Nitrogen Fixation Genes Research Articles

Metagenomic analysis reveals the microbial response to petroleum contamination in oilfield soils

The response of the microbes to total petroleum hydrocarbons (TPHs) in three types of oilfield soils was researched using metagenomic analysis. The ranges of TPH concentrations in the grassland, abandoned well, working well soils were 1.16 × 102–3.50 × 102 mg/kg, 1.14 × 103–1.62 × 104 mg/kg, and 5.57 × 103–3.33 × 104 mg/kg, respectively. The highest concentration of n-alkanes and 16 PAHs were found in the working well soil of Shengli (SL) oilfield compared with those in Nanyang (NY) and Yanchang (YC) oilfields. The abandoned well soils showed a greater extent of petroleum biodegradation than the grassland and working well soils. Α-diversity indexes based on metagenomic taxonomy showed higher microbial diversity in grassland soils, whereas petroleum-degrading microbes Actinobacteria and Proteobacteria were more abundant in working and abandoned well soils. RDA demonstrated that low moisture content (MOI) in YC oilfield inhibited the accumulation of the petroleum-degrading microbes. Synergistic networks of functional genes and Spearman's correlation analysis showed that heavy petroleum contamination (over 2.10 × 104 mg/kg) negatively correlated with the abundance of the nitrogen fixation genes nifHK, however, in grassland soils, low petroleum content facilitated the accumulation of nitrogen fixation genes. A positive correlation was observed between the abundance of petroleum-degrading genes and denitrification genes (bphAa vs. nirD, todC vs. nirS, and nahB vs. nosZ), whereas a negative correlation was observed between alkB (alkane- degrading genes) and amo (ammonia oxidation), hao (nitrification). The ecotoxicity of petroleum contamination, coupled with petroleum hydrocarbons (PH) degradation competing with nitrifiers for ammonia inhibited ammonia oxidation and nitrification, whereas PH metabolism promoted the denitrification process. Moreover, positive correlations were observed between the abundance of amo gene and MOI, as well as between the abundance of the dissimilatory nitrate reduction gene nirA and clay content. Thus, improving the soil physicochemical properties is a promising approach for decreasing nitrogen loss and alleviating petroleum contamination in oilfield soils.

The Salinity Survival Strategy of Chenopodium quinoa: Investigating Microbial Community Shifts and Nitrogen Cycling in Saline Soils.

Quinoa is extensively cultivated for its nutritional value, and its exceptional capacity to endure elevated salt levels presents a promising resolution to the agricultural quandaries posed by salinity stress. However, limited research has been dedicated to elucidating the correlation between alterations in the salinity soil microbial community and nitrogen transformations. To scrutinize the underlying mechanisms behind quinoa's salt tolerance, we assessed the changes in microbial community structure and the abundance of nitrogen transformation genes across three distinct salinity thresholds (1 g·kg-1, 3 g·kg-1, and 6 g·kg-1) at two distinct time points (35 and 70 days). The results showed the positive effect of quinoa on the soil microbial community structure, including changes in key populations and its regulatory role in soil nitrogen cycling under salt stress. Choroflexi, Acidobacteriota, and Myxococcota were inhibited by increased salinity, while the relative abundance of Bacteroidota increased. Proteobacteria and Actinobacteria showed relatively stable abundances across time and salinity levels. Quinoa possesses the ability to synthesize or modify the composition of keystone species or promote the establishment of highly complex microbial networks (modularity index > 0.4) to cope with fluctuations in external salt stress environments. Furthermore, quinoa exhibited nitrogen (N) cycling by downregulating denitrification genes (nirS, nosZ), upregulating nitrification genes (Archaeal amoA (AOA), Bacterial amoA (AOB)), and stabilizing nitrogen fixation genes (nifH) to absorb nitrate-nitrogen (NO3-_N). This study paves the way for future research on regulating quinoa, promoting soil microbial communities, and nitrogen transformation in saline environments.

Grass-legume mixtures maintain forage biomass under microbial diversity loss via gathering Pseudomonas in root zone soil.

