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Microbiology (Reading, England) Apr 2018Azotobacter vinelandii has been studied for over 100 years since its discovery as an aerobic nitrogen-fixing organism. This species has proved useful for the study of... (Review)
Review
Azotobacter vinelandii has been studied for over 100 years since its discovery as an aerobic nitrogen-fixing organism. This species has proved useful for the study of many different biological systems, including enzyme kinetics and the genetic code. It has been especially useful in working out the structures and mechanisms of different nitrogenase enzymes, how they can function in oxic environments and the interactions of nitrogen fixation with other aspects of metabolism. Interest in studying A. vinelandii has waned in recent decades, but this bacterium still possesses great potential for new discoveries in many fields and commercial applications. The species is of interest for research because of its genetic pliability and natural competence. Its features of particular interest to industry are its ability to produce multiple valuable polymers - bioplastic and alginate in particular; its nitrogen-fixing prowess, which could reduce the need for synthetic fertilizer in agriculture and industrial fermentations, via coculture; its production of potentially useful enzymes and metabolic pathways; and even its biofuel production abilities. This review summarizes the history and potential for future research using this versatile microbe.
Topics: Azotobacter vinelandii; Biofuels; Biopolymers; Hydrogen; Metabolic Engineering; Metabolic Networks and Pathways; Nitrogen; Nitrogenase; Oxidoreductases; Oxygen
PubMed: 29533747
DOI: 10.1099/mic.0.000643 -
Journal of Biological Inorganic... Mar 2015Nitrogenase catalyzes biological nitrogen fixation, a key step in the global nitrogen cycle. Three homologous nitrogenases have been identified to date, along with... (Review)
Review
Nitrogenase catalyzes biological nitrogen fixation, a key step in the global nitrogen cycle. Three homologous nitrogenases have been identified to date, along with several structural and/or functional homologs of this enzyme that are involved in nitrogenase assembly, bacteriochlorophyll biosynthesis and methanogenic process, respectively. In this article, we provide an overview of the structures and functions of nitrogenase and its homologs, which highlights the similarity and disparity of this uniquely versatile group of enzymes.
Topics: Azotobacter vinelandii; Bacteriochlorophylls; Catalysis; Molybdenum; Nitrogen; Nitrogen Fixation; Nitrogenase; Structure-Activity Relationship
PubMed: 25491285
DOI: 10.1007/s00775-014-1225-3 -
The FEBS Journal Oct 2017The flavodoxin-like fold is a protein architecture that can be traced back to the universal ancestor of the three kingdoms of life. Many proteins share this α-β... (Review)
Review
The flavodoxin-like fold is a protein architecture that can be traced back to the universal ancestor of the three kingdoms of life. Many proteins share this α-β parallel topology and hence it is highly relevant to illuminate how they fold. Here, we review experiments and simulations concerning the folding of flavodoxins and CheY-like proteins, which share the flavodoxin-like fold. These polypeptides tend to temporarily misfold during unassisted folding to their functionally active forms. This susceptibility to frustration is caused by the more rapid formation of an α-helix compared to a β-sheet, particularly when a parallel β-sheet is involved. As a result, flavodoxin-like proteins form intermediates that are off-pathway to native protein and several of these species are molten globules (MGs). Experiments suggest that the off-pathway species are of helical nature and that flavodoxin-like proteins have a nonconserved transition state that determines the rate of productive folding. Folding of flavodoxin from Azotobacter vinelandii has been investigated extensively, enabling a schematic construction of its folding energy landscape. It is the only flavodoxin-like protein of which cotranslational folding has been probed. New insights that emphasize differences between in vivo and in vitro folding energy landscapes are emerging: the ribosome modulates MG formation in nascent apoflavodoxin and forces this polypeptide toward the native state.
Topics: Azotobacter vinelandii; Escherichia coli; Escherichia coli Proteins; Flavodoxin; Gene Expression; Methyl-Accepting Chemotaxis Proteins; Models, Molecular; Protein Biosynthesis; Protein Conformation, alpha-Helical; Protein Conformation, beta-Strand; Protein Folding; Protein Isoforms; Thermodynamics
PubMed: 28380286
DOI: 10.1111/febs.14077 -
Bioengineered 2015Glycerol is an interesting feedstock for biomaterials such as biofuels and bioplastics because of its abundance as a by-product during biodiesel production. Here we...
