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Biochimica Et Biophysica Acta.... Aug 2024Azotobacter vinelandii is a genetically tractable Gram-negative proteobacterium able to fix nitrogen (N2) under aerobic growth conditions. This narrative describes how... (Review)
Review
Azotobacter vinelandii is a genetically tractable Gram-negative proteobacterium able to fix nitrogen (N2) under aerobic growth conditions. This narrative describes how biochemical-genetic approaches using A. vinelandii to study nitrogen fixation led to the formulation of the "scaffold hypothesis" for the assembly of both simple and complex [Fe-S] clusters associated with biological nitrogen fixation. These studies also led to the discovery of a parallel, but genetically distinct, pathway for maturation of [Fe-S] proteins that support central metabolic processes.
Topics: Azotobacter vinelandii; Iron-Sulfur Proteins; Nitrogen Fixation; Bacterial Proteins
PubMed: 38762171
DOI: 10.1016/j.bbamcr.2024.119750 -
Nature Communications May 2024Due to the complexity of the catalytic FeMo cofactor site in nitrogenases that mediates the reduction of molecular nitrogen to ammonium, mechanistic details of this...
Due to the complexity of the catalytic FeMo cofactor site in nitrogenases that mediates the reduction of molecular nitrogen to ammonium, mechanistic details of this reaction remain under debate. In this study, selenium- and sulfur-incorporated FeMo cofactors of the catalytic MoFe protein component from Azotobacter vinelandii are prepared under turnover conditions and investigated by using different EPR methods. Complex signal patterns are observed in the continuous wave EPR spectra of selenium-incorporated samples, which are analyzed by Tikhonov regularization, a method that has not yet been applied to high spin systems of transition metal cofactors, and by an already established grid-of-error approach. Both methods yield similar probability distributions that reveal the presence of at least four other species with different electronic structures in addition to the ground state E. Two of these species were preliminary assigned to hydrogenated E states. In addition, advanced pulsed-EPR experiments are utilized to verify the incorporation of sulfur and selenium into the FeMo cofactor, and to assign hyperfine couplings of S and Se that directly couple to the FeMo cluster. With this analysis, we report selenium incorporation under turnover conditions as a straightforward approach to stabilize and analyze early intermediate states of the FeMo cofactor.
Topics: Electron Spin Resonance Spectroscopy; Azotobacter vinelandii; Nitrogenase; Molybdoferredoxin; Selenium; Sulfur; Bacterial Proteins
PubMed: 38740794
DOI: 10.1038/s41467-024-48271-8 -
Journal of Microbiological Methods Jul 2024The present study was carried out to valorise cereal (rice and wheat) bran for the development of low-cost liquid consortium bioformulation. Different concentrations of...
Novel cereal bran based low-cost liquid medium for enhanced growth, multifunctional traits and shelf life of consortium biofertilizer containing Azotobacter chroococcum, Bacillus subtilis and Pseudomonas sp.
The present study was carried out to valorise cereal (rice and wheat) bran for the development of low-cost liquid consortium bioformulation. Different concentrations of bran-based liquid media formulations were evaluated for the growth of consortium biofertilizer cultures (Azotobacter chroococcum, Bacillus subtilis and Pseudomonas sp.). Among the bran-based formulations, wheat bran-based formulation WB5, exhibited the highest viable cell of 10.68 ± 0.09 Log CFU/ml and 12.63 ± 0.04 Log CFU/ml for Azotobacter chroococcum and Bacillus subtilis whereas for Pseudomonas sp., rice bran based bioformulation RB5 recorded maximum viability (12.71 ± 0.05 Log CFU/ml) after 72 h of incubation. RB5 and WB5liquid formulations were further optimized for enhanced shelf life using 5, 10 and 15 mM of trehalose, 0.05 and 0.1% carboxymethyl cellulose, and 0.5 and 1.0% glycerol. Following the peak growth at 72 h of incubation, a gradual decrease in the viable population of consortium biofertilizer cultures was observed in all the liquid formulations. The WB5 and RB5 formulations with 15 mM trehalose and 0.1% CMC, not only recorded significantly highest cell count of consortium biofertilizer cultures, but also maximally supported multi-functional traits i.e., phosphate and zinc solubilization, ammonia and IAA production up to 150 days. Further evaluation of seedling emergence and growth of wheat (PBW 826) under axenic conditions recorded WB5 amended with 15 mM trehalose-based consortium bioformulation to exhibit maximum emergence and growth of wheat seedlings. This low-cost liquid formulation can be used for large-scale biofertilizer production as a cost-effective liquid biofertilizer production technology.
