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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 -
Biochemistry Oct 2009The molybdenum nitrogenase is responsible for most biological nitrogen fixation, a prokaryotic metabolic process that determines the global biogeochemical cycles of...
The molybdenum nitrogenase is responsible for most biological nitrogen fixation, a prokaryotic metabolic process that determines the global biogeochemical cycles of nitrogen and carbon. Here we describe the trafficking of molybdenum for nitrogen fixation in the model diazotrophic bacterium Azotobacter vinelandii. The genes and proteins involved in molybdenum uptake, homeostasis, storage, regulation, and nitrogenase cofactor biosynthesis are reviewed. Molybdenum biochemistry in A. vinelandii reveals unexpected mechanisms and a new role for iron-sulfur clusters in the sequestration and delivery of molybdenum.
Topics: Amino Acids; Azotobacter vinelandii; Bacterial Proteins; Gases; Gene Expression Regulation, Bacterial; Homeostasis; Kinetics; Models, Biological; Models, Molecular; Molybdenum; Molybdoferredoxin; Nitrogen Fixation; Nitrogenase; Proteins; Soil; Transcription Factors
PubMed: 19772354
DOI: 10.1021/bi901217p -
Scientific Reports Mar 2022Since nitrogenase is irreversibly inactivated within a few minutes after exposure to oxygen, current studies on the heterologous expression of nitrogenase are limited to...
Since nitrogenase is irreversibly inactivated within a few minutes after exposure to oxygen, current studies on the heterologous expression of nitrogenase are limited to anaerobic conditions. This study comprehensively identified genes showing oxygen-concentration-dependent expression only under nitrogen-fixing conditions in Azotobacter vinelandii, an aerobic diazotroph. Among the identified genes, nafU, with an unknown function, was greatly upregulated under aerobic nitrogen-fixing conditions. Through replacement and overexpressing experiments, we suggested that nafU is involved in the maintenance of nitrogenase activity under aerobic nitrogenase activity. Furthermore, heterologous expression of nafU in nitrogenase-producing Escherichia coli increased nitrogenase activity under aerobic conditions by 9.7 times. Further analysis of NafU protein strongly suggested its localization in the inner membrane and raised the possibility that this protein may lower the oxygen concentration inside the cells. These findings provide new insights into the mechanisms for maintaining stable nitrogenase activity under aerobic conditions in A. vinelandii and provide a platform to advance the use of nitrogenase under aerobic conditions.
Topics: Azotobacter; Azotobacter vinelandii; Escherichia coli; Nitrogen; Nitrogen Fixation; Nitrogenase; Oxygen
PubMed: 35264690
DOI: 10.1038/s41598-022-08007-4 -
Microbiology (Reading, England) Jun 2011Azotobacter vinelandii is a soil bacterium that undergoes differentiation to form cysts that are resistant to desiccation. Upon induction of cyst formation, the...
Azotobacter vinelandii is a soil bacterium that undergoes differentiation to form cysts that are resistant to desiccation. Upon induction of cyst formation, the bacterium synthesizes alkylresorcinols that are present in cysts but not in vegetative cells. Alternative sigma factors play important roles in differentiation. In A. vinelandii, AlgU (sigma E) is involved in controlling the loss of flagella upon induction of encystment. We investigated the involvement of the sigma factor RpoS in cyst formation in A. vinelandii. We analysed the transcriptional regulation of the rpoS gene by PsrA, the main regulator of rpoS in Pseudomonas species, which are closely related to A. vinelandii. Inactivation of rpoS resulted in the inability to form cysts resistant to desiccation and to produce cyst-specific alkylresorcinols, whereas inactivation of psrA reduced by 50 % both production of alkylresorcinols and formation of cysts resistant to desiccation. Electrophoretic mobility shift assays revealed specific binding of PsrA to the rpoS promoter region and that inactivation of psrA reduced rpoS transcription by 60 %. These results indicate that RpoS and PsrA are involved in regulation of encystment and alkylresorcinol synthesis in A. vinelandii.
Topics: Azotobacter vinelandii; Bacterial Proteins; DNA-Binding Proteins; Desiccation; Electrophoretic Mobility Shift Assay; Gene Expression Regulation, Bacterial; Resorcinols; Sigma Factor; Transcription Factors
PubMed: 21454367
DOI: 10.1099/mic.0.046268-0 -
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 -
Scientific Reports Jul 2020Bacterial alginate initially consists of 1-4-linked β-D-mannuronic acid residues (M) which can be later epimerized to α-L-guluronic acid (G). The family of AlgE...
