Regulation of hydrogen utilisation in Rhizobium japonicum by cyclic AMP.

Utilisation (uptake) of hydrogen gas by whole cells of Rhizobium japonicum was found to be influenced by the carbon source(s) present in the growth medium, with activity being highest in a medium containing sugars. Tricarboxylic acid cycle intermediates, such as malate, significantly reduced H2 utilisation. No reduction in the hydrogenase activity is observed when the enzyme is assayed directly by the tritium exchange method, indicating that the decrease in hydrogen uptake activity is not due to repression of hydrogenase biosynthesis. Cyclic AMP was found to alleviate the inhibition of H2 uptake by malate, and this requires new protein synthesis. Addition of chloramphenicol or rifampicin simultaneously with cyclic AMP eliminated the stimulation of H2 uptake in the malate medium. These results show that in R. japonicum cyclic AMP plays a major role in the regulation of H2 metabolism.     

Properties of the hydrogenase system in Rhizobium japonicum bacteroids

The hydrogenase system which catalyzes the oxyhydrogen reaction in soybean nodules produced by strains of $\text{Rhizobium japonicum}$ is located in the bacteroids. The hydrogenase complex in intact bacteroids has an apparent K_m for H₂ of 2.8 μM and an apparent K_m for O₂ of 1.3 μM. The addition of hydrogen to bacteroids increases oxygen uptake but decreases respiratory CO₂ production, indicating a conservation of endogenous substrates. After correction for the effect of hydrogen on endogenous respiration a ratio of 1.9 ± 0.1 for H₂ to O₂ uptake was determined. Bacteroids from greenhouse or field-grown soybeans that evolved hydrogen showed no measurable oxyhydrogen reaction activity whereas consistent activity was demonstrated by bacteroids from soybean nodules that evolved little or no H₂.     

Increased Nitrogenase-Dependent H2 Photoproduction by hup Mutants of Rhodospirillum rubrum

Transposon Tn5 mutagenesis was used to isolate mutants of Rhodospirillum rubrum which lack uptake hydrogenase (Hup) activity. Three Tn5 insertions mapped at different positions within the same 13-kb EcoRI fragment (fragment E1). Hybridization experiments revealed homology to the structural hydrogenase genes hupSLM from Rhodobacter capsulatus and hupSL from Bradyrhizobium japonicum in a 3.8-kb EcoRI-ClaI subfragment of fragment E1. It is suggested that this region contains at least some of the structural genes encoding the nickel-dependent uptake hydrogenase of R. rubrum. At a distance of about 4.5 kb from the fragment homologous to hupSLM, a region with homology to a DNA fragment carrying hypDE and hoxXA from B. japonicum was identified. Stable insertion and deletion mutations were generated in vitro and introduced into R. rubrum by homogenotization. In comparison with the wild type, the resulting hup mutants showed increased nitrogenase-dependent H₂ photoproduction. However, a mutation in a structural hup gene did not result in maximum H₂ production rates, indicating that the capacity to recycle H₂ was not completely lost. Highest H₂ production rates were obtained with a mutant carrying an insertion in a nonstructural hup-specific sequence and with a deletion mutant affected in both structural and nonstructural hup genes. Thus, besides the known Hup activity, a second, previously unknown Hup activity seems to be involved in H₂ recycling. A single regulatory or accessory gene might be responsible for both enzymes. In contrast to the nickel-dependent uptake hydrogenase, the second Hup activity seems to be resistant to the metal chelator EDTA.     

Hydrogen-stimulated CO2 fixation and coordinate induction of hydrogenase and ribulosebiphosphate carboxylase in a H2-uptake positive strain of Rhizobium japonicum

H₂-uptake positive strains (122 DES and SR) and H₂-uptake negative strains SR2 and SR3 of Rhizobium japonicum were examined for ribulosebisphosphate (RuBP) carboxylase and H₂-uptake activities during growth conditions which induced formation of the hydrogenase system. The rate of ¹⁴CO₂ uptake by hydrogenase-derepressed cells was about 6-times greater in the presence than in the absence of H₂. RuBP carboxylase activity was observed in free-living R. japonicum strains 122 DES or SR only when the cells were derepressed for their hydrogenase system. Hydrogenase and RuBP carboxylase activities were coordinately induced by H₂ and both were repressed by added succinate. Hydrogenase-negative mutant strains SR2 and SR3 derived from R. japonicum SR showed no detecyable RuBP carboxylase activities under hydrogenase derepression conditions. No detectable RuBP carboxylase was observed in bacteroids formed by H₂-uptake positive strains R. japonicum 122 DES or SR. Propionyl CoA carboxylase activity was consistently observed in extracts of cells from free-living cultures of R. japonicum but activity was not appreciably influenced by the addition of H₂. Neither phosphoenolpyruvate carboxylase nor phosphoenolpyruvate carboxykinase activity was detected in extracts of R. japonicum.Abbreviations RuBP Ribulose 1,5-bisphosphate - (Na₂EDTA) (Ethylenedinitrilo)-tetraacetic acid, disodium salt - (propionyl CoA) Propionyl coenzyme A - (PEP) Phosphoenolpyruvate - (GSH) Reduced glutathione - (Tricine) N-tris(hydroxymethyl)-methylglycine     

