Nitrogen-fixing Organisms Research Articles

Environmental conditions play a key role in controlling the composition and diversity of Colombian biocrust microbiomes.

Drylands soils worldwide are naturally colonized by microbial communities known as biocrusts. These soil microbiomes render important ecosystem services associated with soil fertility, water holding capacity, and stability to the areas they cover. Because of the importance of biocrusts in the global cycling of nutrients, there is a growing interest in describing the many microbial configurations these communities display worldwide. However, comprehensive 16S rRNA genes surveys of biocrust communities do not exist for much of the planet: for example, in the continents of South America and the northern part of Africa. The absence of a global understanding of biocrust biodiversity has lead us to assign a general importance to community members that may, in fact, be regional. Here we report for the first time the presence of biocrusts in Colombia (South America) through 16S rRNA genes surveys across an arid, a semi-arid and a dry subtropical region within the country. Our results constitute the first glance of the Bacterial/Archaeal communities associated with South American biocrust microbiomes. Communities where cyanobacteria other than Microcoleus vaginatus prevail, despite the latter being considered a key species elsewhere, illustrate differentiable results in these surveys. We also find that the coastal biocrust communities in Colombia include halo-tolerant and halophilic species, and that niche preference of some nitrogen fixing organisms deviate from previously described global trends. In addition, we identified a high proportion (ranging from 5 to 70%, in average) of cyanobacterial sequences that did not match any formally described cyanobacterial species. Our investigation of Colombian biocrusts points to highly diverse communities with climatic regions controlling taxonomic configurations. They also highlight an extensive local diversity to be discovered which is central to better design management and restoration strategies for drylands soils currently undergoing disturbances due to land use and global warming. Finally, this field study highlights the need for an improved mechanistic understanding of the response of key biocrust community members to changes in moisture and temperature.

Microalgal and Nitrogen-Fixing Bacterial Consortia: From Interaction to Biotechnological Potential.

Microalgae are used in various biotechnological processes, such as biofuel production due to their high biomass yields, agriculture as biofertilizers, production of high-value-added products, decontamination of wastewater, or as biological models for carbon sequestration. The number of these biotechnological applications is increasing, and as such, any advances that contribute to reducing costs and increasing economic profitability can have a significant impact. Nitrogen fixing organisms, often called diazotroph, also have great biotechnological potential, mainly in agriculture as an alternative to chemical fertilizers. Microbial consortia typically perform more complex tasks than monocultures and can execute functions that are challenging or even impossible for individual strains or species. Interestingly, microalgae and diazotrophic organisms are capable to embrace different types of symbiotic associations. Certain corals and lichens exhibit this symbiotic relationship in nature, which enhances their fitness. However, this relationship can also be artificially created in laboratory conditions with the objective of enhancing some of the biotechnological processes that each organism carries out independently. As a result, the utilization of microalgae and diazotrophic organisms in consortia is garnering significant interest as a potential alternative for reducing production costs and increasing yields of microalgae biomass, as well as for producing derived products and serving biotechnological purposes. This review makes an effort to examine the associations of microalgae and diazotrophic organisms, with the aim of highlighting the potential of these associations in improving various biotechnological processes.

Nitrogen-fixing sulfate reducing bacteria in shallow coastal sediments under simulated resuspension

Nitrogen (N2) fixation by heterotrophic non-cyanobacterial diazotrophs is common in marine deep-sea sediments. However, in shallow coastal areas, where resuspension of sediments is extensive, the magnitude of sediment-associated N2 fixation during resuspension is unknown. We examined nitrogen fixation in dark slurry incubations with sediments (0–5 cm and 0–10 cm depths) under simulated resuspension from five shallow stations (water depth <1 m) and an anoxic site in the Bay of Gdansk (109 m) in the Baltic Sea. Abiotic variables and the composition of nitrogen fixing organisms (diazotrophs) were measured at the study sites. To estimate the contribution of nitrogen fixing sulfate reducing bacteria to total nitrogen fixation, parallel incubations with sodium molybdate as inhibitor were performed. Our data show low but variable nitrogen fixation rates (n.d. - 23.7 nmol N g−1 d−1), promoted in small-grained sediments associated with increased organic carbon content and high nutrient concentrations in pore waters. Highest nitrogen fixation at the shallow sites was encountered in the upper 0–5 cm of the sediments while rates were negligible below. Sulfate reducing bacteria (e.g. Desulfobacterales and Desulfovibrionales) were responsible for most of the heterotrophic nitrogen fixation and appear as key players for pelagic N2 fixation during resuspension. Our study reveals an important sediment – water coupling, which may be accentuated by the increased storms and resuspension events predicted for the Baltic Sea region.

