Calcofluor- and lectin-binding exocellular polysaccharides of Azospirillum brasilense and Azospirillum lipoferum.

Extracellular polysaccharides synthesized by Azospirillum brasilense and A. lipoferum were shown on agar plates and liquid flocculating cultures. The six strains used in this work expressed a mucoid phenotype, yielding positive calcofluor fluorescence under UV light. The calcofluor-binding polysaccharides were distributed between the capsular and exopolysaccharide fractions, suggesting exocellular localization. No calcofluor fluorescence was observed in residual cells after separation of the capsular and exopolysaccharide fractions. Cellulose content was significantly higher in flocculating than in nonflocculating cultures. Failure to induce flocculation by addition of cellulose (100 mg/ml) to nonflocculating cultures, together with the sensitivity of flocs to cellulase digestion, suggested that cellulose is involved in maintenance of floc stability. Different A. brasilense and A. lipoferum strains bound to a wheat lectin (fluorescein isothiocyanate-wheat germ agglutinin), indicating the occurrence of specific sugar-bearing receptors for wheat germ agglutinin on the cell surface. The biochemical specificity of the reaction was shown by hapten inhibition with N-acetyl-D-glucosamine. All six strains failed to recognize fluorescein isothiocyanate-soybean seed lectin under our experimental conditions. We conclude that azospirilla produce exocellular polysaccharides with calcofluor- and lectin-binding properties.

Cyst production and brown pigment formation in aging cultures of Azospirillum brasilense ATCC 29145.

Encystation in Azospirillum brasilense ATCC 29145 was observed by using routine laboratory staining and phase-contrast and electron microscopy. Encystment occurred in liquid and in solid or semisolid media containing fructose (8 mM) and KNO3 (0.5 mM). The encysted forms consisted of a central body filled with poly-beta-hydroxybutyric acid granules, an electron-transparent intinelike region, and a thick outer layer. Enlarged giant encysted forms with multiple central bodies were also observed during the germination of a desiccated brown colony. Morphogenetically different forms in an aging culture could be resolved by sucrose density gradient centrifugation. The dense encysted forms along with numerous granules in a fibrillar network pelleted at 70% sucrose, while empty saclike envelopes along with vegetative cells and coccoid bodies pelleted at 55% sucrose. Different media induced various degrees of pigmentation in A. brasilense ATCC 29145 after aging. The pigment possessed several of the properties reported for microbial melanins, including insolubility in water and organic solvents, solubility in cold and hot alkali, and bleaching in hydrogen peroxide. The UV absorption maxima of the alkali extract were at 280 and 310 nm. Electron micrographs of the brown pigment showed that it occurred as aggregated granules surrounding the encysting cells as well as being excreted into the medium in an aging culture. It is concluded that A. brasilense ATCC 29145 produces compounds that form a brown pigment similar to melanin and are expressed under the influence of certain cultural conditions conducive for encystment.

Flocculation in Azospirillum brasilense and Azospirillum lipoferum: exopolysaccharides and cyst formation.

The phenomena of flocculation and floc formation by Azospirillum brasilense Sp7 (ATCC 29145) and Azospirillum lipoferum Sp59b (ATCC 29707) were studied in aerobic liquid cultures. Carbon sources representative of various entry pathways in combination with various nitrogen sources induced flocculation in both species of azospirilla. Noticeably, the combination of fructose and nitrate was the most effective in terms of floc yields. Phase-contrast microscopic observations revealed a transition in cell morphology from freely motile, vibrioid cells to nonmotile, highly refractile encysting forms during the formation of flocs. The nonmotile forms in flocs appeared to be entangled within a fibrillar matrix, and the cells were highly resistant to desiccation. Dried flocs kept for almost 6 months still maintained the highly refractile encysting forms, and their viability was confirmed by pellicle formation and acetylene reduction in semisolid malate medium. Electron microscopic observations of the desiccated flocs revealed the presence of cell forms containing abundant poly beta-hydroxybutyrate granules within a central body and surrounded by a thick layer of exopolysaccharides. The latter were characterized by alkali and acid digestion, crude cellulase hydrolysis, and calcofluor staining. It was concluded that the overproduction of exocellular polymers induces the flocculent growth and is associated with the concomitant transformation of vegetative cells to the desiccation-resistant encysting forms under limiting cultural conditions.

Expression of Nitrate and Nitrite Reductase Activities under Various Forms of Nitrogen Nutrition in Phaseolus vulgaris L.

