Masaharu Ishii

The University of Tokyo | Todai · Department of Biotechnology

Microalgae have emerged as a promising, next-generation sustainable resource with versatile applications, particularly as expression platforms and green cell factories. They possess the ability to overcome the limitations of terrestrial plants, such non-arable land, water scarcity, time-intensive growth, and seasonal changes. However, the heterolog...

The obligate chemolithoautotrophic bacterium, Hydrogenovibrio marinus MH-110, has three ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) isoenzymes, CbbM, CbbLS-1, and CbbLS-2, which differ in CO2/O2 specificity factor values. Expressions of CbbM and CbbLS-1 are regulated differently by transcriptional regulators of the LysR family, CbbRm...

Leptolyngbya sp. KC45 is a cyanobacterium isolated from a hot spring in the southern region of Thailand. This thermophilic and alkaliphilic strain can be cultured in an open pond without contamination under high pH growth condition. Additionally, its large trichome size makes it convenient for harvesting. Thus, Leptolyngbya sp. KC45 is a promising...

We report here the complete genome sequence of the acetic acid bacterium Acetobacter aceti type strain NBRC 14818. The genome comprises a chromosome of 3,596,270 bp with 57.1% GC content and four plasmids/phages of 63,279 bp, 25,755 bp, 4,858 bp, and 2,964 bp, with an average GC content of 57.0%.

This chapter provides an overview of microbial intracellular electron flow and its physiological contribution to metabolism. Oxidative and reductive reactions are involved in most cellular processes, including energy conservation, anabolic biosynthesis, maintenance of cellular redox balance, and antioxidant defense. The respiratory electron transpo...

Hydrogenophilus spp., which are moderately thermophilic aerobic betaproteobacteria, are widely distributed in geothermal environments. They fix carbon dioxide via the Calvin-Benson-Bassham cycle and exhibit rapid autotrophic growth using hydrogen as an energy source. Here, we report the complete genome sequence of Hydrogenophilus thermoluteolus str...

Dissimilatory nitrate reductase (NAR) and assimilatory nitrate reductase (NAS) serve as key enzymes for nitrogen catabolism and anabolism in many organisms. We purified NAR and NAS from H. thermophilus, a hydrogen-oxidizing chemolithoautotroph belonging to the phylogenetically deepest branch in the Bacteria domain. Physiological contribution of the...

Pseudomonas aeruginosa has one A-type (caa3) and multiple C-type (cbb3) cytochrome c oxidases as well as two quinol oxidases for aerobic respiration. The caa3 oxidase is highly efficient in creating a proton gradient across the cell membrane, but it is not expressed under normal growth conditions and its physiological role has not been investigated...

Schematic representation of the method used for constructing the transcriptional and translational fusion plasmids employed in the coxB promoter assay. (PDF)

Schematic representation of the promoter and regulatory regions of the transcriptional and translational fusion plasmids used for the coxB promoter assay. (PDF)

Bacterial strains and plasmids used in this study. (PDF)

Cytochrome c oxidase activity in the transformed strains visualized in blue color by the Nadi assay. (PDF)

Primers used in this study. (PDF)

Effect of the deletion of the terminal oxidase genes on the transcriptional activities of the RoxSR-dependent coxA and cioB promoters. The bacterial strains that carry the coxB or cioA promoter fused with lacZ were grown anaerobically in LB medium supplemented with 40 mM sodium nitrate. The overnight cultures were diluted ten times in synthetic med...

ROS generation by overexpression of the cox genes. (PDF)

Mutations identified in QXAaS2. (PDF)

Emergence of suppressor mutants that could grow aerobically in glutamate medium under aerobic conditions. The quadruple mutant QXAa and the quintuple mutant PTO5 were cultivated aerobically in 40 ml of LB medium or glutamate medium in 100-ml Erlenmeyer flasks with shaking at 300 rpm. (PDF)

Significance The cbb 3 cytochrome c oxidase has high affinity for oxygen and is typically used for respiration in many pathogenic bacteria in hypoxic environments. Here we show that the opportunistic human pathogen Pseudomonas aeruginosa has a capability to produce 16 different cbb 3 isoforms by combinations of multiple isosubunits. Utilization of...

