Nitrogenase Activity Research Articles

Degradation of Complex Carbon Sources in Organic Fertilizers Facilitates Nitrogen Fixation.

Biological nitrogen fixation is crucial for agriculture and improving fertilizer efficiency, but organic fertilizers in enhancing this process remain debated. Here, we investigate the impact of organic fertilizers on biological nitrogen fixation through experiments and propose a new model where bacterial interactions with complex carbon sources enhance nitrogen fixation. Field experiments showed that adding organic fertilizers increased the nitrogenase activity by 57.85%. Subculture experiments revealed that organic fertilizer addition enriched genes corresponding to complex carbon and energy metabolism, as well as nifJ involved in electron transfer for nitrogenase. It also enhanced bacterial interactions and enhanced connectors associated with complex carbon degradation. Validation experiments demonstrated that combinations increased nitrogenase activity by 2.98 times compared to the single. Our findings suggest that organic fertilizers promoted nitrogen fixation by enhancing microbial cooperation, improved the degradation of complex carbon sources, and thereby provided utilizable carbon sources, energy, and electrons to N-fixers, thus increasing nitrogenase activity and nitrogen fixation.

Deciphering the Nitrogen Fixation Gene Cluster in Vibrio natriegens: A Study on Optimized Expression and Application of Nitrogenase.

Microbial nitrogen fixation presents a viable alternative to chemical fertilizers, yet the limited colonization and specificity of naturally occurring nitrogen-fixing microorganisms present significant challenges to their widespread application. In this study, we identified a nitrogen fixation gene cluster (VNnif) in Vibrio natriegens (VN) and tested its nitrogenase activity through the acetylene reduction assay. We investigated the potential utilization of nitrogenase by incorporating the nitrogenase gene cluster from VN into plant growth-promoting rhizosphere bacteria Pseudomonas protegens CHA0 and enhancing its activity to 48.16 nmol C2H2/mg/h through promoter replacement and cluster rearrangement. The engineered strain CHA0-PVNnif was found to positively impact the growth of Arabidopsis thaliana col-0 and Triticum aestivum L. (wheat). This study expanded the role of plant growth-promoting rhizobacteria (PGPR) and provided a research foundation for enhancing nitrogenase activity.

Vigna marina as a Potential Leguminous Cover Crop for High Salinity Soils

The beach bean (Vigna marina) exhibits robust growth in habitats characterised by sandy substrates, limited nutrient availability, and elevated saline levels. The utilisation of V. marina, a potentially beneficial leguminous cover crop, allows for its cultivation in regions characterised by soil salinity, hence facilitating the alleviation of environmental stress and the promotion of nitrogen fixation within the soil. A study assessed the feasibility of V. marina as a leguminous cover crop, in which this legume was cultivated in both coastal and inland soils. Pueraria javanica and Mucuna bracteata, widely recognised as established leguminous cover crops, were used as the control in this experiment. The observations involved were total plant biomass, nitrogenase activity, and leaf chlorophyll content of the host plants. The experiment consisted of five replicates arranged in a randomised complete block design, respectively. The effects of commercialised rhizobial compost on the development of the leguminous plants planted in both plots were also investigated. The results indicated that V. marina flourished in coastal and inland soils with the highest leaf chlorophyll concentration throughout the eight weeks of growth. It showed that V. marina has the potential to outperform the other two established leguminous cover crops when planted in highly salinised soils. The results also showed evidence that V. marina was an excellent potential leguminous cover crop, especially for any agricultural plots of high salinity soils, compared to the other two well-established leguminous cover crops, P. javanica and M. bracteata.

