The oxygen consumption rate was measured in the field using a closed chamber equipped with an oxygen electrode. The carbon dioxide emission rate of the peat was measured by an infrared gas analyser in the laboratory under controlled conditions. Results of these measurements were tested by correlation analysis. The rate of mass loss of peat positively correlated with the CO2 emission rate. The cellulose decomposition rate showed significant differences among community types, and it had significant positive correlation with the oxygen consumption rate. Although oxygen consumption measurement is not generally used to estimate peatland soil respiration, the oxygen consumption method can be used for predicting long-term decomposition rate according to different vegetation types within a short time. (C) 2002 Editions scientifiques et medicales Elsevier SAS. All rights reserved.

その他活動・業績

共同研究・競争的資金等の研究課題

  • 淡水資源のヒ素汚染対策における嫌気性ヒ素酸化細菌の有用性の評価
    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 : 2020年04月 -2023年03月 
    代表者 : 小島 久弥
  • 多雪寒冷地域の湿原生態系におけるメタン動態の解明
    日本学術振興会:科学研究費助成事業 基盤研究(B)
    研究期間 : 2018年04月 -2022年03月 
    代表者 : 福井 学, 小島 久弥
     
    メタンは強力な温室効果ガスであり、その大気中濃度は地球環境に重要な影響を及ぼすと考えられている。その発生源の約2割は湿地(湿原)であると推定されているが、メタン動態機構は未解明な部分が多い。特に、アクセスが困難な多雪地帯については研究が進んでいない。本研究では、アクセスが困難な多雪地帯でのメタン動態機構について未解明な部分が多いため、多雪寒冷地域高層湿原である国立公園尾瀬ケ原湿原をモデルとし、メタン生成とメタン消費の主要な担い手である微生物群集に着目してメタンの時空間的動態を解明することを目的とし、泥炭層の上に直接積雪する尾瀬ヶ原湿原見本園及び凍結した湖水表面に積雪する池塘群においてメタン代謝に関わる微生物の群集構造を行った。特に、見本園アカシボ泥炭、上田代池塘アカシボ及び泥炭層よりメタン酸化細菌集積培養系が得られた。これらの集積培養系でのメタンモノオキゲナーゼ遺伝子の解析を行うとともに、純粋培養化を試みた。現在までに純粋培養菌が得られておらず、現在繰り返し純化を継続している。
  • 集水域における炭素-窒素-リンの共役循環が湖のメタン動態に及ぼす影響の解明
    日本学術振興会:科学研究費助成事業 基盤研究(B)
    研究期間 : 2016年04月 -2020年03月 
    代表者 : 岩田 智也, 篠原 隆一郎, 小島 久弥, 田中 健太
     
    平成30年度は「サブテーマ3)ホスホン酸代謝を担う浮遊性細菌の時空間分布解明」、「サブテーマ4)リン飢餓状態における微生物細胞内のホスホン酸生成過程」、「サブテーマ5)栄養塩バランスの異なる湖水における好気的メタン生成カイネティクスの定式化」に関する研究を行った。
    次世代シークエンスによる群集解析の結果、亜表層に顕著なメタン極大が出現する深い湖においては、メタン極大が発達する水温躍層付近にシアノバクテリアであるSynechococcusが特異的に多く分布していることが明らかとなった。SynechococcusはC-Pリアーゼを有すると考えられることやメタン生成能が申請者らの先行研究で確認されていることから、メタン極大の形成に大きく寄与しているものと考えられた。サブテーマ2である微生物細胞内のホスホン酸生成については、湖水から大量捕集した微生物試料について実験条件の検討を行い、解析の準備を完了した。また、培養細胞では実験場件下で特定の微生物が細胞内にホスホン酸を生成・蓄積することが確認された。この微生物については、既知のホスホン酸生合成経路とは異なる反応によるホスホン酸を生成している可能性が示唆された。サブテーマ3については、湖水へのホスホン酸添加実験から、夏季に強いリン飢餓状態となるメタン極大層付近においてC-Pリアーゼ活性が認められた。さらに、カイネティクスの推定も試み、ごく微量ながら湖水中にホスホン酸が存在している可能性を示すことができた。
  • 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 : 2015年04月 -2018年03月 
    代表者 : 小島 久弥
     
    ヒ素化合物は深刻な健康被害を起こす環境汚染物質であり、その環境中での挙動に大きな影響を及ぼすと考えられるのが、嫌気性ヒ素酸化細菌である。環境中での嫌気性ヒ素酸化細菌の多様性と分布を解析するための手法を開発し、実際に野外から得られた試料に適用した。その結果、効率的な手法が確立され、それを用いることによって環境中には従来知られていたよりも多様な嫌気性ヒ素酸化細菌が生息していることを示唆する結果が得られた。
  • 日本学術振興会:科学研究費助成事業 基盤研究(B)
    研究期間 : 2012年04月 -2016年03月 
    代表者 : 奥田 昇, 小島 久弥, 福井 学, 陀安 一郎, 中野 伸一, 伊藤 雅之, 小林 由紀, 藤林 恵, 夏 復國, 謝 志豪, 三木 健, 何 珮綺, 張 俊偉, 中野 伸一, 陀安 一郎
     
