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.

Mapping Geological Events and Nitrogen Fixation Evolution Onto the Timetree of the Evolution of Nitrogen-Fixation Genes.

Nitrogen is essential for all organisms, but biological nitrogen fixation (BNF) occurs only in a small fraction of prokaryotes. Previous studies divided nitrogenase-gene-carrying prokaryotes into Groups I to IV and provided evidence that BNF first evolved in bacteria. This study constructed a timetree of the evolution of nitrogen-fixation genes and estimated that archaea evolved BNF much later than bacteria and that nitrogen-fixing cyanobacteria evolved later than 1,900 MYA, considerably younger than the previous estimate of 2,200 MYA. Moreover, Groups III and II/I diverged ∼2,280 MYA, after the Kenorland supercontinent breakup (∼2,500-2,100 MYA) and the Great Oxidation Event (∼2,400-2,100 MYA); Groups III and Vnf/Anf diverged ∼2,086 MYA, after the Yarrabubba impact (∼2,229 MYA); and Groups II and I diverged ∼1,920 MYA, after the Vredefort impact (∼2,023 MYA). In summary, this study provided a timescale of BNF events and discussed the possible effects of geological events on BNF evolution.

Alanine synthesized by alanine dehydrogenase enables ammonium-tolerant nitrogen fixation in Paenibacillus sabinae T27.

Most diazotrophs fix nitrogen only under nitrogen-limiting conditions, for example, in the presence of relatively low concentrations of NH4+ (0 to 2 mM). However, Paenibacillus sabinae T27 exhibits an unusual pattern of nitrogen regulation of nitrogen fixation, since although nitrogenase activities are high under nitrogen-limiting conditions (0 to 3 mM NH4+) and are repressed under conditions of nitrogen sufficiency (4 to 30 mM NH4+), nitrogenase activity is reestablished when very high levels of NH4+ (30 to 300 mM) are present in the medium. To further understand this pattern of nitrogen fixation regulation, we carried out transcriptome analyses of P. sabinae T27 in response to increasing ammonium concentrations. As anticipated, the nif genes were highly expressed, either in the absence of fixed nitrogen or in the presence of a high concentration of NH4+ (100 mM), but were subject to negative feedback regulation at an intermediate concentration of NH4+ (10 mM). Among the differentially expressed genes, ald1, encoding alanine dehydrogenase (ADH1), was highly expressed in the presence of a high level of NH4+ (100 mM). Mutation and complementation experiments revealed that ald1 is required for nitrogen fixation at high ammonium concentrations. We demonstrate that alanine, synthesized by ADH1 from pyruvate and NH4+, inhibits GS activity, leading to a low intracellular glutamine concentration that prevents feedback inhibition of GS and mimics nitrogen limitation, enabling activation of nif transcription by the nitrogen-responsive regulator GlnR in the presence of high levels of extracellular ammonium.

Nitrogen-fixing bacteria promote growth and bioactive components accumulation of Astragalus mongholicus by regulating plant metabolism and rhizosphere microbiota.

BACKGROUND: The excessive application of chemical fertilizers in the cultivation of Astragalus mongholicus Bunge results in a reduction in the quality of the medicinal plant and compromises the sustainable productivity of the soil. PGPB inoculant is a hot topic in ecological agriculture research. In the cultivation of Astragalus mongholicus, the screened nitrogen-fixing bacteria can promote plant growth, however, whether it can promote the accumulation of main bioactive components remains unknown. In this study, mixed inoculants containing 5 strains of growth promoting bacteria (Rhizobium T16 , Sinorhizobium T21 , Bacillus J1 , Bacillus G4 and Arthrobacter J2) were used in the field experiment. The metabolic substances in the root tissues of Astragalus mongholicus were identified during the harvest period by non-targeted metabolomics method, and the differential metabolites between groups were identified by statistical analysis. Meanwhile, high-throughput sequencing was performed to analyze the changes of rhizosphere soil and endophytic microbial community structure after mixed microbial treatment. RESULTS: The results of non-targeted metabolism indicated a significant increase in the levels of 26 metabolites after treatment including 13 flavonoids, 3 saponins and 10 other components. The contents of three plant hormones (abscisic acid, salicylic acid and spermidine) also increased after treatment, which presumed to play an important role in regulating plant growth and metabolism. Studies on endosphere and rhizosphere bacterial communities showed that Rhzobiaceae, Micromonosporaceae, and Hypomicrobiaceae in endophytic, and Oxalobactereae in rhizosphere were significantly increased after treatment. These findings suggest their potential importance in plant growth promotion and secondary metabolism regulation. CONCLUSIONS: This finding provides a basis for developing nitrogen-fixing bacteria fertilizer and improving the ecological planting efficiency of Astragalus mongholicus.

