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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 ferrihydrite occurs in the cultures of the three thermophilic methanogens only when H2 was supplied as the source of reducing equivalents, even in the cultures of Mst. thermophila that do not grow on and produce CH4 from H2/ CO2. While supplementation of ferrihydrite resulted in complete inhibition or suppression of methanogenesis by the thermophilic methanogens, ferrihydrite reduction by the methanogens at least partially alleviates the inhibitory effects. Microscopic and crystallographic analyses on the ferrihydrite-reducing Msr. thermophila cultures exhibited generation of magnetite on its cell surfaces through partial reduction of ferrihydrite. These findings suggest that at least certain thermophilic methanogens have the ability to extracellularly transfer electrons to insoluble Fe( III) compounds, affecting their methanogenic activities, which would in turn have significant impacts on materials and energy cycles in thermophilic anoxic environments. [ABSTRACT FROM AUTHOR]

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 (H2O2) and organic hydroperoxide in the presence of NADPH and FNR as electron donors. The calculated Km and Vmax values of Fpx for organic hydroperoxides were comparable to that for H2O2, 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 domainswapped structure is well conserved among thermophiles, implying the importance of structural stability of domainswapped 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. [ABSTRACT FROM AUTHOR]

mmonia accumulation is one of the main causes of the loss of methane production observed during fermentation. We investigated the effect of addition of carbon fiber textiles (CFT) to thermophilic methanogenic bioreactors with respect to ammonia tolerance during the process of degradation of artificial garbage slurry, by comparing the performance of the reactors containing CFT with the performance of reactors without CFT. Under total ammonia-N concentrations of 3,000 mg L, the reactors containing CFT were found to mediate stable removal of organic compounds and methane production. Under these conditions, high levels of methanogenic archaea were retained at the CFT, as determined by 16S rRNA gene analysis for methanogenic archaea. In addition, Methanobacterium sp. was found to be dominant in the suspended fraction, and Methanosarcina sp. was dominant in the retained fraction of the reactors with CFT. However, the reactors without CFT had lower rates of removal of organic compounds and production of methane under total ammonia-N concentrations of 1,500 mg L. Under this ammonia concentration, a significant accumulation of acetate was observed in the reactors without CFT (130.0 mM), relative to the reactors with CFT (4.2 mM). Only Methanobacterium sp. was identified in the reactors without CFT. These results suggest that CFT enables stable proliferation of aceticlastic methanogens by preventing ammonia inhibition. This improves the process of stable garbage degradation and production of methane in thermophilic bioreactors that include high levels of ammonia. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The article discusses the results of a study analyzing the gene structure and expression profile of cytochrome bc nitric oxide reductase from the bacterium Hydrogenobacter thermophilus TK-6. Evidence showed that the expression of NorC was induced by nitrate, nitrite or sodium nitroprusside therefore the norCB gene product is a denitrification enzyme. The norC promoter region contains -35 and -10 consensus sequences.

A novel enzyme catalysing citryl-CoA cleavage to acetyI-CoA and oxaloacetate was purified from Hydrogenobacter thermophilusTK-6, and designated citrylCoA lyase (CCL). The citrate cleavage reaction in this organism proceeded by a unique set of two consecutive reactions: (i) citryI-CoA formation by citryI-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 (α3). CitryI-CoA lyase showed Iow 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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 a thermophilic anaerobic digester were investigated by process analyses and 16S rRNA gene-based molecular approaches. The biogas was produced stably without the accumulation of volatile fatty acids (VFAs) at low organic loading rates (OLRs) in the beginning of reactor operation. Increasing OLR in stages disrupted the stable reactor performance, and high OLR conditions continued the deteriorated performance with slight biogas production and high accumulation of VFAs. Thereafter, the gradual decrease of OLR resulted in the recovery from the deterioration, giving rise to the stable performance again. The stable performances before and after the high OLR conditions conducted were associated with compositionally similar but not identical methanogenic consortia. The bacterial and archaeal populations were synchronously changed at both the transient phases toward the deteriorated performance and in recovery process, during which the dynamic shift of aceticlastic and hydrogenotrophic methanogens including the recently identified Methanomassiliicoccus might contribute to the maintenance of the methanogenic activity. The distinctive bacterial population with a high predominance of Methanobacterium formicicum as archaeal member was found for the deteriorated performance. The results in this study indicate the coordinated reorganization of the bacterial and archaeal populations in response to functional states induced by the change of organic loading conditions in the anaerobic digester. [ABSTRACT FROM AUTHOR]