Glycerol is an interesting feedstock for biomaterials such as biofuels and bioplastics because of its abundance as a by-product during biodiesel production. Here we demonstrate glycerol metabolism in the nitrogen-fixing species Azotobacter vinelandii through metabolomics and nitrogen-free bacterial production of biopolymers, such as poly-d-3-hydroxybutyrate (PHB) and alginate, from glycerol. Glycerol-3-phosphate was accumulated in A. vinelandii cells grown on glycerol to the exponential phase, and its level drastically decreased in the cells grown to the stationary growth phase. A. vinelandii also overexpressed the glycerol-3-phosphate dehydrogenase gene when it was grown on glycerol. These results indicate that glycerol was first converted to glycerol-3-phosphate by glycerol kinase. Other molecules with industrial interests, such as lactic acid and amino acids including γ-aminobutyric acid, have also been accumulated in the bacterial cells grown on glycerol. Transmission electron microscopy revealed that glycerol-grown A. vinelandii stored PHB within the cells. The PHB production level reached 33% per dry cell weight in nitrogen-free glycerol medium. When grown on glycerol, alginate-overproducing mutants generated through chemical mutagenesis produced 2-fold the amount of alginate from glycerol than the parental wild-type strain. To the best of our knowledge, this is the first report on bacterial production of biopolymers from glycerol without addition of any nitrogen source.
Topics: Alginates; Azotobacter vinelandii; Bacterial Proteins; Culture Media; Fermentation; Gene Expression Regulation, Bacterial; Glucuronic Acid; Glycerol; Glycerol Kinase; Glycerolphosphate Dehydrogenase; Glycerophosphates; Hexuronic Acids; Hydroxybutyrates; Lactic Acid; Mutation; Nitrogen; Polyesters; gamma-Aminobutyric Acid
PubMed: 25880041
DOI: 10.1080/21655979.2015.1040209 -
Protein Science : a Publication of the... Oct 2017Azotobacter vinelandii flavodoxin II serves as a physiological reductant of nitrogenase, the enzyme system mediating biological nitrogen fixation. Wildtype A. vinelandii...
Azotobacter vinelandii flavodoxin II serves as a physiological reductant of nitrogenase, the enzyme system mediating biological nitrogen fixation. Wildtype A. vinelandii flavodoxin II was electrochemically and crystallographically characterized to better understand the molecular basis for this functional role. The redox properties were monitored on surfactant-modified basal plane graphite electrodes, with two distinct redox couples measured by cyclic voltammetry corresponding to reduction potentials of -483 ± 1 mV and -187 ± 9 mV (vs. NHE) in 50 mM potassium phosphate, 150 mM NaCl, pH 7.5. These redox potentials were assigned as the semiquinone/hydroquinone couple and the quinone/semiquinone couple, respectively. This study constitutes one of the first applications of surfactant-modified basal plane graphite electrodes to characterize the redox properties of a flavodoxin, thus providing a novel electrochemical method to study this class of protein. The X-ray crystal structure of the flavodoxin purified from A. vinelandii was solved at 1.17 Å resolution. With this structure, the native nitrogenase electron transfer proteins have all been structurally characterized. Docking studies indicate that a common binding site surrounding the Fe-protein [4Fe:4S] cluster mediates complex formation with the redox partners Mo-Fe protein, ferredoxin I, and flavodoxin II. This model supports a mechanistic hypothesis that electron transfer reactions between the Fe-protein and its redox partners are mutually exclusive.
Topics: Azotobacter vinelandii; Bacterial Proteins; Crystallography, X-Ray; Electrochemistry; Flavodoxin; Hydrogen-Ion Concentration; Iron; Models, Molecular; Nitrogenase; Protein Conformation
PubMed: 28710816
DOI: 10.1002/pro.3236 -
Antioxidants & Redox Signaling Feb 2020Structural and functional characterization of the globin-coupled sensors (GCSs) from (GReg) and (GReg). Ultraviolet/visible and resonance Raman spectroscopies...
Structural and functional characterization of the globin-coupled sensors (GCSs) from (GReg) and (GReg). Ultraviolet/visible and resonance Raman spectroscopies confirm the presence in GReg and GReg of a globin domain capable of reversible gaseous ligand binding. In GReg, an influence of the transmitter domain on the heme proximal region of the globin domain can be seen, and ' is higher than for other GCSs. The O binding kinetics suggests the presence of an open and a closed conformation. As for GReg, the fully oxygenated GReg show a very high diguanylate cyclase activity. The carbon monoxide rebinding to GReg indicates that intra- and intermolecular interactions influence the ligand binding. The globin domains of both proteins (GReg globin domain and GRegGb with cysteines (Cys16, 45, 114, 154) mutated to serines [GReg-Gb*]) share the same GCS fold, a similar proximal but a different distal side structure. They homodimerize through a G-H helical bundle as in other GCSs. However, GReg-Gb* shows also a second dimerization mode. This article extends our knowledge on the GCS proteins and contributes to a better understanding of the GCSs role in the formation of bacterial biofilms. GReg and GReg conform to the GCS family, share a similar overall structure, but they have different properties in terms of the ligand binding. In particular, GReg shows an open and a closed conformation that in the latter form will very tightly bind oxygen. GReg has only one closed conformation. In both proteins, it is the fully oxygenated GCS form that catalyzes the production of the second messenger.
