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Mike S. M. Jetten - One of the best experts on this subject based on the ideXlab platform.
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Autotrophic and mixotrophic metabolism of an Anammox Bacterium revealed by in vivo ^13C and ^2H metabolic network mapping
The ISME Journal, 2021Co-Authors: Christopher E. Lawson, Mike S. M. Jetten, R.m. De Graaf, Guylaine H. L. Nuijten, Tyler B. Jacobson, Martin Pabst, David M. Stevenson, Daniel R. Noguera, Katherine D. Mcmahon, Daniel Amador-noguezAbstract:Anaerobic ammonium-oxidizing (Anammox) bacteria mediate a key step in the biogeochemical nitrogen cycle and have been applied worldwide for the energy-efficient removal of nitrogen from wastewater. However, outside their core energy metabolism, little is known about the metabolic networks driving Anammox bacterial anabolism and use of different carbon and energy substrates beyond genome-based predictions. Here, we experimentally resolved the central carbon metabolism of the Anammox Bacterium Candidatus ‘Kuenenia stuttgartiensis’ using time-series ^13C and ^2H isotope tracing, metabolomics, and isotopically nonstationary metabolic flux analysis. Our findings confirm predicted metabolic pathways used for CO_2 fixation, central metabolism, and amino acid biosynthesis in K. stuttgartiensis , and reveal several instances where genomic predictions are not supported by in vivo metabolic fluxes. This includes the use of the oxidative branch of an incomplete tricarboxylic acid cycle for alpha-ketoglutarate biosynthesis, despite the genome not having an annotated citrate synthase. We also demonstrate that K. stuttgartiensis is able to directly assimilate extracellular formate via the Wood–Ljungdahl pathway instead of oxidizing it completely to CO_2 followed by reassimilation. In contrast, our data suggest that K. stuttgartiensis is not capable of using acetate as a carbon or energy source in situ and that acetate oxidation occurred via the metabolic activity of a low-abundance microorganism in the bioreactor’s side population. Together, these findings provide a foundation for understanding the carbon metabolism of Anammox bacteria at a systems-level and will inform future studies aimed at elucidating factors governing their function and niche differentiation in natural and engineered ecosystems.
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autotrophic and mixotrophic metabolism of an Anammox Bacterium revealed by in vivo 13 c and 2 h metabolic network mapping
The ISME Journal, 2021Co-Authors: Christopher E. Lawson, Mike S. M. Jetten, R.m. De Graaf, Guylaine H. L. Nuijten, Tyler B. Jacobson, Martin Pabst, David M. Stevenson, Daniel R. NogueraAbstract:Anaerobic ammonium-oxidizing (Anammox) bacteria mediate a key step in the biogeochemical nitrogen cycle and have been applied worldwide for the energy-efficient removal of nitrogen from wastewater. However, outside their core energy metabolism, little is known about the metabolic networks driving Anammox bacterial anabolism and use of different carbon and energy substrates beyond genome-based predictions. Here, we experimentally resolved the central carbon metabolism of the Anammox Bacterium Candidatus ‘Kuenenia stuttgartiensis’ using time-series 13C and 2H isotope tracing, metabolomics, and isotopically nonstationary metabolic flux analysis. Our findings confirm predicted metabolic pathways used for CO2 fixation, central metabolism, and amino acid biosynthesis in K. stuttgartiensis, and reveal several instances where genomic predictions are not supported by in vivo metabolic fluxes. This includes the use of the oxidative branch of an incomplete tricarboxylic acid cycle for alpha-ketoglutarate biosynthesis, despite the genome not having an annotated citrate synthase. We also demonstrate that K. stuttgartiensis is able to directly assimilate extracellular formate via the Wood–Ljungdahl pathway instead of oxidizing it completely to CO2 followed by reassimilation. In contrast, our data suggest that K. stuttgartiensis is not capable of using acetate as a carbon or energy source in situ and that acetate oxidation occurred via the metabolic activity of a low-abundance microorganism in the bioreactor’s side population. Together, these findings provide a foundation for understanding the carbon metabolism of Anammox bacteria at a systems-level and will inform future studies aimed at elucidating factors governing their function and niche differentiation in natural and engineered ecosystems.
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Nutrient Limitation Causes Differential Expression of Transport- and Metabolism Genes in the Compartmentalized Anammox Bacterium Kuenenia stuttgartiensis.
