H2 Uptake System Research Articles

Enhanced production of polyhydroxybutyrate from syngas by using nanoscaled cellulose particles with a syngas-converting enzyme complex immobilized on Ralstonia eutropha

Synthetic gas (syngas) produced from pyrolysis or a blast furnace is a mixture of different ratio of H2, CO, N2, CH4, CO2 and other carbon-containing gas. CO2 and H2 are the key sources for Ralstonia eutropha, which uses CO2 to produce polyhydroxybutyrate (PHB) with a H2 uptake system. For the efficient conversion of syngas to accessible carbon source, nanoscaled cellulose particles with an enzyme complex consisting of carbon monoxide dehydrogenase (CooS), carbon monoxide binding unit (CooA) and carbonic anhydrase (CA) were assembled by minimized scaffoldin (mCbpA). In addition to this assembly, mCbpA-BspA (MB) established to bridge the nanoparticle assembly and R. eutropha was used to provide higher enzyme stability and activity. The highest PHB accumulation after 72 h of fermentation was 14.2 g/L in a closed serum bottle that contained the enzyme assembly immobilized on R. eutropha. This catalytic assembly may be a new potential conversion tool to produce biodegradable PHB from syngas.

Differential Expression of Uptake Hydrogenase Activity in Free Living Cells and Nodule Bacteroids of Azorhizobium caulinodans

An oxygen sensitive mutant of Azorhizobium caulinodans strain IRBG 46 in which N2 fixation ability was affected, was previously isolated by NTG mutagenesis. Now, the mutation has been shown to affect H2- uptake hydrogenase (Hup) activity under symbiotic conditions. However, free living Hup activity remained unaffected. Thus the mutant is Hup- under symbiotic conditions and Hup+ under free living condtions. A possible regulatory link between N2 fixation and H2 uptake system has been discussed.

Comparison of symbiotic effectiveness of Rhizobium sp. with positive hydrogen-uptake activity compared with negative mutants in relation to nitrogen fixation in mungbean (Vigna radiata L.)

H2 uptake activity was well distributed in Rhizobium sp. strains isolated from nodules of mung-bean (Vigna radiata L.). Two effective strains, RMP1 und RMP2, exhibiting significantly higher H2 uptake activity were subjected to mutagenesis with nitrosoguanidine. The respective mutation frequencies were 0.18 and 0.19%. Three Hup- mutants each of RMP1 und RMP2 were compared with the wild-type parent strains under pot culture experiments to evaluate the significance of the H2 uptake system in biological N2 fixation. Nodulation capabilities, plant growth characteristics, and the chlorophyll content of the leaves were significantly reduced in the plants treated with Hup- mutants. Nitrogenase activity in Hup- nodules was reduced by 8–41%. Similarly, N accumulation was also reduced singificantly.

Hydrogenase in Bradyrhizobium japonicum: genetics, regulation and effect on plant growth

A region of 15 kbp DNA located on the chromosome of Bradyrhizobium japonicum is essential for a Hup(+) phenotype and contains the hydrogenase structural genes. During the last few years, other genes with various functions related to hydrogenase oxidation, such as Ni(2+) incorporation into the hydrogenase enzyme, electron transport from hydrogenase to O2, and regulation of hydrogenase expression in free-living conditions, have been identified in this region. A region in front of the hydrogenase structural genes is necessary for transcriptional regulation of hydrogenase expression by O2, H2 and Ni(2+). In addition, the N2 fixation system and the tertiary structure of the chromosome seem to be involved in the expression of the hydrogenase genes. The effect of legume inoculation with Hup(+) rhizobia has been evaluated. The possible benefits of a Hup(+) phenotype, such as the regeneration of chemical energy in the form of ATP or reductants, and the removal of O2 and H2 from the active site of the nitrogenase enzyme where they might inhibit the nitrogenase reaction, are discussed. The data indicate that a H2-uptake system is beneficial in soybean nodules, but host plant and environmental factors may interfere with the effects of H2 cycling in the plant.

Uptake hydrogenase activity in denitrifying Azospirillum brasilense grown anaerobically with nitrous oxide or nitrate.

zospirillum brasilense Sp7 was grown anaerobically with N2O as the terminal electron acceptor and NH4Cl as the nitrogen source. Hydrogen uptake activity (O2-dependent H3H oxidation) was expressed in the presence and absence of 5% H2; it reached its maximum in late logarithmic phase as the malate became limiting. This activity was very stable in stationary phase, even in the absence of exogenous H2, compared with microaerobically grown cultures; this supports the hypothesis that the exclusion of O2 is critical for maintaining the integrity of the H2 uptake system in this organism. Oxygen, as well as methylene blue and N2O, supported H2 uptake, indicating the presence of electron transport components leading to O2 in anaerobically grown A. brasilense. Nitrite (0.5 mM) inhibited H2 uptake. In cultures grown with NO3- as the terminal electron acceptor and NH4Cl as the nitrogen source, in the presence and absence of exogenous H2, only low H2 uptake activity was observed. Methylene blue, O2, N2O, NO3-, and NO2- were all capable of acting as the electron acceptor for H2 oxidation. Nitrite (0.5 mM) did not inhibit H2 uptake in NO3--grown cells, as it did in N2O-grown cells. A. brasilense appears to be one of the few organisms capable of expressing the H2 uptake system under denitrifying conditions in the absence of molecular H2.