Metagenomic insights into nitrogen cycling functional gene responses to nitrogen fixation and transfer in maize-peanut intercropping.

The fixation and transfer of biological nitrogen from peanuts to maize in maize-peanut intercropping systems play a pivotal role in maintaining the soil nutrient balance. However, the mechanisms through which root interactions regulate biological nitrogen fixation and transfer remain unclear. This study employed a 15N isotope labelling method to quantify nitrogen fixation and transfer from peanuts to maize, concurrently elucidating key microorganisms and genera in the nitrogen cycle through metagenomic sequencing. The results revealed that biological nitrogen fixation in peanut was 50 mg and transfer to maize was 230 mg when the roots interacted. Moreover, root interactions significantly increased nitrogen content and the activities of protease, dehydrogenase (DHO) and nitrate reductase in the rhizosphere soil. Metagenomic analyses and structural equation modelling indicated that nrfC and nirA genes played important roles in regulating nitrogen fixation and transfer. Bradyrhizobium was affected by soil nitrogen content and DHO, indirectly influencing the efficiency of nitrogen fixation and transfer. Overall, our study identified key bacterial genera and genes associated with nitrogen fixation and transfer, thus advancing our understanding of interspecific interactions and highlighting the pivotal role of soil microorganisms and functional genes in maintaining soil ecosystem stability from a molecular ecological perspective.

Differential Mechanisms of Microbial As(III) and Sb(III) Oxidation and Their Contribution to Tailings Reclamation.

Mine tailings are extremely oligotrophic environments frequently contaminated with elevated As and Sb, making As(III) and Sb(III) oxidation potentially important energy sources for the tailing microbiome. Although they have been proposed to share similar metabolic pathways, a systemic comparison of the As(III) and Sb(III) oxidation mechanisms and energy utilization efficiencies requires further elucidation. In this study, we employed a combination of physicochemical, molecular, and bioinformatic analyses to compare the kinetic and genetic mechanisms of As(III) and Sb(III) oxidation as well as their respective energy efficiencies for fueling the key nutrient acquisition metabolisms. Thiobacillus and Rhizobium spp. were identified as functional populations for both As(III) and Sb(III) oxidation in mine tailings by DNA-stable isotope probing. However, these microorganisms mediated As(III) and Sb(III) oxidation via different metabolic pathways, resulting in preferential oxidation of Sb(III) over As(III). Notably, both As(III) and Sb(III) oxidation can facilitate nitrogen fixation and phosphate solubilization in mine tailings, with Sb(III) oxidation being more efficient in powering these processes. Thus, this study provided novel insights into the microbial As(III) and Sb(III) oxidation mechanisms and their respective nutrient acquisition efficiencies, which may be critical for the reclamation of mine tailings.

Phototrophic Nitrogen Fixation, a Neglected Biogeochemical Process in Mine Tailings?