Bacterial alginate initially consists of 1-4-linked β-D-mannuronic acid residues (M) which can be later epimerized to α-L-guluronic acid (G). The family of AlgE mannuronan C-5-epimerases from Azotobacter vinelandii has been extensively studied, and three genes putatively encoding AlgE-type epimerases have recently been identified in the genome of Azotobacter chroococcum. The three A. chroococcum genes, here designated AcalgE1, AcalgE2 and AcalgE3, were recombinantly expressed in Escherichia coli and the gene products were partially purified. The catalytic activities of the enzymes were stimulated by the addition of calcium ions in vitro. AcAlgE1 displayed epimerase activity and was able to introduce long G-blocks in the alginate substrate, preferentially by attacking M residues next to pre-existing G residues. AcAlgE2 and AcAlgE3 were found to display lyase activities with a substrate preference toward M-alginate. AcAlgE2 solely accepted M residues in the positions - 1 and + 2 relative to the cleavage site, while AcAlgE3 could accept either M or G residues in these two positions. Both AcAlgE2 and AcAlgE3 were bifunctional and could also catalyze epimerization of M to G. Together, we demonstrate that A. chroococcum encodes three different AlgE-like alginate-modifying enzymes and the biotechnological and biological impact of these findings are discussed.
Topics: Alginates; Amino Acid Sequence; Azotobacter; Azotobacter vinelandii; Bacterial Proteins; Biocatalysis; Carbohydrate Epimerases; Genes, Bacterial; Multigene Family; Sequence Alignment; Substrate Specificity
PubMed: 32719381
DOI: 10.1038/s41598-020-68789-3 -
Microbiology (Reading, England) Jun 2008Azotobacter vinelandii is a nitrogen-fixing soil bacterium that undergoes differentiation to form cysts resistant to desiccation. Upon encystment, this bacterium becomes...
Azotobacter vinelandii is a nitrogen-fixing soil bacterium that undergoes differentiation to form cysts resistant to desiccation. Upon encystment, this bacterium becomes non-motile. As in enteric bacteria, motility in A. vinelandii occurs through the use of peritrichous flagella. Pseudomonas aeruginosa, a phylogenetically close relative of A. vinelandii, possesses a single polar flagellum. The FlhDC proteins are the master regulators of flagella and motility in enterobacteria, whereas FleQ is the master regulator in P. aeruginosa, and it is under AlgU (sigmaE) negative control. At present, nothing is known about the organization and expression of flagella genes in A. vinelandii. Here, we identified the flagella gene cluster of this bacterium. Homologues of the master regulatory genes flhDC and fleQ are present in A. vinelandii. Inactivation of flhDC, but not fleQ, impaired flagella biogenesis and motility. We present evidence indicating that a negative effect of the AlgU sigma factor on flhDC expression causes loss of motility in A. vinelandii, and that CydR (a homologue of Fnr) is under AlgU control and has a negative effect on flhDC expression. Taken together, these results suggest the existence of a cascade consisting of AlgU and CydR that negatively controls expression of flhDC; the results also suggest that the block in flagella synthesis under encystment conditions centres on flhDC repression by the AlgU-CydR cascade.
Topics: Azotobacter vinelandii; Bacterial Proteins; Base Sequence; Binding Sites; Flagella; Gene Expression Regulation, Bacterial; Gene Silencing; Genes, Bacterial; Genetic Complementation Test; Genome, Bacterial; Molecular Sequence Data; Repressor Proteins; Sequence Alignment
PubMed: 18524926
DOI: 10.1099/mic.0.2008/017665-0 -
Scientific Reports Apr 2017Azotobacter vinelandii, a strict aerobic, nitrogen fixing bacterium in the Pseudomonadaceae family, exhibits a preferential use of acetate over glucose as a carbon...
Azotobacter vinelandii, a strict aerobic, nitrogen fixing bacterium in the Pseudomonadaceae family, exhibits a preferential use of acetate over glucose as a carbon source. In this study, we show that GluP (Avin04150), annotated as an H-coupled glucose-galactose symporter, is the glucose transporter in A. vinelandii. This protein, which is widely distributed in bacteria and archaea, is uncommon in Pseudomonas species. We found that expression of gluP was under catabolite repression control thorugh the CbrA/CbrB and Crc/Hfq regulatory systems, which were functionally conserved between A. vinelandii and Pseudomonas species. While the histidine kinase CbrA was essential for glucose utilization, over-expression of the Crc protein arrested cell growth when glucose was the sole carbon source. Crc and Hfq proteins from either A. vinelandii or P. putida could form a stable complex with an RNA A-rich Hfq-binding motif present in the leader region of gluP mRNA. Moreover, in P. putida, the gluP A-rich Hfq-binding motif was functional and promoted translational inhibition of a lacZ reporter gene. The fact that gluP is not widely distributed in the Pseudomonas genus but is under control of the CbrA/CbrB and Crc/Hfq systems demonstrates the relevance of these systems in regulating metabolism in the Pseudomonadaceae family.
