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Biology Nov 2023Iron is an essential nutrient for all life forms. Specialized mechanisms exist in bacteria to ensure iron uptake and its delivery to key enzymes within the cell, while... (Review)
Review
Iron is an essential nutrient for all life forms. Specialized mechanisms exist in bacteria to ensure iron uptake and its delivery to key enzymes within the cell, while preventing toxicity. Iron uptake and exchange networks must adapt to the different environmental conditions, particularly those that require the biosynthesis of multiple iron proteins, such as nitrogen fixation. In this review, we outline the mechanisms that the model diazotrophic bacterium uses to ensure iron nutrition and how it adapts Fe metabolism to diazotrophic growth.
PubMed: 37998022
DOI: 10.3390/biology12111423 -
Scientific Reports Nov 2023Calcareous soils are characterized by a high calcium carbonate content (calcite), which plays a crucial role in the soil structure, plant growth, and nutrient...
Calcareous soils are characterized by a high calcium carbonate content (calcite), which plays a crucial role in the soil structure, plant growth, and nutrient availability. The high content of CaCO leads to the increment of the soil alkalinity, which results in a lowering of the nutrient availability causing a challenge for the agriculture in these soils. In this study, the calcite-solubilizing potential of the diazotrophic Azotobacter salinestris YRNF3 was investigated in vitro as a probable bio-agent for enhancing the calcareous soils properties such as soil pH and nutrient availability. Twelve diazotrophic bacterial strains were isolated from wheat rhizosphere collected from different wheat-cultivated fields in five Egyptian governorates. Using Nessler's reagent, all isolated bacterial strains were found to have the ability to produce ammonia. By amplification of nifH gene, a PCR product of 450 bp was obtained for all isolated bacterial strains. For each isolate, three biological and three technical replicates were applied. All isolated diazotrophic bacteria were qualitatively screened for their calcite-solubilizing ability. To quantitatively investigate the calcite-solubilizing potential of A. salinestris YRNF3 in vitro, changes in the contents of soluble calcium (Ca), bicarbonate (HCO), total nitrogen (TN), total protein (TP), and pH were daily measured in its culture filtrate along 10 days of incubation. The results showed that the pH values in the culture filtrate ranged from 5.73 to 7.32. Concentration of Ca and HCO in the culture filtrate significantly decreased with the increment in the incubation time, while concentration of TN increased along the time. The highest TN concentration (0.0807 gL) was observed on days 4 and 5, compared to that of the day 0 (0.0014 gL). Content of TP in the culture filtrate also significantly increased along the incubation period. The highest TP content was recorded in day 4 (0.0505%), while no TP content was recorded on day 0. Furthermore, data obtained revealed that A. salinestris YRNF3 produced acid phosphatase at low activity (5.4 U mL). HPLC analysis of the culture filtrate indicated production of different organic acids, namely lactic acid (82.57 mg mL), formic acid (46.8 mg mL), while acetic acid was detected in a low quantity (3.11 mg mL). For each analysis, three replicates of each treatment were analyzed. Means of the tested treatments were compared using Tukey's HSD test at p ≤ 0.05. In conclusion, findings of this work suggested that A. salinestris YRNF3 has the potential to be a probable bioagent to be used for the reclamation of the calcareous soils by solubilizing CaCO, improving soil fertility, and promoting plant growth. However, further studies are needed to investigate its field application and their long-term effects on the soil properties and plant productivity. To the best of the author's knowledge, this is the first study reporting the calcite-solubilizing ability of a nitrogen-fixing bacteria. Having these two abilities by one microorganism is a unique feature, which qualifies it as a promising bioagent for reclamation of the calcareous soils.
Topics: Soil; Calcium Carbonate; Azotobacter; Bacteria; Excipients; Soil Microbiology
PubMed: 37996572
DOI: 10.1038/s41598-023-47924-w -
ACS Synthetic Biology Dec 2023The soil environment adjacent to plant roots, termed the rhizosphere, is home to a wide variety of microorganisms that can significantly affect the physiology of nearby...