Biological nitrogen fixation (BNF) has important ecological significance in mine tailing by contributing to the initial accumulation of nitrogen. In addition to chemolithotrophic and heterotrophic BNF, light may also fuel BNF in oligotrophic mine tailings. However, knowledge regarding the occurrence and ecological significance of this biogeochemical process in mine tailings remains ambiguous. The current study observed phototrophic BNF in enrichment cultures established from three primary successional stages (i.e., original tailings, biological crusts, and pioneer plants) of tailings. Notably, phototrophic BNF in tailings may be more active at vegetation stages (i.e., biological crusts and pioneering plants) than in bare tailings. DNA-stable isotope probing identified Roseomonas species as potential aerobic anoxygenic phototrophs responsible for phototrophic BNF. Furthermore, metagenomic binning as well as genome mining revealed that Roseomonas spp. contained essential genes involved in nitrogen fixation, anoxygenic photosynthesis, and carbon fixation, suggesting their genetic potential to mediate phototrophic BNF. A causal inference framework equipped with the structural causal model suggested that the enrichment of putative phototrophic diazotrophic Roseomonas may contribute to an elevated total nitrogen content during primary succession in these mine tailings. Collectively, our findings suggest that phototrophic diazotrophs may play important roles in nutrient accumulation and hold the potential to facilitate ecological succession in tailings.

Developed Rhizobium Strains Enhance Soil Fertility and Yield of Legume Crops in Haryana, India.

Three strains of Gram-negative bacterium, Rhizobium, were developed by gamma (γ)-irradiation random mutagenesis. The developed strains were evaluated for their augmented features for symbiotic association, nitrogen fixation, and crop yield of three leguminous plants-chickpea, field-pea, and lentil-in agricultural fields of the northern Indian state of Haryana. Crops treated with developed mutants exhibited significant improvement in plant features and the yield of crops when compared to the control-uninoculated crops and crops grown with indigenous or commercial crop-specific strains of Rhizobium. This improvement was attributed to generated mutants, MbPrRz1 (on chickpea), MbPrRz2 (on lentil), and MbPrRz3 (on field-pea). Additionally, the cocultured symbiotic response of MbPrRz1 and MbPrRz2 mutants was found to be more pronounced on all three crops. The statistical analysis using Pearson's correlation coefficients revealed that nodulation and plant biomass were the most related parameters of crop yield. Among the effectiveness of developed mutants, MbPrRz1 yielded the best results for all three tested crops. Moreover, the developed mutants enhanced macro- and micronutrients of the experimental fields when compared with fields harboring the indigenous rhizobial community. These developed mutants were further genetically characterized, predominantly expressing nitrogen fixation marker, nifH, and appeared to belong to Mesorhizobium ciceri (MbPrRz1) and Rhizobium leguminosarum (both MbPrRz2 and MbPrRz3). In summary, this study highlights the potential of developed Rhizobium mutants as effective biofertilizers for sustainable agriculture, showcasing their ability to enhance symbiotic relationships, crop yield, and soil fertility.

Overexpression of GmPAP4 Enhances Symbiotic Nitrogen Fixation and Seed Yield in Soybean under Phosphorus-Deficient Condition.

Legume crops establish symbiosis with nitrogen-fixing rhizobia for biological nitrogen fixation (BNF), a process that provides a prominent natural nitrogen source in agroecosystems; and efficient nodulation and nitrogen fixation processes require a large amount of phosphorus (P). Here, a role of GmPAP4, a nodule-localized purple acid phosphatase, in BNF and seed yield was functionally characterized in whole transgenic soybean (Glycine max) plants under a P-limited condition. GmPAP4 was specifically expressed in the infection zones of soybean nodules and its expression was greatly induced in low P stress. Altered expression of GmPAP4 significantly affected soybean nodulation, BNF, and yield under the P-deficient condition. Nodule number, nodule fresh weight, nodule nitrogenase, APase activities, and nodule total P content were significantly increased in GmPAP4 overexpression (OE) lines. Structural characteristics revealed by toluidine blue staining showed that overexpression of GmPAP4 resulted in a larger infection area than wild-type (WT) control. Moreover, the plant biomass and N and P content of shoot and root in GmPAP4 OE lines were also greatly improved, resulting in increased soybean yield in the P-deficient condition. Taken together, our results demonstrated that GmPAP4, a purple acid phosphatase, increased P utilization efficiency in nodules under a P-deficient condition and, subsequently, enhanced symbiotic BNF and seed yield of soybean.

Molecular Mechanisms of Pseudomonas-Assisted Plant Nitrogen Uptake: Opportunities for Modern Agriculture.

