Hydrogen-oxidizing Bacterium Research Articles

Effect of redox potential on the catalytic properties of the NAD-dependent hydrogenase from Alcaligenes eutrophus Z1

The effect of redox potential on the catalytic activities of the soluble hydrogenase from the hydrogen bacterium Alcaligenes eutrophus Z1 was studied. Several transitions were observed on the enzyme catalytic activity vs potential profiles. The coenzyme-dependent activities of the hydrogenase, its diaphorase activity and activity toward NAD, are controlled by the E m –300 mV, while the process of hydrogen evolution from reduced methyl viologen is governed by the midpoint redox potential of –435 mV. This value of E m was independent of pH in the range 5 to 8. The redox potential of the medium appears to be one of the major factors determining the hydrogenase activation, inactivation, and catalytic properties. It is suggested that a change in the redox state of the enzyme electron transport chain is followed by structural rearrangements within the protein affecting both the hydrogenase catalytic activity and stability. The probable mechanism of enzyme activity regulation is discussed.

ChemInform Abstract: Nickel in Metalloproteins

Publisher Summary Nickel has long been suspected to be an essential trace element for living organisms, but the identification of its functions in molecular terms is relatively recent. The first nickel protein to be identified was urease (urea ammonia hydrolase) from jack bean. Certain bacteria—in particular hydrogen bacteria, methanogens, and acetogens—were found to have a relatively high demand for nickel as a trace element. The requirement for nickel was explained when it was found that the enzyme hydrogenase contains nickel. The composition and biological function of hydrogenases have been reviewed recently , and this chapter focuses on the chemistry of those nickel centers. In the methanogenic bacteria, nickel is also involved in a second process, in which a complex series of reactions leads to the release of methane gas. The chapter explains the use of carbon monoxide oxidoreductase. As the principal function of the enzyme is probably to synthesize acetyl-coenzyme A, the name “acetyl-CoA synthase” is also appropriate. It is a major enzyme of acetogenic bacteria, which have a novel pathway for fixation of CO 2 , and it is also found in methanogens. The nickel in urease is nonmagnetic and appears to be in the oxidation state Ni(II).

Isolation ofBacillus schlegelii, a thermophilic, hydrogen oxidizing, aerobic autotroph, from geothermal and nongeothermal environments

Thermophilic hydrogen-oxidizing strains forming round, terminal endospores were isolated from geothermal areas. They were neutrophilic and facultatively autotrophic. They resembledBacillus schlegelii, a thermophilic hydrogen bacterium found so far only in cold environments. Phenotypic similarities, as well as DNA G+C content and DNA:DNA homologies, clearly revealed that the isolated strains belonged to the taxospeciesB. schlegelii. Hence, the strains ofB. schlegelii found in cold environments are probably allochthonous, their origin being geothermal and volcanic areas.

Competition for hydrogen between sulphate-reducing bacteria and methanogenic bacteria from the human large intestine.

Sulphate-reducing activity in human faecal slurries was followed by measuring sulphide production. Sulphate-reducing bacteria (SRB) were found to outcompete methanogenic bacteria (MB) for the mutual substrate hydrogen in faecal slurries from methane- and non-methane-producing individuals mixed together. When molybdate (20 mmol/l) was added to these slurries, sulphate reduction was inhibited and methanogenesis became the major route of electron disposal. Sulphide production was stimulated by the addition of 20 mmol/l sulphate in non-methanogenic but not in methanogenic slurries. In methanogenic slurries that contained the methanogen inhibitor 2-bromoethanesulphonic acid (BES), hydrogen accumulated whilst sulphide levels were unaffected, confirming the absence of SRB in methanogenic faeces. The addition of nitrate (10 mmol/l) to faecal slurries completely inhibited methanogenesis but only slightly reduced sulphate reduction. The sulphated mucopolysaccharides, chondroitin sulphate and mucin, strongly stimulated sulphide production in non-methanogenic faecal slurries only, suggesting that these substances may be a potential source of sulphate in the large gut.

Nickel affects expression of the nickel-containing hydrogenase of Alcaligenes latus.