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 His85 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. [ABSTRACT FROM AUTHOR]

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 functional phosphoserine synthetic pathway. The apparent lack of a PSP ortholog in Hydrogenobacter thermophilus, an obligately chemolithoautotrophic and thermophilic bacterium, represented a missing link in serine anabolism because our previous study suggested that serine should be synthesized from phosphoserine. Here, we detected PSP activity in cell-free extracts of H. thermophilus and purified two proteins with PSP activity. Surprisingly, these proteins belonged to the histidine phosphatase superfamily and had been annotated as cofactor-dependent phosphoglycerate mutase (dPGM). However, because they possessed neither mutase activity nor the residues important for the activity, we defined these proteins as novel-type PSPs. Considering the strict substrate specificity toward L-phosphoserine, kinetic parameters, and PSP activity levels in cell-free extracts, these proteins were strongly suggested to function as PSPs in vivo. We also detected PSP activity from "dPGM-like" proteins of Thermus thermophilus and Arabidopsis thaliana, suggesting that PSP activity catalyzed by dPGM-like proteins may be distributed among a broad range of organisms. In fact, a number of bacterial genera, including Firmicutes and Cyanobacteria, were proposed to be strong candidates for possessing this novel type of PSP. These findings will help to identify the missing link in serine anabolism. [ABSTRACT FROM AUTHOR]

Abstract: 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. [Copyright &y& Elsevier]

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. [ABSTRACT FROM AUTHOR]

Hydrogenobacter thermophilus TK-6, a thermophilic and obligately chemoautotrophic bacterium, assimilates ammonium using glutamine synthetase (GS). GS was purified using three chromatography steps. The purified GS was found to belong to GS type I on the basis of its subunit composition and molecular weight. The Mg2+-dependent activity of this GS significantly increased after incubation with phosphodiesterase, indicating that GS is subject to adenylyl/deadenylyl regulation, a posttranslational modification system reported mainly among enterobacteria. The degree of this posttranslational modification changed depending on growth phase, confirming that adenylyl/deadenylyl regulation functions in vivo. Interestingly, the K m for glutamate of H. thermophilus GS was significantly higher than those of other organisms, suggesting that GS activity is affected by intracellular glutamate concentration. [Copyright &y& Elsevier]

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°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. [Copyright &y& Elsevier]

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. [Copyright &y& Elsevier]

A thermophilic, chemolithoautotrophic hydrogen-oxidizing bacterium, Hydrogenobacter thermophilus TK-6, fixes carbon dioxide via the reductive TCA cycle. 2-Oxoglutarate:ferredoxin oxidoreductase (OGOR) of this strain is one of the key enzymes of the pathway. OGOR of strain TK-6 has been reported to be a two-subunit-type OGOR and encoded by korAB. A gene cluster, forDABGEF, encoding another OGOR was found 148 bp upstream of korAB in the opposite orientation. Five of the for genes (forDABGE) were required for the expression of the active recombinant enzyme in Escherichia coli. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis of the purified enzyme showed five polypeptides corresponding to the forDABGE gene products, suggesting that the enzyme had a novel five-subunit structure. The recombinant enzyme had high substrate specificity toward 2-oxoglutarate as in the case of the gene products of korAB. Primer extension analysis showed that the korA and forD genes were transcribed from one and two transcriptional initiation sites, respectively. The results also suggested that both gene clusters were expressed in the cells of strain TK-6. [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]