Temporal variations of biological nitrogen fixation and diazotrophic communities associated with artificial seaweed farms

Diazotrophic communities contribute inorganic nitrogen for the primary productivity of the marine environment by biological nitrogen fixation (BNF). They play a vital role in the biogeochemical cycle of nitrogen in the marine ecological environment. However, there is still an incomplete understanding of BNF and diazotrophs in artificial seaweed farms. Therefore, this study comprehensively investigated the temporal variations of BNF associated with Gracilariopsis lemaneiformis, as well as the diazotrophic communities associated with macroalgae and its surrounding seawater. Our results revealed that a total of 13 strains belonging to Proteobacteria and Bacteroidetes were identified as N2-fixing bacteria using azotobacter selective solid medium and nifH gene cloning. Subsequently, BNF and diazotrophic communities were characterized using the acetylene reduction method and high-throughput sequencing of the nifH gene, respectively. The results showed that nitrogenase activity and nifH gene abundance of epiphytic bacteria on G. lemaneiformis varied significantly among four different cultivation periods, i.e., Cultivation Jan. (CJ), Cultivation Feb. (CF), Cultivation Mar. (CM), Cultivation Apr. (CA). Among them, the nitrogenase activity and nifH gene abundance of epiphytic bacteria on G. lemaneiformis in CM were significantly higher than those in CJ, CF, and CA, indicating that the BNF of eiphytic bacteria on G. lemaneiformis was markedly enhanced. Combined with the data on environmental factors, it was found that the low concentration of nitrogen and phosphorus in CM might considerably boost the BNF of epiphytic bacteria in G. lemaneiformis. The sequencing results of the nifH gene showed that the α-diversity of diazotrophic communities associated with G. lemaneiformis and seawater in CM was higher than that in other cultivation periods. In addition, the diazotrophic communities on G. lemaneiformis were significantly different in CJ, CF, CM, and CA, and they were significantly diverse from diazotrophic communities in seawater. LEfSe analysis indicated that Rhodobacterales, Hyphomonadaceae, Robiginitomaculum, and Robiginitomaculum antarcticum within α-proteobacteria played a remarkable role in BNF in response to nitrogen nutrient deficiency. Taken together, these results provide a unique insight into the interaction between macroalgae and its epiphytic bacteria and lay a foundation for further research on the mechanism of action of nitrogen-cycling microorganisms associated with macroalgae.

Azotosporobacter soli gen. nov., sp. nov., a novel nitrogen-fixing bacterium isolated from paddy soil.

A nitrogen-fixing strain designated SG130T was isolated from paddy soil in Fujian Province, China. Strain SG130T was Gram-staining-negative, rod-shaped, and strictly anaerobic. Strain SG130T showed the highest 16S rRNA gene sequence similarities with the type strains Dendrosporobacter quercicolus DSM 1736T (91.7%), Anaeroarcus burkinensis DSM 6283T (91.0%) and Anaerospora hongkongensis HKU 15T (90.9%). Furthermore, the phylogenetic and phylogenomic analysis also suggested strain SG130T clustered with members of the family Sporomusaceae and was distinguished from other genera within this family. Growth of strain SG130T was observed at 25-45°C (optimum 30°C), pH 6.0-9.5 (optimum 7.0) and 0-1% (w/v) NaCl (optimum 0.1%). The quinones were Q-8 and Q-9. The polar lipids were phosphatidylserine (PS), phosphatidylethanolamine (PE), glycolipid (GL), phospholipid (PL) and an unidentified lipid (UL). The major fatty acids (> 10%) were iso-C13:0 3OH (26.6%), iso-C17:1 (15.6%) and iso-C15:1 F (11.4%). The genomic DNA G + C content was 50.7%. The average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values between strain SG130T and the most closely related type strain D. quercicolus DSM 1736T (ANI 68.0% and dDDH 20.3%) were both below the cut-off level for species delineation. The average amino acid identity (AAI) between strain SG130T and the most closely related type strain D. quercicolus DSM 1736T was 63.2%, which was below the cut-off value for bacterial genus delineation (65%). Strain SG130T possessed core genes (nifHDK) involved in nitrogen fixation, and nitrogenase activity (106.38μmol C2H4 g-1 protein h-1) was examined using the acetylene reduction assay. Based on the above results, strain SG130T is confirmed to represent a novel genus of the family Sporomusaceae, for which the name Azotosporobacter soli gen. nov., sp. nov. is proposed. The type strain is SG130T (= GDMCC 1.3312T = JCM 35641T).

