Control of nitrogen fixation and ammonia excretion in Azorhizobium caulinodans

Due to the costly energy demands of nitrogen (N) fixation, diazotrophic bacteria have evolved complex regulatory networks that permit expression of the catalyst nitrogenase only under conditions of N starvation, whereas the same condition stimulates upregulation of high-affinity ammonia (NH₃) assimilation by glutamine synthetase (GS), preventing excess release of excess NH₃ for plants. Diazotrophic bacteria can be engineered to excrete NH₃ by interference with GS, however control is required to minimise growth penalties and prevent unintended provision of NH₃ to non-target plants. Here, we tested two strategies to control GS regulation and NH₃ excretion in our model cereal symbiont Azorhizobium caulinodans AcLP, a derivative of ORS571. We first attempted to recapitulate previous work where mutation of both P_II homologues glnB and glnK stimulated GS shutdown but found that one of these genes was essential for growth. Secondly, we expressed unidirectional adenylyl transferases (uATs) in a ΔglnE mutant of AcLP which permitted strong GS shutdown and excretion of NH₃ derived from N₂ fixation and completely alleviated negative feedback regulation on nitrogenase expression. We placed a uAT allele under control of the NifA-dependent promoter PnifH, permitting GS shutdown and NH₃ excretion specifically under microaerobic conditions, the same cue that initiates N₂ fixation, then deleted nifA and transferred a rhizopine nifA_L94Q/D95Q-rpoN controller plasmid into this strain, permitting coupled rhizopine-dependent activation of N₂ fixation and NH₃ excretion. This highly sophisticated and multi-layered control circuitry brings us a step closer to the development of a "synthetic symbioses” where N₂ fixation and NH₃ excretion could be specifically activated in diazotrophic bacteria colonising transgenic rhizopine producing cereals, targeting delivery of fixed N to the crop while preventing interaction with non-target plants.     

Cluster analysis of genes for nitrogen fixation from several diazotrophs

Hierarchical clustering and similarity coefficients of pairwise alignments of the published nucleotide sequences of 27nifH genes suggest thatnif genes are as ancient as the archaebacteria and clostridia. The positions ofnifHl ofMethanococcus thermolithotrophicus, nifH3 ofClostridium pasteurianum, nifH3 ofAzotobacter vinelandii andnifH ofFrankia suggest that a variety of lateral transfers may have occurred during evolution ofnifH gene. The genes for type 3 nitrogenase ofA. vinelandii may have diverged early from methanogens and clostridia. A high similarity coefficient with the derived amino acid sequence of type 3 nitrogenase suggests the presence of a functionally similar enzyme inC. pasteurianum. The type 2 nitrogenase genenifH2 of azotobacters seems to have originated recently from the genenifHl for conventional type I nitrogenase. RhizobialnifH genes comprise two closely related but discrete clusters that are in consonance with the plasmid or chromosomal location ofnif genes. The chromosomal and plasmid locatednifH of rhizobia seem to have evolved independently but contemporaneously.     

Analysis of codon usage in genes for nitrogen fixation from phylogenetically diverse diazotrophs

Summary A cluster analysis based on codon usage in genes for biological nitrogen fixation (nif genes) grouped diazotrophs into three distinct classes: anaerobes, cyanobacteria, and aerobes. In thenif genes ofKlebsiella pneumoniae there was no evidence for selection pressure in favor of highly translatable codons. However, in the nitrogen regulatory operonglnAntrBntrC of enteric bacteria the stoichiometrically high level of glutamine synthetase may be facilitated by the presence of efficiently translatable codons inglnA. Thenif genes of the cyanobacteriumAnabaena showed codon selection in favor of translational efficiency. Computation of codon adaptation indices for expression in heterologous systems indicated that the reading frames most suitable for expression ofnif genes inEscherichia coli, Bacillus subtilis, andSaccharomyces cerevisiae were present in azotobacters, clostridia, and cyanobacteria, respectively. In codon-usage-based cluster analysis, type 3 nitrogenase genes ofAzotobacter vinelandii grouped along with type 1 and type 2 genes. This is in contrast to the nucleotide sequence-based multiple alignment in which type 3 nitrogenase genes ofA. vinelandii have been reported to cluster with entirely unrelated diazotrophs such as methanogens and clostridia. This may be indicative of lateral transfer ofnif genes among widely divergent taxons. The chromosomal- and plasmid-locatednif genes of rhizobia also cluster separately in nucleotide sequence-based analysis but showed similar codon usage. These analyses suggested that the phylogeny ofnif genes drawn on the basis of nucleotide sequence homology was not masked by the taxon-specific pressure on codon usage.     