The main objectives of this work were to study the effect of different N sources on plant growth, N accumulation, and on the expression of nitrate reductase activity in Phaseolus vulgaris L. leaves. Plants were grown under greenhouse conditions (15 to 25 kilolux; 16/8 hour day/night cycles) in plastic pots filled with perlite: vermiculite (1:1) and watered daily with a minus N solution (N(2) plants) or supplemented with either KNO(3), (NH(4))(2)SO(4), or urea as combined N sources.Significant levels of nitrate reductase activity in trifoliolate leaves of N(2)-, NH(4) (+)-, urea-, or NO(3) (-)-dependent plants was demonstrated throughout this work. Leaves from the urea- or NH(4) (+)-grown plants accumulated NO(2) (-) in the dark but not in the light when NO(2) (-) was supplied by vacuum infiltration. These results indicated that the potential for reduction of NO(3) (-) or NO(2) (-) was not impaired by growing the plants on NH(4) (+) or urea and, in addition, provided evidence for the occurrence of a non-nitrate-inducible nitrite reductase. The nitrate reductase activities associated with N(2)-, NH(4) (+)-, or urea-dependent plants are tentatively regarded as ;constitutive' to differentiate from the widely occurring NO(3) (-)-inducible nitrate reductase activity.Plants grown on NO(3) (-) or urea accumulated significantly larger amounts of reduced N and dry matter as compared to NH(4) (+)- and N(2)-dependent plants. Regardless of N treatment, or size of plants, about 50% of the N accumulated by the plant was allocated to the leaves.

Nitrate and Nitrite Reduction in Relation to Nitrogenase Activity in Soybean Nodules and Rhizobium japonicum Bacteroids.

Soybean (Glycine max L. cv Williams) seeds were sown in pots containing a 1:1 perlite-vermiculite mixture and grown under greenhouse conditions. Nodules were initiated with a nitrate reductase expressing strain of Rhizobium japonicum, USDA 110, or with nitrate reductase nonexpressing mutants (NR(-) 108, NR(-) 303) derived from USDA 110. Nodules initiated with either type of strain were normal in appearance and demonstrated nitrogenase activity (acetylene reduction). The in vivo nitrate reductase activity of N(2)-grown nodules initiated with nitrate reductase-negative mutant strains was less than 10% of the activity shown by nodules initiated with the wild-type strain. Regardless of the bacterial strain used for inoculation, the nodule cytosol and the cell-free extracts of the leaves contained both nitrate reductase and nitrite reductase activities. The wild-type bacteroids contained nitrate reductase but not nitrite reductase activity while the bacteroids of strains NR(-) 108 and NR(-) 303 contained neither nitrate reductase nor nitrite reductase activities.Addition of 20 millimolar KNO(3) to bacteroids of the wild-type strain caused a decrease in nitrogenase activity by more than 50%, but the nitrate reductase-negative strains were insensitive to nitrate. The nitrogenase activity of detached nodules initiated with the nitrate reductase-negative mutant strains was less affected by the KNO(3) treatment as compared to the wild-type strain; however, the results were less conclusive than those obtained with the isolated bacteroids.The addition of either KNO(3) or KNO(2) to detached nodules (wild type) suspended in a semisolid agar nutrient medium caused an inhibition of nitrogenase activity of 50% and 65% as compared to the minus N controls, and provided direct evidence for a localized effect of nitrate and nitrite at the nodule level. Addition of 0.1 millimolar sucrose stimulated nitrogenase activity in the presence or absence of nitrate or nitrite. The sucrose treatment also helped to decrease the level of nitrite accumulated within the nodules.

Nitrogenase Activity Associated with Roots and Stems of Field-Grown Corn (Zea mays L.) Plants.

Corn (Zea mays L.) plants were assayed for nitrogenase activity (C(2)H(2) reduction) during early ear development. Hybrid corn and inbred lines were grown separately at two experimental fields in New Jersey. Acetylene-dependent ethylene production was observed a few hours after harvest, from the field, on intact plants, root-soil cores, lower stem segments, and excised roots, all assayed under air and not preincubated previously. Incubation of excised roots at 1% O(2) resulted in lower rates of C(2)H(2) reduction. The time course of C(2)H(2) reduction by excised roots, assayed in air, was similar for all genotypes studied (two hybrids, eight inbreds, and a cross of corn x teosinte) and indicated that a long preincubation at reduced O(2) is not absolutely required for early detection of nitrogenase activity. Isolation of N(2)-fixing bacteria from within the roots and stems, together with the diurnal fluctuation of nitrogenase activity in response to day/night cycles, were indicative of a close association with plant function. Collectively, the results provided strong evidence for the occurrence of nitrogenase activity associated with corn plants growing in a temperate climate and dependent upon indigenous N(2)-fixing bacteria.

