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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 as electric syntrophy). Electric syntrophy has been reported to enhance methanogenic degradation of organic acids by mesophilic communities in soil and anaerobic digester. Here we investigated the effects of supplementation of conductive iron oxides (magnetite) on thermophilic methanogenic microbial communities derived from a thermophilic anaerobic digester. Supplementation of magnetite accelerated methanogenesis from acetate and propionate under thermophilic conditions, while supplementation of ferrihydrite also accelerated methanogenesis from propionate. Microbial community analysis revealed that supplementation of magnetite drastically changed bacterial populations in the methanogenic acetate-degrading cultures, in which Tepidoanaerobacter sp. and Coprothermobacter sp. dominated. These results suggest that supplementation of magnetite induce electric syntrophy between organic acid-oxidizing bacteria and methanogenic archaea and accelerate methanogenesis even under thermophilic conditions. Findings from this study would provide a possibility for the achievement of stably operating thermophilic anaerobic digestion systems with high efficiency for removal of organics and generation of CH4., (Copyright © 2014 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

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 suppressor mutant, indicating that the enzyme counteracts oxidative stress in the absence of ferriperoxin.

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 competition between iron-reducing microorganisms and methanogens was not the most significant cause for the suppression of methanogenesis. Microbial community analyses revealed that the presence of ferrihydrite markedly affected the bacterial composition, but not the archaeal composition. These results indicate that the presence of ferrihydrite directly and indirectly suppresses thermophilic methanogenesis.

Decomposition of lignocelluloses by cooperative microbial actions is an essential process of carbon cycling in nature and provides a basis for biomass conversion to fuels and chemicals in biorefineries. In this study, structurally stable symbiotic aero-tolerant lignocellulose-degrading microbial consortia were obtained from biodiversified microflora present in industrial sugarcane bagasse pile (BGC-1), cow rumen fluid (CRC-1), and pulp mill activated sludge (ASC-1) by successive subcultivation on rice straw under facultative anoxic conditions. Tagged 16S rRNA gene pyrosequencing revealed that all isolated consortia originated from highly diverse environmental microflora shared similar composite phylum profiles comprising mainly Firmicutes, reflecting convergent adaptation of microcosm structures, however, with substantial differences at refined genus level. BGC-1 comprising cellulolytic Clostridium and Acetanaerobacterium in stable coexistence with ligninolytic Ureibacillus showed the highest capability on degradation of agricultural residues and industrial pulp waste with CMCase, xylanase, and β-glucanase activities in the supernatant. Shotgun pyrosequencing of the BGC-1 metagenome indicated a markedly high relative abundance of genes encoding for glycosyl hydrolases, particularly for lignocellulytic enzymes in 26 families. The enzyme system comprised a unique composition of main-chain degrading and side-chain processing hydrolases, dominated by GH2, 3, 5, 9, 10, and 43, reflecting adaptation of enzyme profiles to the specific substrate. Gene mapping showed metabolic potential of BGC-1 for conversion of biomass sugars to various fermentation products of industrial importance. The symbiotic consortium is a promising simplified model for study of multispecies mechanisms on consolidated bioprocessing and a platform for discovering efficient synergistic enzyme systems for biotechnological application.

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 amino acid sequence similarity to dPGMs but lack phosphoglycerate mutase activity. Genes coding dPGM-like phosphatases have been identified in a broad range of organisms; however, predicting the function of the corresponding proteins based on sequence information alone is difficult due to their diverse substrate preferences. Here, we determined the crystal structure of iPSP1 from H. thermophilus in the apo-form and in complex with its substrate L-phosphoserine to find structural units important for its phosphatase activity toward L-phosphoserine. Structural and biochemical characterization of iPSP1 revealed that the side chains of His(85) and C-terminal region characteristic of iPSP1 are responsible for the PSP activity. The importance of these structural units for PSP activity was confirmed by high PSP activity observed in two novel dPGM-like proteins from Cyanobacteria and Chloroflexus in which the two structural units were conserved. We anticipate that our present findings will facilitate understanding of the serine biosynthesis pathways of organisms that lack gene(s) encoding conventional PSPs, as the structural information revealed here will help to identify iPSP from sequence databases.

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 obtained by the sitting-drop vapour-diffusion method using PEG 4000 as the precipitant. Two diffraction data sets with resolution ranges of 45.0-2.50 and 45.0-1.50 Å were collected from a single crystal and were merged to give a highly complete data set. The space group of the crystal was identified as primitive orthorhombic P2(1)2(1)2(1), with unit-cell parameters a = 49.8, b = 73.6, c = 124.3 Å. The calculated Matthews coefficient (V(M) = 2.32 Å(3) Da(-1)) indicated that the crystal contained one iPSP1 complex per asymmetric unit.

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 substituent are preferred over the threo forms and the β-methyl substituent, respectively. Although several mechanisms have been proposed, what determines the substrate preference remains unclear. We first performed quantum mechanical calculations to assess the validity of the reaction mechanisms. The results indicate that the retro-aldol mechanism starting with abstraction of the proton from the β-hydroxyl group is plausible. This also suggests that Cα-Cβ bond cleavage is the rate-limiting step. We next measured the dependence of the rate constants on temperature with four representative substrates and calculated the activation energies and pre-exponential factors from the Arrhenius plots. The activation energies of the erythro forms were lower than those of the threo forms. The β-phenyl substituent lowered the activation energy in the threo form, whereas it did not alter the activation energy but increased the pre-exponential factor in the erythro form. We present a unified model to explain the origin of the substituent and stereochemical preferences by combining the theoretical and experimental results. A possible biological role of the tetrahydrofolate-independent activity in thermophiles is also discussed., (© 2011 The Authors Journal compilation © 2011 FEBS.)

