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Applied and Environmental Microbiology Sep 2000The activity of nitrogenase in the nitrogen-fixing bacterium Azotobacter vinelandii grown diazotrophically under aerobic conditions is generally considered to be...
The activity of nitrogenase in the nitrogen-fixing bacterium Azotobacter vinelandii grown diazotrophically under aerobic conditions is generally considered to be protected against O(2) by a high respiration rate. In this work, we have shown that a high rate of respiration is not the prevailing mechanism for nitrogenase protection in A. vinelandii grown in phosphate-limited nitrogen-free chemostat culture. Instead, the formation of alginate appeared to play a decisive role in protecting the nitrogenase that is required for cell growth in this culture. Depending on the O(2) tension and cell growth rate, the formation rate and composition of alginate released into the culture broth varied significantly. Furthermore, transmission electron microscopic analysis of cell morphology and the cell surface revealed the existence of an alginate capsule on the surface of A. vinelandii. The composition, thickness, and compactness of this alginate capsule also varied significantly. In general, increasing O(2) tension led to the formation of alginate with a higher molecular weight and a greater L-guluronic acid content. The alginate capsule was accordingly thicker and more compact. In addition, the formation of the alginate capsule was found to be strongly affected by the shear rate in a bioreactor. Based on these experimental results, it is suggested that the production of alginate, especially the formation of an alginate capsule on the cell surface, forms an effective barrier for O(2) transfer into the cell. It is obviously the quality, not the quantity, of alginate that is decisive for the protection of nitrogenase.
Topics: Alginates; Azotobacter vinelandii; Bacterial Capsules; Bioreactors; Culture Media; Glucuronic Acid; Hexuronic Acids; Nitrogenase; Oxygen Consumption; Phosphates; Surface Properties
PubMed: 10966426
DOI: 10.1128/AEM.66.9.4037-4044.2000 -
Journal of Bacteriology May 2009During encystment of Azotobacter vinelandii, a family of alkylresorcinols (ARs) and alkylpyrones (APs) are synthesized. In the mature cyst, these lipids replace the...
Isolation and characterization of Azotobacter vinelandii mutants impaired in alkylresorcinol synthesis: alkylresorcinols are not essential for cyst desiccation resistance.
During encystment of Azotobacter vinelandii, a family of alkylresorcinols (ARs) and alkylpyrones (APs) are synthesized. In the mature cyst, these lipids replace the membrane phospholipids and are also components of the layers covering the cyst. In this study, A. vinelandii strains unable to synthesize ARs were isolated after mini-Tn5 mutagenesis. Cloning and nucleotide sequencing of the affected loci revealed the presence of the transposons within the arsA gene of the previously reported arsABCD gene cluster, which encodes a type I fatty acid synthase. A mutant strain (SW-A) carrying an arsA mutation allowing transcription of arsBCD was constructed and shown to be unable to produce ARs, indicating that the ArsA protein is essential for the synthesis of these phenolic lipids. Transcription of arsA was induced 200-fold in cells undergoing encystment, but only 14-fold in aged cultures of A. vinelandii, in accordance with AR synthesis and cyst formation percentages under the two conditions. Although it was previously reported that the inactivation of arsB abolishes AR synthesis and results in a failure in encystment, the arsA mutants were able to form cysts resistant to desiccation. These data indicate that ARs play a structural role in the exine layer of the cysts, but they are not essential for either cyst formation or for desiccation resistance.
Topics: Azotobacter vinelandii; Bacterial Proteins; DNA Transposable Elements; Desiccation; Fatty Acid Synthases; Gene Expression Profiling; Gene Knockout Techniques; Gene Order; Microbial Viability; Mutagenesis, Insertional; Mutation; Operon; Resorcinols; Spores, Bacterial
PubMed: 19270099
DOI: 10.1128/JB.01575-08 -
The Journal of Biological Chemistry Nov 2014The bacterium Azotobacter vinelandii produces a family of seven secreted and calcium-dependent mannuronan C-5 epimerases (AlgE1-7). These epimerases are responsible for...