Frontiers in microbiology, 2020Co-Authors: Marjan J. Smeulders, Mike S. M. Jetten, Huub J. M. Op Den Camp, Guylaine H. L. Nuijten, Theo Van Alen, Stijn H. Peeters, Daan De Bruijckere, Laura Van NiftrikAbstract:Anaerobic ammonium-oxidizing (Anammox) bacteria, members of the "Candidatus Brocadiaceae" family, play an important role in the nitrogen cycle and are estimated to be responsible for about half of the oceanic nitrogen loss to the atmosphere. Anammox bacteria combine ammonium with nitrite and produce dinitrogen gas via the intermediates nitric oxide and hydrazine (Anammox reaction) while nitrate is formed as a by-product. These reactions take place in a specialized, membrane-enclosed compartment called the Anammoxosome. Therefore, the substrates ammonium, nitrite and product nitrate have to cross the outer-, cytoplasmic-, and Anammoxosome membranes to enter or exit the Anammoxosome. The genomes of all Anammox species harbor multiple copies of ammonium-, nitrite-, and nitrate transporter genes. Here we investigated how the distinct genes for ammonium-, nitrite-, and nitrate- transport were expressed during substrate limitation in membrane bioreactors. Transcriptome analysis of Kuenenia stuttgartiensis planktonic cells showed that four of the seven ammonium transporter homologs and two of the nine nitrite transporter homologs were significantly upregulated during ammonium-limited growth, while another ammonium transporter- and four nitrite transporter homologs were upregulated in nitrite limited growth conditions. The two nitrate transporters were expressed to similar levels in both conditions. In addition, genes encoding enzymes involved in the Anammox reaction were differentially expressed, with those using nitrite as a substrate being upregulated under nitrite limited growth and those using ammonium as a substrate being upregulated during ammonium limitation. Taken together, these results give a first insight in the potential role of the multiple nutrient transporters in regulating transport of substrates and products in and out of the compartmentalized Anammox cell.
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autotrophic and mixotrophic metabolism of an Anammox Bacterium revealed by in vivo 13c and 2h metabolic network mapping
bioRxiv, 2020Co-Authors: Christopher E. Lawson, Mike S. M. Jetten, R.m. De Graaf, Guylaine H. L. Nuijten, Tyler B. Jacobson, Martin Pabst, David M. Stevenson, Daniel R. Noguera, Katherine D. Mcmahon, Daniel AmadornoguezAbstract:Anaerobic ammonium-oxidizing (Anammox) bacteria mediate a key step in the biogeochemical nitrogen cycle and have been applied worldwide for the energy-efficient removal of nitrogen from wastewater. However, outside their core energy metabolism, little is known about the metabolic networks driving Anammox bacterial anabolism and mixotrophy beyond genome-based predictions. Here, we experimentally resolved the central carbon metabolism of the Anammox Bacterium Candidatus Kuenenia stuttgartiensis using time-series 13C and 2H isotope tracing, metabolomics, and isotopically nonstationary metabolic flux analysis (INST-MFA). Our findings confirm predicted metabolic pathways used for CO2 fixation, central metabolism, and amino acid biosynthesis in K. stuttgartiensis, and reveal several instances where genomic predictions are not supported by in vivo metabolic fluxes. This includes the use of an oxidative tricarboxylic acid cycle, despite the genome not encoding a known citrate synthase. We also demonstrate that K. stuttgartiensis is able to directly assimilate extracellular formate via the Wood-Ljungdahl pathway instead of oxidizing it completely to CO2 followed by reassimilation. In contrast, our data suggests that K. stuttgartiensis is not capable of using acetate as a carbon or energy source in situ and that acetate oxidation occurred via the metabolic activity of a low-abundance microorganism in the bioreactors side population. Together, these findings provide a foundation for understanding the carbon metabolism of Anammox bacteria at a systems-level and will inform future studies aimed at elucidating factors governing their function and niche differentiation in natural and engineered ecosystems.