Integrating the impact of global change on the niche and physiology of marine nitrogen-fixing cyanobacteria.

Marine nitrogen fixation is a major source of new nitrogen to the ocean, which interacts with climate driven changes to physical nutrient supply to regulate the response of ocean primary production in the oligotrophic tropical ocean. Warming and changes in nutrient supply may alter the ecological niche of nitrogen-fixing organisms, or 'diazotrophs', however, impacts of warming on diazotroph physiology may also be important. Lab-based studies reveal that warming increases the nitrogen fixation-specific elemental use efficiency (EUE) of two prevalent marine diazotrophs, Crocosphaera and Trichodesmium, thus reducing their requirements for the limiting nutrients iron and phosphorus. Here, we coupled a new diazotroph model based upon observed diazotroph energetics of growth and resource limitation to a state-of-the-art global model of phytoplankton physiology and ocean biogeochemistry. Our model is able to address the integrated response of nitrogen fixation by Trichodesmium and Crocosphaera to warming under the IPCC high emission RCP8.5 scenario for the first time. Our results project a global decline in nitrogen fixation over the coming century. However, the regional response of nitrogen fixation to climate change is modulated by the diazotroph-specific thermal performance curves and EUE, particularly in the Pacific Ocean, which shapes global trends. Spatially, the response of both diazotrophs is similar with expansion towards higher latitudes and reduced rates of nitrogen fixation in the lower latitudes. Overall, 95%-97% of the nitrogen fixation climate signal can be attributed to the combined effect of temperature on the niche and physiology of marine diazotrophs, with decreases being associated with a reduced niche and increases resulting due to a combination of expanding niche and temperature driven changes to EUE. Climate change impacts on both the niche and physiology of marine diazotrophs interact to shape patterns of marine nitrogen fixation, which will have important implications for ocean productivity in the future.

The essential bioactive role of nickel in the oceans: Evidence from nickel isotopes

The role of nickel (Ni) in ocean biogeochemical cycles is both under-studied and controversial. Strong correlations between Ni and organic carbon in modern and ancient marine sediments suggest a prominent biogeochemical role over a substantial portion of Earth history. Addition of Ni to culturing and seawater incubation experiments produces strong responses in terms of cell growth, particularly of nitrogen-fixing organisms. But the implied limiting role for phytoplankton growth is inconsistent with observations in the real ocean, specifically that photic zone Ni concentrations never descend to the very low values that characterise other bioactive, and often bio-limiting, metals like iron. These two observations can be reconciled if a large portion of the total dissolved Ni present in open-ocean surface waters is not bio-available on short timescales. Here we present new Ni concentration and stable isotope data from the GEOVIDE transect in the North Atlantic. We interpret these new data in the light of the growing database for Ni stable isotopes in the modern ocean, with implications for the biogeochemical importance of Ni.In the new North Atlantic dataset, the lowest Ni concentrations (1.8-2.6 nmol/L) and highest δ60Ni (up to +1.67‰) are associated with low nitrate, south of the subarctic front (SAF). By contrast, stations at latitudes north of the SAF, with higher surface nitrate, show very subdued variation in Ni concentrations throughout the entire depth of the water column (3.6±0.3 nmol/L, mean and 2SD), and no variation in δ60Ni beyond the narrow global deep-ocean range (+1.33±0.13 ‰). These North Atlantic Ni isotope data also show relationships with nitrogen isotope effects, observed in the same samples, that are suggestive of a link between Ni utilisation, isotope fractionation and nitrogen fixation.The global dataset, including the new data presented here, reveals a biogeochemical divide with Ni isotope fractionation only occurring in low latitude surface waters. A simple observationally constrained three-dimensional model of Ni cycling suggests that the creation of this isotopically heavy, Ni-poor, end-member, together with the physical circulation and remineralisation at depth, can explain the global Ni-δ60Ni systematics. Taken together, these findings hint at Ni-N co-limitation in the modern ocean. We advocate for more extensive and detailed culturing/incubation studies of this neglected metal in order to elucidate its potentially crucial biogeochemical role.