Acetobacter aceti temporarily accumulates acetate as an incomplete oxidation product when cultured in the presence of ethanol. The accumulated acetate is utilized as a carbon and energy source via the tricarboxylic acid (TCA) cycle after the depletion of ethanol by the phenomenon termed acetate overoxidation. In this chapter, we provide an overview...

Factors that increase protein thermostability are of considerable interest in both scientific and industrial fields. Disulfide bonds are one of such factors that increase thermostability, but are rarely found in intracellular proteins because of the reducing environment of the cytosol. Here, we report the first example of an intermolecular disulfid...

NAD(+)-reducing [NiFe] hydrogenases catalyze the oxidoreduction of dihydrogen concomitant with the interconversion of NAD(+) and NADH. Here, the isolation, purification and crystallization of the NAD(+)-reducing [NiFe] hydrogenase from Hydrogenophilus thermoluteolus TH-1 are reported. Crystals of the NAD(+)-reducing [NiFe] hydrogenase were obtained...

Anaerobic digester is one of the attractive technologies for treatment of organic wastes and wastewater, while continuous development and improvements on their stable operation with efficient organic removal are required. Particles of conductive iron oxides (e.g., magnetite) are known to facilitate microbial interspecies electron transfer (termed a...

Organic loading conditions are an important factor influencing reactor performances in methanogenic bioreactors. Yet the underlying microbiological basis of the process stability, deterioration, and recovery remains to be understood. Here, structural responses of the bacterial and archaeal populations to the change of organic loading conditions in...

Acidification results from the excessive accumulation of volatile fatty acids and the breakthrough of buffering capacity in anaerobic digesters. However, little is known about the identity of the acidogenic bacteria involved. Here, we identified an active fermentative bacterium during acidification in a thermophilic anaerobic digester by sequencing...

Nitrite reductase (NIR) catalyzes the reduction of nitrite to nitric oxide (NO) in the denitrification pathway. In Pseudomonas aeruginosa, expression of the gene encoding NIR (nirS)is induced by NO and is under control of the NO-sensing regulator DNR. BecauseDNR is under control of the oxygen-sensing regulator ANR, nirSis expressed only under low o...

The ubiquitous opportunistic pathogen Pseudomonas aeruginosa has five aerobic terminal oxidases: bo3-type quinol oxidase (Cyo), cyanide-insensitive oxidase (CIO), aa3-type cytochrome c oxidase (aa3), and two cbb3-type cytochrome c oxidases (cbb3-1 and cbb3-2). These terminal oxidases are differentially regulated under various growth conditions and...

Ferriperoxin is a novel peroxidase essential for aerobiosis of Hydrogenobacter thermophilus. Although the ferriperoxin-deficient mutant (δfpx) was unable to grow aerobically, a suppressor mutant capable of aerobic growth was obtained after long aerobic cultivation. The alkyl hydroperoxide reductase gene was significantly upregulated in the suppress...

In anaerobic condition, amino acids are oxidatively deaminated, and decarboxylated, resulting in the production of volatile fatty acids. In this process, excess electrons are produced and their consumption is necessary for the accomplishment of amino acid degradation. In this study, we anaerobically constructed leucine-degrading enrichment cultures...

Three thermophilic methanogens (Methanothermobacter thermautotrophicus, Methanosaeta thermophila, and Methanosarcina thermophila) were investigated for their ability to reduce poorly crystalline Fe(III) oxides (ferrihydrite) and the inhibitory effects of ferrihydrite on their methanogenesis. This study demonstrated that Fe(II) generation from ferri...

The addition of ferrihydrite to methanogenic microbial communities obtained from a thermophilic anaerobic digester suppressed methanogenesis in a dose-dependent manner. The amount of reducing equivalents consumed by the reduction of iron was significantly smaller than that expected from the decrease in the production of CH4, which suggested that co...

The effect of a non-cellulolytic bacterium W2-10 (Geobacillus sp.) on the cellulose-degrading activity of a cellulolytic bacterium CTL-6 (Clostridium thermocellum) was determined using cellulose materials (paper and straw) in peptone cellulose solution (PCS) medium under aerobic conditions. The results indicated that in the co-culture, addition of...

The goal of this research was to investigate the effects of oxygen concentration on the composting process in China. The compost system was composed of cattle manure, chicken manure, and wheat straw combined at a ratio 1:3:6 for a period of 60 days. Microaerobic treatment was attained by manual turning, while aerobic treatment was performed by forc...

