- 著者
- Yusuke Okazaki Tuyen Thi Nguyen Arisa Nishihara Hisashi Endo Hiroyuki Ogata Shin-ichi Nakano Hideyuki Tamaki
- 出版者
- Japanese Society of Microbial Ecology / Japanese Society of Soil Microbiology / Taiwan Society of Microbial Ecology / Japanese Society of Plant Microbe Interactions / Japanese Society for Extremophiles
- 雑誌
- Microbes and Environments (ISSN:13426311)
- 巻号頁・発行日
- vol.38, no.1, pp.ME22102, 2023 (Released:2023-03-15)
- 参考文献数
- 20
- 被引用文献数
- 1
We herein propose a fast and easy DNA and RNA co-extraction method for environmental microbial samples. It combines bead beating and phenol-chloroform phase separation followed by the separation and purification of DNA and RNA using the Qiagen AllPrep DNA/RNA mini kit. With a handling time of ~3 h, our method simultaneously extracted high-quality DNA (peak size >10–15 kb) and RNA (RNA integrity number >6) from lake bacterioplankton filtered samples. The method is also applicable to low-biomass samples (expected DNA or RNA yield <50 ng) and eukaryotic microbial samples, providing an easy option for more versatile eco-genomic applications.
- 著者
- Arisa Nishihara Katsumi Matsuura Marcus Tank Shawn E. McGlynn Vera Thiel Shin Haruta
- 出版者
- Japanese Society of Microbial Ecology · The Japanese Society of Soil Microbiology
- 雑誌
- Microbes and Environments (ISSN:13426311)
- 巻号頁・発行日
- pp.ME18041, (Released:2018-11-23)
- 被引用文献数
- 21
The phylum Aquificae comprises chemolithoautotrophic thermophilic to hyperthermophilic bacteria, in which the nitrogenase reductase gene (nifH) has been reported. However, nitrogen-fixing activity has not yet been demonstrated in members of this deeply branching bacterial phylum. We isolated two thermophilic diazotrophic strains from chemosynthetic microbial communities in slightly alkaline hot springs (≥70°C) in Nakabusa, Nagano Prefecture, Japan. A phylogenetic analysis based on 16S rRNA genes identified these strains as members of the genus Hydrogenobacter within Aquificae. Their NifH sequences showed 96.5 and 97.4% amino acid sequence identities to that from Hydrogenobacter thermophilus TK-6. Nitrogenase activity, measured by acetylene reduction, was confirmed in both strains at 70°C. These novel strains grew under semi-aerobic conditions by using CO2 as the sole carbon source and N2 as the sole nitrogen source in media containing hydrogen and/or thiosulfate. To the best of our knowledge, this is the first demonstration of active nitrogen fixation in thermophilic bacteria at 70°C and in the phylum Aquificae.
- 著者
- Yuxin Chen Arisa Nishihara Shin Haruta
- 出版者
- Japanese Society of Microbial Ecology / Japanese Society of Soil Microbiology / Taiwan Society of Microbial Ecology / Japanese Society of Plant Microbe Interactions / Japanese Society for Extremophiles
- 雑誌
- Microbes and Environments (ISSN:13426311)
- 巻号頁・発行日
- vol.36, no.2, pp.ME21018, 2021 (Released:2021-06-10)
- 参考文献数
- 87
- 被引用文献数
- 13
Fermentative nitrogen-fixing bacteria have not yet been examined in detail in thermal environments. In the present study, we isolated the thermophilic fermentative bacterium, strain YA01 from a hot spring. This strain grew at temperatures up to 78°C. A phylogenetic analysis based on its 16S rRNA gene sequence indicated that strain YA01 belonged to the genus Caldicellulosiruptor, which are fermentative bacteria in the phylum Firmicutes, with 97.7–98.0% sequence identity to its closest relatives. Strain YA01 clearly exhibited N2-dependent growth at 70°C. We also confirmed N2-dependent growth in the relatives of strain YA01, Caldicellulosiruptor hydrothermalis 108 and Caldicellulosiruptor kronotskyensis 2002. The nitrogenase activities of these three strains were examined using the acetylene reduction assay. Similar activities were detected for all tested strains, and were slightly suppressed by the addition of ammonium. A genome analysis revealed that strain YA01, as well as other Caldicellulosiruptor, possessed a gene set for nitrogen fixation, but lacked the nifN gene, which encodes a nitrogenase iron-molybdenum cofactor biosynthesis protein that is commonly detected in nitrogen-fixing bacteria. The amino acid sequences of nitrogenase encoded by nifH, nifD, and nifK shared 92–98% similarity in Caldicellulosiruptor. A phylogenetic tree of concatenated NifHDK sequences showed that NifHDK of Caldicellulosiruptor was in the deepest clade. To the best of our knowledge, this is the first study to demonstrate the nitrogen-fixing ability of fermentative bacteria at 70°C. Caldicellulosiruptor may have retained an ancient nitrogen-fixing enzyme system.