Endogenous clock-mediated regulation of intracellular oxygen dynamics is essential for diazotrophic growth of unicellular cyanobacteria

The discovery of nitrogen fixation in unicellular cyanobacteria provided the first clues for the existence of a circadian clock in prokaryotes. However, recalcitrance to genetic manipulation barred their use as model systems for deciphering the clock function. Here, we explore the circadian clock in the now genetically amenable Cyanothece 51142, a unicellular, nitrogen-fixing cyanobacterium. Unlike non-diazotrophic clock models, Cyanothece 51142 exhibits conspicuous self-sustained rhythms in various discernable phenotypes, offering a platform to directly study the effects of the clock on the physiology of an organism. Deletion of kaiA, an essential clock component in the cyanobacterial system, impacted the regulation of oxygen cycling and hindered nitrogenase activity. Our findings imply a role for the KaiA component of the clock in regulating the intracellular oxygen dynamics in unicellular diazotrophic cyanobacteria and suggest that its addition to the KaiBC clock was likely an adaptive strategy that ensured optimal nitrogen fixation as microbes evolved from an anaerobic to an aerobic atmosphere under nitrogen constraints.

Synergistic interaction of Piriformospora indica and microbial inoculants on symbiotic potential, plant nutrition and productivity of chickpea (Cicer arietinum)

The present study was carried out during the winter season of 2011–12 and 2012–13 at Indian Agricultural Re search Institute, New Delhi, to evaluate the efficacy of microbial bioinoculants in chickpea (Cicer arietinum L.) us ing combinatorial approach. A group of microbial inoculants (Mesorhizobium ciceri). Phosphate-solubilizing micro organisms and plant growth-promoting rhizobacteria) in presence and absence of P fertilizer was used to testify its synergistic effect with Piriformospora indica on symbiotic traits, growth and yield of chickpea under field condi tions. Significant differences were observed with respect to symbiotic potential and other associated parameters with combined inoculation. Inoculation with Mesorhizobium ciceri enhanced nitrogenase activity and leghemoglobin content significantly over un-inoculated control. Significant improvement in N and P nutrition with combined inoculation amended with 30 kg P/ha emphasized the beneficial effect of these and similar trend was reflected in productivity enhancement as well.

Microbial consortium with multifunctional attributes for the plant growth of eggplant (Solanum melongena L.).

In the past few decades, the pressure of higher food production to satisfy the demand of ever rising population has inevitably increased the use synthetic agrochemicals which have deterioration effects. Biostimulants containing beneficial microbes (single inoculants and microbial consortium) were found as an ideal substitute of synthetic chemical fertilizers. In recent years, microbial consortium is known as a better bioinoculant in comparison to single inoculant bioformulation because of multifarious plant growth-promoting advantages. Looking at the advantageous effect of consortium, in present investigation, different bacteria were isolated from rhizospheric soil and plant samples collected from the Himalayan mountains on the green slopes of the Shivaliks, Himachal Pradesh. The isolated bacteria were screened for nitrogen (N) fixation, phosphorus (P)solubilization and potassium (K) solubilization plant growth promotingattributes, and efficient strains were identified through 16S rRNA gene sequencingand BLASTn analysis. The bacteria showing a positive effect in NPK uptake were developed as bacterial consortium for the growth promotion of eggplant crop. A total of 188 rhizospheric and endophytic bacteria were sorted out, among which 13 were exhibiting nitrogenase activity, whereas 43 and 31 were exhibiting P and K solubilization traits, respectively. The selected three efficient and potential bacterial strains were identified using 16S rRNA gene sequencing as Enterobacter ludwigii EU-BEN-22 (N-fixer; 35.68 ± 00.9 nmol C2H4 per mg protein per h), Micrococcus indicus EU-BRP-6 (P-solubilizer; 201 ± 0.004 mg/L), and Pseudomonas gessardii EU-BRK-55 (K-solubilizer; 51.3 ± 1.7 mg/mL), and they were used to develop a bacterial consortium. The bacterial consortium evaluation on eggplant resulted in the improvement of growth (root/shoot length and biomass) and physiological parameters (chlorophyll, carotenoids, total soluble sugar, and phenolic content) of the plants with respect to single culture inoculation, chemical fertilizer, and untreated control. A bacterial consortium having potential to promote plant growth could be used as bioinoculant for horticulture crops growing in hilly regions.