Grass-legume mixtures are a common practice for establishing artificial grasslands, directly or indirectly contributing to the improvement of yield. In addition, this method helps maintain soil and plant health by reducing the use of chemical fertilizers. The impact of grass-legume mixtures on yield and its underlying microbial mechanisms have been a focus of scientific investigation. However, the benefits of mixtures in the context of soil microbial diversity loss remain a problem worthy of exploration. In this study, we examined different aboveground and belowground diversity combinations to elucidate the mechanisms by which grass-legume mixtures help maintain stable yields in the face of diversity loss. We identified the significantly enriched Pseudomonas genus microbial ASV53, which was gathered through homogeneous selection and served as a keystone in the co-occurrence network. ASV53 showed a strong positive correlation with biomass and the abundance of nitrogen-fixing genes. These findings provide a new theoretical foundation for utilizing grass-legume mixtures to enhance grass yields and address the challenges posed by diversity loss.

Nitrogen fixation and transcriptome of a new diazotrophic Geomonas from paddy soils.

The ability of Geomonas species to fix nitrogen gas (N2) is an important metabolic feature for its application as a plant growth-promoting rhizobacterium. This research is of great importance as it provides the first comprehensive direct experimental evidence of nitrogen fixation by the genus Geomonas in pure culture. We isolated a number of Geomonas strains from paddy soils and determined that nifH was present in these strains. This study demonstrated that these Geomonas species harbored genes encoding nitrogenase, as do Geobacter and Anaeromyxobacter in the same class of Deltaproteobacteria. We demonstrated N2-dependent growth of Geomonas and determined regulation of gene expression associated with nitrogen fixation. The research establishes and advances our understanding of nitrogen fixation in Geomonas.

Single-nucleus transcriptomes reveal spatiotemporal symbiotic perception and early response in Medicago.

Establishing legume-rhizobial symbiosis requires precise coordination of complex responses in a time- and cell type-specific manner. Encountering Rhizobium, rapid changes of gene expression levels in host plants occur in the first few hours, which prepare the plants to turn off defence and form a symbiotic relationship with the microbes. Here, we applied single-nucleus RNA sequencing to characterize the roots of Medicago truncatula at 30 min, 6 h and 24 h after nod factor treatment. We found drastic global gene expression reprogramming at 30 min in the epidermis and cortex and most of these changes were restored at 6 h. Moreover, plant defence response genes are activated at 30 min and subsequently suppressed at 6 h in non-meristem cells. Only in the cortical cells but not in other cell types, we found the flavonoid synthase genes required to recruit rhizobia are highly expressed 30 min after inoculation with nod factors. A gene module enriched for symbiotic nitrogen fixation genes showed that MtFER (MtFERONIA) and LYK3 (LysM domain receptor-like kinase 3) share similar responses to symbiotic signals. We further found that MtFER can be phosphorylated by LYK3 and it participates in rhizobial symbiosis. Our results expand our understanding of dynamic spatiotemporal symbiotic responses at the single-cell level.

Multi-environment ecogenomics analysis of the cosmopolitan phylum Gemmatimonadota.