Novel-type serine-synthesizing enzymes, termed metal-independent phosphoserine phosphatases (iPSPs), were recently identified and characterized from Hydrogenobacter thermophilus, a chemolithoautotrophic bacterium belonging to the order Aquificales. iPSPs are cofactor-dependent phosphoglycerate mutase (dPGM)-like phosphatases that have significant a...

Background Conversion of industrial processes to more nature-friendly modes is a crucial subject for achieving sustainable development. Utilization of hydrogen-oxidation reactions by hydrogenase as a driving force of bioprocess reaction can be an environmentally ideal method because the reaction creates no pollutants. We expressed NAD-dependent alc...

A cytochrome bc-type complex of Roseobacter denitrificans OCh114 was thought to be a novel cytochrome c oxidase. To determine its function, we deleted the genes encoding the complex. The mutant grew normally by aerobic respiration, but failed to grow by denitrification and lacked nitric oxide reductase activity, indicating that the physiological fu...

Hydrogenobacter thermophilus is a chemolithoautotroph that utilizes not only hydrogen (H(2)) but also thiosulfate as sole source of energy and assimilates carbon dioxide via the reductive tricarboxylic acid (RTCA) cycle. We systematically carried out transcriptome analysis of metabolic enzymes in both H(2)- and thiosulfate-grown H. thermophilus cel...

Wild-type Acetobacter aceti NBRC 14818 possesses genes encoding isocitrate lyase (aceA) and malate synthase (glcB), which constitute the glyoxylate pathway. In contrast, several acetic acid bacteria that are utilized for vinegar production lack these genes. Here, an aceA-glcB knockout mutant of NBRC 14818 was constructed and used for investigating...

Two novel-type phosphoserine phosphatases (PSPs) with unique substrate specificity from the thermophilic and hydrogen-oxidizing bacterium Hydrogenobacter thermophilus TK-6 have previously been identified. Here, one of the PSPs (iPSP1) was heterologously expressed in Escherichia coli, purified and crystallized. Diffraction-quality crystals were obta...

Species belonging to the order Aquificales are believed to be an early branching lineage within the Bacteria. However, the branching order of this group in single-gene phylogenetic trees is highly variable; for example, it has also been proposed that the Aquificales should be grouped with ε-proteobacteria. To investigate the phylogenetic position o...

List of 62 proteins to construct the phylogenetic tree based on 18 whole genomes

Rubrerythrin (Rbr) is a non-heme iron protein composed of two distinctive domains and functions as a peroxidase in anaerobic organisms. A novel Rbr-like protein, ferriperoxin (Fpx), was identified in Hydrogenobacter thermophilus and was found not to possess the rubredoxin-like domain that is present in typical Rbrs. Although this protein is widely...

Phylogenetic tree of 1- and 2-domain types of rubrerythrin-like proteins based on amino acid sequence. One hundred amino acid sequences were obtained by BLAST search using the amino acid sequence of Hydrogenobacter thermophilus Fpx (Hth). The 100 sequences and three additional amino acid sequences of Rbrs (Desulfovibrio vulgaris, Dvu; Pyrococcus fu...

The pH-dependent changes of peroxidase activity of Fpx. In order to determine the optimal pH for peroxidase activity of Fpx, pH-dependency of peroxidase activity of Fpx was analyzed. Peroxidase assays were routinely carried out anaerobically in a 200-μl reaction mixture containing 0.32 µM Fpx, 0.0074 µM FNR, 0.5 mM NADPH, 100 µM hydroperoxides (H2O...

Amino acid sequences of Fpx, SulE, and two Rbr proteins. Residues liganded to metal centers and those conserved among all four sequences are underlined and shown in bold, respectively. Predicted secondary structure of Fpx is shown above the sequences; dotted lines and an arrow represent the α-helix and loop, respectively. Hth, Hydrogenobacter therm...

Characterization of recombinant Fpx, a rubrerythrin-like protein from H. thermophilus. A. SDS-PAGE analysis of the purified recombinant Fpx on a 13% acrylamide gel. Lane 1, 11 µg of purified Fpx, which had an apparent molecular mass of 16 kDa. B. Elution profiles of Fpx and selected molecular standards. Molecular mass of native subunit composition...

