Sterling A. Russell

Hydrogenase in Rhizobium japonicum Increases Nitrogen Fixation by Nodulated Soybeans

Some Rhizobium strains synthesize a unidirectional hydrogenase system in legume nodule bacteroids; this system participates in the recycling of hydrogen that otherwise would be lost as a by-product of the nitrogen fixation process. Soybeans inoculated with Rhizobium japonicum strains that synthesized the hydrogenase system fixed significantly more nitrogen and produced greater yields than plants inoculated with strains lacking hydrogen-uptake capacity. Rhizobium strains used as inocula for legumes should have the capability to synthesize the hydrogenase system as one of their desirable characteristics.

Induction of ammonia monooxygenase and hydroxylamine oxidoreductase mRNAs by ammonium in Nitrosomonas europaea

In Nitrosomonas europaea, ammonia monooxygenase (AMO) and hydroxylamine oxidoreductase (HAO) catalyse the oxidation of ammonia (NH3) to nitrite (NO2−). A transcript of 3500 bases hybridizes to probes for amoA and amoB (genes that code for AMO proteins). A transcript of 2100 bases hybridizes to probes for hao (the gene that codes for HAO). Induction of the mRNAs detected by amo and hao probes required the presence of ammonium (NH4+). To correlate new levels of mRNA with de novo activity, existent mRNA pools and AMO activity were depleted prior to induction by NH4+. The mRNAs of AMO and HAO were depleted by depriving the cells of energy for at least 8 h; AMO activity was inactivated with acetylene (C2H2) after mRNA depletion. In cells treated this way, levels of new AMO mRNA and de novo AMO enzyme activity were correlated with increased NH4+ concentrations up to 1 mM after 3 h of incubation. HAO mRNA also increased in the NH4+‐treated cells. Other proteins and RNAs induced by NH4+ were detected in 14CO2‐labelling experiments. The AMO and HAO mRNAs were preferentially synthesized during energy‐limiting conditions.

Physiological Chemistry of Symbiotic Nitrogen Fixation by Legumes

The beneficial effects of including legumes in crop rotations were realized by early Greek, Roman and Chinese agriculturists centuries before the elementary principles of biological N2 fixation were established (Fred, Baldwin and McCoy, 1932; Stewart, 1966). Although the occurrence of nodules on roots of legumes had been described (Malpighi, 1675), and evidence for N2 fixation by leguminous plants in field plots had been reported (Boussingault, 1838), the basic biological aspects of symbiotic N2 fixation by legumes were not established until the results of the classical experiments of Hellriegel and Wilfarth (1888) were reported. They assumed that nodules on the roots of Pisum sativum were caused by bacteria and that nodulation enabled the plants to fix N2. Furthermore, both non-legumes and leguminous plants without nodules were considered to be dependent upon fixed nitrogen compounds in the soil. Experiments in which legumes and non-legumes were cultured in sterile soil with and without non-sterile extracts of garden soil provided conclusive evidence that the non-sterile extract was needed for nodulation and that nodulation was essential for normal growth without adequate combined nitrogen.

The importance of hydrogen recycling in nitrogen fixation by legumes

In vivo losses of H2 from legume nodules are often much greater than 25% of the nitrogenase electron flux. A minority of strains of Rhizobium form bacteroids with a capability to activate H2 by an hydrogenase and to oxidize this gas via the respiratory electron transport chain. A minority of R. leguminosarum strains but a majority of strains of Bradyrhizobium spp. (cowpea) possess H2 recycling capability. The genetic determinants for hydrogenase expression are present in the rhizobial cells but the effectiveness of the H2 uptake in nodules is influenced by the host and environmental conditions. The majority of trials in which Hup+ and Hup- inocula have been compared have shown growth increases from H2 recycling capability, but most of these comparisons have not utilized genetically defined strains. The genes for hydrogenase expression have been cloned and are being characterized. Progress in the transfer and expression of H2 recycling capability among strains of Rhizobium is discussed.

Inoculation of millet with Azospirillum

SummaryMillet plants (Pennisetum glaucum) were grown at three levels of nitrogen fertilization with and without an inoculum of live nitrogen-fixing Azospirillum cells. The highest average rate of nitrogen fixation as estimated from acetylene reduction by excised preincubated roots was only 23g N2 fixed per ha per day and occurred after treatment with low levels of nitrogen amendment. The average rates of acetylene reduction for intact plants at all treatments were also low. The lack of significant nitrogen fixation due to an Azospirillum-millet association in this study was substantiated by plant dry weight analysis, and determination of the nitrogen content of plants, pot leachate, and soil. There was significant correlation between the total nitrogen content of the plants per pot at the termination of the experiment and the amount of nitrogen fertilizer added initially, but there was no effect of inoculum on final total nitrogen content.