Antenna Modification Leads to Enhanced Nitrogenase Activity in a High Light-Tolerant Cyanobacterium.

ABSTRACTBiological nitrogen fixation is an energy-intensive process that contributes significantly toward supporting life on this planet. Among nitrogen-fixing organisms, cyanobacteria remain unrivaled in their ability to fuel the energetically expensive nitrogenase reaction with photosynthetically harnessed solar energy. In heterocystous cyanobacteria, light-driven, photosystem I (PSI)-mediated ATP synthesis plays a key role in propelling the nitrogenase reaction. Efficient light transfer to the photosystems relies on phycobilisomes (PBS), the major antenna protein complexes. PBS undergo degradation as a natural response to nitrogen starvation. Upon nitrogen availability, these proteins are resynthesized back to normal levels in vegetative cells, but their occurrence and function in heterocysts remain inconclusive. Anabaena 33047 is a heterocystous cyanobacterium that thrives under high light, harbors larger amounts of PBS in its heterocysts, and fixes nitrogen at higher rates compared to other heterocystous cyanobacteria. To assess the relationship between PBS in heterocysts and nitrogenase function, we engineered a strain that retains large amounts of the antenna proteins in its heterocysts. Intriguingly, under high light intensities, the engineered strain exhibited unusually high rates of nitrogenase activity compared to the wild type. Spectroscopic analysis revealed altered PSI kinetics in the mutant with increased cyclic electron flow around PSI, a route that contributes to ATP generation and nitrogenase activity in heterocysts. Retaining higher levels of PBS in heterocysts appears to be an effective strategy to enhance nitrogenase function in cyanobacteria that are equipped with the machinery to operate under high light intensities.

Structural analysis of the reductase component AnfH of iron-only nitrogenase from Azotobacter vinelandii

Biological nitrogen fixation, the conversion of atmospheric dinitrogen into bioavailable ammonium, is exclusively catalyzed by the enzyme nitrogenase that is present in nitrogen-fixing organisms, the diazotrophs. So far, three different nitrogenase variants, encoded in their corresponding, distinct gene clusters, have been found in nature. Each one of these consists of a catalytic dinitrogenase component and a unique, ATP-dependent reductase, the Fe protein. The three variant nitrogenases differ in the composition of the active site and contain either molybdenum, vanadium or only iron in the dinitrogenase component. Here we present the 2.0 Å resolution crystal structure of the ADP-bound reductase component AnfH of the iron-only nitrogenase from the model diazotroph Azotobacter vinelandii. A comparison of this structure with the ones reported for the two other Fe protein homologs NifH and VnfH in the ADP-bound state shows that all are adopting the same conformation. However, cross-reactivity assays with the three nitrogenase homologs revealed AnfH to be compatible with iron-only nitrogenase and to a lesser degree with the vanadium-containing enzyme, but not with molybdenum nitrogenase.

Partial synthetic models of FeMoco with sulfide and carbyne ligands: Effect of interstitial atom in nitrogenase active site

Nitrogen-fixing organisms perform dinitrogen reduction to ammonia at an Fe-M (M = Mo, Fe, or V) cofactor (FeMco) of nitrogenase. FeMco displays eight metal centers bridged by sulfides and a carbide having the MFe7S8C cluster composition. The role of the carbide ligand, a unique motif in protein active sites, remains poorly understood. Toward addressing how the carbon bridge affects the physical and chemical properties of the cluster, we isolated synthetic models of subsite MFe3S3C displaying sulfides and a chelating carbyne ligand. We developed synthetic protocols for structurally related clusters, [Tp*M'Fe3S3X]n-, where M' = Mo or W, the bridging ligand X = CR, N, NR, S, and Tp* = Tris(3,5-dimethyl-1-pyrazolyl)hydroborate, to study the effects of the identity of the heterometal and the bridging X group on structure and electrochemistry. While the nature of M' results in minor changes, the chelating, μ3-bridging carbyne has a large impact on reduction potentials, being up to 1 V more reducing compared to nonchelating N and S analogs.