Topics: Bacillus subtilis; Pseudomonas; Azotobacter; Fertilizers; Dietary Fiber; Culture Media; Oryza; Edible Grain; Microbial Viability
PubMed: 38740286
DOI: 10.1016/j.mimet.2024.106952 -
Plant Physiology and Biochemistry : PPB Jun 2024The consistently increasing use of zinc oxide nanoparticles (ZnONPs) in crop optimization practices and their persistence in agro-environment necessitate expounding...
Amelioration of phytotoxic impact of biosynthesized zinc oxide nanoparticles: Plant growth promoting rhizobacteria facilitates the growth and biochemical responses of Eggplant (Solanum melongena) under nanoparticles stress.
The consistently increasing use of zinc oxide nanoparticles (ZnONPs) in crop optimization practices and their persistence in agro-environment necessitate expounding their influence on sustainable agro-environment. Attempts have been made to understand nanoparticle-plant beneficial bacteria (PBB)- plant interactions; the knowledge of toxic impact of nanomaterials on soil-PBB-vegetable systems and alleviating nanotoxicity using PBB is scarce and inconsistent. This study aims at bio-fabrication of ZnONPs from Rosa indica petal extracts and investigates the impact of PBB on growth and biochemical responses of biofertilized eggplants exposed to phyto-synthesized nano-ZnO. Microscopic and spectroscopic techniques revealed nanostructure, triangular shape, size 32.5 nm, and different functional groups of ZnONPs and petal extracts. Inoculation of Pseudomonas fluorescens and Azotobacter chroococcum improved germination efficiency by 22% and 18% and vegetative growth of eggplants by 14% and 15% under NPs stress. Bio-inoculation enhanced total chlorophyll content by 36% and 14 %, increasing further with higher ZnONP concentrations. Superoxide dismutase and catalase activity in nano-ZnO and P. fluorescens inoculated eggplant shoots reduced by 15-23% and 9-11%. Moreover, in situ experiment unveiled distortion and accumulation of NPs in roots revealed by scanning electron microscope and confocal laser microscope. The present study highlights the phytotoxicity of biosynthesized ZnONPs to eggplants and demonstrates that PBB improved agronomic traits of eggplants while declining phytochemicals and antioxidant levels. These findings suggest that P. fluorescens and A. chroococcum, with NPs ameliorative activity, can be cost-effective and environment-friendly strategy for alleviating NPs toxicity and promoting eggplant production under abiotic stress, fulfilling vegetable demands.
Topics: Zinc Oxide; Solanum melongena; Metal Nanoparticles; Pseudomonas fluorescens; Azotobacter; Stress, Physiological; Chlorophyll; Nanoparticles
PubMed: 38714126
DOI: 10.1016/j.plaphy.2024.108678 -
The FEBS Journal May 2024The nitrogenase reductase NifH catalyses ATP-dependent electron delivery to the Mo-nitrogenase, a reaction central to biological dinitrogen (N) fixation. While NifHs...
The nitrogenase reductase NifH catalyses ATP-dependent electron delivery to the Mo-nitrogenase, a reaction central to biological dinitrogen (N) fixation. While NifHs have been extensively studied in bacteria, structural information about their archaeal counterparts is limited. Archaeal NifHs are considered more ancient, particularly those from Methanococcales, a group of marine hydrogenotrophic methanogens, which includes diazotrophs growing at temperatures near 92 °C. Here, we structurally and biochemically analyse NifHs from three Methanococcales, offering the X-ray crystal structures from meso-, thermo-, and hyperthermophilic methanogens. While NifH from Methanococcus maripaludis (37 °C) was obtained through heterologous recombinant expression, the proteins from Methanothermococcus thermolithotrophicus (65 °C) and Methanocaldococcus infernus (85 °C) were natively purified from the diazotrophic archaea. The structures from M. thermolithotrophicus crystallised as isolated exhibit high flexibility. In contrast, the complexes of NifH with MgADP obtained from the three methanogens are superposable, more rigid, and present remarkable structural conservation with their homologues. They retain key structural features of P-loop NTPases and share similar electrostatic profiles with the counterpart from the bacterial model organism Azotobacter vinelandii. In comparison to the NifH from the phylogenetically distant Methanosarcina acetivorans, these reductases do not cross-react significantly with Mo-nitrogenase from A. vinelandii. However, they associate with bacterial nitrogenase when ADP· is added to mimic a transient reactive state. Accordingly, detailed surface analyses suggest that subtle substitutions would affect optimal binding during the catalytic cycle between the NifH from Methanococcales and the bacterial nitrogenase, implying differences in the N-machinery from these ancient archaea.
