Anoxygenic phototroph of the Chloroflexota uses a type I reaction centre.
Jackson M. Tsuji, Nicolette Shaw, Nagashima S, Jason J. Venkiteswaran, Schiff SL, Watanabe T, Fukui M, Hanada S, Tank M, Josh Neufeld
Nature, 2024年03月13日
研究論文（学術雑誌）, 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 diversified^1,2. 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.

Desulfoferula mesophilus gen. nov. sp. nov., a mesophilic sulfate-reducing bacterium isolated from a brackish lake sediment
Tomohiro Watanabe, Tatsuya Yabe, Jackson M. Tsuji, Manabu Fukui
Archives of Microbiology, 2023年12月
研究論文（学術雑誌）

Thiomicrorhabdus immobilis sp. nov., a mesophilic sulfur-oxidizing bacterium isolated from sediment of a brackish lake in northern Japan.
Hisaya Kojima, Jun Mochizuki, Mamoru Kanda, Tomohiro Watanabe, Manabu Fukui
Archives of microbiology, 204, 10, 605, 605, 2022年09月07日, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

Structures of methanogenic Fdh-Hdr-Fmd complexes give insight into direct electron transfer and electron bifurcation processes
Tomohiro Watanabe, Olivia Pfeil-Gardiner, Seigo Shima, Bonnie J. Murphy
Biochimica et Biophysica Acta (BBA) - Bioenergetics, 1863, 148739, 148739, Elsevier {BV}, 2022年09月
英語, 研究論文（学術雑誌）

Sulfurimonas aquatica sp. nov., a sulfur-oxidizing bacterium isolated from water of a brackish lake.
Hisaya Kojima, Yukako Kato, Tomohiro Watanabe, Manabu Fukui
Archives of microbiology, 204, 9, 559, 559, 2022年08月17日, ［国際誌］
英語, 研究論文（学術雑誌）, 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).

Dissimilatory microbial sulfur and methane metabolism in the water column of a shallow meromictic lake
Tomohiro Watanabe, Kyoko Kubo, Yoshiharu Kamei, Hisaya Kojima, Manabu Fukui
Systematic and Applied Microbiology, 45, 3, 126320, 126320, Elsevier {BV}, 2022年05月, ［国際誌］
英語, 研究論文（学術雑誌）, Lake Harutori is a brackish meromictic lake with a steep physicochemical gradient in shallow water. Anoxic water below the chemocline has been characterized by high concentrations of sulfide (>10 mM) and methane (>1.5 mM). Previously, we reported that uncultured bacteria in the SEEP-SRB1 group were major sulfate reducers in the lake [21], but knowledge of sulfur oxidation and methane metabolism was scarce. In this current study, the Lake Harutori microbial community structure in the mixolimnion (at depths of 1.5 m and 3.0 m), upper chemocline (3.5 m), and monimolimnion (4.5 m) was further investigated by 16S rRNA gene amplicon sequencing and catalyzed reporter deposition fluorescence in situ hybridization (CARD-FISH). Reads of type I and II methanotrophs were retrieved mainly from 3.5 m and above. Methanotrophic bacteria detected by CARD-FISH accounted for 3.1% of DAPI-stained cells at 3.5 m. Detection frequencies of reads affiliated with the genera Sulfurimonas and Thiomicrorhabdus, which are known to comprise sulfur oxidizers, were relatively high at 3.5 m. Methanogenic archaeal reads were retrieved from the monimolimnion and they affiliated with the genus Methanosaeta. CARD-FISH counts indicated that the cells of Methanosaeta/Methanosarcina/Methanomicrobiales accounted for up to 0.8% of the DAPI-stained cells in the monimolimnion. On the other hand, many of the reads retrieved primarily from the monimolimnion were affiliated with phylogenetically novel uncultured groups.

