Nitrogenase activity (acetylene reduction) in root-associated, cold-climate species of Azospirillum, Enterobacter, Klebsiella and Pseudomonas growing at various temperatures

Abstract 23 Strains of diazotrophic root-associated bacteria isolated from various parts of Finland were tested for nitrogenase activity during growth at various temperatures. Nitrogenase activity was optimal at 20–37°C in cultures of Klebsiella pneumoniae , and at 14–20°C in cultures of Klebsiella terrigena and Enterobacter agglomerans . Strains of K. terrigena and E. agglomerans showed no activity at 37°C, and K. pneumoniae only minimal or no activity at 14°C. Azospirillum lipoferum exhibited high nitrogenase activity at both 28–37°C, but less than 25% of optimal activity at 20°C and no activity at 14°C. Pseudomonas sp. expressed nitrogenase activity at 14–28°C. None of the strains manifested nitrogenase activity at 4 or 42°C. There were only small local variations within a species between strains isolated at different locations.     

Effects of inoculation ofPoa pratensis andTriticum aestivum with root-associated,N2-fixing Klebsiella,Enterobacter and Azospirillum

Strains ofKlebsiella pneumoniae, Klebsiella terrigena, Enterobacter agglomerans andAzospirillum lipoferum were compared as diazotrophic inoculants in association withPoa pratensis andTriticum aestivum. Each strain colonized both plants in numbers ranging from 10⁴ to 10⁷ bacteria per root, and electron microscopy and immunofluorescence staining of inoculated roots revealed bacteria mainly on root hairs. Indirect immunofluorescence with specific antifimbriae antibodies showed that the enteric bacteria expressed their fimbria in both associations. All associations were positive in an acetylene reduction test but only in half of them was atmospheric nitrogen transferred to the plant. In the inoculated plants, variable effects in the dry matter and N yields in both hosts were observed and no correlation was found between dry matter, nitrogen content or the amount of fixed nitrogen. In infected plants, the number of root hairs and lateral roots increased and the length of the zone of elongation decreased. The changes in root morphology were more evident in associations with enteric bacteria than with Azospirillum. The results give further evidence on the importance of bacterial adhesion in associative N₂ fixation and suggest that bacteria-induced physiological changes in plant roots may be more important than the amount of nitrogen transferred to the plant.     

Measurement of nitrogenase activity in legume root nodules: In defense of the acetylene reduction assay

The closed acetylene reduction assay has been used as a measure of nitrogenase activity and an indicator of N₂ fixation in Rhizobium/legume symbioses for 25 years. However, starting 10 years ago this assay has come under harsh criticism as being inaccurate. Currently, confusion exists regarding the conditions under which the acetylene reduction assay can be used accurately, or whether it can be used at all as a measure of nitrogenase activity. This article reviews the circumstance that has lead to this confusion. The author argues that under the proper assay conditions and with the appropriate checks, the closed acetylene reduction assay is still a valuable tool in assessing relative differences in nitrogenase activity in Rhizobium/legume symbioses.     

During stationary phase, Beijerinckia derxii shows nitrogenase activity concomitant with the release and accumulation of nitrogenated substances

Beijerinckia derxii, a free-living nitrogen-fixing bacterium, maintained an increasing nitrogenase specific activity during the stationary growth phase. To verify the destination of the nitrogen fixed during this phase, intra and extracellular nitrogenated contents were analyzed. Organic nitrogen and amino acids were detected in the supernatant of the cultures. An increase in intracellular content of both nitrogen and protein occurred. Cytoplasmic granules indicated the presence of arginine. The ability of a non-diazotrophic bacterium (E. coli) to use B. derxii proteins as a source of nitrogen was observed concomitantly with E. coli growth. There is a suggestion that B. derxii contributes to the environment by both releasing nitrogenated substances and accumulating substances capable of being consumed after its death.     