Glutamate production from aerial nitrogen using the nitrogen-fixing bacterium Klebsiella oxytoca

Glutamate is an essential biological compound produced for various therapeutic and nutritional applications. The current glutamate production process requires a large amount of ammonium, which is generated through the energy-consuming and CO2-emitting Haber–Bosch process; therefore, the development of bio-economical glutamate production processes is required. We herein developed a strategy for glutamate production from aerial nitrogen using the nitrogen-fixing bacterium Klebsiella oxytoca. We showed that a simultaneous supply of glucose and citrate as carbon sources enhanced the nitrogenase activity of K. oxytoca. In the presence of glucose and citrate, K. oxytoca strain that was genetically engineered to increase the supply of 2-oxoglutarate, a precursor of glutamate synthesis, produced glutamate extracellularly more than 1 g L−1 from aerial nitrogen. This strategy offers a sustainable and eco-friendly manufacturing process to produce various nitrogen-containing compounds using aerial nitrogen.

Multidimensional engineering of Rhodobacter sphaeroides for enhanced photo-fermentative hydrogen production

The photo-fermentative hydrogen production by purple non-sulfur photosynthetic bacteria (PNSB) is regarded as a promising approach for sustainable hydrogen production. In PNSB, biohydrogen synthesis is catalyzed by nitrogenase which requires two essential cofactors of adenosine triphosphate (ATP) and electron. To avoid the insufficient ATP supply that limits the nitrogenase activity for hydrogen production, we systematically engineered Rhodobacter sphaeroides to improve the hydrogen production by manipulating the light harvesting (LH) complexes, the electron transfer chain (ETC), the adenosine diphosphate (ADP) synthetic pathway, the F0F1-ATPase expression, and the nitrogenase expression. To improve the photosynthetic efficiency, we engineered the LH complexes by eliminating pucBA and overexpressing pufQ and spbA. Then, the ETC was boosted by deleting qxt-type oxidase and integrating the electron carrier cytochrome cy at the site of cyt cbb3 oxidase. In addition, we increased the supply of precursor ADP by overexpressing adk, and the abundance of F0F1-ATPase was efficiently enhanced through overexpressing atpXF. Further transposon engineering with the integration of nifHDK and atpXF resulted in a mutant YH27 with a substantial improvement of performance over the base strain, and its nitrogenase activity and hydrogen production were enhanced by 404 % and 93 %, respectively. Taken together, the multidimensional strategies enabled the engineered R. sphaeroides with a hydrogen productivity of 61.2 mL/(Lh) and a yield of 3.0 mol/mol acetate under the fed-batch condition, which represents one of the most efficient biohydrogen-producing strains.

Graphene quantum dots enhance the biological nitrogen fixation by up-regulation of cellular metabolism and electron transport

Graphene quantum dots (GQDs) are useful nanomaterials of excellent water solubility, biocompatibility and optical stability for various applications. In this study, we achieved the nitrogen fixation activity enhancement of autogenous azotobacters (Azotobacter vinelandii) by GQDs and revealed its mechanism. GQDs (1–10 mg/L) accelerated the logarithmic growth phase of A. vinelandii, making bacteria enter the plateau earlier. Biocompatible GQDs did not cause cell death and oxidative damage of A. vinelandii. The expression levels of metabolism and nitrogen fixation related genes were significantly up-regulated by GQDs, thereby enhancing the activity of nitrogenase. The nitrogen fixation activity of A. vinelandii reached 471.7 % of the control upon the exposure to GQDs. The intracellular polyhydroxyalkanoate vesicles were consumed to provide energy for nitrogen fixation, which involved with a chain reaction of carbohydrate metabolism, lipid metabolism, and energy metabolism pathways accompanied by significant increases in electron transfer rate. The synergistic effect of GQDs on A. vinelandii further facilitated the augmentation of soil nitrogen content in Qinghai-Tibet Plateau. Our results provided an effective approach to enhance the biological nitrogen fixation by GQDs.

