Role of two 2-oxoglutarate:ferredoxin oxidoreductases in Hydrogenobacter thermophilus under aerobic and anaerobic conditions

Hydrogenobacter thermophilus TK-6 is a thermophilic, hydrogen-oxidizing bacterium that fixes carbon dioxide as a sole carbon source via the reductive tricarboxylic acid cycle. 2-Oxoglutarate:ferredoxin oxidoreductase (OGOR) is one of the key enzymes in the pathway. Strain TK-6 has at least two isozymes of OGOR, namely For and Kor. These OGORs showed different expression patterns under aerobic conditions than under anaerobic conditions. In this work, we developed a homologous recombination method for Hydrogenobacter, and constructed a For mutant and a Kor mutant. Observation of phenotypes of the mutant strains showed that Kor was essential for anaerobic growth and that For activity supported robust aerobic growth of the organism.     

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 ATP:citrate lyase from Hydrogenobacter thermophilus TK-6.

ATP:citrate lyase [ATP citrate (pro-3S)-lyase; EC 4.1.3.8] was purified and characterized from the cells of Hydrogenobacter thermophilus, an aerobic, thermophilic, hydrogen-oxidizing bacterium which fixes carbon dioxide by a reductive carboxylic acid cycle. The enzyme was quite stable, even in the absence of sulfhydryl reagents. Optimum pH for reaction was 6.7 to 6.9, and optimum temperature was around 80 degrees C. The molecular weight of native enzyme was estimated to be 260,000 by gel filtration analysis, and that of a subunit was estimated to be 43,000 by sodium dodecyl sulfate-polyacrylamide gel analysis. Km values for reaction components were as follows: citrate, 6.25 mM; ATP, 650 microM; coenzyme A, 40.8 microM; and Mg2+, 8 mM. The enzyme showed citrate synthase activity in the presence of Mg2+, but the reaction rate was very low (less than 1/200 of the lyase activity).     

Complete genome sequence of Hydrogenobacter thermophilus type strain (TK-6)

Hydrogenobacter thermophilus Kawasumi et al. 1984 is the type species of the genus Hydrogenobacter. H. thermophilus was the first obligate autotrophic organism reported among aerobic hydrogen-oxidizing bacteria. Strain TK-6(T) is of interest because of the unusually efficient hydrogen-oxidizing ability of this strain, which results in a faster generation time compared to other autotrophs. It is also able to grow anaerobically using nitrate as an electron acceptor when molecular hydrogen is used as the energy source, and able to aerobically fix CO(2)via the reductive tricarboxylic acid cycle. This is the fifth completed genome sequence in the family Aquificaceae, and the second genome sequence determined from a strain derived from the original isolate. Here we describe the features of this organism, together with the complete genome sequence and annotation. The 1,742,932 bp long genome with its 1,899 protein-coding and 49 RNA genes is a part of the Genomic Encyclopedia of Bacteria and Archaea project.     

Amino acid sequence of cytochrome c-552 from a thermophilic hydrogen-oxidizing bacterium, Hydrogenobacter thermophilus.

The complete amino acid sequence of cytochrome c-552 from an extremely thermophilic hydrogen bacterium, Hydrogenobacter thermophilus TK-6 (IAM 12695), was determined. It is a single polypeptide chain of 80 residues, and its molecular weight, including heme, was calculated to be 7,599. The sequence of cytochrome c-552 from H. thermophilus TK-6 closely resembles that of cytochromes c-551 from Pseudomonas species. Moreover, the tertiary structure of Hydrogenobacter cytochrome c-552 is suggested to be similar to that of cytochrome c-551 from Pseudomonas aeruginosa. The sequence similarity between Hydrogenobacter cytochrome c-552 and that of other bacteria physiologically related to H. thermophilus is not high.     

A novel ferredoxin-dependent glutamate synthase from the hydrogen-oxidizing chemoautotrophic bacterium Hydrogenobacter thermophilus TK-6

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.     