A enzyme complex of methanogenic electron-bifurcating and CO2-fixing enzymes　　　　　　　　　　　　　　　
Tomohiro Watanabe
Bioscience & Industry, 2022年03月10日
研究論文（学術雑誌）

Three-megadalton complex of methanogenic electron-bifurcating and CO 2 -fixing enzymes
Tomohiro Watanabe, Olivia Pfeil-Gardiner, Jörg Kahnt, Jürgen Koch, Seigo Shima, Bonnie J. Murphy
Science, 373, 6559, 1151, 1156, American Association for the Advancement of Science ({AAAS}), 2021年09月03日
研究論文（学術雑誌）

MvhB-type polyferredoxin as an electron-transfer chain in putative redox-enzyme complexes
Tomohiro Watanabe, Seigo Shima
Chemistry Letters, The Chemical Society of Japan, 2020年11月18日
英語, 研究論文（学術雑誌）

Anoxygenic phototrophicChloroflexotamember uses a Type I reaction center
JM Tsuji, NA Shaw, S Nagashima, JJ Venkiteswaran, SL Schiff, T Watanabe, M Fukui, S Hanada, M Tank, JD Neufeld
2020年07月07日

The Bacterial [Fe]-Hydrogenase Paralog HmdII Uses Tetrahydrofolate Derivatives as Substrates
Tomohiro Watanabe, Tristan Wagner, Gangfeng Huang, Jörg Kahnt, Kenichi Ataka, Ulrich Ermler, Seigo Shima
Angewandte Chemie International Edition, 58, 11, 3506, 3510, Wiley, 2019年03月11日
英語, 研究論文（学術雑誌）, [Fe]-hydrogenase (Hmd) catalyzes the reversible hydrogenation of methenyl-tetrahydromethanopterin (methenyl-H4MPT+) with H-2. H4MPT is a C1-carrier of methanogenic archaea. One bacterial genus, Desulfurobacterium, contains putative genes for the Hmd paralog, termed HmdII, and the HcgA-G proteins. The latter are required for the biosynthesis of the prosthetic group of Hmd, the iron-guanylylpyridinol (FeGP) cofactor. This finding is intriguing because Hmd and HmdII strictly use H4MPT derivatives that are absent in most bacteria. We identified the presence of the FeGP cofactor in D. thermolithotrophum. The bacterial HmdII reconstituted with the FeGP cofactor catalyzed the hydrogenation of derivatives of tetrahydrofolate, the bacterial C1-carrier, albeit with low enzymatic activities. The crystal structures show how Hmd recognizes tetrahydrofolate derivatives. These findings have an impact on future biotechnology by identifying a bacterial Hmd paralog.

Genomes of Neutrophilic Sulfur-Oxidizing Chemolithoautotrophs Representing 9 Proteobacterial Species From 8 Genera
Tomohiro Watanabe, Hisaya Kojima, Kazuhiro Umezawa, Chiaki Hori, Taichi E. Takasuka, Yukako Kato, Manabu Fukui
FRONTIERS IN MICROBIOLOGY, 10, FRONTIERS MEDIA SA, 2019年02月
英語, 研究論文（学術雑誌）, 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.

Diversity of anaerobic arsenite-oxidizing bacteria in low-salt environments analyzed with a newly developed PCR-based method
Melody Cabrera Ospino, Hisaya Kojima, Tomohiro Watanabe, Tomoya Iwata, Manabu Fukui
Limnology, 19, 2, 177, 183, Springer Science and Business Media {LLC}, 2018年04月
英語, 研究論文（学術雑誌）, 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.

Dominance of Sulfuritalea species in nitrate-depleted water of a stratified freshwater lake and arsenate respiration ability within the genus
Tomohiro Watanabe, Aya Miura, Tomoya Iwata, Hisaya Kojima, Manabu Fukui
Environmental Microbiology Reports, 9, 5, 522, 527, Wiley, 2017年10月, ［国際誌］
英語, 研究論文（学術雑誌）, 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 × 104 to 2.1 × 105 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.

The complete genome sequences of sulfur-oxidizing Gammaproteobacteria Sulfurifustis variabilis skN76T and Sulfuricaulis limicola HA5T
Kazuhiro Umezawa, Tomohiro Watanabe, Aya Miura, Hisaya Kojima, Manabu Fukui
Standards in Genomic Sciences, 11, 1, Springer Science and Business Media LLC, 2016年12月
英語, 研究論文（学術雑誌）, 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.

Identity of major sulfur-cycle prokaryotes in freshwater lake ecosystems revealed by a comprehensive phylogenetic study of the dissimilatory adenylylsulfate reductase
Tomohiro Watanabe, Hisaya Kojima, Manabu Fukui
Scientific Reports, 6, 1, Springer Science and Business Media LLC, 2016年12月
英語, 研究論文（学術雑誌）, 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.