Cyanobacterial community composition and their functional shifts associated with biocrust succession in the Gurbantunggut Desert.

Cyanobacteria, as key biocrust components, provide a variety of ecosystem functions in drylands. In this study, to identify whether a cyanobacterial community shift is involved in biocrust succession and whether this is linked to altered ecological functions, we investigated cyanobacterial composition, total carbon and nitrogen contents of biocrusts in the Gurbantunggut Desert. Our findings showed that the biocrust cyanobacteria in the Gurbantunggut desert were mostly filamentous, coexisting with abundant unicellular colonial Chroococcidiopsis. Heterocystous Nostoc, Scytonema and Tolypothrix always represented the majority of biocrust nitrogen-fixing organisms, comprising an average of 92% of the nifH gene reads. Community analysis showed a clear shift in prokaryotic community composition associated with biocrust succession from cyanobacteria- to lichen- and moss-dominated biocrusts, and filamentous non-nitrogen-fixing cyanobacteria-dominated communities were gradually replaced by nitrogen-fixing and unicellular colonial communities. Along the succession, there were concomitant reductions in cyanobacterial relative abundance, whereas Chl-a, total carbon and nitrogen contents increased. Concurrently, distinct carbon and nitrogen stores shifts occurred, implying that the main ecological contribution of cyanobacteria in biocrusts changes from carbon- to nitrogen-fixation along with the succession. Our results suggest that any activity that reverses biocrust succession will influence cyanobacterial community composition and eventually lead to large reductions in soil carbon and nitrogen stores.

Thymidylate kinase (TMK) of the photosynthetic, nitrogen-fixing cyanobacterium Nostoc sp. strain PCC7120: Biophysical, biochemical and physiological characterisation

Thymidylate kinase (TMK/TMPK) is an important enzyme in DNA biosynthesis and catalyses the conversion of dTMP to dTDP. Due to its therapeutic potential, the focus has been on characterizing the TMK proteins of pathogens and human origin, with very little information available on the TMK proteins of photosynthetic organisms and agriculturally important nitrogen-fixing organisms. In this work we report the characterisation of TMK in an evolutionarily ancient organism, cyanobacteria. The TMK protein of the photosynthetic, nitrogen-fixing cyanobacterium Nostoc PCC7120 (AnTMK) was found to have low conformational stability, which related to its low Tm of ~46 °C confirmed by Differential Scanning Fluorimetry (DSF) and Differential Scanning Calorimetry (DSC) techniques. The AnTMK protein exhibited substrate specificity for dTMP and ATP with Km of 20.74 ± 1.47 μM and 20.17 ± 2.96 μM respectively. The enzyme kinetics data and the positive co-operativity observed between dTMP and ATP binding correlated well with the data obtained from Isothermal Titration Calorimetry (ITC). Homology model of the enzyme suggested that the binding mode of substrate nucleotides to the enzyme is conserved. When overexpressed constitutively in Nostoc PCC7120 (Antmk+), it supported faster growth measured in terms of chlorophyll a content under normal growth conditions, but exhibited lower photosynthetic efficiency. Compared to the vector control recombinant Nostoc AnpAM, the Antmk + cells exhibited higher photoinhibition at higher light irradiance with more open reaction centres and lower dissipation of heat, indicative of damage to photosynthetic machinery. This indicated that the TMK is likely to have a significant role in photosynthetic organisms.