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 distributed among aerobic organisms, its function remains unknown. In this study, Fpx exhibited ferredoxin:NADPH oxidoreductase (FNR)-dependent peroxidase activity and reduced both hydrogen peroxide (H(2)O(2)) and organic hydroperoxide in the presence of NADPH and FNR as electron donors. The calculated K(m) and V(max) values of Fpx for organic hydroperoxides were comparable to that for H(2)O(2), demonstrating a multiple reactivity of Fpx towards hydroperoxides. An fpx gene disruptant was unable to grow under aerobic conditions, whereas its growth profiles were comparable to those of the wild-type strain under anaerobic and microaerobic conditions, clearly indicating the indispensability of Fpx as an antioxidant of H. thermophilus in aerobic environments. Structural analysis suggested that domain-swapping occurs in Fpx, and this domain-swapped structure is well conserved among thermophiles, implying the importance of structural stability of domain-swapped conformation for thermal environments. In addition, Fpx was located on a deep branch of the phylogenetic tree of Rbr and Rbr-like proteins. This finding, taken together with the wide distribution of Fpx among Bacteria and Archaea, suggests that Fpx is an ancestral type of Rbr homolog that functions as an essential antioxidant and may be part of an ancestral peroxide-detoxification system.

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 cells. The analysis indicated that the expression of hydrogenase genes is repressed under thiosulfate oxidation conditions as compared with H(2) oxidation conditions. This was confirmed by enzyme assay. In contrast, some genes for sulfur metabolism, including sox genes, showed almost the same expression levels under both conditions. In addition, the genes for the RTCA cycle showed high expression levels under both conditions. It was suggested that sulfur metabolism and the RTCA cycle function as forms of basal metabolism, and H(2) oxidation is inducible. Switching of H(2) oxidation can be advantageous for the lifestyle of this bacterium in nature.

Sugarcane bagasse is an important lignocellulosic by-product with potential for conversion to biofuels and chemicals in biorefinery. As a step towards an understanding of microbial diversity and the processes existing in bagasse collection sites, the microbial community in industrial bagasse feedstock piles was investigated. Molecular biodiversity analysis of 16S rDNA sequences revealed the presence of a complex bacterial community. A diverse group of mainly aerobic and facultative anaerobic bacteria was identified reflecting the aerobic and high temperature microenvironmental conditions under the pile surface. The major bacterial taxa present were identified as Firmicutes, Alpha- and Gammaproteobacteria, Acidobacteria, Bacteroidetes, and Actinobacteria. Analysis of the eukaryotic microbial assemblage based on an internal transcribed spacer revealed the predominance of diverse cellulolytic and hemicellulolytic ascomycota. A microbial interaction model is proposed, focusing on lignocellulose degradation and methane metabolism. The insights into the microbial community in this study provide a basis for efficient utilization of bagasse in lignocellulosic biomass-based industries.

Bioelectrochemical reactors (BERs) with a cathodic working potential of -0.6 or -0.8 V more efficiently degraded cellulosic material, i.e., filter paper (57.4-74.1% in 3 days and 95.9-96.3% in 7 days) than did control reactors without giving exogenous potential (15.4% in 3 days and 64.2% in 7 days). At the same time, resultant conversions to methane and carbon dioxide in cathodic working chamber of BERs by application of electrochemical reduction in 3 days of operation were larger than control reactors. However, cumulative methane production in cathodic BERs was similar to those in control reactors after 7 days of operation. Microscopic observation and 16S rRNA gene analysis showed that microbial growth in the entire consortium was higher after 2 days of operation of cathodic BERs as compared with the control reactors. In addition, the number of methanogenic 16S rRNA gene copies in cathodic BERs was higher than in control reactors. Moreover, archaeal community structures constructed in cathodic BERs consisted of hydrogenotrophic methanogen-related organisms and differed from those in control reactors after 2 days of operation. Specifically, the amount of Methanothermobacter species in cathodic BERs was higher within archaeal communities than in those control reactors after 2 days of operation. Electrochemical reduction may be effective for accelerating microbial growth in the start-up period and thereby increasing microbial treatment of cellulosic waste and methane production.

In anoxic rice field soil, ferric iron reduction is one of the most important terminal electron accepting processes, yet little is known about the identity of iron-reducing microorganisms. Here, we identified acetate-metabolizing bacteria by RNA-based stable isotope probing in the presence of iron(III) oxides as electron acceptors. After reduction of endogenous iron(III) for 21 days, isotope probing with (13)C-labeled acetate (2 mM) and added ferric iron oxides (ferrihydrite or goethite) was performed in rice field soil slurries for 48 and 72 h. Ferrihydrite reduction coincided with a strong suppression of methanogenesis (77%). Extracted RNA from each treatment was density resolved by isopycnic centrifugation, and analyzed by terminal restriction fragment length polymorphism, followed by cloning and sequencing of 16S rRNA of bacterial and archaeal populations. In heavy, isotopically labeled RNAs of the ferrihydrite treatment, predominant (13)C-assimilating populations were identified as Geobacter spp. (approximately 85% of all clones). In the goethite treatment, iron(II) formation was not detectable. However, Geobacter spp. (approximately 30%), the delta-proteobacterial Anaeromyxobacter spp. (approximately 30%), and novel beta-Proteobacteria were predominant in heavy rRNA fractions indicating that (13)C-acetate had been assimilated in the presence of goethite, whereas none were detected in the control heavy RNA. For the first time, active acetate-oxidizing iron(III)-reducing bacteria, including novel hitherto unrecognized populations, were identified as a functional guild in anoxic paddy soil.

