Restoration of Escherichia coli hyd B Mutant Hydrogenase by Cultivation in Media Containing Various Metals

Hydrogenase restoration of Escherichia coli hydrogenase deficient mutant HK7, which carries a mutation at hyd B locus, was studied. Anaerobic growth of HK7 in the presence of iron chloride or vanadium chloride resulted in the restoration of hydrogen uptake activity of hydrogenase, but not hydrogen evolution activity. The growth of HK7 in the presence of nickel chloride restored total hydrogenase activity (hydrogen uptake and evolution) as Waugh and Boxer (1986) reported. Therefore, the leniency of HK7 hyd B product might permit the transportation and incorporation of iron chloride or vanadium chloride in hydrogenase, resulting in the alteration of hydrogenase activity.     

Mass-spectrometric kinetic studies of the nitrogenase and hydrogenase activities in in-vivo cultures of Azospirillum brasilense Sp. 7

Acetylene reduction, deuterium uptake and hydrogen evolution were followed in in-vivo cultures of Azospirillum brasilense, strain Sp 7, by a direct mass-spectrometric kinetic method. Although oxygen was needed for nitrogenase functioning, the enzyme was inactivated by a fairly low oxygen concentration in the culture and an equilibrium had to be found between the rate of oxygen diffusion and bacterial respiration. A nitrogenase-mediated hydrogen evolution was observed only in the presence of carbon monoxide inhibiting the uptake hydrogenase activity which normally recycles all the hydrogen produced. However, under anaerobic conditions and in the presence of deuterium, a bidirectional hydrogenase activity was observed, consisting in D₂ uptake and in H₂ and HD evolution. In contrast to the nitrogenase-mediated H₂ production, this anaerobic H₂ and HD evolution was insensitive to the presence of acetylene and was partly inhibited by carbon monoxide. It was moreover relatively unaffected by the deuterium partial pressure. These results suggest that the anaerobic H₂ and HD evolution can be ascribed to a reverse hydrogenase activity under conditions where D₂ is saturating the uptake process and scavenging the electron acceptors. Although the activities of both nitrogenase and hydrogenase were thus clearly differentiated, a close relationship was found between their respective functioning conditions.     

Cyanobacterial H<Subscript>2</Subscript> production — a comparative analysis

Several unicellular and filamentous, nitrogen-fixing and non-nitrogen-fixing cyanobacterial strains have been investigated on the molecular and the physiological level in order to find the most efficient organisms for photobiological hydrogen production. These strains were screened for the presence or absence of hup and hox genes, and it was shown that they have different sets of genes involved in H₂ evolution. The uptake hydrogenase was identified in all N₂-fixing cyanobacteria, and some of these strains also contained the bidirectional hydrogenase, whereas the non-nitrogen fixing strains only possessed the bidirectional enzyme. In N₂-fixing strains, hydrogen was mainly produced by the nitrogenase as a by-product during the reduction of atmospheric nitrogen to ammonia. Therefore, hydrogen production was investigated both under non-nitrogen-fixing conditions and under nitrogen limitation. It was shown that the hydrogen uptake activity is linked to the nitrogenase activity, whereas the hydrogen evolution activity of the bidirectional hydrogenase is not dependent or even related to diazotrophic growth conditions. With regard to large-scale hydrogen evolution by N₂-fixing cyanobacteria, hydrogen uptake-deficient mutants have to be used because of their inability to re-oxidize the hydrogen produced by the nitrogenase. On the other hand, fermentative H₂ production by the bidirectional hydrogenase should also be taken into account in further investigations of biological hydrogen production.Abbreviations Chl chlorophyll - MV methyl viologen     

<Emphasis Type="Italic">Escherichia coli</Emphasis> hydrogenase 3 is a reversible enzyme possessing hydrogen uptake and synthesis activities