PubMed: 38696373
DOI: 10.1111/febs.17148 -
Structural basis for the minimal bifunctional alginate epimerase AlgE3 from Azotobacter chroococcum.FEBS Letters Jun 2024Among the epimerases specific to alginate, some of them in Azotobacter genera convert β-d-mannuronic acid to α-l-guluronic acid but also have lyase activity to degrade...
Among the epimerases specific to alginate, some of them in Azotobacter genera convert β-d-mannuronic acid to α-l-guluronic acid but also have lyase activity to degrade alginate. The remarkable characteristics of these epimerases make it a promising enzyme for tailoring alginates to meet specific demands. Here, we determined the structure of the bifunctional mannuronan C-5 epimerase AlgE3 from Azotobacter chroococcum (AcAlgE3) in complex with several mannuronic acid oligomers as well as in apo form, which allowed us to elucidate the binding manner of each mannuronic acid oligomer, and the structural plasticity, which is dependent on calcium ions. Moreover, a comprehensive analysis of the lyase activity profiles of AcAlgE3 combined with structural characteristics explained the preference for different chain length oligomers.
Topics: Azotobacter; Alginates; Carbohydrate Epimerases; Bacterial Proteins; Hexuronic Acids; Substrate Specificity; Calcium; Models, Molecular; Crystallography, X-Ray; Protein Binding; Catalytic Domain
PubMed: 38649293
DOI: 10.1002/1873-3468.14886 -
NRT1.1B mediates rice plant growth and soil microbial diversity under different nitrogen conditions.AMB Express Apr 2024Interactions between microorganisms and plants can stimulate plant growth and promote nitrogen cycling. Nitrogen fertilizers are routinely used in agriculture to improve...
Interactions between microorganisms and plants can stimulate plant growth and promote nitrogen cycling. Nitrogen fertilizers are routinely used in agriculture to improve crop growth and yield; however, poor use efficiency impairs the optimal utilization of such fertilizers. Differences in the microbial diversity and plant growth of rice soil under different nitrogen application conditions and the expression of nitrogen-use efficiency-related genes have not been previously investigated. Therefore, this study investigates how nitrogen application and nitrogen-use efficiency-related gene NRT1.1B expression affect the soil microbial diversity and growth indices of two rice varieties, Huaidao 5 and Xinhuai 5. In total, 103,463 and 98,427 operational taxonomic units were detected in the soils of the Huaidao 5 and Xinhuai 5 rice varieties, respectively. The Shannon and Simpson indices initially increased and then decreased, whereas the Chao and abundance-based coverage estimator indices decreased after the application of nitrogen fertilizer. Nitrogen fertilization also reduced soil bacterial diversity and richness, as indicated by the reduced abundances of Azotobacter recorded in the soils of both rice varieties. Nitrogen application initially increased and then decreased the grain number per panicle, yield per plant, root, stem, and leaf nitrogen, total nitrogen content, glutamine synthetase, nitrate reductase, urease, and root activities of both varieties. Plant height showed positive linear trends in response to nitrogen application, whereas thousand-grain weights showed a negative trend. Our findings may be used to optimize nitrogen fertilizer use for rice cultivation and develop crop-variety-specific strategies for nitrogen fertilizer application.
PubMed: 38647736
DOI: 10.1186/s13568-024-01683-7 -
Bioresource Technology May 2024Excavating nitrogen-fixing bacteria with high-temperature tolerance is essential for the efficient composting of animal dung. In this study, two strains of thermophilic...
