Growth and nitrogenase activity of Azotobacter vinelandii in the presence of several phenolic acids

Growth and nitrogenase activity (acetylene reduction) of Azotobacter vinelandii in chemically defined N-free media were studied in the presence of p-hydroxybenzoic, vanillic, p-coumaric, and ferulic acids at concentrations from 0.01 to 1% (w/v). Growth and nitrogenase activity were only detected when the microorganism was cultured on p-hydroxybenzoic acid either as sole carbon source or mixed with other phenolic acids, suggesting that p-hydroxybenzoic acid could be utilized as a carbon source by A. vinelandii for growing under certain environmental conditions.     

Nitrogen fixation inpara-nodules of wheat roots by introduced free-living diazotrophs

Nitrogen-fixation (C₂H₂-reduction) was demonstrated in wheat root nodules (p-nodules) induced by 2,4-dichlorophenoxyacetate (2,4-D) and inoculated withA. brasilense. By lowering the O₂ tension it was possible to distinguish the nitrogenase activity of bacteria located within thep-nodule of the wheat root system from that in the rhizosphere. Using cytological evidence, nitrogenase activity was attributed mainly to be coming from the bacteria within thep-nodule. It was also shown that the host plant was able to supply the necessary substrate required for the bacterial N₂-fixation (C₂H₂-reduction) within thep-nodules.     

Analysis of factors affectingin situ nitrogenase (C2H2) activity ofGalega orientalis,Trifolium pratense andMedicago sativa in temperate conditions

Summary A practical fibreglass cylinder-plastic bag system has been designed for making acetylene reduction assays in the field. Thein situ assay was used to determine seasonal patterns of nitrogenase activity for the perennial forage legumesGalega orientalis, Trifolium pratense andMedicago sativa grown under stadard management in southern Filand (60° north). Nitrogenase activity was still detected in the field plots in November, when soil temperature was 1.5°C and air temperature 0.5°C. The acetylene reduction data from weekly measurements were analyzed for correlation with plant growth rate and short-term fluctuations of environmental factors. Generally, there was a good correlation between nitrogenase activity and plant growth rate. Residual fluctuations in activity were only correlated with environmental factors in one case. The nitrogenase activity ofM. sativa was dependent on air temperature in addition to growth rate. Thus, the nitrogen fixing systems in these forage legumes seem to be an integrated part of the plants, being fairly insensitive to short-term environmental changes.Dedicated to Prof. Helge Gyllenberg on the occasion of his 60th birthday.     

Acetylene reduction activity of pearl millet inbred lines grown in soil

Pearl millet inbred lines previously selected for differences in acetylene reduction activity (ARA) in seedling agar tubes were found to support low soil ARA. Lines selected for high ARA gave better performance than lines selected for low ARA. None of the high ARA lines was found to approach the ARA of alfalfa with their average rates over 40 fold lower.     

Expression ofKlebsiella pneumoniae nif genes inProteus mirabilis

Self-transmissible plasmids carryinghis andnif genes fromKlebsiella pneumoniae have been introduced into threehis mutants ofProteus mirabilis: strains 5006-1, WR19 and WR20. Expression ofhis by the transconjugants was unequivocal, if slightly temperature-sensitive, but none was Nif⁺ when tested for acetylene reduction in anaerobic glucose medium using inocula from rich or glucose-minimal aerobic agar cultures. Succinate or pyruvate in place of glucose, low glucose, lower temperature or elevated Na₂MoO₄ did not allownif expression and no nitrogenase MoFe-protein peptide was detected immunologically after exposure to conditions in which diazotrophic enterobacteria, normal or genetically constructed, derepressnif.One strain,P. mirabilis WR19, carrying thehis nif Km^r plasmid pMF250 was examined in detail. Thenif activator genenifA was introduced on the plasmid pCK1. Such derivatives remained Nif^- when tested, after aerobic growth on rich agar media, with normal or low glucose, with succinate or with elevated Mo. However, pre-conditioning by aerobic growth on glucose-minimal agar led to subsequent anaerobic expression ofnif in glucose medium from pMF250 in WR19 carrying pCK1. NH ₄ ⁺ or proline could serve as N-source in the glucose-minimal agar. Maximum activity was about 5% of that ofK. pneumoniae in our assay conditions. Material cross-reacting with anti-serum to the nitrogenase MoFe protein was formed. Nitrogenase activity was not switched off by NH ₄ ⁺ .P. mirabilis WR19 (pCK1) showed NH ₄ ⁺ -constitutive temperature-sensitive kanamycin resistance (anif-related phenotype of this plasmid) in aerobic glucose minimal medium. Expression ofnif inP. mirabilis WR19 (pCK1, pMF250) was NH ₄ ⁺ -repressible despite the constitutivenifA character of pCK1 and introduction of thentrA ⁺ plasmid pMM17 did not alter this phenotype. However, pCK1 did not give rise to NH ₄ ⁺ -constitutive diazotrophy in the wild-typeK. pneumoniae M5al. A construct of WR19 carrying pMF250 and constitutiventrC plasmid (pMD45) remained Nif^- even after pre-growth on glucose-minimal media.We conclude (a) thatP. mirabilis forms a gene product functionally equivalent to that ofntrA inK. pneumoniae, (b) that it forms no functional equivalent of thentrC product in our growth conditions. The need for pre-conditioning on aerobic glucose media remains perplexing.Non-common abbreviation NFDM Nitrogen-free-Davis-Mingioli medium     