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 enzymes, termed For and Kor. We report here a method for measuring the carboxylation of succinyl-CoA catalyzed by OGORs. The method involves the in vitro coupling of OGOR with ferredoxin and pyruvate:ferredoxin oxidoreductase from strain TK-6, and glutamate dehydrogenase from Sulfolobus tokodaii. Using this method, we determined both the apparent maximum velocities and the K (m) values of For and Kor for the carboxylation of succinyl-CoA. This is the first reported kinetic analysis of carbon fixation catalyzed by OGOR enzymes from the rTCA cycle.

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 tandemly coded soxA genes were uniquely found. These results suggest that the Sox system is widespread even in thermo-neutrophilic environments.

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 this cycle, POR acts as pyruvate synthase catalyzing the reverse reaction (i.e. reductive carboxylation of acetyl-CoA) to form pyruvate. The pyruvate synthesis reaction catalyzed by POR is an energetically unfavorable reaction and requires a strong reductant. Moreover, the reducing equivalents must be supplied via its physiological electron mediator, a small iron-sulfur protein ferredoxin. Therefore, the reaction is difficult to demonstrate in vitro and the reaction mechanism has been poorly understood. In the present study, we coupled the decarboxylation of 2-oxoglutarate catalyzed by 2-oxoglutarate: ferredoxin oxidoreductase (EC 1.2.7.3), which generates sufficiently low-potential electrons to reduce ferredoxin, to drive the energy-demanding pyruvate synthesis by POR. We demonstrate that H. thermophilus POR catalyzes pyruvate synthesis from acetyl-CoA and CO(2), confirming the operation of the reductive tricarboxylic acid cycle in this bacterium. We also measured the electron paramagnetic resonance spectra of the POR intermediates in both the forward and reverse reactions, and demonstrate the intermediacy of a 2-(1-hydroxyethyl)- or 2-(1-hydroxyethylidene)-thiamine pyrophosphate radical in both reactions. The reaction mechanism of the reductive carboxylation of acetyl-CoA is also discussed.

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 ferredoxin-NADP(+) reductase (FNR, EC 1.18.1.2), was expressed in Escherichia coli, and the recombinant protein was purified to homogeneity. Unexpectedly, the monomeric Fpr protein contained one molecule of FMN as a prosthetic group, although FNRs from other organisms are known to contain FAD. The FMN-containing Fpr was shown to be a bona fide FNR that catalyzes a reversible redox reaction between NADP(+)/NADPH and ferredoxins.

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 was not detected in the membrane fraction but was found in the soluble fraction. The purified enzyme was demonstrated to be a novel type of FRD, consisting of five subunits. One subunit showed high sequence identity to the catalytic subunits of known FRDs. Although the genes of typical FRDs are assembled in a cluster, the five genes encoding the H. thermophilus FRD were distant from each other in the genome. Furthermore, phylogenetic analysis showed that the H. thermophilus FRD was located in a distinct position from those of known soluble FRDs. This is the first report of a soluble NADH-dependent FRD in Bacteria and of the purification of a FRD that operates in the reductive tricarboxylic acid cycle.

Antibiotic-sensitive bacteria have been found to coexist with antibiotic-producing bacteria in biofilms, but little is known about how the former develop in such an environment. Here we isolated pyocyanin-sensitive bacteria belonging to the genus Brevibacillus from a biofilm derived from soil extract and based on the preestablished biofilm of a pyocyanin producer, Pseudomonas aeruginosa strain P1. In addition, pyocyanin-resistant strains belonging to the genus Raoultella were isolated from the same biofilm. Microbial relationships within biofilms were examined by using three strains, strain P1, Brevibacillus strain S1, and Raoultella strain R1, each of which individually formed a biofilm within 2 days in a flow cell. Strain S1 did not fully develop on the preestablished biofilm of strain P1 during 4 days of cultivation, whereas a mutant of strain P1 which was deficient in pyocyanin production allowed strain S1 to cocolonize within a biofilm. On the other hand, strain R1 developed on the biofilm of strain P1 regardless of pyocyanin production. When mixed 1:1 inocula of strains S1 and R1 were introduced into the strain P1 biofilm, all three species were found in the 4-day biofilm. In the mixed biofilm, strain S1 was surrounded by the layer of strain R1 and seemed to be separated from strain P1 and the outflow solution. However, strain S1 did not survive in a three-species mixed culture under planktonic conditions. These results indicate that the survival of sensitive bacteria in biofilm with a pyocyanin producer is achieved by covering them with a layer of resistant bacteria. We also evaluated the influence of antibiotic production on the producer.

Isocitrate dehydrogenase (ICDH) from Hydrogenobacter thermophilus catalyzes the reduction of oxalosuccinate, which corresponds to the second step of the reductive carboxylation of 2-oxoglutarate in the reductive tricarboxylic acid cycle. In this study, the oxidation reaction catalyzed by H. thermophilus ICDH was kinetically analyzed. As a result, a rapid equilibrium random-order mechanism was suggested. The affinities of both substrates (isocitrate and NAD+) toward the enzyme were extremely low compared to other known ICDHs. The binding activities of isocitrate and NAD+ were not independent; rather, the binding of one substrate considerably promoted the binding of the other. A product inhibition assay demonstrated that NADH is a potent inhibitor, although 2-oxoglutarate did not exhibit an inhibitory effect. Further chromatographic analysis demonstrated that oxalosuccinate, rather than 2-oxoglutarate, is the reaction product. Thus, it was shown that H. thermophilus ICDH is a nondecarboxylating ICDH that catalyzes the conversion between isocitrate and oxalosuccinate by oxidation and reduction. This nondecarboxylating ICDH is distinct from well-known decarboxylating ICDHs and should be categorized as a new enzyme. Oxalosuccinate-reducing enzyme may be the ancestral form of ICDH, which evolved to the extant isocitrate oxidative decarboxylating enzyme by acquiring higher substrate affinities.