Inoculated biocrust cover and functions diverged over a gradient of soil textures and water availability

Restoring biological crust (biocrust) in disturbed drylands is challenging due to the difficult environmental conditions, such as limited soil moisture, low soil nutrients, and extreme temperatures, that impede growth. Understanding how the key components of biocrust—mosses, lichens, and cyanobacteria—react to different environmental factors informs the optimal timing, locations, and species composition for biocrust reintroduction, thereby increasing the likelihood of establishment. Here, we inoculated soils with a diverse range of biocrust organisms, analogous to seeding an area with diverse vascular plant seeds, and varied environmental conditions to observe how these changes influenced the development and functions of reintroduced biocrust. We found that by manipulating soil texture and time spent wet, we can change the proportional cover of biocrust within a restoration‐like setting. Specifically, we found that 4 months after inoculation, finer textured soils that received more water become dominated by moss cover, while coarser textured soils with less water remained dominated by cyanobacteria cover, and the interactions between texture and time spent wet strongly influenced cover. We found biocrust morphological group cover had a small, but detectable, effect on ecosystem functions (soil stability and nitrogenase activity, a proxy for nitrogen fixation), but that environmental conditions had a stronger impact on the functions we measured. Manipulative experiments in controlled environments, like this one, can help elucidate the mechanisms underlying the establishment rate and patterns of biocrusts post‐inoculation, and inform implementation of inoculations in the field.

Characterization of ferredoxins involved in electron transfer pathways for nitrogen fixation implicates differences in electronic structure in tuning 2[4Fe[sbnd]4S] Fd activity

Ferredoxins (Fds) are small proteins which shuttle electrons to pathways like biological nitrogen fixation. Physical properties tune the reactivity of Fds with different pathways, but knowledge on how these properties can be manipulated to engineer new electron transfer pathways is lacking. Recently, we showed that an evolved strain of Rhodopseudomonas palustris uses a new electron transfer pathway for nitrogen fixation. This pathway involves a variant of the primary Fd of nitrogen fixation in R. palustris, Fer1, in which threonine at position 11 is substituted for isoleucine (Fer1T11I). To understand why this substitution in Fer1 enables more efficient electron transfer, we used in vivo and in vitro methods to characterize Fer1 and Fer1T11I. Electrochemical characterization revealed both Fer1 and Fer1T11I have similar redox transitions (−480 mV and − 550 mV), indicating the reduction potential was unaffected despite the proximity of T11 to an iron‑sulfur (FeS) cluster of Fer1. Additionally, disruption of hydrogen bonding around an FeS cluster in Fer1 by substituting threonine with alanine (T11A) or valine (T11V) did not increase nitrogenase activity, indicating that disruption of hydrogen bonding does not explain the difference in activity observed for Fer1T11I. Electron paramagnetic resonance spectroscopy studies revealed key differences in the electronic structure of Fer1 and Fer1T11I, which indicate changes to the high spin states and/or spin-spin coupling between the FeS clusters of Fer1. Our data implicates these electronic structure differences in facilitating electron flow and sets a foundation for further investigations to understand the connection between these properties and intermolecular electron transfer.

Biological activity of sod-podzolic soils at different levels of radioactive contamination

The biological (enzymatic) activity of sod-podzolic soils has been investigated in the zone of mandatory resettlement and in the alienation zone of the Chornobyl Nuclear Power Plant (CNPP) under the influence of ionizing radiation. Gas chromatographic methods were used to determine potential nitrogenfixing (nitrogenase) activity, biochemical methods were applied to study the activity of hydrolytic enzymes (cellulase and protease) responsible for the decomposition of plant residues in the soil, as well as enzymes involved in oxidative-reductive reactions of organic matter transformation (catalase and polyphenoloxidases). Statistical analysis was also employed. The results of the conducted research indicate that a relatively low increase in the total dose rate in the radioactive contamination area (from 0.2 to 1.6 µGy/h) at Polygon No. 1 (the zone of mandatory resettlement near the village of Khrystynivka, Narodychi district, Zhytomyr region) stimulated potential nitrogenase activity of soil diazotrophs and the activity of both hydrolytic enzymes and oxidoreductases. High dose rates (up to 22.2, 61.6, and especially up to 84.0 mGy/h) at Polygon No. 2, located in the alienation zone of the CNPP directly in the area of the former Red Forest, led to a decrease in soil enzymatic activity. Thus, the obtained results unequivocally demonstrate the different nature of the impact of ionizing radiation levels on the biological activity of sod-podzolic soils. The more radioactive soil in the zone of mandatory resettlement stimulates the enzymatic activity of soil microbiota. This could be attributed to adaptive changes in the development and activity of microorganisms or changes in the composition of their communities, with a prevalence of radio-tolerant microbiota representatives. It is also possible that the manifestation of a radiobiological effect, such as radiation stimulation (radiation hormesis), accelerating the growth and development of microorganisms, may occur. In the alienation zone of the CNPP, with high radioactive contamination, the negative influence of ionizing radiation on the metabolism of soil microorganisms is preserved. The obtained results are highly correlated with the indicators of the abundance of nitrogen-fixing microorganisms in the investigated soils, as well as microorganisms representing the saccharolytic (fungi and cellulolytic bacteria) and peptolytic (ammonifiers) pathways of plant residue degradation, as previously demonstrated by us.