Study on Interactive Effects of Different Levels of Lead and Mercury on Nitrogen Fixation of Some Diazotrophs

Researchers have studied the effects of addition of metal elements in combination with nitrogen fixing organisms as inoculants on the plants (growth) predominantly in legumes, however there is a major gap because responses and effects of these proposed micronutrients on the nitrogen fixation activity of these microbes both free living and symbiotic remains sketchy at best. Therefore, the effect of supplementation of lead and Mercury (bioaugmentation) on the nitrogen fixation potential of two (2) diazotrophs was evaluated in this study.&#x0D; Aims: To evaluate the interactive effects of different levels of Lead and Mercury on Nitrogen fixation of both Rhizobium and Xanthobactersppin-vitro.&#x0D; Place and Duration of Study: Sample organisms where collected from Groundnut rhizospheric soil of a farm in Cross River state, Nigeria. The microorganism isolation and nitrogen fixation analysis was further carried out at MacCliff General services Laboratory, Owerri, Nigeria for a duration of 3 months.&#x0D; Study Design: The interactive plots serve to show the effect of one variable (lead) on the value of mercury (the other) and is derived by selecting high and low values for lead (Pb) and entering them into the equation along with the range of values for Mercury (Hg). The values of independent variables (lead and mercury levels) used in the plots were selected by observing the highest concentration (+1) and lowest concentration (-1) values which are able to support nitrogen fixation independently in Rhizobium and Xanthobacter.&#x0D; Methodology: The soil samples were collected from groundnut rhizosphere at a 20 cm depth using sterile soil corer (sterilized with 95% ethanol) and matured Groundnut plants were uprooted with care. From these samples, both Rhizobium and Xanthobacterspp were isolated. The isolated organisms were re-vitalized in Jensen’s nitrogen free broth and standardized to 0.5 McFarland standards. To determine nitrogen fixation, the broth cultures were examined for nitrate nitrogen (NO3-N) and amino nitrogen (Amino-N) levels after ten days of the experiment under continuous airflow using the Jensen’s nitrogen free broth containing the metal salts, Mercury (II) chloride HgCl2 and Lead (II) acetate trihydratePb (CH3COO)2.3H2O). Nitrate nitrogen and amino Nitrogen was obtained using cataldo and ninhydrins methods respectively. The data obtained was made in triplicates and reported as mean values. Interactive effect plots and statistical analysis were done using Minitab 17 software at 5% level of significance (p&lt;0.05).&#x0D; Results: The main effect plots illustrate that to maximize nitrogen fixation in Xanthobacterspthrough the utilization of the selected metals as micronutrient, we should use lead at 6.25 mg/L and mercury at 25mg/L yielding 0.508 mg/L for nitrogen fixation response. The plot also suggests that if lead metals are used at a higher concentration than stated nitrogen fixation will decline. On the interaction plots, the slopes indicate that an interference or antagonistic interaction effect (crossed lines) exist between lead and mercury in the nitrogen fixation activity of Xanthobacter. The R-squared adjusted value suggests that 70.87% of the variations in nitrogen fixation response is explained by the interaction of lead and mercury, hence the model likely fits the data. However, the P-value was not significant at 0.102. For Rhizobium sp. mercury also has a higher fixation magnitude than Lead but relatively at 0.554 mg/L. However, the interaction plot showed parallel lines indicating that there was no interaction effect. Therefore, one can say that the relationship between lead and nitrogen fixation does not depend on the concentration of mercury and vice versa. The model was also statistically insignificant at 0.981.&#x0D; Conclusion: Interactive effect only occurred in the nitrogen fixation of Xanthobactersp. This raises a need for further study combination of metal elements which could be utilized to stimulate nitrogen, phosphorus and potassium production in Diazotrophs both in the field and in-vitro.

Cryoelectron microscopy structure and mechanism of the membrane-associated electron-bifurcating flavoprotein Fix/EtfABCX

The electron-transferring flavoprotein-menaquinone oxidoreductase ABCX (EtfABCX), also known as FixABCX for its role in nitrogen-fixing organisms, is a member of a family of electron-transferring flavoproteins that catalyze electron bifurcation. EtfABCX enables endergonic reduction of ferredoxin (E°' ∼-450 mV) using NADH (E°' -320 mV) as the electron donor by coupling this reaction to the exergonic reduction of menaquinone (E°' -80 mV). Here we report the 2.9 Å structure of EtfABCX, a membrane-associated flavin-based electron bifurcation (FBEB) complex, from a thermophilic bacterium. EtfABCX forms a superdimer with two membrane-associated EtfCs at the dimer interface that contain two bound menaquinones. The structure reveals that, in contrast to previous predictions, the low-potential electrons bifurcated from EtfAB are most likely directly transferred to ferredoxin, while high-potential electrons reduce the quinone via two [4Fe-4S] clusters in EtfX. Surprisingly, EtfX shares remarkable structural similarity with mammalian [4Fe-4S] cluster-containing ETF ubiquinone oxidoreductase (ETF-QO), suggesting an unexpected evolutionary link between bifurcating and nonbifurcating systems. Based on this structure and spectroscopic studies of a closely related EtfABCX, we propose a detailed mechanism of the catalytic cycle and the accompanying structural changes in this membrane-associated FBEB system.

