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Last Updated :2024/11/21

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Master

Affiliation (Master)

  • Institute of Low Temperature Science　Environmental Biology

Affiliation (Master)

  • Institute of Low Temperature Science　Environmental Biology

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Profile and Settings

Affiliation

  • Hokkaido University, Institute of Low Temperature Science, Environmental Biology, Professor

Degree

  • Master of Science（Tokyo Metropolitan University）

Profile and Settings

  • Name (Japanese)

    Fukui

  • Name (Kana)

    Manabu

  • Name

    200901066213259619

Alternate Names

Affiliation

  • Hokkaido University, Institute of Low Temperature Science, Environmental Biology, Professor

Achievement

Research Interests

  • Methane dynamics   Sulfur cycle   系統分類進化   微生物生態学   陸水生態学   Freshwater Ecology   Microbial Ecology   Limnology   

Research Areas

  • Environmental science/Agricultural science / Environmental dynamics
  • Life sciences / Ecology and environmental science

Research Experience

  • 2024/04 - Today Hokkaido University Institute of Low Temperature Science
  • 2004/08 - 2024/03 北海道大学低温科学研究所 教授
  • 2018/04 - 2022/03 北海道大学低温科学研究所 所長
  • 1998 - 2004 Tokyo Metropolitan University
  • 1992 - 1998 通商産業省工業技術院資源環境技術総合研究所 主任研究官
  • 1989 - 1992 通商産業省工業技術公害資源研究所 研究員

Awards

  • 2011 科研費審査委員表彰

Published Papers

  • J M Tsuji, N A Shaw, S Nagashima, J J Venkiteswaran, S L Schiff, T Watanabe, M Fukui, S Hanada, M Tank, J D Neufeld
    Nature 627 (8005) 915 - 922 2024/03 [Refereed]
     
    Scientific exploration of phototrophic bacteria over nearly 200 years has revealed large phylogenetic gaps between known phototrophic groups that limit understanding of how phototrophy evolved and diversified1,2. Here, through Boreal Shield lake water incubations, we cultivated an anoxygenic phototrophic bacterium from a previously unknown order within the Chloroflexota phylum that represents a highly novel transition form in the evolution of photosynthesis. Unlike all other known phototrophs, this bacterium uses a type I reaction centre (RCI) for light energy conversion yet belongs to the same bacterial phylum as organisms that use a type II reaction centre (RCII) for phototrophy. Using physiological, phylogenomic and environmental metatranscriptomic data, we demonstrate active RCI-utilizing metabolism by the strain alongside usage of chlorosomes3 and bacteriochlorophylls4 related to those of RCII-utilizing Chloroflexota members. Despite using different reaction centres, our phylogenomic data provide strong evidence that RCI-utilizing and RCII-utilizing Chloroflexia members inherited phototrophy from a most recent common phototrophic ancestor. The Chloroflexota phylum preserves an evolutionary record of the use of contrasting phototrophic modes among genetically related bacteria, giving new context for exploring the diversification of phototrophy on Earth.
  • Tomohiro Watanabe, Tatsuya Yabe, Jackson M Tsuji, Manabu Fukui
    Archives of microbiology 205 (12) 368 - 368 2023/11/03 [Refereed]
     
    A novel sulfate-reducing bacterium (strain 12FAKT) was isolated from sediment sampled from a brackish lake in Japan. Respiratory growth was observed with formate and pyruvate as an electron donor. Sulfate, thiosulfate, elemental sulfur and dimethyl sulfoxide were utilized as an electron acceptor. The isolate grew over a temperature range of 18-42 °C (optimum 35-37 °C), a NaCl concentration range of 50-450 mM (optimum 150-300 mM) and a pH range of 6.6-7.5. The 12FAKT genome consists of a circular chromosome with a length of 4.5 Mbp and G + C content of 63.6%. Based on 16S rRNA gene sequence similarity, the closest cultured relative was Desulfarculus baarsii 2st14T (92.2%). Genome-based phylogenetic analysis placed strain 12FAKT within the family Desulfarculaceae but did not affiliate the strain with any existing genus. Taken together, we propose a novel species of a novel genus, Desulfoferula mesophilus gen. nov. sp. nov. with the type strain 12FAKT (= DSM 115219T = JCM 39399T).
  • Dmitriy Alekseevsky, Yelizaveta Chernysh, Vladimir Shtepa, Viktoriia Chubur, Lada Stejskalová, Magdalena Balintova, Manabu Fukui, Hynek Roubík
    Water (Switzerland) 15 (21) 2023/11 [Refereed]
     
    This study aimed to improve the control system of the biological stage of wastewater treatment using the quality control information system to support the concept of environmental efficiency management. In this case, the object of the study was the treatment facilities of Sumy city (Ukraine). For automatic control of wastewater quality, pH, oxidation reduction potential (ORP), electrical conductivity, and temperature indicators were taken, as well as hydrobiological analysis of activated sludge and mathematical modelling. The pH of wastewater at the input system has systematically unacceptable values (above 8.5 were recorded). Unacceptable concentrations of sulphur-containing toxicants arrive at the entrance of treatment facilities (0.22–1.3 mg/L). The response of activated sludge biocenosis to increasing concentrations of hydrogen sulphide in wastewater was analysed. Furthermore, a mathematical model of monoculture population growth, with two factors that affect population growth (nutrient concentration and monoculture production concentration), was implemented for the initial assessment of possible negative effects on wastewater treatment. The differential equation of the population dynamics of the i-th species of microorganisms in activated sludge was described. The applied system of automated monitoring of wastewater parameters with expert assessment of activated sludge and a unified mathematical model of approaches allows for a complex system of decision-making support to be realised. However, this requires the construction of mathematical models that would take into account the cause–effect relations that operate under conditions of incomplete technological information and the potential presence of emergencies due to natural disasters and military activities.
  • Kazuhiro Umezawa, Hisaya Kojima, Yukako Kato, Manabu Fukui
    Systematic and Applied Microbiology 45 (6) 0723-2020 2022/11 
    The authors regret that the type of Thermodesulfovibrionales was incorrectly assigned in the original article. According to rule 15 of International Code of Nomenclature of Prokaryotes [1], the type of an order should be a genus, not family. In this case, it must be Thermodesulfovibrio, not Thermodesulfovibrionaceae. The corrected protologues is below. Description of Thermodesulfovibrionales ord. nov. Thermodesulfovibrionales (The.mo.de.sul.fo.vib.ri.o.na'les. N.L. masc. n. Thermodesulfovibrio, type genus of the order; L. fem. pl. suff. -ales, ending to denote a class; N.L. fem. pl. n. Thermodesulfovibrionales, the order of the genus Thermodesulfovibrio.). This order was established on the basis of genome-based phylogeny. Encompasses families Thermodesulfovibrionaceae and Dissulfurispiraceae. Type genus is Thermodesulfovibrio. The species belonging Thermodesulfovibrionales are obligately anaerobic thermophiles. The authors would like to apologise for any inconvenience caused. Reference [1] Parker, C.T., Tindall, B.J., Garrity, G.M. (2019) International Code of Nomenclature of Prokaryotes. Int. J. Syst. Evol. Microbiol. 69, S1-S111.
  • Hisashi Ito, Hideyuki Saito, Manabu Fukui, Ayumi Tanaka, Keita Arakawa
    Plant Science 324 0168-9452 2022/11 [Refereed]
     
    Chlorophyll breakdown is observed during senescence. The first step in chlorophyll breakdown is the removal of central Mg by Mg-dechelatase. This reaction is the rate-limiting step in the chlorophyll breakdown pathway. We evaluated the effect of induced chlorophyll breakdown on abscission through the removal of Mg by Mg-dechelatase. Poplar transformants carrying the dexamethasone-inducible Mg-dechelatase gene were prepared using the Arabidopsis Stay-Green1 cDNA. When leaves were treated with dexamethasone, chlorophyll was degraded, photosynthetic capacity was reduced, and an abscission zone was formed, resulting in leaf abscission. In addition, ethylene, which plays an important role during senescence, was produced in this process. Thus, chlorophyll breakdown induces the phenotype in the same way as commonly observed during leaf senescence. This study suggests a physiological role of chlorophyll breakdown in the leaf abscission of deciduous trees. Furthermore, this study shows that the dexamethasone-inducible gene expression system is an available option for deciduous tree studies.
  • Hisaya Kojima, Miho Watanabe, Naoyuki Miyata, Manabu Fukui
    Archives of Microbiology 204 (10) 0302-8933 2022/10 
    In the original article, strain numbers assigned by BCRC were incorrectly presented. The correct numbers are BCRC 81316T (=J5BT) and BCRC 81317T (= M52T).
  • Miho Watanabe, Ayaka Takahashi, Hisaya Kojima, Naoyuki Miyata, Manabu Fukui
    Archives of microbiology 204 (10) 647 - 647 2022/09/27 [Refereed]
     
    A novel sulfate-reducing bacterium, strain PPLLT, was isolated from marsh soil. Cells of strain PPLLT were rod-shaped with length of 1.5 μm and width of 0.7 μm. Growth was observed at 22-37 °C (optimum 35 °C) and pH 6.8-8.4 (optimum 7.3). Lactate, succinate, fumarate, formate and malate were utilized as electron donors for sulfate reduction. Fermentative growth was not observed on tested organic acids. Besides sulfate, sulfite, thiosulfate and elemental sulfur were utilized as electron acceptors. Hydrogen is used only in the presence acetate or yeast extract. The major fatty acid was C16:0. The complete genome of strain PPLLT was composed of a circular chromosome with length of 4.2 Mbp and G + C content of 57.7 mol%. Sequence analysis of the 16S rRNA gene showed that strain PPLLT was affiliated with the genus Desulfofustis in the family Desulfocapsaceae. On the basis of differences in the phylogenetic and phenotypic properties between the strain and the type strain of the genus Desulfofustis, strain PPLLT (DSM 110475T = JCM 39161T) is proposed as the type strain of a new species, with name of Desulfofustis limnaeus sp. nov.
  • Miho Watanabe, Ayaka Takahashi, Hisaya Kojima, Manabu Fukui
    Archives of microbiology 204 (10) 640 - 640 2022/09/22 [Refereed]
     
    A novel sulfate-reducing bacterium, strain ASN36T, was isolated from sediment of a brackish lake in Japan. Cells of strain ASN36T were not motile and rod-shaped, with length of 2.0-4.9 μm and width of 0.6-0.9 μm. Growth was observed at 5-35 °C with an optimum growth temperature of 25-30 °C. The pH range for growth was 6.6-8.8 with an optimum pH of 7.3. Major fatty acids were C16:1 ω7c and C16:0. Under sulfate-reducing conditions, strain ASN36T utilized lactate, malate, pyruvate, butyrate, ethanol, butanol, glycerol, yeast extract and H2/CO2 as growth substrate. Fermentative growth occurred on malate and pyruvate. The novel isolate used sulfate, sulfite and thiosulfate as electron acceptors. The genome of strain ASN36T is composed of a chromosome with length of 6.3 Mbp and G + C content of 55.1 mol%. Analyses of the 16S rRNA gene indicated that strain ASN36T is related to Desulfoluna species. Overall genome relatedness indices indicated that strain ASN36T does not belong to any existing species. In contrast to the closest relatives, strain ASN36T lacks genes for reductive dehalogenase required for organohalide respiration and does not use halogenated aromatics as electron acceptors. On the basis of its genomic and phenotypic properties, strain ASN36T (= DSM 111985 T = JCM 39257 T) is proposed as the type strain of a new species, Desulfoluna limicola sp. nov.
  • Hisaya Kojima, Jun Mochizuki, Mamoru Kanda, Tomohiro Watanabe, Manabu Fukui
    Archives of microbiology 204 (10) 605 - 605 2022/09/07 [Refereed]
     
    A novel sulfur-oxidizing bacterium, strain Am19T, was isolated from sediment of a brackish lake. Strain Am19T grew chemolithoautotrophically on inorganic sulfur compounds, and heterotrophic growth was not observed. Cells were rod-shaped with length of 1.1-3.0 μm and diameter of 0.5-0.8 μm. Growth was observed at 5-37 °C with an optimum growth temperature of 30 °C. The pH range for growth was 5.6-8.5 with an optimum pH of 6.6-7.0. Major fatty acids were summed feature 3 (C16: 1ω7c and/or C16: 1ω6c), summed feature 8 (C18: 1ω7c and/or C18: 1ω6c) and C16: 0. The sole respiratory quinone was ubiquinone-8. The complete genome of strain Am19T is composed of a circular chromosome with length of 2.5 Mbp and G + C content of 42.7 mol%. Phylogenetic analysis based on genomic data indicated that strain Am19T belongs to the genus Thiomicrorhabdus but is distinct from any existing species. Analysis of the 16S rRNA gene supported creation of a new species to accommodate strain Am19T. On the basis of genomic and phenotypic characteristics, strain Am19T (= NBRC 114602 T = BCRC 81336 T) is proposed as the type strain of a new species, with name of Thiomicrorhabdus immobilis sp. nov.
  • Hisaya Kojima, Miho Watanabe, Naoyuki Miyata, Manabu Fukui
    Archives of microbiology 204 (9) 595 - 595 2022/09/02 [Refereed]
     
    Strains J5BT and M52T are facultatively autotrophic sulfur-oxidizing bacteria isolated from a microbial mat from a hot spring. They were isolated and partially characterized in previous studies, as facultative anaerobes which use nitrate as electron acceptor. In this study, additional characterizations were made to determine their taxonomic status. In both strains, major cellular fatty acids were C16:1 (C16:1ω7c and/or C16:1ω6c) and C16:0. Their chemolithoautotrophic growth was supported by thiosulfate and elemental sulfur. They used some organic acids as growth substrates. Their 16S rRNA gene sequences indicated the highest sequence identities to species in the family Sterolibacteriaceae, but the identities were 95% or lower. Phylogenetic analysis indicated that these strains do not belong to any existing genera. Values of average nucleotide identity and digital DNA-DNA hybridization between strains J5BT and M52T were 87.93% and 34.3%, respectively. On the basis of phenotypic and genomic characteristics, Sulfuricystis multivorans gen. nov. sp. nov., and Sulfuricystis thermophila sp. nov. are proposed, with type strains of J5BT and M52T, respectively. An emended description of the genus Rugosibacter is also proposed, for its reclassification to the family Sterolibacteriaceae.
  • Hisaya Kojima, Yukako Kato, Tomohiro Watanabe, Manabu Fukui
    Archives of microbiology 204 (9) 559 - 559 2022/08/17 [Refereed]
     
    A novel chemolithoautotrophic bacterium, strain H1576T, was isolated from water of a brackish lake. Strain H1576T grew aerobically on inorganic sulfur compounds. Hydrogen gas did not support autotrophic growth, and heterotrophic growth was not observed. Cells were rod shaped, motile, 1.5-2.7 μm in length and 0.6-0.7 μm in width. Growth was observed at 3-22 °C with an optimum growth temperature of 13-15 °C. The pH range for growth was 6.0-7.4 with an optimum pH of 6.6-6.8. Major fatty acids were summed feature 3 (C16: 1ω7c and/or C16: 1ω6c). The complete genome of strain H1576T consists of a circular chromosome and a plasmid, with total length of 2.8 Mbp and G+C content of 46.4 mol%. Phylogenetic analyses indicated that strain H1576T belongs to the genus Sulfurimonas but distinct from representatives of existing species. On the basis of genomic and phenotypic characteristics, a new species named Sulfurimonas aquatica sp. nov. is proposed with the type strain of strain H1576T (= BCRC 81254T = JCM 35004T).
  • Ayaka Takahashi, Hisaya Kojima, Miho Watanabe, Manabu Fukui
    Archives of microbiology 204 (6) 307 - 307 2022/05/09 [Refereed]
     