Reinvestigation of the steady-state kinetics and physiological function of the soluble NiFe-hydrogenase I of Pyrococcus furiosus

Pyrococcus furiosus has two types of NiFe-hydrogenases: a heterotetrameric soluble hydrogenase and a multimeric transmembrane hydrogenase. Originally, the soluble hydrogenase was proposed to be a new type of H₂ evolution hydrogenase, because, in contrast to all of the then known NiFe-hydrogenases, the hydrogen production activity at 80°C was found to be higher than the hydrogen consumption activity and CO inhibition appeared to be absent. NADPH was proposed to be the electron donor. Later, it was found that the membrane-bound hydrogenase exhibits very high hydrogen production activity sufficient to explain cellular H₂ production levels, and this seems to eliminate the need for a soluble hydrogen production activity and therefore leave the soluble hydrogenase without a physiological function. Therefore, the steady-state kinetics of the soluble hydrogenase were reinvestigated. In contrast to previous reports, a low K_m for H₂ (~20 μM) was found, which suggests a relatively high affinity for hydrogen. Also, the hydrogen consumption activity was 1 order of magnitude higher than the hydrogen production activity, and CO inhibition was significant (50% inhibition with 20 μM dissolved CO). Since the K_m for NADP⁺ is ~37 μM, we concluded that the soluble hydrogenase from P. furiosus is likely to function in the regeneration of NADPH and thus reuses the hydrogen produced by the membrane-bound hydrogenase in proton respiration.     

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.     

Hydrogenase mediated nitrite reduction in chlorella

The assay of the hydrogenase of glucose-grown cells of Chlorella pyrenoidosa, strain 7-11-05 by means of nitrite reduction with molecular hydrogen is described. The hydrogenase of Chlorella shows maximum activity immediately after equilibration in the hydrogen atmosphere. The hydrogenase mediated reduction of nitrite to ammonia requires the presence of CO₂. However, at pH 6.4. when the reaction proceeds optimally, there is apparently sufficient retention of metabolic CO₂ to support the reaction, which goes to completion, at near maximum rates.

Reduction of nitrite in the hydrogenase system when CO₂ is present results in the uptake of 3 moles of H₂ per mole of nitrite and ammonia is the product. When CO₂ is absent or limiting, ammonia is also formed from nitrite but with the uptake of less than the stoichiometric amount of H₂. It is concluded that CO₂ is essential for the uptake of H₂, and that in the absence of CO₂ internal hydrogen donors support nitrite reduction.

The possibility that CO₂ exerts a catalytic effect in all reductions mediated by hydrogenase in algae is considered, and a further hypothesis, that hydrogenase arises from that portion of the photosynthetic machinery which also shows a catalytic requirement for CO₂, is proposed.

     

Spectral evidence for a component involved in hydrogen metabolism of soybean nodule bacteroids

A component with a difference spectrum similar to that of b-type cytochromes which becomes reduced upon the addition of H₂ has been demonstrated in soybean nodule bacteroids. This electron carrier, referred to as component 559-H₂, is present in hydrogenase-positive strains of Rhizobium japonicum but has not been detected in mutants that lack hydrogenase activity or in hydrogenase-negative wild-type strains. A positive correlation between concentrations of component 559-H₂ and hydrogenase activities has been established. These results provide further evidence that component 559-H₂ is involved in H₂ metabolism in R. japonicum.     

Determination of Hydrogenase in Free-living Cultures of Rhizobium japonicum and Energy Efficiency of Soybean Nodules

A sensitive tritium exchange assay was applied to the Rhizobium system for measuring the expression of uptake hydrogenase in free-living cultures of Rhizobium japonicum. Hydrogenase was detected about 45 hours after inoculation of cultures maintained under microaerophilic conditions (about 0.1% O₂). The tritium exchange assay was used to screen a variety of different strains of R. japonicum (including major production strains) with the findings that about 30% of the strains expressed hydrogenase activity with identical results being observed using an alternative assay based on uptake of H₂. The relative efficiency of intact soybean nodules inoculated with 10 different rhizobial strains gave results identical to those obtained using free-living cultures. The tritium exchange assay provides an easy, quick, and accurate assessment of H₂ uptake efficiency of intact nodules.     