Phosphate diester utilization by marine diazotrophs Trichodesmium erythraeum and Crocosphaera watsonii

In the phosphate-depleted oligotrophic ocean, microbes utilize various dissolved organic phosphorus (P) compounds as alternative P sources, using enzymes such as alkaline phosphatases. However, knowledge of such P acquisition mechanisms is limited, especially in association with the physiology of nitrogen-fixing organisms, which play a substantial role in marine biogeochemical cycling. We show that nonaxenic clonal cultures of 2 oceanic diazotrophs,Trichodesmium erythraeumandCrocosphaera watsonii, have the ability to utilize phosphate diester as their sole P source, using a model artificial compound—bis-p-nitrophenyl phosphate (bisNPP). Although both diazotroph cultures likely preferred phosphate monoester to diester, the expressed diesterase activity was theoretically sufficient to fulfill their P demands, and they showed significant growth in bisNPP-added media. Interestingly, a distinct difference in their growth trends was observed, with faster onset of growth byC. watsoniiand delayed onset of growth byT. erythraeum. This indicates that theC. watsoniiconsortium can effectively and rapidly assimilatein situdiesters as alternative P sources in the field. Nonetheless, when considering the poor bisNPP utilization reported from other marine phytoplankton taxa, our results indicate that the utilization of particular diester compounds is a notable and advantageous strategy for both diazotroph consortia to alleviate P limitation in the oligotrophic ocean.

Carbon and Macronutrient Budgets in an Alder Plantation Grown on a Reclaimed Combustion Waste Landfill

Combustion waste landfills are unfavorable for revegetation due to nitrogen deficiency, and therefore, the introduction of nitrogen-fixing organisms, such as alder species (Alnus sp.), may be promising for reclamation and restoration of these sites. We investigated the carbon and macronutrient stocks in the combustion waste technosols and biomass of black alder (Alnus glutinosa) and grey alder (Alnus incana) 10 years after introduction onto a combustion waste landfill. The alder species were planted with or without lignite addition in planting holes, the latter acting as control plots. Black alder biomass was higher than that of grey alder. The total macronutrient stocks were higher in the uppermost technosol layer (0–30 cm) than in the biomass nutrient stocks. However, the K and P stocks in the black alder biomass were still greater than the exchangeable K+ and available phosphorus (Pav) stocks in technosols. This is important for the nutrition of the trees planted in combustion waste landfills and confirms the Pav deficit in investigated technosols. The differentiation of nutrients in biomass shows that the largest stock was found in the wood of trunks and branches (40–70% of the stock of individual biomass macronutrients). Although foliage biomass represented approximately 7% of the total tree biomass, the nutrient stocks therein represented a significant proportion of total nutrient stocks: approximately 27–29% nitrogen, 17–22% calcium, 28% magnesium, 7–10% potassium and 12–16% phosphorus. This is particularly important in the context of the turnover of nutrients from litterfall and soil organic matter and the circulation of nutrients in the ecosystem developed on combustion waste technosols.

Identification of stress tolerant rhizobial strains inhabiting &lt;em&gt;Gliricidia sepium&lt;/em&gt; in Polonnaruwa district, Sri Lanka

