The critical problem of oxygen toxicity for nitrogen-fixing organisms may be related to damage caused by oxygen radicals and peroxides. An enzymatic mechanism is described for removal of peroxides in root nodules of soybean ( Glycine max ). The system utilizes ascorbate as an antioxidant and glutathione as a reductant to regenerate ascorbate. The enzymes involved are ascorbate peroxidase (ascorbate:hydrogen-peroxide oxidoreductase, EC 1.11.1.7), dehydroascorbate reductase (glutathione:dehydroascorbate oxidoreductase, EC 1.8.5.1), and glutathione reductase (NADPH:oxidized-glutathione oxidoreductase, EC 1.6.4.2). The reactions are essentially the same as those involving scavenging of H 2 O 2 in chloroplasts. Glutathione peroxidase (glutathione:hydrogenperoxide oxidoreductase, EC 1.11.1.9) was not detected. During the course of early nodule development, ascorbate peroxidase and dehydroascorbate reductase activities and total glutathione contents of nodule extracts increased strikingly and were positively correlated with acetylene reduction rates and nodule hemoglobin contents. The evidence indicates an important role of glutathione, ascorbate, ascorbate peroxidase, dehydroascorbate reductase, and glutathione reductase as components of a peroxide-scavenging mechanism in soybean root nodules.

A medium is described on which selected Rhizobium japonicum strains express hydrogenase (H2 uptake) activity under free-living conditions. Low concentrations of carbon substrates, decreased oxygen tension, and the quantity of combined nitrogen in the medium were major factors influencing hydrogenase expression. Hydrogenase activity was dependent upon a preincubation period in the presence of H2 under conditions such that the cels did not exhibit nitrogenase activity. H2 uptake rates were easily measured amperometrically in aerobically or anaerobically prepared suspensions from free-living cultures. Six R. japonicum strains that formed nodules with the ability to utilize H2 oxidized this gas when grown in free-living cultures. In comparison six randomly chosen strains forming nodules that lost H2 in air either showed no or low capacity to take up H2 under ree-living conditions. The reduction of triphenyltetrazolium chloride in an agar medium was used to detect strains capable of oxidizing H2. This method has enabled us to isolate a spontaneous R. japonicum mutant strain that has lost the ability to utilize H2. This mutant strain forms nodules that evolve H2 but other symbiotic characteristics appear normal. This strain will be useful in evaluating the importance of the hydrogenase system in the nitrogen-fixing process of legumes.In addition to catalysis of N2 reduction, nitrogenases from all known sources catalyze ATP-dependent H2 evolution. Apparently, H2 evolution during N2 fixation is an inherent property of the nitrogenase reaction. Energy loss through nitrogenase-dependent H2 evolution is important because four or five ATP molecules are consumed per pair of electrons transferred (1, 2) regardless of whether N2 or H+ is the electron acceptor. The significance of ATP-dependent evolution of H2 by nodules as an energy-wasteful process during N2 fixation by legumes has been pointed out by Dixon (3), Schubert and Evans (4) The H2-uptake medium is a further modification of the modified LNB5 medium used to detect acetylene reduction activity in free-living cultures of R. japonicum (11). The H2-uptake medium contains the following in 1 liter of distilled water: NaH2PO4-H20, 150 mg; CaCl2-2H20, 150 mg; MgSO4-7H20, 250 mg; iron EDTA, 28 mg; MnSO4-H20, 10 mg; H3BO3, 3 mg; ZnSO4-7H20, 2 mg; NaMoO4-2H20, 0.25 mg; CuSO4-5H20, 0.04 mg; CoC12-6H20, 0.025 mg; KI, 0

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.

