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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 -
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 -
Proceedings of the National Academy of... Mar 2021Nitrogenases utilize Fe-S clusters to reduce N to NH The large number of Fe sites in their catalytic cofactors has hampered spectroscopic investigations into their...
Nitrogenases utilize Fe-S clusters to reduce N to NH The large number of Fe sites in their catalytic cofactors has hampered spectroscopic investigations into their electronic structures, mechanisms, and biosyntheses. To facilitate their spectroscopic analysis, we are developing methods for incorporating Fe into specific sites of nitrogenase cofactors, and we report herein site-selective Fe labeling of the L-cluster-a carbide-containing, [FeSC] precursor to the Mo nitrogenase catalytic cofactor. Treatment of the isolated L-cluster with the chelator ethylenediaminetetraacetate followed by reconstitution with Fe results in Fe labeling of the terminal Fe sites in high yield and with high selectivity. This protocol enables the generation of L-cluster samples in which either the two terminal or the six belt Fe sites are selectively labeled with Fe. Mössbauer spectroscopic analysis of these samples bound to the nitrogenase maturase NifX reveals differences in the primary coordination sphere of the terminal Fe sites and that one of the terminal sites of the L-cluster binds to H35 of NifX. This work provides molecular-level insights into the electronic structure and biosynthesis of the L-cluster and introduces postbiosynthetic modification as a promising strategy for studies of nitrogenase cofactors.
Topics: Azotobacter vinelandii; Electron Spin Resonance Spectroscopy; Molybdoferredoxin; Nitrogenase; Protein Precursors; Spectroscopy, Mossbauer
PubMed: 33836573
DOI: 10.1073/pnas.2015361118 -
Journal of Applied Microbiology Oct 2011To characterize the complementary production of two types of siderophores in Azotobacter vinelandii.
AIM
To characterize the complementary production of two types of siderophores in Azotobacter vinelandii.
METHODS AND RESULTS
In an iron-insufficient environment, nitrogen-fixing A. vinelandii produces peptidic (azotobactin) and catechol siderophores for iron uptake to be used as a nitrogenase cofactor. Molybdenum, another nitrogenase cofactor, was also found to affect the production level of siderophores. Wild-type cells excreted azotobactin into molybdenum-supplemented and iron-insufficient medium, although catechol siderophores predominate in molybdenum-free environments. Two gene clusters were identified to be involved in the production of azotobactin and catechol siderophores through gene annotation and disruption. Azotobactin-deficient mutant cells produced catechol siderophores under the molybdenum-supplemented and iron-insufficient conditions, whereas catechol siderophore-deficient mutant cells extracellularly secreted excess azotobactin under iron-deficient condition independent of the concentration of molybdenum. This evidence suggests that a complementary siderophore production system exists in A. vinelandii.
CONCLUSIONS
Molybdenum was found to regulate the production level of two types of siderophores. Azotobacter vinelandii cells are equipped with a complementary production system for nitrogen fixation in response to a limited quantity of metals.
SIGNIFICANCE AND IMPACT OF THE STUDY
This is the first study identifying A. vinelandii gene clusters for the biosynthesis of two types of siderophores and clarifying the relationship between them.
Topics: Azotobacter vinelandii; Catechols; Culture Media; Gene Targeting; Genes, Bacterial; Iron; Molybdenum; Multigene Family; Mutation; Peptides; Siderophores
PubMed: 21794033
DOI: 10.1111/j.1365-2672.2011.05109.x -
Biochemistry Feb 2018Photoinduced charge-transfer dynamics and the influence of cluster size on the dynamics were investigated using five iron-sulfur clusters: the 1Fe-4S cluster in...
Photoinduced charge-transfer dynamics and the influence of cluster size on the dynamics were investigated using five iron-sulfur clusters: the 1Fe-4S cluster in Pyrococcus furiosus rubredoxin, the 2Fe-2S cluster in Pseudomonas putida putidaredoxin, the 4Fe-4S cluster in nitrogenase iron protein, and the 8Fe-7S P-cluster and the 7Fe-9S-1Mo FeMo cofactor in nitrogenase MoFe protein. Laser excitation promotes the iron-sulfur clusters to excited electronic states that relax to lower states. The electronic relaxation lifetimes of the 1Fe-4S, 8Fe-7S, and 7Fe-9S-1Mo clusters are on the picosecond time scale, although the dynamics of the MoFe protein is a mixture of the dynamics of the latter two clusters. The lifetimes of the 2Fe-2S and 4Fe-4S clusters, however, extend to several nanoseconds. A competition between reorganization energies and the density of electronic states (thus electronic coupling between states) mediates the charge-transfer lifetimes, with the 2Fe-2S cluster of Pdx and the 4Fe-4S cluster of Fe protein lying at the optimum leading to them having significantly longer lifetimes. Their long lifetimes make them the optimal candidates for long-range electron transfer and as external photosensitizers for other photoactivated chemical reactions like solar hydrogen production. Potential electron-transfer and hole-transfer pathways that possibly facilitate these charge transfers are proposed.
