Expression of Rhizobial Nitrogenase: Influence of Plant Cell-Conditioned Medium

Conditioned medium was obtained from suspension cultures of soybean (Glycine max L. Merrit) cells after incubating them for 4 to 8 days with rhizobia which were separated from the soybean cells by two dialysis bags, one within another. This conditioned medium from the plant cell side (PCM) of the two membranes was used to elicit and influence nitrogenase activity (acetylene reduction) in rhizobia. When conditions for obtaining PCM from the soybean cell suspension cultures were varied, it could be shown that freshly grown rhizobia were able to induce active compounds in the PCM. These compounds caused acetylene reduction activity in test rhizobia under conditions where control rhizobia, containing various substrates, showed little or no acetylene reduction activity. Rhizobia that were already capable of acetylene reduction could not induce such compounds in the PCM when this was included with test rhizobia. The PCM from soybean cultures was also found to aid the expression of nitrogenase activity in suspension cultures of rhizobia normally associated with either peas, lupins, broad beans, or clovers. This is the first communication indicating nitrogenase activity in freeliving cultures for various species of rhizobia.     

Acetylene reduction by transfilter suspension cultures of Rhizobium japonicum.

Cultures of Rhizobium japonicum were grown in a defined medium and then placed in a transfilter-apparatus. Suspension cultures of soybean root cells were grown in Gamborg's B-5 defined medium and then were placed in a second chamber of this apparatus. The plant-cell medium was renewed under conditions shown to give partial synchrony in soybean cell cultures. Sampling of rhizobia showed that acetylene reduction activity could be obtained after approximately four days in the transfilter-apparatus. Criteria for precluding contaminations have been listed. This is the first report on the activation of Rhizobium japonicum in transfilter suspension cultures using defined media.     

Hydrogen (h(2)) evolution by rhizobia after synergetic culture with soybean cell suspensions

Rhizobium japonicum cells were grown in liquid suspension cultures and separated from soybean plant cells by two to three bacterial membrane filters. Under these conditions, the plant cells elaborated materials into the medium which aided in the expression of a major rhizobial phenotype, namely, nitrogenase activity (acetylene reduction). The evolution of H₂ was also measured and this activity relative to acetylene reduction, was influenced by: (a) O₂; (b) the quantity of conditioned plant medium; and (c) ammonia. It is concluded that plant substances are of major importance in the H₂ evolution and nitrogenase activities of free-living rhizobia in suspension cultures.     

Nitrogen fixation by a marine non‐heterocystous cyanobacterium requires a heterotrophic bacterial consort

Cultures of the non‐heterocystous cyanobacterium, Leptolyngbya nodulosa, could be grown indefinitely in media devoid of combined nitrogen. Acetylene reduction assays showed that these cultures fixed nitrogen in the dark period of a diurnal cycle under micro‐oxygenic or anaerobic conditions. Addition of DCMU to cultures induced much higher rates of nitrogenase activity, most of which occurred in the light. Measurements of activity in the presence of chloramphenicol indicated that nitrogenase is synthesized in darkness and probably destroyed in the subsequent light period. Neither the dark‐mediated nitrogenase in the absence of DCMU nor light‐mediated activity in the presence of DCMU could be sustained for more than 3 days without a photoperiodic light/dark cycle. Axenic cultures could not be grown in the absence of combined nitrogen and did not demonstrate any acetylene reduction activity. An identical nifH gene sequence was found in axenic and non‐axenic cultures of L. nodulosa. RT‐PCR demonstrated that this gene was expressed only in non‐axenic cultures. Western blotting showed that the Fe‐protein of nitrogenase is absent in cultures that are incapable of acetylene reduction, indicating that the lack of nitrogenase activity is likely due to the absence of the enzyme. These observations strongly indicate that L. nodulosa contains a functional nitrogenase which is not expressed in the absence of heterotrophic bacteria.     