Gemmatimonadota is a diverse bacterial phylum commonly found in environments such as soils, rhizospheres, fresh waters, and sediments. So far, the phylum contains just six cultured species (five of them sequenced), which limits our understanding of their diversity and metabolism. Therefore, we analyzed over 400 metagenome-assembled genomes (MAGs) and 5 culture-derived genomes representing Gemmatimonadota from various aquatic environments, hydrothermal vents, sediments, soils, and host-associated (with marine sponges and coral) species. The principal coordinate analysis based on the presence/absence of genes in Gemmatimonadota genomes and phylogenomic analysis documented that marine and host-associated Gemmatimonadota were the most distant from freshwater and wastewater species. A smaller genome size and coding sequences (CDS) number reduction were observed in marine MAGs, pointing to an oligotrophic environmental adaptation. Several metabolic pathways are restricted to specific environments. For example, genes for anoxygenic phototrophy were found only in freshwater, wastewater, and soda lake sediment genomes. There were several genomes from soda lake sediments and wastewater containing type IC/ID ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO). Various genomes from wastewater harbored bacterial type II RuBisCO, whereas RuBisCO-like protein was found in genomes from fresh waters, soil, host-associated, and marine sediments. Gemmatimonadota does not contain nitrogen fixation genes; however, the nosZ gene, involved in the reduction of N2O, was present in genomes from most environments, missing only in marine water and host-associated Gemmatimonadota. The presented data suggest that Gemmatimonadota evolved as an organotrophic species relying on aerobic respiration and then remodeled its genome inventory when adapting to particular environments. IMPORTANCE Gemmatimonadota is a rarely studied bacterial phylum consisting of a handful of cultured species. Recent culture-independent studies documented that these organisms are distributed in many environments, including soil, marine, fresh, and waste waters. However, due to the lack of cultured species, information about their metabolic potential and environmental role is scarce. Therefore, we collected Gemmatimonadota metagenome-assembled genomes (MAGs) from different habitats and performed a systematic analysis of their genomic characteristics and metabolic potential. Our results show how Gemmatimonadota have adapted their genomes to different environments.

The response of microbial compositions and functions to chronic single and multiple antibiotic exposure by batch experiment

Understanding the response of the microbial community to external disturbances such as micropollutants is vital for ecological risk evaluation. In this study, the effect of chronic antibiotic exposure on community compositions and functions was investigated by two batch experiments. The first experiment investigated the effect of chronic sulfamethoxazole (SMX) exposure, while the second investigated the combined effect of dissolved organic matter (DOM) sources and multi-antibiotic exposure. The results showed that the community responses to chronic antibiotic exposure depended on the dynamic balance among community resistance, adaptation, recovery, and selection, leading to nonlinear composition diversity variations. The disturbance strength of chronic SMX exposure increased with concentration (0.5–50 μg/L). However, complex sources and structures of coexisting organic matter might delay the disturbance by elevating metabolic activity and generating functional redundancy. Especially, when nutrient was a limiting factor, the disturbance strength by DOM source was greater than that by chronic antibiotic exposure. The resistance of abundant taxa to external distributions resulted in a low explanation of community diversity, while rare taxa played key roles in response to community variation and thereby affected community assembly. Long-term SMX exposure reduced the number of key species and favored the deterministic assembly process by 21%. However, elevated community adaptability might weaken the influence of antibiotic selection. Chronic SMX exposure elevated the relative abundance of sulfonamide resistance genes (sul1, sul2) by a factor of 1.2–4.3, while that of nitrogen-fixing genes (nifH, nifK) and the metabolic pathways related to the toluene, ethylbenzene, and dioxin degradation decreased. However, the combined influence of DOM sources and multi-antibiotic exposure barely caused the difference in the genes linking to element metabolism and drug resistance of microbial communities between blank and exposed groups. This study suggested that more concern should be given to the chronic environmental effect of organic micropollutants.

Exploring the nitrogen fixing strategy of bacterial communities in nitrogen cycling by adding calcium superphosphate at various periods during composting

Chicken manure, as an organic solid waste with a high nitrogen content, generates large amounts of ammonia during composting, which leads to pollution of the surrounding environment, and causes a reduction in the quality of the compost product. Nitrogen is transformed through the nitrogen cycle and bacterial communities are the main contributors to the transformation of the nitrogen cycle. The microbial composition changes dramatically at different stages during composting. Therefore, calcium superphosphate (SSP) was added to compost as a nitrogen-fixing agent to elucidate the strategy and function of the bacterial community involved in the nitrogen cycle. The results showed that the addition of SSP at the initial, high temperature and cooling stages increased the inorganic nitrogen (NH4+-N, NO3−-N) content by 51.99 %, 202.72 % and 173.37 % compared to CK, respectively. In addition, nitrogen cycle functional genes (gdh, nifH, pmoA-amoA, hao, nxrA, nirK, napA, nosZ, narG) abundance were determined by real-time qPCR. The nitrogen cycle genetic results showed that SSP addition at high temperature phase resulted in a 62.43 % down-regulation of ammonification genes, while nitrogen fixation and nitrification genes were enhanced. Random forests revealed a shift in the participation strategy of bacterial communities (e.g., Mycobacterium, Izemoplasmatales, Paracoccus, Ruminococcus) within the nitrogen cycle, leading to altered importance rankings despite involvement in different nitrogen cycle pathways. Moreover, Regression analysis and structural equation modelling revealed that SSP addition at high temperature stage stimulated the bacterial community engaged in nitrogen fixation and nitrification, resulting in increased nitrogen accumulation as NO3−-N during composting. This paper offers the potential to yield novel scientific insights into the impact of microbially mediated nitrogen transformation processes and reduce gaseous pollution.