Topics: Azotobacter vinelandii; Bacterial Proteins; Binding Sites; Bordetella pertussis; Globins; Heme-Binding Proteins; Protein Structure, Quaternary; Protein Structure, Tertiary; Structure-Activity Relationship
PubMed: 31559835
DOI: 10.1089/ars.2018.7690 -
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 -
Journal of Bacteriology Nov 2021Azotobacter vinelandii is a nitrogen-fixing free-living soil microbe that has been studied for decades in relation to biological nitrogen fixation (BNF). It is highly...
Azotobacter vinelandii is a nitrogen-fixing free-living soil microbe that has been studied for decades in relation to biological nitrogen fixation (BNF). It is highly amenable to genetic manipulation, helping to unravel the intricate importance of different proteins involved in the process of BNF, including the biosynthesis of cofactors that are essential to assembling the complex metal cofactors that catalyze the difficult reaction of nitrogen fixation. Additionally, A. vinelandii accomplishes this feat while growing as an obligate aerobe, differentiating it from many of the nitrogen-fixing bacteria that are associated with plant roots. The ability to function in the presence of oxygen makes A. vinelandii suitable for application in various potential biotechnological schemes. In this study, we employed transposon sequencing (Tn-seq) to measure the fitness defects associated with disruptions of various genes under nitrogen-fixing dependent growth, versus growth with extraneously provided urea as a nitrogen source. The results allowed us to probe the importance of more than 3,800 genes, revealing that many genes previously believed to be important, can be successfully disrupted without impacting cellular fitness. These results provide insights into the functional redundancy in A. vinelandii, while also providing a direct measure of fitness for specific genes associated with the process of BNF. These results will serve as a valuable reference tool in future studies to uncover the mechanisms that govern this process.
Topics: Azotobacter vinelandii; Bacterial Proteins; Base Sequence; DNA Transposable Elements; Gene Expression Regulation, Bacterial; Genetic Fitness; Molybdenum; Nitrogen; Urea
PubMed: 34570624
DOI: 10.1128/JB.00404-21 -
Molecular Microbiology Jul 2022All diazotrophic bacteria and archaea isolated so far utilise a nitrogenase enzyme-containing molybdenum in the active site co-factor to fix atmospheric dinitrogen to...
All diazotrophic bacteria and archaea isolated so far utilise a nitrogenase enzyme-containing molybdenum in the active site co-factor to fix atmospheric dinitrogen to ammonia. However, in addition to the Mo-dependent nitrogenase, some nitrogen-fixing prokaryotes also express genetically distinct alternative nitrogenase isoenzymes, namely the V-dependent and Fe-only nitrogenases, respectively. Nitrogenase isoenzymes are expressed hierarchically according to metal availability and catalytic efficiency. In proteobacteria, this hierarchy is maintained via stringent transcriptional regulation of gene clusters by dedicated bacterial enhancer-binding proteins (bEBPs). The model diazotroph Azotobacter vinelandii contains two paralogs of the vanadium nitrogenase activator VnfA (henceforth, VnfA1), designated VnfA2 and VnfA3, with unknown functions. Here we demonstrate that the VnfA1 and VnfA3 bEBPs bind to the same target promoters in the Azotobacter vinelandii genome and co-activate a subset of genes in the absence of V, including the structural genes for the Fe-only nitrogenase. Co-activation is inhibited by the presence of V and is dependent on an accessory protein VnfZ that is co-expressed with VnfA3. Our studies uncover a plethora of interactions between bEBPs required for nitrogen fixation, revealing the unprecedented potential for fine-tuning the expression of alternative nitrogenases in response to metal availability.
Topics: Azotobacter vinelandii; Bacterial Proteins; DNA-Binding Proteins; Isoenzymes; Metals; Molybdenum; Nitrogen Fixation; Nitrogenase
PubMed: 35718936
DOI: 10.1111/mmi.14955 -
International Journal of Molecular... Jan 2023Nitrogen-fixing bacteria execute biological nitrogen fixation through nitrogenase, converting inert dinitrogen (N) in the atmosphere into bioavailable nitrogen.... (Review)
Review
Nitrogen-fixing bacteria execute biological nitrogen fixation through nitrogenase, converting inert dinitrogen (N) in the atmosphere into bioavailable nitrogen. Elaborating the molecular mechanisms of orderly and efficient biological nitrogen fixation and applying them to agricultural production can alleviate the "nitrogen problem". is a well-established model bacterium for studying nitrogen fixation, utilizing nitrogenase encoded by the gene cluster to fix nitrogen. In , the NifA-NifL system fine-tunes the gene cluster transcription by sensing the redox signals and energy status, then modulating nitrogen fixation. In this manuscript, we investigate the transcriptional regulation mechanism of the gene in autogenous nitrogen-fixing bacteria. We discuss how autogenous nitrogen fixation can better be integrated into agriculture, providing preliminary comprehensive data for the study of autogenous nitrogen-fixing regulation.
Topics: Nitrogen Fixation; Transcription Factors; Bacterial Proteins; Nitrogenase; Azotobacter vinelandii; Genes, Bacterial; Nitrogen; Gene Expression Regulation, Bacterial
PubMed: 36674420
DOI: 10.3390/ijms24020907