Control of dinitrogen fixation in ammonium-assimilating cultures of Azotobacter vinelandii

Azotobacter vinelandii was grown at constant growth rate in a chemostat with different molar ratios of sucrose to ammonium (C/N) in the influent media. Both compounds were consumed at essentially the same ratios as were present in the influent media. At low (C/N)-ratios, the cultures were ammonium-limited. At increased (C/N)-ratio ammonium-assimilating cultures additionally began to fix dinitrogen. The (C/N)-ratio at which nitrogenase activity became measurable, increased when the ambient oxygen concentration was increased. Immunoblotting revealed the appearance of nitrogenase proteins when the activity became detectable. Nitrogenase activity as determined either by acetylene reduction or by total nitrogen fixation gave constant relative activities of 1:3.8 (mol of N₂ fixed per mol of acetylene reduced) under all sets of conditions used in this investigation. In spite of the oxygen dependent variation of the (C/N)-ratio, nitrogenase became active when the ammonium supply was less than about 14 nmol of ammonium per g of protein. This suggests that oxygen was not directly involved in the onset of dinitrogen fixation.     

Inoculationin vitro of wheat seedlings and wheat straw cultures withBacillus azotofixans

Bacillus azotofixans is a recently described species capable of fixing molecular nitrogen efficiently.Ecological studies performed in monoxenic wheat cultures, both in 0.7% agar and in vermiculite-sand mixture, showed that no acetylene reduction occurred and that this bacteria did not grow when supplied only with the wheat plant root exudates. However, after glucose addition to the 0.7% agar cultures, acetylene reduction ability (ARA) was detected. Comparing ARA for media with glucose both with and without plants, it was observed that the plants supply some component leading to the increase of the nitrogenase activity, since the ARA doubled in the samples containing plants.In wheat straw cultures a fast growth of the bacteria was observed in the first 24 hours after inoculation, but no acetylene reduction was detected. After glucose addition to the media with and without straw, nitrogenase activity was detected.     

Nitrogen fixation (Acetylene reduction) by free-livingRhizobium meliloti

Evidence is presented thatRhizobium meliloti is able to fix nitrogen (as scored by acetylene reduction) while growing in the absence of a plant host. The highest nitrogen fixation rate was obtained when bacteria were grown in defined liquid medium under normal atmosphere in sealed tubes. The nitrogen fixation rates obtained are of the same order as those reported for other rhizobia.     

Nitrogenase activity in the non-heterocystous cyanobacterium Oscillatoria sp. grown under alternating light-dark cycles

The non-heterocystous cyanobacterium Oscillatoria sp. strain 23 fixes nitrogen under aerobic conditions. If nitrate-grown cultures were transferred to a medium free of combined nitrogen, nitrogenase was induced within about 1 day. The acetylene reduction showed a diurnal variation under conditions of continuous light. Maximum rates of acetylene reduction steadily increased during 8 successive days. When grown under alternating light-dark cycles, Oscillatoria sp. fixes nitrogen preferably in the dark period. For dark periods longer than 8 h, nitrogenase activity is only present during the dark period. For dark periods of 8 h and less, however, nitrogenase activity appears before the beginning of the dark period. This is most pronounced in cultures grown in a 20 h light – 4 h dark cycle. In that case, nitrogenase activity appears 3–4 h before the beginning of the dark period. According to the light-dark regime applied, nitrogenase activity was observed during 8–11 h. Oscillatoria sp. grown under 16 h light and 8 h dark cycle, also induced nitrogenase at the usual point of time, when suddenly transferred to conditions of continuous light. The activity appeared exactly at the point of time where the dark period used to begin. No nitrogenase activity was observed when chloramphenicol was added to the cultures 3 h before the onset of the dark period. This observation indicated that for each cycle, de novo nitrogenase synthesis is necessary.     

The induction of nitrogenase activity in Rhizobium by non-legume plant cells

Summary Nitrogen fixation was induced in a strain of cowpea rhizobia, 32Hl, when it was grown in association with cell cultures of the non-legume, tobacco (Nicotiana tabacum). Rhizobia grown alone on the various media examined did not show nitrogenase activity, indicating the involvement of particular plant metabolites in nitrogenase induction. Nitrogenase activity, as measured by C₂H₂ reduction, was maximized at an O₂ concentration of 20% and at an assay temperature of 30°C, the conditions under which the plant cell-rhizobia associations developed. Glutamine, as a nitrogen source, could be replaced by other organic nitrogen sources, but NH₄ ⁺ and NO₃ ^- repressed nitrogenase activity. Nitrogenase activity induced in rhizobia when cultured adjacent to, but not in contact with, the plant cells could be stimulated by providing succinate in the medium. At least 12 other strains of rhizobia also reduced C₂H₂ in association with tobacco cells; the highest levels of activity were found among cowpea strains.     