Rhizobia are well-reputed biological nitrogen fixing organisms due to their proficiency in symbiotic root nodule formation with leguminous plants. Gliricidia sepium is a widely distributed legume which hosts a wide range of rhizobia. Few studies have been carried out to identify the stress tolerant rhizobial populations inhabiting G. sepium in Sri Lanka. This study was focused on the characterization and identification of stress tolerant rhizobial strains residing in the root nodules of G. sepium growing in seven selected locations of Polonnaruwa district, Sri Lanka. Isolated rhizobial strains were separately subjected to stress conditions (pH, temperature, salinity, and drought) and to a combination of given stress conditions. Most of the strains were tolerant to 25 - 45oC temperature range, extreme drought conditions and 1.5 % and 2.0 % salinity levels. However all the strains were adversely affected by acidic pH. Among the fifteen strains that were selected from the statistical analysis, fourteen displayed higher survival capacity when the combinations of physiological conditions were applied. There was great genetic diversity within these isolates and they belonged to 10 clusters. Cross inoculation of these isolated stress tolerant strains into crop legumes can be used as an ideal alternative for chemical nitrogen fertilizers.

Nutrient Management in Organic Baby Corn Production: A Review

The concept of organic agriculture is receiving increased attention and organic food markets are also expanding quite fast in many parts of the world including India. Development of production technology particularly appropriate nutrient management strategy for organic baby corn is necessary for realizing higher yield and economic returns as well as soil health aspect. Most of the organic manures are low in nutrient contents, which are not sufficient to meet the nutritional requirement of the crops with smaller quantities, especially when inorganic fertilizers are not applied. Under such circumstances, enrichment of organic manures and composts with permitted additives like rock phosphate and beneficial microbial cultures is a feasible option for nutrient supplementation in organic food production. Microbial enrichment technique with bio-inoculants to composting material had been shown to improve the quality of compost. The enrichment of the organic manures with beneficial microbial cultures will further contribute to the enhancement of N and P contents through nitrogen fixation and phosphate solubilization. Hence, the enriched organic manures and their combination provide an ideal nutrition strategy for the crop. There is the possibility of increasing the nitrogen content of compost by inoculation with nitrogen-fixing organisms and the phosphorus content by the inoculation with phosphate solubilizing bacteria. Improved manures will play a major role in organic farming since the method of production is simple and easy.

Complete Genome Sequence of Kosakonia radicincitans GXGL-4A, a Nitrogen-Fixing Bacterium with Capability to Degrade TEX.

Kosakonia radicincitans GXGL-4A, a free-living nitrogen-fixing (NF) bacterial strain isolated from maize (Zea mays L.) roots was found to have ability to degrade aromatic hydrocarbons. In this study, we describe the main morphological characteristics of bacterium, aromatic hydrocarbon-degrading capability, and the complete genome of K. radicincitans GXGL-4A. The genome is consisted of only one 5,687,681bp linear chromosome with a G + C content of 53.96%. The strain has two genetically distinct nitrogenase systems, one based on molybdenum (Mo) similar to nitrogenase isolated from a wide range of nitrogen-fixing organisms, and the other contains iron (Fe). The differences in transcriptional level of several important nitrogen fixation (nif) genes between LB (nitrogen-rich, NR) and A15 nitrogen-free (nitrogen-limited, NL) culture conditions were detected using Real-time Quantitative Reverse Transcription PCR (qRT-PCR). The bacterial cells of GXGL-4A can grow well in LB liquid medium containing 1% toluene, ethylbenzene or xylene, suggesting a good resistance to the tested aromatic hydrocarbons. The results of GC-MS analysis showed that K. radicincitans GXGL-4A has a good capability to degrade toluene, ethylbenzene, and xylene (TEX). Completion of the genome sequencing will no doubt contribute to the deep exploration and comprehensive utilization of this NF bacterium in sustainable agriculture and bioremediation of aromatic pollutants.

Artificial activation of nif gene expression in nodule bacteria Ex Planta

Background. Rhizobia are the most effective nitrogen-fixing organisms that can fix nitrogen only in symbiosis with leguminous plants. The general transcriptional activator of nitrogen fixation genes in diazotrophic bacteria is NifA. In this work, the possibility of modifying the regulation of nitrogen fixation in the nodule bacteria Mesorhizobium, Ensifer and Rhizobium was studied by introducing an additional copy of the nifA gene into the bacterial genomes during the regulation of induced bacterial promoters.&#x0D; Materials and methods. A series of expression genetic constructs with NifA genes of nodule bacteria strains under the control of an inducible promoter Pm were created. The resulting constructs were transformed into strains of nodule bacteria. The obtained recombinant strains were investigated for the appearance of their nitrogen-fixing activity in the free-living state.&#x0D; Results. It was shown that the expression of nifA in recombinant cells of all three genera of bacteria leads to the appearance of insignificant nitrogenase activity. At the same time, the level of nitrogenase activity does not have a correlation with the level of expression of the introduced nifA gene, which, most likely, is a consequence of the multilevel regulation of nitrogen fixation.&#x0D; Conclusion. The possibility of artificial activation of nitrogenase activity in nodule bacteria in the free-living state by introducing the NifA regulatory protein gene into bacteria was shown.