- 著者
- Arisa Nishihara Vera Thiel Katsumi Matsuura Shawn E. McGlynn Shin Haruta
- 出版者
- Japanese Society of Microbial Ecology · The Japanese Society of Soil Microbiology
- 雑誌
- Microbes and Environments (ISSN:13426311)
- 巻号頁・発行日
- pp.ME18030, (Released:2018-11-07)
- 被引用文献数
- 23
Chemosynthetic microbial communities develop and form dense cell aggregates in slightly alkaline sulfidic hot springs in the temperature range of 70–86°C at Nakabusa, Japan. Nitrogenase activity has recently been detected in the microbial communities collected. To identify possible members capable of nitrogen fixation, we examined the diversities of 16S rRNA and nitrogenase reductase (NifH) gene sequences in four types of chemosynthetic communities with visually different colors and thicknesses. The results of a 16S rRNA gene analysis indicated that all four microbial communities had similar bacterial constituents; the phylum Aquificae was the dominant member, followed in abundance by Thermodesulfobacteria, Firmicutes, and Thermotogae. Most of the NifH sequences were related to sequences reported in hydrothermal vents and terrestrial hot springs. The results of a phylogenetic analysis of NifH sequences revealed diversity in this gene among the communities collected, distributed within 7 phylogenetic groups. NifH sequences affiliated with Aquificae (Hydrogenobacter/Thermocrinis) and Firmicutes (Caldicellulosiruptor) were abundant. At least two different energy metabolic pathways appeared to be related to nitrogen fixation in the communities analyzed; aerobic sulfur/hydrogen-oxidizing bacteria in Aquificae and fermentative bacteria in Firmicutes. The metabolic characteristics of these two dominant phyla differed from those previously inferred from nitrogenase activity assays on chemosynthetic communities, which were associated with hydrogen-dependent autotrophic sulfate reduction. These assays may correspond to the observed NifH sequences that are distantly related to the known species of Thermodesulfovibrio sp. (Nitrospirae) detected in the present study. The activities of nitrogen-fixing organisms in communities may depend on redox states as well as the availability of electron donors, acceptors, and carbon sources.
- 著者
- Joval N. Martinez Arisa Nishihara Mads Lichtenberg Erik Trampe Shigeru Kawai Marcus Tank Michael Kühl Satoshi Hanada Vera Thiel
- 出版者
- Japanese Society of Microbial Ecology · The Japanese Society of Soil Microbiology
- 雑誌
- Microbes and Environments (ISSN:13426311)
- 巻号頁・発行日
- pp.ME19047, (Released:2019-11-02)
- 被引用文献数
- 17
Phototrophic microbial mats are assemblages of vertically layered microbial populations dominated by photosynthetic microorganisms. In order to elucidate the vertical distribution and diversity of phototrophic microorganisms in a hot spring-associated microbial mat in Nakabusa (Japan), we analyzed the 16S rRNA gene amplicon sequences of the microbial mat separated into five depth horizons, and correlated them with microsensor measurements of O2 and spectral scalar irradiance. A stable core community and high diversity of phototrophic organisms dominated by the filamentous anoxygenic phototrophs, Roseiflexus castenholzii and Chloroflexus aggregans were identified together with the spectral signatures of bacteriochlorophylls (BChls) a and c absorption in all mat layers. In the upper mat layers, a high abundance of cyanobacteria (Thermosynechococcus sp.) correlated with strong spectral signatures of chlorophyll a and phycobiliprotein absorption near the surface in a zone of high O2 concentrations during the day. Deeper mat layers were dominated by uncultured chemotrophic Chlorobi such as the novel putatively sulfate-reducing “Ca. Thermonerobacter sp.”, which showed increasing abundance with depth correlating with low O2 in these layers enabling anaerobic metabolism. Oxygen tolerance and requirements for the novel phototroph “Ca. Chloroanaerofilum sp.” and the uncultured chemotrophic Armatimonadetes member type OS-L detected in Nakabusa hot springs, Japan appeared to differ from previously suggested lifestyles for close relatives identified in hot springs in Yellowstone National Park, USA. The present study identified various microenvironmental gradients and niche differentiation enabling the co-existence of diverse chlorophototrophs in metabolically diverse communities in hot springs.
