Hydrogenobacter Thermophilus TK-6 Research Articles

Nitrogenase Activity in Thermophilic Chemolithoautotrophic Bacteria in the Phylum Aquificae Isolated under Nitrogen-Fixing Conditions from Nakabusa Hot Springs.

The phylum Aquificae comprises chemolithoautotrophic thermophilic to hyperthermophilic bacteria, in which the nitrogenase reductase gene (nifH) has been reported. However, nitrogen-fixing activity has not yet been demonstrated in members of this deeply branching bacterial phylum. We isolated two thermophilic diazotrophic strains from chemosynthetic microbial communities in slightly alkaline hot springs (≥70°C) in Nakabusa, Nagano Prefecture, Japan. A phylogenetic analysis based on 16S rRNA genes identified these strains as members of the genus Hydrogenobacter within Aquificae. Their NifH sequences showed 96.5 and 97.4% amino acid sequence identities to that from Hydrogenobacter thermophilus TK-6. Nitrogenase activity, measured by acetylene reduction, was confirmed in both strains at 70°C. These novel strains grew under semi-aerobic conditions by using CO2 as the sole carbon source and N2 as the sole nitrogen source in media containing hydrogen and/or thiosulfate. To the best of our knowledge, this is the first demonstration of active nitrogen fixation in thermophilic bacteria at 70°C and in the phylum Aquificae.

Structural Units Important for Activity of a Novel-type Phosphoserine Phosphatase from Hydrogenobacter thermophilus TK-6 Revealed by Crystal Structure Analysis

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.

Purification of three aminotransferases from Hydrogenobacter thermophilus TK-6 - novel types of alanine or glycine aminotransferase

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, which has been reported to possess unique features in its carbon and nitrogen anabolism. AT1, AT2 and AT3 exhibited glutamate:oxaloacetate aminotransferase, glutamate:pyruvate aminotransferase and alanine:glyoxylate aminotransferase activities, respectively. In addition, both AT1 and AT2 catalysed a glutamate:glyoxylate aminotransferase reaction. Interestingly, phylogenetic analysis showed that AT2 belongs to aminotransferase family IV, whereas known glutamate:pyruvate aminotransferases and glutamate:glyoxylate aminotransferases are members of family Igamma. In contrast, AT3 was classified into family I, distant from eukaryotic alanine:glyoxylate aminotransferases which belong to family IV. Although Thermococcus litoralis alanine:glyoxylate aminotransferase is the sole known example of family I alanine:glyoxylate aminotransferases, it is indicated that this alanine:glyoxylate aminotransferase and AT3 are derived from distinct lineages within family I, because neither high sequence similarity nor putative substrate-binding residues are shared by these two enzymes. To our knowledge, this study is the first report of the primary structure of bacterial glutamate:glyoxylate aminotransferase and alanine:glyoxylate aminotransferase, and demonstrates the presence of novel types of aminotransferase phylogenetically distinct from known eukaryotic and archaeal isozymes.

A Soluble NADH-Dependent Fumarate Reductase in the Reductive Tricarboxylic Acid Cycle of Hydrogenobacter thermophilus TK-6

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.

Sequencing and Reverse Transcription-Polymerase Chain Reaction (RT-PCR) Analysis of Four Hydrogenase Gene Clusters from an Obligately Autotrophic Hydrogen-Oxidizing Bacterium, Hydrogenobacter thermophilus TK-6

A thermophilic, chemolithoautotrophic hydrogen-oxidizing bacterium, Hydrogenobacter thermophilus TK-6, fixes carbon dioxide via the reductive tricarboxylic acid cycle and grows rapidly with a generation time of about 1 h. TK-6 is believed to have an efficient hydrogen-oxidizing ability to support such rapid growth. We cloned hydrogenase genes from TK-6 and found that this strain has at least four clusters of hydrogenase genes. Reverse transcription-polymerase chain reaction (RT-PCR) analyses showed that all four hydrogenase gene clusters were transcribed under aerobic condition at hydrogen concentrations of 45% and 60%. One of them was not transcribed at a hydrogen concentration of 20%. All the four hydrogenase gene clusters were expressed under anaerobic denitrifying condition at a hydrogen concentration of 75%.

Purification and properties of glutamine synthetase from Hydrogenobacter thermophilus TK-6

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 Km for glutamate of H. thermophilus GS was significantly higher than those of other organisms, suggesting that GS activity is affected by intracellular glutamate concentration.

A novel oxalosuccinate‐forming enzyme involved in the reductive carboxylation of 2‐oxoglutarate in Hydrogenobacter thermophilus TK‐6

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.

Purification, characterization, and gene cloning of thermophilic cytochrome cd1 nitrite reductase from Hydrogenobacter thermophilus TK-6

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.