Accessory Proteins of the Nitrogenase Assembly, NifW, NifX/NafY, and NifZ, Are Essential for Diazotrophic Growth in the Nonheterocystous Cyanobacterium Leptolyngbya boryana.

Since nitrogenase is extremely vulnerable to oxygen, aerobic or micro-aerobic nitrogen-fixing organisms need to create anaerobic microenvironments in the cells for diazotrophic growth, which would be one of the major barriers to express active nitrogenase in plants in efforts to create nitrogen-fixing plants. Numerous cyanobacteria are able to fix nitrogen with nitrogenase by coping with the endogenous oxygen production by photosynthesis. Understanding of the molecular mechanisms enabling to the coexistence of nitrogen fixation and photosynthesis in nonheterocystous cyanobacteria could offer valuable insights for the transfer of nitrogen fixation capacity into plants. We previously identified the cnfR gene encoding the master regulator for the nitrogen fixation (nif) gene cluster in the genome of a nonheterocystous cyanobacterium Leptolyngbya boryana, in addition to initial characterization of the nif gene cluster. Here we isolated nine mutants, in which the nif and nif-related genes were individually knocked out in L. boryana to investigate the individual functions of (1) accessory proteins (NifW, NifX/NafY, and NifZ) in the biosynthesis of nitrogenase metallocenters, (2) serine acetyltransferase (NifP) in cysteine supply for iron-sulfur clusters, (3) pyruvate formate lyase in anaerobic metabolism, and (4) NifT and HesAB proteins. ΔnifW, ΔnifXnafY, and ΔnifZ exhibited the most severe phenotype characterized by low nitrogenase activity (<10%) and loss of diazotrophic growth ability. The phenotypes of ΔnifX, ΔnafY, and ΔnifXnafY suggested that the functions of the homologous proteins NifX and NafY partially overlap. ΔnifP exhibited significantly slower diazotrophic growth than the wild type, with lower nitrogenase activity (22%). The other four mutants (ΔpflB, ΔnifT, ΔhesA, and ΔhesB) grew diazotrophically similar to the wild type. Western blot analysis revealed a high correlation between nitrogenase activity and NifD contents, suggesting that NifD is more susceptible to proteolytic degradation than NifK in L. boryana. The phenotype of the mutants lacking the accessory proteins was more severe than that observed in heterotrophic bacteria such as Azotobacter vinelandii, which suggests that the functions of NifW, NifX/NafY, and NifZ are critical for diazotrophic growth of oxygenic photosynthetic cells. L. boryana provides a promising model for studying the molecular mechanisms that produce active nitrogenase, to facilitate the creation of nitrogen-fixing plants.

Genomic Manipulations of the Diazotroph Azotobacter vinelandii.

The biological reduction of nitrogen gas to ammonia is limited to a select group of nitrogen-fixing prokaryotes. While nitrogenase is the catalyst of nitrogen fixation in these biological systems, a consortium of additional gene products is required for the synthesis, activation, and catalytic competency of this oxygen-sensitive metalloenzyme. Thus, the biochemical complexity of this process often requires functional studies and isolation of gene products from the native nitrogen-fixing organisms. The strict aerobe Azotobacter vinelandii is the best-studied model bacterium among diazotrophs. This chapter provides a description of procedures for targeted genomic manipulation and isolation of A. vinelandii strains. These methods have enabled identification and characterization of gene products with roles in nitrogen fixation and other related aspects of metabolism. The ability to modify and control expression levels of targeted sequences provides a biotechnological tool to uncover molecular details associated with nitrogen fixation, as well as toexploit this model system as a host for expression of oxygen-sensitive proteins.