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.     

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.     

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.     

Biological Nitrogen Fixation is not a Major Contributor to the Nitrogen Demand of a Commercially Grown South African Sugarcane Cultivar

It has previously been reported that endophytic diazotrophic bacteria contribute significantly to the nitrogen budgets of some graminaceous species. In this study the contribution of biological nitrogen fixation to the N-budget of a South African sugarcane cultivar was evaluated using ¹⁵N natural abundance, acetylene reduction and ¹⁵N incorporation. Plants were also screened for the presence of endophytic diazotrophic bacteria using acetylene reduction and nifH-gene targeted PCR with the pure bacterial strains. ¹⁵N natural abundance studies on field-grown sugarcane indicated that the plants did not rely extensively on biological nitrogen fixation. Furthermore, no evidence was found for significant N₂-fixation or nitrogenase activity in field-grown or glasshouse-grown plants using ¹⁵N incorporation measurements and acetylene reduction assays. Seven endophytic bacterial strains were isolated from glasshouse-grown and field-grown plants and cultured on N-free medium. The diazotrophic character of these seven strains could not be confirmed using acetylene reduction and PCR screening for nifH. Thus, although biological nitrogen fixation may occur in South African sugarcane varieties, the contribution of this N-source in the tested cultivar was not significant.     

Cinnamate toxicity expression on phenylalanine ammonia-lyase activity,germination and growth of cucumber (Cucumis sativis) seedlings

Low pH (5.2) decreased nodule number and acetylene reduction. Aluminium further depressed those parameters in theRhizobium leguminosarum-Pisum sativum associations examined. In the Al-treated plants nodule formation by strains 128C53 and 128C30 was not affected by 3 or 15 and 30 or 60 μM Al, respectively, as compared with the number of nodules on plants grown at pH 5.2 in the absence of Al. However, improved nodulation rates by those strains did not enhance plant dry weight or reduced nitrogen content. No differences in nitrogenase activity were found among strains of nodulating plants grown at the same aluminium level. These results suggest that Al-ions affected specifically nitrogenase activity and that this effect was primarily responsible for the reduction in plant growth.     

Characteristics of nitrogen-fixingKlebsiella oxytoca isolated from wheat roots

Summary Of 45 fermentative gram negative bacterial isolates examined from wheat roots, three were capable of fixing atmospheric nitrogen as determined by the acetylene reduction technique and by protein contents of cells. A gram negative non-motile facultatively anaerobic bacterial strain capable of N₂ fixation was identified asKlebsiella oxytoca ZMK-2.Optimal growth and N₂ fixation occurred at pH 6.5. The optimum temperatures for growth under anaerobic conditions ranged between 30°–37°C. Acetylene reduction by intact cells was strikingly inhibited by 0.1 atm. or greater partial pressure of O₂. Furthermore, the accumulation of H₂ in the gas phase over cultures ofKlebsiella oxytoca ZMK-2 at partial pressures greater than 0.02 atm. resulted in a striking inhibition in the rate of C₂H₂ reduction. The addition of suspensions of eitherKlebsiella oxytoca ZMK-2 orAzotobacter vinelandii or a mixed culture of these two organisms to axenic cultures of wheat plants produced no significant increase in plant growth as measured by plant dry weight or nitrogen content of plants.     