An anaerobic packed-bed reactor using carbon fiber textiles (CFT) as the supporting material was continuously operated using an artificial garbage slurry. 16S rRNA gene analysis showed that many bacteria in the biomass adhering to CFT were closely related to those observed from other anaerobic environments, although a wide variety of unidentified bacteria were also found. Dot blot hybridization results clarified that 16S rRNA levels of methanogens in the adhering biomass were higher than those in the effluent. Based on microscopic observation, the adhering biomass consisted of microorganisms, organic material, and void areas. Bacteria and Archaea detected by fluorescence in situ hybridization were distributed from the surface to the inner regions of the adhering biomass. Methanosarcina sp. tended to be more abundant in the inner part of the adhering biomass than at the surface. This is the first report to elucidate the structure of the microbial community on CFT in a packed-bed reactor.

Glutamate synthases are classified according to their specificities for electron donors. Ferredoxin-dependent glutamate synthases had been found only in plants and cyanobacteria, whereas many bacteria have NADPH-dependent glutamate synthases. In this study, Hydrogenobacter thermophilus, a hydrogen-oxidizing chemoautotrophic bacterium, was shown to possess a ferredoxin-dependent glutamate synthase like those of phototrophs. This is the first observation, to our knowledge, of a ferredoxin-dependent glutamate synthase in a nonphotosynthetic organism. The purified enzyme from H. thermophilus was shown to be a monomer of a 168-kDa polypeptide homologous to ferredoxin-dependent glutamate synthases from phototrophs. In contrast to known ferredoxin-dependent glutamate synthases, the H. thermophilus glutamate synthase exhibited glutaminase activity. Furthermore, this glutamate synthase did not react with a plant-type ferredoxin (Fd3 from this bacterium) containing a [2Fe-2S] cluster but did react with bacterial ferredoxins (Fd1 and Fd2 from this bacterium) containing [4Fe-4S] clusters. Interestingly, the H. thermophilus glutamate synthase was activated by some of the organic acids in the reductive tricarboxylic acid cycle, the central carbon metabolic pathway of this organism. This type of activation has not been reported for any other glutamate synthases, and this property may enable the control of nitrogen assimilation by carbon metabolism.

We have previously demonstrated that the reductive carboxylation of 2-oxoglutarate in Hydrogenobacter thermophilus TK-6 is not simply a reversal of the oxidative decarboxylation catalysed by isocitrate dehydrogenase (ICDH). The reaction involves a novel biotin protein (carboxylating factor for ICDH-CFI) and ATP. In this study, we have analysed the ICDH/CFI system responsible for the carboxylation reaction. Sequence analysis revealed a close relationship between CFI and pyruvate carboxylase. Rather unexpectedly, the rate of ATP hydrolysis was greater than that of isocitrate formation or NADH oxidation. Furthermore, ATP hydrolysis catalysed by CFI was dependent on 2-oxoglutarate but not on ICDH, suggesting that a carboxylated product is formed in the absence of ICDH. The product, which was detectable only at low temperatures, was identified as oxalosuccinate. Thus, CFI was confirmed to be a novel enzyme that catalyses the carboxylation of 2-oxoglutarate to form oxalosuccinate, which corresponds to the first step of the reductive carboxylation from 2-oxoglutarate to isocitrate. The CFI-ICDH system may also be present in mammals, where it could play a significant role in modulating central metabolism.

Characterization of microbial communities using single-strand conformation polymorphism (SSCP) was compared with that using denaturing gradient gel electrophoresis (DGGE). This comparison was based on the V3-4 region (Escherichia coli positions: 341-806) of 16S rRNA gene of bacterial or archaeal communities obtained from a methanogenic bioreactor. Significant differences in the bacterial banding profiles were observed while attempting to detect the diversity of the community and its succession during the reactor operation. The SSCP produced a number of sharp bands and differentiated the bacterial community structures to which the DGGE gave an identical pattern. On the other hand, the SSCP and DGGE provided similar succession patterns for archaeal community.

The genes for a nitric oxide reductase-like cytochrome bc complex were cloned from a thermophilic, chemolithoautotrophic hydrogen-oxidizing bacterium, Hydrogenobacter thermophilus TK-6. The structural genes norC and norB, which encode cytochrome c and cytochrome b subunits of the complex respectively, are probably transcribed as a tricistronic operon with a following gene encoding a putative membrane protein. NorC has, unusually, two hydrophobic transmembrane spans in its N-terminus. Immunoblot analysis showed that expression of NorC was induced by nitrate, nitrite, or sodium nitropurusside, suggesting that the norCB gene product is a denitrification enzyme, nitric oxide reductase. The consensus sequences for the DNR/NnrR-type or the NorR/FhpR-type nitric oxide-sensing regulators of proteobacteria were not found in the norC promoter region, but consensus -35 and -10 sequences were found in this region. These results indicate that strain TK-6 has a nitrogen oxide-sensing regulatory system that differs from proteobacterial systems.

A thermophilic, chemolithoautotrophic hydrogen-oxidizing bacterium, Hydrogenobacter thermophilus TK-6 can grow autotrophically under anaerobic conditions by denitrification. One of the denitrification enzymes, cytochrome cd(1) nitrite reductase, was isolated and its gene was cloned from strain TK-6. The subunit molecular mass of the purified enzyme was 61.5 kDa and the isoelectric point was determined to be 9.3. The optimum temperature and pH for the enzymatic reaction were 70-75 degrees C and 6.5-7.0, respectively. The structural gene for the enzyme, nirS, is probably transcribed as a hexacistronic operon with the following genes encoding a putative diheme cytochrome c and the proteins required for biosynthesis of heme d(1). The NirS sequence was phylogenetically distinct from those of proteobacteria. The consensus -35 and -10 sequences were found in the putative nirS promoter region, but the consensus sequences for the DNR/NnrR-type or the NorR/FhpR-type nitric oxide sensing regulators were not found in this region.

