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Microorganisms May 2024To optimize the application of plant growth-promoting rhizobacteria (PGPR) in field trials, tracking methods are needed to assess their shelf life and to determine the...
To optimize the application of plant growth-promoting rhizobacteria (PGPR) in field trials, tracking methods are needed to assess their shelf life and to determine the elements affecting their effectiveness and their interactions with plants and native soil microbiota. This work developed a real-time PCR (qtPCR) method which traces and quantifies bacteria when added as microbial consortia, including five PGPR species: , , , , and Through a literature search and in silico sequence analyses, a set of primer pairs which selectively tag three bacterial species (, and ) was retrieved. The primers were used to trace these microbial species in a field trial in which the consortium was tested as a biostimulant on two wheat varieties, in combination with biochar and the mycorrhizal fungus . The qtPCR assay demonstrated that the targeted bacteria had colonized and grown into the soil, reaching a maximum of growth between 15 and 20 days after inoculum. The results also showed biochar had a positive effect on PGPR growth. In conclusion, qtPCR was once more an effective method to trace the fate of supplied bacterial species in the consortium when used as a cargo system for their delivery.
PubMed: 38792831
DOI: 10.3390/microorganisms12051002 -
Foods (Basel, Switzerland) May 2024Due to climate change and exacerbated population growth, the search for new sustainable strategies that allow for greater food productivity and that provide greater...
Due to climate change and exacerbated population growth, the search for new sustainable strategies that allow for greater food productivity and that provide greater nutritional quality has become imperative. One strategy for addressing this problem is the combined use of fertilization with a reduced dose of nitrogen and biostimulants. Celery processing produces a large amount of waste with its concomitant pollution. Therefore, it is necessary to address the valorization of its byproducts. Our results revealed reductions in the biomass, Na, P, Mn, B, sugars, and proteins in the byproducts and increased lipid peroxidation, Fe (all celery parts), and K (byproducts) when the N supplied was reduced. Plants inoculated with obtained a greater biomass, a higher accumulation of K (byproducts), a build-up of sugars and proteins, reduced concentrations of P, Cu, Mn, B, Fe (petioles), and Zn (byproducts), and reduced lipid peroxidation. The application of Se at 8 μM reinforced the beneficial effect obtained after inoculation with . In accordance with our results, edible celery parts are recommended as an essential ingredient in the daily diet. Furthermore, the valorization of celery byproducts with health-promoting purposes should be considered.
PubMed: 38790737
DOI: 10.3390/foods13101437 -
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 -
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 -
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 -
Plants (Basel, Switzerland) Feb 2024The use of nitrogen as a fertilizer can be highly risky when used excessively, and it is therefore necessary to find novel techniques to reduce its use. Aquaponics...
The use of nitrogen as a fertilizer can be highly risky when used excessively, and it is therefore necessary to find novel techniques to reduce its use. Aquaponics reduces the use of synthetic fertilizers and water, and the leaching of nitrate into the environment. One way to avoid problems due to a reduction in nitrogen availability could be the use of plant growth promoting rhizobacteria (PGPR). This study examines the effect of PGPR on kohlrabi plants grown with a traditional nutrient solution (100S), in combination with "fish water" (50F/50D), or with a supplement of synthetic fertilizers (50F/50D + S). Two formulations were used: T1 ( and ) and T2 (). Irrigation with 50F/50D caused a reduction in several of the measured parameters. The combined application of 50F/50D with T1 attenuated the negative effects. T2 did not present significant effects on the parameters measured. The results obtained with 50F/50D + S hardly showed differences with the 100S. Thus, by irrigating with 50F/50D + S, we were able to maintain the yields while reducing fertilizer use and water. The combined use of T1 and 50F/50D was also positive; however, it would be necessary to continue adjusting the amount of nitrate supplied to maintain production.
PubMed: 38475442
DOI: 10.3390/plants13050595 -
Scientific Reports Mar 2024The 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 functional dynamic activity of diazotrophs in vitro and in situ. Our experiments involved three different diazotrophs (Sinorhizobium meliloti, Azotobacter vinelandii, and Rahnella aquatilis) cultured on media with amendments of carbon and nitrogen sources. Over 5 days, we observed increased dynamics in nutrient-amended media. These results suggest that the observed bacterial dynamics correlate with their metabolic activity. Furthermore, we applied qOBM to visualize microbial dynamics within the root cap and elongation zone of Arabidopsis thaliana 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 dynamics and provide insights into plant-microbe interactions in situ, offering a valuable tool for advancing our understanding of sustainable agriculture.