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a nitric oxide binding heterodimeric cytochrome c complex from the Anammox Bacterium kuenenia stuttgartiensis binds to hydrazine synthase
Journal of Biological Chemistry, 2019Co-Authors: Mohd Akram, Mike S. M. Jetten, Boran Kartal, Andreas Dietl, Joachim Reimann, Andreas Menzel, Wouter Versantvoort, Thomas R. M. BarendsAbstract:Anaerobic ammonium oxidation (Anammox) is a microbial process responsible for significant nitrogen loss from the oceans and other ecosystems. The redox reactions at the heart of Anammox are catalyzed by large multiheme enzyme complexes that rely on small cytochrome c proteins for electron shuttling. Among the most highly abundant of these cytochromes is a unique heterodimeric complex composed of class I and class II c-type cytochromes called NaxLS, which has distinctive biochemical and spectroscopic properties. Here, we present the 1.7 A resolution crystal structure of this complex from the Anammox organism Kuenenia stuttgartiensis (KsNaxLS). The structure reveals that the heme irons in each subunit exhibit a rare His/Cys ligation, which, as we show by substitution, causes the observed unusual spectral properties. Unlike its individual subunits, the KsNaxLS complex binds nitric oxide (NO) only at the distal heme side, forming 6cNO adducts. This is likely due to steric immobilization of the proximal heme-binding motifs upon complex formation, a finding that may be of functional relevance, because NO is an intermediate in the central Anammox metabolism. Pulldown experiments with K. stuttgartiensis cell-free extract showed that the KsNaxLS complex binds specifically to one of the central Anammox enzyme complexes, hydrazine synthase, which uses NO as one of its substrates. It is therefore possible that the KsNaxLS complex plays a role in binding the volatile NO to retain it in the cell for transfer to hydrazine synthase. Alternatively, we propose that KsNaxLS may shuttle electrons to this enzyme complex.
Kenji Furukawa - One of the best experts on this subject based on the ideXlab platform.
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Unique hexameric structure of copper-containing nitrite reductase of an Anammox Bacterium KSU-1.
Biochemical and biophysical research communications, 2020Co-Authors: Daisuke Hira, Kenji Furukawa, Misa Matsumura, Ryuji Kitamura, Takao FujiiAbstract:Abstract Anaerobic ammonium oxidation (Anammox) and denitrification are two different microbial reactions that form nitrogen gas. The initial step in the Anammox reaction—reduction of nitrite to nitric oxide—is thought to be catalyzed by homologs of dissimilatory nitrite reductase, which is known to be involved in denitrification. Here, we reveal the crystal structure of the copper-containing nitrite reductase (CuNIR) of strain KSU-1, an Anammox Bacterium. CuNIR had a unique homohexameric structure with three disulfide bridges between homotrimers, although the trimer was similar to that of known CuNIRs. Kinetic and mutagenesis analyses suggested that the hexameric structure is important for the electron transfer reaction.
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Anammox Organism KSU-1 Expresses a Novel His/DOPA Ligated Cytochrome c
Journal of molecular biology, 2018Co-Authors: Daisuke Hira, Kenji Furukawa, Ryuji Kitamura, Teruya Nakamura, Yuriko Yamagata, Takao FujiiAbstract:Abstract Anammox is a bacterial energy metabolic process that forms N2 gas from nitrite and ammonium ions. The enzymatic mechanisms of Anammox have been gradually revealed; however, the electron transport chain in Anammox bacteria remains poorly understood. In the present study, we purified and characterized two low-molecular-weight c-type cytochromes from an enriched culture of the Anammox Bacterium strain, KSU-1. Their genes, KSU1_B0428 and KSU1_C0855, were identified in the KSU-1 genome, and their recombinant proteins were characterized. KSU1_B0428 is a typical c-type cytochrome with a His/Met coordinated heme, acting as an electron transfer protein. In contrast, KSU1_C0855 could not be assigned as a known cytochrome and its heme was suggested to have an uncommon axial ligand set. Crystal structural analyses of C0855 clearly showed that its heme iron is coordinated by His15 as a fifth ligand. Moreover, the sixth coordination site is occupied by the aromatic ring of Tyr60, and an unassignable electron density that is inseparable with that of aromatic carbon of Tyr60 was found. The additional electron density was assigned to an O atom by molecular mass analyses. Therefore, Tyr60 would be chemically modified to 3,4-dihydroxyphenylalanine and bound to the Fe atom. We revealed that an Anammox Bacterium strain KSU-1 expresses a novel cytochrome c having an unprecedented His/3,4-dihydroxyphenylalanine coordinating heme. The expression of the novel c-type cytochrome might be required for the redox reaction of the Anammox process.