Kinetic characterization of the inactivation of ammonia monooxygenase in Nitrosomonas europaea by alkyne, aniline and cyclopropane derivatives

The kinetic mechanisms of seven inactivators of ammonia oxidation activity in cells of the nitrifying bacterium, Nitrosomonas europaea were investigated. The effects of the inactivators were specific for ammonia monooxygenase (AMO) which oxidizes ammonia to hydroxylamine. The aniline derivatives, 1,3-phenylenediamine and p-anisidine, were potent inactivators of AMO while other derivatives were ineffective as inactivators. Two cyclopropane derivatives, 1, 2-dimethylcyclopropane and cyclopropyl bromide, were inactivators while cyclopropane was not an inactivator. The mechanisms of three alkynes, 1-hexyne, 3-hexyne, and acetylene, were also examined. For all seven compounds, the inactivation of AMO was irreversible, time-dependent, first-order, and dependent on catalytic turnover. Saturation of the rate of inactivation was indicated for p-anisidine (kinact=2.85 min-1; KI=1.0 mM) and cyclopropyl bromide (kinact=4.4 min-1; KI=97 microM), but not for any of the remaining five inactivators, including acetylene. Ammonia slowed the rate of inactivation for acetylene and cyclopropyl bromide, but enhanced the rate of inactivation for the remaining inactivators. All seven compounds appear to be mechanism-based inactivators of AMO.

An endogenous electron carrier for the nitrogenase system of Rhizobium bacteroids.

Abstract A partly purified electron carrier isolated from an extract of Rhizobium bacteroids (from soybean root nodules) mediated the transfer of reducing power generated by illuminated spinach chloroplasts to the nitrogenase of Rhizobium bacteroids or of Azotobacter vinelandii . The electron carrier is reducible by dithionite and, according to preliminary evidence, is similar to the recently reported Azotobacter type of ferredoxin.

THE PRESENT STATUS OF HYDROGEN RECYCLING IN LEGUMES

ABSTRACT A brief discussion is presented of recent information concerning (a) factors influencing extent of N2, loss during N2 fixation by legumes; (b) electron carriers involved in the oxyhydrogen reaction of Rhizobium japonicum bacteroids; and (c) progress made in evaluating H2 recycling advantages. A major factor determining whether H2 is evolved from legume nodules is the presence of an active uptake hydrogenase which participates in the oxidation of H2 that is evolved as a by-product of the nitrogenase reaction. The extent of H2 evolution from the nitrogenase reaction is affected by those factors that influence the nitrogenase turnover rate. These include the supply of ATP and reductant and the ratio of the Fe protein to the MoFe protein component of nitrogenase. Oxidation of H2 in Rhizobium bacteroids is catalyzed by a series of enzymes located in bacteroid membranes. In addition to the hydrogenase per se, carriers so far shown to be involved in the process include cytochromes of the b and c types a...

Characterization, Significance and Transfer of Hydrogen Uptake Genes from Rhizobium Japonicum

Nitrogenases from different sources exhibit ATP-dependent formation of both NH4 + and H2. Many N2-fixing microorganisms are able to recycle the nitrogenase-mediated H2 using a membrane-bound hydrogenase, thereby recovering some of the energy utilized in the formation of H2. The physiology, biochemistry and energetics of H2 recycling have been extensively reviewed (Dixon, 1978; Robson, Postgate, 1980; Adams et al., 1981; Eisbrenner, Evans, 1983; Evans et al., 1985).

Investigations into the pathway of electron transport to the nitrogenase from nodule bacteroids

SummaryA series of investigations were conducted with the objective of elucidating natural pathways of electron transport from respiratory processes to the site of N2 fixation in nodule bacteroids. A survey of dehydrogenase activities in a crude extract of soybean nodule bacteroids revealed relatively high activities of NAD-specific β-hydroxybutyrate and glyceraldehyde-3-phosphate dehydrogenases. Moderate activities of NADP-specific isocitrate and glucose-6-phosphate dehydrogenases were observed. By use of the ATP-dependent acetylene reduction reaction catalyzed by soybean bacteroid nitrogenase, and enzymes and cofactors from bacteroids and other sources, the following sequences of electron transport to bacteroid nitrogenase were demonstrated: (1) H2 to bacteroid nitrogenase in presence of a nitrogenase-free extract ofC. pasteurianum; (2) β-hydroxybutyrate to bacteroid nitrogenase in a reaction containing β-hydroxybutyrate dehydrogenase, NADH dehydrogenase, NAD and benzyl viologen; (3) β-hydroxybutyrate dehydrogenase, to nitrogenase in reaction containing NADH dehydrogenase, NAD and either FMN or FAD; (4) light-dependent transfer of electrons from ascorbate to bacteroid nitrogenase in a reaction containing photosystem I from spinach chloroplasts, 2,6-dichlorophenolindophenol, and either azotoflavin from Azotobacter or non-heme iron protein from bacteroids; (5) glucose-6-phosphate to bacteroid nitrogenase in a system that included glucose-6-phosphate dehydrogenase, NADP, NADP-ferredoxin reductase from spinach, azotoflavin from Azotobacter and bacteroid non-heme iron protein. The electron transport factors, azotoflavin and bacteroid non-heme iron protein, failed to function in the transfer of electrons from an NADH-generating system to bacteroid nitrogenase. When FMN or FAD were added to systems containing azotoflavin and bacteroid non-heme iron protein, electrons apparently were transferred to the flavin-nucleotides and then nitrogenase without involvement of azotoflavin and bacteroid non-heme iron protein.Evidence is available indicating that nodule bacteroids contain flavoproteins analogous to Azotobacter, azotoflavin, and spinach ferredoxin-NADP reductase. It is concluded that physiologically important systems involved in transport of electrons from dehydrogenases to nitrogenase in bacteroids very likely will include relatively specific electron transport proteins such as bacteroid non-heme iron protein and a flavoprotein from bacteroids that is analogous to azotoflavin.