    湖沼は、高い温室効果をもつメタンの主要な自然放出源である。湖底で生成されるメタンを好気・嫌気的に同化するメタン酸化細菌を起点としたメタン栄養食物網は、メタン由来炭素を湖沼生態系の炭素循環に組み込み、メタンの大気放出を抑制する「炭素リサイクルシステム」とみなすことができる。本研究は、亜熱帯に位置する台湾・翡翠水庫のメタン・プロファイルおよびメタン酸化細菌叢の時空間動態が一回循環湖と部分循環湖の双方の特徴を併せ持つこと、そして、湖沼の栄養フローにおけるメタン栄養食物網の相対的重要性に影響する湖沼物理メカニズムとして台風による温度成層の攪乱と深水層のメタン貯留の消失が重要であることを明らかにした。
  • 日本学術振興会:科学研究費助成事業 若手研究(B)
    研究期間 : 2012年04月 -2014年03月 
    代表者 : 小島 久弥
     
    強力な温室効果ガスであるメタンの大気中への放出量に影響を持つ、環境中に生息するメタン酸化細菌の低酸素条件下での生態に関する研究を行った。これまで知見が無かった亜熱帯おける調査では、ダム湖の水中から無酸素条件下でメタンを酸化する能力を持った細菌と近縁な微生物が数多く検出された。積雪中のメタン酸化細菌に関する研究では、メタン酸化細菌と鉄還元菌の相互作用の可能性が両者の微視的な空間分布から示唆された。
  • 日本学術振興会:科学研究費助成事業 基盤研究(B)
    研究期間 : 2010年04月 -2014年03月 
    代表者 : 福井 学, 小島 久弥
     
    寒冷圏水界における微生物学的硫黄循環は、炭素及び窒素循環とも深く関連した生態系における重要なプロセスであるが、依然不明な点が多い。本研究は、環境から微生物由来のゲノム及びタンパク質を直接解析する環境オミクス手法により、微生物学的硫黄循環の潜在的な代謝経路および活性を網羅的に解明することを目的とした。地理的に異なる淡水湖沼を調査した結果、新属の硫黄酸化細菌Sulfuricella denitrificansが硫黄代謝に鍵となる主であることが判明し、ゲノム解析からその代謝経路を明らかにした。さらに同菌種において低温環境特有のタンパク質発現パターンもプロテオーム解析から明らかになった。
  • 日本学術振興会:科学研究費助成事業 若手研究(B)
    研究期間 : 2007年 -2008年 
    代表者 : 小島 久弥
     
    細胞内に硝酸イオンを蓄積する能力を持つ硫黄酸化細菌は、生体を構成する主要な元素の循環において重要な役割を担っていると考えられている。純粋培養できないこれらの細菌について、遺伝子解析からの機能推定を行った。淡水性の種が持つことが判明していた遺伝子を特異的に定量する手法を新たに確立した。国内外の淡水・汽水湖沼で採取された試料に対し硫黄酸化に関わる遺伝子の解析を行い、配列を特定した。
  • 水界の硫黄循環システムと微生物群集の共進化に関する分子生態学的研究
    日本学術振興会:科学研究費助成事業 基盤研究(B)
    研究期間 : 2004年 -2007年 
    代表者 : 福井 学, 小島 久弥
     
    微生物学的硫酸還元は、無酸素環境で硫酸塩を硫化物に還元する反応であるが、34.7億年前の地球ですでに行われていることが示されている。しかし、現在の水界生態系で観察される、多様な硫黄循環システムとそれを担う微生物群集が対古代からどのようなメカニズムで共進化して来たかは未解明である。そこで、本研究は、水界の硫黄循環システムと微生物群集の共進化を分子生態学的に解明とすることを目的とする。現場環境で成立している微生物群集の系統遺伝子及び機能遺伝子の解析と物質代謝データを包括的に行うことにより、生物による硫黄酸化及び硫酸還元の起源を探り、硫黄循環システムとそれを担う微生物の相互作用が地球の酸化-還元、温度、光条件等の変化でいかに変遷して来たかを明らかとした。70〜80℃の陸上温泉で発達する微生物マットでは、好熱性硫酸還元菌であるThermodesulfobacterium様微生物及び好熱性硫黄酸化細菌であるSurifurihydrogenium様微生物で構成されていた。微生物マット内では、両者が硫黄循環に関与していることが確認された。両菌は分子系統進化学上生命の共通祖先に比較的近いことから、太古代の地球では、両菌による硫黄循環システムが原始タイプであることが推察される。一方、60〜65℃で発達する微生物マットでは、両菌に加え、好熱性光合成細菌であるChloroflexus様微生物(原始的光合成細菌)で構成され、硫黄循環システムに光合成が組み込まれることが明らかとなった。さらに、中温型の部分循環湖である貝池や淡水湖である琵琶湖では、プロテオバクテリアに属する多種の微生物が硫黄循環に関与し、その代謝経路は複雑化していることが明らかとなった。以上のように、地球環境の温度が減少していく過程で硫黄循環システムも複雑化していくことが示唆された。

教育活動情報

主要な担当授業

  • 分子生物学基礎論
    開講年度 : 2019年
    課程区分 : 修士課程
    開講学部 : 環境科学院
    キーワード : 分子生物学,転写,翻訳,微生物の群集構造解析,微生物の呼吸活性,電子顕微鏡観察,遺伝子操作,バイオフィルム形成,植物ストレス耐性,バイオセンサー,昆虫免疫系,昆虫体表脂質, 動物細胞 molecular biology, transcription, translation, bacterial culture, bacterial community structure analysis, bacterial respiration activity, electron microscopic observation, genetic manipulation, biofilm formation, stress tolerance of plants, biosensor, insect immune system, insect body surface lipids, animal cells


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