The soil environment adjacent to plant roots, termed the rhizosphere, is home to a wide variety of microorganisms that can significantly affect the physiology of nearby plants. Microbes in the rhizosphere can provide nutrients, secrete signaling compounds, and inhibit pathogens. These processes could be manipulated with synthetic biology to enhance the agricultural performance of crops grown for food, energy, or environmental remediation, if methods can be implemented in these nonmodel microbes. A common first step for domesticating nonmodel organisms is the development of a set of genetic engineering tools, termed a synthetic biology toolbox. A toolbox comprises transformation protocols, replicating vectors, genome engineering (e.g., CRISPR/Cas9), constitutive and inducible promoter systems, and other gene expression control elements. This work validated synthetic biology toolboxes in three nitrogen-fixing soil bacteria: , (), and a new isolate of . All three organisms were amenable to transformation and reporter protein expression, with several functional inducible systems available for each organism. and showed more reliable plasmid-based expression, resulting in successful Cas9 recombineering to create scarless deletions and insertions. Using these tools, we generated mutants with inducible nitrogenase activity and introduced heterologous genes to produce resorcinol products with relevant biological activity in the rhizosphere.
Topics: Nitrogen; Soil; Synthetic Biology; Plasmids; Genetic Engineering; CRISPR-Cas Systems
PubMed: 37988619
DOI: 10.1021/acssynbio.3c00414 -
Communications Chemistry Nov 2023The reduction of dinitrogen to ammonia catalyzed by nitrogenase involves a complex series of events, including ATP hydrolysis, electron transfer, and activation of metal...
The reduction of dinitrogen to ammonia catalyzed by nitrogenase involves a complex series of events, including ATP hydrolysis, electron transfer, and activation of metal clusters for N reduction. Early evidence shows that an essential part of the mechanism involves transducing information between the nitrogenase component proteins through conformational dynamics. Here, millisecond time-resolved hydrogen-deuterium exchange mass spectrometry was used to unravel peptide-level protein motion on the time scale of catalysis of Mo-dependent nitrogenase from Azotobacter vinelandii. Normal mode analysis calculations complemented this data, providing insights into the specific signal transduction pathways that relay information across protein interfaces at distances spanning 100 Å. Together, these results show that conformational changes induced by protein docking are rapidly transduced to the active site, suggesting a specific mechanism for activating the metal cofactor in the enzyme active site.
PubMed: 37980448
DOI: 10.1038/s42004-023-01046-6 -
PloS One 2023In the Pseduomonadacea family, the extracytoplasmic function sigma factor AlgU is crucial to withstand adverse conditions. Azotobacter vinelandii, a closed relative of...
In the Pseduomonadacea family, the extracytoplasmic function sigma factor AlgU is crucial to withstand adverse conditions. Azotobacter vinelandii, a closed relative of Pseudomonas aeruginosa, has been a model for cellular differentiation in Gram-negative bacteria since it forms desiccation-resistant cysts. Previous work demonstrated the essential role of AlgU to withstand oxidative stress and on A. vinelandii differentiation, particularly for the positive control of alginate production. In this study, the AlgU regulon was dissected by a proteomic approach under vegetative growing conditions and upon encystment induction. Our results revealed several molecular targets that explained the requirement of this sigma factor during oxidative stress and extended its role in alginate production. Furthermore, we demonstrate that AlgU was necessary to produce alkyl resorcinols, a type of aromatic lipids that conform the cell membrane of the differentiated cell. AlgU was also found to positively regulate stress resistance proteins such as OsmC, LEA-1, or proteins involved in trehalose synthesis. A position-specific scoring-matrix (PSSM) was generated based on the consensus sequence recognized by AlgU in P. aeruginosa, which allowed the identification of direct AlgU targets in the A. vinelandii genome. This work further expands our knowledge about the function of the ECF sigma factor AlgU in A. vinelandii and contributes to explains its key regulatory role under adverse conditions.
Topics: Sigma Factor; Regulon; Azotobacter vinelandii; Proteomics; Heat-Shock Proteins; Alginates; Bacterial Proteins; Gene Expression Regulation, Bacterial; Pseudomonas aeruginosa
PubMed: 37967103
DOI: 10.1371/journal.pone.0286440 -
Research Square Nov 2023The increasing global demand for food, coupled with concerns about the environmental impact of synthetic fertilizers, underscores the urgency of developing sustainable...
The increasing global demand for food, coupled with concerns about the environmental impact of synthetic fertilizers, underscores the urgency of developing sustainable agricultural practices. Nitrogen-fixing bacteria, known as diazotrophs, offer a potential solution by converting atmospheric nitrogen into bioavailable forms, reducing the reliance on synthetic fertilizers. However, a deeper understanding of their interactions with plants and other microbes is needed. In this study, we introduce a recently developed label-free 3D quantitative phase imaging technology called dynamic quantitative oblique back-illumination microscopy (DqOBM) to assess the dynamic activity of diazotrophs and . Our experiments involved three different diazotrophs (, , and ) cultured on media with amendments of carbon and nitrogen sources. Over five days, we observed increased dynamic activity in nutrient-amended media. These results suggest that the observed bacterial dynamics correlate with their metabolic activity. Furthermore, we applied qOBM to visualize bacterial activity within the root cap and elongation zone of primary roots. This allowed us to identify distinct areas of microbial infiltration in plant roots without the need for fluorescent markers. Our findings demonstrate that DqOBM can effectively characterize microbial activity and provide insights into plant-microbe interactions , offering a valuable tool for advancing our understanding of sustainable agriculture.