    A novel mesophilic and neutrophilic sulfate-reducing bacterium, strain SF6T, was isolated from sediment of a brackish lake in Japan. Cells of strain SF6T were motile and rod-shaped with length of 1.2-2.5 μm and width of 0.6-0.9 μm. Growth was observed at 10-37 °C with an optimum growth temperature of 28 °C. The pH range for growth was 5.8-8.2 with an optimum pH of 7.0. The most predominant fatty acid was anteiso-C15:0. Under sulfate-reducing conditions, strain SF6T utilized lactate, ethanol and glucose as growth substrate. Chemolithoautotrophic growth on H2 was not observed, although H2 was used as electron donor. Fermentative growth occurred on pyruvate. As electron acceptor, sulfate, sulfite, thiosulfate and nitrate supported heterotrophic growth of the strain. The complete genome of strain SF6T is composed of a circular chromosome with length of 3.8 Mbp and G+C content of 54 mol%. Analyses of the 16S rRNA gene and whole genome sequence indicated that strain SF6T belongs to the genus Pseudodesulfovibrio but distinct form all existing species in the genus. On the basis of its genomic and phenotypic properties, strain SF6T (= DSM111931T = NBRC 114895T) is proposed as the type strain of a new species, with name of Pseudodesulfovibrio sediminis sp. nov.
  • Tomohiro Watanabe, Kyoko Kubo, Yoshiharu Kamei, Hisaya Kojima, Manabu Fukui
    Systematic and Applied Microbiology 126320 - 126320 0723-2020 2022/03 [Refereed]
  • Yelizaveta Chernysh, Magdalena Balintova, Vladimir Shtepa, Polina Skvortsova, Maksym Skydanenko, Manabu Fukui
    Ecological Engineering and Environmental Technology 23 (1) 110 - 124 2022 [Refereed]
     
    Large-scale disasters at nuclear power plants (NPPs) and their consequences are still the subject of discussion by the world scientific community, which makes mankind recognize the unsolved problem of radiation pollution. Accordingly, the search for new effective biocomposite materials with high sorption capacity to eliminate harmful effects associated with radiation contamination of large territories is an urgent task on a global scale. This paper is devoted to the study of the decontamination processes of the areas contaminated with radionuclides, the search for new mechanisms of fixation of radionuclides and heavy metals in the soil using the matrix material of different origin. In order to intensify the process of radionuclide fixation in the soil-plant system the method consisting of introducing into the soil the organic-mineral biocomposite based on sewage sludge and phosphogypsum after anaerobic fermentation was proposed. It is necessary to further study the processes of sorption and radionuclides solubilization due to complexation with organic agents present in matrix materials of different nature. The mechanisms of radionuclide and heavy metal fixation using matrix material of different origin were analyzed and a general model was formed. The direction of integration of radionuclide-contaminated soil decontamination technologies into the process of ecological, social, and economic development of the territories under rehabilitation after the accidents at the Chernobyl NPP and Fukushima-1 NPP is proposed.
  • Miho Watanabe, Hisaya Kojima, Kunihiro Okano, Manabu Fukui
    International journal of systematic and evolutionary microbiology 71 (12) 2021/12 [Refereed]
     
    A novel strictly anaerobic chemoorganotrophic bacterium, designated Mahy22T, was isolated from sulfidic bottom water of a shallow brackish meromictic lake in Japan. Cells of the strain were Gram-stain-negative, non-motile and coccoid in shape with diameters of about 600-800 nm. The temperature range for growth was 15-37 °C, with optimum growth at 30-32 °C. The pH range for growth was pH 6.2-8.9, with optimum growth at pH 7.2-7.4. The strain grew with NaCl concentrations of 5% or below (optimum, 2-3%). Growth of the strain was enhanced by the addition of thiosulfate. The major cellular fatty acids were C16:0 and anteiso-C15:0. Respiratory quinones were not detected. The complete genome sequence of strain Mahy22T possessed a 1 885 846 bp circular chromosome and a 12 782 bp circular genetic element. The G+C content of the genome sequence was 30.1 mol%. Phylogenetic analysis based on the 16S rRNA gene revealed that the novel strain belonged to the family Acholeplasmataceae, class Mollicutes. The closest relative of strain Mahy22T with a validly published name was Acholeplasma palmae J233T with a 16S rRNA gene sequence similarity of 90.5%. Based on the results of polyphasic analysis, the name Mariniplasma anaerobium gen. nov., sp. nov. is proposed to accommodate strain Mahy22T, along with reclassification of some Acholeplasma species into Alteracholeplasma gen. nov., Haploplasma gen. nov. and Paracholeplasma gen. nov.
  • Takeshi Miyadera, Hisaya Kojima, Manabu Fukui
    ARCHIVES OF MICROBIOLOGY 0302-8933 2021/09 [Refereed]
     
    A novel methylotrophic bacterium, strain Zm11(T), was isolated from reddish brown snow collected in a moor in Japan. Cells of the isolate were Gram-stain-negative, motile, and rod-shaped (0.6-0.7 x 1.2-2.7 mu m). Growth was observed at 5-32 degrees C with an optimum growth temperature of 25-28 degrees C. The pH range for growth was 5.4-7.8 with an optimum pH of 6.8. The strain utilized only methanol as carbon and energy sources for aerobic growth. The major cellular fatty acids (> 40% of total) were summed feature 3 (C-16:1 omega 7c and/or C-16:1 omega 6c) and C-16: 0. The predominant quinone was Q-8, and major polar lipids were phosphatidylethanolamine and phosphatidylglycerol. The complete genome of strain Zm11(T) is composed of a circular chromosome (2,800,413 bp), with G + C content of 46.4 mol%. Phylogenetic analyses were conducted based on the 16S rRNA gene sequence and conserved proteins encoded in the genome. The results of analyses indicate that strain Zm11(T) is a member of the family Methylophilaceae but does not belong to any existing genus. On the basis of its genomic and phenotypic properties, strain Zm11(T) (= DSM111909(T) = NBRC114766(T)) is proposed as the type strain of a new species in a new genus, Methyloradius palustris gen. nov., sp. nov.
  • Toshihide Hirao, Masanori Fujii, Nobuhiko Shigyo, Hisaya Kojima, Manabu Fukui
    PEDOBIOLOGIA 87-88 0031-4056 2021/08 [Refereed]
     
    Understanding how the community structures and functions of soil microbes respond to environmental changes is essential for predicting the dynamics of ecosystem functions. Elevational gradients are an ideal setting for "natural experiments" on the relationships between soil microbes and environmental factors. However, little is known about elevational patterns of soil microbial functions and their interactions with aboveground vegetation. We examined community structures and nitrogen cycling gene abundance of soil bacteria in 20 vegetation survey quadrats in cool-temperate and sub-alpine forests along an elevational gradient (1050-2000 m above sea level) in central Japan. The diversity of soil bacterial communities was investigated based on terminal restriction fragment length polymorphisms (T-RFLP) in the 16S rRNA gene. The abundance of soil bacteria and key functional groups involved in nitrogen cycling was determined by quantifying the 16S rRNA and bacterial genes related to nitrogen fixation (nifH), ammonia oxidation (amoA), and denitrification (nosZ). The terminal restriction fragment (T-RF) richness of soil bacteria showed no relationship with elevation, but was negatively associated with soil nitrate-nitrogen and the species richness of understory vegetation. The abundance of all bacteria and nitrogen functional groups showed hump-shaped relationships with elevation, and was positively associated with soil water content and understory stem density. These results highlight that community structures and nitrogen cycling gene abundance of soil microbes along the elevational gradient can be explained by soil properties associated with understory vegetation. This suggests that vegetation decline caused by environmental changes, such as climate warming, land use change, and increased herbivore density, may deteriorate soil ecosystem functions through aboveground-belowground interactions.
  • Kazuhiro Umezawa, Hisaya Kojima, Yukako Kato, Manabu Fukui
    Systematic and Applied Microbiology 44 (2) 126184 - 126184 0723-2020 2021/04 [Refereed]
     
    Recently, presence of sulfur-disproportionating bacterial species belonging to the phylum Nitrospirota was indicated by an enrichment culture-based study. In the present study, a strain representing that species was isolated and characterized. The strain, strain T55JT, was isolated from a microbial mat of a hot spring. The cells were motile, and rods or spiral forms with width of 0.32-0.49 μm. The strain grew autotrophically, only by disproportionation of thiosulfate or elemental sulfur. Growth was observed at a temperature range of 25-60°C, with optimum growth at 53-57°C. The pH range for growth was 5.5-9.0, with optimum pH of 7.0-8.0. The complete genome of strain T55JT is composed of a circular chromosome (2,370,875 bp), with G+C content of 38.7%. Thermodesulfovibrio hydrogeniphilus Hdr5T showed the highest sequence similarity of the 16S rRNA gene to strain T55JT, but it was only 88.2%. On the basis of the phylogenetic and physiologic properties, strain T55JT (= DSM 110365T=NBRC 114245T) is proposed as type strain of a novel species in a new genus, Dissulfurispira thermophila gen. nov., sp. nov. To assign the new genus to family and higher taxa, its taxonomic position was assessed by genome-based phylogeny. As a result, it was shown that the novel genus and Thermodesulfovibrio belong to different families. It was also shown that Thermodesulfovibrio should be reclassified at levels from class to family and creation of some novel taxa is required. Based on these results, Thermodesulfovibrionia class. nov., Thermodesulfovibrionales ord. nov., Thermodesulfovibrionaceae fam. nov., and Dissulfurispiraceae fam. nov. are proposed.
  • Yelizaveta Chernysh, Igor Roy, Viktoriia Chubur, Manabu Fukui, Ivan Koziy
    Lecture Notes in Mechanical Engineering 319 - 328 2195-4356 2021 
    This paper focused on determining the regularities of modern trends in the field of stimulation of wastewater and sewage sludge (SS) components transformation in anaerobic conditions with particular attention to the theoretical substantiation of the magnetic fields’ mechanisms field influence on these bioprocesses. The network system’s visualization and clustering of research areas of stimulation of anaerobic fermentation of wastewater and SS were carried out. The clusters were formed through a network: the red cluster conducted with the area of enzyme activity and use of various biostimulants for microorganism growth under anaerobic digestion and ecosystem bioremediation; the green cluster associated with general fermentation processes at the junction of aerobic and anaerobic conditions using different co-substrates; the blue cluster has generalized the use of methods of genetic modification of microorganisms used in anaerobic processes, including the digestion of SS, as well as the influence of minerals on the stimulation of fermentation. The analysis of visualization clusters was also devoted to studies on biostimulation of bacterial activity processes using a magnetic field. The generalized model of the magnetic field’s stimulating effect on bioprocesses was formed, which can be used in anaerobic digestion of SS and wastewater bio-treatment to produce useful bio-based products.
  • Jun Mochizuki, Hisaya Kojima, Manabu Fukui
    ARCHIVES OF MICROBIOLOGY 203 (3) 951 - 957 0302-8933 2020/10 [Refereed]
     
    Aerobic, Gram-stain-negative, obligately chemolithoautotrophic thiosulfate-oxidizing bacteria, strains AkT22(T)and aks77(T)were isolated from a brackish lake in Japan. Strains AkT22(T)and aks77(T)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 degrees C or higher temperature, with optimum growth at 22 degrees C. Strain AkT22(T)grew at a pH range of 5.8-8.0, with optimum growth at pH 6.7-7.8. Strain aks77(T)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 AkT22(T)were C-16:1, C-18:1, and C-16:0. The sole respiratory quinone was ubiquinone-8 in both strains. The genome of strain AkT22(T)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 aks77(T)were ca. 2.7 Mbp and 45.5%, respectively. Among cultured bacteria,Thiomicrorhabdus aquaedulcisHaS4(T)showed the highest sequence identities of the 16S rRNA gene, to strains AkT22(T)(94%) and aks77(T)(95%). On the basis of these results,Thiosulfativibrio zosteraegen. nov., sp. nov. andThiosulfatimonas sediminisgen. nov., sp. nov. are proposed, with type strains of AkT22(T)(= BCRC 81184(T) = NBRC 114012(T) = DSM 109948(T)) and aks77(T)(= BCRC 81183(T) = NBRC 114013(T)), respectively.
  • Hisaya Kojima, Mamoru Kanda, Kazuhiro Umezawa, Manabu Fukui
    ARCHIVES OF MICROBIOLOGY 203 (1) 317 - 323 0302-8933 2020/09 [Refereed]
     
    A facultatively anaerobic sulfur-oxidizing bacterium, strain skT11(T), was isolated from anoxic lake water of a stratified freshwater lake. As electron donor for chemolithoautotrophic growth, strain skT11(T)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 degrees C, with optimum growth at 28 degrees C. Strain skT11(T)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 C(16:1)and C-16:0. The complete genome of strain skT11(T)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 skT11(T)is related to sulfur-oxidizing bacteria of the generaSulfuricella,Sulfurirhabdus,andSulfuriferula, with sequence identities of 95.4% or lower. The analysis also indicated that these three genera should be excluded from the familyGallionellaceae, as members of another family. On the basis of its genomic and phenotypic properties, strain skT11(T)(= DSM 110711( T) = NBRC 114323( T)) is proposed as the type strain of a new species in a new genus,Sulfurimicrobium lacusgen. nov., sp. nov. In addition, emended descriptions of the familiesGallionellaceaeandSulfuricellaceaeare proposed to declare thatSulfuricellaceaeis not a later synonym ofGallionellaceae.
  • Kazuhiro Umezawa, Hisaya Kojima, Yukako Kato, Manabu Fukui
    SYSTEMATIC AND APPLIED MICROBIOLOGY 43 (5) 0723-2020 2020/09 [Refereed]
     
    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. (C) 2020 Elsevier GmbH. All rights reserved.
  • Miho Watanabe, Yuriko Higashioka, Hisaya Kojima, Manabu Fukui
    SYSTEMATIC AND APPLIED MICROBIOLOGY 43 (5) 0723-2020 2020/09 [Refereed]
     
    Strain 28bB2T(T) 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 28bB2T(T) were performed to establish the taxonomic status of the strain. Cells of strain 28bB2T(T) were short oval-shaped (0.8-1.2 x 1.2-2.7 mu m), motile, and Gram-negative. For growth, the optimum pH was pH 6.5-7.0 and the optimum temperature was 28-32 degrees C. Strain 28bB2T(T) 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 53.8 mol%. The genome consisted of an approximately 8.3 Mb of chromosome and two extrachromosomal elements. On the basis of 16S rRNA gene analysis, strain 28bB2T(T) was revealed to belong to the genus Desulfosarcina, with high sequence identities to Desulfosarcina ovata oXyS1(T) (99.5%) and Desulfosarcina cetonica DSM 7267(T) (98.7%). Results of Average Nucleotide Identity (ANI) calculation and digital DNA-DNA hybridization (dDDH) analysis showed that the strain 28bB2T(T) should be classified as a subspecies under D. ovata. Based on physiological and phylogenetic data, strain 28bB2T(T) (=NBRC 106234 =DSM 23484) is proposed as the type strain of a novel species in genus Desulfosarcina, Desulfosarcina ovata subsp. sediminis subsp. nov. (C) 2020 Elsevier GmbH. All rights reserved.
  • Miho Watanabe, Hisaya Kojima, Manabu Fukui
    ARCHIVES OF MICROBIOLOGY 202 (5) 1069 - 1076 0302-8933 2020/07 [Refereed][Not invited]
     
    An aerobic bacterium, designated strain Dysh456(T), was isolated from a crude oil-contaminated soil. Cells of strain Dysh456(T) were rod-shaped, motile, and Gram-stain-negative. Strain Dysh456(T) grew at 13-48 degrees C and pH 4.3-7.9. Major cellular fatty acids were iso-C-15:0 (42.5%), iso-C-17:0 (15.3%) and summed feature 9 (iso-C-17:1 omega 9c/C-16:0 10-methyl [13.7%]). Major respiratory quinone was ubiquinone-8. The genome of strain Dysh456(T) 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 Dysh456(T) 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 Dysh456(T) (= NBRC 112897(T) = DSM 105662(T)) 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.
  • Miho Watanabe, Hisaya Kojima, Manabu Fukui
    ANTONIE VAN LEEUWENHOEK INTERNATIONAL JOURNAL OF GENERAL AND MOLECULAR MICROBIOLOGY 113 (3) 349 - 355 0003-6072 2020/03 [Refereed][Not invited]
     
    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
    INTERNATIONAL JOURNAL OF SYSTEMATIC AND EVOLUTIONARY MICROBIOLOGY 70 (12) 6408 - 6413 1466-5026 2020 [Refereed]
     
    A novel facultatively anaerobic, nitrate-reducing bacterium, designated MeG22(T), was isolated from a freshwater lake in Japan. Cells of the strain were straight rods (0.8x2.5-10 mu m), motile, and Gram-stain-negative. For growth, the optimum NaCl concentration was 0% and the optimum temperature was 30 degrees C. Under anoxic conditions, strain MeG22(T) reduced nitrate to nitrite. Major cellular fatty acids were C-15:1 omega 6c (13.6 %), C-17:0 (11.9 %), anteiso-C-15:0 (10.6 %) and iso-C-15:0 (10.6 %). The major respiratory quinone was menaquinone-7. The genome sequence of strain MeG22(T) consists of 5 712 279 bp with a G+C content of 40.3mol%. 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 MeG22(T) was Sunxiuqinia faeciviva strain JAM-BA0302(T) 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 MeG22(T) (=NBRC 112896(T)=DSM 106262(T)).
  • Hisaya Kojima, Jun Mochizuki, Manabu Fukui
    INTERNATIONAL JOURNAL OF SYSTEMATIC AND EVOLUTIONARY MICROBIOLOGY 70 (5) 3273 - 3277 1466-5026 2020 [Refereed][Not invited]
     