Hydrogenase system in legume nodules: a mechanism of providing nitrogenase with energy and protection from oxygen damage.

Some strains of rhizobia possess a hydrogenase system which catalyzes the oxidation of the H₂ that is evolved from nitrogenase during N₂ fixation. Oxidation of H₂ by a hydrogen uptake positive strain of $\text{Rhizobium japonicum}$ provides energy for support of the N₂ fixation reactions and protects nitrogenase from O₂ damage     

Role of uptake hydrogenase in providing reductant for nitrogenase in Rhizobium leguminosarum bacteroids

The role of uptake hydrogenase in providing reducing power to nitrogenase was investigated in Rhizobium leguminosarum bacteroids from nodules of Pisum sativum L. (cv. Homesteader). H₂ increased the rate of C₂H₂ reduction in the absence of added substrates. Malate also increased nitrogenase (C₂H₂) activity while decreasing the effect of H₂. At exogenous malate concentrations above 0.05 mM no effect of H₂ was seen. Malate appeared to be more important as a source of reductant than of ATP. When iodoacetate was used to minimize the contribution of endogenous substrates to nitrogenase activity in an isolate in which H₂ uptake was not coupled to ATP formation, H₂ increased the rate of C₂H₂ reduction by 77%. In the presence of iodoacetate, an ATP-generating system did not enhance C₂H₂ reduction, but when H₂ was also included, the rate of C₂H₂ reduction was increased by 280% over that with the ATP-generating system alone. The data suggest that, under conditions of substrate starvation, the uptake hydrogenase in R. leguminosarum could provide reductant as well as ATP in an isolate in which the H₂ uptake is coupled to ATP formation, to the nitrogenase complex.     

Occurrence of H(2)-Uptake Hydrogenases in Bradyrhizobium sp. (Lupinus) and Their Expression in Nodules of Lupinus spp. and Ornithopus compressus

Fifty-four strains of Bradyrhizobium sp. (Lupinus) from worldwide collections were screened by a colony hybridization method for the presence of DNA sequences homologous to the structural genes of the Bradyrhizobium japonicum hydrogenase. Twelve strains exhibited strong colony hybridization signals, and subsequent Southern blot hybridization experiments showed that they fell into two different groups on the basis of the pattern of EcoRI fragments containing the homology to the hup probe. All strains in the first group (UPM860, UPM861, and 750) expressed uptake hydrogenase activity in symbiosis with Lupinus albus, Lupinus angustifolius, Lupinus luteus, and Ornithopus compressus, but both the rate of H₂ uptake by bacteroids and the relative efficiency of N₂ fixation (RE = 1 - [H₂ evolved in air/acetylene reduced]) by nodules were markedly affected by the legume host. L. angustifolius was the less permissive host for hydrogenase expression in symbiosis with the three strains (average RE = 0.76), and O. compressus was the more permissive (average RE = 1.0). None of the strains in the second group expressed hydrogenase activity in lupine nodules, and only one exhibited low H₂-uptake activity in symbiosis with O. compressus. The inability of these putative Hup⁺ strains to induce hydrogenase activity in lupine nodules is discussed on the basis of the legume host effect. Among the 42 strains showing no homology to the B. japonicum hup-specific probe in the colony hybridization assay, 10 were examined in symbiosis with L. angustifolius. The average RE for these strains was 0.51. However, one strain, IM43B, exhibited high RE values (higher than 0.80) and high levels of hydrogenase activity in symbiosis with L. angustifolius, L. albus, and L. luteus. In Southern blot hybridization experiments, no homology was detected between the B. japonicum hup-specific DNA probe and total DNA from vegetative cells or bacteroids from strain IM43B even under low stringency hybridization conditions. We conclude from these results that strain IM43B contains hup DNA sequences different from those in B. japonicum and in other lupine rhizobia strains.     