PubMed: 37961396
DOI: 10.21203/rs.3.rs-3517586/v1 -
PeerJ 2023The sustainability of paddy production systems in South Asia has recently been affected by a decline in soil health and excessive water usage. As a response to the...
Conservation tillage and fertiliser management strategies impact on basmati rice ( L): crop performance, crop water productivity, nutrient uptake and fertility status of the soil under rice-wheat cropping system.
BACKGROUND
The sustainability of paddy production systems in South Asia has recently been affected by a decline in soil health and excessive water usage. As a response to the global energy crisis, escalating costs of synthetic fertilisers, and growing environmental concerns, the utilization of organic plant-nutrient sources has gained considerable attention. Emerging adaptation technologies, including conservation tillage and innovative approaches to fertilizer management, present practical choices that can significantly contribute to the long-term preservation of soil fertility.
METHODS
The two year-long field experiment was completed in sandy loam soil during rainy () seasons in 2019 and 2020 at the crop research centre farm of Sardar Vallabhbhai Patel University of Agricultural & Technology, Meerut, Uttar Pradesh to analyze the impacts of different tillage establishment of the crop and its methodologies as well as integrated nutritional management approaches on rice growth, yield, productivity of water, nutrient uptake, and fertility status of soil under a rice-wheat rotation system. The experiment was set up in a factorial randomized block design and replicated three times in a semi-arid subtropical environment.
RESULTS
The conventionally transplanted rice puddled (CT-TPR) grew substantially better taller plants, and higher dry matter buildup leads to increased yields than transplanted rice under raised wide bed (WBed-TPR). WBed-TPR plots had more tillers, LAI, CGR, RGR, and yield characteristics of the rice in two year study. CT-TPR increased grain yield by 4.39 and 4.03% over WBed-TPR in 2019 and 2020, while WBed-TPR produced the highest water productivity (0.44 kg m) than CT-TPR, respectively. The 100% RDF+ ZnSO4 25 kg ha + FYM (5 t ha) + PSB (5 kg ha) + Azotobacter 20 kg ha (N6) treatment outperformed the other fertiliser management practices in terms of crop growth parameters, yields of grain (4,903 and 5,018 kg ha), nutrient uptake and NPK availability, organic soil carbon. Among the fertilizer management practices, with the direct applications of the recommended dose of fertilizer (RDF), farm yard manure (FYM), phosphate solubilizing bacteria (PSB), Azatobactor and zinc worked synergistically and increased grain yields by 53.4, 51.3, 47.9 and 46.2% over their respective control treatment.
CONCLUSIONS
To enhance rice productivity and promote soil health, the study suggests that adopting conservation tillage-based establishment practices and implementing effective fertilizer management techniques could serve as practical alternatives. It is concluded that the rice yield was improved by the inclusive use of inorganic fertiliser and organic manure (FYM). Additionally, the study observed that the combination of conventional puddled transplanted rice (CT-TPR) and N6 nitrogen application resulted in enhanced rice crop productivity and improved soil health.
Topics: Soil; Fertilizers; Oryza; Agriculture; Triticum; Manure; Crops, Agricultural; Edible Grain; Phosphates
PubMed: 37927793
DOI: 10.7717/peerj.16271 -
MBio Nov 2023The functional versatility of the Fe protein, the reductase component of nitrogenase, makes it an appealing target for heterologous expression, which could facilitate...
