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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 -
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 -
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 -
Journal of Inorganic Biochemistry Apr 2024The light-driven reduction of dinitrogen (N) to ammonia (NH) catalyzed by a cadmium sulfide (CdS) nanocrystal‑nitrogenase MoFe protein biohybrid is dependent on a...
The light-driven reduction of dinitrogen (N) to ammonia (NH) catalyzed by a cadmium sulfide (CdS) nanocrystal‑nitrogenase MoFe protein biohybrid is dependent on a range of different factors, including an appropriate hole-scavenging sacrificial electron donor (SED). Here, the impact of different SEDs on the overall rate of N reduction catalyzed by a CdS quantum dot (QD)-MoFe protein system was determined. The selection of SED was guided by several goals: (i) molecules with standard reduction potentials sufficient to reduce the oxidized CdS QD, (ii) molecules that do not absorb the excitation wavelength of the CdS QD, and (iii) molecules that could be readily reduced by sustainable processes. Earlier studies utilized buffer molecules or ascorbic acid as the SED. The effectiveness of ascorbic acid as SED was compared to dithionite (DT), triethanolamine (TEOA), and hydroquinone (HQ) across a range of concentrations in supporting N reduction to NH in a CdS QD-MoFe protein photocatalytic system. It was found that TEOA supported N reduction rates comparable to those observed for dithionite and ascorbic acid. HQ was found to support significantly higher rates of N reduction compared to the other SEDs at a concentration of 50 mM. A comparison of the rates of N reduction by the biohybrid complex to the standard reduction potential (E) of the SEDs reveals that E is not the only factor impacting the efficiency of hole-scavenging. These findings reveal the importance of the SED properties for improving the efficiency of hole-scavenging in the light-driven N reduction reaction catalyzed by a CdS QD-MoFe protein hybrid.
Topics: Nitrogenase; Molybdoferredoxin; Oxidation-Reduction; Dithionite; Catalysis; Ascorbic Acid; Azotobacter vinelandii; Sulfides; Cadmium Compounds
PubMed: 38219407
DOI: 10.1016/j.jinorgbio.2024.112484 -
MBio Feb 2024Biological nitrogen fixation, the conversion of inert N to metabolically tractable NH, is only performed by certain microorganisms called diazotrophs and is catalyzed by...
Biological nitrogen fixation, the conversion of inert N to metabolically tractable NH, is only performed by certain microorganisms called diazotrophs and is catalyzed by the nitrogenases. A [7Fe-9S-C-Mo--homocitrate]-cofactor, designated FeMo-co, provides the catalytic site for N reduction in the Mo-dependent nitrogenase. Thus, achieving FeMo-co formation in model eukaryotic organisms, such as , represents an important milestone toward endowing them with a capacity for Mo-dependent biological nitrogen fixation. A central player in FeMo-co assembly is the scaffold protein NifEN upon which processing of NifB-co, an [8Fe-9S-C] precursor produced by NifB, occurs. Prior work established that NifB-co can be produced in mitochondria. In the present work, a library of genes from diverse diazotrophs was expressed in , targeted to mitochondria, and surveyed for their ability to produce soluble NifEN protein complexes. Many such NifEN variants supported FeMo-co formation when heterologously produced in the diazotroph . However, only three of them accumulated in soluble forms in mitochondria of aerobically cultured . Of these, two variants were active in the FeMo-co synthesis assay. NifEN, NifB, and NifH proteins from different species, all of them produced in and purified from mitochondria, were combined to establish successful FeMo-co biosynthetic pathways. These findings demonstrate that combining diverse interspecies nitrogenase FeMo-co assembly components could be an effective and, perhaps, the only approach to achieve and optimize nitrogen fixation in a eukaryotic organism.IMPORTANCEBiological nitrogen fixation, the conversion of inert N2 to metabolically usable NH3, is a process exclusive to diazotrophic microorganisms and relies on the activity of nitrogenases. The assembly of the nitrogenase [7Fe-9S-C-Mo--homocitrate]-cofactor (FeMo-co) in a eukaryotic cell is a pivotal milestone that will pave the way to engineer cereals with nitrogen fixing capabilities and therefore independent of nitrogen fertilizers. In this study, we identified NifEN protein complexes that were functional in the model eukaryotic organism . NifEN is an essential component of the FeMo-co biosynthesis pathway. Furthermore, the FeMo-co biosynthetic pathway was recapitulated using only proteins expressed in . FeMo-co biosynthesis was achieved by combining nitrogenase FeMo-co assembly components from different species, a promising strategy to engineer nitrogen fixation in eukaryotic organisms.
Topics: Nitrogenase; Saccharomyces cerevisiae; Molybdoferredoxin; Bacterial Proteins; Mitochondria; Nitrogen; Tricarboxylic Acids; Iron Compounds
PubMed: 38126768
DOI: 10.1128/mbio.03088-23 -
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 -
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