Autotrophic growth of strains ofRhizobium and properties of isolated hydrogenase

Four strains ofRhizobium (R. trifolii RCL10,R. japonicum S19 and SB16, andRhizobium sp. NEA4) were demonstrated to grow lithoautotrophically with molecular hydrogen as sole electron donor and with ammonium or with N₂ as N source. All of them showed ribulose-1,5-bisphosphate carboxylase activity and hydrogenase (H₂-uptake) activity with methylene blue and oxygen as electron acceptors. ForR. japonicum SB 16, a doubling time under autotrophic conditions of 30 h and a specific hydrogenase activity (methylene blue reduction) in crude extracts of 1.4 U/mg protein were calculated.Rhizobium hydrogenase is a membrane-bound enzyme. It is mainly detectable in particulate cell fractions, it cross-reacts with the antibodies of the membrane-bound hydrogenase ofAlcaligenes eutrophus, and is unable to reduce NAD. The isolated hydrogenase is a relatively oxygen-sensitive enzyme with a half-life of three days when stored at 4°C under air.     

Mutants of Alcaligenes eutrophus defective in autotrophic metabolism

Forty-four mutants of Alcaligenes eutrophus H 16 were isolated which grew poorly or not at all under autotrophic conditions. Four types were characterized with respect to their defects and their physiological properties. One mutant lacked both enzymes specific for autotrophic CO₂ fixation, another one lacked both hydrogenases, and two mutants lacked either the membrane-bound or the soluble hydrogenase. Comparing the results of studies on these mutant types, the following conclusions were drawn: the lack of each hydrogenase enzyme could be partially compensated by the other one; the lack of membrane-bound hydrogenase did not affect autotrophic growth, whereas the lack of the soluble hydrogenase resulted in a decreased autotrophic growth rate. When pyruvate as well as hydrogen were supplied to the wild-type, the cell yield was higher than in the presence of pyruvate alone. Mutant experiments under these conditions indicated that either of both hydrogenases was able to add to the energy supply of the cell. Only the soluble hydrogenase was involved in the control of the rate of hydrogen oxidation by carbon dioxide; the mutant lacking this enzyme did not respond to the presence or absence of CO₂. The suppression of growth on fructose by hydrogen could be mediated by either of both hydrogenases alone.     

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     

In vivo inactivation of soluble hydrogenase of Alcaligenes eutrophus

The soluble, NAD⁺-reducing hydrogenase in intact cells of Alcaligenes eutrophus was inactivated by oxygen when electron donors such as hydrogen or pyruvate were available. The sole presence of either oxygen or oxidizable substrates did not lead to inactivation of the enzyme. Inactivation occurred similarly under autotrophic growth conditions with hydrogen, oxygen and carbon dioxide. The inactivation followed first order reaction kinetics, and the half-life of the enzyme in cells exposed to a gas atmosphere of hydrogen and oxygen (8:2, v/v) at 30° C was 1.5 h. The process of inactivation did not require ATP-synthesis. There was no experimental evidence that the inactivation is a reversible process catalyzed by a regulatory protein. The possibility is discussed that the inactivation is due to superoxide radical anions (O ₂ ^- ) produced by the hydrogenase itself.     

Hydrogen as an energy source for the human pathogen <Emphasis Type="Italic">Bilophila wadsworthia</Emphasis>

The gram-negative anaerobic gut bacterium Bilophila wadsworthia is the third most common isolate in perforated and gangrenous appendicitis, being also found in a variety of other infections. This organism performs a unique kind of anaerobic respiration in which taurine, a major organic solute in mammals, is used as a source of sulphite that serves as terminal acceptor for the electron transport chain. We show here that molecular hydrogen, one of the major products of fermentative bacteria in the colon, is an excellent growth substrate for B. wadsworthia. We have quantified the enzymatic activities associated with the oxidation of H₂, formate and pyruvate for cells obtained in different growth conditions. The cell extracts present high levels of hydrogenase activity, and up to five different hydrogenases can be expressed by this organism. One of the hydrogenases appears to be constitutive, whereas the others show differential expression in different growth conditions. Two of the hydrogenases are soluble and are recognised by antibodies against a [FeFe] hydrogenase of a sulphate reducing bacterium. One of these hydrogenases is specifically induced during fermentative growth on pyruvate. Another two hydrogenases are membrane-bound and show increased expression in cells grown with hydrogen. Further work should be carried out to reveal whether oxidation of hydrogen contributes to the virulence of B. wadsworthia.     