Organelle-dependent polyprotein designs enable stoichiometric expression of nitrogen fixation components targeted to mitochondria

Introducing nitrogen fixation (nif ) genes into eukaryotic genomes and targeting Nif components to mitochondria or chloroplasts is a promising strategy for engineering nitrogen-fixing plants. A prerequisite for achieving nitrogen fixation in crops is stable and stoichiometric expression of each component in organelles. Previously, we designed a polyprotein-based nitrogenase system depending on Tobacco Etch Virus protease (TEVp) to release functional Nif components from five polyproteins. Although this system satisfies the demand for specific expression ratios of Nif components in Escherichia coli, we encountered issues with TEVp cleavage of polyproteins targeted to yeast mitochondria. To overcome this obstacle, a version of the Nif polyprotein system was constructed by replacing TEVp cleavage sites with minimal peptide sequences, identified by knowledge-based engineering, that are susceptible to cleavage by the endogenous mitochondrial-processing peptidase. This replacement not only further reduces the number of genes required, but also prevents potential precleavage of polyproteins outside the target organelle. This version of the polyprotein-based nitrogenase system achieved levels of nitrogenase activity in E. coli, comparable to those observed with the TEVp-based polyprotein nitrogenase system. When applied to yeast mitochondria, stable and balanced expression of Nif components was realized. This strategy has potential advantages, not only for transferring nitrogen fixation to eukaryotic cells, but also for the engineering of other metabolic pathways that require mitochondrial compartmentalization.

Complete genome sequence of Rhizobium leguminosarum bv. viciae SRDI969, an acid-tolerant, efficient N2-fixing microsymbiont of Vicia faba.

We report the complete genome sequence of Rhizobium leguminosarum bv. viciae SRDI969, an acid-tolerant, efficient nitrogen-fixing microorganism of Vicia faba. The 6.8 Mbp genome consists of a chromosome and four plasmids, with the symbiosis and nitrogen fixation genes encoded on the chromosome.

Structural insights into redox signal transduction mechanisms in the control of nitrogen fixation by the NifLA system

NifL is a conformationally dynamic flavoprotein responsible for regulating the activity of the σ54-dependent activator NifA to control the transcription of nitrogen fixation (nif) genes in response to intracellular oxygen, cellular energy, or nitrogen availability. The NifL-NifA two-component system is the master regulatory system for nitrogen fixation. NifL serves as a sensory protein, undergoing signal-dependent conformational changes that modulate its interaction with NifA, forming the NifL-NifA complex, which inhibits NifA activity in conditions unsuitable for nitrogen fixation. While NifL-NifA regulation is well understood, these conformationally flexible proteins have eluded previous attempts at structure determination. In work described here, we advance a structural model of the NifL dimer supported by a combination of scattering techniques and mass spectrometry (MS)-coupled structural analyses that report on the average structure in solution. Using a combination of small angle X-ray scattering-derived electron density maps and MS-coupled surface labeling, we investigate the conformational dynamics responsible for NifL oxygen and energy responses. Our results reveal conformational differences in the structure of NifL under reduced and oxidized conditions that provide the basis for a model for modulating NifLA complex formation in the regulation of nitrogen fixation in response to oxygen in the model diazotroph, Azotobacter vinelandii.

