Transfer of fixed-N from N2-fixing cyanobacteria associated with the moss Sphagnum riparium results in enhanced growth of the moss

Background

Despite the general assumption that nitrogen fixed by associated cyanobacteria will be readily utilised for growth by the Sphagnum, no empirical evidence is available in the literature. Therefore the effects of nitrogen transfer from cyanobacteria associated with S. riparium were investigated.

Methods

Cultivation of S. riparium with and without cyanobacteria was performed under laboratory conditions for 57 days.

Results

We show that nitrogen fixation by cyanobacteria associated with Sphagnum mosses, influences moss growth by transfer of fixed nitrogen to the moss. More than 35 % of the nitrogen fixed by cyanobacteria was transferred to the newly formed moss biomass and resulted in an increase in the growth of Sphagnum biomass compared to the controls. The variation in the increase of nitrogen content explained 76 % of the biomass increment.

Conclusion

Hence, nitrogen fixation will have immediate effect on the carbon fixation by Sphagnum. This shows that factors regulating nitrogen fixation will have a direct effect on the role of Sphagnum dominated ecosystems with respect to carbon cycling.     

The uptake and metabolism of methylamine by N2-fixing cyanobacteria

The cyanobacteria Anabaena variabilis and Nostoc CAN showed a biphasic pattern of ¹⁴CH₃NH ₃ ⁺ uptake at external pH values of 7.0 and 9.0. The initial phase of uptake, which was independent of metabolism of ¹⁴CH₃NH ₃ ⁺ , was attributed to uptake via a CH₃NH ₃ ⁺ (NH ₄ ⁺ ) transport system at pH 7.0 and probably to passive diffusion of uncharged CH₃NH₂ and trapping by protonation at pH 9.0. The second slower phase of uptake was attributed to metabolism of CH₃NH ₃ ⁺ via glutamine synthetase to form -methylglutamine which accumulates. Anabaena cylindrica showed an initial rapid uptake at pH 7.0 and pH 9.0 but metabolism of ¹⁴CH₃NH ₃ ⁺ was undetectable at pH 7.0 and was barely detectable at pH 9.0. Pretreatment of A. variabilis with l-methionine-d,l-sulphoximine to inactivate glutamine synthetase, inhibited the second phase of ¹⁴CH₃NH ₃ ⁺ uptake at both pH 7.0 and pH 9.0 and the accumulation of -methylglutamine but had no effect on the first phase of uptake. Following transfer of A. variabilis to darkness the initial phase of ¹⁴CH₃NH ₃ ⁺ uptake at pH 7.0 and 9.0 was unaffected but the subsequent metabolism via glutamine synthetase was inhibited.Abbreviations MSX l-methionine-d,l-sulphoximine - GS glutamine synthetase     

Photoproduction of amino acids by mutant strains of N2-fixing cyanobacteria

Summary Mutant strains of the N₂-fixing cyanobacterium bacterium Anabaena variabilis resistant to 6-fluorotryptophan or to ethionine were isolated. Many of these strains liberated amino acids into their media in the absence of 6-fluorotryptophan and ethionine. Nitrogenase activity was higher in mutant strains than in the parent strain. Mutant strains were immobilised in calcium alginate and sustained photoproduction of amino acids has been demonstrated.Abbreviations ETH ethionine - FT 6-fluorotryptophan - Hepes 4-(2-hydroxyethyl)-1, piperazine ethanesulphonic acid - PEP phosphoenolpyruvate - DAHP 3-deoxy-d-arabinoheptulosonate 7-phosphate - chl a chlorophyll a     

The nitrogen physiology of the marine N2-fixing cyanobacteria Trichodesmium spp

Trichodesmium spp. have proved to be enigmatic organisms, and their ecology and physiology are unusual among diazotrophs. Recent research shows that they can simultaneously fix N2 and take up combined nitrogen. The co-occurrence of these two processes is thought to be incompatible, but they could be obligatory in Trichodesmium spp. if only a small fraction of cells within a colony or along a filament are capable of N2 fixation. Combined nitrogen is released from cells during periods of active growth and N2 fixation, and concomitantly taken up by Trichodesmium spp. or cells living in association with colonies. Although the nitrogenase of Trichodesmium spp. is affected by high concentrations of combined nitrogen, it might be relatively less sensitive to low concentrations of combined nitrogen typical of the oligotrophic ocean and culture conditions. Nitrogenase activity and synthesis exhibits an endogenous rhythm in Trichodesmium spp. cultures, which is affected by the addition of nitrogen.     