A cellulose-degrading defined mixed culture (designated SF356) consisting of five bacterial strains (Clostridium straminisolvens CSK1, Clostridium sp. strain FG4, Pseudoxanthomonas sp. strain M1-3, Brevibacillus sp. strain M1-5, and Bordetella sp. strain M1-6) exhibited both functional and structural stability; namely, no change in cellulose-degrading efficiency was observed, and all members stably coexisted through 20 subcultures. In order to investigate the mechanisms responsible for the observed stability, "knockout communities" in which one of the members was eliminated from SF356 were constructed. The dynamics of the community structure and the cellulose degradation profiles of these mixed cultures were determined in order to evaluate the roles played by each eliminated member in situ and its impact on the other members of the community. Integration of each result gave the following estimates of the bacterial relationships. Synergistic relationships between an anaerobic cellulolytic bacterium (C. straminisolvens CSK1) and two strains of aerobic bacteria (Pseudoxanthomonas sp. strain M1-3 and Brevibacillus sp. strain M1-5) were observed; the aerobes introduced anaerobic conditions, and C. straminisolvens CSK1 supplied metabolites (acetate and glucose). In addition, there were negative relationships, such as the inhibition of cellulose degradation by producing excess amounts of acetic acid by Clostridium sp. strain FG4, and growth suppression of Bordetella sp. strain M1-6 by Brevibacillus sp. strain M1-5. The balance of the various types of relationships (both positive and negative) is thus considered to be essential for the stable coexistence of the members of this mixed culture.

The capability of degrading rice straw of lignocellulolytic composite microbial system MC1 was investigated under different methods of preservation and temperatures treatments of 80 degrees C to 95 degrees C, and stability of composite microbial system MC1 was studied through the method of Denaturing Gradient Gel Electrophoresis (DGGE). The results indicate that the rice straw of 2% dry weight of medium can be degraded completely at 50 degrees C within 10 days under static culture. After 9 days inoculating MC1, the dry weight of rice straw, cellulose, hemicellulose and lignin content was degraded by 81% , 99%, 74% and 51%, respectively. Capability of cellulose degrading and stability of composite microbial system MC1 is sustained under 4 years of continuing subculture, 4 years of dry preservation at room temperature, 4 years of preservation at - 20 degrees C, 1 year of liquid preservation at room temperature and at 4 degrees C, and treatment of 90 degrees C for 30 min, respectively. Plate culture results show that composite microbial system MC1 are consisted of bacteria. The main DNA bands are not changed by the method of 16S rDNA PCR-DGGE after culture of six months so that microbial composition of MC1 is very stable.

A thermophilic, obligately chemolithoautotrophic hydrogen-oxidizing bacterium, Hydrogenobacter thermophilus TK-6, assimilates carbon dioxide via the reductive tricarboxylic acid cycle. A gene encoding a ferredoxin involved in this cycle as an electron donor (HtFd1) was cloned and sequenced. Interestingly, another ferredoxin gene (encoding HtFd2) was found in tandem with the HtFd1 gene. These two ferredoxin genes overlapped by four bp, and transcriptional analysis revealed that they are co-transcribed as an operon. The deduced amino acid sequences of HtFd1 and HtFd2 were 42.9% identical and each contained four cysteine residues that serve as probable ligands to an iron-sulfur cluster. Spectroscopic analyses of the purified recombinant ferredoxins heterologously expressed in Escherichia coli indicated that each ferredoxin contains a single [4Fe-4S]2+/1+ cluster.

We attempted to purify ATP citrate lyase (ACL) from Hydrogenobacter thermophilus by following the citrate-, ATP- and CoA-dependent formation of an acyl-CoA species that was detected as hydroxamate. However, citryl-CoA rather than acetyl-CoA was found, indicating that the purified enzyme was a novel citryl-CoA synthetase (CCS) rather than ACL. Because the reaction catalysed by CCS corresponds to the first half of that mediated by ACL, CCS may be responsible for citrate cleavage in H. thermophilus. Thus, a novel citrate cleavage pathway, which does not involve ACL, appears to exist in this organism. Citryl-CoA synthetase is composed of two different polypeptides: a large beta subunit of 46 kDa and a small alpha subunit of 36 kDa. The corresponding genes were cloned and sequenced. The deduced amino acid sequences of the two subunits of CCS display significant similarity to those of succinyl-CoA synthetase (SCS) in the database. As a comparison, SCS was also purified from H. thermophilus and the corresponding genes were cloned and sequenced. Citryl-CoA synthetase and SCS were homologous, but showed different substrate specificity. The deduced amino acid sequences of the CCS subunits show similarity to part of the ACL sequence. The evolutionary relationship between CCS, SCS and ACL is discussed.

A novel enzyme catalysing citryl-CoA cleavage to acetyl-CoA and oxaloacetate was purified from Hydrogenobacter thermophilus TK-6, and designated citryl-CoA lyase (CCL). The citrate cleavage reaction in this organism proceeded by a unique set of two consecutive reactions: (i). citryl-CoA formation by citryl-CoA synthetase (CCS) and (ii). citryl-CoA cleavage by CCL. Purified CCL gave a single 30 kDa band in SDS-PAGE and gel filtration chromatography indicated that the native state of the enzyme exists as a trimer (alpha(3)). Citryl-CoA lyase showed low citrate synthase (CS) activity. Using an oligonucleotide probe, the corresponding gene was cloned and sequenced. The gene was expressed in Escherichia coli and recombinant CCL was also purified. The CCL protein sequence showed similarity to the C-terminal regions of ATP citrate lyase (ACL) and CS sequences in the database. By further sequence comparisons, the phylogenetic relationship between CCS, CCL, ACL and CS was investigated.