In the past, it has been difficult to discriminate between hydrogen synthesis and uptake for the three active hydrogenases in Escherichia coli (hydrogenase 1, 2, and 3); however, by combining isogenic deletion mutations from the Keio collection, we were able to see the role of hydrogenase 3. In a cell that lacks hydrogen uptake via hydrogenase 1 (hyaB) and via hydrogenase 2 (hybC), inactivation of hydrogenase 3 (hycE) decreased hydrogen uptake. Similarly, inactivation of the formate hydrogen lyase complex, which produces hydrogen from formate (fhlA) in the hyaB hybC background, also decreased hydrogen uptake; hence, hydrogenase 3 has significant hydrogen uptake activity. Moreover, hydrogen uptake could be restored in the hyaB hybC hycE and hyaB hybC fhlA mutants by expressing hycE and fhlA, respectively, from a plasmid. The hydrogen uptake results were corroborated using two independent methods (both filter plate assays and a gas-chromatography-based hydrogen uptake assay). A 30-fold increase in the forward reaction, hydrogen formation by hydrogenase 3, was also detected for the strain containing active hydrogenase 3 activity but no hydrogenase 1 or 2 activity relative to the strain lacking all three hydrogenases. These results indicate clearly that hydrogenase 3 is a reversible hydrogenase.     

Interactions of H2 and carbon metabolism in moderating nitrogenase activity of the Gunnera/Nostoc symbiosis

Nitrogenase activity in the Gunnera Nostoc symbiosis is shown to respond dramatically to the addition of glucose. H₂ can replace glucose in stimulating nitrogenase activity, but there is no H₂ stimulation in the presence of excess glucose. Net hydrogen evolution is strongly stimulated by addition of glucose. We postulate that carbohydrate supply and uptake hydrogenase can moderate the apparent activity of nitrogenase by supplying reductant and/or ATP. The recycling of a large proportion of the electron flux in nitrogenase through uptake hydrogenase maintains a high level of potential nitrogenase ready to take advantage of an influx of carbohydrate.     

Biodiversity of Hydrogenases in Frankia

Eighteen Frankia strains originally isolated from nine different host plants were used to study the biodiversity of hydrogenase in Frankia. In the physiological analysis, the activities of uptake hydrogenase and bidirectional hydrogenase were performed by monitoring the oxidation of hydrogen after supplying the cells with 1% hydrogen and the evolution of hydrogen using methyl viologen as an electron donor, respectively. These analyses were supported with a study of the immunological relationship between Frankia hydrogenase and other different known hydrogenases from other microorganisms. Uptake hydrogenase activity was recorded from all the Frankia strains investigated. A methyl-viologen-mediated hydrogen evolution was recorded from only four Frankia strains irrespective of the source of Frankia. From the immunological and physiological studies, we here report that there are at least three types of hydrogenases in Frankia: Ni-Fe uptake hydrogenase, hydrogen-evolving hydrogenase, and [Fe]-hydrogenase. An immunogold localization study, by cryosection technique, of the effect of nickel on the intercellular distribution of hydrogenase proteins in Frankia indicated that nickel affects the transfer of hydrogenase proteins into the membrane.     

Photoevolution of hydrogen during oxygenic photosynthesis of cyanobacteriumNostoc muscorum

Summary Nitrogen fixing cultures of the cyanobacteriumNostoc muscorum lacked hydrogen evolution but cultures infected with cyanophage N-1 showed significant hydrogen evolution and inactive nitrogenase, suggesting that nitrogenase activity is not responsible for the observed oxygen-resistant photoproduction of hydrogen. Significant oxygen-resistant hydrogen production by nitrate or ammonium assimilating cultures deficient in both nitrogenase and uptake hydrogenase activity supports this conclusion. These findings suggest a role of uptake hydrogenase in blocking the production of hydrogen during aerobic photosynthetic conditions.     