    A chemolithoautotrophic sulfur-oxidizing bacterium, strain SGTM(T) was isolated from snow collected in Japan. As electron donors for growth, SGTM(T) 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 degrees C, with optimum growth at 18 degrees C. SGTM(T) 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 (C-16:1 omega 7c and/or C-16:1 omega 6c) and C-16:0. The complete genome of SGTM(T) consisted of a circular chromosome of approximately 3.4 Mbp and two plasmids. Phylogenetic analysis based on the 16S rRNA gene indicated that SGTM(T) represented a member of the genus Sulfuriferula, and its closest relative is Sulfuriferula thiophila mst6(T) with a sequence identity of 98%. A comparative genome analysis showed dissimilarity between the genomes of SGTM(T) and S. thiophila mst6(T), 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. SGTM(T) (=DSM 109609(T)=BCRC 81185(T)) 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.
  • Santona Khatun, Tomoya Iwata, Hisaya Kojima, Manabu Fukui, Takuya Aoki, Seito Mochizuki, Azusa Naito, Ai Kobayashi, Ryo Uzawa
    SCIENCE OF THE TOTAL ENVIRONMENT 696 0048-9697 2019/12 [Refereed][Not invited]
     
    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. (C) 2019 Elsevier B.V. All rights reserved.
  • Hisaya Kojima, Manabu Fukui
    INTERNATIONAL JOURNAL OF SYSTEMATIC AND EVOLUTIONARY MICROBIOLOGY 69 (9) 2849 - 2853 1466-5026 2019/09 [Refereed][Not invited]
     
    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 1664-302X 2019/05 [Refereed][Not invited]
     
    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 1758-2229 2019/04 [Refereed][Not invited]
     
    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.
  • Tatsunori Nakagawa, Yuki Tsuchiya, Shingo Ueda, Manabu Fukui, Reiji Takahashi
    MICROBES AND ENVIRONMENTS 34 (1) 13 - 22 1342-6311 2019/03 [Refereed][Not invited]
     
    Nitrous oxide (N2O) is a powerful greenhouse gas; however, limited information is currently available on the microbiomes involved in its sink and source in seagrass meadow sediments. Using laboratory incubations, a quantitative PCR (qPCR) analysis of N2O reductase (nosZ) and ammonia monooxygenase subunit A (amoA) genes, and a metagenome analysis based on the nosZ gene, we investigated the abundance of N2O-reducing microorganisms and ammonia-oxidizing prokaryotes as well as the community compositions of N2O-reducing microorganisms in in situ and cultivated sediments in the non-eelgrass and eelgrass zones of Lake Akkeshi, Japan. Laboratory incubations showed that N2O was reduced by eelgrass sediments and emitted by non-eelgrass sediments. qPCR analyses revealed that the abundance of nosZ gene clade II in both sediments before and after the incubation as higher in the eelgrass zone than in the non-eelgrass zone. In contrast, the abundance of ammonia-oxidizing archaeal amoA genes increased after incubations in the non-eelgrass zone only. Metagenome analyses of nosZ genes revealed that the lineages Dechloromonas-Magnetospirillum-Thiocapsa and Bacteroidetes (Flavobacteriia) within nosZ gene clade II were the main populations in the N2O-reducing microbiome in the in situ sediments of eelgrass zones. Sulfur-oxidizing Gammaproteobacteria within nosZ gene clade II dominated in the lineage Dechloromonas-Magnetospirillum-Thiocapsa. Alphaproteobacteria within nosZ gene clade I were predominant in both zones. The proportions of Epsilonproteobacteria within nosZ gene clade II increased after incubations in the eelgrass zone microcosm supplemented with N2O only. Collectively, these results suggest that the N2O-reducing microbiome in eelgrass meadows is largely responsible for coastal N2O mitigation.
  • Miho Watanabe, Hisaya Kojima, Kazuhiro Umezawa, Manabu Fukui
    FRONTIERS IN MICROBIOLOGY 10 1664-302X 2019/02 [Refereed][Not invited]
     
    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 1664-302X 2019/02 [Refereed][Not invited]
     
    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 1466-5026 2018/09 [Refereed][Not invited]
     
    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.
  • 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) 1083 - 1084 0912-3814 2018/09 [Refereed][Not invited]
     
    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, Hisaya Kojima, Manabu Fukui
    MARINE GENOMICS 39 1 - 2 1874-7787 2018/06 [Refereed][Not invited]
     
    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 1439-8621 2018/04 [Refereed][Not invited]
     
    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.
  • Junji Matsuo, Shinji Nakamura, Torahiko Okubo, Manabu Fukui, Hiroyuki Yamaguchi
    PARASITOLOGY RESEARCH 117 (3) 937 - 941 0932-0113 2018/03 [Refereed][Not invited]
     
    A free-living amoeba, Naegleria is ubiquitously distributed in various natural environments. Since some Naegleria spp. are exclusively distributed in the Arctic and sub-Antarctic regions, we hypothesized that the amoeba may be useful to determine long-term survival of Naegleria in laboratory conditions at 4 A degrees C. The main objective of the study is to determine that a species of an environmental amoebal isolated can live at low temperatures after a long time. Here, we therefore show long-term survival of an amoeba, Naegleria polaris isolated from a sediment sample, which was collected from Antarctica 10 years ago, and since stored at 4 A degrees C. The sample was put on non-nutrient agar plates with heat-killed Escherichia coli, and then the plate was incubated at 4, 15, or 30 A degrees C. Motile amoebae were seen only when the plate was incubated at 15 A degrees C. The sequencing of ribosomal DNA including internal transcribed spacers (ITS) 1, 5.8S rDNA, and ITS2 region revealed the amoebae to be N. polaris, which is exclusively distributed in the Arctic and sub-Antarctic regions. Scanning electron microscopic observation showed that no typical sucker-like structure was seen on the surface of N. polaris, but the cysts were similar to those of Naegleria fowleri. Thus, our result shows, for the first time, that N. polaris can survive after 10 years of storage at 4 A degrees C. This finding may help us understand the still undescribed effects of environmental samples on viability of amoebae.
  • Miho Watanabe, Riho Tokizawa, Hisaya Kojima, Manabu Fukui
    STANDARDS IN GENOMIC SCIENCES 12 1944-3277 2017/12 [Refereed][Not invited]
     
    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%.
  • Rei Okamoto, Hisaya Kojima, Manabu Fukui
    INTERNATIONAL JOURNAL OF SYSTEMATIC AND EVOLUTIONARY MICROBIOLOGY 67 (11) 4773 - 4776 1466-5026 2017/11 [Refereed][Not invited]
     
    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.
  • 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 0912-3814 2017/11 [Refereed][Not invited]
     
    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.
  • Tomohiro Watanabe, Aya Miura, Tomoya Iwata, Hisaya Kojima, Manabu Fukui
    ENVIRONMENTAL MICROBIOLOGY REPORTS 9 (5) 522 - 527 1758-2229 2017/10 [Refereed][Not invited]
     
    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 1466-5026 2017/09 [Refereed][Not invited]
     
    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, Hisaya Kojima, Manabu Fukui
    INTERNATIONAL JOURNAL OF SYSTEMATIC AND EVOLUTIONARY MICROBIOLOGY 67 (8) 2679 - 2682 1466-5026 2017/08 [Refereed][Not invited]
     
    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, Yuriko Higashioka, Hisaya Kojima, Manabu Fukui
    INTERNATIONAL JOURNAL OF SYSTEMATIC AND EVOLUTIONARY MICROBIOLOGY 67 (8) 2994 - 2997 1466-5026 2017/08 [Refereed][Not invited]
     
    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 1664-302X 2017/08 [Refereed][Not invited]
     
    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
    STANDARDS IN GENOMIC SCIENCES 12 1944-3277 2017/06 [Refereed][Not invited]
     
    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 1466-5026 2017/05 [Refereed][Not invited]
     
    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)).
  • Yuki Kobayashi, Hisaya Kojima, Masayuki Itoh, Noboru Okuda, Manabu Fukui, Fuh-Kwo Shiah
    PLANKTON & BENTHOS RESEARCH 11 (4) 144 - 146 1880-8247 2016/11 [Refereed][Not invited]
     
    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.
  • Tomohiro Watanabe, Hisaya Kojima, Manabu Fukui
    SCIENTIFIC REPORTS 6 2045-2322 2016/11 [Refereed][Not invited]
     
    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 1466-5026 2016/11 [Refereed][Not invited]
     
    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 1466-5026 2016/09 [Refereed][Not invited]
     
    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 1944-3277 2016/09 [Refereed][Not invited]
     
    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, Aya Miura, Arisa Shinohara, Hisaya Kojima, Manabu Fukui
    INTERNATIONAL JOURNAL OF SYSTEMATIC AND EVOLUTIONARY MICROBIOLOGY 66 1986 - 1989 1466-5026 2016/05 [Refereed][Not invited]
     
    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)).
  • Tomohiro Watanabe, Hisaya Kojima, Manabu Fukui
    INTERNATIONAL JOURNAL OF SYSTEMATIC AND EVOLUTIONARY MICROBIOLOGY 66 2041 - 2045 1466-5026 2016/05 [Refereed][Not invited]
     
    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.
  • 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 2016/04/01 [Refereed][Not invited]
  • Hisaya Kojima, Kazuhiro Umezawa, Manabu Fukui
    INTERNATIONAL JOURNAL OF SYSTEMATIC AND EVOLUTIONARY MICROBIOLOGY 66 1828 - 1831 1466-5026 2016/04 [Refereed][Not invited]
     
    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 1466-5026 2016/03 [Refereed][Not invited]
     
    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.
  • Rachel C. Rudd, Jonathan J. Tyler, John Tibby, Yusuke Yokoyama, Ines Tavernier, Elie Verleyen, Manabu Fukui, Yoshinori Takano
    JOURNAL OF QUATERNARY SCIENCE 31 (2) 114 - 125 0267-8179 2016/02 [Refereed][Not invited]
     
    Decadal-centennial-scale climate variability in coastal Antarctica remains poorly understood due to the limited number of highly resolved, well-dated records. We present a 900-year, decadal-scale reconstruction based on sedimentary diatoms from Lake Abi in Lutzow-Holm Bay, East Antarctica. Hydrological change is inferred from diatom ecological preferences in conjunction with an existing regional training set and implies that lake water specific conductivity, depth and nitrogen availability are the key drivers of diatom assemblage change. Lake Abi underwent a series of subtle environmental changes related to these environmental variables, possibly driven by changes in catchment snow melt and the duration of seasonal ice cover. Ordination is used to trace the major patterns of change in the diatom community, with notable shifts identified between 470 and 400 and at approximate to 350 cal a BP (where present=CE 1950). The frequency of environmental variability at Lake Abi is broadly consistent with a record of the Interdecadal Pacific Oscillation during the last millennium, but contrasts with the apparent climate stability elsewhere in eastern Antarctica. Further research is required to constrain the limnological and ecological responses of lakes in coastal Antarctica to obtain more rigorous palaeoclimate reconstructions from these sites of immense potential. Copyright (C) 2016 John Wiley & Sons, Ltd.
  • 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 [Refereed][Not invited]
  • Hisaya Kojima, Tomohiro Watanabe, Manabu Fukui
    INTERNATIONAL JOURNAL OF SYSTEMATIC AND EVOLUTIONARY MICROBIOLOGY 66 266 - 270 1466-5026 2016/01 [Refereed][Not invited]
     
    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 1466-5026 2016/01 [Refereed][Not invited]
     
    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)).
  • Yuriko Higashioka, Hisaya Kojima, Tomohiro Watanabe, Manabu Fukui
    MICROBIOLOGY RESOURCE ANNOUNCEMENTS 3 (6) 2576-098X 2015/11 [Refereed]
     
    Desulfatitalea tepidiphila S28bF(T) is a sulfate-reducing bacterium closely related to Desulfosarcina species. Here, the draft genome sequence of strain S28bF(T) is reported.
  • Hisaya Kojima, Arisa Shinohara, Manabu Fukui
    INTERNATIONAL JOURNAL OF SYSTEMATIC AND EVOLUTIONARY MICROBIOLOGY 65 3709 - 3713 1466-5026 2015/10 [Refereed][Not invited]
     
    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) 2576-098X 2015/09 [Refereed][Not invited]
     
    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).
  • Ikumi Oyabu, Yoshinori Iizuka, Hubertus Fischer, Simon Schuepbach, Gideon Gfeller, Anders Svensson, Manabu Fukui, Jorgen Peder Steffensen, Margareta Hansson
    JOURNAL OF GEOPHYSICAL RESEARCH-ATMOSPHERES 120 (18) 9789 - 9813 2169-897X 2015/09 [Refereed][Not invited]
     
    This study reports the chemical composition of particles present along Greenland's North Greenland Eemian Ice Drilling (NEEM) ice core, back to 110,000years before present. Insoluble and soluble particles larger than 0.45 mu m were extracted from the ice core by ice sublimation, and their chemical composition was analyzed using scanning electron microscope and energy dispersive X-ray spectroscopy and micro-Raman spectroscopy. We show that the dominant insoluble components are silicates, whereas NaCl, Na2SO4, CaSO4, and CaCO3 represent major soluble salts. For the first time, particles of CaMg(CO3)(2) and Ca(NO3)(2)center dot 4H(2)O are identified in a Greenland ice core. The chemical speciation of salts varies with past climatic conditions. Whereas the fraction of Na salts (NaCl+Na2SO4) exceeds that of Ca salts (CaSO4+CaCO3) during the Holocene (0.6-11.7kyr B.P.), the two fractions are similar during the BOlling-AllerOd period (12.9-14.6kyr B.P.). During cold climate such as over the Younger Dryas (12.0-12.6kyr B.P.) and the Last Glacial Maximum (15.0-26.9kyr B.P.), the fraction of Ca salts exceeds that of Na salts, showing that the most abundant ion generally controls the salt budget in each period. High-resolution analyses reveal changing particle compositions: those in Holocene ice show seasonal changes, and those in LGM ice show a difference between cloudy bands and clear layers, which again can be largely explained by the availability of ionic components in the atmospheric aerosol body of air masses reaching Greenland.
  • Miho Watanabe, Hisaya Kojima, Manabu Fukui
    INTERNATIONAL JOURNAL OF SYSTEMATIC AND EVOLUTIONARY MICROBIOLOGY 65 2378 - 2384 1466-5026 2015/08 [Refereed][Not invited]
     
    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 2775 - 2775 1466-5026 2015/08 [Refereed][Not invited]
  • Masayuki Itoh, Yuki Kobayashi, Tzong-Yueh Chen, Takeshi Tokida, Manabu Fukui, Hisaya Kojima, Takeshi Miki, Ichiro Tayasu, Fuh-Kwo Shiah, Noboru Okuda
    JOURNAL OF GEOPHYSICAL RESEARCH-BIOGEOSCIENCES 120 (7) 1246 - 1261 2169-8953 2015/07 [Refereed][Not invited]
     
    Although freshwaters are considered to be substantial natural sources of atmospheric methane (CH4), in situ processes of CH4 production and consumption in freshwater ecosystems are poorly understood, especially in subtropical areas, leading to uncertainties in the estimation of global CH4 emissions. To improve our understanding of physical and biogeochemical factors affecting CH4 dynamics in subtropical lakes, we examined vertical and seasonal profiles of dissolved CH4 and its carbon isotope ratio (C-13) and conducted incubation experiments to assess CH4 production and oxidation in the deep subtropical Fei-Tsui Reservoir (FTR; Taiwan). The mixing pattern of the FTR is essentially monomixis, but the intensity of winter vertical mixing changes with climatic conditions. In years with incomplete vertical mixing (does not reach the bottom) and subsequent strong thermal stratification resulting in profundal hypoxia, we observed increases in sedimentary CH4 production and thus profundal CH4 storage with the development of reducing conditions. In contrast, in years with strong winter vertical mixing to the bottom of the reservoir, CH4 production was suppressed under NO3--rich conditions, during which denitrifiers have the competitive advantage over methanogens. Diffusive emission from profundal CH4 storage appeared to be negligible due to the efficiency of CH4 oxidation during ascent through methane-oxidizing bacteria (MOB) activity. Most of the profundal CH4 was rapidly oxidized by MOB in both oxic and anoxic layers, as characterized by its carbon isotope signature. In contrast, aerobic CH4 production in the subsurface layer, which may be enhanced under high temperatures in summer, may account for a large portion of atmospheric CH4 emissions from this reservoir. Our CH4 profiling results provide valuable information for future studies predicting CH4 emissions from subtropical lakes with the progress of global warming.
  • Miho Watanabe, Hisaya Kojima, Manabu Fukui
    INTERNATIONAL JOURNAL OF SYSTEMATIC AND EVOLUTIONARY MICROBIOLOGY 65 1902 - 1907 1466-5026 2015/06 [Refereed][Not invited]
     