Isocitrate dehydrogenase was purified from Hydrogenobacter thermophilus, and the corresponding gene was cloned and sequenced. The enzyme had similar structural properties to the isocitrate dehydrogenase of Escherichia coli, but differed in its catalytic properties, such as coenzyme specificity, pH dependency and kinetic parameters. Notably, the enzyme catalysed the oxidative decarboxylation of isocitrate, but not the reductive carboxylation of 2-oxoglutarate. The carboxylation reaction required the addition of cell extract and ATP-Mg, suggesting the existence of additional carboxylation factor(s). Further analysis of the carboxylation factor(s) resulted in the purification of two polypeptides. N-terminal amino acid sequencing revealed that the two polypeptides are homologues of pyruvate carboxylase with a biotinylated subunit, but do not catalyse pyruvate carboxylation. Pyruvate carboxylase was also purified, but was not active in stimulating isocitrate dehydrogenase. Isocitrate dehydrogenase, the novel biotin protein, ATP-Mg and NADH were essential for the reductive carboxylation of 2-oxoglutarate. These observations indicate that the novel biotin protein is an ATP-dependent factor, which is involved in the reverse (carboxylating) reaction of isocitrate dehydrogenase.

A thermophilic, chemolithoautotrophic hydrogen-oxidizing bacterium, Hydrogenobacter thermophilus TK-6, fixes carbon dioxide via the reductive TCA cycle. 2-Oxoglutarate:ferredoxin oxidoreductase (OGOR) is one of the key enzymes of this cycle. Strain TK-6 has two distinct OGOR enzymes termed For and Kor. These enzymes were purified and characterized following heterologous expression in Escherichia coli. The specific activity of For was approximately one-tenth of that of Kor. Additionally, For showed higher thermo-stability than Kor under both aerobic and anaerobic conditions. Western blot analysis showed that both of For and Kor were expressed when strain TK-6 was grown under aerobic conditions. In contrast, only Kor was expressed when the strain was grown under anaerobic conditions using nitrate as a terminal electron acceptor. These results indicate that For supports the optimal growth of strain TK-6 in the presence of oxygen.

Cytochrome c is widely distributed in bacterial species, from mesophiles to thermophiles, and is one of the best-characterized redox proteins in terms of biogenesis, folding, structure, function, and evolution. Experimental molecular biology techniques (gene cloning and expression) have become applicable to cytochrome c, enabling its engineering and manipulation. Heterologous expression systems for cytochromes c in bacteria, for use in mutagenesis studies, have been established by extensive investigation of the biological process by which the functional structure is formed. Mutagenesis and structure analyses based on comparative studies using a thermophile Hydrogenobacter thermophilus cytochrome c-552 and its mesophilic counterpart have provided substantial clues to the mechanism underlying protein stability at the amino-acid level. The molecular mechanisms underlying protein maturation, folding, and stability in bacterial cytochromes c are beginning to be understood.

Thermal stability was measured for variants of cytochrome c-551 (PA c-551) from a mesophile, Pseudomonas aeruginosa, and a thermophilic counterpart, Hydrogenobacter thermophilus cytochrome c-552 (HT c-552), by differential scanning calorimetry (DSC) at pH 3.6. The mutated residues in PA c-551, selected with reference to the corresponding residues in HT c-552, were located in three spatially separated regions: region I, Phe7 to Ala/Val13 to Met; region II, Glu34 to Tyr/Phe43 to Tyr; and region III, Val78 to Ile. The thermodynamic parameters determined indicated that the mutations in regions I and III caused enhanced stability through not only enthalpic but also entropic contributions, which reflected improved packing of the side chains. Meanwhile, the mutated region II made enthalpic contributions to the stability through electrostatic interactions. The obtained differences in the Gibbs free energy changes of unfolding [Delta(DeltaG)] showed that the three regions contributed to the overall stability in an additive manner. HT c-552 had the smallest heat capacity change (DeltaC(P)), resulting in higher DeltaG values over a wide temperature range (0-100 degrees C), compared to the PA c-551 variants; this contributed to the highest stability of HT c-552. Our DSC measurement results, in conjunction with mutagenesis and structural studies on the homologous mesophilic and thermophilic cytochromes c, provided an extended thermodynamic view of protein stabilization.

Four groups of microbial mix culture with considerable capability of cellulose degradation were selected from four compost heaps, by combining the four groups, a composite microbial system MC1 with highly efficient cellulose degradation was obtained. It was found that 0.48 g of filter paper, 0.49 g of absorbent cotton, 0.19 g of wheat straw, or 0.08 g of wood residue can be degraded by 100 mL of MC1 at 50 degrees C with in 72 hours under static culture. The CM1 saccharification activity was much higher at 24th hours when degrading filter paper that was 122.3 U.mL-1. This capability of degradation maintained more than 20 days when the substrates were continually added. MC1 could be inoculated in a wide pH rang, from 4 to 10; however, the final pH would be changed to neutrality after incubation. The pH would be stability with filter paper between 6.0 and 6.5, and between 8.0 and 8.5 without the filter paper.