Previous research from this laboratory has demonstrated C02-fixing and H2-uptake capacities of certain strains of Rhizobium japonicum. In this report we have shown that SR, a H2euptake-positive (Hup+) strain of R. japonicum, is capable of autotrophic growth with H2 as the energy source. Growth occurred on mineral salts/vitamins/Noble agar, mineral salts/vitamins liquid medium (0.27 jig of C as vitamins per ml), and in mineral salts liquid medium with no added vitamins when cultures were provided with NH4CI and incubated in an atmosphere containing H2, CO2, 02, and N2. Little or no growth occurred when either H2 or CO2 was omitted from the atmosphere or when the culture was inoculated with SR3, a Hupmutant of SR. Growth was measured by protein synthesis, fixed organic carbon, and increase in cell number in liquid cultures. The organism that grew autotrophically was verified as B.japonicum by (i) apparent purity on streak plates; (ii) retention of the double antibiotic resistance markers; and (iii) its capability to nodulate soybeans. H2-and CO2-supported growth was demonstrated for three additional Hup+ wild type L japonicum strains (USDA 136, 311b 6, and 3I1b 143), while three Hupwild-type strains (USDA 120, 3Ilb 144, and USDA 117) were incapable of growth on the Noble agar medium containing mineral salts/vitamins in the H2/CO2/O2/N2 atmosphere. This demonstrated capability of Hup B. japonicum strains to grow autotrophically requires revision of current concepts regarding conditions for survival and competition of these bacteria in the soil and their relationships to other microorganisms. Beijerinck in 1888 (1) isolated nodule-forming bacteria from the root nodules of several different leguminous plants and referred to them as "Bacillus radicola." Since this pioneering discovery the microorganisms that nodulate roots of legumes have been classified into several species of Rhizobium primarily on the basis of their capacities to form N2-fixing nodules on the roots of certain groups of leguminous hosts (2). All rhizobia have been considered as aerobic chemoorganotrophs that grow best on complex media (3). Pentoses and hexoses are usually supplied as sources of carbon and energy for culture of the slow-growing species of which R. japonicum is an example (3).A hydrogenase system involved in H2 oxidation in root nodules of Pisum sativum was identified by Phelps and Wilson (4) and rediscovered by Dixon (5, 6). Nodules of P. sativum formed by R. leguminosarum strains ONA 311 and 314 consumed H2, but nodules produced by other strains of this species lacked this capability (7). H2-uptake activity of nodules formed by R. japonicum 3Ilb 110 was observed by Schubert et al. (8,9); however, only 7 of 32 R. japonicum strains surveyed by Carter et al. (10) synthesized sufficient hydrogenase in nodule bacteroids to recycle the major portion of the H2 produced during N2 fixation. The H2-uptake reaction in soybean nodule bacteroids was characterized by McCrae et al. (11). Recently, Emerich et al. (12) showed that H2 oxidat...

Soybean plants and Rhizobium japonicum 122 DES, a hydrogen uptake-positive strain, were cultured in media purified to remove Ni. Supplemental Ni had no significant effect on the dry matter or total N content of plants. However, the addition of Ni to both nitrate-grown and symbiotically grown plants resulted in a 7-to 10-fold increase in urease activity (urea amidohydrolase, EC 3.5.1.5) in leaves and significantly increased the hydrogenase activity (EC 1.18.3.1) in isolated nodule bacteroids. When cultured under chemolithotrophic conditions, free-living R. japonicum required Ni for growth and for the expression of hydrogenase activity. Hydrogenase activity was minimal or not detectable in cells incubated either without Ni or with Ni and chloramphenicol. Ni is required for derepression of hydrogenase activity and apparently protein synthesis is necessary for the participation of Ni in hydrogenase expression. The addition of Cr, V, Sn, and Pb in place of Ni failed to stimulate the activity of hydrogenase in R. japonhcum and urease in soybean leaves. The evidence indicates that Ni is an important micronutrient element in the biology of the soybean plant and R. japonicum.Nickel is an essential element in several biological processes, including H2 oxidation in some bacteria and urea hydrolysis by plants (1). Although its function is not completely understood, Ni is a constituent of urease (urea amidohydrolase, EC 3.5.1.5) from jackbean (2) and soybean (3) seeds, CO dehydrogenase from Clostridium thermoaceticum (4), and factor F4w from certain species of Methanobacterium (5, 6). Additionally, hydrogenases (EC 1.18.3.1) from Methanobacterium thermoautotrophicum (7), Alcaligenes eutrophus (8, 9), Desulfovibrio gigas (10), and Vibrio succinogenes (11) contain Ni, which appears to participate in an oxidation-reduction reaction during catalysis (10,12). Ni is a required micronutrient for the synthesis of functional hydrogenase in several other microorganisms (13,14) The potential importance of energy loss via H2 evolution from nitrogenase in nodules has been discussed in a review (19). H2 uptake via the hydrogenase system is important not only in bacteroids but also for chemolithotrophic growth of free-living rhizobia (20, 21). This study was conducted to determine whether the addition of Ni affected (i) the growth of soybean plants cultured either symbiotically or with nitrate, (ii) the expression of urease activity in soybean leaves, and (iii) the expression of hydrogenase activity in nodule bacteroids. In addition, we have examined the effect of adding Ni on the growth of and derepression of hydrogenase in R1. japonicum under chemolithotrophic conditions. MATERIALS AND METHODS Source of Chemicals. All chemicals, except otherwise indicated, were reagent grade and, as indicated below, some were purified to remove Ni. Fe, Co, Zn (as metals), MnCl2, CuCl2, PbF2, V205, SnO2, and Cr2O5 were Speepure grade and purchased directly from Johnson, Matthey (London) or through a United States supplier (Alfa-Ventron, Danvers,...