    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) 2576-098X 2015/05 [Refereed][Not invited]
     
    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.
  • Tomohiro Watanabe, Hisaya Kojima, Manabu Fukui
    INTERNATIONAL JOURNAL OF SYSTEMATIC AND EVOLUTIONARY MICROBIOLOGY 65 1504 - 1508 1466-5026 2015/05 [Refereed][Not invited]
     
    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.
  • 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 1751-7362 2015/05 [Refereed][Not invited]
     
    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.
  • Yoshinori Takano, Hisaya Kojima, Eriko Takeda, Yusuke Yokoyama, Manabu Fukui
    PROGRESS IN EARTH AND PLANETARY SCIENCE 2 2197-4284 2015/04 [Refereed][Not invited]
     
    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.
  • Tomohiro Watanabe, Hisaya Kojima, Manabu Fukui
    Genome Announcements 3 (5) 2169-8287 2015 [Refereed][Not invited]
     
    Strain skMP5T 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 skMP5T.
  • Tomohiro Watanabe, Hisaya Kojima, Manabu Fukui
    Genome Announcements 3 (3) 2169-8287 2015 [Refereed][Not invited]
     
    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.
  • Ikumi Oyabu, Yoshinori Iizuka, Hubertus Fischer, Simon Schüpbach, Gideon Gfeller, Anders Svensson, Manabu Fukui, Jørgen Peder Steffensen, Margareta Hansson
    Journal of Geophysical Research 120 (18) 9789 - 9813 2156-2202 2015 [Refereed][Not invited]
     
    This study reports the chemical composition of particles present along Greenland’s North Greenland Eemian Ice Drilling (NEEM) ice core, back to 110,000 years before present. Insoluble and soluble particles larger than 0.45 µm were extracted from the ice core by ice sublimation, and their chemical composition was analyzed using scanning electron microscope and energy dispersive X-ray spectroscopy and micro-Raman spectroscopy. We show that the dominant insoluble components are silicates, whereas NaCl, Na2SO4, CaSO4, and CaCO3 represent major soluble salts. For the first time, particles of CaMg(CO3)2 and Ca(NO3)2•4H2O are identified in a Greenland ice core. The chemical speciation of salts varies with past climatic conditions. Whereas the fraction of Na salts (NaCl + Na2SO4) exceeds that of Ca salts (CaSO4 + CaCO3) during the Holocene (0.6-11.7 kyr B.P.), the two fractions are similar during the Bølling-Allerød period (12.9-14.6 kyr B.P.). During cold climate such as over the Younger Dryas (12.0-12.6 kyr B.P.) and the Last Glacial Maximum (15.0-26.9 kyr B.P.), the fraction of Ca salts exceeds that of Na salts, showing that the most abundant ion generally controls the salt budget in each period. High-resolution analyses reveal changing particle compositions: those in Holocene ice show seasonal changes, and those in LGM ice show a difference between cloudy bands and clear layers, which again can be largely explained by the availability of ionic components in the atmospheric aerosol body of air masses reaching Greenland.
  • Yuriko Higashioka, Hisaya Kojima, Tomohiro Watanabe, Manabu Fukui
    Genome Announcements 3 (6) 2169-8287 2015 [Refereed][Not invited]
     
    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 0948-3055 2015 [Refereed][Not invited]
     
    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.
  • Hisaya Kojima, Riho Tokizawa, Manabu Fukui
    INTERNATIONAL JOURNAL OF SYSTEMATIC AND EVOLUTIONARY MICROBIOLOGY 64 3983 - 3987 1466-5026 2014/12 [Refereed][Not invited]
     
    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 1462-2912 2014/11 [Refereed][Not invited]
     
    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 0723-2020 2014/10 [Refereed][Not invited]
     
    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 0723-2020 2014/09 [Refereed][Not invited]
     
    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 1466-5026 2014/08 [Refereed][Not invited]
     
    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 2045-2322 2014/07 [Refereed][Not invited]
     
    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 1466-5026 2014/05 [Refereed][Not invited]
     
    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) 1932-6203 2014/04 [Refereed][Not invited]
     
    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 1466-5026 2013/10 [Refereed][Not invited]
     
    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 0723-2020 2013/09 [Refereed][Not invited]
     
    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.
  • Akiko Kamono, Marianne Meyer, Thomas Cavalier-Smith, Manabu Fukui, Anna Maria Fiore-Donno
    FEMS MICROBIOLOGY ECOLOGY 84 (1) 98 - 109 0168-6496 2013/04 [Refereed][Not invited]
     
    In spite of the ecological importance of protists, very little data is available on their distribution in soil. This investigation is the first of its kind on what could be the major components of the soil protistan community, the Myxomycetes, or plasmodial slime-moulds, a monophyletic class in the phylum Amoebozoa. Myxomycetes have a complex life cycle culminating in the formation of mainly macroscopic fruiting bodies, highly variable in shape and colour, which can be found in every terrestrial biome. Despite their prevalence, they are paradoxically absent from environmental DNA sampling studies. We obtained myxomycete SSU rRNA gene sequences from soil-extracted RNAs using specific primers. Soil samples were collected in three mountain ranges (France, Scotland and Japan). Our study revealed an unexpectedly high diversity of dark-spored Myxomycetes, with the recovery of 74 phylotypes. Of these, 74% had <98% identity with known sequences, showing a hidden diversity; there was little overlap between localities, implying biogeographical patterns. Few phylotypes were dominant and many were unique, consistent with the rare biosphere' phenomenon. Our study provides the first detailed insight into the community composition of this ecologically important group of protists, establishing means for future studies of their distribution, abundance and ecology.
  • Takato Matsui, Hisaya Kojima, Manabu Fukui
    ESTUARINE COASTAL AND SHELF SCIENCE 119 139 - 144 0272-7714 2013/03 [Refereed][Not invited]
     
    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 1466-5026 2013/02 [Refereed][Not invited]
     
    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 Iizuka, Barbara Delmonte, Ikumi Oyabu, Torbjorn Karlin, Valter Maggi, Samuel Albani, Manabu Fukui, Takeo Hondoh, Margareta Hansson
    TELLUS SERIES B-CHEMICAL AND PHYSICAL METEOROLOGY 65 (1) 1 - 9 0280-6509 2013 [Refereed][Not invited]
     
    Antarctic ice cores preserve the record of past aerosols, an important proxy of past atmospheric chemistry. Here we present the aerosol compositions of sulphate and chloride particles in the Talos Dome (TD) ice core from the Holocene and Last Glacial Period. We find that the main salt types of both periods are NaCl, Na2SO4 and CaSO4, indicating that TD ice contains relatively abundant sea salt (NaCl) from marine primary particles. By evaluating the molar ratio of NaCl to Na2SO4, we show that about half of the sea salt does not undergo sulphatisation during late Holocene. Compared to in inland Antarctica, the lower sulphatisation rate at TD is probably due to relatively little contact between sea salt and sulphuric acid. This low contact rate can be related to a reduced time of reaction for marine-sourced aerosol before reaching TD and/or to a reduced post-depositional effect from the higher accumulation rate at TD. Many sulphate and chloride salts are adhered to silicate minerals. The ratio of sulphate-adhered mineral to particle mass and the corresponding ratio of chloride-adhered mineral both increase with increasing dust concentration. Also, the TD ice appears to contain Ca(NO3)(2) or CaCO3 particles, thus differing from aerosol compositions in inland Antarctica, and indicating the proximity of peripheral regions to marine aerosols.
  • 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 0883-2927 2012/12 [Refereed][Not invited]
     
    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 0099-2240 2012/09 [Refereed][Not invited]
     
    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 1342-6311 2012/09 [Refereed][Not invited]
     
    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 0723-2020 2012/06 [Refereed][Not invited]
     
    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 0095-3628 2012/04 [Refereed]
     
    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.
  • Anna Maria Fiore-Donno, Akiko Kamono, Marianne Meyer, Martin Schnittler, Manabu Fukui, Thomas Cavalier-Smith
    PLOS ONE 7 (4) 1932-6203 2012/04 [Refereed][Not invited]
     
    The phylogenetic position of the slime-mould genus Lamproderma (Myxomycetes, Amoebozoa) challenges traditional taxonomy: although it displays the typical characters of the order Stemonitales, it appears to be sister to Physarales. This study provides a small subunit (18S or SSU) ribosomal RNA gene-based phylogeny of Lamproderma and its allies, with new sequences from 49 specimens in 12 genera. We found that the order Stemonitales and Lamproderma were both ancestral to Physarales and that Lamproderma constitutes several clades intermingled with species of Diacheopsis, Colloderma and Elaeomyxa. We suggest that these genera may have evolved from Lamproderma by multiple losses of fruiting body stalks and that many taxonomic revisions are needed. We found such high genetic diversity within three Lamproderma species that they probably consist of clusters of sibling species. We discuss the contrasts between genetic and morphological divergence and implications for the morphospecies concept, highlighting the phylogenetically most reliable morphological characters and pointing to others that have been overestimated. In addition, we showed that the first part (similar to 600 bases) of the SSU rDNA gene is a valuable tool for phylogeny in Myxomycetes, since it displayed sufficient variability to distinguish closely related taxa and never failed to cluster together specimens considered of the same species.
  • Masazumi Tsutsumi, Hisaya Kojima, Manabu Fukui
    MICROBES AND ENVIRONMENTS 27 (1) 67 - 71 1342-6311 2012/03 [Refereed]
     
    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 0948-3055 2012 [Refereed][Not invited]
     
    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 0095-3628 2011/11 [Refereed]
     
    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 1466-5026 2011/07 [Refereed]
     
    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.
  • Kyoko Kubo, Katrin Knittel, Rudolf Amann, Manabu Fukui, Katsumi Matsuura
    SYSTEMATIC AND APPLIED MICROBIOLOGY 34 (4) 293 - 302 0723-2020 2011/06 [Refereed]
     
    At the Nakabusa hot spring, Japan, dense olive-green microbial mats develop in regions where the slightly alkaline, sulfidic effluent has cooled to 65 degrees C. The microbial community of such mats was analyzed by focusing on the diversity, as well as the in situ distribution and function of bacteria involved in sulfur cycling. Analyses of 16S rRNA and functional genes (aprA, pufM) suggested the importance of three thermophilic bacterial groups: aerobic chemolithotrophic sulfide-oxidizing species of the genus Sulfurihydrogenibium (Aquificae), anaerobic sulfate-reducing species of the genera Thermodesulfobacterium/Thermodesulfatator, and filamentous anoxygenic photosynthetic species of the genus Chloroflexus. A new oligonucleotide probe specific for Sulfurihydrogenibium was designed and optimized for catalyzed reporter deposition fluorescence in situ hybridization (CARD-FISH). In situ hybridizations of thin mat sections showed a heterogeneous vertical distribution of Sulfurihydrogenibium and Chloroflexus. Sulfurihydrogenibium dominated near the mat surface (50% of the total mat biovolume), while Chloroflexus dominated in deeper layers (up to 64% of the total mat biovolume). Physiological experiments monitoring in vitro changes of sulfide concentration indicated slight sulfide production by sulfate-reducing bacteria under anoxic-dark conditions, sulfide consumption by photosynthetic bacteria under anoxic-light conditions and strong sulfide oxidation by chemolithotrophic members of Aquificae under oxic-dark condition. We therefore propose that Sulfurihydrogenibium spp. act as highly efficient scavengers of oxygen from the spring water, thus creating a favorable, anoxic environment for Chloroflexus and Thermodesulfobacterium/Thermodesulfatator in deeper layers. (C) 2011 Elsevier GmbH. All rights reserved.
  • Masazumi Tsutsumi, Tomoya Iwata, Hisaya Kojima, Manabu Fukui
    FRESHWATER BIOLOGY 56 (2) 342 - 351 0046-5070 2011/02 [Refereed]
     
    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.
  • Hisaya Kojima, Manabu Fukui
    INTERNATIONAL JOURNAL OF SYSTEMATIC AND EVOLUTIONARY MICROBIOLOGY 60 2862 - 2866 1466-5026 2010/12 [Refereed]
     
    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 0095-3628 2010/10 [Refereed]
     
    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 0095-3628 2010/04 [Refereed]
     
    "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.
  • Anna Maria Fiore-Donno, Akiko Kamono, Ema E. Chao, Manabu Fukui, Thomas Cavalier-Smith
    JOURNAL OF EUKARYOTIC MICROBIOLOGY 57 (2) 189 - 196 1066-5234 2010/03 [Refereed]
     
    The genus Hyperamoeba Alexeieff, 1923 was established to accommodate an aerobic amoeba exhibiting three life stagesamoeba, flagellate, and cyst. As more species/strains were isolated, it became increasingly evident from small subunit (SSU) gene phylogenies and ultrastructure that Hyperamoeba is polyphyletic and its species occupy different positions within the class Myxogastria. To pinpoint Hyperamoeba strains within other myxogastrid genera we aligned numerous myxogastrid sequences: whole small subunit ribosomal (SSU or 18S rRNA) gene for 50 dark-spored (i.e. Stemonitida and Physarida) Myxogastria (including a new "Hyperamoeba''/Didymium sequence) and a similar to 400-bp SSU fragment for 147 isolates assigned to 10 genera of the order Physarida. Phylogenetic analyses show unambiguously that the type species Hyperamoeba flagellata is a Physarum (Physarum flagellatum comb. nov.) as it nests among other Physarum species as robust sister to Physarum didermoides. Our trees also allow the following allocations: five Hyperamoeba strains to the genus Stemonitis; Hyperamoeba dachnaya, Pseudodidymium cryptomastigophorum, and three other Hyperamoeba strains to the genus Didymium; and two further Hyperamoeba strains to the family Physaridae. We therefore abandon the polyphyletic and redundant genus Hyperamoeba. We discuss the implications for the ecology and evolution of Myxogastria, whose amoeboflagellates are more widespread than previous inventories supposed, being now found in freshwater and even marine environments.
  • Takano, Y, Yokoyama, Y, Tyler, J.J, Fukui, M, Sato, T, Ogawa, N.O, Suzuki, N, Kitazato, H, Ohkouchi, N
    Biogeosciences Discuss (7) 4341 - 4384 2010 [Refereed][Not invited]
  • Hisaya Kojima, Haruo Fukuhara, Manabu Fukui
    SYSTEMATIC AND APPLIED MICROBIOLOGY 32 (6) 429 - 437 0723-2020 2009/09 [Refereed]
     
    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.
  • Hisaya Kojima, Tomoya Iwata, Manabu Fukui
    FRESHWATER BIOLOGY 54 (7) 1501 - 1509 0046-5070 2009/07 [Refereed]
     
    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.
  • Yuriko Higashioka, Hisaya Kojima, Tatsunori Nakagawa, Shinya Sato, Manabu Fukui
    BIODEGRADATION 20 (3) 383 - 390 0923-9820 2009/06 [Refereed]
     
    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.
  • Akiko Kamono, Jun Matsumoto, Hisaya Kojima, Manabu Fukui
    SOIL BIOLOGY & BIOCHEMISTRY 41 (6) 1324 - 1330 0038-0717 2009/06 [Refereed]
     
    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.
  • Masazumi Tsutsumi, Hisaya Kojima, Shigeru Uemura, Kiyomi Ono, Akihiro Sumida, Toshihiko Hara, Manabu Fukui
    SOIL BIOLOGY & BIOCHEMISTRY 41 (2) 403 - 408 0038-0717 2009/02 [Refereed]
     
    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 0028-1042 2009/01 [Refereed][Not invited]
     
    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.
  • Tatsunori Nakagawa, Shinya Sato, Manabu Fukui
    BIODEGRADATION 19 (6) 909 - 913 0923-9820 2008/11 [Refereed]
     