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ORIGINAL PAPER Development of a fixed-bed Anammox reactor with high treatment potential
2016Co-Authors: Hiroyuki Okamoto, Takao Fujii, Kimito Kawamura, Takashi Nishiyama, Kenji FurukawaAbstract:Abstract A plug-flow type anaerobic ammonium oxidation (Anammox) reactor was developed using malt ceramics (MC) produced from carbonized spent grains as the biomass carriers for Anammox sludge. Partial nitrified effluent of the filtrate from the sludge dehydrator of a brewery company was used as influent to a 20 L Anammox reactor using MC. An average volumetric nitrogen removal rate (VNR) of 8.78 kg-N/m3/day was maintained stably for 76 days with 1 h of HRT. In a larger Anammox reactor (400 L), an average VNR of 4.84 kg-N/m3/day could be main-tained for 86 days during the treatment of low strength synthetic inorganic wastewater. As a result of bacterial community analysis for the 20 L Anammox reactor, Asahi BRW1, probably originating from the waste-water collected at Asahi Breweries, was detected as the dominant Anammox Bacterium. These Anammox reactors were characterized by a high NH4-N removal capacity for low strength wastewater with a short hydraulic retention time
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Reduction of nitric oxide catalyzed by hydroxylamine oxidoreductase from an Anammox Bacterium
Journal of bioscience and bioengineering, 2014Co-Authors: Tatsuya Irisa, Kenji Furukawa, Daisuke Hira, Takao FujiiAbstract:The hydroxylamine oxidoreductase (HAO) from the Anammox Bacterium, Candidatus Kuenenia stuttgartiensis has been reported to catalyze the oxidation of hydroxylamine (NH2OH) to nitric oxide (NO) by using bovine cytochrome c as an oxidant. In contrast, we investigated whether the HAO from Anammox Bacterium strain KSU-1 could catalyze the reduction of NO with reduced benzyl viologen (BVred) and the NO-releasing reagent, NOC 7. The reduction proceeded, resulting in the formation of NH2OH as a product. The oxidation rate of BVred was proportional to the concentration of BVred itself for a short period in each experiment, a situation that was termed quasi-steady state. The analyses of the states at various concentrations of HAO allowed us to determine the rate constant for the catalytic reaction, (2.85 ± 0.19) × 105 M−1 s−1, governing NO reduction by BVred and HAO, which was comparable to that reported for the HAO from the ammonium oxidizer, Nitrosomonas with reduced methyl viologen. These results suggest that the Anammox HAO functions to adjust Anammox by inter-conversion of NO and NH2OH depending on the redox potential of the physiological electron transfer protein in Anammox bacteria.
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Development of a fixed-bed Anammox reactor with high treatment potential
Biodegradation, 2013Co-Authors: Hiroyuki Okamoto, Takao Fujii, Kimito Kawamura, Takashi Nishiyama, Kenji FurukawaAbstract:A plug-flow type anaerobic ammonium oxidation (Anammox) reactor was developed using malt ceramics (MC) produced from carbonized spent grains as the biomass carriers for Anammox sludge. Partial nitrified effluent of the filtrate from the sludge dehydrator of a brewery company was used as influent to a 20 L Anammox reactor using MC. An average volumetric nitrogen removal rate (VNR) of 8.78 kg-N/m^3/day was maintained stably for 76 days with 1 h of HRT. In a larger Anammox reactor (400 L), an average VNR of 4.84 kg-N/m^3/day could be maintained for 86 days during the treatment of low strength synthetic inorganic wastewater. As a result of bacterial community analysis for the 20 L Anammox reactor, Asahi BRW1, probably originating from the wastewater collected at Asahi Breweries, was detected as the dominant Anammox Bacterium. These Anammox reactors were characterized by a high NH_4-N removal capacity for low strength wastewater with a short hydraulic retention time.
Takao Fujii - One of the best experts on this subject based on the ideXlab platform.
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Unique hexameric structure of copper-containing nitrite reductase of an Anammox Bacterium KSU-1.
Biochemical and biophysical research communications, 2020Co-Authors: Daisuke Hira, Kenji Furukawa, Misa Matsumura, Ryuji Kitamura, Takao FujiiAbstract:Abstract Anaerobic ammonium oxidation (Anammox) and denitrification are two different microbial reactions that form nitrogen gas. The initial step in the Anammox reaction—reduction of nitrite to nitric oxide—is thought to be catalyzed by homologs of dissimilatory nitrite reductase, which is known to be involved in denitrification. Here, we reveal the crystal structure of the copper-containing nitrite reductase (CuNIR) of strain KSU-1, an Anammox Bacterium. CuNIR had a unique homohexameric structure with three disulfide bridges between homotrimers, although the trimer was similar to that of known CuNIRs. Kinetic and mutagenesis analyses suggested that the hexameric structure is important for the electron transfer reaction.
