Identification and characterization of plasmids in hydrogen uptake positive and hydrogen uptake negative strains ofRhizobium japonicum

Modifications were made of published procedures to allow routine isolation of plasmids fromRhizobium japonicum. The plasmid profiles of a series of H₂ uptake positive and H₂ uptake negative strains were compared. None of the strains ofR. japonicum with high H₂ uptake activities exhibited discernible plasmids, while most of the strains, with little or no H₂ uptake activity, showed plasmids with molecular weights ranging from approximately 49–290 x10⁶. An examination of H₂ uptake negative mutants derived from an H₂ uptake positive parent revealed two discernible plasmid bands in nonrevertible mutants but no detectable plasmids in revertible mutants or in the parent strain from which mutants were derived.     

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.     

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     

Viability of Rhizobium bacteroids isolated from soybean nodule protoplasts

Bacteriods isolated from protoplasts taken from Rhizobium japonicum induced root nodule of Glycine max L. showed complete viability when plated onto a conventional rhizobial growth medium supplemented with 0.2 M Mannitol. The same medium but without extra mannitol resulted in the absence of colony formation. The protoplast isolation method eliminated the possibility of contaminant bacteria from infection threads to be scored. The redifferentiated bacteroid clones have the same genetical characteristics as the orginal inoculum strain. This and other recent findings of bacteroid viability are discussed in the light of the existing belief that bacteroids are non-viable.     

Uptake and evolution of H2 and reduction of C2H2 by root nodules and nodule homogenates ofAlnus glutinosa

Summary In the growing season no net H₂ evolution is detected when root nodules ofAlnus glutinosa are incubated in air or in argon containing 20% O₂. Due to the hydrogenase activity, N₂-fixing root nodules consume added H₂ at a rate of about 1.4 moles H₂.g fresh nodule^–1.h^–1. The uptake of H₂ is only found in summer. At the end of the season, in autumn, nodules evolve significant quantities of H₂ although the nodules still continue to fix nitrogen. In-vitro studies with fractionated homogenates of summer-harvested nodules show that the recovery of the hydrogenase is high when using methylene-blue or phenazine metasulfate as electron acceptors. No hydrogenase activity is detected in homogenates of autumn-harvested nodules.The hydrogenase is localised in the microsymbiont.     

The synthesis of aspartic acid inRhizobium lupini bacteroids

Summary InRhizobium lupini bacterioids enzymes catalysing biosynthesis of aspartic acid have been found. The first enzyme termed aspartate dehydrogenase catalyses synthesis of aspartate from oxaloacetic acid and ammonia in the presence of NADH. The second enzyme, aspartase (L-aspartate ammonialyase, EC 4.3.1.1.), catalyses synthesis of aspartate from fumaric acid and ammonia. These data show that ammonia can be assimilated not only in the plant part of nodules but also in bacteroids. Biosynthesis of aspartate plays a very important role in the assimilation of ammonia in nodules.     

Utilization of nitrate by bacteroids of Bradyrhizobium japonicum in the soybean root nodule

Bacteroids of Bradyrhizobium japonicum strain CB1809, unlike CC705, do not have a high level of constitutive nitrate reductase (NR; EC 1.7.99.4) in the soybean (Glycine max. Merr.) nodule. Ex planta both strains have a high activity of NR when cultured on 5 mM nitrate at 2% O₂ (v/v). Nitrite reductase (NiR) was active in cultured cells of bradyrhizobia, but activity with succinate as electron donor was not detected in freshly-isolated bacteroids. A low activity was measured with reduced methyl viologen. When bacteroids of CC705 were incubated with nitrate there was a rapid production of nitrite which resulted in repression of NR. Subsequently when NiR was induced, nitrite was utilized and NR activity recovered. Nitrate reductase was induced in bacteroids of strain CB1809 when they were incubated in-vitro with nitrate or nitrite. Increase in NR activity was prevented by rifampicin (10 g· ml^-1) or chloramphenicol (50 g·ml^-1). Nitrite-reductase activity in bacteroids of strain CB1809 was induced in parallel with NR. When nitrate was supplied to soybeans nodulated with strain CC705, nitrite was detected in nodule extracts prepared in aqueous media and it accumulated during storage (1°C) and on further incubation at 25°C. Nitrite was not detected in nodule extracts prepared in ethanol. Thus nitrite accumulation in nodule tissue appears to occur only after maceration and although bacteroids of some strains of B. japonicum have a high level of a constitutive NR, they do not appear to reduce nitrate in the nodule because this anion does not gain access to the bacteroid zone. Soybeans nodulated with strains CC705 and CB1809 were equally sensitive to nitrate inhibition of N₂ fixation.Abbreviations NR nitrate reductase - NiR nitrite reductase - Tris 2-amino-2-(hydroxymethyl)-1,3-propanediol     