Effects of inorganic nitrogen compounds on the activity and synthesis of nitrogenase in Gloeothece (Nägeli) sp. ATCC 27152

Addition of 2 mM nitrite or ammonium to aerobically incubated cultures of Gloeothece rapidly inhibited N₂ fixation (measured as acetylene reduction). In contrast, 2 mM nitrate inhibited N₂ fixation less rapidly and less extensively, and often temporarily stimulated nitrogenase activity. The inhibitory effects of both nitrate and ammonium could be prevented by addition of 3 mM L-methionine-DL-sulphoximine, suggesting that the true inhibitor of N₂ fixation was an assimilatory product of ammonium rather than either ammonium or nitrate itself. The inhibition of N₂ fixation by nitrite could not, however, be prevented by addition of L-methionine-DL- sulphoximine. On the other hand, nitrite (unlike nitrate and ammonium) did not inhibit N₂ fixation in cultures incubated under a gas phase lacking oxygen. These findings suggest that the mechanism whereby nitrite inhibits N₂ fixation in Gloeothece differs from that of either nitrate or ammonium. The inhibitory effect of nitrite on N₂ fixation did not involve reduction of nitrite to nitric oxide, though nitric oxide was a potent inhibitor of nitrogenase activity in Gloeothece . Nitrate and nitrite inhibited the synthesis of nitrogenase in Gloeothece , while ammonium not only inhibited nitrogenase synthesis but also stimulated degradation of the enzyme. In addition, all three compounds favoured the appearance of the Fe-protein of nitrogenase in its larger, presumed inactive, form.     

Factors affecting vesicle formation and acetylene reduction (nitrogenase activity) in Frankia sp. CpI1

Vesicle formation and acetylene reduction (nitrogenase activity) were observed when washed hyphae from cultures of Frankia sp. CpI1 were transferred to a nitrogen-free medium containing ethylenediaminetetraacetic acid and succinate. Succinate could be replaced by malate or fumarate, but not other carbon sources. Maximum acetylene reduction and vesicle numbers were observed at a pH of 6.0-6.5, at 25-30 degrees Centigrade, and at atmos pheric Po2 or somewhat less (5-20 kPa). Addition of 1 mM NH4Cl almost completely inhibited vesicle formation and acetylene-reducing activity, but did not immediately inhibit such reducing activity by cultures with preexisting vesicles. Acetylene-reducing activity was never observed in the absence of vesicle formation.     

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.     

Effects of water stress on nitrogenase activity in Abuts incana

Tolerance to water stress was studied in plants of grey alder, Alnus incana (L.) Moench, grown in a climate chamber in pots of sand supplied with a nitrogen-free nutrient solution. The plants were subjected to a single drying and recovery cycle, during which acetylene reduction, transpiration and stomatal resistance were measured. At different stress levels the plants were placed in a closed system to equilibrate the water potential in the plant-soil system. The water potential of the plants was determined, after which they were watered and their recovery studied. Nitrogenase activity showed low tolerance to water deficit. At moderate stress (−0.6 to −0.8 MPa) acetylene reduction was reduced by half, and at more severe stress, (< −1 MPa) activity was near zero. There was a rapid decrease in nitrogenase activity coincident with stomatal closure, which indicates a continuous need for photoassimilates for nitrogenase activity. Nodules or nitrogenase activity seemed to be weak sinks for assimilates compared with root pressure bleeding. Measurements of nitrogenase activity in root nodule homogenates supplied with ATP and reductant suggested a loss of active nitrogenase in the nodules in response to water stress. The recovery from moderate stress or long dark treatment took several days, and recovery from severe stress took still longer. Shortage of assimilates and disturbances in oxygen and nitrogen balances in the nodules are discussed as reasons for the reduced nitrogenase activity in response to water stress.     

A metabolic connection between nitrogenase activity and the synthesis of ureides in nodulated soybean

Application of allopurinol (AP; 1H-pyrazolo-[3,5- d ]pyrimidine-4-o1) to intact nodulated roots of ureide-forming legumes causes rapid inhibition of NAD:xanthine dehydrogenase (XDH: EC 1.2.1.37), cessation of ureide synthesis and, subsequently, severe nitrogen deficiency (Atkins et al. 1988. Plant Physiology 88: 1229–1234). Nitrogen deficiency is a result of inhibited nitrogenase (EC 1.7.99.2) activity. Using an open gas exchange system to measure H₂ and CO₂ evolution, short term effects of AP application were examined in a Hup ⁻ soybean symbiosis [ Glycine max (L.) Merr. cv. Harosoy: USDA 16]. The onset of inhibition of nitrogenase was detected after ca 2 h exposure of the roots to AP. At the same time xanthine began to accumulate and ureide levels declined in nodules as a result of inhibition of XDH. The decline in H₂ evolution following AP application was not due to altered electron allocation between N₂ and H⁺ by nitrogenease but was coincident with increased gaseous diffusive resistance of nodules and a decline in intracellular oxygen concentration. A possible scheme for the intermediary metabolism of soybean nodules which might account for a direct connection between nitrogenase activity and ureide synthesis is proposed. The suggested mechanism envisages coupling production of reducing power by cytosolic enzymes of purine oxidation to synthesis of dicarboxylic acid substrates (malate and succinate) required for bacteroid respiration.     