Thermithiobacillus plumbiphilus sp. nov., a sulfur-oxidizing bacterium isolated from lead sulfide
Tomohiro Watanabe, Aya Miura, Arisa Shinohara, Hisaya Kojima, Manabu Fukui
International Journal of Systematic and Evolutionary Microbiology, 66, 5, 1986, 1989, Microbiology Society, 2016年05月01日, ［国際誌］
英語, 研究論文（学術雑誌）, A novel sulfur oxidizer, strain wk12T, 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 (C16 : 1ω7c and/or C16 : 1ω6c), C16 : 0 and summed feature 8 (C18 : 1ω7c and/or C18 : 1ω6c). The strain oxidized lead sulfide, thiosulfate and tetrathionate as electron donors to support autotrophic growth. Cells of strain wk12T were motile, rod-shaped (0.5-1.0 × 0.7-2.2 μm), and Gram-stain-negative. For growth, the temperature range was 5-37 °C, and optimum growth was observed at 28-32 °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 3134T, with a 16S rRNA gene sequence similarity of 96 %. On the basis of phylogenetic and phenotypic properties, strain wk12T represents a novel species of the genus Thermithiobacillus, for which the name Thermithiobacillus plumbiphilus sp. nov. is proposed. The type strain is wk12T ( = NBRC 111508T = DSM 101799T).

Sulfuriferula thiophila sp nov., a chemolithoautotrophic sulfur-oxidizing bacterium, and correction of the name Sulfuriferula plumbophilus Watanabe, Kojima and Fukui 2015 to Sulfuriferula plumbiphila corrig.
Watanabe Tomohiro, Kojima Hisaya, Fukui Manabu
International journal of systematic and evolutionary microbiology, 66, 5, 2041, 2045, Society for General Microbiology, 2016年05月01日
英語, 研究論文（学術雑誌）, 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.

Sulfuricaulis limicola gen. nov., sp nov., a sulfur oxidizer isolated from a lake
Kojima Hisaya, Watanabe Tomohiro, Fukui Manabu
International journal of systematic and evolutionary microbiology, 66, 1, 266, 270, the International Union of Microbiological Societies, 2016年01月01日
英語, 研究論文（学術雑誌）, 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)).

Draft Genome Sequence of Desulfatitalea tepidiphila S28bFT
Y. Higashioka, H. Kojima, T. Watanabe, M. Fukui
Genome Announcements, 3, 6, American Society for Microbiology, 2015年11月12日
英語, 研究論文（学術雑誌）, 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.

Sulfurirhabdus autotrophica gen. nov., sp. nov., isolated from a freshwater lake.
Tomohiro Watanabe
International journal of systematic and evolutionary microbiology, 66, 1, 113, 117, Microbiology Society, 2015年10月15日
研究論文（学術雑誌）, A novel sulfur-oxidizing bacterium, designated strain BiS0T, was isolated from a sediment sample collected from a freshwater lake in Japan. The cells were rod-shaped, 1.4-4.6 × 0.4-0.7 μm and Gram-stain-negative. The G+C content of the genomic DNA was around 44 mol%. The isolate possessed summed feature 3 (C16 : 1ω7c and/or C16 : 1ω6c), C16 : 0 and C10 : 0 3-OH as major cellular fatty acids. Strain BiS0T 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 °C (optimum, 15-22 °C), an NaCl concentration range of 0-546.4 mM (optimum 0-66.7 mM) and a pH range of 5.2-8.1 (optimum 6.1-6.3). Phylogenetic analysis, based on 16S rRNA gene sequences, indicated that strain BiS0T belongs to the family Sulfuricellaceae in the class Betaproteobacteria. The closest cultured relatives were Sulfuricella denitrificans skB26T and Sulfuricella sp. T08, with 16S rRNA gene sequence similarities of 96.3 %. On the basis of the data obtained in this study, strain BiS0T represents a novel species of a novel genus, for which the name Sulfurirhabdus autotrophica gen. nov., sp. nov. is proposed. The type strain is BiS0T ( = NBRC 110941T = DSM 100309T).

Draft Genome Sequence of Mizugakiibacter sediminis skMP5(T)
Tomohiro Watanabe, Hisaya Kojima, Manabu Fukui
MICROBIOLOGY RESOURCE ANNOUNCEMENTS, 3, 5, AMER SOC MICROBIOLOGY, 2015年09月, ［査読有り］
英語, 研究論文（学術雑誌）, 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).