Regulation of hydrogenase in Rhizobium japonicum: analysis of mutants altered in regulation by carbon substrates and oxygen

The synthesis of the H2 uptake system in free-living Rhizobium japonicum SR is repressed both by oxygen and by carbon substrates. Mutants selected for the ability to express hydrogenase in 10.0% partial pressure O2 were also less sensitive than the wild type to repression by carbon substrates such as arabinose, glycerol, gluconate, and succinate. The H2 uptake system in another class of mutants, previously shown to be hypersensitive to repression by O2, is also more sensitive to repression by carbon substrates. The oxygen- and carbon-insensitive mutants express the hydrogen uptake system during heterotrophic growth in the absence of hydrogen and thus can be considered constitutive (Hupc). The amount of cytochromes in the Hupc mutants is similar to that in the wild-type strain; however, the Hupc mutants contain greater methylene blue-dependent and O2-dependent hydrogenase activity, both as free-living cells and as bacteroids. Two-dimensional polyacrylamide gel electrophoresis revealed that during heterotrophic growth the Hupc mutant strain SR470 synthesized at least six peptides not found in the wild-type strain. The concentrations of cyclic AMP and guanosine tetraphosphate were similar in strain SR and the Hupc mutants during heterotrophic growth.

Hydrogen Uptake (HUP) Activity of Rhizobium japonicum from Major U.S. Soybean Production Areas

The purpose of these experiments was to determine the distribution of the hydrogen uptake (Hup) system among Rhizobium japonicum isolated from major soybean production areas of the USA. Soybean nodules were collected from approximately 100 different locations during the 1979 and 1980 growing seasons. The major conclusion is that a majority (> 75 %) of the Rhizobium japonicum strains isolated from major soybean producing areas are missing H2 uptake activity. The role of the H2 uptake system in energy efficiency of nitrogen fixation is discussed.

Rhizobium japonicum mutants that are hypersensitive to repression of H2 uptake by oxygen.

The synthesis of an H2 oxidation system in free-living Rhizobium japonicum wild-type strain SR is repressed by oxygen. Maximal H2 uptake rates were obtained in strain SR after derepression in 11 microM or less dissolved oxygen. Oxygen levels above 45 microM completely repressed H2 uptake in strain SR. Five R. japonicum mutant strains that are hypersensitive to repression or H2 oxidation by oxygen were derived from strain SR. The mutants were obtained by screening H2 uptake-negative mutants that retained the ability to oxidize H2 as bacteroids from soybean nodules. As bacteroids, the five mutant strains were capable of H2 oxidation rates comparable to that of the wild type. The mutants did not take up H2 when derepressed in 22 microM dissolved oxygen, whereas strain SR had substantial activity at this oxygen concentration. The O2 repression of H2 uptake in both the wild-type and two mutant strains, SR174 and SR200, was rapid and was similar to the effect of inhibiting synthesis of H2 uptake system components with rifampin. None of the mutant strains was able to oxidize H2 when the artificial electron acceptors methylene blue or phenazine methosulfate were provided. The mutant strains were not sensitive to killing by oxygen, they took up O2 at rates similar to strain SR, and they did not produce an H2 uptake system that was oxygen labile. Cyclic AMP levels were comparable in strain SR and the five mutant strains after subjection of the cultures to the derepression conditions.

Regulation of hydrogenase in Rhizobium japonicum

Factors that regulate the expression of an H2 uptake system in free-living cultures of Rhizobium japonicum have been investigated. Rapid rates of H2 uptake by R. japonicum were obtained by incubation of cell suspensions in a Mg-phosphate buffer under a gas phase of 86.7% N2, 8.3% H2, 4.2% CO2, and 0.8% O2. Cultures incubated under conditions comparable with those above, with the exception that Ar replaced H2, showed no hydrogenase activity. When H2 was removed after initiation of hydrogenase derepression, further increase in hydrogenase activity ceased. Nitrogenase activity was not essential for expression of hydrogenase activity. All usable carbon substrates tested repressed hydrogenase formation, but none of them inhibited hydrogenase activity. No effect on hydrogenase formation was observed from the addition of KNO3 or NH4Cl at 10 mM. Oxygen repressed hydrogenase formation, but did not inhibit activity of the enzyme in whole cells. The addition of rifampin or chloramphenicol to derepressed cultures resulted in inhibition of enzyme formation similar to that observed by O2 repression. The removal of CO2 during derepression caused a decrease in the rate of hydrogenase formation. No direct effect of CO2 on hydrogenase activity was observed.

Characteristics of the H2 oxidizing system in soybean nodule bacteroids

A derivative of Rhizobium japonicum (strain 122 DES) has been isolated which forms nodules on soybeans that evolve little or no H2 in air and efficiently fixes N2. Bacteroids isolated from nodules formed by strain 122 DES took up H2 with O2 as the physiological acceptor and appeared to be typical of those R. japonicum strains that possess the H2 uptake system. The hydrogenase system in soybean nodules is located within the bacteroids and activity in macerated bacteroids is concentrated in a particulate fraction. The pH optimum for the reaction is near 8.0 and apparent Km values for H2 and O2 are 2 μM and 1 μM, respectively. The H2 oxidizing activity of a suspension of 122 DES bacteroids was stable at 4°C for at least 4 weeks and was not particularly sensitive to O2. Neither C2H2 nor CO inhibited O2 dependent H2 uptake activity.