    Anaerobic degradation of p-xylene was studied with sulfate-reducing enrichment culture. The enrichment culture was established with sediment-free sulfate-reducing consortium on crude oil. The crude oil-degrading consortium prepared with marine sediment revealed that toluene, and xylenes among the fraction of alkylbenzene in the crude oil were consumed during the incubation. The PCR-denaturing gradient gel electrophoresis (DGGE) analysis of 16S rRNA gene for the p-xylene degrading sulfate-reducing enrichment culture showed the presence of the single dominant DGGE band pXy-K-13 coupled with p-xylene consumption and sulfide production. Sequence analysis of the DGGE band revealed a close relationship between DGGE band pXy-K-13 and the previously described marine sulfate-reducing strain oXyS1 (similarity value, 99%), which grow anaerobically with o-xylene. These results suggest that microorganism corresponding to pXy-K-13 is an important sulfate-reducing bacterium to degrade p-xylene in the enrichment culture.
  • Natsuko Hamamura, Manabu Fukui, David M. Ward, William P. Inskeep
    ENVIRONMENTAL SCIENCE & TECHNOLOGY 42 (20) 7580 - 7586 0013-936X 2008/10 [Refereed]
     
    The effect of temperature as a determinant for selecting microbial populations associated with alkane-degradation was examined in crude oil-amended soil microcosms. After a 30-day incubation, >95% of n-alkane components in the crude-oil were depleted and approximately 40 and 60% of added [C-14] hexadecane was converted to (CO2)-C-14 at 4-10 and 25 degrees C, respectively. Concomitant with crude-oil depletion, 16S rRNA gene sequence analysis revealed the emergence of a prominent Rhodococcus-like 16S rRNA sequence at all temperatures and a prominent Pseudomonas-like sequence at 4 and 10 degrees C. The diversity of alkane hydroxylase genes (AB) associated with the amendments was examined using group-specific alkB-PCR primers targeting phylogenetically distinct groups of alkane-degrading bacteria and subsequent cloning, denaturing gradient gel electrophoresis and sequencing analyses. Diverse Rhodococcus-alkB genes were detected at all temperatures, while a single prominent Pseudomonas-alkB genotype was detected only at lower temperatures. Two isolates obtained from the microcosms were shown to have 16S rRNA and AB genes identical to those observed and were used to examine growth as a function of temperature. The Pseudomonas isolate exhibited a substantially higher growth rate at 4 and 10 degrees C than the Rhodococcus isolate, consistent with the inference that differences in adaptation to low temperature explain the observed shift in populations. High resolution analysis of alkB genes enabled the differentiation of distinct alkane-degrading populations responding to crude-oil amendment from other closely related, well-studied strains with different temperature adaptations.
  • Hisaya Kojima, Takuo Nakajima, Manabu Fukui
    FEMS MICROBIOLOGY ECOLOGY 59 (1) 23 - 31 0168-6496 2007/01 [Refereed]
     
    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.
  • Akiko Kamono, Manabu Fukui
    JOURNAL OF MICROBIOLOGICAL METHODS 67 (3) 496 - 506 0167-7012 2006/12 [Refereed]
     
    Ecological studies of myxomycetes have been limited by the absence of universal cultivation techniques and the lack of life stage independent identification methods. We designed a novel PCR primer pair for the specific amplification of small subunit ribosomal RNA gene of Didymiaceae and Physaraceae. The primers produced amplicons from 192 fruiting body samples belonging to 10 genera. Twenty-four samples yielded longer fragments and sequence analysis revealed the presence of intron(s). As for the exonic regions, while sequence heterogeneities within a single species/varietas/forma were frequently observed, identical sequences were obtained only from identical species/varietas. The effectiveness of this primer pair in the analysis of morphologically unidentifiable samples was confirmed with the applications to samples of environmental plasmodium/sclerotium and soil. Denaturing gradient gel electrophoresis analysis was also tested with the soil samples. The results presented here demonstrate this PCR-based method can facilitate further ecological studies of Physaraceae and Didymiaceae in the environment. (c) 2006 Elsevier B.V. All rights reserved.
  • Hisaya Kojima, Yoshikazu Koizumi, Manabu Fukui
    MICROBIAL ECOLOGY 52 (4) 765 - 773 0095-3628 2006/11 [Refereed]
     
    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.
  • FUKUHARA Haruo, OHTAKA Akifumi, KIMURA Naoya, KIKUCHI Yoshiaki, YAMAMOTO Yoko, OCHIAI Masahiro, FUKUI Manabu, NOHARA Seiichi, Oze Akashibo Research Group
    Japanese Journal of Limnology (Rikusuigaku Zasshi) The Japanese Society of Limnology 67 (2) 81 - 93 0021-5104 2006/08/20 [Refereed]
     
    Various invertebrates, nematodes, oligochaetes, harpacticoids, tardigrades, cladocerans, mites, larvae of dipteran insects (tipulids, ceratopogonids and chironomids), appeared internivean and supranivean in Akashibo snow, a kind of red snow in the Ozegahara Mire, an extensive marshland in Central Japan (altitude 1400 m, area 7.6 km2). Densities of total animals were 2.4-3.6×104 ind m-2 in internivean and 0.3-2.2×103 ind m-2 on supranivean. These invertebrates seemed to migrate up from the peat surface into snow due to a decline of decrease in oxygen concentrations at subnivean environments and in/on snow during the development of Akashibo. The food web on Akashibo snow is discussed from the viewpoint of snow ecology.
  • FUJIMOTO Junji, FUKUI Manabu
    BIFIDUS--Flores,Fructus et Semina JAPAN BIFIDUS FOUNDATION 19 (1) 47 - 52 1343-0882 2005/01/01 [Refereed]
  • Maruyama Akihiko, Sunamura Michinari, Fukui Manabu, Kurusu Yasuro
    Journal of the Oceanographical Society of Japan The Oceanographic Society of Japan 14 (2) 309 - 318 0916-8362 2005 [Refereed]
     
    Microbial ecosystems associated with hydrothermally active layers (e.g., hydrothermal plume, venting seafloor and sub-vent layers) inside the caldera of Suiyo Seamount (ca. 1, 000∿1, 400 m in the depth), Izu-Bonin Arc, were investigated in the framework of Archaean Park Project using a portable marine driller, manned and unmanned submersibles, water samplers, in situ filtration systems, and in situ growth chambers. Through quantitative population analysis of microbes in the hydrothermal plume, we found that this caldera has functioned as a natural continuous incubator for only two phylotypes of Bacteria in the deep sea. We also discovered several new phylotypes of microbes belonging to the epsilon-Proteobacteria, photosynthetic alpha-Proteobacteria, and chemoautotrophic thermophilic Archaea. These were found in samples collected from natural vents and boreholes using a newly developed catheter-type in situ growth chamber. From one of the growth chamber samples, a new dissimilatory sulfite reductase gene sequence was obtained. We also succeeded in identifying a gene region which regulates optimal growth temperatures of microbes, and we tried to apply this gene information to analysis of natural microbial communities.
  • Y Koizumi, H Kojima, M Fukui
    APPLIED AND ENVIRONMENTAL MICROBIOLOGY 70 (8) 4930 - 4940 0099-2240 2004/08 [Refereed][Not invited]
     
    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.
  • Tatsunori Nakagawa, Jun-Ichiro Ishibashi, Akihiko Maruyama, Toshiro Yamanaka, Yusuke Morimoto, Hiroyuki Kimura, Tetsuro Urabe, Manabu Fukui
    Applied and Environmental Microbiology 70 (1) 393 - 403 0099-2240 2004/01 [Refereed][Not invited]
     
    This study describes the occurrence of unique dissimilatory sulfite reductase (DSR) genes at a depth of 1,380 m from the deep-sea hydrothermal vent field at the Suiyo Seamount, Izu-Bonin Arc, Western Pacific, Japan. The DSR genes were obtained from microbes that grew in a catheter-type in situ growth chamber deployed for 3 days on a vent and from the effluent water of drilled holes at 5°C and natural vent fluids at 7°C. DSR clones SUIYOdsr-A and SUIYOdsr-B were not closely related to cultivated species or environmental clones. Moreover, samples of microbial communities were examined by PCR-denaturing gradient gel electrophoresis (DGGE) analysis of the 16S rRNA gene. The sequence analysis of 16S rRNA gene fragments obtained from the vent catheter after a 3-day incubation revealed the occurrence of bacterial DGGE bands affiliated with the Aquificae and γ- and ε-Proteobacteria as well as the occurrence of archaeal phylotypes affiliated with the Thermococcales and of a unique archaeon sequence that clustered with "Nanoarchaeota." The DGGE bands obtained from drilled holes and natural vent fluids from 7 to 300°C were affiliated with the δ-Proteobacteria, genus Thiomicrospira, and Pelodictyon. The dominant DGGE bands retrieved from the effluent water of casing pipes at 3 and 4°C were closely related to phylotypes obtained from the Arctic Ocean. Our results suggest the presence of microorganisms corresponding to a unique DSR lineage not detected previously from other geothermal environments.
  • Tatsunori Nakagawa, Manabu Fukui
    Applied and Environmental Microbiology 69 (12) 7044 - 7057 0099-2240 2003/12 [Refereed][Not invited]
     
    Community structures of submerged microbial slime streamers (SMSS) in sulfide-containing hot springs at 72 to 80°C at Nakabusa and Yumata, Japan, were investigated by molecular analysis based on the 16S rRNA gene. The SMSS were classified into two consortia consortium I occurred at lower levels of sulfide in the hot springs (less than 0.1 mM), and consortium II dominated when the sulfide levels were higher (more than 0.1 mM). The dominant cell morphotypes in consortium I were filamentous and small rod-shaped cells. The filamentous cells hybridized with fluorescent oligonucleotide probes for the domain Bacteria, the domain Archaea, and the family Aquificaceae. Our analysis of the denaturing gradient gel electrophoresis (DGGE) bands by using reverse transcription (RT)-PCR amplification with two primer sets (Eub341-F with the GC clamp and Univ907R for the Bacteria and Eub341-F with the GC clamp and Arch915R) indicated that dominant bands were phylogenetically related to microbes in the genus Aquifex. On the other hand, consortium II was dominated by long, small, rod-shaped cells, which hybridized with the oligonucleotide probe S-*-Tdes-0830-a-A-20 developed in this study for the majority of as-yet-uncultivated microbes in the class Thermodesulfobacteria. The dominant DGGE band obtained by PCR and RT-PCR was affiliated with the genus Sulfurihydrogenibium. Moreover, our analysis of dissimilatory sulfite reductase (DSR) gene sequences retrieved from both consortia revealed a high frequency of DSR genes corresponding to the DSR of Thermodesulfobacteria-like microorganisms. Using both sulfide monitoring and 35SO42- tracer experiments, we observed microbial sulfide production and consumption by SMSS, suggesting that there is in situ sulfide production by as-yet-uncultivated Thermodesulfobacteria-like microbes and there is in situ sulfide consumption by Sulfurihydrogenibium-like microbes within the SMSS in the Nakabusa and Yumata hot springs.
  • Y Nakajima, H Okada, K Oguri, H Suga, H Kitazato, Y Koizumi, M Fukui, N Ohkouchi
    ENVIRONMENTAL MICROBIOLOGY 5 (11) 1103 - 1110 1462-2912 2003/11 [Refereed][Not invited]
     
    We investigated the distribution of chloropigments in a small meromictic lake, Lake Kaiike, south-west Japan. In the water-column, concentrations of Chl a related to cyanobacteria, BChl a related to purple sulphur bacteria, and three types of BChl e homologues (BChls e(1), e(2) and e(3)) related to brown-coloured green sulphur bacteria, were maximal at the redox boundary. Below the redox boundary, absolute concentrations of Chl a and BChl a gradually decreased with depth, whereas BChls e remained rather constant. Suspended particulate matter (SPM) at the deeper region of the anoxic water-column was enriched in highly alkylated BChl e homologues compared with SPM at the redox boundary. The shift in the relative content of highly alkylated BChl e homologues beneath the boundary was associated with community related adaptation of brown-coloured green sulphur bacteria to changes in light quality/quantity, resulting from the optical absorption and reflectance of SPMs in the overlying water-column. Benthic microbial mats were characterized by high abundances of BChls e, in which highly alkylated homologues were substantially abundant. This suggests that the BChls e in the microbial mat may be derived from the low-light adapted brown-coloured green sulphur bacteria forming the bacterial mat.
  • Y Koizumi, H Kojima, M Fukui
    FEMS MICROBIOLOGY ECOLOGY 46 (2) 147 - 157 0168-6496 2003/11 [Refereed][Not invited]
     
    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.
  • H Kojima, M Fukui
    WATER RESEARCH 37 (13) 3216 - 3223 0043-1354 2003/07 [Refereed][Not invited]
     
    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.
  • Toshiya Katano, Manabu Fukui
    Phycological Research 51 (2) 71 - 76 1322-0829 2003 [Refereed][Not invited]
     
    Quantitative accuracy based on the fluorescent intensity of bands in a denaturing gradient gel electrophoresis (DGGE) profile of polymerase chain reaction (PCR)-amplified 16S rRNA gene fragments was evaluated for the molecular inference of dominant populations using a cyanobacterial primer pair in a picocyanobacterial community. A serial dilution technique of the template prior to PCR of extracted nucleic acids allowed for elimination of minor strains (less than 10% of the whole cell number) using a cell mixture of three known cultured Synechococcus species with different ratios. When the most abundant strain among the three accounted for more than 80% of the cells, the single band derived from the most abundant one was detected exclusively after the template dilution. In the case of two or three strains evenly distributed in the sample, all strains remained as bands after template dilution. The technique used in the present study was also applied to lake water samples collected from depths of 1 and 5 m on 27 August 1999. The same dominant Synechococcus population was detected in both samples. Thus, the template-dilution technique prior to PCR is useful to determine dominant picocyanobacterial populations in the DGGE profiling.
  • H Kojima, A Teske, M Fukui
    APPLIED AND ENVIRONMENTAL MICROBIOLOGY 69 (1) 390 - 398 0099-2240 2003/01 [Refereed][Not invited]
     
    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.
  • Tatsunori Nakagawa, Manabu Fukui
    Journal of General and Applied Microbiology 48 (4) 211 - 222 0022-1260 2002 [Refereed][Not invited]
     
    Dense microbial mats and streamers of various colors developed in an alkaline-hot spring water at 48-76°C and ∼0.077 mM sulfide in Nakabusa, Japan. The microbial community structures with a thermal gradient were compared by denaturing gradient gel electrophoresis (DGGE) analysis of the PCR-amplified 16S ribosomal RNA gene fragments. The sequence analysis revealed that a predominant cyanobacterial DGGE band phylogenetically related to Synechococcus elongatus was detected only from green mats at 48°C. Four DGGE bands were detected commonly from green mats at 48°C, orange mats at 58°C and brown mats at 60°C. The sequence analysis revealed that these were phylogenetically related to Chloroflexaceae group, Rhodothermus group, a candidate division OP10, and an unclassified bacterium. On the other hand, Aquificae-, Thermodesulfobacteria-, Thermus group-, and Crenarchaeota-like sequences were detected as a predominant component of DGGE profiling from the streamers only at temperatures over 66°C, but no phototrophic bacterial bands were detected. Thus, the microbial community structure above 60°C was drastically different from that at the lower temperatures. After the addition of hydrogen into in vitro gray streamers with in situ spring water, sulfide production markedly occurred in the presence of ambient sulfate at 66°C. This result suggests that in situ sulfide is partly produced by Thermodesulfobacteria-like sulfate-reducing bacteria in the streamers.
  • Hiroya Shinozaki, Manabu Fukui
    Journal of General and Applied Microbiology 48 (3) 173 - 176 0022-1260 2002 [Refereed][Not invited]
  • Yoshikazu Koizumi, John J. Kelly, Tatsunori Nakagawa, Hidetoshi Urakawa, Saïd El-Fantroussi, Saleh Al-Muzaini, Manabu Fukui, Yoshikuni Urushigawa, David A. Stahl
    Applied and Environmental Microbiology 68 (7) 3215 - 3225 0099-2240 2002 [Refereed][Not invited]
     