In situ acetylene-ethylene assay of biological nitrogen fixation in lowland rice soils

Summary An in situ device for assaying biological nitrogen fixation in flooded rice soils, using the acetylene reduction method, was developed. Diurnal variations in acetylene reduction by an inoculated field plot and by laboratory-grown cultures of nitrogen-fixing algae showed a prominent single-peak pattern of nitrogenase activity. The peak occurred at mid-day for laboratory-grown algae and at late afternoon for the algae grown in the field plot. Some nitrogenase activity was noted during the night. Acetylene reduction studies in rice fields of Albay province, Philippines, showed an estimated fixation of 18.5 to 33.3 kg N/ha each cropping season for the fields of Puro soil and 2.3 to 5.7 kg N/ha each cropping season for the fields of Santo Domingo soil. re]19751202     

Enhancement of nitrogen fixation inAnabaena flos-aquae (Cyanobacteria) via low-dose ultrasonic treatment

Low-dose (50 W), short-duration (5 min) ultrasonic treatments were delivered to log phase batch cultures of the cyanobacteriumAnabaena flos-aquae grown in nitrogen-free medium. Near the mid-point of nine-day experimental periods, ultrasonically-treated cultures exhibited higher growth rates, enhanced chlorophylla content, and greater acetylene reduction capacity when compared to cultures grown with reciprocal shaking alone. Heterocyst frequency was markedly higher in ultrasonically-treated cultures over the duration of five-day experimental periods.Author for correspondence     

Nitrogen fixation (acetylene reduction) and cross inoculation in 12 Prosopis (mesquite) species

The leguminous tree mesquite (Prosopis spp) exists on millions of hectares of semi-arid regions of the world. No whole plant acetylene reductions for mesquite have been reported in the literature and nodulation has only been reported for three of the forty-four species. We report greenhouse studies in which 12Prosopis species representing African and North and South American germplasm (1) became nodulated when inoculated with rhizobia strain isolated from a North American mesquite, (2) grew on a nitrogen free nutrient media, (3) reduced acetylene to ethylene, and (4) had a positive significant correlation between the acetylene reduction rates and above ground dry matter. The capability of mesquite to fix nitrogen must now be considered firmly established.     

Analysis of the Symbiotic Performance of Bradyrhizobium japonicum USDA 110 and Its Derivative I-110 and Discovery of a New Mannitol-Utilizing, Nitrogen-Fixing USDA 110 Derivative

Previously, Bradyrhizobium japonicum USDA 110 was shown to contain colony morphology variants which differed in nitrogen-fixing ability. Mannitol-utilizing derivatives L1-110 and L2-110 have been shown to be devoid of symbiotic nitrogen fixation ability, and non-mannitol-utilizing derivatives I-110 and S-110 have been shown to be efficient at nitrogen fixation. The objectives of this study were to determine the effect of media carbon sources on the symbiotic N₂-fixing ability of strain USDA 110 and to compare the effectiveness of strain USDA 110 and derivative I-110. Based on acetylene reduction activity and the nitrogen content of 41-day-old soybean plants, neither derivative I-110 nor cultures of USDA 110 grown in media favoring non-mannitol-using derivatives had symbiotic nitrogen fixation that was statistically superior to that of cultures of USDA 110 grown in media favoring mannitol-using derivatives. In another experiment 200 individual nodules formed by strain USDA 110 grown in yeast extract gluconate were screened for colony morphology of occupying variant(s) and acetylene reduction activity. Nodules occupied by mannitol-using derivatives (large colony type on 0.1% yeast extract-0.05% K₂HPO₄-0.08% MgSO₄ · 7H₂O-0.02% NaCl-0.001% FeCl₃ · 6H₂O [pH 6.7] with 1% mannitol [YEM] plates) had a mean acetylene reduction activity equal to that of nodules occupied by non-mannitol-using derivatives (small colony type on YEM plates). A total of 20 large colonial derivatives and 10 small colonial derivatives (I-110-like) were isolated and purified by repeated culture in YEM and YEG (same as YEM except 1% gluconate instead of 1% mannitol) media, respectively, followed by dilution in solutions containing 0.05% Tween 40. After 25 days of growth, soybean plants inoculated with the large colony isolates had mean whole-plant acetylene reduction activity, whole-plant dry weight, and whole-plant nitrogen contents equal to or better than those of plants inoculated with either the small colony isolates (I-110-like) or the I-110 (non-mannitol-using) derivative. Hence, the existence of a mannitol-utilizing derivative that fixes nitrogen in a culture of strain USDA 110 obtained from the U.S. Department of Agriculture, Beltsville, Md., was established. This new USDA 110 derivative was designated as MN-110 because it was a mannitol-utilizing nitrogen-fixing USDA 110 derivative. This derivative was morphologically indistinguishable from the non-nitrogen-fixing derivative L2-110 found in cultures obtained earlier from the U.S. Department of Agriculture, Beltsville. DNA-DNA homology and restriction enzyme analyses indicated that MN-110 is genetically related to other USDA 110 derivatives that have been characterized previously.     