Hydrogenase activity in nitrate-grown cells of the unicellular cyanobacterium Cyanothece PCC 7822

The activity of hydrogenase in intact cells of the unicellular cyanobacterium Cyanothece PCC 7822 was investigated using a mass spectrometer with a permeable membrane inlet. A small hydrogenase-catalyzed hydrogen production was observed with nitrate-grown cells under anoxic conditions in the dark. The same cells were also capable of a much greater rate of hydrogen uptake, induced by oxygen as well as light. Light-induced hydrogen uptake was inhibited by uncoupler. In contrast, addition of uncoupler caused a four-fold stimulation of anoxic hydrogen production in the dark. It is suggested that anoxic hydrogen production is the result of fermentative metabolism.Cyanobacteria are generally considered to have at least two distinct hydrogenases (Houchins 1984). One is a membrane-bound uptake hydrogenase which appears to be associated with nitrogen fixation, removing the hydrogen produced by nitrogenase with the concomitant production of reductant or ATP (Eisbrenner et al. 1978). The second is a reversible hydrogenase located in the cytoplasm and not closely linked to nitrogen metabolism. The reversible character of this enzyme can be demonstrated in the presence of suitable electron donors or acceptors; hydrogen consumption and evolution occur at similar rates (Lambert and Smith 1980).A reversible hydrogenase capable of reducing protons with the artificial electron donor couple dithionite and methyl viologen is widely distributed amongst cyanobacteria. However its physiological role remains unclear. The enzyme appears to be sensitive to oxygen, and consequently in vivo activity can only be demonstrated under anoxic conditions (Houchins 1984).On the basis of in vivo measurements with tritium and the observed low K _m for hydrogen, the function of the reversible hydrogenase of the heterocystous cyanobacterium Anabaena has been proposed to be the uptake of hydrogen as a means of collecting additional reducing power during growth in light-limited anoxic environments (Spiller et al. 1983; Houchins 1984). However, Hallenbeck et al. (1981) reported a modest production of hydrogen by intact filaments of Anabaena.An example of a function of the reversible hydrogenase in the production of hydrogen is provided by the nonheterocystous filamentous cyanobacterium Oscillatoria limnetica. This organism is capable of shifting between oxygenic and anoxygenic photosynthesis (Oren and Padan 1978). In the latter case sulfide is the electron donor supporting photoreduction of CO₂ via photosystem I only. However when CO₂ is limiting, excess reducing equivalents are removed by a reversible hydrogenase (Belkin and Padan 1978). This hydrogen production probably enables the organism to continue photophosphorylation under these conditions.We recently reported that the unicellular cyanobacterium Cyanothece 7822 is capable of hydrogenase-catalyzed hydrogen production in vivo, without the addition of artificial reductants (Van der Oost et al. 1987). In this paper we have investigated the in vivo activity of the hydrogenase in Cyanothece by monitoring the concentrations of dissolved H₂ and O₂ in the cell suspension using a mass spectrometer with a permeable membrane inlet.Abbreviations DBMIB 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone - DCMU N-(3,4-dichlorophenyl) N,N-dimethylurea - FCCP carbonylcyanide-p-trifluoromethoxy phenylhydrazone - PBQ phenyl benzoquinone     

Function of periplasmic hydrogenases in the sulfate-reducing bacterium Desulfovibrio vulgaris Hildenborough

The sulfate-reducing bacterium Desulfovibrio vulgaris Hildenborough possesses four periplasmic hydrogenases to facilitate the oxidation of molecular hydrogen. These include an [Fe] hydrogenase, an [NiFeSe] hydrogenase, and two [NiFe] hydrogenases encoded by the hyd, hys, hyn1, and hyn2 genes, respectively. In order to understand their cellular functions, we have compared the growth rates of existing (hyd and hyn1) and newly constructed (hys and hyn-1 hyd) mutants to those of the wild type in defined media in which lactate or hydrogen at either 5 or 50% (vol/vol) was used as the sole electron donor for sulfate reduction. Only strains missing the [Fe] hydrogenase were significantly affected during growth with lactate or with 50% (vol/vol) hydrogen as the sole electron donor. When the cells were grown at low (5% [vol/vol]) hydrogen concentrations, those missing the [NiFeSe] hydrogenase suffered the greatest impairment. The growth rate data correlated strongly with gene expression results obtained from microarray hybridizations and real-time PCR using mRNA extracted from cells grown under the three conditions. Expression of the hys genes followed the order 5% hydrogen>50% hydrogen>lactate, whereas expression of the hyd genes followed the reverse order. These results suggest that growth with lactate and 50% hydrogen is associated with high intracellular hydrogen concentrations, which are best captured by the higher activity, lower affinity [Fe] hydrogenase. In contrast, growth with 5% hydrogen is associated with a low intracellular hydrogen concentration, requiring the lower activity, higher affinity [NiFeSe] hydrogenase.     