Arbuscular mycorrhizal fungi and rhizobia accelerate plant growth and N accumulation and contribution to soil total N in white clover by difficultly extractable glomalin-related soil protein

Arbuscular mycorrhizal fungi and rhizobia are soil symbiotic microorganisms involved in plant nitrogen (N) acquisition, whereas it is unclear how single or combined inoculation of both contributes to soil total N in legume crops. This study analyzed the effects of single or combined inoculation with arbuscular mycorrhizal fungus (Paraglomus occultum) and rhizobium (Rhizobium leguminosarum bv. trifolii) on growth performance, root soluble protein, leghemoglobin, NH4+-N, NO3−-N, and N concentrations, nitrogenase activities, soil total N contents, N contents in glomalin-related soil protein (GRSP), as well as the contribution of N in GRSP to soil total N in white clover. Twelve weeks after inoculation, P. occultum promoted nodule formation, and R. leguminosarum bv. trifolii accelerated root mycorrhizal colonization. Single or combined inoculation with P. occultum and R. leguminosarum bv. trifolii improved biomass, root total length, surface area and volume, root soluble protein concentrations, nodule leghemoglobin concentrations, and nitrogenase activities to varying degrees, with the trend of improvement being more pronounced with combined inoculation. R. leguminosarum bv. trifolii and/or P. occultum significantly increased NO3−-N, NH4+-N, and total N concentrations in nodules and roots, as well as soil total N concentrations. The N in easily extractable GRSP (EE-GRSP) and difficultly extractable GRSP (DE-GRSP) was 4.37–5.64 mg/g and 6.69–7.83 mg/g, respectively, contributing 2.68–3.26 % and 2.28–3.10 % of soil total N. P. occultum and combined inoculation accelerated the production of EE-GRSP and DE-GRSP and N in EE-GRSP. R. leguminosarum bv. trifolii not only significantly increased DE-GRSP production but also reduced N in DE-GRSP. R. leguminosarum bv. trifolii or P. occultum significantly increased the contribution of N in DE-GRSP, not EE-GRSP, to soil total N. It was concluded that arbuscular mycorrhizal fungus and rhizobium accelerated N accumulation and contribution to soil total N in white clover by DE-GRSP.

Comparing the benthic nitrogenase activity and diazotrophic community assembly of three large river-connected freshwater lakes in eastern China

Biological nitrogen fixation (BNF) is a crucial process that provides bioavailable nitrogen and supports primary production in freshwater lake ecosystems. However, the characteristics of diazotrophic community and nitrogenase activity in freshwater lake sediments remain poorly understood. Here, we investigated the diazotrophic communities and nitrogenase activities in the sediments of three large river-connected freshwater lakes in eastern China using 15N-isotope tracing and nifH sequencing. The sediments in these lakes contained diverse nitrogenase genes that were phylogenetically grouped into Clusters I and III. The diazotrophic communities in the sediments were dominated by stochastic processes in Hongze Lake and Taihu Lake, which had heterogeneous habitats and shallower water depths, while in Poyang Lake, which had deeper water and a shorter hydraulic retention time, the assembly of the diazotrophic community in the sediments was dominated by homogeneous selection processes. Temperature and water depth were also found the key environmental factors affecting the sediment diazotrophic communities. Sediment nitrogenase activities varied in the three lakes and within distinct regions of an individual lake, ranging from 0 to 14.58 nmol/(kg·hr). Nitrogenase activity was significantly correlated with ferric iron, total phosphorus, and organic matter contents. Our results suggested that freshwater lake sediment contain high diversity of nitrogen-fixing microorganisms with potential metabolic diversity, and the community assembly patterns and nitrogenase activities varied with the lake habitat.