Frankia in the rhizosphere of nonhost plants: A comparison with root-associated N2-fixing Enterobacter,Klebsiella and Pseudomonas

Bacterial growth in the rhizosphere and resulting changes in plant growth parameters were studied in small aseptic seedlings of birch (Betula pendula and B. pubescens) and grasses (Poa pratensis and Festuca rubra). The seedlings were inoculated with three Frankia strains (Ai1a and Ag5b isolated from native Alnus root nodules and Ai17 from a root nodule induced by soil originating from a Betula pendula stand), and three associative N₂-fixing bacteria (Enterobacter agglomerans, Klebsiella pneumoniae and Pseudomonas sp., isolated from grass roots). Microscopic observations showed that all the Frankia strains were able to colonize and grow on the root surface of the plants tested without addition of an exogenous carbon source. No net growth of the associative N₂-fixers was observed in the rhizosphere, although inoculum viable counts were maintained over the experimental period. Changes in both the biomass and morphology of plant seedlings in response to bacterial inoculation were recorded, which were more dependent on the plant species than on the bacterial strain.     

Nitrogenase activity (acetylene reduction) associated with leaves of different cultivars of paddy (Oryza sativa L.)

Summary An investigation was conducted to study the levels of nitrogen fixation on the leaf or sheath surfaces of four cultivars of paddy plants by using acetylene reduction technique. Varying levels of positive nitrogenase activity were observed on all the leaf surfaces. Sheath of IET 1991 cultivar showed a higher rate of fixation than the leaf surface. All the nitrogen-fixing organisms on the leaf or sheath surfaces belonged to the genus Beijerinckia. There was no correlation between the bacterial density and the level of fixation. Scanning electron microscopic data revealed that the upper surface of IET 1991 leaf was highly silicified and the microflora was either scanty or nil while the lower surface appeared quite different and harboured more micro-organisms. Similarly, the inner surface of sheath was devoid of silicification and showed the presence of micro-organisms.     

Evidence for the occurrence of the alternative, vanadium-containing nitrogenase in the cyanobacterium Anabaena variabilis

Abstract Anabaena variabilis can be grown with dependence on either molybdenum (Mo) or vanadium (V) in the medium with essentially the same growth rates. Vanadium cultures reduce C₂H₂ to C₂H₄ and partly (to 2–3%) to C₂H₆. These C₂H₄ and C₂H₆ formations can be shown to be strictly light dependent, proving that the gases are formed by the cyanobacterium. C₂H₄ and C₂H₆ productions are accompanied by a H₂ formation which is much higher than in Mo cultures. Maximal C₂H₂-formation rates are 2/3 lower in V-grown cells compared to Mo control cultures. This is the first demonstration of a light-dependent ethane formation and of the occurrence of the alternative nitrogenase in any phototroph.     

An intact plant assay for estimating nitrogenase activity (C2H2 reduction) of sorghum and millet plants grown in pots

Summary A non destructive intact-plant assay for estimating nitrogenase activity (C₂H₂ reduction) of pot-grown sorghum and millet plants is described. Plants with intact shoots sustained more activity than plants whose tops were removed prior to the assay. With this technique individual plants can be assayed several times during their life cycle. The C₂H₂ reduction was linear up to 16h incubation in this assay procedure. More rapid diffusion of C₂H₂ was achieved by injection through a Suba seal in the bottom of the pot. The equlibration of injected C₂H₂ in the gas phase of the pots filled with sand and sand:FYM media was completed within 1 h. Significantly higher nitrogenase activity and better growth of sorghum and millet plants occurred when plants were grown in a mixture of sand and farmyard manure (FYM) than when plants were grown in vermiculite, soil, or sand + soil medium. Nitrogenase activity and plant growth were greater in a mixture of sand with 2 and 3% FYM than with 0.5 and 1% FYM. Activity was higher when the plants were incubated at 33°C and 40°C than at 27°C. Activity also increased with increasing soil moisture. There were significant differences amongst 15 sorghum cultivars screened for associated nitrogenase activity. This new technique has good prospects for screening cultivars of millet, sorghum and other grain crops for their nitrogen-fixing ability.Submitted as Journal article No. 358 by the International Crops Research Institute for the Semi-Arid Tropics (ICRISAT).     