Topics: Fertilizers; Lighting; Microscopy; Plants; Arabidopsis; Nitrogen; Nitrogen Fixation
PubMed: 38461279
DOI: 10.1038/s41598-024-56443-1 -
MSystems Mar 2024A grand challenge for the next century is in facing a changing climate through bioengineering solutions. Biological nitrogen fixation, the globally consequential,...
A grand challenge for the next century is in facing a changing climate through bioengineering solutions. Biological nitrogen fixation, the globally consequential, nitrogenase-catalyzed reduction of atmospheric nitrogen to bioavailable ammonia, is a vital area of focus. Nitrogen fixation engineering relies upon extensive understanding of underlying genetics in microbial models, including the broadly utilized gammaproteobacterium, (). Here, we report the first CRISPR interference (CRISPRi) system for targeted gene silencing in that integrates genomically via site-specific transposon insertion. We demonstrate that CRISPRi can repress transcription of an essential nitrogen fixation gene by ~60%. Further, we show that nitrogenase genes are suitably expressed from the transposon insertion site, indicating that CRISPRi and engineered nitrogen fixation genes can be co-integrated for combinatorial studies of gene expression and engineering. Our established CRISPRi system fills an important gap for engineering microbial nitrogen fixation for desired purposes.IMPORTANCEAll life on Earth requires nitrogen to survive. About 78% of the atmosphere alone is nitrogen, yet humans cannot use it directly. Instead, we obtain the nitrogen we need for our survival through the food we eat. For more than 100 years, a substantial portion of agricultural productivity has relied on industrial methods for nitrogen fertilizer synthesis, which consumes significant amounts of nonrenewable energy resources and exacerbates environmental degradation and human-induced climate change. Promising alternatives to these industrial methods rely on engineering the only biological pathway for generating bioaccessible nitrogen: microbial nitrogen fixation. Bioengineering strategies require an extensive understanding of underlying genetics in nitrogen-fixing microbes, but genetic tools for this critical goal remain lacking. The CRISPRi gene silencing system that we report, developed in the broadly utilized nitrogen-fixing bacterial model, , is an important step toward elucidating the complexity of nitrogen fixation genetics and enabling their manipulation.
Topics: Humans; Nitrogen Fixation; Clustered Regularly Interspaced Short Palindromic Repeats; Nitrogenase; Nitrogen; Base Sequence; Azotobacter vinelandii
PubMed: 38376168
DOI: 10.1128/msystems.00155-24 -
Journal of Proteome Research Mar 2024The value of synthetic microbial communities in biotechnology is gaining traction due to their ability to undertake more complex metabolic tasks than monocultures....
The value of synthetic microbial communities in biotechnology is gaining traction due to their ability to undertake more complex metabolic tasks than monocultures. However, a thorough understanding of strain interactions, productivity, and stability is often required to optimize growth and scale up cultivation. Quantitative proteomics can provide valuable insights into how microbial strains adapt to changing conditions in biomanufacturing. However, current workflows and methodologies are not suitable for simple artificial coculture systems where strain ratios are dynamic. Here, we established a workflow for coculture proteomics using an exemplar system containing two members, and . Factors affecting the quantitative accuracy of coculture proteomics were investigated, including peptide physicochemical characteristics such as molecular weight, isoelectric point, hydrophobicity, and dynamic range as well as factors relating to protein identification such as varying proteome size and shared peptides between species. Different quantification methods based on spectral counts and intensity were evaluated at the protein and cell level. We propose a new normalization method, named "LFQRatio", to reflect the relative contributions of two distinct cell types emerging from cell ratio changes during cocultivation. LFQRatio can be applied to real coculture proteomics experiments, providing accurate insights into quantitative proteome changes in each strain.
Topics: Proteome; Coculture Techniques; Microbiota; Molecular Weight; Proteomics
PubMed: 38354288
DOI: 10.1021/acs.jproteome.3c00714