Amelioration of NaCl stress on germination, growth, and nitrogen fixation of Viciafaba at isosmotic Na–Ca combinations and Rhizobium

Main conclusionThe Na+/Ca2+ ratio of 1/5 ameliorated the inhibitory action of NaCl and improved the germination and growth of Vicia faba. Addition of Rhizobium also enhanced nodulation and nitrogen fixation.Casting light upon the impact of salinity stress on growth and nitrogen fixation of Vicia faba supplemented with Rhizobium has been traced in this work. How Ca2+ antagonizes Na+ toxicity and osmotic stress of NaCl was also targeted in isosmotic combinations of NaCl and CaCl2 having various Na+:Ca2+ ratios. Growth of Viciafaba (cultivar Giza 3) was studied at two stages: germination and seedling. At both experiments, seeds or seedlings were exposed to successively increasing salinity levels (0, 50, 100, 150, and 200 mM NaCl) as well as isosmotic combinations of NaCl and CaCl2 (Na+:Ca2+ of 1:1, 1:5, 1:10, 1:15, 1:18, and 1: 20), equivalent to 150 mM NaCl. Inocula of the local nitrogen-fixing bacteria, Rhizobium leguminosarum (OP715892) were supplemented at both stages. NaCl salinity exerted a negative impact on growth and metabolism of Vicia faba; inhibition was proportional with increasing salinity level up to the highest level of 200 mM. Seed germination, shoot and root lengths, fresh and dry weights, chlorophyll content, and nodules (number, weight, leghemoglobin, respiration, and nitrogenase activity) were inhibited by salinity. Ca2+ substitution for Na+, particularly at a Na/Ca ratio of 1:5, was stimulatory to almost all parameters at both stages. Statistical correlations between salinity levels and Na/Ca combinations proved one of the four levels (strong- or weak positive, strong- or weak negative) with most of the investigated parameters, depending on the parameter.

Restoration of the Functional nif Gene Cluster by Complex Recombination Events during Heterocyst Development in the Nitrogen-Fixing Cyanobacterium Calothrix sp. NIES-4101.

In the genome of the heterocystous cyanobacterium Calothrix sp. NIES-4101 (NIES-4101), the four genes essential for nitrogen fixation (nifB, nifH, nifD and nifK) are highly fragmented into 13 parts in a 350-kb chromosomal region, and four of these parts are encoded in the reverse strand. Such a complex fragmentation feature makes it difficult to restore the intact nifBHDK genes by the excision mechanism found in the nifD gene of the Anabaena sp. PCC 7120 heterocyst. To examine the nitrogen-fixing ability of NIES-4101, we confirmed that NIES-4101 grew well on a combined nitrogen-free medium and showed high nitrogenase activity, which strongly suggested that the complete nifBHDK genes are restored by a complex recombination process in heterocysts. Next, we resequenced the genome prepared from cells grown under nitrogen-fixing conditions. Two contigs covering the complete nifHDK and nifB genes were found by de novo assembly of the sequencing reads. In addition, the DNA fragments covering the nifBHDK operon were successfully amplified by PCR. We propose that the process of nifBHDK restoration occurs as follows. First, the nifD-nifK genes are restored by four excision events. Then, the complete nifH and nifB genes are restored by two excision events followed by two successive inversion events between the inverted repeat sequences and one excision event, forming the functional nif gene cluster, nifB-fdxN-nifS-nifU-nifH-nifD-nifK. All genes coding recombinases responsible for these nine recombination events are located close to the terminal repeat sequences. The restoration of the nifBHDK genes in NIES-4101 is the most complex genome reorganization reported in heterocystous cyanobacteria.

Pontiella agarivorans sp. nov., a novel marine anaerobic bacterium capable of degrading macroalgal polysaccharides and fixing nitrogen.