    A mesophilic toluene-degrading consortium (TDC) and an ethylbenzene-degrading consortium (EDC) were established under sulfate-reducing conditions. These consortia were first characterized by denaturing gradient gel electrophoresis (DGGE) fingerprinting of PCR-amplified 16S rRNA gene fragments, followed by sequencing. The sequences of the major bands (T-1 and E-2) belonging to TDC and EDC, respectively, were affiliated with the family Desulfobacteriaceae. Another major band from EDC (E-1) was related to an uncultured non-sulfate-reducing soil bacterium. Oligonucleotide probes specific for the 16S rRNAs of target organisms corresponding to T-1, E-1, and E-2 were designed, and hybridization conditions were optimized for two analytical formats, membrane and DNA microarray hybridization. Both formats were used to characterize the TDC and EDC, and the results of both were consistent with DGGE analysis. In order to assess the utility of the microarray format for analysis of environmental samples, oil-contaminated sediments from the coast of Kuwait were analyzed. The DNA microarray successfully detected bacterial nucleic acids from these samples, but probes targeting specific groups of sulfate-reducing bacteria did not give positive signals. The results of this study demonstrate the limitations and the potential utility of DNA microarrays for microbial community analysis.
  • Tatsunori Nakagawa, Satoshi Hanada, Akihiko Maruyama, Katsumi Marumo, Tetsuro Urabe, Manabu Fukui
    FEMS Microbiology Ecology 41 (3) 199 - 209 0168-6496 2002 [Refereed][Not invited]
     
    The distribution and diversity of thermophilic sulfate-reducing bacteria at the Cu-Pb-Zn Toyoha underground mine, Japan, were investigated using denaturing gradient gel electrophoresis analysis based on the 16S rRNA gene, and sequence analysis of the dissimilatory sulfite reductase gene. Hydrothermal waters from different boreholes penetrating the Cu-Pb-Zn sulfide veins were collected and concentrated with a sterile filter (pore size: 0.2 μm) at sites A (64°C), B (71°C), and C (48°C). Microbial mats developed at sites A (53°C), B (66°C), and D (73°C) were harvested. The denaturing gel electrophoresis analysis showed 17 bacterial and three archaeal bands including two of spore-forming, Gram-positive sulfate-reducing bacteria, Desulfotomaculum-like 16S rDNA sequences from site B. The phylogenetic analysis of 16 clone families of dissimilatory sulfite reductase genes indicated that they are Desulfotomaculum-, Thermodesulforhabdus-like sequences, and unresolved sequences. We obtained evidence of the diversity and distribution of microbes related to thermophilic sulfate-reducing bacteria within effluent-hydrothermal groundwater and microbial mats in the thermophilic subsurface environment of the Toyoha Mine. © 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.
  • Tatsunori Nakagawa, Shinya Sato, Yoko Yamamoto, Manabu Fukui
    Water Research 36 (11) 2813 - 2823 0043-1354 2002 [Refereed][Not invited]
     
    The microbial community structure and successive changes in a mesophilic ethylbenzene-degrading sulfate-reducing consortium were for the first time clarified by the denaturing gradient gel electrophoresis (DGGE) analysis of the PCR amplified 16S rRNA gene fragments. At least ten bands on the DGGE gel were detected in the stationary phase. Phylogenetic analysis of the DGGE bands revealed that the consortium consisted of different eubacterial phyla including the δ subgroup of Proteobacteria, the order Sphingobacteriales, the order Spirochaetales, and the unknown bacterium. The most abundant band C was closely related to strain mXyS1, an m-xylene-degrading sulfate-reducing bacterium (SRB), and occurred as a sole band on DGGE gels in the logarithmic growth phase that 40% ethylbenzene was consumed accompanied by sulfide production. During further prolonged incubation, the dominancy of band C did not change. These results suggest that SRB corresponds to the most abundant band C and contributes mainly to the degradation of ethylbenzene coupled with sulfate reduction. © 2002 Elsevier Science Ltd. All rights reserved.
  • Oguri Kazumasa, Itou Masashi, Hirano Satoshi, Hisamitsu Toshio, Sakai Saburo, Murayama Masafumi, Kitazato Hiroshi, Koizumi Yoshikazu, Fukui Manabu, Taira Asahiko
    Jour. Geol. Soc. Japan The Geological Society of Japan 108 (12) XXIII - XXIV 0016-7630 2002 [Refereed][Not invited]
     
    鹿児島県上甑島に位置する貝池は, 礫洲によって外海と隔てられた汽水湖である. 湖の表層水は陸水の影響を受け約20PSUの低塩分を呈するが, 水深約3m以深は礫洲を通した海水の侵入により30PSUを越える. このため湖水は恒常的な密度成層状態にあり, 水深5mから湖底までの溶存酸素量はOml/1を示した. 水深4.5～5.0m には光合成バクテリアと思われる微生物の濃密な分布が確認され, 堆積物最表層部分には繊維状の微生物による赤黒色のマットが形成されていることが明らかになった. 不撹乱採取された堆積物コアから, 厚さ1mm未満のマット状の構造が約3cm にわたり積層していることが確認された. これらのマットは赤黒色で繊維質, 青緑色で均質, 黄黒色で繊維質など, 各々の構造は異なる. また堆積物内部には細かいラミナが何枚も観察された. 今後の研究によって無酸素環境におけるバクテリア活動と, 形成されたマットが堆積物中に保存されていく過程の解明が期待される.
  • Kousuke Ishii, Manabu Fukui
    Applied and Environmental Microbiology 67 (8) 3753 - 3755 0099-2240 2001/08 [Refereed][Not invited]
     
    To reduce PCR bias derived from a primer mismatch, the effect of the annealing temperature on the product ratio was investigated by denaturing gradient gel electrophoresis analysis of PCR products from a mixture of perfect-match and one-mismatch templates. These templates were generated by PCR from Pediococcus acidilactici for one mismatch and Micrococcus luteus for the perfect match. PCRs showed that the bias was reduced at lower temperatures. An environmental sample was also examined.
  • Toshiya Katano, Manabu Fukui, Yasunori Watanabe
    Limnology 2 (3) 213 - 218 1439-8621 2001 [Refereed][Not invited]
     
    Eight cultured strains (OK01, OK02, OK03, OK05, OK07, OK08, OK09, and OK10) of picocyanobacteria were isolated from Lake Okutama. Five cyanobacterial DNA fragments (DGGE bands B4, B5, B6, B7, and B8) were obtained from the lake water samples by denaturing gradient gel electrophoresis (DGGE) after polymerase chain reaction (PCR) amplification of 16S ribosomal genes. To classify the picocyanobacterial strains and the DGGE bands, a partial sequence of 16S rDNA was used. Among seven strains, OK01, OK07, and OK09 were identified as the genus Synechococcus and OK02 and OK05 as the genus Phormidium. OK03 was identified as the genus Oscillatoria and was closely related to B4 (100% homology). B5, B6, B7, and B8 were related to the genus Synechococcus. These results revealed that the picocyanobacteria in the lake are phylogenetically diverse. PCR-DGGE analysis is a useful tool to determine picocyanobacterial community structure in freshwater environments.
  • Manabu Fukui, Jung-In Suh, Yoshikuni Urushigawa
    Water Research 34 (5) 1515 - 1524 0043-1354 2000/04 [Refereed][Not invited]
     
    To clarify the interaction between sulfidogens and methanogens in municipal anaerobic digesters, in situ substrate utilization by the two microbial groups was characterized by the accumulation rates of substrates after the addition of specific inhibitors 20 mM of molybdate for sulfidogens and 0.01% (v/v) of chloroform for methanogens in three anaerobic sludge digesters with different concentrations of sulfate (A, 1.2 B, 1.8 C, 2.7 mM). The rates of sulfate reduction and methane production in sludge C were the highest and the lowest, respectively among the three sludges. The major substrates for methanogens were acetate (35-58%), and propionate (17-22%) in all the sludges. Propionate should be utilized by methanogens after conversion to acetate and H2 by syntrophic hydrogen-producing acetogens, as no methanogens are known to directly utilize propionate. Detectable accumulated substrates for sulfidogens (< 20%) were propionate in sludges A and B and acetate in C. Still approximately 80% of substrates for sulfidogens were unknown. These results show that acetate or propionate is a common substrate for sulfidogens and methanogens but sulfidogens can mainly grow on the other substrates in the municipal anaerobic sludges, implying nutritional segregation between sulfidogens and methanogens. (C) 2000 Elsevier Science Ltd.
  • 石井 浩介, 中川 達功, 福井 学
    Microbes and environments 日本微生物生態学会 15 (1) 59 - 73 1342-6311 2000/03/31 [Refereed]
     
    The use of Denaturing Gradient Gel Electrophoresis (DGGE) has been widely recognized in microbial ecology and its importance has increased recently. DNA extracted from the environment is amplified by PCR using a set of primers in which one of the two contains the 40 base GC-rich sequence at the 5'-end prior to DGGE. The PCR amplified DNA fragment migrates in a DGGE gel depending on its melting behavior. Moreover, the DNA resolved by DGGE can be promoted for further analyses, such as hybridization and sequencing without any cloning step. For these reasons, PCR-DGGE is one of the powerful tools available for the detection and phylogenetic analysis of environmental microbes. Although this method is easy and reproducible, some knowledge and technique are required to obtain reliable data. This technical series introduces protocols for the PCR-DGGE method with the theory presented to beginners as comprehensibly as possible. In addition, we discussed the application and limitation of the PCR-DGGE approach to microbial ecology.
  • K. Ishii, M. Fukui, S. Takii
    Journal of Applied Microbiology 89 (5) 768 - 777 1364-5072 2000 [Refereed][Not invited]
     
    Microbial succession during a laboratory-scale composting process of garbage was analysed by denaturing gradient gel electrophoresis (DGGE) combined with measurement of physicochemical parameters such as temperature, pH, organic acids, total dissolved organic carbon and water-soluble humic substance. From the temperature changes, a rapid increase from 25 to 58°C and then a gradual decrease, four phases were recognized in the process as follows mesophilic (S), thermophilic (T), cooling (C) and maturing (M). The polymerase chain reaction-amplified 16S rDNA fragments with universal (907R) and eubacterial (341F with GC clamp) primers were subjected to DGGE analysis. Consequently, the DGGE band pattern changed during the composting process. The direct sequences from DGGE bands were related to those of known genera in the DNA database. The microbial succession determined by DGGE was summarized as follows: in the S phase some fermenting bacteria, such as lactobacillus, were present with the existing organic acids in the T phase thermophilic bacillus appeared and, after the C phase, bacterial populations were more complex than in previous phases and the phylogenetic positions of those populations were relatively distant from strains so far in the DNA database. Thus, the DGGE method is useful to reveal microbial succession during a composting process.
  • Manabu Fukui, Andreas Teske, Bernhard Aßmus, Gerard Muyzer, Friedrich Widdel
    Archives of Microbiology 172 (4) 193 - 203 0302-8933 1999 [Refereed][Not invited]
     
    Microscopy of organic-rich, sulfidic sediment samples of marine and freshwater origin revealed filamentous, multicellular microorganisms with gliding motility. Many of these neither contained sulfur droplets such as the Beggiatoa species nor exhibited the autofluorescence of the chlorophyll- containing cyanobacteria. A frequently observed morphological type of filamentous microorganism was enriched under anoxic conditions in the dark with isobutyrate plus sulfate. Two strains of filamentous, gliding sulfate- reducing bacteria, Tokyo 01 and Jade 02, were isolated in pure cultures. Both isolates oxidized acetate and other aliphatic acids. Enzyme assays indicated that the terminal oxidation occurs via the anaerobic C1 pathway (carbon monoxide dehydrogenase pathway). The 16S rRNA genes of the new isolates and of the two formerly described filamentous species of sulfate-reducing bacteria, Desulfonema limicola and Desulfonema magnum, were analyzed. All four strains were closely related to each other and affiliated with the δ- subclass of Proteobacteria. Another close relative was the unicellular Desulfococcus multivorans. Based on phylogenetic relationships and physiological properties, Strains Tokyo 01 and Jade 02 are assigned to a new species, Desulfonema ishimotoi. A new, fluorescently labeled oligonucleotide probe targeted against 16S rRNA was designed so that that it hybridized specifically with whole cells of Desulfonema species. Filamentous bacteria that hybridized with the same probe were detected in sediment samples and in association with the filamentous sulfur-oxidizing bacterium Thioploca in its natural habitat. We conclude that Desulfonema species constitute an ecologically significant fraction of the sulfate-reducing bacteria in organic-rich sediments and microbial mats.
  • Andreas Teske, Niels B. Ramsing, Kirsten Habicht, Manabu Fukui, Jan Küver, Bo Barker Jørgensen, Yehuda Cohen
    Applied and Environmental Microbiology 64 (8) 2943 - 2951 0099-2240 1998/08 [Refereed][Not invited]
     
    The sulfate-reducing bacteria within the surface layer of the hypersaline cyanobacterial mat of Solar Lake (Sinai, Egypt) were investigated with combined microbiological, molecular, and biogeochemical approaches. The diurnally oxic surface layer contained between 106 and 107 cultivable sulfate-reducing bacteria ml-1 and showed sulfate reduction rates between 1,000 and 2,200 nmol ml-1 day-1, both in the same range as and sometimes higher than those in anaerobic deeper mat layers. In the oxic surface layer and in the mat layers below, filamentous sulfate-reducing Desulfonema bacteria were found in variable densities of 104 to 106 cells ml-1. A Desulfonema-related, diurnally migrating bacterium was detected with PCR and denaturing gradient gel electrophoresis within and below the oxic surface layer. Facultative aerobic respiration, filamentous morphology, motility, diurnal migration, and aggregate formation were the most conspicuous adaptations of Solar Lake sulfate-reducing bacteria to the mat matrix and to diurnal oxygen stress. A comparison of sulfate reduction rates within the mat and previously published photosynthesiS rates showed that CO2 from sulfate reduction in the upper 5 mm accounted for 7 to 8% of the total photosynthetic CO2 demand of the mat.
  • MacHiko Nishino, Manabu Fukui, Takuo Nakajima
    Water Research 32 (3) 953 - 957 0043-1354 1998/03/01 [Refereed][Not invited]
     
    Dense mats of Thioploca spp., gliding filamentous sulfur-oxidizing bacteria were first found in Japan in the sediments of the profundal areas of Lake Biwa, the largest lake in Japan. Thioploca spp. were widely distributed in the northern basin of Lake Biwa and were morphologically similar to Thioploca ingrica. The biomass of Thioploca was estimated and compared between the upper (0-2 cm) and the lower (deeper than 2 cm) layer sediments of three profundal stations. It was one to two orders of magnitude larger in the upper layer for all stations. Upper layer biomass was also compared among five stations of different depths from 3 to 90 m deep, and the most dense trichomes, 5.4 m per cm2 exhibited as total trichome length, were observed at the profundal station of 90 m deep, larger by three orders of magnitude than in the shallow stations. Several facts indicate that Thioploca has recently increased on the profundal bottoms of the lake.
  • Manabu Fukui, Jungin Suh, Yoshitaka Yonezawa, Yoshikuni Urushigawa
    Ecological Research 12 (2) 201 - 209 0912-3814 1997 [Refereed][Not invited]
     
    To clarify the anaerobic microbial interactions in the process of carbon mineralization in marine eutrophic environments, the microbial sulfate reduction and methane production rates were examined in coastal marine sediments of Ise Bay, Japan, in autumn 1990. Sulfate reduction rates (51-210 nmol m1-1 day-1 at 24°C) were much higher than the methane production ones (< 1.78 nmol m1-1 day-1) in the surface sediments (top 2 cm) at the six stations surveyed (water depth: 10.7-23.3 m). Substrates for sulfate-reducing bacteria (SRB) were estimated after the addition of a specific inhibitor for SRB (20 mmol 1-1 molybdate) into the sediment slurry, from the substrate accumulation rates. In the presence of the inhibitor, sulfate reduction was completely stopped and volatile fatty acids (mainly acetate) were accumulated, although hydrogen was not. Methane production occurred markedly accompanied by consumption of the accumulated acetate from the third day after the addition of molybdate. The maximum rate of methane production was 1.2-1.9 μmol ml-1 day-1, which was similar to those in highly polluted freshwater sediments such as the Tama River, Tokyo, Japan. These results show that acetate is a common major substrate for sulfate reduction and methane production, and SRB competitively inhibit potential acetoclastic methanogenesis in coastal sediments. Methanogens may potentially inhabit the sediments at low levels of population density and activity.
  • Ralf Rabus, Manabu Fukui, Heinz Wilkes, Friedrich Widdel
    Applied and Environmental Microbiology 62 (10) 3605 - 3613 0099-2240 1996/10 [Refereed][Not invited]
     