Utilization of cathodically-produced hydrogen from mild steel byDesulfovibrio species with different types of hydrogenases

Summary Desulfovibrio (D.) vulgaris Hildenborough with a highly active Fe-containing periplasmic hydrogenase,D. salexigens British Guiana with a Fe–Ni–Se periplasmic hydrogenase, andD. multispirans with a Fe–Ni cytoplasmic hydrogenase utilized cathodically-produced hydrogen from mild steel as the only energy source for activity and growth. Changes on the mild steel surface occurred during growth of these bacteria. The concentration of iron sulfide, a corrosion product of mild steel, increased over time, andDesulfovibrio species had an active hydrogenase when they were grown in lactate/sulfate media. This hydrogenase may be any of the three types found in the genus,Desulfovibrio. The concentration of iron in the media affected the production and activity of the Fe-hydrogenase fromD. vulgaris Hildenborough. With an iron-limited medium, the specific activity and the total amount of the periplasmic hydrogenase was less than found with a non-iron limited media.     

The Involvement of Hydrogenases 1 and 2 in the Hydrogen-Dependent Nitrate Respiration of Escherichia coli

The study of Escherichia coli mutants synthesizing either hydrogenase 1 (HDK203) or hydrogenase 2 (HDK103) showed that the nitrate-dependent uptake of hydrogen by E. coli cells can be accomplished through the action of either of these hydrogenases. The capability of the cells for hydrogen-dependent nitrate respiration was found to depend on the growth conditions. E. coli cells grown anaerobically without nitrate in the presence of glucose were potentially capable of nitrate-dependent hydrogen consumption. The cells grown anaerobically in the presence of nitrate exhibited a much lower capability for nitrate-dependent hydrogen consumption. The inhibitory effect of nitrate on this capability of bacterial cells was either weak (the mutant HDK203) or almost absent (the mutant HDK103) when the cells were grown in the presence of peptone and hydrogen. Hydrogen stimulated the growth of the wild-type strain and the mutant HDK103 (but not the mutant HDK203) cultivated in the medium with nitrate and peptone. These data suggest that hydrogenase 2 is much more active in catalyzing nitrate-dependent hydrogen consumption than hydrogenase 1.     

Dual organism design cycle reveals small subunit substitutions that improve [NiFe] hydrogenase hydrogen evolution

Background

Photosynthetic microorganisms that directly channel solar energy to the production of molecular hydrogen are a potential future biofuel system. Building such a system requires installation of a hydrogenase in the photosynthetic organism that is both tolerant to oxygen and capable of hydrogen production. Toward this end, we have identified the [NiFe] hydrogenase from the marine bacterium Alteromonas macleodii “Deep ecotype” that is able to be heterologously expressed in cyanobacteria and has tolerance to partial oxygen. The A. macleodii enzyme shares sequence similarity with the uptake hydrogenases that favor hydrogen uptake activity over hydrogen evolution. To improve hydrogen evolution from the A. macleodii hydrogenase, we examined the three Fe-S clusters found in the small subunit of many [NiFe] uptake hydrogenases that presumably act as a molecular wire to guide electrons to or from the active site of the enzyme. Studies by others altering the medial cluster of a Desulfovibrio fructosovorans hydrogenase from 3Fe-4S to 4Fe-4S resulted in two-fold improved hydrogen evolution activity.