Nitrogenase activity in extracts of heterocystous and non-heterocystous blue-green algae

Summary Cell-free extracts capable of acetylene reduction and cyanide reduction have been prepared from heterocystous (Anabaena cylindrica) and non-heterocystous (Plectonema boryanum 594) blue-green algae. Extracts from Anabaena were obtained from cultures grown in blulk under aerobic conditions, while the Plectonema cultures were grown in bulk on nitrate-nitrogen, then washed free from nitrate and sparged with A/CO₂ for 40 h after which time maximum nitrogenase activity was detected. The nitrogenases of both algae are similar and resemble in many respects nitrogenases from bacteria and legumes. Activity is located primarily in a 40,000xgx15 min supernatant fraction and the rate of C₂H₂ reduction observed is about 10 per cent of whole cell activity. ATP and a source of reducing power (Na₂S₂O₄) are required for efficient functioning of the enzyme. ATP-dependent hydrogen evolution occurs, the extracts are cold labile and highly sensitive to oxygen and the oxygen inhibition is irreversible.     

Nodulation and nitrogen fixation (Acetylene reduction) in desert ironwood (Olneya tesota)

Summary Acacia greggi, Cercidium floridium, and Olneya tesota seeds were inoculated with soil from beneath mature native desert trees and grown in the greenhouse on a nitrogen free media. Olneya tesota seedlings nodulated and reduced acetylene to ethylene. Nodulation or acetylene reduction was not observed in A. greggi or C. floridium. This is the first report of nodulation and nitrogen fixation in Olneya tesota.     

Neotropical Legume Tree Inga edulis Forms N2-fixing Symbiosis with Fast-growing Bradyrhizobium Strains

Inga edulis Mart. is a tropical legume tree used for shade in coffee and cacao plantations and as a hedgerow in alley-cropping practices. Little information can be found concerning N₂ fixation in this species. This study was conducted to characterize the rhizobia of I. edulis and determine if it is capable of fixing substantial amounts of N₂. Four strains of fast-growing, Gram-negative rhizobia-type bacteria were isolated from I. edulis nodules. The strains were identified by sequencing of partial 16S–23S rDNA internal spacer region. Nitrogenase activity was determined using acetylene reduction assay (ARA). Dinitrogen fixation was measured under controlled conditions by the ¹⁵N isotope dilution technique using two non-N₂-fixing reference species, Vochysia guatemalensis Donn. Sm, and Gmelina arborea Roxb. ex. Sm. Seedlings were grown in three growth media: native soil and naturally N-depleted sand amended to a low and high N level. The four strains of symbiotic bacteria were closely related to Bradyrhizobium japonicum and to Bradyrhizobium liaoningense. Nodules demonstrated nitrogenase activity as measured by ARA. Vochysia guatemalensis was a better non-N₂-fixing reference than G. arborea. When V. guatemalensis was used as the non-N₂-fixing reference, the estimate of the percentage of N fixed from atmosphere out of total N in I. edulis seedlings was ca. 40 in the two sand media treatments and 10 in the native soil.     

Emerson enhancement of carbon fixation but not of acetylene reduction (nitrogenase activity) in Anabaena cylindrica

Summary Enhancement of carbon fixation was demonstrated in the bluegreen alga, Anabaena cylindrica, grown in either aerobic or microaerobic conditions. Under identical conditions no enhancement of acetylene reduction was observed. Light absorbed by photosystem I supported relatively more acetylene reduction than carbon fixation. No competition between the two processes was observed under light-limiting conditions. The findings suggest that carbon fixation and acetylene reduction may depend on different pools of reductant and ATP. When aerobically grown cells were placed in the dark or at limiting light intensities, acetylene reduction was higher in air than under argon. In contrast, carbon fixation was lower in air than in argon.     