Free-living and symbiotic characteristics of chlorate resistant mutants of Rhizobium.

This work investigated the usefulness of chlorate resistance as a method for the selection of nitrate reductase negative (NR-) strains from Rhizobium japonicum (61A76) and evaluated the symbiotic, characteristics of these strains. Chlorate resistent strains were selected from populations seeded on CS 7 agar containing 10 or 20 mM KC10, and incubated in 2% air- 98% N2-CO2 (95:5). Over 200 resistant strains were isolated, 58% of which lacked the dissimilatory nitrate reductase. In 12 selected isolates, some strains had also lost the assimilatory nitrate reductase, but all retained hydrogenase activity. Chlorate resistant strains inoculated to soybean seedlings were equal to or better than the parent strain in terms of nodule mass and acetylene reduction. Those strains lacking both assimilatory and dissimilatory nitrate reductase showed the best symbiotic characteristics, suggesting that chlorate resistance in R. japonicum could be a useful method for the selection of strains with superiod nitrogen-fixing characteristics.

Seasonal Patterns of Nitrate Reductase and Nitrogenase Activities in Phaseolus vulgaris L.

The patterns of nitrate reductase activity (NRA) in the leaves (in vivo assay) and root nodule nitrogenase activity (C(2)H(2) reduction) were investigated throughout the season in field-grown Phaseolus vulgaris plants.Maximal NRA (per g fresh weight) occurred at early stages of leaf development but total activity (per leaf) was maximal when the leaf reached full size. In mature plants, most NRA was associated with the upper leaves. Nitrogenase activity was initiated about 2 weeks after sowing, reached a maximum at flowering (5 weeks after sowing) and declined rapidly thereafter. Nitrogenase activity followed the pattern of nodule development. After flowering, P. vulgaris was apparently able to take up and assimilate NO(-) (3) as evidenced by the increase in NO(-) (3) content of the stem and the high levels of NRA in the leaves. Total plant NRA was maximal after flowering and addition of NH(4)NO(3) to the soil at flowering resulted in even higher levels of NRA through most of the pod-filling period, thus resulting in higher seed yields (59% over control).It is proposed that P. vulgaris can benefit from both N(2) fixation and NO(-) (3) assimilation and that nitrate reductase plays an important role in the assimilation of nitrogen after flowering.

Pathway for Nitrate Assimilation in Corn (Zea mays L.) Leaves: Cellular Distribution of Enzymes and Energy Sources for Nitrate Reduction.

The localization of enzymes responsible for nitrate assimilation and the generation of NADH for nitrate reduction were studied in corn (Zea mays L.) leaf blades. The techniques used effectively separated mesophyll and bundle sheath cells as judged by microscopic observations, enzymic assays, chlorophyll a/b ratios and photochemical activities. Nitrate reductase, nitrite reductase, and the nitrate content of leaf blades were localized primarily in the mesophyll cells, although some nitrite reductase was found in the bundle sheath cells. Glutamine synthetase, NAD-malate dehydrogenase, NAD-glyceraldehyde-3-phosphate dehydrogenase, and NADP-glutamate dehydrogenase were found in both types of cells, however, more NADP-glutamate dehydrogenase was found in the bundle sheath cells than in the mesophyll cells. These data indicate that the mesophyll cells are the major site for nitrate assimilation in the leaf blade because they contained an ample supply of nitrate and the enzymes considered essential for the assimilation of nitrate into amino acids. Because the specific activity of nitrate reductase was severalfold lower than the other enzymes involved in nitrate assimilation, nitrate reduction is indicated as the rate-limiting step in situ. A sequence of reactions is proposed for nitrate assimilation in the mesophyll cells of corn leaves as related to the C-4 pathway of photosynthesis.

Nitrate and nitrite reductase negative mutants of N2-fixing Azospirillum spp.