Brytella acorum gen. nov., sp. nov., a novel acetic acid bacterium from sour beverages

Polyphasic taxonomic and comparative genomic analyses revealed that a series of lambic beer isolates including strain LMG 32668T and the kombucha isolate LMG 32879 represent a novel species among the acetic acid bacteria, with Acidomonas methanolica as the nearest phylogenomic neighbor with a valid name. Overall genomic relatedness indices and phylogenomic and physiological analyses revealed that this novel species was best classified in a novel genus for which we propose the name Brytella acorum gen. nov., sp. nov., with LMG 32668T (=CECT 30723T) as the type strain. The B. acorum genomes encode a complete but modified tricarboxylic acid cycle, and complete pentose phosphate, pyruvate oxidation and gluconeogenesis pathways. The absence of 6-phosphofructokinase which rendered the glycolysis pathway non-functional, and an energy metabolism that included both aerobic respiration and oxidative fermentation are typical metabolic characteristics of acetic acid bacteria. Neither genome encodes nitrogen fixation or nitrate reduction genes, but both genomes encode genes for the biosynthesis of a broad range of amino acids. Antibiotic resistance genes or virulence factors are absent.

Characterization of Plebeiobacterium marinum gen. nov., sp. nov. and Plebeiobacterium sediminum sp. nov., revealing the potential nitrogen fixation capacity of the order Marinilabiliales

Biological nitrogen fixation plays a crucial role in the marine nitrogen cycle, impacting global marine productivity and related carbon fluxes. The strains were analyzed by gene annotation, growth conditions and phylogenetic analysis of 16S rRNA gene sequences.These two strains were isolated from the coastal sediment at Xiaoshi Island in Weihai, China. The strains were analyzed by gene annotation, growth conditions and phylogenetic analysis of 16S rRNA gene sequences. It was revealed that strains D04T and AATT contain a set of nif gene clusters responsible for nitrogen fixation. Cell are yellow-colored, Gram-stain-negative, facultatively anaerobic, and rod-shaped bacteria. The optimal growth conditions for strain D04T were found to be at 33 °C, pH 7.0, and in 2% (w/v) NaCl, while strain AATT prefers growth conditions at 33 °C, pH 6.5, and in 3% (w/v) NaCl. The highest similarity of strains D04T and AATT was to Saccharicrinis fermentans NBRC 15936T, with a similarity of 94.1% and 94.8%, respectively. The 16S rRNA gene sequence similarity between the two strains was 96.6%. These novel strains were found to represent new taxa of the Marinilabiliaceae family, and we propose the names Plebeiobacterium marinum gen. nov., sp. nov. and Plebeiobacterium sediminum sp. nov. with type strains D04T (MCCC 1H00493T = KCTC 92026T) and AATT (MCCC 1H00485T = KCTC 92028T), respectively. In this study, nitrogen fixation genes were predicted for 53 strains from the whole order Marinilabiliales and it was found that nitrogen fixation gene clusters were present in 26 strains. These gene clusters were found in every family in the order, highlighting that the presence of nitrogen-fixing gene clusters in the order is common. Nitrogen-fixing bacteria in sediments play an important role in various biogeochemical cycles. Thus, understanding the oceanic nitrogen cycle can provide insights into the energy flow of marine systems.

Polyethylene microplastic and soil nitrogen dynamics: Unraveling the links between functional genes, microbial communities, and transformation processes