The bacterium Azotobacter vinelandii produces a family of seven secreted and calcium-dependent mannuronan C-5 epimerases (AlgE1-7). These epimerases are responsible for the epimerization of β-D-mannuronic acid (M) to α-L-guluronic acid (G) in alginate polymers. The epimerases display a modular structure composed of one or two catalytic A-modules and from one to seven R-modules having an activating effect on the A-module. In this study, we have determined the NMR structure of the three individual R-modules from AlgE6 (AR1R2R3) and the overall structure of both AlgE4 (AR) and AlgE6 using small angle x-ray scattering. Furthermore, the alginate binding ability of the R-modules of AlgE4 and AlgE6 has been studied with NMR and isothermal titration calorimetry. The AlgE6 R-modules fold into an elongated parallel β-roll with a shallow, positively charged groove across the module. Small angle x-ray scattering analyses of AlgE4 and AlgE6 show an overall elongated shape with some degree of flexibility between the modules for both enzymes. Titration of the R-modules with defined alginate oligomers shows strong interaction between AlgE4R and both oligo-M and MG, whereas no interaction was detected between these oligomers and the individual R-modules from AlgE6. A combination of all three R-modules from AlgE6 shows weak interaction with long M-oligomers. Exchanging the R-modules between AlgE4 and AlgE6 resulted in a novel epimerase called AlgE64 with increased G-block forming ability compared with AlgE6.
Topics: Alginates; Amino Acid Sequence; Azotobacter vinelandii; Bacterial Proteins; Binding Sites; Calcium-Binding Proteins; Calorimetry; Carbohydrate Epimerases; Catalysis; Escherichia coli; Glucuronic Acid; Hexuronic Acids; Magnetic Resonance Spectroscopy; Molecular Sequence Data; Plasmids; Protein Engineering; Protein Structure, Secondary; Protein Structure, Tertiary; Scattering, Radiation; Sequence Homology, Amino Acid; X-Rays
PubMed: 25266718
DOI: 10.1074/jbc.M114.567008 -
Microbiology (Reading, England) Oct 2019is a soil bacterium that is able to synthesize poly-β-hydroxybutyrate (PHB), a polymer used to produce biodegradable plastic. PHB is stored in the cytoplasm as...
is a soil bacterium that is able to synthesize poly-β-hydroxybutyrate (PHB), a polymer used to produce biodegradable plastic. PHB is stored in the cytoplasm as granules surrounded by several proteins such as the major phasin PhbP, PHB synthase and PHB depolymerase, among others. Many studies have reported the presence of membrane proteins on PHB granules due to contamination during the polymer extraction procedures. Previously, the outer membrane protein I (OprI) was detected on the polymer granules in . In this study, by using random transposon mutagenesis, we identified that a mutation in the gene diminished PHB accumulation in on solid medium. Electron microscopy confirmed the low polymer production by the mutant. Analysis of PHB granules by Tricine-SDS-PAGE revealed that the absence of OprI affected the protein profile of the granules, suggesting that OprI could have a structural role in . Thus, some membrane proteins on PHB granules may not be artefacts as previously described.
Topics: Amino Acid Sequence; Azotobacter vinelandii; Bacterial Proteins; Biopolymers; Culture Media; Cytoplasmic Granules; Hydroxybutyrates; Lipoproteins; Mutation; Polyesters; Protein Binding
PubMed: 31329095
DOI: 10.1099/mic.0.000837 -
Microbiological Research Sep 2018In bacteria, the 5'-end-dependent RNA degradation is triggered by the RNA pyrophosphohydrolase RppH converting tri/diphosphate to monophosphate transcripts. This study...
In bacteria, the 5'-end-dependent RNA degradation is triggered by the RNA pyrophosphohydrolase RppH converting tri/diphosphate to monophosphate transcripts. This study shows that in the soil bacterium Azotobacter vinelandii, inactivation of rppH gene negatively affected the production of bioplastic poly-β-hydroxybutyrate (PHB) by reducing the expression at the translational level of PhbR, the specific transcriptional activator of the phbBAC biosynthetic operon. The effect of RppH on the translation of phbR seemed to be exerted through the translational repressor RsmA, as the inactivation of rsmA in the rppH mutant restored the phbR expression. Interestingly, in Escherichia coli inactivation of rppH also affected the expression of CsrA, the RsmA homolog. The level of the csrA transcript was higher and more stable in the E. coli rppH mutant than in the wild type strain. Additionally, and in contrast to the csrA mutants that are known to have a defective swimming phenotype, the E. coli rppH mutant showed a hyper-swimming phenotype that was suppressed by a csrA mutation, and the AvRppH restored to wild type level the swimming phenotype to the E. coli rppH mutant. We propose that in both A. vinelandii and E. coli, RppH activity plays a role in the expression of the translational regulator protein RsmA/CsrA.