A Comparative Study of the Physiology of Symbioses Formed by Rhizobium japonicum with Glycine max, Vigna unguiculata, and Macroptilium atropurpurem

Although Rhizobium japonicum nodulates Vigna unguiculata and Macroptilium atropurpurem, little is known about the physiology of these symbioses. In this study, strains of R. japonicum of varying effectiveness on soybean were examined. The nonhomologous hosts were nodulated by all the strains tested, but effectiveness was not related to that of the homologous host. On siratro, compared to soybean, many strains reversed their relative effectiveness ranking. Both siratro and cowpea produced more dry matter with standard cowpea rhizobia CB756 and 176A22 than with the strains of R. japonicum. Strains USDA33 and USDA74 were more effective with siratro and cowpea than with soybean. The strain USDA122 expressed high rates of hydrogenase activity in symbiosis with the cowpea as well as the soybean host. The strains USDA61 and USDA74 expressed low levels of hydrogenase activity in symbiosis with cowpea, but no activity was found with soybean. Our results indicate host influence for the expression of hydrogenase activity, and suggest the possibility of host influence of nitrogenase for the allocation of electrons to N₂ and H⁺.     

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.     

Novel,Oxygen-Insensitive Group 5 [NiFe]-Hydrogenase in Ralstonia eutropha

Recently, a novel group of [NiFe]-hydrogenases has been defined that appear to have a great impact in the global hydrogen cycle. This so-called group 5 [NiFe]-hydrogenase is widespread in soil-living actinobacteria and can oxidize molecular hydrogen at atmospheric levels, which suggests a high affinity of the enzyme toward H₂. Here, we provide a biochemical characterization of a group 5 hydrogenase from the betaproteobacterium Ralstonia eutropha H16. The hydrogenase was designated an actinobacterial hydrogenase (AH) and is catalytically active, as shown by the in vivo H₂ uptake and by activity staining in native gels. However, the enzyme does not sustain autotrophic growth on H₂. The AH was purified to homogeneity by affinity chromatography and consists of two subunits with molecular masses of 65 and 37 kDa. Among the electron acceptors tested, nitroblue tetrazolium chloride was reduced by the AH at highest rates. At 30°C and pH 8, the specific activity of the enzyme was 0.3 μmol of H₂ per min and mg of protein. However, an unexpectedly high Michaelis constant (K_m) for H₂ of 3.6 ± 0.5 μM was determined, which is in contrast to the previously proposed low K_m of group 5 hydrogenases and makes atmospheric H₂ uptake by R. eutropha most unlikely. Amperometric activity measurements revealed that the AH maintains full H₂ oxidation activity even at atmospheric oxygen concentrations, showing that the enzyme is insensitive toward O₂.     

Variation in nitrogenase and hydrogenase activity of alaska pea root nodules

Hydrogenase activity of root nodules in the symbiotic association between Pisum sativum L. and Rhizobium leguminosarum was determined by incubating unexcised nodules with tritiated H₂ and measuring tissue HTO. Hydrogenase activity saturated at 0.50 millimolar H₂ and was not inhibited by the presence of 0.10 atmosphere C₂H₂, which prevented H₂ evolution from nitrogenase. Total H₂ production from nitogenase was estimated as net H₂ evolution in air plus H₂ exchange in 0.10 atmosphere C₂H₂. Although such an estimate of nitrogenase function may not be quantitatively exact, due to uncertain relationships between H₂ exchange and H₂ uptake activity of hydrogenase, differences observed in H₂ exchange under various conditions represent an indication of changes in hydrogenase activity. Hydrogenase activity was lower in associations grown under higher photosynthetic photon flux densities and decreased relative to total H₂ production by nitrogenase. Total H₂ production and hydrogenase activity were maximum 28 days after planting. Thereafter, hydrogenase activity and H₂ production declined, but the potential proportion of nitrogenase-produced H₂ recovered by the uptake hydrogenase system increased. Of five R. leguminosarum strains tested two possessed hydrogenase activity. Strains which had the potential to reassimilate H₂ had significantly higher rates of N₂ reduction than those which did not exhibit hydrogenase activity.     

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     

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.     

Selection of symbiotically energy efficient strains of Rhizobium japonicum by their ability to induce a H2-uptake hydrogenase in the free-living state

The capacity of inducing a H₂-uptake hydrogenase in free-living cultures was examined in 21 strains of Rhizobium japonicum. Four strains were found to take up H₂ at rapid rates after 3 days of growth on agar slants inside sealed vials provided with an atmosphere of 5% H₂ in air. Soybean nodules from these strains lost little or no H₂ in air and their bacteroids oxidized H₂ at rates that were similar to those observed in free-living cultures. In contrast, three randomly chosen strains of R. japonicum that showed no H₂-uptake capacity in free-living state produced nodules which lost large amounts of H₂ and the corresponding bacteroids had no hydrogenase activity. A screening procedure is described for the selection of Rhizobium strains producing high energy-efficient nodules based on a test of their ability to induce a H₂-uptake hydrogenase in asymbiotic conditions.