Microplastics (MPs) have emerged as pollutants of growing concern due to their potential threat to soil ecosystems. While some studies have investigated the effects of MPs on soil nitrogen content, the underlying physicochemical and microbial driving mechanisms still need to be explored. In this study, a six-month incubation experiment was conducted with varying polyethylene MP addition rates: CK (0%, mass ratio), MP0.5 (0.5%), MP1 (1%), MP2 (2%), MP4 (4%), and MP8 (8%). The experiment aimed to examine the effects of MPs on soil nitrogen content, physicochemical properties, nitrogen cycling-related genes, microorganisms, and gross nitrogen transformation rates. The results revealed no significant changes in soil total nitrogen and dissolved total nitrogen. However, dissolved organic nitrogen significantly decreased by 16.00–54.60% following MP addition, while ammonium (NH4+-N, 45.71–271.43%) and nitrate (NO3--N, 43.15–209.54%) nitrogen and microbial biomass nitrogen (46.02–123.70%) significantly increased. Soil pH, bulk density, and soil porosity decreased after MP addition, while soil carbon contents, water-stable macroaggregates, and redox potential increased. The soil microbial community structure changed significantly, and microbial diversity increased under MP treatment. MP addition significantly altered the abundance of soil nitrogen cycling functional genes. The relative abundance of nitrogen fixation and denitrification genes decreased with increasing MP addition rates, while organic degradation and synthesis genes increased. The soil nitrogen cycling functional microbial composition shifted dramatically with increased MP addition. Networks with high addition rates (MP2 +MP4 +MP8) exhibited more total nodes, total links, negative links, node degrees, and modules but shorter average path distances and lower modularity than those with low addition rates (CK +MP0.5 +MP1), reflecting increased network complexity induced by MPs. The gross ammonification rate, NH4+-N consumption and immobilization rates, and NO3--N immobilization rate increased, while the gross nitrification rate and net nitrification rate exhibited an initial increase followed by a decrease with increasing MP addition rates, peaking at MP2. Furthermore, redundancy analysis and structural equation modeling demonstrated that soil physicochemical properties significantly affected soil nitrogen cycling genes and microorganisms, ultimately altering nitrogen content. In conclusion, polyethylene MPs promoted soil nitrogen mineralization and transformation and changed the related functional microorganism community structure, exhibiting a noticeable dose-effect relationship. This study provides deeper insight into the effects of MPs on soil nitrogen cycling.

Ensifer canadensis sp. nov. strain T173T isolated from Melilotus albus (sweet clover) in Canada possesses recombinant plasmid pT173b harbouring symbiosis and type IV secretion system genes apparently acquired from Ensifer medicae.

A bacterial strain, designated T173T, was previously isolated from a root-nodule of a Melilotus albus plant growing in Canada and identified as a novel Ensifer lineage that shared a clade with the non-symbiotic species, Ensifer adhaerens. Strain T173T was also previously found to harbour a symbiosis plasmid and to elicit root-nodules on Medicago and Melilotus species but not fix nitrogen. Here we present data for the genomic and taxonomic description of strain T173T. Phylogenetic analyses including the analysis of whole genome sequences and multiple locus sequence analysis (MLSA) of 53 concatenated ribosome protein subunit (rps) gene sequences confirmed placement of strain T173T in a highly supported lineage distinct from named Ensifer species with E. morelensis Lc04T as the closest relative. The highest digital DNA-DNA hybridization (dDDH) and average nucleotide identity (ANI) values of genome sequences of strain T173T compared with closest relatives (35.7 and 87.9%, respectively) are well below the respective threshold values of 70% and 95-96% for bacterial species circumscription. The genome of strain T173T has a size of 8,094,229 bp with a DNA G + C content of 61.0 mol%. Six replicons were detected: a chromosome (4,051,102 bp) and five plasmids harbouring plasmid replication and segregation (repABC) genes. These plasmids were also found to possess five apparent conjugation systems based on analysis of TraA (relaxase), TrbE/VirB4 (part of the Type IV secretion system (T4SS)) and TraG/VirD4 (coupling protein). Ribosomal RNA operons encoding 16S, 23S, and 5S rRNAs that are usually restricted to bacterial chromosomes were detected on plasmids pT173d and pT173e (946,878 and 1,913,930 bp, respectively) as well as on the chromosome of strain T173T. Moreover, plasmid pT173b (204,278 bp) was found to harbour T4SS and symbiosis genes, including nodulation (nod, noe, nol) and nitrogen fixation (nif, fix) genes that were apparently acquired from E. medicae by horizontal transfer. Data for morphological, physiological and symbiotic characteristics complement the sequence-based characterization of strain T173T. The data presented support the description of a new species for which the name Ensifer canadensis sp. nov. is proposed with strain T173T (= LMG 32374T = HAMBI 3766T) as the species type strain.

Whole-genome assembly of A02 bacteria involved in nitrogen fixation within cassava leaves.