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Anammox Organism KSU-1 Expresses a Novel His/DOPA Ligated Cytochrome c
Journal of molecular biology, 2018Co-Authors: Daisuke Hira, Kenji Furukawa, Ryuji Kitamura, Teruya Nakamura, Yuriko Yamagata, Takao FujiiAbstract:Abstract Anammox is a bacterial energy metabolic process that forms N2 gas from nitrite and ammonium ions. The enzymatic mechanisms of Anammox have been gradually revealed; however, the electron transport chain in Anammox bacteria remains poorly understood. In the present study, we purified and characterized two low-molecular-weight c-type cytochromes from an enriched culture of the Anammox Bacterium strain, KSU-1. Their genes, KSU1_B0428 and KSU1_C0855, were identified in the KSU-1 genome, and their recombinant proteins were characterized. KSU1_B0428 is a typical c-type cytochrome with a His/Met coordinated heme, acting as an electron transfer protein. In contrast, KSU1_C0855 could not be assigned as a known cytochrome and its heme was suggested to have an uncommon axial ligand set. Crystal structural analyses of C0855 clearly showed that its heme iron is coordinated by His15 as a fifth ligand. Moreover, the sixth coordination site is occupied by the aromatic ring of Tyr60, and an unassignable electron density that is inseparable with that of aromatic carbon of Tyr60 was found. The additional electron density was assigned to an O atom by molecular mass analyses. Therefore, Tyr60 would be chemically modified to 3,4-dihydroxyphenylalanine and bound to the Fe atom. We revealed that an Anammox Bacterium strain KSU-1 expresses a novel cytochrome c having an unprecedented His/3,4-dihydroxyphenylalanine coordinating heme. The expression of the novel c-type cytochrome might be required for the redox reaction of the Anammox process.
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Enrichment and physiological characterization of an anaerobic ammonium-oxidizing Bacterium 'Candidatus Brocadia sapporoensis'.
Systematic and applied microbiology, 2017Co-Authors: Yuko Narita, Takao Fujii, Lei Zhang, Muhammad Ali, Zen-ichiro Kimura, Satoshi OkabeAbstract:We successfully enriched a novel anaerobic ammonium-oxidizing (Anammox) Bacterium affiliated with the genus 'Candidatus Brocadia' with high purity (>90%) in a membrane bioreactor (MBR). The enriched Bacterium was distantly related to the hitherto characterized 'Ca. Brocadia fulgida' and 'Ca. Brocadia sinica' with 96% and 93% of 16S ribosomal RNA gene sequence identity, respectively. The Bacterium exhibited the common structural features of Anammox bacteria and produced hydrazine in the presence of hydroxylamine under anoxic conditions. The temperature range of Anammox activity was 20-45°C with a maximum activity at 37°C. The maximum specific growth rate (μmax) was 0.0082h-1 at 37°C, corresponding to a doubling time of 3.5 days. The half-saturation constant (KS) for nitrite was 5±2.5μM. The Anammox activity was inhibited by nitrite (IC50=11.6mM) but not by formate and acetate. The major respiratory quinone was identified to be menaquinone-7 (MK-7). The enriched Anammox Bacterium shared nearly half of genes with 'Ca. Brocadia sinica' and 'Ca. Brocadia fulgida'. The enriched Bacterium showed all known physiological characteristics of Anammox bacteria and can be distinguished from the close relatives by its 16S rRNA gene sequence. Therefore, we proposed the name 'Ca. Brocadia sapporoensis' sp. nov.
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ORIGINAL PAPER Development of a fixed-bed Anammox reactor with high treatment potential
2016Co-Authors: Hiroyuki Okamoto, Takao Fujii, Kimito Kawamura, Takashi Nishiyama, Kenji FurukawaAbstract:Abstract A plug-flow type anaerobic ammonium oxidation (Anammox) reactor was developed using malt ceramics (MC) produced from carbonized spent grains as the biomass carriers for Anammox sludge. Partial nitrified effluent of the filtrate from the sludge dehydrator of a brewery company was used as influent to a 20 L Anammox reactor using MC. An average volumetric nitrogen removal rate (VNR) of 8.78 kg-N/m3/day was maintained stably for 76 days with 1 h of HRT. In a larger Anammox reactor (400 L), an average VNR of 4.84 kg-N/m3/day could be main-tained for 86 days during the treatment of low strength synthetic inorganic wastewater. As a result of bacterial community analysis for the 20 L Anammox reactor, Asahi BRW1, probably originating from the waste-water collected at Asahi Breweries, was detected as the dominant Anammox Bacterium. These Anammox reactors were characterized by a high NH4-N removal capacity for low strength wastewater with a short hydraulic retention time