Pseudomonas spp. make up 1.6% of the bacteria in the soil and are found throughout the world. More than 140 species of this genus have been identified, some beneficial to the plant. Several species in the family Pseudomonadaceae, including Azotobacter vinelandii AvOP, Pseudomonas stutzeri A1501, Pseudomonas stutzeri DSM4166, Pseudomonas szotifigens 6HT33bT, and Pseudomonas sp. strain K1 can fix nitrogen from the air. The genes required for these reactions are organized in a nitrogen fixation island, obtained via horizontal gene transfer from Klebsiella pneumoniae, Pseudomonas stutzeri, and Azotobacter vinelandii. Today, this island is conserved in Pseudomonas spp. from different geographical locations, which, in turn, have evolved to deal with different geo-climatic conditions. Here, we summarize the molecular mechanisms behind Pseudomonas-driven plant growth promotion, with particular focus on improving plant performance at limiting nitrogen (N) and improving plant N content. We describe Pseudomonas-plant interaction strategies in the soil, noting that the mechanisms of denitrification, ammonification, and secondary metabolite signaling are only marginally explored. Plant growth promotion is dependent on the abiotic conditions and differs at sufficient and deficient N. The molecular controls behind different plant responses are not fully elucidated. We suggest that superposition of transcriptome, proteome, and metabolome data and their integration with plant phenotype development through time will help fill these gaps. The aim of this review is to summarize the knowledge behind Pseudomonas-driven nitrogen fixation and to point to possible agricultural solutions. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.

Phylogenomics Uncovers Evolutionary Trajectory of Nitrogen Fixation in Cyanobacteria.

Biological nitrogen fixation (BNF) by cyanobacteria is of significant importance for the Earth's biogeochemical nitrogen cycle but is restricted to a few genera that do not form monophyletic group. To explore the evolutionary trajectory of BNF and investigate the driving forces of its evolution, we analyze 650 cyanobacterial genomes and compile the database of diazotrophic cyanobacteria based on the presence of nitrogen fixation gene clusters (NFGCs). We report that 266 of 650 examined genomes are NFGC-carrying members, and these potentially diazotrophic cyanobacteria are unevenly distributed across the phylogeny of Cyanobacteria, that multiple independent losses shaped the scattered distribution. Among the diazotrophic cyanobacteria, two types of NFGC exist, with one being ancestral and abundant, which have descended from diazotrophic ancestors, and the other being anaerobe-like and sparse, possibly being acquired from anaerobic microbes through horizontal gene transfer. Interestingly, we illustrate that the origin of BNF in Cyanobacteria coincide with two major evolutionary events. One is the origin of multicellularity of cyanobacteria, and the other is concurrent genetic innovations with massive gene gains and expansions, implicating their key roles in triggering the evolutionary transition from nondiazotrophic to diazotrophic cyanobacteria. Additionally, we reveal that genes involved in accelerating respiratory electron transport (coxABC), anoxygenic photosynthetic electron transport (sqr), as well as anaerobic metabolisms (pfor, hemN, nrdG, adhE) are enriched in diazotrophic cyanobacteria, representing adaptive genetic signatures that underpin the diazotrophic lifestyle. Collectively, our study suggests that multicellularity, together with concurrent genetic adaptations contribute to the evolution of diazotrophic cyanobacteria.

Interactions between paralogous bacterial enhancer-binding proteins enable metal-dependent regulation of alternative nitrogenases in Azotobacter vinelandii.