Chlorate resistant spontaneous mutants of Azospirillum spp. (syn. Spirillum lipoferum) were selected in oxygen limited, deep agar tubes with chlorate. Among 20 mutants from A. brasilense and 13 from A. lipoferum all retained their functional nitrogenase and 11 from each species were nitrate reductase negative (nr-). Most of the mutants were also nitrite reductase negative (nir-), only 3 remaining nir+. Two mutants from nr+ nir+ parent strains lost only nir and became like the nr+ nir- parent strain of A. brasilense. No parent strain or nr+ mutant showed any nitrogenase activity with 10 mM NO3-. In all nr- mutants, nitrogenase was unaffected by 10 mM NO3-. Nitrite inhibited nitrogenase activity of all parent strains and mutants including those which were nir-. It seems therefore, that inhibition of nitrogenase by nitrate is dependent on nitrate reduction. Under aerobic conditions, where nitrogenase activity is inhibited by oxygen, nitrate could be used as sole nitrogen source for growth of the parent strains and one mutant (nr- nir-) and nitritite of the parent strains and 10 mutants (all types). This indicates the loss of both assimilatory and dissimilatory nitrate reduction but only dissimilatory nitrite reduction in the mutants selected with chlorate.

Denitrification by N2-fixing Sprillum lipoferum.

Forty-nine N2-fixing strains of Spirillum lipoferum isolated from a wide range of plant roots and soils were examined for reduction of NO3-. All strains reduced NO3-to NO2-. Thirty of the strains further reduced NO2-with production of gas. Examiniation of representative strains of the putative denitrifiers showed that they produced both N2O and C2H4 in the presence of 0.1 atm of C2H2. Strains which did not reduce NO2-with production of gas produced C2H4 but ont N2O in the presence of C2H2. This is the first report of a N2-fixing bacterium able to bring about denitrification of NO3.

Nitrate reduction nitrogenase activity in Spirillum lipoferum1.

Nitrate and nitrite reduction under aerobic, microaerophillic, and anaerobic conditions was demonstrated in Spirillum lipoferum (ATCC 29145). Nitrite did not accumulated during assimilatory nitrate reduction in air. The nitrite produced during dissimilatory nitrate reduction accumulated in the medium but not in the cells. On exposure of the bacteria to nitrate and anaerobiosis, a low initial rate (lag) was followed by accelerated rates of nitrite accumulation. A 3-h anaerobic pretreatment, in the absence of nitrate, did not a void the lag phase. No nitrate reductase activity (NRA) developed in the presence of chloramphenicol. The data suggest that induction of anaerobic NRA in S. lipoferum required nitrate and protein synthesis. Anaerobic N2-ase by S. lipoferum was greatly stimulated in the presence of nitrate. The time course of nitrate reduction was coincidental with the pattern of nitrate-stimulated N2-ase activity inidcating that a relationship exists between these two processes.

Relationships between Carbon Dioxide, Malate, and Nitrate Accumulation and Reduction in Corn (Zea mays L.) Seedlings.

The observation that exposure of the leaf canopy to increasing concentrations of CO(2) (100-400 mul/l) decreases the influx of nitrate to the leaf blades, but not to the roots or stalks (largely leaf sheaths), was reconfirmed using (15)NO(3) (-). Decreases in leaf nitrate supply were associated with decreases in induction of nitrate reductase, thus supporting the view that the influx of nitrate to a tissue is a major factor in regulation of the level of nitrate reductase. The whole plant (15)N distribution data show that the CO(2) effects were due to decreased influx of nitrate into the leaf blade rather than CO(2)-enhanced nitrate reduction. The decreases in nitrate accumulation by the leaf blade with increases in CO(2) concentration were only partially accounted for by differences in transpiration. Because the initial malate concentration of root tissue (detopped plants) had no subsequent effect on nitrate uptake, it seems unlikely that high levels of malate induced by CO(2) were responsible for the exclusion of nitrate from the leaf blades.Time course changes in nitrate and malate concentrations in root tissue (detopped plants) during nitrate uptake showed that oxidation of extra malate does not stimulate nitrate uptake and that malate is not specifically required as an energy source at the ion carrier level.The observation that nitrate and malate concentrations in corn leaf blades were negatively correlated was reconfirmed with 25 additional corn genotypes. However, using the same tissue, a higher correlation was obtained between malate plus aconitate and nitrate, suggesting that organic acids other than malate could be involved. The proposal that reduction of nitrate in the leaf is stoichiometrically related to malate production is a valid explanation of the relationship only if malate oxidation does not provide NADH for nitrate reduction. However, addition of malate and NAD to crude extracts (in vitro assay) or malate to leaf blade sections (in vivo assay) caused nitrate reduction. Because of these observations and the known intracellular location of NAD-malate dehydrogenase and nitrate reductase, we believe that malate oxidation is one of the major sources of NADH for nitrate reduction in corn leaf blades in situ.