Nitrogenase — hydrogenase relationships in Rhizobium japonicum

The conditions necessary for coordinate derepression of nitrogenase and O₂-dependent hydrogenase activities in free-living cultures of Rhizobium japonicum were studied. Carbon sources were screened for their ability to support nitrogenase, and then hydrogenase activities. There was a positive correlation between the level of nitrogenase and corresponding hydrogenase activities among the various carbon substrates. The carbon substrate -ketoglutarate was able to support the highest levels of both nitrogenase and hydrogenase activities. When cells were incubated in -ketoglutarate-containing medium, without added H₂ but in the presence of acetylene (to block H₂ evolution from nitrogenase) significant hydrogenase activity was still observed. Complete inhibition of nitrogenase-dependent H₂ evolution by acetylene was verified by the use of a Hup^- mutant. Hydrogen is therefore not required to induce hydrogenase. The presence of 10% acetylene inhibited derepression of hydrogenase. Constitutive (Hup^c) mutants were isolated which contained up to 9 times the level of hydrogenase acitivity than the wild type in nitrogenase induction medium. These mutants did not have greater nitrogenase activities than the wild type.This is contribution number 1254 from the Department of Biology and the McCollum-Pratt Institute Abbreviations: -Ketoglutarate-containing medium (LOKG) and pre-adaptation medium (SRM) as described in Materials and methods     

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     

Carbon catabolism in continuous cultures and bacteroids of Rhizobium leguminosarum MNF 3841

Bacteroids of R. leguminosarum MNF3841 isolated from pea nodules using Percoll gradients had activities of TCA cycle enzymes up to 6-fold higher than those measured in free-living cells grown on fumarate or sucrose. Activities of sugar catabolic enzymes on the other hand were 2–14-fold lower in isolated bacteroids than in sucrose-grown free-living cells. In continuous culture, cells of strain MNF3841 grown on sucrose under P _i limitation had 2–3-fold higher activities of invertase, glucose-6-phosphate dehydrogenase, the Entner-Doudoroff enzymes and 6-phosphogluconate dehydrogenase, than cells grown on fumarate. With one exception O₂ limited cultures had similar activities of the carbon catabolic enzymes to P _i-limited cultures grown in the same substrate. Glucose-6-phosphate dehydrogenase in O₂-limited cells grown of fumarate was 50% lower than in P _i-limited cells. Co-utilization of fumarate and sucrose occurred with chemostat cultures supplied with both under a variety of conditions.Abbreviations E-D Entner-Doudoroff - EMP Embden-Meyerhof-Parnas - PEPCK phosphoenolpyruvate carboxy kinase - HEPES N-[2-hydroxyethyl]piperazine-N-[2-ethanesulphonic acid]     

Hydrogen accumulation by H2-uptake negative strains of Rhizobium

Summary Hydrogen evolution from root nodules has been reported to decrease the efficiency of the nitrogen fixing system. Mutants ofRhizobium meliloti andRhizobium leguminosarum were selected which were deficient in H₂-uptake capacity (Hup^–). The relative efficiency of the nitrogen fixation for both species assessed with C₂H₂ reduction was 0.66.The hydrogen production was monitored using a simple root incubation method. As such, hydrogen production up to 3.83 and 15.57 ml.day^–1.g^–1 plant dry weight were recorded forPisum sativum — Rhizobium leguminosarum 4.20 Hup^– andMedicago sativa — Rhizobium meliloti 1.5 Hup^– respectively. In a closed container (250 ml), hydrogen concentrations up to 20% (v/v) could be reached in the root phase ofMedicago sativa in a time period of 320 hours.     