ROLE OF THE NITROGEN-FIXING BACTERIAL MICROFLORA IN THE EPIPHYTISM OF TILLANDSIA (BROMELIACEAE)

Nitrogen-fixing activity in the phyllosphere of 12 species of Tillandsia from different Mexican habitats was evaluated by the acetylene reduction assay, and nitrogen-fixing microorganisms were isolated and characterized. The leaves from eight of the 12 Tillandsia species examined exhibited nitrogenase activity in enrichment cultures. Among the microorganisms implicated—Agrobacterium, Bacillus, Erwinia, Pseudomonas, Rahnella, Vibrio, and Xanthomonas—only Bacillus megatherium reduced acetylene in pure culture. Our findings suggest that nitrogen fixation in the phyllosphere of the sampled epiphytes occurs under suitable conditions and that most of the bacteria involved are primarily soil and water inhabitants. The results also suggest a relationship between the composition of the nitrogen-fixing microbial communities grown on the leaf and the different development of the leaf area in Tillandsia due to the aerial components (wings) of the trichomes.     

Effect of oxygen on batch and continuous cultures of a nitrogen-fixing Arthrobacter sp.

Growth, acetylene reduction, and respiration rate were studied in batch and continuous cultures of Arthrobacter fluorescents at different oxygen partial pressures. The optimum pO2 values for growth and acetylene reduction were 0.05 and 0.025 atm, respectively, but microorganisms can tolerate higher pO2 values. The growth of cultures provided with combined nitrogen was dependent on oxygen availability, and strict anaerobic conditions did not support growth. Acetylene reduction of a population grown in continuous culture and adapted to low pO2 (0.02 atm) was much more sensitive to oxygenation than that of a population adapted to high pO2 (0.4 atm). Their maximum nitrogenase activity, at their optimal pO2 values, were quite different. The respiratory activity of nitrogen-fixing cultures increased with increasing oxygen tensions until a pO2 of 0.2 atm. At higher pO2 values, the respiration rate began to decrease.     

Comparison of N(2) Fixation and Yields in Cajanus cajan between Hydrogenase-Positive and Hydrogenase-Negative Rhizobia by In Situ Acetylene Reduction Assays and Direct N Partitioning

Pigeon peas [Cajanus cajan (L.) Millsp.] were grown in soil columns containing ¹⁵N-enriched organic matter. Seasonal N₂ fixation activity was determined by periodically assaying plants for reduction of C₂H₂. N₂ fixation rose sharply from the first assay period at 51 days after planting to a peak of activity between floral initiation and fruit set. N₂ fixation (acetylene reduction) activity dropped concomitantly with pod maturation but recovered after pod harvests. Analysis of ¹⁵N content of plant shoots revealed that approximately 91 to 94% of plant N was derived from N₂ fixation. The effect of inoculation with hydrogenase-positive and hydrogenase-negative rhizobia was examined. Pigeon peas inoculated with strain P132 (hydrogenase-positive) yielded significantly more total shoot N than other inoculated or uninoculated treatments. However, two other hydrogenase-positive strains did not yield significantly more total shoot N than a hydrogenase-negative strain. The extent of nodulation by inoculum strains compared to indigenous rhizobia was determined by typing nodules according to intrinsic antibiotic resistance of the inoculum strains. The inoculum strains were detected in almost all typed nodules of inoculated plants.

Gas samples were taken from soil columns several times during the growth cycle of the plants. H₂ was never detected, even in columns containing pigeon peas inoculated with hydrogenase-negative rhizobia. This was attributed to H₂ consumption by soil bacteria. Estimation of N₂ fixation by acetylene reduction activity was closest to the direct ¹⁵N method when ethylene concentrations in the gas headspace (between the column lid and soil surface) were extrapolated to include the soil pore space as opposed solely to measurement in the headspace. There was an 8-fold difference between the two acetylene reduction assay methods of estimation. Based on a planting density of 15,000 plants per hectare, the direct ¹⁵N fixation rates ranged from 67 (noninoculated) to 134 kilograms per hectare, while grain yields ranged from 540 to 825 kilograms per hectare. Grain yields were not increased with N fertilizer.