Results

We adopted a strategy of screening for improved hydrogenase constructs using an Escherichia coli expression system before testing in slower growing cyanobacteria. From the A. macleodii enzyme, we created a mutation in the gene encoding the hydrogenase small subunit that in other systems is known to convert the 3Fe-4S medial cluster to 4Fe-4S. The medial cluster substitution did not improve the hydrogen evolution activity of our hydrogenase. However, modifying both the medial cluster and the ligation of the distal Fe-S cluster improved in vitro hydrogen evolution activity relative to the wild type hydrogenase by three- to four-fold. Other properties of the enzyme including thermostability and tolerance to partial oxygen did not appear to be affected by the substitutions.

Conclusions

Our results show that substitution of amino acids altering the ligation of Fe-S clusters in the A. macleodii [NiFe] uptake hydrogenase resulted in increased hydrogen evolution activity. This activity can be recapitulated in multiple host systems and with purified protein. These results validate the approach of using an E. coli-cyanobacteria shuttle system for enzyme expression and improvement.

     

A simplified method for assay of hydrogenase activities of H<Subscript>2</Subscript> evolution and uptake in <Emphasis Type="Italic">Enterobacter aerogenes</Emphasis>

Assay of hydrogenase activity pertaining to H₂ production needs anaerobic conditions. To establish a simplified method for assay of hydrogenase activities by using intact cells of Enterobater aerogenes, different chemicals capable of enhancing the cell-wall permeability to electron mediators were examined. As a result, Triton X-100 and CTAB were found to be appropriate for H₂ uptake and evolution activities of the intact cells, respectively. This method enabled H₂ uptake and evolution activities of the intact cells to be easily detected. This is also the first report of the presence of H₂ uptake hydrogenase activity in E. aerogenes.Revisions requested 2 March 2005 and 21 April 2005; Revisions received 12 April 2005 and 17 May 2005     

The purification of hydrogenase II (uptake hydrogenase) from the anaerobic N2-fixing bacterium Clostridium pasteurianum

The H₂ uptake activity (units/mg protein) of Clostridium pasteurianum cells with methylene blue as the electron acceptor increases with cell density independent of the growth conditions. The H₂ evolution activity (units/mg protein) of the same cells with reduced methyl viologen as the electron donor remains fairly constant under all growth conditions tested. Cells grown under N₂-fixing conditions have the highest H₂ uptake activity and were used for the purification of hydrogenase II (uptake hydrogenase). Attempts to separate hydrogenase II from hydrogenase I (bidirectional hydrogenase) by a previously published method were unreliable. We report here a new large-scale purification procedure which employs a rapid membrane filtration system to fractionate cell-free extracts. Hydrogenases I and II were easily filtered into the low-molecular-weight fraction (M_r less than 100 000), and from this, hydrogenase II was further purified to a homogeneous state. Hydrogenase II is a monomeric iron-sulfur protein of molecular weight 53 000 containing eight iron atoms and eight acid-labile sulfur atoms per molecule. Hydrogenase II catalyzes both H₂ oxidation and H₂ evolution at rates of 3000 and 5.9 μmol H₂ consumed or evolved/min per mg protein, respectively. The purification procedure for hydrogenase II using the filtration system described greatly facilitates the large-scale purification of hydrogenase I and other enzymes from cell-free extracts of C. pasteurianum.     