Topics: Acid Anhydride Hydrolases; Azotobacter vinelandii; Escherichia coli; Escherichia coli Proteins; Gene Deletion; Gene Expression Regulation, Bacterial; Protein Biosynthesis; RNA-Binding Proteins; Repressor Proteins
PubMed: 30031486
DOI: 10.1016/j.micres.2018.05.013 -
Extremophiles : Life Under Extreme... Nov 2019A cold-adapted monomeric isocitrate dehydrogenase from a psychrophilic bacterium, Psychromonas marina (PmIDH), showed a high degree of amino acid sequential identity...
Effects of the substituted amino acid residues on the thermal properties of monomeric isocitrate dehydrogenases from a psychrophilic bacterium, Psychromonas marina, and a mesophilic bacterium, Azotobacter vinelandii.
A cold-adapted monomeric isocitrate dehydrogenase from a psychrophilic bacterium, Psychromonas marina (PmIDH), showed a high degree of amino acid sequential identity (64%) to a mesophilic one from a mesophilic bacterium, Azotobacter vinelandii (AvIDH). In this study, eight corresponding amino acid residues were substituted between them by site-directed mutagenesis, and several thermal properties of the mutated IDHs were examined. In the PmIDH mutants, PmL735F, substituted Leu735 of PmIDH by the corresponding Phe of AvIDH, showed higher specific activity and thermostability of activity than wild-type PmIDH, while the H600Y and N741P mutations of PmIDH resulted in decreased specific activity and thermostability of activity. On the other hand, among the AvIDH mutants, AvP718T showed lower optimum temperature and thermostability of activity than wild-type AvIDH. In PmIDH variously combined the H600Y, L735F and N741P mutations, PmH600YL735F, including the H600Y and L735F mutations, showed higher specific activity than PmH600Y and similar optimum temperature and thermostability of activity to PmH600Y. Furthermore, PmL735FN741P exhibited higher specific activity and thermostability of activity than PmN741P. These results indicated that the effects of the three mutations of PmIDH are additive on the specific activity of both PmH600YL735F and PmL735FN741P and on thermostability of PmL735FN741P.
Topics: Amino Acid Sequence; Amino Acids; Azotobacter vinelandii; Enzyme Stability; Gammaproteobacteria; Isocitrate Dehydrogenase; Isocitrates; Kinetics
PubMed: 31595369
DOI: 10.1007/s00792-019-01137-0 -
Applied and Environmental Microbiology Jan 2016In this study, we performed a detailed characterization of the siderophore metabolome, or "chelome," of the agriculturally important and widely studied model organism...
In this study, we performed a detailed characterization of the siderophore metabolome, or "chelome," of the agriculturally important and widely studied model organism Azotobacter vinelandii. Using a new high-resolution liquid chromatography-mass spectrometry (LC-MS) approach, we found over 35 metal-binding secondary metabolites, indicative of a vast chelome in A. vinelandii. These include vibrioferrin, a siderophore previously observed only in marine bacteria. Quantitative analyses of siderophore production during diazotrophic growth with different sources and availabilities of Fe showed that, under all tested conditions, vibrioferrin was present at the highest concentration of all siderophores and suggested new roles for vibrioferrin in the soil environment. Bioinformatic searches confirmed the capacity for vibrioferrin production in Azotobacter spp. and other bacteria spanning multiple phyla, habitats, and lifestyles. Moreover, our studies revealed a large number of previously unreported derivatives of all known A. vinelandii siderophores and rationalized their origins based on genomic analyses, with implications for siderophore diversity and evolution. Together, these insights provide clues as to why A. vinelandii harbors multiple siderophore biosynthesis gene clusters. Coupled with the growing evidence for alternative functions of siderophores, the vast chelome in A. vinelandii may be explained by multiple, disparate evolutionary pressures that act on siderophore production.