Relationship between abundance of N2-fixing cyanobacteria and environmental features of Spanish rice fields

In order to estimate the potential utilization of N₂-fixing (heterocystous) cyanobacteria as natural biofertilizers in the Valencian rice fields (Spain), the distribution and seasonal variation of these microorganisms in water and sediment samples were evaluated, and the relationships among cyanobacterial abundance and physical and chemical characteristics of soil and water were investigated. N₂-fixing cyanobacteria were present in all the samples analyzed (25 sampling points sampled three times per year during two years). The relative cyanobacterial abundance in soil and water followed contrasting patterns, maximum presence in soil coincided with minimum abundance in water. Correlation analysis showed that cyanobacterial abundance in the two phases (water and sediment) was influenced more by water than by soil properties. Salinity, mineralization variables, and soluble reactive phosphate (SRP) correlated positively with heterocystous cyanobacteria presence. Furthermore, dissolved inorganic nitrogen (DIN) and the ratio DIN: SRP correlated negatively with cyanobacterial abundance. However DIN: SRP ratio better described the cyanobacterial distribution, with a threshold effect: below the Redfield ratio value (7.2 in mass units) cyanobacterial abundance was clearly higher. Correspondence to: A. Quesada.     

Physiological responses of the freshwater N2-fixing cyanobacterium Raphidiopsis raciborskii to Fe and N availabilities

The cyanobacterium Raphidiopsis raciborskii is of environmental and social concern in view of its toxicity, bloom-forming characteristics and increasingly widespread occurrence. However, while availability of macronutrients and micronutrients such as N and Fe are critically important for the growth and metabolism of this organism, the physiological response of toxic and non-toxic strains of R. raciborskii to varying Fe and N availabilities remains unclear. By determining physiological parameters as a function of Fe and N availability, we demonstrate that R. raciborskii growth and N₂-fixing activity are facilitated at higher Fe availability under N₂-limited conditions with faster growth of the CS-506 (cylindrospermopsin-producing) strain compared with that of CS-509 (the non-toxic) strain. Radiolabelled Fe uptake assays indicated that R. raciborskii acclimated under Fe-limited conditions acquires Fe at significantly higher rates than under Fe replete conditions, principally via unchelated Fe(II) generated as a result of photoreduction of complexed Fe(III). While N₂-fixation of both strains occurred during both day and night, the CS-506 strain overall exhibited higher N₂-fixing and Fe uptake rates than the CS-509 strain under N-deficient and Fe-limited conditions. The findings of this study highlight that Fe availability is of significance for the ecological advantage of CS-506 over CS-509 in N-deficient freshwaters.     

N2-fixing cyanobacteria: Why they do not become dominant in shallow hypertrophic lakes