Cyanobacterial blooms are a worldwide problem, especially in freshwaters. As one of the most abundant co-existing organisms of algae, bacteria play critical roles in cyanobacteria growth, particularly the cyanobactericidal bacteria which can efficiently kill cyanobacteria. Recent years, cyanobactericidal bacteria are highly recognized as a method that could potentially block cyanobacterial blooms. Many studies have been conducted to assess their effects on the termination of cyanobacteria blooms and explore their cyanobactericidal mechanisms, e.g., attacking by cell to cell or releasing specific compounds, the physiological, metabolic, and transcriptional disturbance on cyanobacteria. In this review, the present state of research on cyanobactericidal bacteria for the bloom-causing cyanobacteria species is summarized. The challenges in applying cyanobactericidal bacteria in the control of natural cyanobacterial blooms are discussed. [ABSTRACT FROM AUTHOR]

In anoxic rice field soil, ferric iron reduction is one of the most important terminal electron accepting processes, yet little is known about the identity of iron-reducing microorganisms. Here, we identified acetate-metabolizing bacteria by RNA-based stable isotope probing in the presence of iron(III) oxides as electron acceptors. After reduction of endogenous iron(III) for 21 days, isotope probing with 13C-labeled acetate (2 mM) and added ferric iron oxides (ferrihydrite or goethite) was performed in rice field soil slurries for 48 and 72 h. Ferrihydrite reduction coincided with a strong suppression of methanogenesis (77%). Extracted RNA from each treatment was density resolved by isopycnic centrifugation, and analyzed by terminal restriction fragment length polymorphism, followed by cloning and sequencing of 16S rRNA of bacterial and archaeal populations. In heavy, isotopically labeled RNAs of the ferrihydrite treatment, predominant 13C-assimilating populations were identified as Geobacter spp. (∼85% of all clones). In the goethite treatment, iron(II) formation was not detectable. However, Geobacter spp. (∼30%), the δ-proteobacterial Anaeromyxobacter spp. (∼30%), and novel β-Proteobacteria were predominant in heavy rRNA fractions indicating that 13C-acetate had been assimilated in the presence of goethite, whereas none were detected in the control heavy RNA. For the first time, active acetate-oxidizing iron(III)-reducing bacteria, including novel hitherto unrecognized populations, were identified as a functional guild in anoxic paddy soil. [ABSTRACT FROM AUTHOR]

In nature, microorganisms live by interacting with each other. Microbiological studies that only consider pure cultures are not sufficient to adequately describe the natural behaviour of microbes. Several microbial interactions have been recognized to affect the growth or metabolism of others; e.g. syntrophic cometabolism, competition, production of inhibitors or activators, and predation. It is believed that third-party organisms easily affect the two-species relationships and these relationships form the basis of interspecies networks within microbial communities. A microbial network contributes to ‘functional redundancy’ or ‘structural diversity’ and the microbial communities effectively act as a multicellular organism. It is necessary to understand not only the physiological activity of members within microbial communities but also their roles to regulate the activity or population of others. To access the microbial network, we require (i) comprehensive determination of all possible interspecies relationships among microbes, (ii) knock-out experiments by which certain members can be removed or suppressed, and (iii) supplemental addition of microbes or activation of certain members. Microbial network studies have started using defined microbial communities, i.e. a mixed culture that is composed of three or four species. In order to expand these studies to microflora in nature, microbial ecology requires the help of mathematical biology. [ABSTRACT FROM AUTHOR]

A cytochrome c-552 gene from a thermophilic hydrogen-oxidizing bacterium, Hydrogenobacter thermophilus, was cloned by using two oligonucleotide probes, which had been synthesized based on the known amino acid sequence of the protein. A 780-bp PstI-SphI fragment of the cloned DNA was sequenced and found to contain the entire structural gene coding for cytochrome c-552 bracketed by apparent Escherichia coli consensus sequences for initiation and termination of transcription. Cytochrome c-552 is synthesized in vivo as a precursor having an N-terminal signal sequence consisting of 18 amino acid residues. The cloned cytochrome c-552 gene without its own signal sequence was introduced into the pKK223-3 vector and expressed in E. coli upon induction with isopropyl β-D-thiogalactoside. An expressed cytochrome c-552 protein had a methionine residue at the N-terminus since an initiation signal was introduced before the first amino acid residue of the mature cytochrome c-552. The heme c was attached to apo-type cytochrome c-552 in the cytoplasm of E. coli and the holoprotein had spectral properties, similar to the authentic cytochrome c-552 from H. thermophilus. [ABSTRACT FROM AUTHOR]

Acetate is the most abundant intermediate of organic matter degradation in anoxic rice field soil and is converted to CH4 and/or CO2. Aceticlastic methanogens are the primary microorganisms dissimilating acetate in the absence of sulfate and reducible ferric iron. In contrast, very little is known about bacteria capable of assimilating acetate under methanogenic conditions. Here, we identified active acetate-assimilating microorganisms by using a combined approach of frequent label application at a low concentration and comparative RNA-stable isotope probing with 13C-labeled and unlabeled acetate. Rice field soil was incubated anaerobically at 25°C for 12 days, during which 13C-labeled acetate was added at a concentration of 500 μM every 3 days. 13C-labeled CH4 and CO2 were produced from the beginning of the incubation and accounted for about 60% of the supplied acetate 13C. RNA was extracted from the cells in each sample taken and separated by isopycnic centrifugation according to molecular weight. Bacterial and archaeal populations in each density fraction were screened by reverse transcription-PCR-mediated terminal restriction fragment polymorphism analysis. No differences in the bacterial populations were observed throughout the density fractions of the unlabeled treatment. However, in the heavy fractions of the 13C treatment, terminal restriction fragments (T-RFs) of 161 bp and 129 bp in length predominated. These T-RFs were identified by cloning and sequencing of 16S rRNA as from a Geobacter sp. and an Anaeromyxobacter sp., respectively. Apparently these bacteria, which are known as dissimilatory iron reducers, were able to assimilate acetate under methanogenic conditions, i.e., when CO2 was the predominant electron acceptor. We hypothesize that ferric iron minerals with low bioavailability might have served as electron acceptors for Geobacter spp. and Anaeromyxobacter spp. under these conditions. [ABSTRACT FROM AUTHOR]