Non-symbiotic nitrogen fixation in soil

Summary 1. The fixation of N¹⁵-labeled nitrogen in small vessels of California soil under various conditions of pH, substrate level, oxygen tension, and other soil conditions was observed.2. Nitrate concentrations greater than 1.0–1.5 microequivalents per gram soil were found to suppress nitrogen fixation but not the growth ofAzotobacter.3. Large amounts of nitrogen were fixed when soluble organic substrates (e.g. glucose or sucrose) were added to the soil.4. Moderate fixation also resulted from the inversion of a disc of sod.5. Fixed nitrogen appeared largely in the nitrate and ammonia-amide fractions with that in the nitrate fraction probably representing nitrification of more reduced initial products of fixation.6. Under conditions of these experiments growing grass did not enhance fixation. At higher light intensities, however, such an enhancement might be observed.7. The incorporation of grass cuttings, straw or alfalfa meal into the soil caused only a slight increase in fixation.8. The inoculation of soils with large populations ofAzotobacter did not result in increased fixation.This investigation was supported in part by a contract with the United States Atomic Energy Commission.     

Non-cyanobacterial diazotrophs mediate dinitrogen fixation in biological soil crusts during early crust formation

Biological soil crusts (BSCs) are key components of ecosystem productivity in arid lands and they cover a substantial fraction of the terrestrial surface. In particular, BSC N₂-fixation contributes significantly to the nitrogen (N) budget of arid land ecosystems. In mature crusts, N₂-fixation is largely attributed to heterocystous cyanobacteria; however, early successional crusts possess few N₂-fixing cyanobacteria and this suggests that microorganisms other than cyanobacteria mediate N₂-fixation during the critical early stages of BSC development. DNA stable isotope probing with ¹⁵N₂ revealed that Clostridiaceae and Proteobacteria are the most common microorganisms that assimilate ¹⁵N₂ in early successional crusts. The Clostridiaceae identified are divergent from previously characterized isolates, though N₂-fixation has previously been observed in this family. The Proteobacteria identified share >98.5% small subunit rRNA gene sequence identity with isolates from genera known to possess diazotrophs (for example, Pseudomonas, Klebsiella, Shigella and Ideonella). The low abundance of these heterotrophic diazotrophs in BSCs may explain why they have not been characterized previously. Diazotrophs have a critical role in BSC formation and characterization of these organisms represents a crucial step towards understanding how anthropogenic change will affect the formation and ecological function of BSCs in arid ecosystems.     

Temperature control of nitrogen fixation in Klebsiella pneumoniae

At growth temperatures above 37°C, Klebsiella pneumoniae does not grow in a medium containing N₂ or NO ₃ ^- as nitrogen sources. However, both the growth in the presence of other nitrogen sources as well as the in vitro nitrogenase activity are not affected at this temperature. The inability to fix N₂ at high temperature is due to the failure of the cells to synthesize nitrogenase and other nitrogen fixation (nif) gene encoded proteins. When cells grown under nitrogen fixing conditions at 30°C were shifted to 39°C, there was a rapid decrease of the rate of de novo biosynthesis of nitrogenase (component 1), nitrogenase reductase (component 2), and the nifJ gene product. There was no degradation of nitrogenase at the elevated temperature since preformed enzyme remained stable over a period of at least 3 h at 39°C. Thus, temperature seems to represent a third control system, besides NH ₄ ⁺ and O₂, governing the expression of nif genes of K. pneumoniae.     