The endophytic nitrogen (N)-fixing bacterium A02 belongs to the genus Curtobacterium (Curtobacterium sp.) and is crucial for the N metabolism of cassava ( Manihot esculenta Crantz). We isolated the A02 strain from cassava cultivar SC205 and used the 15N isotope dilution method to study the impacts of A02 on growth and accumulation of N in cassava seedlings. Furthermore, the whole genome was sequenced to determine the N-fixation mechanism of A02. Compared with low N control (T1), inoculation with the A02 strain (T2) showed the highest increase in leaf and root dry weight of cassava seedlings, and 120.3 nmol/(mL·h) was the highest nitrogenase activity recorded in leaves, which were considered the main site for colonization and N-fixation. The genome of A02 was 3,555,568 bp in size and contained a circular chromosome and a plasmid. Comparison with the genomes of other short bacilli revealed that strain A02 showed evolutionary proximity to the endophytic bacterium NS330 (Curtobacterium citreum) isolated from rice (Oryza sativa) in India. The genome of A02 contained 13 nitrogen fixation (nif) genes, including 4 nifB, 1 nifR3, 2 nifH, 1 nifU, 1 nifD, 1 nifK, 1 nifE, 1 nifN, and 1 nifC, and formed a relatively complete N fixation gene cluster 8-kb long that accounted for 0.22% of the whole genome length. The nifHDK of strain A02 (Curtobacterium sp.) is identical to the Frankia alignment. Function prediction showed high copy number of the nifB gene was related to the oxygen protection mechanism. Our findings provide exciting information about the bacterial genome in relation to N support for transcriptomic and functional studies for increasing N use efficiency in cassava.

Plant growth regulators improve nitrogen metabolism, yield, and quality of soybean–rhizobia symbiosis

PurposeIn recent years, considerable research has been conducted on the use of plant growth regulators (PGRs) to improve crop yields. Large amounts of PGRs are applied to regulate crop growth. However, few studies have assessed the impact of PGRs, which leave soil residues, on soil microorganisms, especially rhizobia.MethodsIn this study, the influence of inoculation of soybean roots with Sinorhizobium fredii and Bradyrhizobium japonicum exposed to gibberellin A3 (GA3) and mepiquat chloride (MC) on nodule number, nitrogenase activity, and a symbiotic nitrogen fixation gene (fixA) expression was studied. The nitrate content, free amino acids, and nitrate reductase activity of the roots, and seed yield and quality of soybean were measured.ResultNodulation was promoted to some extent, whereas nitrogenase activity and fixA expression were inhibited to some extent by treatment with GA3; however, these effects were reversed by treatment with MC. In a pot experiment to study phenotypic characteristics, rhizobia treatment in combination with GA3 or MC significantly improved soybean yield and seed nitrogen content, and increased the root nitrate content, free amino acid content, and nitrate reductase activity.ConclusionThe results indicated that PGRs, which leave soil residues, have significant positive effects on the growth and activity of soybean and rhizobia.

A novel strictly anaerobic sulfate-reducing diazotrophic bacterium Fundidesulfovibrio terrae sp. nov., isolated from paddy soil.

A strictly anaerobic sulfate-reducing strain, designated SG127T, was isolated from paddy soil. SG127T showed the highest 16S rRNA gene sequence similarity to the type strain of Fundidesulfovibrio magnetotacticus (98.2 %). A phylogenetic tree based on 16S rRNA gene sequences indicated that SG127T clustered with members of the genus Fundidesulfovibrio. Growth of SG127T was observed at 20-37 °C (optimum, 30 °C), pH 5.5-9.0 (optimum, 7.0-8.0) and with 0-0.2 % (w/v) NaCl (optimally without NaCl). SG127T contained MK-7 as the only menaquinone and anteiso-C15 : 0, anteiso-C17 : 1ω9c, C18 : 0, iso-C14 : 0, iso-C15 : 0, iso-C16:0, iso-C16 : 1H, iso-C18 : 1H and summed feature nine as the major fatty acids. The genomic DNA G+C content of SG127T was 64.6 %. The average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values between SG127T and the closely related Fundidesulfovibrio magnetotacticus were 78.5% and 23.2 %, respectively, which were lower than the cut-off values (ANI 95-96% and dDDH 70 %) for prokaryotic species delineation. SG127T had desulfoviridin, possessed nitrogen fixation genes (nifHDK) and actively fixed nitrogen according to the acetylene reduction assay. On the basis of these results, strain SG127T represents a novel species of the genus Fundidesulfovibrio, for which the name Fundidesulfovibrio terrae sp. nov. is proposed. The type strain is SG127T (= GDMCC 1.3137T = JCM 35589T).