All diazotrophic bacteria and archaea isolated so far utilise a nitrogenase enzyme-containing molybdenum in the active site co-factor to fix atmospheric dinitrogen to ammonia. However, in addition to the Mo-dependent nitrogenase, some nitrogen-fixing prokaryotes also express genetically distinct alternative nitrogenase isoenzymes, namely the V-dependent and Fe-only nitrogenases, respectively. Nitrogenase isoenzymes are expressed hierarchically according to metal availability and catalytic efficiency. In proteobacteria, this hierarchy is maintained via stringent transcriptional regulation of gene clusters by dedicated bacterial enhancer-binding proteins (bEBPs). The model diazotroph Azotobacter vinelandii contains two paralogs of the vanadium nitrogenase activator VnfA (henceforth, VnfA1), designated VnfA2 and VnfA3, with unknown functions. Here we demonstrate that the VnfA1 and VnfA3 bEBPs bind to the same target promoters in the Azotobacter vinelandii genome and co-activate a subset of genes in the absence of V, including the structural genes for the Fe-only nitrogenase. Co-activation is inhibited by the presence of V and is dependent on an accessory protein VnfZ that is co-expressed with VnfA3. Our studies uncover a plethora of interactions between bEBPs required for nitrogen fixation, revealing the unprecedented potential for fine-tuning the expression of alternative nitrogenases in response to metal availability.

Nitrogen fixation by common beans in crop mixtures is influenced by growth rate of associated species.

BACKGROUND: Legumes can fix atmospheric nitrogen (N) and facilitate N availability to their companion plants in crop mixtures. However, biological nitrogen fixation (BNF) of legumes in intercrops varies largely with the identity of the legume species. The aim of our study was to understand whether BNF and concentration of plant nutrients by common bean is influenced by the identity of the companion plant species in crop mixtures. In this greenhouse pot study, common beans were cultivated with another legume (chickpea) and a cereal (Sorghum). We compared BNF, crop biomass and nutrient assimilation of all plant species grown in monocultures with plants grown in crop mixtures. RESULTS: We found beans to exhibit low levels of BNF, and to potentially compete with other species for available soil N in crop mixtures. The BNF of chickpeas however, was enhanced when grown in mixtures. Furthermore, biomass, phosphorous and potassium values of chickpea and Sorghum plants were higher in monocultures, compared to in mixtures with beans; suggesting competitive effects of beans on these plants. Concentration of calcium, magnesium and zinc in beans was higher when grown with chickpeas than with Sorghum. CONCLUSIONS: It is generally assumed that legumes benefit their companion plant species. Our study highlights the contrary and shows that the specific benefits of cereal-legume mixtures are dependent on the growth rate of the species concerned. We further highlight that the potential of legume-legume mixtures is currently undervalued and may play a strong role in increasing N use efficiency of intercrop-based systems.

Screening of high-efficiency nitrogen-fixing bacteria from the traditional Chinese medicine plant Astragalus mongolicus and its effect on plant growth promotion and bacterial communities in the rhizosphere.

BACKGROUND: Astragalus mongolicus Bunge is used in traditional Chinese medicine and is thus cultivated in bulk. The cultivation of A. mongolicus requires a large amount of nitrogen fertilizer, increasing the planting cost of medicinal materials and polluting the environment. Isolation and screening of plant growth-promoting rhizobacteria (PGPR) and exploring the nitrogen fixation potential of A. mongolicus rhizosphere microorganisms would effectively reduce the production cost of A. mongolicus. RESULTS: This study used A. mongolicus roots and rhizosphere soil samples from Longxi County of Gansu Province, Jingle County, and Hunyuan County of Shanxi Province, China, to isolate and identify nitrogen-fixing bacteria. Through nitrogen fixation efficiency test, single strain inoculation test, and plant growth-promoting characteristics, three strains, Bacillus sp. J1, Arthrobacter sp. J2, and Bacillus sp. G4 were selected from 86 strains of potential nitrogen-fixing bacteria, which were the most effective in promoting the A. mongolicus growth and increasing the nitrogen, phosphorus, and potassium content in plants. The antagonistic test showed that these bacteria could grow smoothly under the co-culture conditions. The J1, J2, and G4 strains were used in a mixed inoculum and found to enhance the biomass of A. mongolicus plants and the accumulation of the main medicinal components in the field experiment. Mixed bacterial agent inoculation also increased bacterial diversity and changed the structure of the bacterial community in rhizosphere soil. Meanwhile, the relative abundance of Proteobacteria increased significantly after inoculation, suggesting that Proteobacteria play an important role in plant growth promotion. CONCLUSIONS: These findings indicate that specific and efficient PGPRs have a significant promoting effect on the growth of A. mongolicus, while also having a positive impact on the structure of the host rhizosphere bacteria community. This study provides a basis for developing a nitrogen-fixing bacterial fertilizer and improving the ecological planting efficiency of A. mongolicus.