Liberation of ammonia during nitrogen fixation by a facultatively heterotrophic cyanobacterium

Nitrogen fixation (acetylene reduction) and ammonia liberation were studied in a facultatively heterotrophic cyanobacterium. Autotrophically grown cells lost acetylene reduction activity when incubated under anaerobic conditions; the activity was maintained in the presence of methionine sulfoximine; or by pretreatment of the cells with a carbon supply. Heterotrophically grown cells maintained acetylene reduction activity anaerobically in the absence of methionine sulfoximine. Both cell types required light for maintenance of activity. The data indicate that methionine sulfoximine preserves the intracellular pool of reductant needed for nitrogenase. Autotrophs and heterotrophs both liberated ammonia when treated with methionine sulfoximine under nitrogen-fixing conditions. However, on treatment with methionine sulfoximine under anaerobiosis, heterotrophs also accumulated large amounts of intracellular ammonia in a pool which was diminished by the Photosystem II inhibitor, 3(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU). DCMU enhanced ammonia liberation without affecting acetylene reduction activity, and hence changed the ratio of acetylene reduced to ammonia formed by the heterotrophs. These data suggest a role for Photosystem II in ammonia liberation by the cyanobacteria.     

Nutrient feedbacks to soil heterotrophic nitrogen fixation in forests

Multiple nutrient cycles regulate biological nitrogen (N) fixation in forests, yet long-term feedbacks between N-fixation and coupled element cycles remain largely unexplored. We examined soil nutrients and heterotrophic N-fixation across a gradient of 24 temperate conifer forests shaped by legacies of symbiotic N-fixing trees. We observed positive relationships among mineral soil pools of N, carbon (C), organic molybdenum (Mo), and organic phosphorus (P) across sites, evidence that legacies of symbiotic N-fixing trees can increase the abundance of multiple elements important to heterotrophic N-fixation. Soil N accumulation lowered rates of heterotrophic N-fixation in organic horizons due to both N inhibition of nitrogenase enzymes and declines in soil organic matter quality. Experimental fertilization of organic horizon soil revealed widespread Mo limitation of heterotrophic N-fixation, especially at sites where soil Mo was scarce relative to C. Fertilization also revealed widespread absence of P limitation, consistent with high soil P:Mo ratios. Responses of heterotrophic N-fixation to added Mo (positive) and N (negative) were correlated across sites, evidence that multiple nutrient controls of heterotrophic N-fixation were more common than single-nutrient effects. We propose a conceptual model where symbiotic N-fixation promotes coupled N, C, P, and Mo accumulation in soil, leading to positive feedback that relaxes nutrient limitation of overall N-fixation, though heterotrophic N-fixation is primarily suppressed by strong negative feedback from long-term soil N accumulation.     

Relationship of ammonia excretion,dinitrogen fixation and ammonia assimilatory enzymes to encystation in a continuous culture ofAzospirillum brasilense

Continuous culture studies have been carried out on a strain ofA. brasilense to study ammonia excretion, dinitrogen fixation and ammonia assimilatory enzymes (glutamate-ammonia ligase and glutamate synthase (NADPH)) in encysted conditions. High glutamate synthase (NADPH) and low ammonia excretion was observed at the time of induction of cyst formation. Low and oscillating nitrogenase activity was observed throughout the experiment.     

No stimulation of nitrogen fixation by non‐filamentous diazotrophs under elevated CO2 in the South Pacific

Nitrogen fixation by diazotrophic cyanobacteria is a critical source of new nitrogen to the oligotrophic surface ocean. Research to date indicates that some diazotroph groups may increase nitrogen fixation under elevated pCO₂. To test this in natural plankton communities, four manipulation experiments were carried out during two voyages in the South Pacific (30–35^oS). High CO₂ treatments, produced using 750 ppmv CO₂ to adjust pH to 0.2 below ambient, and ‘Greenhouse’ treatments (0.2 below ambient pH and ambient temperature +3 °C), were compared with Controls in trace metal clean deckboard incubations in triplicate. No significant change was observed in nitrogen fixation in either the High CO₂ or Greenhouse treatments over 5 day incubations. qPCR measurements and optical microscopy determined that the diazotroph community was dominated by Group A unicellular cyanobacteria (UCYN‐A), which may account for the difference in response of nitrogen fixation under elevated CO₂ to that reported previously for Trichodesmium. This may reflect physiological differences, in that the greater cell surface area:volume of UCYN‐A and its lack of metabolic pathways involved in carbon fixation may confer no benefit under elevated CO₂. However, multiple environmental controls may also be a factor, with the low dissolved iron concentrations in oligotrophic surface waters limiting the response to elevated CO₂. If nitrogen fixation by UCYN‐A is not stimulated by elevated pCO₂, then future increases in CO₂ and warming may alter the regional distribution and dominance of different diazotroph groups, with implications for dissolved iron availability and new nitrogen supply in oligotrophic regions.     