Nitrogenase activity in Rhodopseudomonas sulfidophila

The marine purple nonsulfur bacterium, Rhodopseudomonas sulfidophila, strain W4, was capable of photosynthetic growth on dinitrogen and malate. Higher growth rates were observed when either glutamate or ammonia replaced dinitrogen as nitrogen source and when bicarbonate was omitted from the culture medium. Although ammonia was released from cells growing on malate and N₂, no nitrogenase activity could be detected unless -ketoglutarate was added to the culture medium. No nitrogenase activity was found in cultures grown in the presence of NH ₄ ⁺ . In cultures grown on glutamate as nitrogen source, nitrogenase and hydrogenase activities were found to be 5.4 nmol C₂H₂ reduced · min^-1 · mg^-1 dry weight and 50 nmol methylene blue reduced · min^-1 · mg^-1 dry weight respectively. Such activities are significantly lower than those observed for other members of the Rhodospirillaceae e.g. Rhodopseudomonas capsulata. However, the hydrogenase activity would be sufficient to recycle all H₂ produced by nitrogenase. It was indeed observed that growing cells did not evolve molecular hydrogen during photoheterotrophic growth and that H₂ stimulated nitrogenase activity in resting cells of R. sulfidophila. The nitrogenase from this bacterium proved to be extremely sensitive to low concentrations of oxygen, half-inhibition occurring at between 1–1.5% O₂ in the gas phase, depending on the bacterial concentration. Light was essential for nitrogenase activity. No activity was found during growth in the dark under extremely low oxygen concentrations (1–2% O₂), which are still sufficient to support good growth. Resting cell suspensions prepared from such cultures were unable to reduce acetylene upon illumination. Optimum nitrogenase activities were broadly defined over the temperature range, 30–38°C, and between pH 6.9 and 8.0. The results are discussed in comparison with the non-marine purple nonsulfur bacterium, R. capsulata, which somewhat resembles R. sulfidophila.     

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     

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.     

A new facultatively autotrophic hydrogen- and sulfur-oxidizing bacterium from an alkaline environment

An alkaliphilic bacterium, strain AHO 1, was isolated from an enrichment culture with hydrogen at pH 10 inoculated with a composite sample of sediments from five highly alkaline soda lakes (Kenya). This bacterium is a gram-negative, nonmotile, rod-shaped, obligately aerobic, and facultatively autotrophic hydrogen-oxidizing organism. It was able to oxidize reduced sulfur compounds to sulfate during heterotrophic growth. It utilized a wide range of organic compounds as carbon and energy sources and grew mixotrophically with hydrogen and acetate. With sulfur compounds, mixotrophic growth was observed only in acetate-limited continuous culture. The normal pH range for autotrophic growth with hydrogen was pH 8.0–10.25, with a pH optimum at 9–9.5. Growth at pH values lower than 8.0 was extremely slow. Heterotrophic growth with acetate was optimal at pH 10.0. The hydrogen-oxidizing activity of whole cells was maximal at pH 9.0 and still substantial up to pH 11. NAD-dependent hydrogenase activity was found in the soluble fraction of the cell-free extract, but no methylene blue-dependent activity in either the soluble or membrane fractions was observed. On the basis of its pH profile, the soluble hydrogenase of strain AHO 1 was a typical pH-neutral enzyme. Phylogenetic analysis revealed that strain AHO 1 belongs to the α-3 subgroup of the Proteobacteria with a closest relation to a recently described alkaliphilic aerobic bacteriochlorophyll a-containing bacterium "Roseinatronobacter thiooxidans." Received: February 29, 2000 / Accepted: April 3, 2000     

Chemolithotrophic growth and regulation of hydrogenase formation in the coryneform hydrogen bacterium strain 11/x