Gold-FISH enables targeted NanoSIMS analysis of plant-associated bacteria.

Bacteria colonize plant roots and engage in reciprocal interactions with their hosts. However, the contribution of individual taxa or groups of bacteria to plant nutrition and fitness is not well characterized due to a lack of in situ evidence of bacterial activity. To address this knowledge gap, we developed an analytical approach that combines the identification and localization of individual bacteria on root surfaces via gold-based in situ hybridization with correlative NanoSIMS imaging of incorporated stable isotopes, indicative of metabolic activity. We incubated Kosakonia strain DS-1-associated, gnotobiotically grown rice plants with 15 N-N2 gas to detect in situ N2 fixation activity. Bacterial cells along the rhizoplane showed heterogeneous patterns of 15 N enrichment, ranging from the natural isotope abundance levels up to 12.07 at% 15 N (average and median of 3.36 and 2.85 at% 15 N, respectively, n = 697 cells). The presented correlative optical and chemical imaging analysis is applicable to a broad range of studies investigating plant-microbe interactions. For example, it enables verification of the in situ metabolic activity of host-associated commercialized strains or plant growth-promoting bacteria, thereby disentangling their role in plant nutrition. Such data facilitate the design of plant-microbe combinations for improvement of crop management.

The outstanding diversity of rhizobia microsymbionts of common bean (Phaseolus vulgaris L.) in Mato Grosso do Sul, central-western Brazil, revealing new Rhizobium species.

Common bean is considered a legume of great socioeconomic importance, capable of establishing symbioses with a wide variety of rhizobial species. However, the legume has also been recognized for its low efficiency in fixing atmospheric nitrogen. Brazil is a hotspot of biodiversity, and in a previous study, we identified 13 strains isolated from common bean (Phaseolus vulgaris) nodules in three biomes of Mato Grosso do Sul state, central-western Brazil, that might represent new phylogenetic groups, deserving further polyphasic characterization. The phylogenetic tree of the 16S rRNA gene split the 13 strains into two large clades, seven in the R. etli and six in the R. tropici clade. The MLSA with four housekeeping genes (glnII, gyrB, recA, and rpoA) confirmed the phylogenetic allocation. Genomic comparisons indicated eight strains in five putative new species and the remaining five as R. phaseoli. The average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) comparing the putative new species and the closest neighbors ranged from 81.84 to 92.50% and 24.0 to 50.7%, respectively. Other phenotypic, genotypic, and symbiotic features were evaluated. Interestingly, some strains of both R. etli and R. tropici clades lost their nodulation capacity. The data support the description of the new species Rhizobium cerradonense sp. nov. (CNPSo 3464T), Rhizobium atlanticum sp. nov. (CNPSo 3490T), Rhizobium aureum sp. nov. (CNPSo 3968T), Rhizobium pantanalense sp. nov. (CNPSo 4039T), and Rhizobium centroccidentale sp. nov. (CNPSo 4062T).

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

BACKGROUND: Biological 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. RESULTS: Twenty-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. CONCLUSIONS: The 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.

Chemolithotrophic Biological Nitrogen Fixation Fueled by Antimonite Oxidation May Be Widespread in Sb-Contaminated Habitats.