Sulfuriferula multivorans gen. nov., sp. nov., isolated from a freshwater lake, reclassification of ‘thiobacillus plumbophilus’ assulfuriferula plumbophilus sp. nov., and description of sulfuricellaceae fam. nov. and sulfuricellales ord. nov
Tomohiro Watanabe, Hisaya Kojima, Manabu Fukui
International Journal of Systematic and Evolutionary Microbiology, 65, 5, 1504, 1508, Society for General Microbiology, 2015年06月27日
英語, 研究論文（学術雑誌）, A sulfur-oxidizing bacterium, strain TTN<
sup>
T<
/sup>
, 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<
sup>
T<
/sup>
. Cells were rods, motile, and Gram-stain-negative. The G+C content of the genomic DNA was approximately 66 mol%. Strain TTN<
sup>
T<
/sup>
grew over a temperature range of 8-32 °C (optimum 22-25 °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 TTN<
sup>
T<
/sup>
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 TTN<
sup>
T<
/sup>
represents a novel species of a novel genus, for which the name Sulfuriferula multivorans gen. nov., sp. nov. is proposed. The type strain is TTN<
sup>
T<
/sup>
(=NBRC 110683<
sup>
T<
/sup>
=DSM 29343<
sup>
T<
/sup>
). Along with this, the reclassification of ‘Thiobacillus plumbophilus’ asSulfuriferula plumbophilus sp. nov. (type strain Gro7<
sup>
T<
/sup>
=NBRC 107929<
sup>
T<
/sup>
=DSM 6690<
sup>
T<
/sup>
) is proposed. Based on the data obtained in this study, we describe the designations Sulfuricellaceae fam. nov. and Sulfuricellales ord. nov.

Ecophysiology of Thioploca ingrica as revealed by the complete genome sequence supplemented with proteomic evidence
Hisaya Kojima, Yoshitoshi Ogura, Nozomi Yamamoto, Tomoaki Togashi, Hiroshi Mori, Tomohiro Watanabe, Fumiko Nemoto, Ken Kurokawa, Tetsuya Hayashi, Manabu Fukui
The ISME Journal, 9, 5, 1166, 1176, Springer Science and Business Media LLC, 2015年05月
英語, 研究論文（学術雑誌）, 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.

Draft Genome Sequence of a Sulfur-Oxidizing Autotroph, Sulfuricella sp. Strain T08, Isolated from a Freshwater Lake
Tomohiro Watanabe, Hisaya Kojima, Manabu Fukui
MICROBIOLOGY RESOURCE ANNOUNCEMENTS, 3, 3, AMER SOC MICROBIOLOGY, 2015年05月
英語, 研究論文（学術雑誌）, 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.

Draft genome sequence of Mizugakiibacter sediminis skMP5T
Tomohiro Watanabe, Hisaya Kojima, Manabu Fukui
Genome Announcements, 3, 5, American Society for Microbiology, 2015年
英語, 研究論文（学術雑誌）, 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.

Complete genomes of freshwater sulfur oxidizers Sulfuricella denitrificans skB26 and Sulfuritalea hydrogenivorans sk43H: genetic insights into the sulfur oxidation pathway of betaproteobacteria.
Tomohiro Watanabe
Systematic and applied microbiology, 37, 6, 387, 395, Elsevier BV, 2014年06月24日
研究論文（学術雑誌）, 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.

Identification of major planktonic sulfur oxidizers in stratified freshwater lake
Hisaya Kojima, Tomohiro Watanabe, Tomoya Iwata, Manabu Fukui
PLoS ONE, 9, 4, Public Library of Science, 2014年04月02日
英語, 研究論文（学術雑誌）, 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. © 2014 Kojima et al.

Diversity of sulfur-cycle prokaryotes in freshwater lake sediments investigated using aprA as the functional marker gene
Tomohiro Watanabe, Hisaya Kojima, Yoshinori Takano, Manabu Fukui
Systematic and Applied Microbiology, 36, 6, 436, 443, Elsevier BV, 2013年09月
英語, 研究論文（学術雑誌）, 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.

Draft Genome Sequence of a Psychrotolerant Sulfur-Oxidizing Bacterium, Sulfuricella denitrificans skB26, and Proteomic Insights into Cold Adaptation
Tomohiro Watanabe, Hisaya Kojima, Manabu Fukui
Applied and Environmental Microbiology, 78, 18, 6545, 6549, American Society for Microbiology, 2012年09月15日
英語, 研究論文（学術雑誌）, Except for several conspicuous cases, very little is known about sulfur oxidizers living in natural freshwater environments.Sulfuricella denitrificansskB26 is a psychrotolerant sulfur oxidizer recently isolated from a freshwater lake as a representative of a new genus in the classBetaproteobacteria. 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°C and 5°C, proteins were extracted for analysis by nano-liquid chromatography–electrospray ionization–tandem mass spectrometry. In the cells cultured at 5°C, relative abundances of ribosomal proteins, cold shock proteins, and DEAD/DEAH box RNA helicases were increased in comparison to those at 22°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.