- 著者
- Joval N. Martinez Arisa Nishihara Mads Lichtenberg Erik Trampe Shigeru Kawai Marcus Tank Michael Kühl Satoshi Hanada Vera Thiel
- 出版者
- Japanese Society of Microbial Ecology / Japanese Society of Soil Microbiology / Taiwan Society of Microbial Ecology / Japanese Society of Plant Microbe Interactions / Japanese Society for Extremophiles
- 雑誌
- Microbes and Environments (ISSN:13426311)
- 巻号頁・発行日
- vol.34, no.4, pp.374-387, 2019 (Released:2019-12-27)
- 参考文献数
- 72
- 被引用文献数
- 17
Phototrophic microbial mats are assemblages of vertically layered microbial populations dominated by photosynthetic microorganisms. In order to elucidate the vertical distribution and diversity of phototrophic microorganisms in a hot spring-associated microbial mat in Nakabusa (Japan), we analyzed the 16S rRNA gene amplicon sequences of the microbial mat separated into five depth horizons, and correlated them with microsensor measurements of O2 and spectral scalar irradiance. A stable core community and high diversity of phototrophic organisms dominated by the filamentous anoxygenic phototrophs, Roseiflexus castenholzii and Chloroflexus aggregans were identified together with the spectral signatures of bacteriochlorophylls (BChls) a and c absorption in all mat layers. In the upper mat layers, a high abundance of cyanobacteria (Thermosynechococcus sp.) correlated with strong spectral signatures of chlorophyll a and phycobiliprotein absorption near the surface in a zone of high O2 concentrations during the day. Deeper mat layers were dominated by uncultured chemotrophic Chlorobi such as the novel putatively sulfate-reducing “Ca. Thermonerobacter sp.”, which showed increasing abundance with depth correlating with low O2 in these layers enabling anaerobic metabolism. Oxygen tolerance and requirements for the novel phototroph “Ca. Chloroanaerofilum sp.” and the uncultured chemotrophic Armatimonadetes member type OS-L detected in Nakabusa hot springs, Japan appeared to differ from previously suggested lifestyles for close relatives identified in hot springs in Yellowstone National Park, USA. The present study identified various microenvironmental gradients and niche differentiation enabling the co-existence of diverse chlorophototrophs in metabolically diverse communities in hot springs.
- 著者
- Arisa Nishihara Katsumi Matsuura Marcus Tank Shawn E. McGlynn Vera Thiel Shin Haruta
- 出版者
- Japanese Society of Microbial Ecology / Japanese Society of Soil Microbiology / Taiwan Society of Microbial Ecology / Japanese Society of Plant Microbe Interactions / Japanese Society for Extremophiles
- 雑誌
- Microbes and Environments (ISSN:13426311)
- 巻号頁・発行日
- vol.33, no.4, pp.394-401, 2018 (Released:2018-12-28)
- 参考文献数
- 48
- 被引用文献数
- 7 21
The phylum Aquificae comprises chemolithoautotrophic thermophilic to hyperthermophilic bacteria, in which the nitrogenase reductase gene (nifH) has been reported. However, nitrogen-fixing activity has not yet been demonstrated in members of this deeply branching bacterial phylum. We isolated two thermophilic diazotrophic strains from chemosynthetic microbial communities in slightly alkaline hot springs (≥70°C) in Nakabusa, Nagano Prefecture, Japan. A phylogenetic analysis based on 16S rRNA genes identified these strains as members of the genus Hydrogenobacter within Aquificae. Their NifH sequences showed 96.5 and 97.4% amino acid sequence identities to that from Hydrogenobacter thermophilus TK-6. Nitrogenase activity, measured by acetylene reduction, was confirmed in both strains at 70°C. These novel strains grew under semi-aerobic conditions by using CO2 as the sole carbon source and N2 as the sole nitrogen source in media containing hydrogen and/or thiosulfate. To the best of our knowledge, this is the first demonstration of active nitrogen fixation in thermophilic bacteria at 70°C and in the phylum Aquificae.
- 著者
- Arisa Nishihara Shin Haruta Shawn E. McGlynn Vera Thiel Katsumi Matsuura
- 出版者
- 日本微生物生態学会・日本土壌微生物学会
- 雑誌
- Microbes and Environments (ISSN:13426311)
- 巻号頁・発行日
- pp.ME17134, (Released:2018-01-24)
- 被引用文献数
- 21
The activity of nitrogen fixation measured by acetylene reduction was examined in chemosynthetic microbial mats at 72–75°C in slightly-alkaline sulfidic hot springs in Nakabusa, Japan. Nitrogenase activity markedly varied from sampling to sampling. Nitrogenase activity did not correlate with methane production, but was detected in samples showing methane production levels less than the maximum amount, indicating a possible redox dependency of nitrogenase activity. Nitrogenase activity was not affected by 2-bromo-ethane sulfonate, an inhibitor of methanogenesis. However, it was inhibited by the addition of molybdate, an inhibitor of sulfate reduction and sulfur disproportionation, suggesting the involvement of sulfate-reducing or sulfur-disproportionating organisms. Nitrogenase activity was affected by different O2 concentrations in the gas phase, again supporting the hypothesis of a redox potential dependency, and was decreased by the dispersion of mats with a homogenizer. The loss of activity that occurred from dispersion was partially recovered by the addition of H2, sulfate, and carbon dioxide. These results suggested that the observed activity of nitrogen fixation was related to chemoautotrophic sulfate reducers, and fixation may be active in a limited range of ambient redox potential. Since thermophilic chemosynthetic communities may resemble ancient microbial communities before the appearance of photosynthesis, the present results may be useful when considering the ancient nitrogen cycle on earth.