The list of organisms used for the phylogenetic tree of Rbr. (PDF)

Many aerobic microorganisms can colonize at the air-liquid interface and form a multicellular structure, known as a pellicle. In this study, the involvement of motility and attachment traits in the Pseudomonas aeruginosa pellicle formation process was investigated. Flagella- and flagellar-motor-deficient mutants exhibited delayed pellicle formation...

Phosphoserine phosphatase (PSP) catalyzes the dephosphorylation of phosphoserine to serine and inorganic phosphate. PSPs, which have been found in all three domains of life, belong to the haloacid dehalogenase-like hydrolase superfamily. However, certain organisms, particularly bacteria, lack a classical PSP gene, although they appear to possess a...

This study aimed to understand characteristics of thermostable phycoerythrin from hot spring cyanobacteria Leptolyngbya sp. KC45. Phycoerythrin was purified with the purification index at 17.38 of A565/A280 ratio and demonstrated as two protein bands of 21 and 18 kDa under SDS-PAGE analysis. The native protein was assumed to be hexamer with a molec...

Serine hydroxymethyltransferase catalyzes the cleavage of β-hydroxyamino acids into glycine and aldehydes in the absence of tetrahydrofolate. The enzyme accepts various β-hydroxyamino acids as the substrate of this reaction. The reaction rate varies depending on the substituent and stereochemistry at the Cβ atom: the erythro forms and the β-phenyl...

Acetobacter aceti NBRC 14818 shows a diauxic growth profile and temporarily accumulates acetate when grown in medium containing ethanol. However, the mechanisms underlying the metabolic switching between the incomplete oxidation of ethanol and overoxidation of acetate, and the control of stress resistance systems in A. aceti cells grown on ethanol...

Four cyanobacterial strains including Cyanosarcina sp. SK40, Phormidium sp. PD40-1, Scytonema sp. TP40 and Leptolyngbya sp. KC45 were selected and investigated for the phycobiliprotein (PBP) content and thermostable antioxidant activity of their cell-free extracts. The highest content of 181.63 mg/g dry weight phycobiliprotein was found in Leptolyn...

We developed a cross-linking far-western blotting (WB) technique to detect weak protein-protein interactions. In addition, by comparison of the banding patterns resulting from the conventional and cross-linking methods, weak interactions can be identified by monitoring the enhancement of band intensities.

Syntrophic oxidation of acetate, so-called reversed reductive acetogenesis, is one of the most important degradation steps in anaerobic digesters. However, little is known about the genetic diversity of the micro-organisms involved. Here we investigated the activity and composition of potentially acetate-oxidizing syntrophs using a combinatorial ap...

An efficient cellulose degrading bacteria exists in the thermophilic wheat straw-degrading community, WDC2. However, this strain cannot be isolated and cultured using conventional separation techniques under strict anaerobic conditions. We successfully isolated a strain of effective cellulose degrading bacteria CTL-6 using a wash, heat shock, and s...

The air-liquid interface is a selectively advantageous niche for aerobes due to the accessibility to oxygen. Various species of aerobes form a biofilm-like structure at air-liquid interfaces, known as a pellicle. Although the pellicle is one of the major growth modes of microorganisms, the metabolic features of pellicle cells and the determinative...

Acidification is one of the most common and serious problems inducing process failure in anaerobic digesters. The production of volatile fatty acids (VFAs) mainly triggers acidic shock. However, little is known about the bacteria involved in the processes of acidogenic metabolism, such as fermentation and reductive acetogenesis. Here, the metabolic...

We focused on bacterial interspecies relationships at the air-liquid interface where the formation of pellicles by aerobes was observed. Although an obligate aerobe (Brevibacillus sp. M1-5) was initially dominant in the pellicle population, a facultative aerobe (Pseudoxanthomonas sp. M1-3) emerged and the viability of M1-5 rapidly decreased due to...

The draft genome sequence of Acetobacter aceti NBRC 14818 was determined by whole-genome shotgun sequencing and the transcriptome profile in cells exponentially grown on ethanol, acetate or glucose was analysed by using a DNA microarray. The genes for all enzymes that constitute the complete tricarboxylic acid (TCA) cycle and glyoxylate pathway wer...