1. The present paper deals with the chemolithotrophic growth of a Gram-positive hydrogen bacterium strain 11/x which shows the characteristic features of some coryneform bacteria.
2. Like other hydrogen bacteria, the strain 11/x is a facultative chemolithotroph and grows on many organic substrates faster than in a mineral medium under an atmosphere of knallgas+CO₂. Fully induced, autotrophically grown cells, subcultured mixotrophically on fructose show additive growth.
3. Cell-free extracts of autotrophically grown cells are able to reduce methylene blue, dichlorophenolindophenol, phenazine methosulphate, menadione, and FMN with hydrogen. Conditions for direct NAD(P) reduction could not be found.
4. Hydrogenase is formed under autotrophic as well as mixotrophic conditions. In the latter case the rate of hydrogenase formation is diminished depending on the organic substrate. Heterotrophically grown cells do not have any detectable hydrogenase activity. For the induction of hydrogenase in those cells a nitrogen source is a prerequisite.
5. The formation of ribulose-1,5-diphosphate carboxylase and phosphoribulokinase seems to be regulated in a way similar to that of hydrogenase: the enzymes could only be detected in autotrophically and mixotrophically grown cells but not in those grown heterotrophically.

     

Pseudomonas siderophores: A mechanism explaining disease-suppressive soils

The membrane-bound hydrogenase ffomPseudomonas pseudoflava GA3 was purified up to a specific activity of 172 μmol H₂ oxidized/min and mg protein and a yield of 31%. The enzyme has a molecular weight of 98,000, consists of two different subunits (65,000 and 30,000), and contains 6 atoms iron and 6 molecules of acid-labile sulfide per molecule of enzyme. The isoelectric point was determined to be 6.5. The enzyme was stable under nitrogen, oxygen, and air atmosphere and unstable under hydrogen. The purified hydrogenase was able to reduce only a few of artificial electron acceptors, i.e., pyocyanine, methylene blue, phenazinemethosulfate, benzylviologen, and dichlorophenolindophenol.     

HYDROGENASE REACTIONS IN RHODOPSEUDOMONAS PALUSTRIS

1. The hydrogenase reactions in a purple non-sulfur bacterium,Rhodopseudornonas palustris, were investigated. Under photoheterotrophicculturing conditions, the photosynthetic activity of the cellswas found to be closely paralleled by the activity of hydrogenase.It was also revealed that the bacterium can grow under suchconditions even withont both photosynthetic and the enzyme activities. 2. The enzyme was revealed to be localized in the particulatefraction (presumably chromatophores) of the disintegrated cells.The properties of the enzyme in the cell-free preparation andin intact cells were described. 3. Among various hydrogen acceptors tested, p-benzoquinone wasmost rapidly hydrogenated. Some heat-labile factor was shownto be involved in the reduction of quinone, which was not requiredin the reduction of methylene blue. 4. The reactions of hydrogenase, both in cell-free state (quinone-and MB-reduction) and in intact cells (oxyhydrogen reaction),were markedly inhibited by molecular oxygen. The inhibitionwas noncompetitive with respect to H₂. A reversible mole-to-molecombination of the enzyme and O₂ was suggested as the mechanismof the inhibition. 5. Carbon monoxide inhibition was suggested also to be causedby a reversible mole-to-mole combination of the enzyme and theinhibitor. This inhibition was competitive with respect to H₂. 6. Rhodopseudomonas palustris hydrogenase was found to be refractorytoward cyanide, azide and sulfhydryl reagents. 7. Light markedly suppressed the oxyhydrogen reaction (intactcells) whereas other hydrogenation reactions (intact cells andcell- free preparations) were not affected by illumination. ¹Present address: Laboratory of Biological Chemistry, TokyoInstitute of Technology, Meguro-ku, Tokyo. (Received August 21, 1961; )     

The regulation of hydrogenase formation as a differentiating character of strains of Paracoccus denitrificans

Paracoccus denitrificans strains Stanier 381 (DSM 65), Morris (DSM 413), and Vogt 11 (DSM 415) and eleven newly isolated strains were compared with respect to the localization of hydrogenase and its regulation. In all strains hydrogenase was found to be membrane-bound and not able to reduce pyridine nucleotides.The enzyme was inducible in strain 381 and was found only in cells grown with hydrogen as the sole hydrogen donor; in cells grown under mixotrophic or heterotrophic conditions the hydrogenase activity was zero.In all other strains hydrogenase was constitutive and was present in cells grown under autotrophic, mixotrophic and heterotrophic conditions. Under the latter conditions the specific hydrogenase activity was even higher than under mixotrophic conditions.     