Nitrogen (N) deficiency in mining-contaminated habitats usually hinders plant growth and thus hampers tailing revegetation. Biological N fixation (BNF) is an essential biogeochemical process that contributes to the initial accumulation of N in oligotrophic mining-contaminated regions. Previous studies reported that chemolithotrophic rather than heterotrophic diazotrophs frequently dominated in the mining-contaminated regions. Chemolithotrophic diazotrophs may utilize elements abundant in such habitats (e.g., sulfur (S), arsenic (As), and antimony (Sb)) as electron donors to fix N2. BNF fueled by the oxidation of S and As has been detected in previous studies. However, BNF fueled by Sb(III) oxidation (Sb-dependent BNF) has never been reported. The current study observed the presence of Sb-dependent BNF in slurries inoculated from Sb-contaminated habitats across the South China Sb belt, suggesting that Sb-dependent BNF may be widespread in this region. DNA-stable isotope probing identified bacteria associated with Rhodocyclaceae and Rhizobiaceae as putative microorganisms responsible for Sb-dependent BNF. Furthermore, metagenomic-binning demonstrated that Rhodocyclaceae and Rhizobiaceae contained essential genes involved in Sb(III) oxidation, N2 fixation, and carbon fixation, suggesting their genetic potential for Sb-dependent BNF. In addition, meta-analysis indicated that these bacteria are widespread among Sb-contaminated habitats with different niche preferences: Rhodocyclaceae was enriched in river sediments and tailings, while Rhizobiaceae was enriched only in soils. This study may broaden our fundamental understanding of N fixation in Sb-mining regions.

Not just passengers, but co-pilots! Non-rhizobial nodule-associated bacteria promote cowpea growth and symbiosis with (brady)rhizobia.

AIMS: To isolate and characterize non-rhizobial nodule-associated bacteria (NAB) from cowpea root-nodules regarding their performance of plant-growth-promoting mechanisms and their ability to enhance cowpea growth and symbiosis when co-inoculated with bradyrhizobia. METHODS AND RESULTS: Sixteen NAB were isolated, identified, and in vitro evaluated for plant growth promotion traits. The ability to promote cowpea growth was analyzed when co-inoculated with Bradyrhizobium pachyrhizi BR 3262 in sterile and non-sterile substrates. The 16S rRNA gene sequences analysis revealed that NAB belonged to the genera Chryseobacterium (4), Bacillus (3), Microbacterium (3), Agrobacterium (1), Escherichia (1), Delftia (1), Pelomonas (1), Sphingomonas (1), and Staphylococcus (1). All strains produced different amounts of auxin siderophores and formed biofilms. Twelve out of the 16 strains carried the nifH, a gene associated with nitrogen fixation. Co-inoculation of NAB (ESA 424 and ESA 29) with Bradyrhizobium pachyrhizi BR 3262 significantly promoted cowpea growth, especially after simultaneous inoculation with the three strains. CONCLUSIONS: NAB are efficient cowpea growth promoters and can improve the efficiency of the symbiosis between cowpea and the N2-fixing microsymbiont B. pachyrhizi BR 3262, mainly under a specific triple microbial association.

Gut symbiotic bacteria are involved in nitrogen recycling in the tephritid fruit fly Bactrocera dorsalis.

BACKGROUND: Nitrogen is considered the most limiting nutrient element for herbivorous insects. To alleviate nitrogen limitation, insects have evolved various symbiotically mediated strategies that enable them to colonize nitrogen-poor habitats or exploit nitrogen-poor diets. In frugivorous tephritid larvae developing in fruit pulp under nitrogen stress, it remains largely unknown how nitrogen is obtained and larval development is completed. RESULTS: In this study, we used metagenomics and metatranscriptomics sequencing technologies as well as in vitro verification tests to uncover the mechanism underlying the nitrogen exploitation in the larvae of Bactrocera dorsalis. Our results showed that nitrogenous waste recycling (NWR) could be successfully driven by symbiotic bacteria, including Enterobacterales, Lactobacillales, Orbales, Pseudomonadales, Flavobacteriales, and Bacteroidales. In this process, urea hydrolysis in the larval gut was mainly mediated by Morganella morganii and Klebsiella oxytoca. In addition, core bacteria mediated essential amino acid (arginine excluded) biosynthesis by ammonium assimilation and transamination. CONCLUSIONS: Symbiotic bacteria contribute to nitrogen transformation in the larvae of B. dorsalis in fruit pulp. Our findings suggest that the pattern of NWR is more likely to be applied by B. dorsalis, and M. morganii, K. oxytoca, and other urease-positive strains play vital roles in hydrolysing nitrogenous waste and providing metabolizable nitrogen for B. dorsalis.