Metabolism of poly-\-hydroxybutyric acid in bacteroids ofRhizobium lupini in connection with nitrogen fixation and photosynthesis

Summary The darkening of lupin plants grown in a sand culture on a nitrogen-free medium at a stage of initial flowering led to a sharply decreased nitrogen fixation intensity which eventually ceased. Decreased intensity of nitrogen fixation in bacteroids was accompanied by an accumulation of poly--hydroxybutyric acid (PHB); in the course of 10–20 h (depending upon temperature) its content increased by 2.5–3.0 times. If, following darkening, the plants were once again exposed to light, an abrupt increase of nitrogen fixation intensity was observed and a simultaneous decrease of PHB content.It has been shown that lupin's exposure to light in¹⁴CO₂ atmosphere lasting 19 h resulted in the latter's incorporation into PHB, bacteroids and into the entire nodule; these processes developed almost in parallel.During the early period of vegetation growth prior to flowering, the PHB content of bacteroids decreased from 13–14 to 3–4% of dry weight, whereas the intensity of nitrogen fixation was raised. Concurrently increase of the activity of some enzymes connected with the PHB metabolism (acetoacetyl-CoA-reductase, acetyl-CoA-acetyl transferase, PHB-depolymerase, CoA-transferase of 3-ketoacids) occured. The plants' subsequent ageing and reduction of nitrogen fixation intensity led to a noticeable increase of PHB content and a decrease of the above mentioned enzymes' activity. The specific activity of \-hydroxybutyric dehydrogenase involved with PHB catabolism was high and was maintained at a constant level throughout the entire vegetative period.     

Soil H2-uptake in relation to soil properties and rhizobial H2-production

Summary The biological nature of soil H₂-consumption has been investigated. Soil microorganisms were capable to remove H₂ present in the gas phase at concentrations in the range of 200 ppm at rates varying between 0.2 and 1.0 l.min^–1. 100 g^–1. Free soil enzymes did not contribute significantly at the H₂ concentrations tested. Oxygen seemed to be the predominant electron acceptor. The influence of microbiological and physical soil properties on the H₂-uptake activity was examined for 38 soils.A highly significant correlation between biomass-C and H₂-uptake rate of the soil was noted, suggesting that the latter parameter might be useful as an indirect estimation of soil microbial biomass. The correlation was however not applicable for soils recently grown with legumes. Indeed, soya plants nodulated with aRhizobium strain with a weak hydrogen uptake capability, strongly increased the hydrogen oxidizing capability of the surrounding soil.     

Agglutination ofRhizobium japonicum 3I1b110 by soybean lectin

Summary Conditions leading to agglutination ofRhizobium japonicum 3I1b110 with soybean seed lectin were examined. Ability of cells to be agglutinated was transient and was optimal for cultures grown for 4–5 days on yeast extract mannitol plates. Similar lectin-binding results were obtained with cells from the same cultures using fluorescence microscopy with fluorescein isothiocyanate-labelled lectin. These results revise the previous model for soybean lectin-R. japonicum interactions, since it was based on the inability of soybean lectin to agglutinate these bacteria.     

Nitrogenase activity of pea bacteroids as affected by carbohydrates and ammonium chloride

Summary Regulation and efficiency of the nitrogen-fixing system of the rhizobium-pea symbiosis were investigated. Acetylene reduction of detached root nodules was measured with various substrates added. Succinate, fumarate and malate were most effective in stimulating nitrogenase activity; glucose, pyruvate and citrate were also active. Acetylene reducing activity of detached nodules was inhibited by the addition of NH₄Cl, irrespective of the substrate present. Nitrogenase activity of isolated bacteroids was not influenced by NH₄Cl.Respiration of detached nodules was not significantly stimulated by the addition of substrates. Ammonium chloride did not influence respiration. With detached nodules and isolated bacteroids a consumption of about 16 g of carbohydrate per g of nitrogen fixed could be calculated. Detached nodules produced more hydrogen relative to the acetylene reduced than did isolated bacteroids and intact plants.Results obtained indicate that the regulation of nitrogenase activity and the efficiency of substrate consumption depend on environmental conditions.     