Summary Phytoplankton species shifts and succession phenomenona in lakes of increasing trophic state were considered, using the basic information on the growth kinetics of the species involved. One of the most obvious signs of advanced eutrophication is the dominance of cyanobacteria (blue-green algae). Striking examples are the shallow, hypertrophic Dutch lakes The Veluwerandmeren (e.g., Wolderwijd and Veluwemeer), whereOscillatoria agardhii, a non-N₂-fixing cyanobacterium, has become dominant over the green algae, diatoms and N₂-fixing cyanobacteria (BERGER, 1975).We have studied the natural population ofO.agardhii during the growing season, by using physiological indicators, and could adduce that the natural population was successively growing under phosphorus, light, or nitrogen limitation (ZEVENBOOM and MUR, 1978a,b; ZEVENBOOMet al., 1982). One might expect that during the period of nitrogen limitation the N₂-fixing speciesAphanizomenon flos-aquae would be favoured and would be able to outgrow the nitrogen-limitedO.agardhii. However, in these lakes,A. flos-aquae was present only in few numbers and a succession fromO. agardhii toA. flos-aquae did not occur. Although field observations may give some indication, they cannot give decisive answers to the question which factor is triggering the observed species shifts and species dominance in natural waters. Such answers can only be obtained from growth kinetic and physiological data of the species involved. In our opinion, the most important factor to consider is the availability of light energy, which decreases with increasing eutrophication.The hypothesis was proposed by Mur and coworkers (MURet al., 1978) that in hypertrophic lakes the prevailing light conditions (low light irradiance) are more favourable forO.agardhii, since this species has a much lower requirement of light energy for growth than green algae as a consequence of its lower specific maintenance rate constant, _e (VAN LIERE, 1979; GONS, 1977). Competition experiments, performed withO. agardhii andScenedesmus protuberans under lightlimiting conditions, confirmed the hypothesis (MURet al., 1978), Continuous culture experiments withA. flos-aquae showed that also this species had a higher energy requirement thanO. agardhii (ZEVENBOOM, 1980). The differences were not found in the value of _e, but in the growth efficiency. The higher energy requirement ofA.flos-aquae was expected, since energy is needed for heterocyst production and N₂ fixation. Under light-limiting conditions and nutrient sufficiency (including nitrogen-nitrate) it can thus be expected that the N₂-fixer will be outcompeted by the non-N₂-fixing cyanobacterium. This was indeed observed (ZEVENBOOM et al., 1981).We further investigated the competitive interactions betweenA.flos-aquae, O. agardhii andS. protuberans under different sets of irradiance values and nitrate concentrations. We used the growth kinetic data of the species involved, which were obtained by means of continuous culture experiments (GONS, 1977; VAN LIERE. 1979; VAN LIERE and MUR, 1979; GONS and MUR, 1980; ZEVENBOOM and MUR, 1980; ZEVENBOOMet al., 1980; ZEVENBOOMet al., 1981). The competing species could be placed along the gradients of light irradiance values and nitrate concentrations, their positions being defined by their energy requirements and half-saturation constants for nitrate-limited growth, respectively. Distinct niches for the three species were found with respect to light and nitrate. Under conditions of low irradiance values and low (realistic) nitrate concentrations, nitrogen-limitedO.agardhii was able to outgrowA. flos-aquae andS. protuberans as a consequence of its low energy requirement and its high affinity for nitrate. The growth rates of the last two species were restricted by the limited availability of light. However, at high irradiance values,O.agardhii was inhibited in its growth rate and therefore failed to outgrow the other two species. The competition was then restricted to nitrogen-limitedS.protuberans and light-limitedA.flos-aquae; the latter could dominate at low nitrate concentrations. The results of competition experiments withO.agardhii andA.flos-aquae under different sets of irradiance values and nitrate concentrations agreed well with the niche-model described above (Zevenboom, unpubl. results).In conclusion, kinetic data of growth, obtained with continuous culture experiments, can provide basic information to explain species shifts and dominance in lakes with increasing eutrophication. Nitrogen-limiting conditions favour N₂-fixing cyanobacteria only when sufficient light is available for their growth (in less hypertrophic waters). The trophic state is thus of major importance and decisive with regard to which species will dominate.     

Remarkable N2-fixing bacterial diversity detected in rice roots by molecular evolutionary analysis of nifH gene sequences.

To demonstrate the extent of phylogenetic diversity of diazotrophic bacteria associated with rice roots, we characterized phylogenetically 23 nifH gene sequences obtained by PCR amplification of mixed organism DNA extracted directly from rice roots without culturing the organisms. The analyses document the presence of eight novel NifH types, which appear to be a variety of significant components of the diazotrophic community, dominated mainly by proteobacteria.     