The effects of nickel on the expression of hydrogenase in the hydrogen-oxidizing bacterium Alcaligenes latus were studied. In the absence of added nickel, both hydrogenase activity, measured as O2-dependent H2 uptake, and hydrogenase protein, measured in a Western immunoblot, were very low compared with the levels in cells induced for hydrogenase in the presence of nickel. Hydrogenase activity and protein levels were dependent on the added nickel concentration and were saturated at 30 nM added Ni2+. The amount of hydrogenase protein in a culture at a given nickel concentration was calculated from the H2 uptake activity of the culture at that Ni2+ concentration. Between 0 and 30 nM added Ni2+, the amount of hydrogenase protein (in nanomoles) was stoichiometric with the amount of added Ni2+. Thus, all of the added Ni2+ could be accounted for in hydrogenase. Between 0 and 50 nM added Ni2+, all the Ni present in the cultures was associated with the cells after 12 h; above 50 nM added Ni2+, some Ni remained in the medium. No other divalent metal cations tested were able to substitute for Ni2+ in the formation of active hydrogenase. We suggest two possible mechanisms for the regulation of hydrogenase activity and protein levels by nickel.

Bioenergetic Conditions of Butyrate Metabolism by a Syntrophic, Anaerobic Bacterium in Coculture with Hydrogen-Oxidizing Methanogenic and Sulfidogenic Bacteria

The butyrate-oxidizing, proton-reducing, obligately anaerobic bacterium NSF-2 was grown in batch cocultures with either the hydrogen-oxidizing bacterium Methanospirillum hungatei PM-1 or Desulfovibrio sp. strain PS-1. Metabolism of butyrate occurred in two phases. The first phase exhibited exponential growth kinetics (phase a) and had a doubling time of 10 h. This value was independent of whether NSF-2 was cultured with a methanogen or a sulfate reducer and likely represents the maximum specific growth rate of NSF-2. This exponential growth phase was followed by a second phase with a nearly constant rate of degradation (phase b) which dominated the time course of butyrate degradation. The specific activity of H(2) uptake by the hydrogen-oxidizing bacterium controlled the bioenergetic conditions of metabolism in phase b. During this phase both the Gibbs free energy (DeltaG') and the butyrate degradation rate (v) were greater for NSF-2-Desulfovibrio sp. strain PS-1 (DeltaG' = -17.0 kJ/mol; v = 0.20 mM/h) than for NSF-2-M. hungatei PM-1 (DeltaG' = -3.8 kJ/mol, v = 0.12 mM/h). The DeltaG' value remained stable and characteristic of the two hydrogen oxidizers during phase b. The stable DeltaG' resulted from the close coupling of the rates of butyrate and H(2) oxidation. The addition of 2-bromoethanesulfonate to a NSF-2-methanogen coculture resulted in the total inhibition of butyrate degradation; the inhibition was relieved when Desulfovibrio sp. strain PS-1 was added as a new H(2) sink. When the specific activity of H(2) consumption was increased by adding higher densities of the Desulfovibrio sp. to 2-bromoethanesulfonate-inhibited NSF-2-methanogen cocultures, lower H(2) pool sizes and higher rates of butyrate degradation resulted. Thus, it is the kinetic parameters of H(2) consumption, not the type of H(2) consumer per se, that establishes the thermodynamic conditions which in turn control the rate of fatty acid degradation. The bioenergetic homeostasis we observed in phase b was a result of the kinetics of the coculture members and the feedback inhibition by hydrogen which prevents butyrate degradation rates from reaching their theoretical V(max).

Translational system of the hydrogen-oxidizing bacterium Alcaligenes eutrophus

Some structural and functional properties of ribosomes from the hydrogen-oxidizing bacterium Alcaligenes eutrophus were studied in order to investigate the background of expression of genetic information at the translational level. Ribosomal proteins from 30S subunits of A. eutrophus H16 were separated by two-dimensional gel electrophoresis into 21 spots, those from 50S subunits into 32 spots. While electrophoretic mobilities of several ribosomal proteins differed markedly from those of Escherichia coli, proteins sharing common immunological determinants with E. coli ribosomal proteins S1 and L7/L12 were found in A. eutrophus. Shifting from heterotrophic to autotrophic conditions of growth had no influence on the ribosomal protein pattern. Ribosomes of A. eutrophus had similar requirements for Mg2+ and poly(U) concentrations for optimum polyphenylalanine synthesis as those of E. coli. Protein synthesis elongation factors Tu from A. eutrophus and E. coli were immunologically similar. Efficiency of the A. eutrophus polyphenylalanine-synthesizing system was comparable to that of an analogous system derived from E. coli. This suggests that A. eutrophus could be employed for efficient expression of recombinant DNA.