Topics: Azotobacter vinelandii; Bacterial Proteins; Catabolite Repression; Glucose; Histidine Kinase; Host Factor 1 Protein; Monosaccharide Transport Proteins; Pseudomonas
PubMed: 28404995
DOI: 10.1038/s41598-017-00980-5 -
Journal of Bacteriology Dec 2000Azotobacter vinelandii produces the exopolysaccharide alginate, which is essential for its differentiation to desiccation-resistant cysts. In different bacterial...
Azotobacter vinelandii produces the exopolysaccharide alginate, which is essential for its differentiation to desiccation-resistant cysts. In different bacterial species, the alternative sigma factor sigma(E) regulates the expression of functions related to the extracytoplasmic compartments. In A. vinelandii and Pseudomonas aeruginosa, the sigma(E) factor (AlgU) is essential for alginate production. In both bacteria, the activity of this sigma factor is regulated by the product of the mucA, mucB, mucC, and mucD genes. In this work, we studied the transcriptional regulation of the A. vinelandii algU-mucABCD gene cluster, as well as the role of the mucA and mucC gene products in alginate production. Our results show the existence of AlgU autoregulation and show that both MucA and MucC play a negative role in alginate production.
Topics: Alginates; Azotobacter vinelandii; Bacterial Proteins; Base Sequence; Carbohydrate Dehydrogenases; DNA, Bacterial; Escherichia coli; Gene Expression Regulation, Bacterial; Glucuronic Acid; Heat-Shock Proteins; Hexuronic Acids; Molecular Sequence Data; Multigene Family; Mutagenesis; Serine Endopeptidases; Sigma Factor; Transcription Factors; Transcription, Genetic; Transcriptional Activation
PubMed: 11073894
DOI: 10.1128/JB.182.23.6550-6556.2000 -
European Journal of Biochemistry Jul 1992The 10 C-terminal residues are not visible in the crystal structure of lipoamide dehydrogenase from Azotobacter vinelandii, but can be observed in the crystal structures...
The 10 C-terminal residues are not visible in the crystal structure of lipoamide dehydrogenase from Azotobacter vinelandii, but can be observed in the crystal structures of the lipoamide dehydrogenases from Pseudomonas putida and Pseudomonas fluorescens. In these structures, the C-terminus folds back towards the active site and is involved in interactions with the other subunit. The function of the C-terminus of lipoamide dehydrogenase from A. vinelandii was studied by deletion of 5, 9 and 14 residues, respectively. Deletion of the last 5 residues does not influence the catalytic properties and conformational stability (thermoinactivation and unfolding by guanidinium hydrochloride). Removal of 9 residues results in an enzyme (enzyme delta 9) showing decreased conformational stability and high sensitivity toward inhibition by NADH. These features are even more pronounced after deletion of 14 residues (enzyme delta 14). In addition Tyr16, conserved in all lipoamide dehydrogenases sequenced thus far, and shown from the other structures to be likely to be involved in subunit interaction, was replaced by Phe and Ser. Mutation of Tyr16 also results in a strongly increased sensitivity toward inhibition by NADH. The conformational stability of both Tyr16-mutated enzymes is comparable to enzyme delta 9. The results strongly indicate that a hydrogen bridge between tyrosine of one subunit (Tyr16 in the A. vinelandii sequence) and histidine of the other subunit (His470 in the A. vinelandii sequence), exists in the A. vinelandii enzyme. In the delta 9 and delta 14 enzymes this interaction is abolished. It is concluded that this interaction mediates the redox properties of the FAD via the conformation of the C-terminus containing residues 450-470.
Topics: Azotobacter vinelandii; DNA Mutational Analysis; Dihydrolipoamide Dehydrogenase; Genes, Bacterial; Kinetics; Oxidation-Reduction; Protein Conformation; Restriction Mapping; Spectrometry, Fluorescence; Spectrum Analysis; Structure-Activity Relationship; Tryptophan
PubMed: 1633805
DOI: 10.1111/j.1432-1033.1992.tb17076.x