Marine macroalgae produce abundant and diverse polysaccharides, which contribute substantially to the organic matter exported to the deep ocean. Microbial degradation of these polysaccharides plays an important role in the turnover of macroalgal biomass. Various members of the Planctomycetes-Verrucomicrobia-Chlamydia (PVC) superphylum are degraders of polysaccharides in widespread anoxic environments. In this study, we isolated a novel anaerobic bacterial strain NLcol2T from microbial mats on the surface of marine sediments offshore Santa Barbara, CA, USA. Based on 16S ribosomal RNA (rRNA) gene and phylogenomic analyses, strain NLcol2T represents a novel species within the Pontiella genus in the Kiritimatiellota phylum (within the PVC superphylum). Strain NLcol2T is able to utilize various monosaccharides, disaccharides, and macroalgal polysaccharides such as agar and ɩ-carrageenan. A near-complete genome also revealed an extensive metabolic capacity for anaerobic degradation of sulfated polysaccharides, as evidenced by 202 carbohydrate-active enzymes (CAZymes) and 165 sulfatases. Additionally, its ability of nitrogen fixation was confirmed by nitrogenase activity detected during growth on nitrogen-free medium, and the presence of nitrogenases (nifDKH) encoded in the genome. Based on the physiological and genomic analyses, this strain represents a new species of bacteria that may play an important role in the degradation of macroalgal polysaccharides and with relevance to the biogeochemical cycling of carbon, sulfur, and nitrogen in marine environments. Strain NLcol2T (= DSM 113125T = MCCC 1K08672T) is proposed to be the type strain of a novel species in the Pontiella genus, and the name Pontiella agarivorans sp. nov. is proposed.IMPORTANCEGrowth and intentional burial of marine macroalgae is being considered as a carbon dioxide reduction strategy but elicits concerns as to the fate and impacts of this macroalgal carbon in the ocean. Diverse heterotrophic microbial communities in the ocean specialize in these complex polymers such as carrageenan and fucoidan, for example, members of the Kiritimatiellota phylum. However, only four type strains within the phylum have been cultivated and characterized to date, and there is limited knowledge about the metabolic capabilities and functional roles of related organisms in the environment. The new isolate strain NLcol2T expands the known substrate range of this phylum and further reveals the ability to fix nitrogen during anaerobic growth on macroalgal polysaccharides, thereby informing the issue of macroalgal carbon disposal.

Species Diversity, Nitrogen Fixation, and Nutrient Solubilization Activities of Endophytic Bacteria in Pea Embryos

Endophytic bacteria, especially those that participate in nitrogen fixation, play critical roles in supplying essential nutrients for legume plant growth. Despite that there have been numerous investigations targeting bacterial microbiomes in legume roots and nodules, little is known about embryonic bacteria that facilitate plant nutrient utilization after seed germination. Here, we collected and investigated endophytic bacterial microbiome in edible pea (Pisum sativum) embryos using five representative cultivars and a pea sprout (shoot of pea [SHP]) control. Twenty-six nitrogen-fixing bacteria (NFB) were isolated from pea embryos, with three strains found in fresh grain pea (FGP) and snow pea (SP) exhibiting the strongest nitrogenase activity of above 85 nmol C2H4/mL/h. Some NFB isolates are also potassium-solubilizing bacteria (KSB) or phosphorus-solubilizing bacteria (PSB) utilizing inorganic and/or organic phosphorus. All 26 NFB showed variable levels (0.41 to 7.10 μg/mL) of indole-3-acetic acid (IAA) secretion. The nutrient-solubilizing NFB identified in our research are potential targets for biofertilizer development. They could be useful in converting nitrogen, potassium, and/or phosphorus into usable forms for the plants. At the microbiome level, high-throughput 16S ribosomal RNA (rRNA) sequencing of 40 bacterial collections from pea embryos generated 4234 operational taxonomic units (OTUs) using 97% identity as the threshold for clustering high-quality effective reads (valid tags). Analysis of OTU annotation results revealed similar species community structures, abundance, and diversity in most samples. Our embryo-derived endophytic bacterial pool provides a microbiome platform for seed dormancy and germination research of edible peas, as well as for digging new biofertilizer resources in general.