Abstract: 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 lower the pH of the broth and produce high levels of lactic acid. Using a combination of plate isolation, denaturing gradient gel electrophoresis (DGGE) and 16S rDNA sequencing, the microbial composition of the SFC-2 was classified into Lactobacillus, mainly comprised of L. fermentum, L. plantarum and L. paracacei. An evaluation of the fermentation effect of SFC-2 on rice straw showed that it lowered the pH and significantly (P <0.05) increased lactic acid concentration in the straw. Further analysis with DGGE indicated that L. plantarum, L. fermentum and L. paracasei were the dominant species during fermentation. [Copyright &y& Elsevier]

The microbial community in a garbage-decomposing system was analyzed using denaturing gradient gel electrophoresis (DGGE) on the basis of 16S rDNA. The system treated 1 kg of garbage everyday for two months at ambient temperature with almost constant decomposition efficiency, although a transient pH increase occurred. Succession of the banding pattern of the DGGE profile suggested that the bacterial community was not directly affected by the continuous addition of non-sterilized garbage into the open system, but changed with the fluctuation of pH. These resistance and resilience characteristics of the community structure may be effective to keep the decomposition efficiency stable. The analyses of the DNA sequences from the DGGE bands suggested the existence of uncultured or novel bacteria as well as Lactobacillus sp., Corynebacterium spp., Enterococcus spp., and Staphylococcus sp. A specific PCR detection was performed to evaluate the existence of Escherichia coli within the community. E. coli 16S rDNAs were not detected from the decomposing system. [Copyright &y& Elsevier]

A self-heating field-scale composter treating agro-industrial wastes within a period of 30 d was analyzed by denaturing gradient gel electrophoresis (DGGE) (Pedro et al., J. Biosci. Bioeng., 91, 159–165, 2001). Three major bands were derived from Propionibacterium acnes, Methylobacterium mesophilicum or M. radiotolerans, and Bacillus thermocloacae. Strains MSP09A and MSP06G with close affiliation to P. acnes and B. thermocloacae, respectively, were successfully isolated. Based on quantitative-PCR results, the relative population of MSP09A increased towards the end of the composting process (mesophilic stage) while MSP06G seemed to predominate during the middle period (thermophilic stage). These results correlated highly with their growth temperatures. MSP09A and MSP06G had different metabolic profiles which were largely affected by culture conditions. MSP09A was able to utilize large complex molecules of lipids and proteins. An interspecies relationship in terms of metabolites such as propionic acid was expected between the two microorganisms. [Copyright &y& Elsevier]

The identification of dominant microbes in anaerobic mercury (Hg) methylation, methylmercury (MeHg) demethylation, and methane oxidation as sulfate-reducing bacteria, methanogens or, probably, anaerobic methanotrophic archaea (ANMEs) is of great interest. To date, however, the interrelationship of bacteria and archaea involved in these processes remains unclear. Here, we demonstrated the dynamics of microorganisms participating in these processes. Anaerobic fixed-bed reactors were operated with swine manure and sludge to produce methane stably, and then, sulfate (reactor C), sulfate and Hg(II) (reactor H), and sulfate and MeHg (reactor M) were added, and the reactors were operated for 120 d, divided equally into four periods, P1–P4. The bacterial compositions changed nonsignificantly, whereas Methanosaeta in reactors H and M decreased significantly, revealing that it was irrelevant for Hg transformation. The abundances of Syntrophomonadaceae , Methanoculleus , Candidatus Methanogranum and Candidatus Methanoplasma increased continuously with time; these species probably functioned in these processes, but further evidence is needed. Desulfocella and Desulfobacterium dominated first but eventually almost vanished, while the dominant archaeal genera Methanogenium , Methanoculleus and Methanocorpusculum were closely related to ANME–1 and ANME–2. PLS-DA results indicated that both bacteria and archaea in different periods in the three reactors were clustered separately, implying that the microbial compositions in the same periods were similar and changed markedly with time. [Display omitted] • Methanosaeta decreased significantly after adding Hg and was unrelated to Hg transformation. • Desulfocella and Desulfobacterium dominated first but finally almost vanished. • The dominant archaeal genera were closely related to ANME–1 and ANME–2. • Microbes in different periods clustered separately, showing a significant change with time. [ABSTRACT FROM AUTHOR]

The introduction of an exogenous cellulolytic bacterium into a microbial community that was degrading rice straw effectively was evaluated. A stable coexistence of the indigenous and exogenous cellulolytic bacteria was achieved by adjusting the cultivation conditions. The obtained community required several subcultures to reach the highest degradation efficiency. [Copyright &y& Elsevier]

The population of methanogenic archaea in a packed-bed reactor was determined. A difference between populations on the supporting material and in effluent was observed at operation under a high organic loading rate and a short hydraulic retention time. This difference was characterized by the predominance of Methanosarcina sp. on the supporting material. [Copyright &y& Elsevier]

The microbial community in a thermophilic anaerobic packed-bed reactor, which had been successfully operated to convert acetic and butyric acids to methane at a short hydraulic retention time (from 24 h to 1.9 h), was investigated. Archaea closely related to known methanogens were detected by 16S rRNA gene analyses of the effluents, together with diverse types of unidentified bacteria. [Copyright &y& Elsevier]