    A mesophilic sulfate-reducing enrichment culture growing anaerobically on crude oil was used as a model system to study which nutritional types of sulfate-reducing bacteria may develop on original petroleum constituents in oil wells, tanks, and pipelines. Chemical analysis of oil hydrocarbons during growth revealed depletion of toluene and o-xylene within 1 month and of m- xylene, o-ethyltoluene, m-ethyltoluene, m-propyltoluene, and m- isopropyltoluene within approximately 2 months. In anaerobic counting series, the highest numbers of CFU (6 x 106 to 8 x 106 CFU ml-1) were obtained with toluene and benzoate. Almost the same numbers were obtained with lactate, a substrate often used for detection of the vibrio-shaped, incompletely oxidizing Desulfovibrio sp. In the present study, however, lactate yielded mostly colonies of oval to rod-shaped, completely oxidizing, sulfate-reducing bacteria which were able to grow slowly on toluene or crude oil. Desulfovibrio species were detected only at low numbers (3 x 105 CFU ml-1). In agreement with this finding, a fluorescently labeled, 16S rRNA- targeted oligonucleotide probe described in the literature as specific for members of the Desulfovibrionaceae (suggested family) hybridized only with a small portion (< 5%) of the cells in the enrichment culture. These results are consistent with the observation that known Desulfovibrio species do not utilize aromatic hydrocarbons, the predominant substrates in the enrichment culture. All known sulfate-reducing bacteria which utilize aromatic compounds belong to a separate branch, the Desulfobacteriaceae (suggested family). Most members of this family are complete oxidizers. For specific hybridization with members of this branch, the probe had to be modified by a nucleotide exchange. Indeed, this modified probe hybridized with more than 95% of the cells in the enrichment culture. The results show that completely oxidizing, alkylbenzene-utilizing sulfate-reducing bacteria rather than Desulfovibrio species have to be considered in attempts to understand the microbiology of sulfide production in oil wells, tanks, and pipelines when no electron donors other than the indigenous oil constituents are available.
  • Partitioning of chlorobenzenes between suspended particulates and water in coastal water
    Shigeki Masunaga, Yoshitaka Yonezawa, Yoshikuni Urushigawa, Manabu Fukui
    Journal of Environmental Science and Health A31 887 - 903 1996 [Refereed]
  • Manabu Fukui, Susumu Takii
    Ecological Research 11 (3) 257 - 267 0912-3814 1996 [Refereed][Not invited]
     
    To clarify the ecological significance of the association of sulfate- reducing bacteria (SRB) with sediment particle size, SRB utilizing lactate (l-SRB), propionate (p-SRB) and acetate (a-SRB) were examined with different sizes of sediment particles in a hypertrophic freshwater lake using the anaerobic plate count method. The numbers of l-SRB and a-SRB were 104-105 colony forming units (CFU) per ml in the 0-3 cm layer and 102-103 CFU ml- 1 in the 10-13 cm layer, while the numbers of p-srb were one or two orders lower than those of l-SRB and a-SRB. A sediment suspension was fractionated into four fractions (< 1, 1-10, 10-94 and > 94 μm). The highest proportions of l-SRB and a-SRB were found in the 10-94 μm fraction: 66-97% for l-SRB and 53-98% for a-SRB. The highest proportion of p-SRB was found in the > 94 μm fraction (70-74%). These results indicate that most SRB were associated with sediment particles. One isolate from an acetate-utilizing enrichment culture was similar to Desulfotomaculum acetoxidans, a spore-forming sulfate-reducing bacterium. When lactate and sulfate were added to sediment samples, l-SRB and a-SRB in the < 10 μm-fraction grew more rapidly than those in whole sediment for the first 2 days. This result suggests that nutrients uptake by free- living and small particle-associated (< 10 μm) SRB is higher than that by SRB associated with larger particles.
  • Manabu Fukui, Yuichi Suwa, Yoshikuni Urushigawa
    FEMS Microbiology Ecology 19 (1) 17 - 25 0168-6496 1996 [Refereed][Not invited]
     
    Exponentially grown Desulfobacter latus cells were transferred to anaerobically prepared minimum medium without a carbon or energy substrate and incubated under anaerobic conditions. Changes in 16S ribosomal RNA (rRNA) of individual cells and the viable fraction in a population were monitored. The cell preparation was stained with a phylogenetic DNA probe labelled with fluorescent dye and the fluorescence of each cell was determined with confocal scanning laser microscope. Viable cells were defined as those capable of reducing a tetrazolium salt (the INT method [1]). The viability of a Desulfobacter starvation culture decreased to 85% in 48 h, but further decrease was not observed during prolonged starvation. The mean amount of 16S rRNA in individual cells decreased exponentially for 48 h to 30% the mean value obtained for exponentially glowing cells, but did not decrease by prolonged starvation. About 30% of the mean content of 16S rRNA in growing cells was found in the starved cell population, suggesting that most individual cells in the starved population were not metabolically active. The difference between gross pixel intensity of cells having < 8% of 16S rRNA in growing cells and those with a negative control probe was not significant. Thus, non-viable cells may not show positive signals by phylogenetic staining.
  • S. Takii, M. Fukui
    Hydrobiologia 319 (1) 37 - 45 0018-8158 1996 [Refereed][Not invited]
  • Kiyoshi Takeda, Manabu Fukui
    Bulletin of Japanese Society of Microbial Ecology Japanese Society of Microbial Ecology / The Japanese Society of Soil Microbiology 10 (1) 1 - 7 0911-7830 1995 [Refereed]
     
    Complete conversion of lactate to methane and CO2 by triculture with a sulfate reducer, strain F-1, a hydrogen-utilizing methanogen, strain HM-1, and an acetate-utilizing methanogen, strain AM-1, was studied with sulfate-free medium under N2 and CO2. Strain F-1 was motile, Gram-negative and vibrio-shaped cells which utilized H2, lactate and ethanol as electron donors. Strain F-1 were hybridized with the oligodeoxynucleotide probe complementary to specific regions of mesophilic Gram-negative sulfate-reducing bacteria (SRB probe). The G+C content of the DNA was 63.2mol%. Strain F-1 was tentatively classified into Desulfovibrio vulgaris. Strain F-1 was unable to degrade lactate in the sulfate-free medium under N2 and CO2. However, it degraded lactate in the dual culture with strain HM-1 in sulfate-free medium. Lactate was completely converted to methane and carbon dioxide by triculture with strain F-1, strain HM-1 and strain AM-1 which was a filamentous acetateutilizing methanogen. The triculture formed flocs and grew well on the bottom of culture flasks. Acetate accumulated in the dual culture was used concurrently with its production in the triculture. Acetate conversion to methane by strain AM-1 was not inhibited by hydrogen. Lactate was converted to methane and CO2 stoichiometrically by the triculture in a sulfate-free minimal medium. The 16S rRNA-targeted oligodeoxynucleotide fluorescent probe in situ hybridization method was used for identification and enumeration of strain F-1 in the microbial triculture ecosystem. A large number of strain F-1 cells hybridized with the SRB probe were observed while lactate was consumed at the early stage of incubation.
  • 益永茂樹, 米澤義尭, 漆川芳國, 福井学
    環境化学 4 (3) 619-629  1994/09 [Refereed][Not invited]
  • Toshifumi Konda, Susumu Takii, Manabu Fukui, Yasushi Kusuoka, Genki I. Matsumoto, Tetsuya Torii
    Japanese Journal of Limnology The Japanese Society of Limnology 55 (3) 185 - 192 0021-5104 1994 [Refereed]
     
    Vertical distributions of bacterial numbers, cell sizes, and biomass in the water of Lake Fryxell in the McMurdo Dry Valleys of the Antarctic were examined by the acridine orange direct count (AODC) method. Total bacterial numbers increased with increasing depth from 5.4×105 cells⋅ml-1 in the surface water (5.0 m depth) to 1.7×107 cells⋅ml-1 in the bottom water (19.0 m depth). Bacteria from waters at all sampling depths were dominated by rod-shaped cells having the average values in the ranges of 0.37-0.64 μm width and 1.04-1.85 μm length. However, those from waters with depth of between 10.0 and 12.5 m had relatively large mean cell sizes. Bacterial biomass estimated from cell volumes ranged from 0.017 to 0.29 mgC⋅-1, corresponding to from 0.75 to 5.9% of total organic carbon. A total of 80 isolates from the stored water samples were identified as Pseudomonas, Acinetobacter-Moraxella and Caulobacter. Sulfate-reducing bacteria were recovered from the anaerobic bottom water.
  • Yoshitaka Yonezawa, Manabu Fukui, Shigeki Masunaga, Yoshikuni Urushigawa
    Chemosphere 28 (12) 2179 - 2184 0045-6535 1994 [Refereed][Not invited]
     
    The relation between dechlorination activities of 1,2,4-trichlorobenzene and anaerobic microbial activity were studied in the sediment collected at three sites in Ise Bay in Japan. The degradation rate of spiked 1,2,4-trichlorobenzene (3nmol ml-1) ranged from 15 to 35 pmol day-1 ml-1 wet sediment and about 1 3 to 1 2 of degraded the trichlorobenzene was recovered as dechlorinated products. Among the dichlorobenzenes, the 1,2-isomer had the highest and 1,3-isomer had the lowest production rate. Comparing the three sampling sites, the trichlorobenzene degradation and dichlorobenzenes production rates were related to the sulfate reducing activity for the unit number of sulfate reducing bacteria. Production rates of dichlorobenzenes were completely inhibited by adding molybdate (20 mM), nitrate (60 mM), and formaldehyde solution (4 %). These results indicated that dechlorination activity in the Ise Bay sediment was supported by sulfate reduction activity in the sediment, and not supported by any other anaerobic microbial activity. © 1994.
  • Manabu Fukui, Susumu Takii
    FEMS Microbiology Ecology 13 (4) 241 - 247 1574-6941 1994 [Refereed][Not invited]
     
    Effects on sulfate respiration of association of sulfate-reducing bacteria (SRB) with solid particles (anion exchange resin and FeS-precipitate) were examined using Desulfovibrio desulfuricans. The rates of sulfide production by resin- and FeS-associated cells were 2-3% and 19-56% of that by free-living ones, respectively, under sulfate- and lactate-rich conditions. On the other hand, under sulfate-poor (less than 50 μM) and lactate-rich conditions the rate by FeS-associated cells was higher than that by free-living ones. The values of Km (μM), half saturation constant of the Michaelis-Menten model, for sulfate were 244 for free-living cells, 8.96 for resin-associated ones and 8.42 for FeS-associated ones. Under lactate-poor and sulfate-rich conditions the rate by FeS-associated cells was similar to that by free-living ones. These results suggest that FeS-associated SRB are more advantageous than free-living ones under sulfate-poor environments such as freshwater sediments. © 1994.
  • Yoshitaka Yonezawa, Manabu Fukui, Toshiaki Yoshida, Akiko Ochi, Toshihiro Tanaka, Yoshiyuki Noguti, Takao Kowata, Yuji Sato, Shigeki Masunaga, Yoshikuni Urushigawa
    Chemosphere 29 (6) 1349 - 1356 0045-6535 1994 [Refereed][Not invited]
     
    The vertical distribution patterns of the transofmration products of tri-n-butyltin (TBT) in sediment cores collected from 6 sites in Ise Bay, Japan indicated that TBT was transformed by two pathways: methylation and debutylation. Anaerobic incubation of sediment spiked with TBT showed that the methylation was mainly supported by sulfate reducing activity and debutylation was mainly supported by nitrate reducing activity. These results showed that contribution of the two transformation reactions of TBT varied according to the dominant microbial activities in the sediment. The two types of vertical distribution of butyltins observed in the sediment cores appear to be the result of different dominant microbial activity in the sediment. © 1994.
  • SUH Jung-In, FUKUI Manabu, YAMAGISHI Takao, URUSHIGAWA Yoshikuni, MORI Tadahiro
    Japan journal of water pollution research Japan Society on Water Environment 16 (9) 645 - 651 0916-8958 1993 [Refereed]
     
    Effects of sulfate load on substrates utilization for sulfidogens and methanogens were investigated in anaerobic sludge that had been acclimated with glucose under various sulfate loads (0 to 0.475mmol SO42-·l-1·d-1). Methanogenesis was decreased with increasing sulfate load and the methane production rate was decreased by 23% at 0.475mmol SO42-·l-1·d-1 compared with no sulfate load sludge. When sulfate reduction was inhibited by the addition of molybdate in sulfate load sludge, methane production rate was the same as that in no sulfate load sludge. At the same time, the utilization rates of propionate and acetate by sulfidogens were increased, but that of acetate, hydrogen and formate by methanogens were decreased with increasing sulfate load. The utilization rate of propionate by syntrophic hydrogen-producing acetogens also decreased. These results showed that sulfidogens competed for acetate with methanogens and for propionate with syntrophic hydrogen-producing acetogens in high sulfate load condition.
  • SUH Jung-In, FUKUI Manabu, URUSHIGAWA Yoshikuni, MORI Tadahiro
    Japan journal of water pollution research Japan Society on Water Environment 16 (9) 633 - 637 0916-8958 1993 [Refereed]
     
    Effects of sulfate and sulfide on methanogenesis were examined in a sulfate-poor anaerobic sludge that had been acclimated with glucose as a carbon source for more than 3 years. Bacterial sulfide production occurred immediately when sulfate was added into the sludge. High density of sulfidogens (1010-1011 cells per g VSS) should inhabit in the sludge. At the same time methanogenesis was inhibited and the maximum inhibition was 53% of the initial methanogenesis at 0.6mM of sulfate. Up to 3mM of sulfide stimulated the methanogenesis, but more than 5mM of sulfide inhibited markedly the methanogenesis. These results show that high density of sulfidogens maintained by their fermentation from substrates under sulfate-poor environments and inhibited partially methanogenesis under sulfate-rich environment because of sulfidogens's predominant utilization of competitive substrates between sulfidogens and methanogens.
  • Analysis of substrates for methanogenesis in anaerobic sludges using specific inhibitors
    JungIn Suh, Manabu Fukui, Yuichi Suwa, Takao Yamagishi, Yoshikuni Urushigawa, Tadahiro Mori
    Water Science and Technology 26 847 - 856 1992 [Refereed]
  • Susumu Takii, Manabu Fukui
    Bulletin of Japanese Society of Microbial Ecology 6 9 - 17 1991 [Refereed]
  • Manabu Fukui, Susumu Takii
    Japanese Journal Of Limnology (Rikusuigaku Zasshi) 51 (2) 63 - 71 1882-4897 1990/02/01 [Refereed][Not invited]
     
    Seasonal variations in population density of sulfate-reducing bacteria (SRB) and sulfate reduction rates were examined for sediments of an offshore and a reed site of Lake Teganuma (freshwater, hypertrophic), Chiba Prefecture, Japan. The numbers of SRB (colony forming units•ml-1) in the surface sediments (0~2 cm) were at a level of 105at the reed site and a level of 104at the offshore one, and they remained ralatively constant throughout the year. The activities at both sites increased from summer to autumn, and the reed sediments showed higher activities (1.08-16.6 times) than the offshore ones in this period the maximum rates (September) (nmol•ml-1•day-1) in the offshore and reed sediments were 290 in the 0~3 cm layer and 1990 in the 3~6 cm layer, respectively. In situ temperature may have greatly affected sulfate reduction. © 1990, The Japanese Society of Limnology. All rights reserved.
  • Toshi Nagata, Takashi Someya, Toshifumi Konda, Takayuki Yamamoto, Kazuko Morikawa, Manabu Fukui, Noburo Kuroda, Kazushi Takahashi, Suwa Oh, Masayuki Mori, Shigehiro Mori, Kenji Kato
    Bulletin of Japanese Society of Microbial Ecology Japanese Society of Microbial Ecology / The Japanese Society of Soil Microbiology 4 (2) 89 - 99 0911-7830 1990 [Refereed]
     
    Intercalibration for counting planktonic bacteria by the acridine orange direct count (AODC) method was made among ten investigators of seven laboratories in Japan. The first calibration was made for one freshwater and two seawater samples sent to seven investigators belonging to five laboratories. Each investigator estimated bacterial abundance in these samples by the AODC method according to routine procedures of each laboratory. For the freshwater sample the estimates differed by a factor of 2.3 [coefficient of variation (CV), 36%], while for the seawater samples they varied greatly by factors of 4.0 and 11 (CV's, 46 and 94%). In the second calibration nine investigators counted bacteria on the same AODC slides under the same optical condition. The estimates of nine investigators for three freshwater samples differed by factors of 1.8-2.5 (CV's, 24-29%), while for a seawater sample they differed by a factor of 17 (CV, 89%). Sampling error associated with contagious distribution of bacteria on a filter, and difference in the criterion for discriminating bacteria from nonbacterial particles were considered to be possible sources of variation in counting bacteria.
  • Manabu Fukui, Susumu Takii
    FEMS Microbiology Letters 73 (4) 317 - 322 1574-6968 1990 [Refereed][Not invited]
     