The methanogenic pathway and microbial community in a thermophilic anaerobic digestion process of organic solid waste were investigated in a continuous-flow stirred-tank reactor using artificial garbage slurry as a feedstock. The decomposition pathway of acetate, a significant precursor of CH(4) and a key intermediate metabolite in the anaerobic di...

The thermo-neutrophilic hydrogen-oxidizing bacterium Hydrogenobacter thermophilus constitutively oxidizes thiosulfate. Soluble extracts of it showed temperature-dependent thiosulfate oxidation activity with an optimum at 60 degrees C. A gene cluster for sox (sulfur oxidation) is presumably responsible for this activity. Among the cluster, two tande...

Aminotransferases catalyse synthetic and degradative reactions of amino acids, and serve as a key linkage between central carbon and nitrogen metabolism in most organisms. In this study, three aminotransferases (AT1, AT2 and AT3) were purified and characterized from Hydrogenobacter thermophilus, a hydrogen-oxidizing chemolithoautotrophic bacterium,...

Hydrogenobacter thermophilus is a thermophilic, obligately chemolithoautotrophic and aerobic hydrogen-oxidizing bacterium. It is unique in its ability to fix carbon dioxide via the reductive tricarboxylic acid cycle under aerobic conditions. It utilizes molecular hydrogen, elemental sulfur, or thiosulfate as the sole energy source. Here, we report...

Pyruvate: ferredoxin oxidoreductase (POR; EC 1.2.7.1) catalyzes the thiamine pyrophosphate-dependent oxidative decarboxylation of pyruvate to form acetyl-CoA and CO(2). The thermophilic, obligate chemolithoautotrophic hydrogen-oxidizing bacterium, Hydrogenobacter thermophilus TK-6, assimilates CO(2) via the reductive tricarboxylic acid cycle. In th...

Hydrogenobacter thermophilus TK-6 is a thermophilic, chemolithoautotrophic, hydrogen-oxidizing bacterium that fixes carbon dioxide via the reductive tricarboxylic acid (rTCA) cycle. 2-Oxoglutarate:ferredoxin oxidoreductase (OGOR) is the key enzyme in this cycle that fixes carbon dioxide. The genome of strain TK-6 encodes at least two distinct OGOR...

Pseudomonas aeruginosa has five terminal oxidases for aerobic respiration. Two of them, the bo(3) oxidase (Cyo) and the cyanide-insensitive oxidase (CIO), are quinol oxidases and the other three, the cbb(3)-1 oxidase (Cbb3-1), the cbb(3)-2 oxidase (Cbb3-2) and the aa(3) oxidase (Aa3), are cytochrome c oxidases. The expression pattern of the genes f...

The thermophilic, obligately chemolithoautotrophic hydrogen-oxidizing bacterium, Hydrogenobacter thermophilus TK-6, assimilates carbon dioxide via the reductive tricarboxylic acid cycle. Small iron-sulfur proteins, ferredoxins, play a central role as low-potential electron donors for this cycle. The fpr gene of this bacterium, encoding a putative f...

To study the microbial community responsible for the reduction of the polluting load during aerobic digestion of pig slurry. We analysed bacterial succession by nonculture-based methods and determined the physicochemical parameters and polluting substances during 6 days of aerobic digestion. The bacterial subpopulations evolved by aeration, predomi...

Fumarate reductase (FRD) is an enzyme that reduces fumarate to succinate. In many organisms, it is bound to the membrane and uses electron donors such as quinol. In this study, an FRD from a thermophilic chemolithoautotrophic bacterium, Hydrogenobacter thermophilus TK-6, was purified and characterized. FRD activity using NADH as an electron donor w...

To speed up the conversion of rice straw into feeds in a low-temperature region, a start culture used for ensiling rice straw at low temperature was selected by continuous enrichment cultivation. During the selection, the microbial source for enrichment was rice straw and soil from two places in Northeast China. Lab-scale rice straw fermentation at...

To efficiently utilize rice straw and lessen its disposal problem on the environment, a lactic acid bacteria community, SFC-2 was developed from natural fermentation products of rice straw by continuous enrichment with the MRS-S broth (MRS broth with sucrose), and used to accelerate the fermentation of air-dried straws. The SFC-2 could rapidly lowe...