Topics: Azotobacter vinelandii; Bacterial Proteins; Biosynthetic Pathways; Chromatography, Liquid; Mass Spectrometry; Metabolome; Siderophores
PubMed: 26452553
DOI: 10.1128/AEM.03160-15 -
Acta Crystallographica. Section F,... Nov 2021Azotobacter vinelandii is a model diazotroph and is the source of most nitrogenase material for structural and biochemical work. Azotobacter can grow in...
Azotobacter vinelandii is a model diazotroph and is the source of most nitrogenase material for structural and biochemical work. Azotobacter can grow in above-atmospheric levels of oxygen, despite the sensitivity of nitrogenase activity to oxygen. Azotobacter has many iron-sulfur proteins in its genome, which were identified as far back as the 1960s and probably play roles in the complex redox chemistry that Azotobacter must maintain when fixing nitrogen. Here, the 2.1 Å resolution crystal structure of the [2Fe-2S] protein I (Shethna protein I) from A. vinelandii is presented, revealing a homodimer with the [2Fe-2S] cluster coordinated by the surrounding conserved cysteine residues. It is similar to the structure of the thioredoxin-like [2Fe-2S] protein from Aquifex aeolicus, including the positions of the [2Fe-2S] clusters and conserved cysteine residues. The structure of Shethna protein I will provide information for understanding its function in relation to nitrogen fixation and its evolutionary relationships to other ferredoxins.
Topics: Azotobacter vinelandii; Crystallography, X-Ray; Ferredoxins; Iron-Sulfur Proteins; Nitrogenase
PubMed: 34726179
DOI: 10.1107/S2053230X21009936 -
ELife Jul 2022The nitrogenase Fe protein mediates ATP-dependent electron transfer to the nitrogenase MoFe protein during nitrogen fixation, in addition to catalyzing MoFe...
The nitrogenase Fe protein mediates ATP-dependent electron transfer to the nitrogenase MoFe protein during nitrogen fixation, in addition to catalyzing MoFe protein-independent substrate (CO) reduction and facilitating MoFe protein metallocluster biosynthesis. The precise role(s) of the Fe protein FeS cluster in some of these processes remains ill-defined. Herein, we report crystallographic data demonstrating ATP-dependent chalcogenide exchange at the FeS cluster of the nitrogenase Fe protein when potassium selenocyanate is used as the selenium source, an unexpected result as the Fe protein cluster is not traditionally perceived as a site of substrate binding within nitrogenase. The observed chalcogenide exchange illustrates that this FeS cluster is capable of core substitution reactions under certain conditions, adding to the Fe protein's repertoire of unique properties.
Topics: Adenosine Triphosphate; Azotobacter vinelandii; Cyanates; Molybdoferredoxin; Nitrogenase; Oxidoreductases; Protein Conformation; Selenium Compounds
PubMed: 35904245
DOI: 10.7554/eLife.79311 -
Molecular Microbiology Mar 2011Nitrogen fixation by the free-living organism Azotobacter vinelandii can occur through the activity of three different systems that are genetically distinct but...
Nitrogen fixation by the free-living organism Azotobacter vinelandii can occur through the activity of three different systems that are genetically distinct but mechanistically related. A combination of bioinformatic and biochemical-genetic studies has revealed that at least 82 different genes are likely to be associated with the formation and regulation of these systems. Studies performed over many years have established that cross-talk occurs between the various nitrogen fixation systems, and that expression and fine-tuning of their activities are integrated with overall cellular physiology. Martinez-Noel and co-workers now report another newly discovered aspect of the process. Evidence is presented to suggest that a nitrogen fixation-specific paralogue of ClpX is used to control the accumulation of proteins involved in formation of a metal-sulphur cluster that provides a nitrogenase active site. The intriguing aspect of this work is that it indicates that the nitrogen fixation-associated ClpX must recruit ClpP, for which a paralogue is not duplicated within any of the nitrogen fixation regions of the genome, to achieve its function related to nitrogen fixation. Inspection of the A. vinelandii genome indicates that such recruitment of cellular housekeeping components is a common feature used to integrate nitrogen fixation with global cellular physiology.
Topics: Azotobacter vinelandii; Bacterial Proteins; Gene Expression Regulation, Bacterial; Nitrogen Fixation; Nitrogenase
PubMed: 21338415
DOI: 10.1111/j.1365-2958.2011.07541.x