    Microhabitats and survival of sulfate-reducing bacteria (SRB) in an oxic surface sediment of a seawater lake were examined. The size of fractionation of the sediment suspension showed that most of SRB were associated with sediment particles larger than 10 μm. The D values (time in h required to destroy 90% of the initial viable population) for SRB in the whole sediment suspension and for SRB i n the < μm and the < 5 μm fractions were, respectively, 23.7, 10 and 4 when the SRB were exposed to air. Survival of the FeS-associated Desulfovibrio desulfuricans (D value, 9.3) was higher than that of the free-living ones (D value, 1.8). These results show that particle-associated SRB are more protected against oxygen than free-living ones in oxic sediments. © 1990.
  • Manabu Fukui, Susumu Takii
    FEMS Microbiology Letters 73 (1) 85 - 89 1574-6968 1990 [Refereed][Not invited]
     
    Free-living cells produced colonies more rapidly than particle-associated ones when a pure culture of Desulfovibrio desulfuricans containing sediment particles was inoculated on an agar medium. The time required for the appearance of the first colony, tr of the first order reation model [4] was 53 h for free-living cells and 112 h for particle-associated ones. The value of tr may be determined from the growth rate and sulfate reduction activity because the activity of free-living cells was much higher than that of aggregating ones. © 1990.
  • Manabu Fukui, Susumu Takii
    Bulletin of Japanese Society of Microbial Ecology 3 67 - 71 1989 [Refereed]
  • Manabu Fukui, Susumu Takii
    FEMS Microbiology Letters 62 (1) 13 - 19 1574-6968 1989 [Refereed][Not invited]
     
    The reduction of tetrazolium salts by the sulfate-reducing bacteria, Desulfovibrio desulfuricans and Desulfotomaculum orientis, was examined. D. desulfuricans and D. orientis reduced triphenyltetrazolium chloride (TTC) and 2-(p-iodophenyl)-3-(p-nitrophenyl)-5-phenyltetrazolium chloride (INT) forming intracellular formazan deposits. The reduction rate of INT was higher than that of TTC. INT reduction was not inhibited by the addition of sulfate or molybdate, and sulfate uptake was inhibited by the addition of both INT and molybdate. The ratio of intracellular formazan forming cells to acridine orange direct counts in both strains decreased with culture age and starvation time. © 1989.
  • Manabu Fukui, Susumu Takii
    Japanese Journal of Limnology 48 (4) 249 - 256 1882-4897 1987 [Refereed][Not invited]
     
    Distribution of sulfate-reducing bacteria (SRB) utilizing lactate (1-SRB), propionate (p-SRB) and acetate (a-SRB) was examined along with some physico-chemical environmental factors in various aquatic environments with various trophic levels and various salinities. In marine environments a-SRB showed a tendency to dominate SRB, except for several samples. On the other hand, in freshwater environments 1-SRB tended to dominate SRB. However, a-SRB were found abundantly as well as 1-SRB in hypertrophic freshwater sediments. The relationship between distribution of 1-, p- or a-SRB and environmental factors was discussed. © 1987, The Japanese Society of Limnology. All rights reserved.
  • Colony formation on agar plates by sulfate-reducing bacteria in water and sediment of lake
    Manabu Fukui, Haruo Fukuhara
    Bulletin of Japanese Society of Microbial Ecology 1 75 - 78 1986 [Refereed]

MISC

Books etc

  • 分子でよむ環境汚染
    東海大学出版会 2009

Association Memberships

  • International Society for Microbial Ecology   日本微生物生態学会   日本陸水学会   日本生態学会   

Research Projects

  • 日本学術振興会：科学研究費助成事業
    Date (from‐to) : 2020/04 -2023/03 
    Author : 小島 久弥, 福井 学
     
    淡水資源のヒ素汚染対策に嫌気性ヒ素酸化細菌を利用するための基盤整備として、モデル微生物候補であるM52株についての研究を進めた。M52株の生育可能なpH範囲を培養実験によって確認し、中性を好むことを確認した。M52株は分類学上の位置が未確定であり、学名が付与されていない。M52株が新属新種であることは過去の研究から強く示唆されていたが、これを正式に確定させるためには国外の菌株保存機関へ寄託が必須の状況にあった。先方との種々の調整を経て必要な手続きが完了したため、M52株および近縁種であるJ5B株を記載して学名を付与するための論文を執筆、これを専門誌に投稿した。この論文を完成させるにあたり、基礎的性状として記述が要求される両株の菌体脂肪酸組成も明らかにした。 M52株およびJ5B株は、特徴的なヒ素酸化酵素であるArxをコードするarxA遺伝子を有している。arxA遺伝子は嫌気性ヒ素酸化細菌を検出するためのマーカーとして利用できることが示唆されている。arxA遺伝子を有する微生物にどのようなものがいるのかを、最新の公共データベースを利用して改めて解析した。その結果、研究代表者によって発見された淡水性硫黄酸化細菌であるSulfurimicrobium lacus skT11株がarxA遺伝子を有していることを新たに確認した。skT11株の遺伝子配列を考慮に入れたうえで、arxA遺伝子解析用の新たなプライマーを設計した。従来のプライマーは、あらゆるarxA遺伝子をカバーするために混合塩基を数多く含んでおり、非特異的な増幅が起こりやすいという問題があった。そこで、多様なarxA遺伝子のなかから、中性に近いpHを好む淡水性細菌に特徴的にみられる系統のものを選び出した。この系統に特化することにより、淡水資源のヒ素汚染対策を目標とした研究により適したプライマーを設計することができた。
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for JSPS Fellows
    Date (from‐to) : 2020/11 -2023/03 
    Author : 福井 学, TSUJI JACKSON
     
    In FY2021, we continued to characterize the ecophysiology of “Candidatus Chlorohelix allophototropha”, a highly novel phototrophic bacterium that we enriched from an iron-rich lake in Canada. We established a stable co-culture of “Ca. Chx. allophototropha” with a putative iron-reducing bacterium. By analyzing this co-culture using HPLC and absorption spectroscopy, we provided strong evidence that “Ca. Chx. allophototropha” uses bacteriochlorophyll c and chlorosomes to perform phototrophy. We also sequenced the complete genome of “Ca. Chx. allophototropha”. Alongside physiological approaches, we sequenced metagenomes of samples collected from the iron-rich marshes of Oze National Park (Fukushima Prefecture), and we recovered the partial genome of a novel species related to “Ca. Chx. allophototropha”. This novel species is the first known representative of the “Ca. Chlorohelix” clade in Japan, and its discovery sheds light on the global ecology of the clade. We also analyzed metagenomes and metatranscriptomes of samples we collected from Lake Harutori in FY2020 and, although “Ca. Chx. allophototropha” relatives were not detected, we recovered multiple novel microbial genomes that we will analyze further in FY2022. Overall, this research is providing an important foundation to use “Ca. Chx. allophototropha” as a model organism to understand the evolution and biochemistry of photosynthesis.
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B)
    Date (from‐to) : 2018/04 -2022/03 
    Author : Fukui Manabu
     
    Methane emissions from mires and wetlands are a major natural source in the global budget of atmospheric methane. A large fraction of these emissions result from diffusive methane emission from the surface of sediments and/or snow. Methane is typically produced by methanogens in anoxic peat sediments and oxidized in the oxic surface, but still unknown in the snow on the peat sediments. Reddish-brown colored snow so called “Akashibo”, containing spherical brown particles has been observed in Ozegahara mire at the time of melting snow. This study clarified that the microbial consortia consisted of methanotrophs, methylotrophs and iron-metabolizing bacteria. Such microbial consortia in snow may contribute to the consumption of methane to reduce the emission into the atmosphere at the time of melting snow.
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B)
    Date (from‐to) : 2012/04 -2016/03 
    Author : OKUDA Noboru, KOJIMA Hisaya, FUKUI Manabu, Tayasu Ichiro, NAKANO Shin-ichi, ITOH Masayuki, KOBAYASHI Yuki, FUJIBAYASHI Megumu, SHIAH Fuh-Kwo, HSIEH Chih-hao, MIKI Takeshi, HO Pei-Chi, CHANG Chun-Wei
     
    Recent studies suggest that freshwaters are the primary source of atmospheric methane emitted from natural ecosystems. Methane oxidizing bacteria (MOB) which assimilate dissolved methane aerobically or anaerobically have great impacts on methane cycling in freshwater ecosystems. Methanotrophic food webs (MFWs), in which methane-derived carbon is trophically transferred through the MOB and embedded within a food web, serve as a carbon recycling unit in lake ecosystems. In a deep subtropical Fei-Tsui Reservoir which is located in Taipei, methane profiles and MOB community composition showed characteristics intermediate between subtropical monomictic and tropical meromictic lakes. Our four-year monitoring revealed that typhoon-induced disturbance of thermal stratification and the subsequent decrease in profundal methane storage due to methane oxidation can be the primary limno-physical mechanism to affect the relative contribution of MFWs to the overall trophic carbon flows.
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (A)
    Date (from‐to) : 2011/04 -2015/03 
    Author : IMURA Satoshi, OHTANI Shuji, KAGOSHIMA Hiroshi, KUROSAWA Norio, SUZUKI Tadashi, TOJYO Motoaki, BAN Shuhei, FUKUI Manabu, HOSHINO Tamotsu, NAGANUMA Takeshi, INOUE Genki
     
    Metagenome analysis on prokaryotes and eukaryotes was almost completed on moss pillars in Antarctic lakes. By understandings the biodiversity mainly on microbes, and reconstruction of material cycle by them, the meaning of moss pillars as a type- ecosystem for further studies was thought to be established. On mosses, diatoms and fungi, the diversity in wide area of Antarctica was clarified, and the studies on comparative analysis between the area and inter-specific relations were carried out. Especially on tardigrades, the diversity in Syowa Station area were completed and the researches on reproductive ecology by using culture system has developed and their characteristic features were clarified. On alien species, the origin of human-induced seed plant found in Langhovde area was clarified and risk on Antarctic biodiversity was estimated.
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Challenging Exploratory Research
    Date (from‐to) : 2012/04 -2014/03 
    Author : MURAKAMI Masashi, FUKUI Manabu, HIRAO Toshihide
     
    There is a common hypothesis "Everything is everywhere, but the environment selects" for the distribution and functioning of microbes. To confirm this hypothesis, the analyses based on data base search and also field samplings were carried out. The targeted assemblages were bacteria in freshwater ecosystem, and green algae on cryosphere. The analyses revealed the simultaneous existence of cosmopolitan- and endemic- clades within closely related microbial assemblages. These results suggested the synergetic effects of dispersal and local environmental niche for the diversity and structure of microbial assemblages. The relative importance of these factors should be further examined in future studies.
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B)
    Date (from‐to) : 2010/04 -2014/03 
    Author : FUKUI Manabu, KOJIMA Hisaya
     
    Microbial sulfur cycle in aquatic systems of cryosphere is still unclear although the significance of systems linking carbon and nitrogen cycles has been pointed out. Analysis of aprA gene, which encodes a key enzyme in dissimilatory sulfate reduction and sulfur oxidation, showed that newly isolated Sulfuricella denitrificans-like sulfur oxidizer is a key species from geographically distant lakes in Antarctica and Japan. The complete genome sequence analysis indicated their unique sulfur metabolic pathway adapting freshwater environment. The proteomics results suggest that maintenance of proper translation is critical for growth under low-temperature.
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B)
    Date (from‐to) : 2008 -2010 
    Author : KASAHARA Yasuhiro, FUKUI Manabu, SHIMANO Satoshi
     
    In the forest soil, warming of 5 degrees for 5 years, the amount of total soil carbon and total nitrogen slightly were decreased. However, the warming disturbance was considered to be little effect on the structure of microbial community in soils. We developed a molecular technique, and established PCR-DGGE analysis to analyze a structure of protist community. For the function analysis of microbial communities in soil, we were able to be established the platform of environmental metaproteomics.
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research
    Date (from‐to) : 2006 -2009 
    Author : NAGANUMA Takeshi, IMANAKA Tadayuki, IMURA Satoshi, UCHIDA Masaki, OHTANI Syuji, KANDA Hiroshi, KUROSAWA Norio, KOHSHIMA Shiro, TAKANO Yoshinori, TOJO Motoaki, BAN Syuhei, FUKUI Manabu, HOSHINO Tamotsu, MIYASHITA Hideaki, YOSHIMURA Yoshitaka
     
    This study was conducted as a core program of the International Polar Year (IPY) 2007-2008. Among the global environmental changes, global warming affects the most polar regions, and microorganisms would respond to warming the most promptly. This study thus targeted at ecological aspects of microorganisms living in polar and alpine regions ; and, resulted in an IPY legacy of the microbiological-ecological dataset with community structures, abundances, and occurrences of endemic and cosmopolite species. The IPY legacy should serve as a basic reference for future monitorings.
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B)
    Date (from‐to) : 2006 -2009 
    Author : IMURA Satoshi, KANDA Hiroshi, KUDOU Sakae, FUKUI Manabu, NAGANUMA Takeshi, INOUE Genki, BAN Shuhei, OHTANI Shuji
     
    Monitoring system of limnological data on physical and chemical characteristics of lake water was established to understand the long-term change of Antarctic lake environment. Biodiversity on many groups of bacteria, cyanobacteria, algae, mosses were almost totally clarified. Sediment analysis on several lakes will provide valuable data set to reconstruct the paleo-environment in Antarctica.
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B)
    Date (from‐to) : 2004 -2007 
    Author : FUKUI Manabu, KOJIMA Hisaya
     
    Microbial sulfate reduction is recorded in 〜3.47-Gyr-old barites from North Pole, Australia (Shen et al., 2001: Nature 401, 77-81), indicating that emergence of sulfate-reducing bacteria was earlier than we thought. However, it is unclear how various types of sulfur cycle and sulfur-metabolizing microorganism co-evolve in the present aquatic ecosystems. Aims of this study was first to compare phylogenetic relationship among sulfur-metabolizing microorganisms inhabiting high-temperature terrestrial hot springs, a moderate-temperature meromictic saline lake and a moderate-temperature freshwater oligotrophic lake, second to infer the origin of microbial sulfur cycle, and finally to understand the successive development of the sulfur cycle systems throughout Earth history. By phylogenetic and functional gene analysis, two dominant members were observed in the microbial dens mat at 70 to 80℃ of hot springs; thermophilic sulfate-reducing Thermodesulfobacteria and thermophilic sulfur-oxidizing Sulfurihydrogenibium. Both members are phylogenetically near to the origin of life. Tracer and incubation experiments using the mat showed that both members contributed to the internal sulfur cycle. These results suggest that the microbial sulfur cycle in the hot spring at 70 to 80℃ is one of ancient types. On the other hand, sulfur-metabolizing bacteria from a moderate-temperature saline and a freshwater lake are phylogenetically affiliated to proteobacteria and more diverse than those from high-temperature terrestrial hot springs. The sulfur cycle and metabolic pathways were complicated as compared to the hot springs. Thus, the process of global temperature decrease on Earth seems to be a key factor for diverse sulfur cycle and the microbial community in aquatic ecosystems.
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B)
    Date (from‐to) : 2000 -2002 
    Author : FUKUI Manabu, TAKII Susumu
     
    Molecular ecological studies were done for sulfur-metabolizing microorganisms in various aquatic environments. Among these, freshwater 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 characterized morphologically, ecologically and phylogenetically. The Lake Biwa Thioploca resembled morphologically Thioploca ingrica, a brackish water species from a Danish fjord. The diameter of individual trichomes was 3-5.6 μm ; the diameter of complete Thioploca filaments was in the range of 18-75 μm. Cell lengths ranged from 1.2 to 3.8 μm. In TEM specimens stained with uranyl acetate, dense intracellular particles were found, which did not show any positive signal of phosphorus and sulfur in the X-ray analysis. The 16S rRNA gene of Thioploca from Lake Biwa was amplified using newly designed Thioploca-specific primers (706-Thioploca, Biwa160F and Biwa829R) in combination with general bacterial primers, to avoid unspecific amplification of contaminating bacterial DNA. DGGE analysis of all three overlapping PCR products resulted in single DGGE bands, indicating that a single 16S rRNA gene has been amplified. With the same method, Thioploca from Lake Constance was examined. The 16S rRNA sequence was verified with FISH hybridization targeted at specific motifs of Lake Biwa Thioploca. Positive signals were obtained for the bacterial probe EUB-338, the γ-proteobacterial probe GAM42a, and the probe Biwa829 targeting Lake Biwa Thioploca. Based on the near-complete 16S rRNA sequence and on morphological similarities, Thioploca from Lake Biwa and Lake Constance are closely related to Thioploca ingrica and to each other.
  • 低温環境における物質循環に関わる微生物の生理生態学的研究

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