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physiological characterization of anaerobic ammonium oxidizing Bacterium candidatus jettenia caeni
Environmental Microbiology, 2015Co-Authors: Muhammad Ali, Takanori Awata, Tomonori Kindaichi, Daisuke Hira, Mamoru Oshiki, Hisashi Satoh, Zen-ichiro Kimura, Kazuo Isobe, Hiroaki Yoshikawa, Takao FujiiAbstract:To date, six candidate genera of anaerobic ammonium-oxidizing (Anammox) bacteria have been identified, and numerous studies have been conducted to understand their ecophysiology. In this study, we examined the physiological characteristics of an Anammox Bacterium in the genus 'Candidatus Jettenia'. Planctomycete KSU-1 was found to be a mesophilic (20-42.5°C) and neutrophilic (pH 6.5-8.5) Bacterium with a maximum growth rate of 0.0020 h(-1) . Planctomycete KSU-1 cells showed typical physiological and structural features of Anammox bacteria; i.e. (29) N2 gas production by coupling of (15) NH4 (+) and (14) NO2 (-) , accumulation of hydrazine with the consumption of hydroxylamine and the presence of Anammoxosome. In addition, the cells were capable of respiratory ammonification with oxidation of acetate. Notably, the cells contained menaquinone-7 as a dominant respiratory quinone. Proteomic analysis was performed to examine underlying core metabolisms, and high expressions of hydrazine synthase, hydrazine dehydrogenase, hydroxylamine dehydrogenase, nitrite/nitrate oxidoreductase and carbon monoxide dehydrogenase/acetyl-CoA synthase were detected. These proteins require iron or copper as a metal cofactor, and both were dominant in planctomycete KSU-1 cells. On the basis of these experimental results, we proposed the name 'Ca. Jettenia caeni' sp. nov. for the bacterial clade of the planctomycete KSU-1.
Daisuke Hira - One of the best experts on this subject based on the ideXlab platform.
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Unique hexameric structure of copper-containing nitrite reductase of an Anammox Bacterium KSU-1.
Biochemical and biophysical research communications, 2020Co-Authors: Daisuke Hira, Kenji Furukawa, Misa Matsumura, Ryuji Kitamura, Takao FujiiAbstract:Abstract Anaerobic ammonium oxidation (Anammox) and denitrification are two different microbial reactions that form nitrogen gas. The initial step in the Anammox reaction—reduction of nitrite to nitric oxide—is thought to be catalyzed by homologs of dissimilatory nitrite reductase, which is known to be involved in denitrification. Here, we reveal the crystal structure of the copper-containing nitrite reductase (CuNIR) of strain KSU-1, an Anammox Bacterium. CuNIR had a unique homohexameric structure with three disulfide bridges between homotrimers, although the trimer was similar to that of known CuNIRs. Kinetic and mutagenesis analyses suggested that the hexameric structure is important for the electron transfer reaction.
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Anammox Organism KSU-1 Expresses a Novel His/DOPA Ligated Cytochrome c
Journal of molecular biology, 2018Co-Authors: Daisuke Hira, Kenji Furukawa, Ryuji Kitamura, Teruya Nakamura, Yuriko Yamagata, Takao FujiiAbstract:Abstract Anammox is a bacterial energy metabolic process that forms N2 gas from nitrite and ammonium ions. The enzymatic mechanisms of Anammox have been gradually revealed; however, the electron transport chain in Anammox bacteria remains poorly understood. In the present study, we purified and characterized two low-molecular-weight c-type cytochromes from an enriched culture of the Anammox Bacterium strain, KSU-1. Their genes, KSU1_B0428 and KSU1_C0855, were identified in the KSU-1 genome, and their recombinant proteins were characterized. KSU1_B0428 is a typical c-type cytochrome with a His/Met coordinated heme, acting as an electron transfer protein. In contrast, KSU1_C0855 could not be assigned as a known cytochrome and its heme was suggested to have an uncommon axial ligand set. Crystal structural analyses of C0855 clearly showed that its heme iron is coordinated by His15 as a fifth ligand. Moreover, the sixth coordination site is occupied by the aromatic ring of Tyr60, and an unassignable electron density that is inseparable with that of aromatic carbon of Tyr60 was found. The additional electron density was assigned to an O atom by molecular mass analyses. Therefore, Tyr60 would be chemically modified to 3,4-dihydroxyphenylalanine and bound to the Fe atom. We revealed that an Anammox Bacterium strain KSU-1 expresses a novel cytochrome c having an unprecedented His/3,4-dihydroxyphenylalanine coordinating heme. The expression of the novel c-type cytochrome might be required for the redox reaction of the Anammox process.
