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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 -
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 -
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 -
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 -
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 -
Applied and Environmental Microbiology Aug 2018selectively utilizes three types of nitrogenase (molybdenum, vanadium, and iron only) to fix N, with their expression regulated by the presence or absence of different...
selectively utilizes three types of nitrogenase (molybdenum, vanadium, and iron only) to fix N, with their expression regulated by the presence or absence of different metal cofactors in its environment. Each alternative nitrogenase isoenzyme is predicted to have different electron flux requirements based on measurements, with the molybdenum nitrogenase requiring the lowest flux and the iron-only nitrogenase requiring the highest. Here, prior characterized strains, derepressed in nitrogenase synthesis and also deficient in uptake hydrogenase, were further modified to generate new mutants lacking the ability to produce poly-β-hydroxybutyrate (PHB). PHB is a storage polymer generated under oxygen-limiting conditions and can represent up to 70% of the cells' dry weight. The absence of such granules facilitated the study of relationships between catalytic biomass and product molar yields across different adaptive respiration conditions. The released hydrogen gas observed during growth, due to the inability of the mutants to recapture hydrogen, allowed for direct monitoring of nitrogenase activity for each isoenzyme. The data presented here show that increasing oxygen exposure limits equally the activities of all nitrogenase isoenzymes, while under comparative conditions, the Mo nitrogenase enzyme evolves more hydrogen per unit of biomass than the alternative isoenzymes. has been a focus of intense research for over 100 years. It has been investigated for a variety of functions, including agricultural fertilization and hydrogen production. All of these endeavors are centered around 's ability to fix nitrogen aerobically using three nitrogenase isoenzymes. The majority of research up to this point has targeted measurements of the molybdenum nitrogenase, and robust data contrasting how oxygen impacts the activity of each nitrogenase isoenzyme are lacking. This article aims to provide nitrogenase activity data using a real-time evaluation of hydrogen gas released by derepressed nitrogenase mutants lacking an uptake hydrogenase and PHB accumulation.
Topics: Azotobacter vinelandii; Bacterial Proteins; Hydrogen; Hydroxybutyrates; Iron; Molybdenum; Nitrogen; Nitrogen Fixation; Nitrogenase; Oxidation-Reduction; Oxygen; Polyesters; Vanadium
PubMed: 29915110
DOI: 10.1128/AEM.01208-18 -
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 -
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 -
Applied and Environmental Microbiology Feb 2022The structure and functional properties of alginates are dictated by the monomer composition and molecular weight distribution. Mannuronan C-5-epimerases determine the...
The structure and functional properties of alginates are dictated by the monomer composition and molecular weight distribution. Mannuronan C-5-epimerases determine the monomer composition by catalyzing the epimerization of β-d-mannuronic acid (M) residues into α-l-guluronic acid (G) residues. The molecular weight is affected by alginate lyases, which catalyze a β-elimination mechanism that cleaves alginate chains. The reaction mechanisms for the epimerization and lyase reactions are similar, and some enzymes can perform both reactions. These dualistic enzymes share high sequence identity with mannuronan C-5-epimerases without lyase activity. The mechanism behind their activity and the amino acid residues responsible for it are still unknown. We investigate mechanistic determinants involved in the bifunctional epimerase and lyase activity of AlgE7 from Azotobacter vinelandii. Based on sequence analyses, a range of AlgE7 variants were constructed and subjected to activity assays and product characterization by nuclear magnetic resonance (NMR) spectroscopy. Our results show that calcium promotes lyase activity, whereas NaCl reduces the lyase activity of AlgE7. By using defined polymannuronan (polyM) and polyalternating alginate (polyMG) substrates, the preferred cleavage sites of AlgE7 were found to be M|XM and G|XM, where X can be either M or G. From the study of AlgE7 mutants, R148 was identified as an important residue for the lyase activity, and the point mutant R148G resulted in an enzyme with only epimerase activity. Based on the results obtained in the present study, we suggest a unified catalytic reaction mechanism for both epimerase and lyase activities where H154 functions as the catalytic base and Y149 functions as the catalytic acid. Postharvest valorization and upgrading of algal constituents are promising strategies in the development of a sustainable bioeconomy based on algal biomass. In this respect, alginate epimerases and lyases are valuable enzymes for tailoring the functional properties of alginate, a polysaccharide extracted from brown seaweed with numerous applications in food, medicine, and material industries. By providing a better understanding of the catalytic mechanism and of how the two enzyme actions can be altered by changes in reaction conditions, this study opens further applications of bacterial epimerases and lyases in the enzymatic tailoring of alginate polymers.
Topics: Alginates; Azotobacter vinelandii; Carbohydrate Epimerases; Hexuronic Acids; Polysaccharide-Lyases
PubMed: 34878812
DOI: 10.1128/AEM.01836-21 -
The Journal of Biological Chemistry Jan 1996We have identified the molecular basis for the nitrogenase negative phenotype exhibited by Azotobacter vinelandii UW97. This strain was initially isolated following... (Comparative Study)
Comparative Study
We have identified the molecular basis for the nitrogenase negative phenotype exhibited by Azotobacter vinelandii UW97. This strain was initially isolated following nitrosoguanidine mutagenesis. Recently, it was shown that this strain lacks the Fe protein activity, which results in the synthesis of a FeMo cofactor-deficient apodinitrogenase. Activation of this apodinitrogenase requires the addition of both MgATP and wild-type Fe protein to the crude extracts made by A. vinelandii UW97 (Allen, R.M., Homer, M.J., Chatterjee R., Ludden, P.W., Roberts, G.P., and Shah, V.K. (1993) J. Biol. Chem. 268 23670-23674). Earlier, we proposed the sequence of events in the MoFe protein assembly based on the biochemical and spectroscopic analysis of the purified apodinitrogenase from A. vinelandii DJ54 (Gavini, N., Ma, L., Watt, G., and Burgess, B.K. (1994) Biochemistry 33, 11842-11849). Taken together, these results imply that the assembly process of apodinitrogenase is arrested at the same step in both of these strains. Since A. vinelandii DJ54 is a delta nifH strain, this strain is not useful in identifying the features of the Fe protein involved in the MoFe protein assembly. Here, we report a systematic analysis of an A. vinelandii UW97 mutant and show that, unlike A. vinelandii DJ54, the nifH gene of A. vinelandii UW97 has no deletion in either coding sequence or the surrounding sequences. The specific mutation responsible for the Nif- phenotype of A. vinelandii UW97 is the substitution of a non-conserved serine at position 44 of the Fe protein by a phenylalanine as shown by DNA sequencing. Furthermore, oligonucleotide site-directed mutagenesis was employed to confirm that the Nif- phenotype in A. vinelandii UW97 is exclusively due to the substitution of the Fe protein residue serine 44 by phenylalanine. By contrast, replacing Ser-44 with alanine did not affect the Nif phenotype of A. vinelandii. Therefore, it seems that the Nif- phenotype of A. vinelandii UW97 is caused by a general structural disturbance of the Fe protein due to the presence of the bulky phenylalanine at position 44.
Topics: Amino Acid Sequence; Azotobacter vinelandii; Consensus Sequence; Genes, Bacterial; Molecular Sequence Data; Molybdoferredoxin; Mutagenesis, Site-Directed; Nitrogen Fixation; Nitrogenase; Sequence Alignment; Sequence Homology, Amino Acid; Structure-Activity Relationship
PubMed: 8567634
DOI: 10.1074/jbc.271.4.1884