Sequence analysis of the Alcaligenes eutrophus chromosomally encoded ribulose bisphosphate carboxylase large and small subunit genes and their gene products

The nucleotide sequence of the chromosomally encoded ribulose bisphosphate carboxylase/oxygenase (RuBPCase) large (rbcL) and small (rbcS) subunit genes of the hydrogen bacterium Alcaligenes eutrophus ATCC 17707 was determined. We found that the two coding regions are separated by a 47-base-pair intergenic region, and both genes are preceded by plausible ribosome-binding sites. Cotranscription of the rbcL and rbcS genes has been demonstrated previously. The rbcL and rbcS genes encode polypeptides of 487 and 135 amino acids, respectively. Both genes exhibited similar codon usage which was highly biased and different from that of other organisms. The N-terminal amino acid sequence of both subunit proteins was determined by Edman degradation. No processing of the rbcS protein was detected, while the rbcL protein underwent a posttranslational loss of formylmethionyl. The A. eutrophus rbcL and rbcS proteins exhibited 56.8 to 58.3% and 35.6 to 38.5% amino acid sequence homology, respectively, with the corresponding proteins from cyanobacteria, eucaryotic algae, and plants. The A. eutrophus and Rhodospirillum rubrum rbcL proteins were only about 32% homologous. The N- and C-terminal sequences of both the rbcL and the rbcS proteins were among the most divergent regions. Known or proposed active site residues in other rbcL proteins, including Lys, His, Arg, and Asp residues, were conserved in the A. eutrophus enzyme. The A. eutrophus rbcS protein, like those of cyanobacteria, lacks a 12-residue internal sequence that is found in plant RuBPCase. Comparison of hydropathy profiles and secondary structure predictions by the method described by Chou and Fasman (P. Y. Chou and G. D. Fasman, Adv. Enzymol. 47:45-148, 1978) revealed striking similarities between A. eutrophus RuBPCase and other hexadecameric enzymes. This suggests that folding of the polypeptide chains is similar. The observed sequence homologies were consistent with the notion that both the rbcL and rbcS genes of the chemoautotroph A. eutrophus and the thus far characterized rbc genes of photosynthetic organisms have a common origin. This suggests that both subunit genes have a very ancient origin. The role of quaternary structure as a determinant of the rate of accepted amino acid substitution was examined. It is proposed that the sequence of the dimeric R. rubrum RuBPCase may be less conserved because there are fewer structural constraints for this RuBPCase than there are for hexadecameric enzymes.

Small Angle X-Ray Study on the Structure of Active and Inactive Ribulose-1,5-bisphosphate Carboxylase-Oxygenase from Spinach. Evidence for a Configurational Change

Small angle X-ray scattering studies on ribulose-1,5-bisphosphate carboxylase-oxygenase (Rubisco) from spinach reveal a configurational change in its quaternary structure upon the transition of the molecule from the activated form occurring in the presence of CO2 and Mg2+ to the deactivated form obtained when CO2 and Mg2+ are removed by extensive dialysis under nitrogen. Present structural models are comparable to models which were postulated previously for the same enzyme but isolated from the hydrogen bacterium Alcaligenes eutrophus [O. Meisen- berger, I. Pilz, B. Bowien, G. P. Pal, and W. Saenger, J. Biol. Chem. 259, 4463-4465 (1984)]. The radius of gyration is R = 47.5 ±0.2 nm for the active spinach Rubisco. Upon deactivation, R changes to 49.2 ± 0.2 nm, suggesting a more elongated quaternary structure. The observed differ­ence in deactivation behaviour in ambient and in nitrogen atmosphere indicates a higher affinity of this spinach enzyme to CO2 with respect to the same enzyme from Alcaligenes eutrophus.

Regulation of H2 oxidation activity and hydrogenase protein levels by H2, O2, and carbon substrates in Alcaligenes latus.