Natural 15N abundance of presumed N2-fixing and non-N2-fixing plants from selected ecosystems

Summary The ¹⁵N/¹⁴N ratios of plant and soil samples from Northern California ecosystems were determined by mass spectrometry. The ¹⁵N abundance of 176 plant foliar samples averaged 0.0008 atom % ¹⁵N excess relative to atmospheric N₂ and ranged from-0.0028 to 0.0064 atom % ¹⁵N excess relative to atmospheric N₂. Foliage from reported N₂-fixing species had significantly lower mean ¹⁵N abundance (relative to atmospheric N₂ and total soil N) and significantly higher N concentration (% N dry wt.) than did presumed non-N₂-fixing plants growing on the same sites. The mean difference between N₂-fixing species and other plants was 0.0007 atom % ¹⁵N. N₂-fixing species had lower ¹⁵N abundance than the other plants on most sites examined despite large differences between sites in vegetation, soil, and climate. The mean ¹⁵N abundance of N₂-fixing plants varied little between sites and was close to that of atmospheric N₂. The ¹⁵N abundance of presumed non-N₂-fixing species was highest at coastal sites and may reflect an input of marine spray N having relatively high ¹⁵N abundance. The ¹⁵N abundance of N₂-fixing species was not related to growth form but was for other plants. Annual herbaceous plants had highest ¹⁵N abundance followed in decreasing order by perennial herbs, shrubs, and trees. Several terrestrial ferns (Pteridaceae) had ¹⁵N abundances comparable to N₂-fixing legumes suggesting N₂-fixation by these ferns. On sites where the ¹⁵N abundance of soil N differs from that of the atmosphere, N₂-fixing plants can be identified by the natural ¹⁵N abundance of their foliage. This approach can be useful in detecting and perhaps measuring N₂-fixation on sites where direct recovery of nodules is not possible.     

Response of n(2)-fixing cyanobacteria to salt

The effect of salt on photosynthetic activity, acetylene reduction, and related activities was examined in two species of cyanobacteria, Nostoc muscorum and Calothrix scopulorum. Photosynthesis was more resistant to high salt concentration than was N(2) fixation. The salt resistance of both activities increased after a period of exposure of the cells to salinity. The transfer of electrons via ferredoxin and ferredoxin-nicotinamide adenine dinucleotide phosphate reductase was found to be extremely sensitive to salt. In comparison, the transfer of reducing power by glucose-6-phosphate dehydrogenase, isocitric dehydrogenase, and photosystem 1 was less affected by NaCl, whereas glutamine synthetase exhibited higher tolerance to salt.     

Simultaneous occurrence of two different glyceraldehyde-3-phosphate dehydrogenases in heterocystous N(2)-fixing cyanobacteria

Enzyme activity determinations and Western and Northern blot analyses have shown the presence of two catalytically different glyceraldehyde-3-phosphate dehydrogenases (GAPDH) in both vegetative cells and heterocysts of several N(2)-fixing Anabaena strains: (a) the gap2-encoded NAD(P)-dependent GAPDH2 (EC 1.2.1.59), the enzyme involved in the photosynthetic carbon assimilation pathway, which is present at higher levels in vegetative cells, and (b) the gap3-encoded NAD-dependent GAPDH3 (EC 1.2.1.12), presumably involved in carbohydrate anabolism and catabolism, which is the predominant GAPDH in heterocysts. In contrast, the gap1-encoded GAPDH1, which is the other NAD-dependent cyanobacterial GAPDH, is virtually absent in both cell types. These findings are discussed in the context of carbon metabolism of heterocystous N(2)-fixing cyanobacteria.     

Response of feather moss associated N2 fixation and litter decomposition to variations in simulated rainfall intensity and frequency

