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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 -
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 -
Molecular Plant-microbe Interactions :... Sep 2023spp. make up 1.6% of the bacteria in the soil and are found throughout the world. More than 140 species of this genus have been identified, some beneficial to the...
spp. make up 1.6% of the bacteria in the soil and are found throughout the world. More than 140 species of this genus have been identified, some beneficial to the plant. Several species in the family Pseudomonadaceae, including AvOP, A1501, DSM4166, 6HT33bT, and sp. strain K1 can fix nitrogen from the air. The genes required for these reactions are organized in a nitrogen fixation island, obtained via horizontal gene transfer from , , and . Today, this island is conserved in spp. from different geographical locations, which, in turn, have evolved to deal with different geo-climatic conditions. Here, we summarize the molecular mechanisms behind -driven plant growth promotion, with particular focus on improving plant performance at limiting nitrogen (N) and improving plant N content. We describe -plant interaction strategies in the soil, noting that the mechanisms of denitrification, ammonification, and secondary metabolite signaling are only marginally explored. Plant growth promotion is dependent on the abiotic conditions and differs at sufficient and deficient N. The molecular controls behind different plant responses are not fully elucidated. We suggest that superposition of transcriptome, proteome, and metabolome data and their integration with plant phenotype development through time will help fill these gaps. The aim of this review is to summarize the knowledge behind -driven nitrogen fixation and to point to possible agricultural solutions. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.
PubMed: 36989040
DOI: 10.1094/MPMI-10-22-0223-CR -
Microbiology Spectrum Sep 2023Biological nitrogen fixation, the microbial reduction of atmospheric nitrogen to bioavailable ammonia, represents both a major limitation on biological productivity and...
Biological nitrogen fixation, the microbial reduction of atmospheric nitrogen to bioavailable ammonia, represents both a major limitation on biological productivity and a highly desirable engineering target for synthetic biology. However, the engineering of nitrogen fixation requires an integrated understanding of how the gene regulatory dynamics of host diazotrophs respond across sequence-function space of its central catalytic metalloenzyme, nitrogenase. Here, we interrogate this relationship by analyzing the transcriptome of engineered with a phylogenetically inferred ancestral nitrogenase protein variant. The engineered strain exhibits reduced cellular nitrogenase activity but recovers wild-type growth rates following an extended lag period. We find that expression of genes within the immediate nitrogen fixation network is resilient to the introduced nitrogenase sequence-level perturbations. Rather the sustained physiological compatibility with the ancestral nitrogenase variant is accompanied by reduced expression of genes that support trace metal and electron resource allocation to nitrogenase. Our results spotlight gene expression changes in cellular processes adjacent to nitrogen fixation as productive engineering considerations to improve compatibility between remodeled nitrogenase proteins and engineered host diazotrophs. IMPORTANCE is a key model bacterium for the study of biological nitrogen fixation, an important metabolic process catalyzed by nitrogenase enzymes. Here, we demonstrate that compatibilities between engineered strains and nitrogenase variants can be modulated at the regulatory level. The engineered strain studied here responds by adjusting the expression of proteins involved in cellular processes adjacent to nitrogen fixation, rather than that of nitrogenase proteins themselves. These insights can inform future strategies to transfer nitrogenase variants to non-native hosts.
PubMed: 37702481
DOI: 10.1128/spectrum.02815-23 -
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 -
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 -
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 -
Microorganisms Sep 2023Genomic DNA has high phosphate content; therefore, monoploid prokaryotes need an external phosphate source or an internal phosphate storage polymer for replication and...
One Advantage of Being Polyploid: Prokaryotes of Various Phylogenetic Groups Can Grow in the Absence of an Environmental Phosphate Source at the Expense of Their High Genome Copy Numbers.
Genomic DNA has high phosphate content; therefore, monoploid prokaryotes need an external phosphate source or an internal phosphate storage polymer for replication and cell division. For two polyploid prokaryotic species, the halophilic archaeon and the cyanobacterium PCC 6803, it has been reported that they can grow in the absence of an external phosphate source by reducing the genome copy number per cell. To unravel whether this feature might be widespread in and typical for polyploid prokaryotes, three additional polyploid prokaryotic species were analyzed in the present study, i.e., the alphaproteobacterium , the gammaproteobacterium , and the haloarchaeon Polyploid cultures were incubated in the presence and in the absence of external phosphate, growth was recorded, and genome copy numbers per cell were quantified. Limited growth in the absence of phosphate was observed for all three species. Phosphate was added to phosphate-starved cultures to verify that the cells were still viable and growth-competent. Remarkably, stationary-phase cells grown in the absence or presence of phosphate did not become monoploid but stayed oligoploid with about five genome copies per cell. As a negative control, it was shown that monoploid cultures did not exhibit any growth in the absence of phosphate. Taken together, all five polyploid prokaryotic species that have been characterized until now can grow in the absence of environmental phosphate by reducing their genome copy numbers, indicating that cell proliferation outperforms other evolutionary advantages of polyploidy.
PubMed: 37764113
DOI: 10.3390/microorganisms11092267 -
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 -
Faraday Discussions Jul 2023Nitrogenases catalyse the 6-electron reduction of dinitrogen to ammonia, passing through a series of redox and protonation levels during catalytic substrate reduction....
Nitrogenases catalyse the 6-electron reduction of dinitrogen to ammonia, passing through a series of redox and protonation levels during catalytic substrate reduction. The molybdenum-iron nitrogenase is the most well-studied, but redox potentials associated with proton-coupled transformations between the redox levels of the catalytic MoFe protein have proved difficult to pin down, in part due to a complex electron-transfer pathway from the partner Fe protein, linked to ATP-hydrolysis. Here, we apply electrochemical control to the MoFe protein of nitrogenase, using europium(III/II)-ligand couples as low potential redox mediators. We combine insight from the electrochemical current response with data from gas chromatography and infrared spectroscopy, in order to define potentials for the binding of a series of inhibitors (carbon monoxide, methyl isocyanide) to the metallo-catalytic site of the MoFe protein, and the onset of catalytic transformation of alternative substrates (protons and acetylene) by the enzyme. Thus, we associate potentials with the redox levels for inhibition and catalysis by nitrogenase, with relevance to the elusive mechanism of biological nitrogen fixation.
Topics: Nitrogenase; Molybdoferredoxin; Oxidation-Reduction; Proteins; Nitrogen Fixation
PubMed: 37060162
DOI: 10.1039/d2fd00170e