Genus Sulfuricella　　　　　　　　　　　　　　　
Tomohiro Watanabe, Hisaya Kojima, Bergey's manual of Systematics of Archaea and Bacteria, 2019年07月, ［査読有り］, ［招待有り］, ［筆頭著者］

Hydrogenotrophic methanogenesis
Wagner, T, Watanabe, T, Shima, S, Biogenesis of hydrocarbons. Handbook of hydrocarbon and lipid microbiology, 1, 29, 2018年

P14-11 新目Sulfuricella目に属する新たな淡水性硫黄酸化細菌の分離と特徴付け(ポスター発表)
渡邉 友浩, 小島 久弥, 福井 学, 日本微生物生態学会講演要旨集, 2014, 152, 152, 2014年
日本微生物生態学会, 日本語

OA-001 淡水湖沼の硫黄酸化細菌群集における主要構成種の特定とゲノム解析(水圏生態系,口頭発表)
渡邉 友浩, 小島 久弥, 福井 学, 日本微生物生態学会講演要旨集, 29, 81, 81, 2013年
日本微生物生態学会, 日本語

祖先的な亜硫酸生成反応の起源に迫る酵素の探索
科学研究費助成事業
2021年04月01日 - 2024年03月31日
渡邉 友浩
太古代地球における生命のエネルギー代謝のひとつには、亜硫酸の酸化還元反応が関与していたと考えられている。この反応を触媒する酵素は生命進化を研究する上で重要である。本研究では、硫黄化合物を酸化してエネルギーを獲得する微生物（硫黄酸化菌）の一部が、今まで知られていなかった酵素を使って亜硫酸合成反応を触媒する可能性を検証している。情報生物学と生化学の知見を総合すると、未知酵素の触媒反応にはリポ酸の誘導体が関与すると考えられる。このため、リポ酸を利用して硫黄化合物からエネルギーを獲得する硫黄酸化菌を実験材料に用いた。対象の硫黄酸化菌を、複数の硫黄化合物あるいは水素ガスをエネルギー源とする条件で培養し、それぞれの条件で発現する遺伝子を同定した（トランスクリプトーム解析）。この結果、硫黄化合物をエネルギー源とする際に、リポ酸の誘導体を生じる酵素の遺伝子発現量が有意に増加することを見出した。この遺伝子と一緒に発現量が変化する遺伝子の中に、亜硫酸を合成する未知酵素遺伝子が含まれると考えられる。現在、各遺伝子の配列特徴を詳細に解析している。これと同時に、硫黄酸化菌を大量に培養するための実験系を確立した。今後、大量培養で得られた細胞から細胞粗酵素液を調整して、未知酵素が触媒すると考えられる酵素反応の検証実験を行う。この際、リポ酸結合タンパク質が反応基質の１つとして関与すると考えられる。このため、硫黄酸化菌由来のリポ酸結合タンパク質を大腸菌で合成し、これをアフィニティークロマトグラフィー等で精製した。
日本学術振興会, 若手研究, 北海道大学, 21K14025

未知の電子バイフリケーション反応の検証ー新たな遺伝子資源の開拓ー
科学研究費助成事業
2020年09月11日 - 2022年03月31日
渡邉 友浩
ヘテロジスルフィド還元酵素が触媒する電子分岐反応は、電子対の一方の電子を高エネルギー状態にする。これは、持続可能な社会の技術基盤として応用できる可能性が指摘されている。本研究では、硫黄酸化菌が合成する機能未知のヘテロジスルフィド還元酵素（sHdr）を研究した。遺伝子発現解析の結果、sHdrは硫黄化合物からのエネルギー合成に関与することが示唆された。sHdrを合成する硫黄酸化菌を大量培養することに成功した。細胞粗酵素液を各種クロマトグラフィーで分画した。紫外可視吸収スペクトル、SDS-PAGE、Native-PAGEによってsHdrの精製条件を検討し、得られた画分の質量分析を進めている。
日本学術振興会, 研究活動スタート支援, 北海道大学, 20K22559