Can mushrooms fix atmospheric nitrogen?

It is generally reported that fungi likePleurotus spp. can fix nitrogen (N₂). The way they do it is still not clear. The present study hypothesized that only associations of fungi and diazotrophs can fix N₂. This was testedin vitro. Pleurotus ostreatus was inoculated with a bradyrhizobial strain nodulating soybean andP. ostreatus with no inoculation was maintained as a control. At maximum mycelial colonization by the bradyrhizobial strain and biofilm formation, the cultures were subjected to acetylene reduction assay (ARA). Another set of the cultures was evaluated for growth and nitrogen accumulation. Nitrogenase activity was present in the biofilm, but not when the fungus or the bradyrhizobial strain was alone. A significant reduction in mycelial dry weight and a significant increase in nitrogen concentration were observed in the inoculated cultures compared to the controls. The mycelial weight reduction could be attributed to C transfer from the fungus to the bradyrhizobial strain, because of high C cost of biological N₂ fixation. This needs further investigations using¹⁴C isotopic tracers. It is clear from the present study that mushrooms alone cannot fix atmospheric N₂. But when they are in association with diazotrophs, nitrogenase activity is detected because of the diazotrophic N₂ fixation. It is not the fungus that fixes N₂ as reported earlier. Effective N₂ fixing systems, such as the present one, may be used to increase protein content of mushrooms. Our study has implications for future identification of as yet unidentified N₂ systems occurring in the environment.     

The physiology of spore-negative and spore-positive nodules ofMyrica gale

The physiology of spore-negative and spore-positive root nodules was investigated inMyrica gale L. grown in water culture in a growth chamber. Spore(–) nodules were induced withFrankia cultures and spore(+) nodules with crushed nodules. Gas exchange was measured in a flow-through system.The time course of acetylene reduction following addition of acetylene was essentially the same in both spore(–) and spore(+) nodules with a stable maximum between 2 and 4 minutes followed by a steep decline to a minimum (37% of the maximum) between 9 and 30 minutes depending on the plant. The minimum was followed by a partial recovery. Nodule CO₂ evolution showed a similar pattern but the minimum rate (83% of the maximum) was not nearly as low.Plants nodulated with one spore(–) and one spore(+) strain were compared at 6, 8 and 10 weeks after inoculation. At 6 weeks the spore(–) plants had 52% greater specific nitrogenase activity and 46% more biomass than the spore(+) plants. At 8 and 10 weeks, however, the differences between plants with spore(–) and spore(+) nodules became smaller.Plants nodulated with 4 spore(–) and 5 spore(+) strains were compared at 8 weeks after inoculation. Collectively the spore(–) plants exhibited a 32% greater specific nitrogenase activity, a 15% lower energy cost of nitrogenase activity (CO₂/C₂H₄), and invested 31% less biomass in nodules than the spore(+) plants. The spore(–) plants also produced 16% more biomass indicating that spore(–) strains are generally more desirable than spore(+) strains. However, two spore(+) strains were as effective as the spore(–) strains.     

Effect of seed inoculation,mycorrhizal infection and organic amendment on wheat growth

Summary The effect of seed inoculation withAzotobacter spp. orAzospirillum spp., and garbage amendment (0.5%), on the growth of wheat was studied in a field experiment under sub-tropical conditions. Two levels of N fertilizer were applied, the usual field rate (150 kg N ha^–1) and half this amount. Tillering of plants, dry matter contents and nitrogenase activity were determined 30, 60 and 90 days after sowing. At the end of the experimental period, spore numbers and percentage of mycorrhizal infection were observed in the rhizosphere and root systems of plants. Straw and grain yields were also determined. The results of this study showed that seed inoculation and/or organic amendment stimulated plant growth, nitrogenase activity and mycorrhizal infection. This was more noticeable withAzotobacter than withAzospirillum. Inoculation withAzotobacter together with 1/2 N dose and organic amendment was the most effective application (19.75 and 10.70 t ha^–1 were recorded for straw and grain yield, respectively).     