Feather mosses in boreal forests form a dense ground‐cover that is an important driver of both nutrient and carbon cycling. While moss growth is highly sensitive to moisture availability, little is known about how moss effects on nutrient and carbon cycling are affected by the dynamics of moisture input to the ecosystem. We experimentally investigated how rainfall regimes affected ecosystem processes driven by the dominant boreal feather moss Pleurozium schreberi by manipulating total moisture amount, frequency of moisture addition and moss presence/absence. Moisture treatments represented the range of rainfall conditions that occur in Swedish boreal forests as well as shifts in rainfall expected through climate change. We found that nitrogen (N) fixation by cyanobacteria in feather mosses (the main biological N input to boreal forests) was strongly influenced by both moisture amount and frequency, and their interaction; increased frequency had greater effects when amounts were higher. Within a given moisture amount, N fixation varied up to seven‐fold depending on how that amount was distributed temporally. We also found that mosses promoted vascular litter decomposition rates, concentrations of litter nutrients, and active soil microbial biomass, and reduced N release into soil solution. These effects were usually strongest under low moisture amount and/or frequency, and revealed a buffering effect of mosses on the decomposer subsystem under moisture limitation. These results highlight that both the amount and temporal distribution of rainfall, determine the effect of feather mosses on ecosystem N input and the decomposer subsystem. They also emphasize the role of feather mosses in mediating moisture effects on decomposer processes. Finally, our results suggest that projected shifts in precipitation in the Swedish boreal forest through climate change will result in increased moss growth and N₂ fixation but a reduced dependency of the decomposer subsystem on feather moss cover for moisture retention.     

Composition and diversity of nifH genes of nitrogen-fixing cyanobacteria associated with boreal forest feather mosses

Recent studies have revealed that nitrogen fixation by cyanobacteria living in association with feather mosses is a major input of nitrogen to boreal forests. We characterized the community composition and diversity of cyanobacterial nifH phylotypes associated with each of two feather moss species (Pleurozium schreberi and Hylocomium splendens) on each of 30 lake islands varying in ecosystem properties in northern Sweden. Nitrogen fixation was measured using acetylene reduction, and nifH sequences were amplified using general and cyanobacterial selective primers, separated and analyzed using density gradient gel electrophoresis (DGGE) or cloning, and further sequenced for phylogenetic analyses. Analyses of DGGE fingerprinting patterns revealed two host-specific clusters (one for each moss species), and sequence analysis showed five clusters of nifH phylotypes originating from heterocystous cyanobacteria. For H. splendens only, N(2) fixation was related to both nifH composition and diversity among islands. We demonstrated that the cyanobacterial communities associated with feather mosses show a high degree of host specificity. However, phylotype composition and diversity, and nitrogen fixation, did not differ among groups of islands that varied greatly in their availability of resources. These results suggest that moss species identity, but not extrinsic environmental conditions, serves as the primary determinant of nitrogen-fixing cyanobacterial communities that inhabit mosses.     

腾格里沙漠东南缘藻结皮与藓结皮放线菌多样性及其潜在代谢功能

放线菌作为干旱、半干旱环境中生物土壤结皮(Biological Soil Crusts,BSCs)组成的重要生命存在形式之一,不仅是潜在临床有用天然产物化学多样性的重要来源,也是该生态系统物质循环与能量流动的重要参与者。以腾格里沙漠东南缘广泛分布的藻结皮和藓结皮为研究对象,通过宏基因组测序比较分析两种BSCs放线菌种群的分布特征、组成及其潜在代谢功能。结果表明,腾格里沙漠东南缘藻结皮与藓结皮土壤微生物组主要形成以地嗜皮菌属、红色杆菌属、类诺卡氏菌属、游动放线菌属、芽生球菌属、链霉菌属、贫养杆菌属、糖丝菌属、土壤红杆菌属、假诺卡氏菌属、小单孢子菌属、康奈斯氏杆菌属、大理石雕菌属、小月菌属以及弗兰克氏菌属等为主要类群的放线菌群落结构,在两种BSCs类型之间各属分布存在差异。藓结皮中放线菌参与的氨基糖与核苷酸糖代谢、原核生物中的碳固定途径、丁酸代谢、丙酸代谢、丙氨酸/天门冬氨酸和谷氨酸代谢、甲烷代谢、2-羰基羧酸代谢、肽聚糖生物合成、淀粉与蔗糖代谢以及缬氨酸/亮氨酸与异亮氨酸降解显著高于藻结皮。藓结皮中地嗜皮菌属和红色杆菌属对相对丰度前10的代谢功能分类的贡献度显著低于藻结皮,而类诺卡氏菌属...     

