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Angewandte Chemie (International Ed. in... May 2024Nitrogenase reduces N to NH at its active-site cofactor. Previous studies of an N-bound Mo-nitrogenase from Azotobacter vinelandii suggest binding of three N species via...
Nitrogenase reduces N to NH at its active-site cofactor. Previous studies of an N-bound Mo-nitrogenase from Azotobacter vinelandii suggest binding of three N species via asymmetric belt-sulfur displacements in the two cofactors of its catalytic component (designated Av1*), leading to the proposal of stepwise N reduction involving all cofactor belt-sulfur sites; yet, the evidence for the existence of multiple N species on Av1* remains elusive. Here we report a study of ATP-independent, Eu/SO -driven turnover of Av1* using GC-MS and frequency-selective pulse NMR techniques. Our data demonstrate incorporation of D-derived D by Av1* into the products of CH- and H-reduction, and decreased formation of NH by Av1* concomitant with the release of N under H; moreover, they reveal a strict dependence of these activities on SO . These observations point to the presence of distinct N species on Av1*, thereby providing strong support for our proposed mechanism of stepwise reduction of N via belt-sulfur mobilization.
Topics: Nitrogenase; Azotobacter vinelandii; Nitrogen; Adenosine Triphosphate
PubMed: 38527309
DOI: 10.1002/anie.202400273 -
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 -
Frontiers in Plant Science 2023In terms of providing additional feeds and improving the soil fertility, planting forage crops during the fallow seasons is an effective strategy to promote resource...
In terms of providing additional feeds and improving the soil fertility, planting forage crops during the fallow seasons is an effective strategy to promote resource utilization. The objective of this research was to compare the effects of planting different forage crops on the yields and nutritive compositions of forage and soil properties of winter fallow paddy in southern China. Five forage crops, including alfalfa (, AF), common vetch (, CV), milk vetch (, MV), smooth vetch (, SV) and Italian ryegrass (, IR), were planted by monoculture on the winter fallow paddy in 2017-2018 (season 1) and 2018-2019 (season 2), respectively. The dry matter yield of IR was significantly higher than those of AF, CV, SV and MV (<0.05). The crude protein yield of IR was significantly higher than those of AF, CV and MV (<0.05). The neutral detergent fiber and acid detergent fiber contents of CV, SV and IR were significantly lower than those of AF and MV (<0.05). Forage crops significantly affected the culturable microbial population of soils (<0.05). The bacteria, actinomyces and fungi numbers on IR were the highest, while azotobacter number was the lowest. The catalase, acid-phosphatase and invertase activities of IR soil were the lowest. The numbers of bacteria, actinomyces and fungi of IR soil were the highest. IR and SV were the best crops to obtain forage and improve the soil. When producers pursue higher forage yield, we recommend planting Italian ryegrass. If the producers want to improve soil characteristics, smooth vetch is the most suitable plant. These results provide useful information to rice growers for cropping management when growing forage crops (based on the yield and nutritional value) or green manure (based on improving the soil fertility) as an alternative to late rice harvest.
PubMed: 38293627
DOI: 10.3389/fpls.2023.1292114 -
International Journal of Biological... Mar 2024The production of poly-3-hydroxybutyrate (PHB) on an industrial scale remains a major challenge due to its higher production cost compared to petroleum-based plastics....
The production of poly-3-hydroxybutyrate (PHB) on an industrial scale remains a major challenge due to its higher production cost compared to petroleum-based plastics. As a result, it is necessary to develop efficient fermentative processes using low-cost substrates and identify high-value-added applications where biodegradability and biocompatibility properties are of fundamental importance. In this study, grape residues, mainly grape skins, were used as the sole carbon source in Azotobacter vinelandii OP cultures for PHB production and subsequent nanoparticle synthesis based on the extracted polymer. The grape residue pretreatment showed a high rate of conversion into reducing sugars (fructose and glucose), achieving up to 43.3 % w w without the use of acid or external heat. The cultures were grown in shake flasks, obtaining a biomass concentration of 2.9 g L and a PHB accumulation of up to 37.7 % w w. PHB was characterized using techniques such as Fourier transform infrared spectroscopy (FTIR), thermogravimetric analysis (TGA), and differential scanning calorimetry (DSC). The formation of emulsified PHB nanoparticles showed high stability, with a particle size between 210 and 240 nm and a zeta potential between -12 and - 15 mV over 72 h. Owing to these properties, the produced PHB nanoparticles hold significant potential for applications in drug delivery.
Topics: Vitis; Carbon; Polyesters; Polymers; Hydroxybutyrates; Polyhydroxybutyrates
PubMed: 38266847
DOI: 10.1016/j.ijbiomac.2024.129649 -
Journal of Hazardous Materials Mar 2024Vanadium dioxide (VO) has been used in a variety of products due to its outstanding phase transition properties. However, as potential heavy metal contaminants, the...