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physiological characterization of anaerobic ammonium oxidizing Bacterium candidatus jettenia caeni
Environmental Microbiology, 2015Co-Authors: Muhammad Ali, Takanori Awata, Tomonori Kindaichi, Daisuke Hira, Mamoru Oshiki, Hisashi Satoh, Zen-ichiro Kimura, Kazuo Isobe, Hiroaki Yoshikawa, Takao FujiiAbstract:To date, six candidate genera of anaerobic ammonium-oxidizing (Anammox) bacteria have been identified, and numerous studies have been conducted to understand their ecophysiology. In this study, we examined the physiological characteristics of an Anammox Bacterium in the genus 'Candidatus Jettenia'. Planctomycete KSU-1 was found to be a mesophilic (20-42.5°C) and neutrophilic (pH 6.5-8.5) Bacterium with a maximum growth rate of 0.0020 h(-1) . Planctomycete KSU-1 cells showed typical physiological and structural features of Anammox bacteria; i.e. (29) N2 gas production by coupling of (15) NH4 (+) and (14) NO2 (-) , accumulation of hydrazine with the consumption of hydroxylamine and the presence of Anammoxosome. In addition, the cells were capable of respiratory ammonification with oxidation of acetate. Notably, the cells contained menaquinone-7 as a dominant respiratory quinone. Proteomic analysis was performed to examine underlying core metabolisms, and high expressions of hydrazine synthase, hydrazine dehydrogenase, hydroxylamine dehydrogenase, nitrite/nitrate oxidoreductase and carbon monoxide dehydrogenase/acetyl-CoA synthase were detected. These proteins require iron or copper as a metal cofactor, and both were dominant in planctomycete KSU-1 cells. On the basis of these experimental results, we proposed the name 'Ca. Jettenia caeni' sp. nov. for the bacterial clade of the planctomycete KSU-1.
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Reduction of nitric oxide catalyzed by hydroxylamine oxidoreductase from an Anammox Bacterium
Journal of bioscience and bioengineering, 2014Co-Authors: Tatsuya Irisa, Kenji Furukawa, Daisuke Hira, Takao FujiiAbstract:The hydroxylamine oxidoreductase (HAO) from the Anammox Bacterium, Candidatus Kuenenia stuttgartiensis has been reported to catalyze the oxidation of hydroxylamine (NH2OH) to nitric oxide (NO) by using bovine cytochrome c as an oxidant. In contrast, we investigated whether the HAO from Anammox Bacterium strain KSU-1 could catalyze the reduction of NO with reduced benzyl viologen (BVred) and the NO-releasing reagent, NOC 7. The reduction proceeded, resulting in the formation of NH2OH as a product. The oxidation rate of BVred was proportional to the concentration of BVred itself for a short period in each experiment, a situation that was termed quasi-steady state. The analyses of the states at various concentrations of HAO allowed us to determine the rate constant for the catalytic reaction, (2.85 ± 0.19) × 105 M−1 s−1, governing NO reduction by BVred and HAO, which was comparable to that reported for the HAO from the ammonium oxidizer, Nitrosomonas with reduced methyl viologen. These results suggest that the Anammox HAO functions to adjust Anammox by inter-conversion of NO and NH2OH depending on the redox potential of the physiological electron transfer protein in Anammox bacteria.
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Anammox organism KSU-1 expresses a NirK-type copper-containing nitrite reductase instead of a NirS-type with cytochrome cd1.
FEBS letters, 2012Co-Authors: Daisuke Hira, Takashi Nishiyama, Kenji Furukawa, Hidehiro Toh, Catharina T. Migita, Hiroki Okubo, Masahira Hattori, Takao FujiiAbstract:Anaerobic ammonium oxidation (Anammox) and denitrification are two distinct microbial reactions relevant to the global nitrogen cycle. The proposed initial step of the Anammox reactions, reduction of nitrite to nitric oxide, has been postulated to be identical to that in denitrification catalyzed by the dissimilatory nitrite reductase of the cytochrome cd1-type. Here, we characterized the copper-containing nitrite reductase homolog encoded by nirK detected in the genome of an Anammox Bacterium strain KSU-1. We hypothesize that this NirK-type nitrite reductase, rather than a nitrite reductase of the cytochrome cd1-type (NirS), is likely to catalyze nitrite reduction in Anammox organism KSU-1.
Akiyoshi Ohashi - One of the best experts on this subject based on the ideXlab platform.
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Effects of Salts on the Activity and Growth of "Candidatus Scalindua sp.", a Marine Anammox Bacterium.