Reversible hydrogenase in Anabaena variabilis ATCC 29413

The presence and localization of a reversible hydrogenase in non-N₂-fixing cells of the filamentous cyanobacterium Anabaena variabilis were investigated by in vitro activity measurements, native-PAGE/activity stain, SDS-PAGE/Western immunoblots, and immunogold localization. Reversible hydrogenase activity was induced approximately 100-fold by sparging the cell suspensions with a mixture of 99% argon and 1% CO₂ for 20–26 h. Native-PAGE/activity stain demonstrated the presence of an in vitro functional enzyme with an apparent molecular mass of 118 kDa. Native-PAGE/Western immunoblots, using polyclonal antisera directed against purified hydrogenase from the purple sulphur bacterium Thiocapsa roseopersicina, detected two native proteins with molecular masses of 118 and 133 kDa, respectively. SDS-PAGE/Western immunoblots confirmed the presence of a single polypeptide with a molecular mass of approximately 40 kDa in both induced and non-induced cells. Immunocytolocalization experiments using ultrathin sections again demonstrated the presence of hydrogenase in both induced and non-induced cells. A higher specific labeling was associated with the thylakoid regions, which, using an image analyzer, was calculated to be approximately 4 x higher per cell area compared to in the centroplasm. It is suggested that anaerobic incubation induces higher reversible hydrogenase activity, regulated mainly at the level of activating (pre)existing form(s) of inactive enzyme(s)/protein(s), maybe in combination with synthesis of additional subunit(s).     

The activities of hydrogenase and enoate reductase in two Clostridium species,their interrelationship and dependence on growth conditions

The enzyme activities of Clostridium La 1 and Clostridium kluyveri involved in the stereospecific hydrogenation of ,-unsaturated carbonyl compounds with hydrogen gas were measured. In C. La 1 the specific activities of hydrogenase and enoate reductase depended heavily on the growth phase and the composition of the medium. During growth in batch cultures on 70 mM crotonate the specific activity of hydrogenase increased and then dropped to about 10% of its maximum value, whereas the activity of enoate reductase reached its maximum in cells of the stationary phase. Under certain conditions during growth the activity ratio hydrogenase: enoate reductase changed from 120 to 1. Thus, the rate limiting enzyme for the hydrogenation can be either the hydrogenase or the enoate reductase, depending on the growth conditions of the cells.The specific activities of ferredoxin-NAD reductase and butyryl-CoA dehydrogenase increased 3-4-fold during growth on crotonate. By turbidostatic experiments it was shown that at constant input of high crotonate concentrations (200 mM) the enoate reductase activity was almost completely suppressed; it increased steadily with decreasing crotonate down to an input concentration of 35 mM.Glucose as carbon source led to high hydrogenase and negligible enoate reductase activities. The latter could be induced by changing the carbon source of the medium from glucose to crotonate. Tetracycline inhibited the formation of enoate reductase.A series of other carbon sources was tested. They can be divided into ones which result in high hydrogenase and rather low enoate reductase activities and others which cause the reverse effect.When the Fe²⁺ concentration in crotonate medium was growth limiting, cells with relatively high hydrogenase activity and very low enoate reductase activity in the stationary phase were obtained. At Fe²⁺ concentrations above 3·10^-7 M enoate reductase increased and hydrogenase activity reached its minimum. The ratio of activities changes by a factor of about 200. In a similar way the dependence of enzyme activities on the concentration of sulfate was studied.In batch cultures of Clostridium kluyveri a similar opposite time course of enoate reductase and hydrogenase was found.The possible physiological significance of this behavior is discussed.Non Standard Abbreviations O.D.₅₇₈ Optical density at 578 nm Dedicated to Professor Dr. O. Kandler on the occasion of his 60th birthday     

On chemolithotrophy and hydrogenase of a gram-positive knallgas bacterium

Summary A newly isolated gram-positive knallgas bacterium is described. In contrast to some Hydrogenomonas strains, the presence or absence of CO₂ had no noticeable influence on the oxidation of hydrogen. Cell-free extracts reduced methylene blue and oxygen with hydrogen but no physiological acceptors such as NAD(P), FMN and FAD. The majority of the hydrogenase is bound to relatively large particles. Extracts contained an ribulose-1,5-diphosphate carboxylating enzyme.     