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 alpha, beta, gamma, and delta 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 enzymatic system against oxidative stress in the thermophilic hydrogen-oxidizing bacterium Hydrogenobacter thermophilus

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.     

Carbamoylphosphate synthetase activity is essential for the optimal growth of Streptococcus thermophilus in milk

Aim:  The aim of the study was to study the role of carbon dioxide metabolism in Streptococcus thermophilus through investigation of the phenotype of a carbamoylphosphate synthetase-negative mutant.
Methods and results:  The effect of carbon dioxide on the nutritional requirements of Strep. thermophilus DSM20617^T and its derivative, carbamoylphosphate synthetase-negative mutant A17( ΔcarB ), was investigated by cultivating the strain in a chemically defined medium under diverse gas compositions and in milk. The results obtained revealed that CO₂ depletion or carB gene inactivation determined the auxotrophy of Strep. thermophilus for l -arginine and uracil. In addition, the parent strain grew faster than the mutant, even when milk was supplemented with uracil or arginine.
Conclusions:  Milk growth experiments underlined that carbamoylphosphate synthetase activity was essential for the optimal growth of Strep. thermophilus in milk.
Significance and impact of the study:  The study of the carbon dioxide metabolism in Strep. thermophilus revealed new insights with regard to the metabolism of this species, which could be useful for the optimization of dairy fermentation processes.     

Carboxylation reaction catalyzed by 2-oxoglutarate:ferredoxin oxidoreductases from Hydrogenobacter thermophilus

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.     

2-Methylthio-1,4-naphthoquinone, a unique sulfur-containing quinone from a thermophilic hydrogen-oxidizing bacterium, Hydrogenobacter thermophilus

A quinone was extracted and purified from the cells of an extremely thermophilic hydrogen bacterium, Hydrogenobacter thermophilus TK-6 (IAM 12695). Its chemical structure was determined as 2-methylthio-3-VI, VII-tetrahydromultiprenyl-1,4-naphthoquinone by elemental analysis, 1H nuclear magnetic resonance spectroscopy, mass spectroscopy, and infrared spectroscopy of the quinone and by gas-liquid chromatography and gas chromatography-mass spectroscopy analysis of the ozonolysis products of the quinone. It was shown that the other five strains of H. thermophilus have the same quinone system. We named the quinone with the 2-methylthio-1,4-naphthoquinone nucleus "methionaquinone." The abbreviation of MTK is recommended for this class of quinone.     

The drug resistance of Hydrogenobacter thermophilus strain TK-6

Abstract Hydrogenobacter thermophilus is an extremely thermophilic and obligately autotrophic hydrogen-oxidising bacterium with various unusual properties and believed to occupy a unique taxonomic position. Inhibitory patterns of various antibiotics on the cell growth of H. thermophilus strain TK-6 clearly showed that the bacterium possessed prokaryote-type systems of DNA, RNA and protein syntheses. Effect of ionophore antibiotics supported that the bacterium was a Gram-negative bacterium, but high sensitivities against macrolide and some other antibiotics and insensitivity against polymyxin B were unusual as a Gram-negative eubacterium.
Growth inhibition by cell wall synthesis inhibitors revealed the existence of peptidoglycan on the surface of H. thermophilus , but ineffectiveness of cell wall lytic enzymes (lysozyme and lysostaphin) on intact cells and purified cell wall strongly suggested the uniqueness of the cell wall structure of the bacterium.     