Nitrate uptake and induction of nitrate reductase in excised corn roots.

The characteristics of nitrate uptake and induction of nitrate reductase were studied in excised roots of corn (Zea mays L.). Upon initial exposure to nitrate, the low initial rate of nitrate uptake gradually increased until a steady uptake rate was achieved in 1 to 2 hours depending on the NO(3) (-) concentration. The pattern was observed over a wide range (0.2-5 mm) of nitrate concentrations and was independent of the accompanying cation.The nitrate uptake pattern as a function of increasing external nitrate concentrations (0.2-50 mm) followed saturation type kinetics. The reciprocal plot of the data was not linear but hyperbolic, indicating that more than one Km for nitrate uptake can be resolved from the data. This suggests the existence of either one carrier system with changing kinetic constants or the existence of dual uptake systems. The pattern of induction of nitrate reductase was coincident with the pattern of nitrate uptake as a function of time and increasing nitrate concentrations. The rate of induction of nitrate reductase was regulated by the rate of nitrate flux.Washing the roots for 2 hours enhances nitrate uptake by 2.5-fold over the nonwashed tissue. The presence of nitrate in the washing solution leads to further (3.5-fold over control) increases in the rate of nitrate uptake supporting the contention that nitrate plays a specific role in the induction of the inducible nitrate carrier independent of the washing effect.

Dependence of nitrite reduction on electron transport chloroplasts.

Methyl viologen and phenazine methosulfate (photosystem I electron acceptors), 3-(3,4-dichlorophenyl)-1, 1-dimethylurea (DCMU, electron-transport inhibitor), and methylamine (photophosphorylation uncoupler) were used to study the dependence of nitrite reduction on electron transport in chloroplasts.DCMU, methyl viologen, and phenazine methosulfate markedly inhibited, whereas methylamine stimulated NO(2) (-) reduction in isolated, intact spinach (Spinacia oleracea L.) chloroplasts. The addition of DCMU to leaf sections of spinach and corn, (Zea mays L. var. XL81), incubated with No(3) (-), caused no inhibition of nitrate reduction but inhibited nitrite reduction leading to the accumulation of NO(2) (-) in the light. The addition of methylamine to comparable leaf sections did not affect either nitrate or nitrite reduction.WE CONCLUDED THAT: (a) nitrite reduction is functionally associated with the electron transport arising from the light reactions of the chloroplast and this provides additional support for the localization of nitrite reductase in the chloroplast; (b) nitrite reduction is associated with photosystem I and ferredoxin is the most likely donor in leaf tissue; and (c) ATP is not involved directly in nitrite reduction. However, ATP synthesis, by regulating electron flow to photosystem I, can affect nitrite reduction in the light.

Nitrite assimilation and amino nitrogen synthesis in isolated spinach chloroplasts.

The assimilation of nitrite leading to de novo synthesis of amino nitrogen in a chloroplast-enriched fraction isolated from freshly harvested young spinach (Spinacia oleracea L.) leaves was demonstrated. The preparations showed approximately 55% intact chloroplasts as determined by light scattering properties and fixed CO(2) at rates of approximately 100 mumoles hr(-1) mg chlorophyll(-1).The chloroplast-enriched fraction contained the enzymes, nitrite reductase and NADPH-glutamate dehydrogenase, needed for the reduction of nitrite and incorporation of ammonia into glutamate. Kinetic studies showed that the reduction of nitrite by the chloroplast-enriched fraction is light-dependent, and the process proceeds at rates of 6 to 12 mumoles hr(-1) mg chlorophyll(-1). The addition of nitrite to the chloroplast preparation caused a 3-fold increase in the production of alpha-amino nitrogen when compared with the control without nitrite. There was a stoichiometric relation between amino-nitrogen synthesis and nitrite disappearance from the medium. The ratio of amino-nitrogen: NO(2) (-) ranged from 0.6 to 0.9. The initial rate of amino-nitrogen production was faster when (alpha)-ketoglutarate was added to the nitrite reducing chloroplast medium than when it was omitted. However, these high rates were not sustained and the total amino-nitrogen production at the end of a 30-minute period was only slightly higher. These data show that chloroplasts are functionally able and contain the enzyme complement necessary to utilize light energy for the reduction of nitrite to amino nitrogen. Thus, chloroplasts should be considered as a major site for in vivo amino-nitrogen synthesis in green plants.