Regulation of H2 oxidation activity and hydrogenase protein levels in the free-living hydrogen bacterium Alcaligenes latus was investigated. Hydrogenase activity was induced when heterotrophically grown cells were transferred to chemolithoautotrophic conditions, i.e., in the presence of H2 and absence of carbon sources, with NH4Cl as the N source. Under these conditions, H2 oxidation activity was detectable after 30 min of incubation and reached near-maximal levels by 12 h. The levels of hydrogenase protein, as measured by a Western blot (immunoblot) assay of the hydrogenase large subunit, increased in parallel with activity. This increase suggested that the increased H2 oxidation activity was due to de novo synthesis of hydrogenase protein. H2 oxidation activity was controlled over a surprisingly wide range of H2 concentrations, between 0.001 and 30% in the gas phase. H2 oxidation activity was induced to high levels between 2 and 12.5% O2, and above 12.5% O2, H2 oxidation activity was inhibited. Almost all organic carbon sources studied inhibited the expression of hydrogenase, although none repressed hydrogenase synthesis completely. In all cases examined, hydrogenase protein, as detected by Western blot, paralleled the level of H2 oxidation activity, suggesting that control of hydrogenase activity was mediated through changes in hydrogenase protein levels.

Xanthobacter agilis sp. nov., a Motile, Dinitrogen-Fixing, Hydrogen-Oxidizing Bacterium

Xanthobacter agilis sp. nov. is described. It is the third described species in the genus Xantobacter , and comprises dinitrogen-fixing, motile, non-pleomorphic, hydrogen-oxidizing bacteria. Four strains from three different habitats were investigated. The type strain is SA 35 and is deposited in the Deutsche Sammlung von Mikroorganismen (access number DSM 3770).

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.

Purification and properties of a protein linked to the soluble hydrogenase of hydrogen-oxidizing bacteria.

In Alcaligenes eutrophus, the formation of the hydrogenases and of five new peptides is subject to the hydrogenase control system. Of these, the B peptide was purified to homogeneity. This protein (Mr, 37,500) was composed of two identical subunits (Mr, 18,800). Antibodies against the B protein were used for its quantification by rocket immunoelectrophoresis. About 4% of the total protein consisted of the B protein; its molar ratio to the NAD-linked hydrogenase was about 4:1. The B protein appeared to be associated with the NAD-linked hydrogenase, as shown by gel filtration analysis with Sephadex G-200. The B protein was not detected in cells that had not expressed the hydrogenase proteins or that lacked the genetic information of the hydrogen-oxidizing character; it was also not detected in Tn5 insertional mutants that were unable to form soluble hydrogenase antigens. Immunochemical analysis of other species and genera than A. eutrophus revealed that only strains able to form a NAD-linked hydrogenase also formed B-protein antigens. The B protein is not required for the catalytic activity of soluble hydrogenase in vitro; its function is at present unknown.

Structure of D-ribulose-l,5-bisphosphate carboxylase/oxygenase from Alcaligenes eutrophyus H16

RuBPcase, D-ribulose-1,5-bisphosphate carboxylase/oxygenase (EC4.1.1.39) is the key enzyme of the reductive pentose phosphate cycle. Because of its biological significance, many structural studies on a number of plant and bacterial RuBPCases have been undertaken, including the enzyme isolated from the autotrophic hydrogen-oxidizing bacterium Alcaligenes eutrophus H16 (refs 2-6). Although both the higher plant enzyme and the A. eutrophus enzyme consist of eight large and eight small subunits (L(8)S(8)), no model describing the quaternary structure is generally accepted. Here we present a model for the A. eutrophus RuBPCase derived from X-ray crystallography of three-dimensional (3D) crystals, and electron microscopy and image analysis of two-dimensional (2D) crystals of the enzyme. The X-ray electron density of RuBPCase in the presence of HCO(-)(3), Mg(2+), and the transition state analogue 2-carboxyarabinitol-1,5-bisphosphate (CABP) shows an L(8)S(8) molecule in which the L(4)S(4) half molecules have local 4-fold symmetry (C4). The local 4-fold axes of the two L(4)S(4) halves do not coincide but are shifted by 36 Å and are related by a crystallographic 2-fold axis perpendicular to and between the local 4-fold axes. Electron microscope data of the enzyme without CABP, which can be perfectly modelled using the X-ray densities, do not show this shift and the low-resolution point group of the molecules in the 2D crystals is D4. Both structures are presented.