Fructose metabolism of the purple non-sulfur bacterium Rhodospirillum rubrum: effect of carbon dioxide on growth, and production of bacteriochlorophyll and organic acids

During fermentative metabolism, carbon dioxide fixation plays a key role in many bacteria regarding growth and production of organic acids. The present contribution, dealing with the facultative photosynthetic bacterium Rhodospirillum rubrum, reveals not only the strong influence of ambient carbon dioxide on the fermentative break-down of fructose but also a high impact on aerobic growth with fructose as sole carbon source. Both growth rates and biomass yield increased with increasing carbon dioxide supply in chemoheterotrophic aerobic cultures. Furthermore, intracellular metabolite concentration measurements showed almost negligible concentrations of the tricarboxylic acid cycle intermediates succinate, fumarate and malate under aerobic growth, in contrast to several metabolites of the glycolysis. In addition, we present a dual phase fed-batch process, where an aerobic growth phase is followed by an anaerobic production phase. The biosynthesis of bacteriochlorophyll and the secretion of organic acids were both affected by the carbon dioxide supply, the pH value and by the cell density at the time of switching from aerobic to anaerobic conditions. The formation of pigmented photosynthetic membranes and the amount of bacteriochlorophyll were inversely correlated to the secretion of succinate. Accounting the high biotechnological potential of R. rubrum, optimization of carbon dioxide supply is important because of the favored application of fructose-containing fermentable feedstock solutions in bio-industrial processes.     

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

Abstract 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.     

An obligatory intermediate in the folding pathway of cytochrome c552 from Hydrogenobacter thermophilus

The folding mechanism of many proteins involves the population of partially organized structures en route to the native state. Identification and characterization of these intermediates is particularly difficult, as they are often only transiently populated and may play different mechanistic roles, being either on-pathway productive species or off-pathway kinetic traps. Following different spectroscopic probes, and employing state-of-the-art kinetic analysis, we present evidence that the folding mechanism of the thermostable cytochrome c552 from Hydrogenobacter thermophilus does involve the presence of an elusive, yet compact, on-pathway intermediate. Characterization of the folding mechanism of this cytochrome c is particularly interesting for the purpose of comparative folding studies, because H. thermophilus cytochrome c552 shares high sequence identity and structural homology with its homologue from the mesophilic bacterium Pseudomonas aeruginosa cytochrome c551, which refolds through a broad energy barrier without the accumulation of intermediates. Analysis of the folding kinetics and correlation with the three-dimensional structure add new evidence for the validity of a consensus folding mechanism in the cytochrome c family.     

Thermostability of cytochrome c-552 from the thermophilic hydrogen-oxidizing bacterium Hydrogenobacter thermophilus

The denaturation of the c-type cytochrome of the thermophilic bacterium Hydrogenobacter thermophilus cytochrome c-552 by heat and guanidine hydrochloride was studied by measuring the change in circular dichroic spectra. The melting temperature (T1/2) of cytochrome c-552 in the presence of 1.5 M guanidine hydrochloride was 34 degrees C higher than that of the c-type cytochrome of Pseudomonas aeruginosa cytochrome c-551. Hydrogenobacter cytochrome c-552 is a much more stable protein than cytochrome c-551 of the mesophilic bacterium P. aeruginosa, even though their amino acid sequences are 56% identical and they have numerous other similarities. However, notwithstanding these similarities between the sequences of the cytochromes c-552 and c-551 that were compared, it is very likely that these differences in stability could be due to some heretofore undefined differences in their spatial structures. It has been suggested that alpha-helix structure and electrostatic interaction could be the source of the stable spatial structure of cytochrome c-552.     

Crystallization and preliminary X-ray diffraction study of cytochrome c552 from Hydrogenobacter thermophilus.

Cytochrome c552 from a thermophilic hydrogen-oxidizing bacterium, Hydrogenobacter thermophilus, exhibits remarkable thermostability. The oxidized cytochrome c552 has been crystallized in an ethanol/water mixture by means of the vapor diffusion method. The crystals belong to the orthorhombic system, space group P2(1)2(1)2, with unit cell dimensions of a = 93.4 A, b = 52.9 A, and c = 32.4 A. Most probably the asymmetric unit contains two molecules of cytochrome c552. The crystals diffract X-rays to better than 2.5 A resolution and are stable to X-ray irradiation.     

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.     

Adaptation of Hydrogenobacter thermophilus toward oxidative stress triggered by high expression of alkyl hydroperoxide reductase

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.