Microbes and environments, 2018Co-Authors: Amin Mojiri, Takanori Awata, Akiyoshi Ohashi, Noriatsu Ozaki, Kazuma Nishimoto, Yoshiteru Aoi, Tomonori KindaichiAbstract:Four salts, SEALIFE (a synthetic sea salt), NaCl, Na2SO4, and NaCl+KCl, were applied to monitor the effects of salinity on "Candidatus Scalindua sp.", a marine anaerobic ammonium oxidation (Anammox) Bacterium. The highest ammonium consumption of 10 μmol mg protein-1 d-1 was observed at 88 mmol L-1 of Na in the presence of NaCl. The highest inorganic carbon uptake of 0.6 μmol mg protein-1 d-1 was observed at 117 mmol L-1 of Na and at 16 mmol L-1 of K in the presence of NaCl+KCl. Thus, Na and K are both important for maintaining a high growth rate of "Candidatus Scalindua sp."
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biomass yield efficiency of the marine Anammox Bacterium candidatus scalindua sp is affected by salinity
Microbes and Environments, 2015Co-Authors: Takanori Awata, Tomonori Kindaichi, Noriatsu Ozaki, Akiyoshi OhashiAbstract:The growth rate and biomass yield efficiency of anaerobic ammonium oxidation (Anammox) bacteria are markedly lower than those of most other autotrophic bacteria. Among the Anammox bacterial genera, the growth rate and biomass yield of the marine Anammox Bacterium “Candidatus Scalindua sp.” is still lower than those of other Anammox bacteria enriched from freshwater environments. The activity and growth of marine Anammox bacteria are generally considered to be affected by the presence of salinity and organic compounds. Therefore, in the present study, the effects of salinity and volatile fatty acids (VFAs) on the Anammox activity, inorganic carbon uptake, and biomass yield efficiency of “Ca. Scalindua sp.” enriched from the marine sediments of Hiroshima Bay, Japan, were investigated in batch experiments. Differences in VFA concentrations (0–10 mM) were observed under varying salinities (0.5%–4%). Anammox activity was high at 0.5%–3.5% salinity, but was 30% lower at 4% salinity. In addition, carbon uptake was higher at 1.5%–3.5% salinity. The results of the present study clearly demonstrated that the biomass yield efficiency of the marine Anammox Bacterium “Ca. Scalindua sp.” was significantly affected by salinity. On the other hand, the presence of VFAs up to 10 mM did not affect Anammox activity, carbon uptake, or biomass yield efficiency.
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Biomass Yield Efficiency of the Marine Anammox Bacterium, “Candidatus Scalindua sp.,” is Affected by Salinity
Microbes and Environments, 2015Co-Authors: Takanori Awata, Tomonori Kindaichi, Noriatsu Ozaki, Akiyoshi OhashiAbstract:The growth rate and biomass yield efficiency of anaerobic ammonium oxidation (Anammox) bacteria are markedly lower than those of most other autotrophic bacteria. Among the Anammox bacterial genera, the growth rate and biomass yield of the marine Anammox Bacterium “Candidatus Scalindua sp.” is still lower than those of other Anammox bacteria enriched from freshwater environments. The activity and growth of marine Anammox bacteria are generally considered to be affected by the presence of salinity and organic compounds. Therefore, in the present study, the effects of salinity and volatile fatty acids (VFAs) on the Anammox activity, inorganic carbon uptake, and biomass yield efficiency of “Ca. Scalindua sp.” enriched from the marine sediments of Hiroshima Bay, Japan, were investigated in batch experiments. Differences in VFA concentrations (0–10 mM) were observed under varying salinities (0.5%–4%). Anammox activity was high at 0.5%–3.5% salinity, but was 30% lower at 4% salinity. In addition, carbon uptake was higher at 1.5%–3.5% salinity. The results of the present study clearly demonstrated that the biomass yield efficiency of the marine Anammox Bacterium “Ca. Scalindua sp.” was significantly affected by salinity. On the other hand, the presence of VFAs up to 10 mM did not affect Anammox activity, carbon uptake, or biomass yield efficiency.
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physiological characterization of an anaerobic ammonium oxidizing Bacterium belonging to the candidatus scalindua group
Applied and Environmental Microbiology, 2013Co-Authors: Takanori Awata, Tomonori Kindaichi, Akiyoshi Ohashi, Noriatsu Ozaki, Mamoru Oshiki, Satoshi OkabeAbstract:The phylogenetic affiliation and physiological characteristics (e.g., Ks and maximum specific growth rate [μmax]) of an anaerobic ammonium oxidation (Anammox) Bacterium, “Candidatus Scalindua sp.,” enriched from the marine sediment of Hiroshima Bay, Japan, were investigated. “Candidatus Scalindua sp.” exhibits higher affinity for nitrite and a lower growth rate and yield than the known Anammox species.