Induction and regulation of ribulose bisphosphate carboxylase activity in Rhizobium japonicum during formate-dependent growth

Rhizobium japonicum CJ1 was capable of growing using formate as the sole source of carbon and energy. During aerobic growth on formate a cytoplasmic NAD⁺-dependent formate dehydrogenase and ribulose bisphosphate carboxylase activity was demonstrated in cell-free extracts, but hydrogenase enzyme activity could not be detected. Under microaerobic growth conditions either formate or hydrogen metabolism could separately or together support ribulose bisphosphate carboxylase-dependent CO₂ fixation. A number of R. japonicum strains defective in hydrogen uptake activity were shown to metabolise formate and induce ribulose bisphosphate carboxylase activity. The induction and regulation of ribulose bisphosphate carboxylase is discussed.Abbreviations hup hydrogen uptake - MOPS 3-(N-morpholino)-propanesulphonate - TSA tryptone soya agar - RuBP ribulose 1,5-bisphosphate - FDH formate dehydrogenase     

Hydrogen-dependent growth of Escherichia coli in anaerobic respiration and the presence of hydrogenases with different functions.

E. coli K10 was found to grow anaerobically on molecular hydrogen by reducing nitrate, fumarate, and trimethylamine N-oxide when peptone was added to the culture medium. Molar growth yields based on consumed hydrogen estimated from the amounts of reduction products were all 7.8 g cells/mol, suggesting that 1 mol of ATP was produced in the oxidation of 1 mol of hydrogen. Hydrogenase activity measured in terms of hydrogen evolution was several times higher in cells grown on glucose than in cells grown on hydrogen in the presence of fumarate and trimethylamine N-oxide, while hydrogenase activity measured in terms of hydrogen uptake was unchanged in both cases. The ratio of hydrogenase activities measured in terms of hydrogen uptake and evolution was also high in the extract and centrifugal fractions from cells grown in hydrogen. The soluble fraction and trypsin digest of the precipitate at 100,000 X g were subjected to polyacrylamide disc gel electrophoresis and hydrogenase bands were stained by reduction of benzyl viologen with hydrogen and by oxidation of reduced methyl viologen. The resulting patterns suggest that multiple forms of hydrogenase are present and that the amounts of forms functioning in hydrogen evolution were greatly decresed in cells grown on hydrogen in the presence of acceptors.     

Physiological characteristics and growth behavior of single and double hydrogenase mutants of Desulfovibrio fructosovorans

The presence of one periplasmic [NiFe] hydrogenase, one periplasmic [Fe] hydrogenase, and one cytoplasmic NADP-reducing hydrogenase has been previously established in Desulfovibrio fructosovorans. In the present work, marker-exchange mutagenesis was performed to determine the function of the tetrameric NADP-reducing hydrogenase encoded by the hndA, B, C, and D genes. The mutations performed were not lethal to the cells, although the H₂-dependent NADP reduction was completely abolished. The double-mutated DM4 (ΔhynABC, ΔhndD) strain was still able to grow on hydrogen plus sulfate as the sole energy source. The growth may have occurred under these culture conditions because of the presence of the remaining [Fe] hydrogenase. The cells grew differently on various substrates depending on whether fructose, lactate, or pyruvate was used in the presence of sulfate. The (hnd mutant growth rates were 25–70% lower than those of the wild-type strain, although the molar growth yield remained unchanged. By contrast, mutants devoid of both [NiFe] hydrogenase and NADP-reducing hydrogenase had 24-38% lower growth yields and showed a corresponding drop in the growth rates. We concluded that each of the three hydrogenases may contribute to the energy supply in D. fructosovorans and that the loss of one enzyme might be compensated for by another. However, the loss of two hydrogenases affected the phosphorylation accompanying the metabolism of fructose, lactate, and pyruvate. Received: 17 September 1996 / Accepted: 5 November 1996     

Evidence for a Third Uptake Hydrogenase Phenotype among the Soybean Bradyrhizobia