Anabolic five subunit-type pyruvate:ferredoxin oxidoreductase from Hydrogenobacter thermophilus TK-6

The thermophilic, obligately chemolithoautotrophic hydrogen-oxidizing bacterium, Hydrogenobacter thermophilus TK-6, assimilates carbon dioxide via the reductive tricarboxylic acid cycle. A gene cluster, porEDABG, encoding pyruvate:ferredoxin oxidoreductase (POR), which plays a key role in this cycle, was cloned and sequenced. The nucleotide sequence and the gene organization were similar to those of the five subunit-type 2-oxoglutarate:ferredoxin oxidoreductase from this strain, although the anabolic POR had been previously reported to consist of four subunits. A small protein (8 kDa) encoded by porE, which had not been detected in the previous work, was identified in the purified recombinant POR expressed in Escherichia coli, indicating that the enzyme is also a five-subunit type. Incorporation of PorE in the wild-type POR enzyme was confirmed by immunological analysis. PorA, PorB, PorG, and PorE were similar to the α, β, γ, and δ subunits of the four subunit-type 2-oxoacid oxidoreductases, respectively, and had conserved specific motifs. PorD had no specific motifs but was essential for the expression of the active enzyme.

A novel enzyme, citryl-CoA synthetase, catalysing the first step of the citrate cleavage reaction in Hydrogenobacter thermophilus TK-6.

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, citryl-CoA lyase, catalysing the second step of the citrate cleavage reaction in Hydrogenobacter thermophilus TK-6.

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.

Characterization of two different 2-oxoglutarate:ferredoxin oxidoreductases from Hydrogenobacter thermophilus TK-6

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.

Purification and characterization of pyruvate:ferredoxin oxidoreductase from Hydrogenobacter thermophilus TK-6

Pyruvate:ferredoxin oxidoreductase was purified to electrophoretic homogeneity from an aerobic, thermophilic, obligately chemolithoautotrophic, hydrogen-oxidizing bacterium, Hydrogenobacter thermophilus TK-6, by precipitation with ammonium sulfate and fractionation by DEAE-Sepharose CL-6B, polyacrylate-quaternary amine, hydroxyapatite, and Superdex-200 chromatography. The native enzyme had a molecular mass of 135 kDa and was composed of four different subunits with apparent molecular masses of 46, 31.5, 29, and 24.5 kDa, respectively, indicating that the enzyme has an alphabetagammadelta-structure. The activity was detected with pyruvate, coenzyme A, and one of the following electron acceptors in substrate amounts: ferredoxin isolated from H. thermophilus, FAD, FMN, triphenyltetrazolium chloride, or methyl viologen. NAD, NADP, and ferredoxins from Chlorella spp. and Clostridium pasteurianum were ineffective as the electron acceptor. The temperature optimum for pyruvate oxidation was approximately 80 degrees C. The pH optimum was 7.6-7.8. The apparent Km values for pyruvate and coenzyme A at 70 degrees C were 3.45 mM and 54 microM, respectively. The enzyme was extremely thermostable under anoxic conditions; the time for a 50% loss of activity (t50%) at 70 degrees C was approximately 8 h.

NADH:ferredoxin reductase and NAD-reducing hydrogenase activities in Hydrogenobacter thermophilus strain TK-6

NADH:ferredoxin reductase (EC 1.18.1.3) and NAD-reducing hydrogenase (EC 1.12.1.2) activities were detected in the cytoplasm of Hydrogenobacter thermophilus TK-6. NADH:ferredoxin reductase activity was detected using metronidazole, an artificial electron acceptor, which reacts specifically with reduced ferredoxin. Soluble NAD-reducing hydrogenase activity was detected after extended preincubation. The lag disappeared when cell-free extract was incubated anaerobically for more than 30 min. The electron transport system of this chemolithoautotrophic bacterium is discussed.

Effect of organic compounds on the growth of an obligately autotrophic hydrogen-oxidizing bacterium, Hydrogenobacter thermophilus TK-6.

In the cell-free extracts and intact cells of an obligately autotrophic, extremely thermophilic, aerobic hydrogen-oxidizing bacterium, Hydrogenobacter thermophilus TK-6, eleven different organic compounds tested were only slightly oxidized compared with NADH or H2. Glucose, acetate and pyruvate could be incorporated into intact cells of strain TK-6 only in the presence of hydrogen and oxygen in an atmosphere. These two findings, both weak oxidation of organic compounds and the requirement of energy for incorporation of organic compounds, presented a partial explanation for the obligate autotrophy of Hydrogenobacter thermophilus TK-6. The addition of a small amount of pyruvate, acetate and malate to the medium, stimulated the autotrophic growth of this strain, which suggests that these three compounds may play important roles in the carbon metabolism of the strain.