Vanadium dioxide (VO) has been used in a variety of products due to its outstanding phase transition properties. However, as potential heavy metal contaminants, the environmental hazards and risks of VO should be systematically investigated. Biological nitrogen fixation is one of the most dominant processes in biogeochemical cycle, which is associated with nitrogen-fixing bacteria. In this study, we reported the environmental bio-effects of VO micro/nanoparticles on the nitrogen-fixing bacterium Azotobacter vinelandii. VO at 10 and 30 mg/L caused severe hazards to A. vinelandii, such as cell apoptosis, oxidative damage, physical damage, genotoxicity, and the loss of nitrogen fixation activity. The up-regulated differentially expressed genes of A. vinelandii were related to stress response, and the down-regulated genes were mainly related to energy metabolism. Surprisingly, VO of 10 mg/L decreased the nif gene expression but elevated the vnf gene expression, which enhanced the ability of A. vinelandii to reduce acetylene in anaerobic environment. In addition, under tested conditions, VO nanoparticles exhibited insignificantly higher toxicity than VO microparticles.
Topics: Azotobacter vinelandii; Nitrogen-Fixing Bacteria; Nitrogen Fixation; Nitrogen
PubMed: 38266589
DOI: 10.1016/j.jhazmat.2024.133553 -
Environmental Science and Pollution... Feb 2024The degradation of biodegradable plastics poses a significant environmental challenge and requires effective solutions. In this study, an esterase derived from a...
The degradation of biodegradable plastics poses a significant environmental challenge and requires effective solutions. In this study, an esterase derived from a phyllosphere yeast Pseudozyma antarctica (PaE) enhanced the degradation and mineralization of poly(butylene succinate-co-adipate) (PBSA) film in soil. PaE was found to substitute for esterases from initial degraders and activate sequential esterase production from soil microbes. The PBSA film pretreated with PaE (PBSA-E) rapidly diminished and was mineralized in soil until day 55 with high CO production. Soil with PBSA-E maintained higher esterase activities with enhancement of microbial abundance, whereas soil with inactivated PaE-treated PBSA film (PBSA-inact E) showed gradual degradation and time-lagged esterase activity increases. The fungal genera Arthrobotrys and Tetracladium, as possible contributors to PBSA-film degradation, increased in abundance in soil with PBSA-inact E but were less abundant in soil with PBSA-E. The dominance of the fungal genus Fusarium and the bacterial genera Arthrobacter and Azotobacter in soil with PBSA-E further supported PBSA degradation. Our study highlights the potential of PaE in addressing concerns associated with biodegradable plastic persistence in agricultural and environmental contexts.
Topics: Polyesters; Esterases; Saccharomyces cerevisiae; Soil; Biodegradable Plastics; Microbiota; Plastics
PubMed: 38265596
DOI: 10.1007/s11356-024-31994-y -
Frontiers in Microbiology 2023Genus is a large assemblage of diverse microorganisms, not sharing a common evolutionary history. To clarify their evolutionary relationships and classification, we...
Genus is a large assemblage of diverse microorganisms, not sharing a common evolutionary history. To clarify their evolutionary relationships and classification, we have conducted comprehensive phylogenomic and comparative analyses on 388 genomes. In phylogenomic trees, species formed 12 main clusters, apart from the "Aeruginosa clade" containing its type species, . In parallel, our detailed analyses on protein sequences from genomes have identified 98 novel conserved signature indels (CSIs), which are uniquely shared by the species from different observed clades/groups. Six CSIs, which are exclusively shared by species from the "Aeruginosa clade," provide reliable demarcation of this clade corresponding to the genus in molecular terms. The remaining 92 identified CSIs are specific for nine other species clades and the genera and which branch in between them. The identified CSIs provide strong independent evidence of the genetic cohesiveness of these species clades and offer reliable means for their demarcation/circumscription. Based on the robust phylogenetic and molecular evidence presented here supporting the distinctness of the observed species clades, we are proposing the transfer of species from the following clades into the indicated novel genera: Alcaligenes clade - gen. nov.; Fluvialis clade - gen. nov.; Linyingensis clade - gen. nov.; Oleovorans clade - gen. nov.; Resinovorans clade - gen. nov.; Straminea clade - gen. nov.; and Thermotolerans clade - gen. nov. In addition, descriptions of the genera , , , and are emended to include information for the CSIs specific for them. The results presented here should aid in the development of a more reliable classification scheme for species.
PubMed: 38249459
DOI: 10.3389/fmicb.2023.1273665