The existence of a hydrogen uptake host-regulated (Hup-hr) phenotype was established among the soybean bradyrhizobia. The Hup-hr phenotype is characterized by the expression of uptake hydrogenase activity in symbiosis with cowpea but not soybean. Uptake hydrogenase induction is not possible under free-living cultural conditions by using techniques developed for uptake hydrogenase-positive (Hup⁺) Bradyrhizobium japonicum. Hydrogen oxidation by Hup-hr phenotype USDA 61 in cowpea symbioses was significant because hydrogen evolution from nitrogen-fixing nodules was not detected. An examination for uptake hydrogenase activity in soybean and cowpea with 123 strains diverse in origin and serology identified 16 Hup⁺ and 28 Hup-hr phenotype strains; the remainder appeared to be Hup⁻. The Hup-hr phenotype was associated with serogroups 31, 76, and 94, while strains belonging to serogroups 6, 31, 110, 122, 123, and 38/115 were Hup⁺. Hup⁺ strains of the 123 serogroup typed positive with USDA 129-specific antiserum. The presence of the uptake hydrogenase protein in cowpea bacteroids of Hup⁺ strains was demonstrated with immunoblot analyses by using antibodies against the 65-kDa subunit of uptake hydrogenase purified from strain SR470. However, the hydrogenase protein of Hup-hr strains was not detected. Results of Southern hybridization analyses with pHU1 showed the region of DNA with hydrogenase genes among Hup⁺ strains to be similar. Hybridization was also obtained with Hup-hr strains by using a variety of cloned DNA as probes including hydrogenase structural genes. Both hydrogenase structural genes also hybridized with the DNA of four Hup⁻ strains.     

Hydrogen Oxidation by the Host-Controlled Uptake Hydrogenase Phenotype of Bradyrhizobium japonicum in Symbiosis with Soybean Host Plants

Symbioses between uptake hydrogenase host-regulated (Hup-hr) phenotypes of Bradyrhizobium japonicum and exotic, agronomically unadapted soybean germ plasm were examined for expression of uptake hydrogenase activity. Determinations for hydrogen evolution and uptake hydrogenase activity identified five plant introduction (PI) lines which formed hydrogen-oxidizing symbioses with strains USDA 61 and PA3 6c. Hup-hr strains belonging to serogroup 94 expressed uptake hydrogenase activity in symbioses with PI 181696 and PI 219655 at rates sufficient to prevent hydrogen from escaping the nodules. The identification of soybean germ plasm forming hydrogen-oxidizing symbioses with Hup-hr bradyrhizobia potentially has implications for enhancing nitrogen fixation efficiency in soybean production.     

Autotrophic growth of nitrogen-fixingAzospirillum species and partial characterization of hydrogenase from strain CC

Out of 15 strains ofAzospirillum spp. isolated from the roots of different plants, only 4 (CY, M, CC, and AM) were able to grow autotrophically with H₂ and CO₂. All of them showed H₂ uptake in the presence of oxygen or methylene blue and ribulose-1,5-bisphosphate carboxylase activity. Among the four strains, strain CC isolated from the roots ofCenchrus cilliaris showed maximum H₂+O₂ uptake (32.5 l/min. mg protein) as well as H₂ uptake in the presence of methylene blue (41.4 l/min·mg protein) and also the maximum activity of ribulose-1,5-bisphosphate carboxylase (17 units [U]/g protein). The doubling time of this strain under autotrophic growth conditions and at low oxygen concentration (2.5%, vol/vol) was 10 h. At the same O₂ concentration the maximal rates of H₂+O₂ uptake were reached. The distribution of hydrogenase activity among soluble and particulate protein fractions revealed that the hydrogenase ofAzospirillum strain CC is a membrane-bound enzyme. It showed cross-reaction with antibodies raised against the membrane-bound hydrogenase ofAlcaligenes eutrophus. The hydrogenase in intact cells and crude extracts reacted with methylene blue, phenazine methosulfate, and ferricyanide, but not with NAD or FMN. The specific hydrogenase activity, with methylene blue as an acceptor, was 5.71 U/mg protein in crude extract at 9.38 U/mg protein in the membrane suspension. Hydrogen evolution from reduced viologen dyes could not be demonstrated. The hydrogenase is oxygen sensitive and can be optimally stabilized by addition of dithionite to H₂-gased samples.