Immediate acetylene reduction by excised grass roots not previously preincubated at low oxygen tensions

Excised roots of Spartina alterniflora Loisel. and corn reduced acetylene in air without the previously reported period of zero activity lasting 8 to 18 hours. The profiles of acetylene-dependent ethylene accumulation by excised roots and intact plants of S. alterniflora were similar. No significant change in the number of bacteria associated with the roots was detectable during the assay. Most of the nitrogenase activity was detected in the roots and rhizomes of the plants. The salt marsh sediment also was capable of reducing acetylene. Additional damage to roots by washing and cutting increased the rate of acetylene reduction with samples incubated in air. Low concentrations of nitrate significantly inhibited the nitrogenase activity associated with the sediment and excised roots, but not with intact plants. Rates of acetylene reduction by excised corn roots were low. Oxidation and endogenous production of ethylene in the absence of acetylene were negligible. Measurements made with excised grass roots as described probably reflect the occurrence and magnitude of nitrogenase activity associated with the plants in the field.     

Contribution of basal portion of shoot to N2 fixation associated with wetland rice

Summary Oryza sativa grown in flooded soil were transferred to water culture solution and acetylene reduction activities (ARA) of intact plants and rootless plants were measured for 5 h. Relative rate of ARA associated with the rootless wetland rice plant as compared with an intact plant varied from 8 to 100 percent, depending on the growth stage and varieties of rice and highest at the early stage (3 weeks after transplanting) for all varieties tested (IR26, Latisail, Khao Lo, and JBS236). ARA of shoots was associated with basal parts of the shoots about 3 cm from the base of wetland cultivated rice andOryza australiensis. Phyllospheric ARA was negligible except for senescent outer leaf sheaths. Microaerophilic N₂-fixing bacteria also inhabited basal parts of shoots (outer leaf sheaths and stems) of wetland rice. These findings suggest that N₂-fixation is partly associated with the shoots of wetland rice plants.     

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.     

木麻黄和桤木离体根瘤和立地土壤的固氮酶与N2O还原酶活性的研究

为了解非豆科固氮树种的固氮酶和N_2O还原酶(Nos)活性,采用乙炔还原法和乙炔抑制技术对细枝木麻黄(Casuarina cunninghamiana)和江南桤木(Alnus trabeculosa)离体根瘤及立地土壤的两种酶活性进行了研究。结果表明,离体根瘤只在厌氧条件下有固氮酶活性,在好氧条件下有Nos活性。根瘤区根际土和非根瘤区根际土的固氮酶活性在好氧条件大于厌氧条件,Nos活性只表现在厌氧条件下。在好氧条件下,根瘤区根际土和非根瘤区根际土的固氮酶活性无显著差异;根瘤区根际土的Nos活性显著大于非根瘤区根际土。除离体根瘤在好氧条件下不表现固氮酶活性外,细枝木麻黄和桤木的离体根瘤、根瘤区根际土和非根瘤区根际土的固氮酶活性均都大于Nos活性。好氧条件下根瘤区根际土的固氮酶活性与非根瘤区根际土的呈极显著正相关,而厌氧条件下根瘤的固氮酶活性与好氧条件下根瘤区根际土和非根瘤区根际土固氮酶活性、好氧条件下根瘤的Nos活性与厌氧条件下根瘤区根际土和非根瘤区根际土Nos活性均呈极显著负相关。这为研究弗兰克氏菌结瘤植物共生固氮体系对N2O汇强度的影响和调控奠定基础。     

Ammonia inhibition of nitrogenase activity in purple and green bacteria

Ammonia reversibly inhibits the nitrogenase activity not only in purple nonsulfur bacteria but in purple (Thiocapsa roseopersicina) and green (Chlorobium limicola forma thiosulfatophilum) sulfur bacteria as well.The complete inhibition of nitrogenase activity (acetylene reduction) is observed about 30 s after addition of NH ₄ ⁺ (2.5×10^-6 M) to cell suspensions. The pattern of ammonia inhibition of acetylene reduction in T. roseopersicina does not differ from the action of tetrabutylammonium and tetraphenylphosphonium (3 · 10^-6-5·10^-5 M) on nitrogenase activity of this bacterium.Simultaneously with the switch-off effect of NH ₄ ⁺ a considerable increase of ATP in cells of Rhodobacter sphaeroides and C. limicola f. thiosulphatophilum was observed.     

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.