Ｃ１微生物の特異的機能とその応用 水素細菌の機能と利用

Ｃ１微生物の特異的機能とその応用 水素細菌の機能と利用

Molecular cloning of structural and regulatory hydrogenase (hox) genes of Alcaligenes eutrophus H16.

A gene bank of the 450-kilobase (kb) megaplasmid pHG1 from the hydrogen-oxidizing bacterium Alcaligenes eutrophus H16 was constructed in the broad-host-range mobilizable vector pSUP202 and maintained in Escherichia coli. hox DNA was identified by screening the E. coli gene bank for restoration of hydrogenase activity in A. eutrophus Hox mutants. Hybrid plasmids that contained an 11.6-kb EcoRI fragment restored soluble NAD-dependent hydrogenase activity when transferred by conjugation into one class of Hos- mutants. An insertion mutant impaired in particulate hydrogenase was partially restored in Hop activity by an 11-kb EcoRI fragment. A contiguous sequence of two EcoRI fragments of 8.6 and 2.0 kb generated Hox+ recombinants from mutants that were devoid of both hydrogenase proteins. hox DNA was subcloned into the vector pVK101. The resulting recombinant plasmids were used in complementation studies. The results indicate that we have cloned parts of the structural genes coding for Hos and Hop activity and a complete regulatory hox DNA sequence which encodes the thermosensitive, energy-dependent derepression signal of hydrogenase synthesis in A. eutrophus H16.

6. 鉱工業用微生物の保存に関する研究(第7報) : 酸素耐性水素細菌について(昭和61年度第32回凍結及び乾燥研究会研究報告)

6. 鉱工業用微生物の保存に関する研究(第7報) : 酸素耐性水素細菌について(昭和61年度第32回凍結及び乾燥研究会研究報告)

Growth of thermophilic obligately autotrophic hydrogen-oxidizing bacteria on thiosulfate

Thermophilic obligately autotrophic H2-oxidizing bacteria from Icelandic hot springs were tested for growth on thiosulfate. Ten strains were tested and all grew on thiosulfate but not on sulfite or sulfur. The product of thiosulfate oxidation was sulfate. The growth rate on thiosulfate was slower (μ=0.12 h-1) than on H2 (μ=0.34 h-1). Washed cells which had been grown on thiosulfate could oxidize thiosulfate rapidly but H2-grown cells oxidized thiosulfate much more slowly and with about a 3 h lag time. The bacteria would not grow on agar medium under H2 but grew on agar medium containing thiosulfate.

Growth of thermophilic, obligatorily chemolithoautotrophic hydrogen-oxidizing bacteria related to Hydrogenobacter with thiosulfate and elemental sulfur as electron and energy source

Strains related to Hydrogenobacter, a genus of thermophilic, obligatorily chemolithoautotrophic bacteria, were able to utilize elemental sulfur or thiosulfate, as well as molecular hydrogen, as sole electron and energy source. Extracellular elemental sulfur was produced as an intermediate during oxidation of thiosulfate. Growth with thiosulfate alone was strongly microaerophilic, whereas no hydrogenase activity was detected. Mixolithotrophic growth with both hydrogen and thiosulfate was faster than with hydrogen alone, and the cells harbored a hydrogenase activity comparable to that of cells grown under hydrogen without thiosulfate.

Conjugal transfer of hydrogen-oxidizing ability of [formula omitted] to [formula omitted

Conjugal transfer of hydrogen-oxidizing ability (Hox) of the hydrogen bacterium Alcaligenes hydrogenophilus was examined. Intraspecific cross of plasmid pHG21-a that encodes hydrogenases that mediate hydrogen oxidation was most frequent at 25 C; the optimal temperature for growth was 30 C. The plasmid could be transferred from A. hydrogenophilus to Pseudomonas oxalaticus OX1 and OX4, and the resulting strains gained the capacity for autotrophic growth with H 2 and CO 2. Plasmid pHG21-a was maintained in P. oxalaticus OX1 and OX4 as stably as in A. hydrogenophilus .