Isolation of pea nodule bacteroids utilizing silica sol (Percoll) density gradient centrifugation

Isolation of bacteroids from effective (Fix⁺) and ineffective (Fix^–) pea nodules, inoculated withRhizobium leguminosarum K, were performed by a density gradient centrifugation method using silica sol (Percoll). Only one zone (=1.064–1.072; n-zone) was recognized in the Fix⁺ nodule which contained typical Y-shaped bacteroids while two zones (n-zone and =1.125–1.145; n'-zone) were obtained from the Fix^– nodule. The cells in the n'-zone, which are long rods differed morphologically from free-living cells at any growth phase (=1.108–1.125; f-zone and =1.074–1.078; f'-zone), and differed from Y-shaped bacteroids by cell density. The esterase isozyme pattern of bacteroids in the n-and n'-zones also showed clear differences from that of f-and f'-zone of free-living cells.     

The role of formate metabolism in nitrogen fixation in Rhizobium Spp.

Formate metabolism supported nitrogen-fixation activity in free-living cultures of Rhizobium japonicum. However, formate0dependent nitrogense activity was observed only in the presence of carbon sources such as glutamate, ribose or aspartate which by themselves were unable to support nitrogenase activity. Formate-dependent nitrogenase activity was not detected in the presence of carbon sources such as malate, gluconate or glycerol which by themselves supported nitrogenase activity. A mutant strain of R. japonicum was isolated that was unable to utilise formate and was shown to lack formate dehydrogenase activity. This mutant strain exhibited no formate-dependent nitrogenase activity. Both the wild-type and mutant strains nodulated soybean plants effectively and there were no significant differences in the plant dry weight or total nitrogen content of the respective plants. Furthermore pea bacteroids lacked formate dehydrogenase activity and exogenously added formate had no stimulatory effect on the endogenous oxygen uptake rate. The role of formate metabolism in symbiotic nitrogen fixation is discussed.Abbreviation FDH formate dehydrogenase     

Effects of the plant host on the detergent sensitivity and viability of Rhizobium bacteroids

Bacteroids prepared from different legume species showed large differences in detergent sensitivity as judged by changes in turbidity and the release of cytochrome c oxidase activity after detergent treatments. There was a strong correlation between the detergent sensitivity and non-viability of bacteroids. Differences in the detergent sensitivity of bacteroids were determined by the plant host rather than the Rhizobium strain or the effectiveness of the symbiosis. The most common level of detergent sensitivity observed amongst bacteroids from 34 legume species was intermediate between lupin bacteroids and brothcultured bacteria.     

毛豆根瘤菌转吸氢基因的研究

通过三亲本杂交将携带豌豆根瘤菌吸氢基因的质粒pAL₆₁₈转移到台湾毛豆根瘤菌292-C中,经抗性筛选、质粒检测和吸氢活性测定,得到能稳定遗传的结合株292-C₂和292-C₃。在自生条件下结合株均表现较高的吸氢活性,而受体株292-C不吸氢,在共生条件下结合株表现为高吸氢,而受体株表现为放氢。     

Nitrite inhibition of nitrogenase from soybean bacteroids

Nitrogenase from soybean bacteroids was purified and used to study NO ₂ ^– effects either as unfractionated enzyme or as reconstituted enzyme from separated nitrogenase components I and II. Partially purified enzyme was strongly inhibited by nitrite at concentrations less than 0.1 mM. This inhibition was typically referred to as competitive with an inhibition constant (K _i) for NO ₂ ^– which was 5.2 mM. Kinetics studies showed an abnormally low apparent constant of association between enzyme and NO ₂ ^– (k _a=60 M^-1·s^-1). Nitrite appeared to bind to the MoFe protein, without any effect on Fe component, giving a completely reversible inhibition. Nitrite was found not to be an alternative substrate for nitrogenase.Abbreviations TES N-tris (hydroxymethyl) methyl-2-aminoethane sulfonic acid - PPG Polypropylene glycol