Relationship between porewater organic carbon content, sulphate reduction and nitrogen fixation (acetylene reduction) in the rhizosphere of Zostera noltii

Depth profiles of nitrogen fixation (acetylene reduction), sulphate reduction, NH ₄ ⁺ concentration and porewater volatile fatty acids concentrations were measured in Zostera noltii colonised sediments in the Bassin d'Arcachon, France in March 1994. Acetylene reduction activity (ARA) was detectable throughout sediment profiles. Addition of sodium molybdate (20 mmol l^–1) a specific inhibitor of sulphate reduction to slurries inhibited ARA by >75% inferring that sulphate-reducing bacteria (SRB) were the dominant component of the nitrogen fixing microflora. The peak of ARA was coincident with that of sulphate reduction and a relatively constant relationship of 40 mole sulphate reduced per mole acetylene reduced was recorded throughout the profiles. From this ratio it was calculated that at least 17% of the ATP yield from sulphate reduction would be required to support the measured rates of nitrogen fixation (acetylene reduction).Acetate was the dominant constituent of the porewater volatile fatty acids pool, accounting for >90% of the total pool as measured by HPLC. Concentrations of porewater acetate recorded by HPLC were compared with those measured using an enzymatic technique and these data indicate that approximately 10% of the total porewater acetate pool was not available to microbial metabolism. Profiles of porewater acetate concentrations measured by both techniques were similar to those recorded for both ARA and sulphate reduction and thus acetate oxidation may fuel these activities.     

Characterization of an Azospirillum brasilense Tn5 mutant with enhanced N(2) fixation: the effect of ORF280 on nifH expression

Disruption of an open reading frame (ORF) of 840 bp (280 amino acids; ORF280) in an Azospirillum brasilense Tn5 mutant resulted in a pleiotrophic phenotype. Besides an enhanced N(2)-fixing capacity and altered expression pattern of a nifH-gusA fusion, growth on the charged polar amino acids glutamate and arginine was severely affected. ORF280, similar to previously identified ORFs present in Bradyrhizobium japonicum (ORF277), Paracoccus denitrificans (ORF278) and Rhodobacter capsulatus (ORF277), exhibits in its C-terminus a significant similarity with the recently defined family of universal stress proteins.     

Effect of lime additions to acid strip-mine spoil on survival,growth and nitrogen fixation (acetylene reduction) of several woody legume and actinomycete-nodulated species

Summary An acid mine spoil in Southern Indiana was amended with lime (CaCO₃) (0.0, 12.5, 25 and 39t/ha) and planted withElaegnus umbellata Thunb.,Alnus glutinosa Gaertn.,Robinia pseudoacacia L.,Robinia fertilis Ashe, Arnot,Myrica pensylvania Lois,Caragana arborescens L. andShepherdia argentea Nutt. Survival and soil data were collected periodically and plants were harvested 15 months after planting. Nodule and top dry weights were determined and acetylene reduction assays performed on the nodules.Addition of lime caused significant increases in pH, and 39 t/ha of lime were required to maintain a pH above 5.5. Survival of plant material was greatest at the highest lime addition, although response of individual species varied.Elaeagnus umbellata, R. pseudoacacia, R. fertilis Arnot, andA. glutinosa appeared more tolerant of the harsh conditions. OnlyC. arborescens showed a linear increase in top dry weight due to lime addition.Alnus glutinosa andS. argentea achieved statistically the same growth regardless of pH, andR. fertilis Arnot andE. umbellata did not increase in top dry weight above an addition of 25 t/ha.Robinia pseudoacacia achieved maximum top dry weight at 25 t/ha, whereasM. pensylvanica growth declined with increasing pH. Nodule dry weights increased with increasing pH; however,S. argentea showed greater nodule dry weights at lower lime levels. Acetylene reduction rates increased with lime addition.Elaegnus umbellata did not respond above 25 t/ha lime, whereasA. glutinosa did not show an increase until this point.