Excavating nitrogen-fixing bacteria with high-temperature tolerance is essential for the efficient composting of animal dung. In this study, two strains of thermophilic nitrogen-fixing bacteria, NF1 (Bacillus subtilis) and NF2 (Azotobacter chroococcum), were added to cow dung compost both individually (NF1, NF2) and mixed together (NF3; mixing NF1 and NF2 at a ratio of 1:1). The results showed that NF1, NF2, and NF3 inoculants increased the total Kjeldahl nitrogen level by 38.43%-55.35%, prolonged the thermophilic period by 1-13 d, increased the seed germination index by 17.81%, and the emissions of NH and NO were reduced by 25.11% and 42.75%, respectively. Microbial analysis showed that Firmicutes were the predominant bacteria at the thermophilic stage, whereas Chloroflexi, Proteobacteria, and Bacteroidetes were the predominant bacteria at the mature stage. These results confirmed that the addition of the isolated strains to cow dung composting improved the bacterial community structure and benefited nitrogen retention.
Topics: Animals; Cattle; Nitrogen; Composting; Manure; Feces; Nitrogen-Fixing Bacteria; Bacillus subtilis; Temperature; Germination
PubMed: 38599350
DOI: 10.1016/j.biortech.2024.130681 -
Plants (Basel, Switzerland) Mar 2024Due to the increasing presence of industrial hemp ( L.) and its multiple possibilities of use, the influence of different light and several biopreparations based on...
Due to the increasing presence of industrial hemp ( L.) and its multiple possibilities of use, the influence of different light and several biopreparations based on beneficial fungi and bacteria on hemp's morphological and physiological properties were examined. Different biopreparations and their combinations were inoculated on hemp seed and/or substrate and grown under blue and white light. A completely randomized block design was conducted in four replications within 30 days. For biopreparation treatment, vesicular arbuscular mycorrhiza (VAM) in combination with and spp. were inoculated only on seed or both on seed and in the substrate. Generally, the highest morphological parameters (stem, root and plant length) were recorded on plants in white light and on treatment with applied spp., both on seed and substrate. Blue light negatively affected biopreparation treatments, resulting in lower values of all morphological parameters compared to control. Leaves pigments were higher under blue light, as compared to the white light. At the same time, 1-diphenyl-2-picrylhydrazyl (DPPH), ferric reducing antioxidant power (FRAP), flavonoids, total flavanol content and phenolic acids were not influenced by light type. Biopreparation treatments did not significantly influence the leaves' pigments content (Chl a, Chl b and Car), nor the phenolic and flavanol content.
PubMed: 38592854
DOI: 10.3390/plants13060840 -
PloS One 2024The stringent response exerted by (p)ppGpp and RNA-polymerase binding protein DksA regulates gene expression in diverse bacterial species. To control gene expression...
The stringent response exerted by (p)ppGpp and RNA-polymerase binding protein DksA regulates gene expression in diverse bacterial species. To control gene expression (p)ppGpp, synthesized by enzymes RelA and SpoT, interacts with two sites within the RNA polymerase; site 1, located in the interphase between subunits β' and ω (rpoZ), and site 2 located in the secondary channel that is dependent on DksA protein. In Escherichia coli, inactivation of dksA results in a reduced sigma factor RpoS expression. In Azotobacter vinelandii the synthesis of polyhydroxybutyrate (PHB) is under RpoS regulation. In this study, we found that the inactivation of relA or dksA, but not rpoZ, resulted in a negative effect on PHB synthesis. We also found that the dksA, but not the relA mutation reduced both rpoS transcription and RpoS protein levels, implying that (p)ppGpp and DksA control PHB synthesis through different mechanisms. Interestingly, despite expressing rpoS from a constitutive promoter in the dksA mutant, PHB synthesis was not restored to wild type levels. A transcriptomic analysis in the dksA mutant, revealed downregulation of genes encoding enzymes needed for the synthesis of acetyl-CoA, the precursor substrate for PHB synthesis. Together, these data indicate that DksA is required for optimal expression of RpoS which in turn activates transcription of genes for PHB synthesis. Additionally, DksA is required for optimal transcription of genes responsible for the synthesis of precursors for PHB synthesis.
Topics: Escherichia coli Proteins; Azotobacter vinelandii; Guanosine Pentaphosphate; Escherichia coli; Gene Expression Regulation, Bacterial; Bacterial Proteins; Polyhydroxybutyrates
PubMed: 38574051
DOI: 10.1371/journal.pone.0299640