Phosphorus regulation of nitrogen fixation in a traditional Mexican agroecosystem

Although nitrogen is considered to be the nutrient that most commonly limits production of natural and managed terrestrial ecosystems, I propose that phosphorus may regulate productivity in many continuously cultivated agroecosystems that do not receive applications of synthetic fertilizers. One way P may limit agroecosystem productivity is by controlling nitrogen fixation of legume crops, thus affecting nitrogen availability in the overall agroecosystem. I tested this hypothesis in two studies by examining the effect of phosphorus nutrition on nitrogen fixation of alfalfa in traditional Mexican agroecosystems. All farms used in the research relied on alfalfa as the primary nitrogen source for maize cultivation and other crops, and had minimal or no reliance on synthetic fertilizers.In one study, I used the natural abundance of¹⁵N to estimate nitrogen fixation in five alfalfa plots with soils representing a wide range of P fertility. I found a correlation of r = 0.85 between foliage P concentrations and nitrogen fixation in the alfalfa plots. Mean nitrogen fixation in alfalfa plots ranged between 232–555 kg ha^–1 yr^–1 as estimated by the¹⁵N-natural abundance method.In a second study, I sampled soils from alfalfa plots on traditional farms located in 5 different physiographic regions of Mexico. Half of each soil sample was augmented with phosphorus in a greenhouse experiment. I grew alfalfa on the fertilized and unfertilized soils from each site and then determined nitrogenase activity (acetylene reduction) of the Rhizobium on the plant roots. Nitrogenase activity increased in the alfalfa grown on all soils with added phosphorus, with two of the five differences being statistically significant at P < 0.01, 0 and one at P < 0.05. Foliage P concentrations and nitrogenase activity were 0 positively correlated (r = 0.81,P < 0.01).0     

Oxygen and the regulation of nitrogen fixation in legume nodules

In N₂-fixing legume nodules, O₂ is required in large amounts for aerobic respiration, yet nitrogenase, the bacterial enzyme that fixes N₂, is O₂ labile. A high rate of O₂ consumptition and a cortical barrier to gas diffusion work together to maintain a low, non-inhibitory O₂ concentration in the central, infected zone of the nodule. At this low O₂ concentration, cytosolic leghemoglobin is required to facilitate the diffusion of O₂ through the infected cell to the bacteria. The resistance of the cortical diffusion barrier is variable and is used by legume nodules to regulate the O₂ concentration in the infected cells such that it limits aerobic respiration and N₂ fixation at all times. The resistance of the diffusion barrier and therefore the degree of O₂ limitation seems to be regulated in response to changes in the O₂ concentration of the central infected zone, the supply of phloem sap to the nodule, and the rate of N assimilation into the end products of fixation.     

Transcriptional regulation of nitrogen fixation genes by DNA supercoiling

DNA gyrase (ATP dependent topoisomerase type II, EC 5.99.1.3) was found to be essential for the expression of the Klebsiella pneumoniae nitrogen fixation gene cluster carried by plasmid pRD1 in Escherichia coli. In the absence of DNA gyrase activity, nitrogen fixation activity could be restored by providing a constitutively expressed nifA function in trans. Our results suggest that nif gene regulation by oxygen may be mediated through the alteration of the superhelical status of the promoter of the nifLA regulatory operon, in addition to the action of the nifL gene product.Communicated by J. Schell