Systematic identification of endogenous strong constitutive promoters from the diazotrophic rhizosphere bacterium Pseudomonas stutzeri DSM4166 to improve its nitrogenase activity

BackgroundBiological nitrogen fixation converting atmospheric dinitrogen to ammonia is an important way to provide nitrogen for plants. Pseudomonas stutzeri DSM4166 is a diazotrophic Gram-negative bacterium isolated from the rhizosphere of cereal Sorghum nutans. Endogenous constitutive promoters are important for engineering of the nitrogen fixation pathway, however, they have not been systematically characterized in DSM4166.ResultsTwenty-six candidate promoters were identified from DSM4166 by RNA-seq analysis. These 26 promoters were cloned and characterized using the firefly luciferase gene. The strengths of nineteen promoters varied from 100 to 959% of the strength of the gentamicin resistance gene promoter. The strongest P12445 promoter was used to overexpress the biological nitrogen fixation pathway-specific positive regulator gene nifA. The transcription level of nitrogen fixation genes in DSM4166 were significantly increased and the nitrogenase activity was enhanced by 4.1 folds determined by the acetylene reduction method. The nifA overexpressed strain produced 359.1 µM of extracellular ammonium which was 25.6 times higher than that produced by the wild-type strain.ConclusionsThe endogenous strong constitutive promoters identified in this study will facilitate development of DSM4166 as a microbial cell factory for nitrogen fixation and production of other useful compounds.

Changes in Soil Rhizobia Diversity and Their Effects on the Symbiotic Efficiency of Soybean Intercropped with Maize

It has been established that maize/soybean intercropping can improve nitrogen use efficiency. However, few studies have addressed how maize/soybean intercropping affects nitrogen-fixing bacterial diversity and N fixation efficiency of intercropped soybean. In this study, nitrogen-fixing bacterial communities, N fixation efficiency, and their relationships with soil properties under three nitrogen fertilization application rates (N0 0 kg/ha, N1 40 kg/ha, N2 80 kg/ha) were explored through field experiments. Nitrogen fixation and nitrogen-fixing bacteria diversity were assessed using 15N natural abundance, Illumina high-throughput sequencing, and nifH (nitrogen fixation) gene copies quantification in the rhizosphere soil of intercropped soybean. The results showed that nitrogen application rates significantly decreased the nitrogen-fixing bacteria diversity, nitrogen fixation efficiency, and nifH gene copies in the rhizosphere soil. Nitrogen fixation efficiency, nodule number, and dry weight of intercropped soybean were highest in the N0 treatment, and nitrogen fixation was the highest in the N1 treatment. The nitrogen-fixing efficiency in N0, N1, and N2 treatments increased by 69%, 59%, and 42% and the nodule number of soybean was 10%, 22%, and 21%, respectively, compared with monocultures. The soybean nitrogen-fixing bacteria diversity in intercropping under N0 and N1 treatments significantly increased compared with monocultures. There was a significant positive correlation between soil nifH gene copies and N fixation efficiency and a negative correlation with soil available nitrogen. Bradyrhizobium abundance in soybean rhizosphere soil decreased significantly with the increase in nitrogen application rates and was significantly correlated with soil AN (available nitrogen) and pH content in the soybean rhizosphere. These results help us to understand the mechanisms by which nitrogen use efficiency was improved, and nitrogen fertilizer could be reduced in legume/Gramineae intercropping, which is important to improve the sustainability of agricultural production.