The functional versatility of the Fe protein, the reductase component of nitrogenase, makes it an appealing target for heterologous expression, which could facilitate future biotechnological adaptations of nitrogenase-based production of valuable chemical commodities. Yet, the heterologous synthesis of a fully active Fe protein of (NifH) in has proven to be a challenging task. Here, we report the successful synthesis of a fully active NifH protein upon co-expression of this protein with IscS/U and NifM in . Our metal, activity, electron paramagnetic resonance, and X-ray absorption spectroscopy/extended X-ray absorption fine structure (EXAFS) data demonstrate that the heterologously expressed NifH protein has a high [FeS] cluster content and is fully functional in nitrogenase catalysis and assembly. Moreover, our phylogenetic analyses and structural predictions suggest that NifM could serve as a chaperone and assist the maturation of a cluster-replete NifH protein. Given the crucial importance of the Fe protein for the functionality of nitrogenase, this work establishes an effective framework for developing a heterologous expression system of the complete, two-component nitrogenase system; additionally, it provides a useful tool for further exploring the intricate biosynthetic mechanism of this structurally unique and functionally important metalloenzyme. IMPORTANCE The heterologous expression of a fully active Fe protein (AvNifH) has never been accomplished. Given the functional importance of this protein in nitrogenase catalysis and assembly, the successful expression of AvNifH in as reported herein supplies a key element for the further development of heterologous expression systems that explore the catalytic versatility of the Fe protein, either on its own or as a key component of nitrogenase, for nitrogenase-based biotechnological applications in the future. Moreover, the "clean" genetic background of the heterologous expression host allows for an unambiguous assessment of the effect of certain nif-encoded protein factors, such as AvNifM described in this work, in the maturation of AvNifH, highlighting the utility of this heterologous expression system in further advancing our understanding of the complex biosynthetic mechanism of nitrogenase.
PubMed: 37909748
DOI: 10.1128/mbio.02572-23 -
International Journal of Biological... Dec 2023Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) has attracted substantial attention as a promising material for industrial applications. In this study, different...
Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) has attracted substantial attention as a promising material for industrial applications. In this study, different PHBV films with distinct 3-hydroxyvalerate (3HV) contents produced by Azotobacter vinelandii OP were evaluated. The 3HV fraction ranged from 18.6 to 36.7 mol%, and the number-average molecular weight (Mn) was between 238 and 434 kDa. In the bioreactor, a 3HV fraction (36.7 mol%) and an Mn value of 409 kDa were obtained with an oxygen transfer rate (OTR) of 12.5 mmol L h. Thermal analysis measurements showed decreased melting (T) and glass transition (T) temperatures, and values with relatively high 3HV fractions indicated improved thermomechanical properties. The incorporation of the 3HV fraction in the PHBV chain improved the thermal stability of the films, reduced the polymer T and affected the tensile strength. PHBV film with 36.7 mol% 3HV showed an increase in its tensile strength (51.8 MPa) and a decrease in its T (170.61 °C) compared with PHB. Finally, scanning electron microscopy (SEM) results revealed that the PHBV film with 32.8 mol% 3HV showed a degradation upon contact with soil, water, or soil bacteria, showing more porous surfaces after degradation. The latter phenomenon indicated that thermomechanical properties played an important role in biodegradation.
Topics: Azotobacter vinelandii; Hydroxybutyrates; Polyesters; Poly A; Soil
PubMed: 37890746
DOI: 10.1016/j.ijbiomac.2023.127681 -
Proceedings of the National Academy of... Oct 2023Nitrogenase is an active target of heterologous expression because of its importance for areas related to agronomy, energy, and environment. One major hurdle for...
Nitrogenase is an active target of heterologous expression because of its importance for areas related to agronomy, energy, and environment. One major hurdle for expressing an active Mo-nitrogenase in is to generate the complex metalloclusters (P- and M-clusters) within this enzyme, which involves some highly unique bioinorganic chemistry/metalloenzyme biochemistry that is not generally dealt with in the heterologous expression of proteins via synthetic biology; in particular, the heterologous synthesis of the homometallic P-cluster ([FeS]) and M-cluster core (or L-cluster; [FeSC]) on their respective protein scaffolds, which represents two crucial checkpoints along the biosynthetic pathway of a complete nitrogenase, has yet to be demonstrated by biochemical and spectroscopic analyses of purified metalloproteins. Here, we report the heterologous formation of a P-cluster-containing NifDK protein upon coexpression of , , , and genes, and that of an L-cluster-containing NifB protein upon coexpression of , and genes alongside the gene, in . Our metal content, activity, EPR, and XAS/EXAFS data provide conclusive evidence for the successful synthesis of P- and L-clusters in a nondiazotrophic host, thereby highlighting the effectiveness of our metallocentric, divide-and-conquer approach that individually tackles the key events of nitrogenase biosynthesis prior to piecing them together into a complete pathway for the heterologous expression of nitrogenase. As such, this work paves the way for the transgenic expression of an active nitrogenase while providing an effective tool for further tackling the biosynthetic mechanism of this important metalloenzyme.
Topics: Nitrogenase; Escherichia coli; Nitrogen Fixation; Oxidoreductases; Azotobacter vinelandii; Metalloproteins; Bacterial Proteins
PubMed: 37871225
DOI: 10.1073/pnas.2314788120