Physiology of nitrogen fixation in Azospirillum lipoferum Br 17 (ATCC 29 709)

Changes of cellular activities during batch cultures with Azospirillum lipoferum strain Br 17 (ATCC 29 709) were observed within the growth cycle, at optimal pO₂ (0.002–0.003 atm). The relative growth rate for cells growing with N₂ as sole nitrogen source during log phase was =0.13 h^-1 and the doubling time was 5.3 h. Nitrogenase activity was not accompanied by hydrogen evolution at any growth stage, and a very active uptake hydrogenase was demonstrated. The hydrogenase activity increased towards the end of the growth period when glucose became limiting and N₂ fixation reached its maximal specific activity. Oxygen consumption and oxygen tolerance at the various growth stages, increased simultaneously with the uptake hydrogenase activity indicating a possible role of this enzyme in an oxygen protection mechanism of A. lipoferum nitrogenase. The efficiency of nitrogen fixation expressed as mg total nitrogen fixed in cells and supernatant per g glucose consumed, was 20 at the early log phase and increased to 48 at the late log phase. About 25% of the total fixed nitrogen was recovered in the culture supernatant.Abbreviations DOT Dissolved oxygen tension - PHB Poly--hydroxybutyric acid - O.D. Optical density (560 nm) - A.T.C.C. American type culture collection - NTA Nitrilotriacetic acid Graduate student of the Universidade Federal Rural do Rio de Janeiro, Brazil     

Chemical Modification of Catalytically Essential Functional Groups of NAD-Dependent Hydrogenase from Ralstonia eutropha H16

Amino acid residues His and Cys of the NAD-dependent hydrogenase from the hydrogen-oxidizing bacterium Ralstonia eutropha H16 were chemically modified with specific reagents. The modification of His residues of the nonactivated hydrogenase resulted in decrease in both hydrogenase and diaphorase activities of the enzyme. Activation of NADH hydrogenase under anaerobic conditions additionally modified a His residue (or residues) significant only for the hydrogenase activity. The rate of decrease in the diaphorase activity was unchanged. The modification of thiol groups of the nonactivated enzyme did not affect the hydrogenase activity. The effect of thiol-modifying agents on the activated hydrogenase was accompanied by inactivation of both diaphorase and hydrogenase activities. The modification degree and changes in the corresponding catalytic activities depended on conditions of the enzyme activation. Data on the modification of cysteine and histidine residues of the hydrogenase suggested that the enzyme activation should be associated with significant conformational changes in the protein globule.     

The occurrence of the hydrogenase in some blue-green algae

Several blue-green algae were surveyed for the occurrence of the hydrogenase which was assayed by the oxyhydrogen or Knallgas reaction in the intact organisms. In aerobically grown cultures, the reaction was detectable in Anabaena cylindrica, Nostoc muscorum and in two Anabaena variabilis species, whereas virtually no activity was observed in Anacystis nidulans and Cyanophora paradoxa. In these latter two algae, the reaction was, however, found after growth under molecular hydrogen for several days, which drastically increased the activity levels with all the algae tested. In the nitrogen fixing species, the activity of the Knallgas reaction was enhanced when all combined nitrogen was omitted from the media. H₂ and hydrogenase could not significantly support the CO₂-fixation in photoreduction experiments with all blue-green algae investigated here. Hydrogenase was assayed by the dithionite and methyl viologen dependent evolution of hydrogen and was found to be present with essentially the same specific activity levels in preparations of both heterocysts and vegetative cells from Anabaena cylindrica. Na₂S₂O₄ as well as H₂ supported the C₂H₂-reduction of the isolated heterocysts. The H₂-dependent C₂H₂-reduction did not require the presence of oxygen but was strictly light-dependent where H₂ served as an electron donor to photosystem I of these cells. It is concluded that hydrogen can be utilized by two different pathways in blue-green algae.Abbreviations Chl chlrophyll - CP creatine phosphate - CP kinase creatine phosphokinase - DCMU N-(3,4-dichlorophenyl)N,N-dimethylurea     

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