Estimation of N2 fixation based on differences in the natural abundance of 15N among freshwater N2-fixing and non-N2-fixing algae

The hypothesis that small mammal burrows can increase the amount of water infiltrating into the soil profile was tested. The amount of water added to the soil profile from spring recharge in areas adjacent to ground squirrel (Spermophilus townsendii and S. elegans) burrows was compared to nearby areas without burrows. Recharge amounts in burrow areas were significantly higher than nonburrow areas. An average of 21% more of the winter precipitation infiltrated into the soil near burrows. The amount of recharge was also found to be positively related to burrow density. Burrows also affected the distribution of the recharge by adding significantly more water to the deeper portions (>50 cm) of the soil profile.     

New, man-made N2-fixing systems

The major inputs of fixed N into the global nitrogen cycle are assessed and compared as indicators of both the need for and the likely basis of new, complementary, man-made N2-fixing processes. The development, since 1964, of the purely chemical, highly reactive systems for the reduction of N2, including those driven electro- and photochemically, is traced, along with the parallel efforts to synthesize metal-N2 complexes (the first step in any likely fixation process) and subsequently protonate them to produce hydrazine or ammonia. These experimental approaches are convergent. Successful cycling or catalysing of some of these N2-binding systems has been achieved. The advantages and limitations of the more successful systems are noted. Approaches to this problem via direct modelling of the nitrogenase active site are outlined, as is the one successful use of such complexes in achieving N2 reduction. This wealth of effort on the reductive approaches contrasts vividly with the almost complete absence of research on N2 oxidation. Currently, only a re-evaluation of the arc discharge process is continuing. Finally, the author's studies of the extruded molybdenum-containing prosthetic group of nitrogenase, the enzymic N2-reducing site, are described in relation to future N2-fixing systems.     

CO2 concentrating mechanisms in cyanobacteria: molecular components,their diversity and evolution

Cyanobacteria have evolved an extremely effective single-cell CO(2) concentrating mechanism (CCM). Recent molecular, biochemical and physiological studies have significantly extended current knowledge about the genes and protein components of this system and how they operate to elevate CO(2) around Rubisco during photosynthesis. The CCM components include at least four modes of active inorganic carbon uptake, including two bicarbonate transporters and two CO(2) uptake systems associated with the operation of specialized NDH-1 complexes. All these uptake systems serve to accumulate HCO(3)(-) in the cytosol of the cell, which is subsequently used by the Rubisco-containing carboxysome protein micro-compartment within the cell to elevate CO(2) around Rubisco. A specialized carbonic anhydrase is also generally present in this compartment. The recent availability of at least nine cyanobacterial genomes has made it possible to begin to undertake comparative genomics of the CCM in cyanobacteria. Analyses have revealed a number of surprising findings. Firstly, cyanobacteria have evolved two types of carboxysomes, correlated with the form of Rubisco present (Form 1A and 1B). Secondly, the two HCO(3)(-) and CO(2) transport systems are distributed variably, with some cyanobacteria (Prochlorococcus marinus species) appearing to lack CO(2) uptake systems entirely. Finally, there are multiple carbonic anhydrases in many cyanobacteria, but, surprisingly, several cyanobacterial genomes appear to lack any identifiable CA genes. A pathway for the evolution of CCM components is suggested.     

Identification of facultatively heterotrophic, N2-fixing cyanobacteria able to receive plasmid vectors from Escherichia coli by conjugation.

Plasmid vectors transferable by conjugation from Escherichia coli to obligately photoautotrophic strains of Anabaena spp. are also transferred to and maintained in heterotrophic, filamentous cyanobacteria of the genus Nostoc. These organisms can be used for the genetic analysis of oxygenic photosynthesis, chromatic adaptation, nitrogen fixation, and heterocyst development.     

Physiological and molecular aspects of the inorganic carbon-concentrating mechanism in cyanobacteria

This paper reviews progress made in elucidating the inorganic carbon concentrating mechanism in cyanobacteria at the physiological and molecular levels. Emphasis is placed on the mechanism of inorganic carbon transport, physiological and genetical analysis of high-CO₂-requiring mutants, the polypeptides induced during adaptation to low CO₂, the functional significance of carboxysomes, and the role of carbonic anhydrase. We also make occasional reference to the green algal inorganic carbon-concentrating mechanism.