- 著者
- Arisa Nishihara Shin Haruta Shawn E. McGlynn Vera Thiel Katsumi Matsuura
- 出版者
- Japanese Society of Microbial Ecology · The Japanese Society of Soil Microbiology
- 雑誌
- Microbes and Environments (ISSN:13426311)
- 巻号頁・発行日
- vol.33, no.1, pp.10-18, 2018 (Released:2018-03-29)
- 参考文献数
- 77
- 被引用文献数
- 21
The activity of nitrogen fixation measured by acetylene reduction was examined in chemosynthetic microbial mats at 72–75°C in slightly-alkaline sulfidic hot springs in Nakabusa, Japan. Nitrogenase activity markedly varied from sampling to sampling. Nitrogenase activity did not correlate with methane production, but was detected in samples showing methane production levels less than the maximum amount, indicating a possible redox dependency of nitrogenase activity. Nitrogenase activity was not affected by 2-bromo-ethane sulfonate, an inhibitor of methanogenesis. However, it was inhibited by the addition of molybdate, an inhibitor of sulfate reduction and sulfur disproportionation, suggesting the involvement of sulfate-reducing or sulfur-disproportionating organisms. Nitrogenase activity was affected by different O2 concentrations in the gas phase, again supporting the hypothesis of a redox potential dependency, and was decreased by the dispersion of mats with a homogenizer. The loss of activity that occurred from dispersion was partially recovered by the addition of H2, sulfate, and carbon dioxide. These results suggested that the observed activity of nitrogen fixation was related to chemoautotrophic sulfate reducers, and fixation may be active in a limited range of ambient redox potential. Since thermophilic chemosynthetic communities may resemble ancient microbial communities before the appearance of photosynthesis, the present results may be useful when considering the ancient nitrogen cycle on earth.
- 著者
- Arisa Nishihara Vera Thiel Katsumi Matsuura Shawn E. McGlynn Shin Haruta
- 出版者
- Japanese Society of Microbial Ecology · The Japanese Society of Soil Microbiology
- 雑誌
- Microbes and Environments (ISSN:13426311)
- 巻号頁・発行日
- vol.33, no.4, pp.357-365, 2018 (Released:2018-12-28)
- 参考文献数
- 79
- 被引用文献数
- 1 23
Chemosynthetic microbial communities develop and form dense cell aggregates in slightly alkaline sulfidic hot springs in the temperature range of 70–86°C at Nakabusa, Japan. Nitrogenase activity has recently been detected in the microbial communities collected. To identify possible members capable of nitrogen fixation, we examined the diversities of 16S rRNA and nitrogenase reductase (NifH) gene sequences in four types of chemosynthetic communities with visually different colors and thicknesses. The results of a 16S rRNA gene analysis indicated that all four microbial communities had similar bacterial constituents; the phylum Aquificae was the dominant member, followed in abundance by Thermodesulfobacteria, Firmicutes, and Thermotogae. Most of the NifH sequences were related to sequences reported in hydrothermal vents and terrestrial hot springs. The results of a phylogenetic analysis of NifH sequences revealed diversity in this gene among the communities collected, distributed within 7 phylogenetic groups. NifH sequences affiliated with Aquificae (Hydrogenobacter/Thermocrinis) and Firmicutes (Caldicellulosiruptor) were abundant. At least two different energy metabolic pathways appeared to be related to nitrogen fixation in the communities analyzed; aerobic sulfur/hydrogen-oxidizing bacteria in Aquificae and fermentative bacteria in Firmicutes. The metabolic characteristics of these two dominant phyla differed from those previously inferred from nitrogenase activity assays on chemosynthetic communities, which were associated with hydrogen-dependent autotrophic sulfate reduction. These assays may correspond to the observed NifH sequences that are distantly related to the known species of Thermodesulfovibrio sp. (Nitrospirae) detected in the present study. The activities of nitrogen-fixing organisms in communities may depend on redox states as well as the availability of electron donors, acceptors, and carbon sources.