Topics: Azotobacter vinelandii; Bacteria; Bacterial Proteins; Catalytic Domain; Electron Transport; Ferredoxins; Iron-Sulfur Proteins; Models, Molecular; Oxidation-Reduction; Oxidoreductases; Protein Conformation; Pseudomonas putida; Pyrococcus furiosus; Rubredoxins
PubMed: 29303562
DOI: 10.1021/acs.biochem.7b01159 -
Applied and Environmental Microbiology Mar 2022The ubiquitous diazotrophic soil bacterium Azotobacter vinelandii has been extensively studied as a model organism for biological nitrogen fixation (BNF). In A....
The ubiquitous diazotrophic soil bacterium Azotobacter vinelandii has been extensively studied as a model organism for biological nitrogen fixation (BNF). In A. vinelandii, BNF is regulated by the NifL-NifA two-component system, where NifL acts as an antiactivator that tightly controls the activity of the nitrogen fixation-specific transcriptional activator NifA in response to redox, nitrogen, and carbon status. While several studies reported that mutations in A. vinelandii resulted in the deregulation of nitrogenase expression and the release of large quantities of ammonium, knowledge about the specific determinants for this ammonium-excreting phenotype is lacking. In this work, we report that only specific disruptions of lead to large quantities of ammonium accumulated in liquid culture (∼12 mM). The ammonium excretion phenotype is associated solely with deletions of NifL domains combined with the insertion of a promoter sequence in the orientation opposite that of transcription. We further demonstrated that the strength of the inserted promoter could influence the amounts of ammonium excreted by affecting gene expression as an additional requirement for ammonium excretion. These ammonium-excreting mutants significantly stimulate the transfer of fixed nitrogen to rice. This work defines discrete determinants that bring about A. vinelandii ammonium excretion and demonstrates that strains can be generated through simple gene editing to provide promising biofertilizers capable of transferring nitrogen to crops. There is considerable interest in the engineering of ammonium-excreting bacteria for use in agriculture to promote the growth of plants under fixed-nitrogen-limiting conditions. This work defines discrete determinants that bring about A. vinelandii ammonium excretion and demonstrates that strains can be generated through simple gene editing to provide promising biofertilizers capable of transferring nitrogen to crops.
Topics: Ammonium Compounds; Azotobacter vinelandii; Bacterial Proteins; Gene Expression Regulation, Bacterial; Nitrogen Fixation; Nitrogenase
PubMed: 35138932
DOI: 10.1128/AEM.01876-21 -
Microbiology (Reading, England) Aug 2012In Azotobacter vinelandii the two-component GacS/GacA system is required for synthesis of polyhydroxybutyrate (PHB) and of the exopolysaccharide alginate. The RsmA...
In Azotobacter vinelandii the two-component GacS/GacA system is required for synthesis of polyhydroxybutyrate (PHB) and of the exopolysaccharide alginate. The RsmA protein was shown to interact with the alginate biosynthetic algD mRNA, acting as a translational repressor, and GacA was found to activate transcription of the rsmZ1 and rsmZ2 genes that encode small RNAs interacting with RsmA to counteract its repressor activity. The phbBAC operon encodes the enzymes of PHB synthesis and is activated by the transcriptional regulator PhbR. This study shows that GacA is required for transcription of one rsmY and seven rsmZ1-rsmZ7 genes present in the A. vinelandii genome, and that inactivation of rsmA results in increased PHB production. Transcriptional and translational phbR-gusA gene fusions were used to show that the gacA mutation negatively affected the expression of the phbR gene at the translational level. We also demonstrated an in vitro interaction of RsmA with RNAs corresponding to phbB and phbR mRNA leaders, and showed that the stability of phbR and phbB mRNAs is increased in the rsmA mutant. Taken together these results indicate that in A. vinelandii, RsmA post-transcriptionally represses the expression of PhbR.
Topics: Amino Acid Sequence; Azotobacter vinelandii; Bacterial Proteins; Biosynthetic Pathways; Gene Expression Regulation, Bacterial; Hydroxybutyrates; Molecular Sequence Data; Operon; Repressor Proteins; Sequence Alignment; Transcription, Genetic
PubMed: 22609755
DOI: 10.1099/mic.0.059329-0 -
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 -
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 -
Cell Stress & Chaperones May 2017Late embryogenesis abundant (LEA) proteins constitute a large protein family that is closely associated with resistance to abiotic stresses in multiple organisms and...
Late embryogenesis abundant (LEA) proteins constitute a large protein family that is closely associated with resistance to abiotic stresses in multiple organisms and protect cells against drought and other stresses. Azotobacter vinelandii is a soil bacterium that forms desiccation-resistant cysts. This bacterium possesses two genes, here named lea1 and lea2, coding for avLEA1 and avLEA2 proteins, both containing 20-mer motifs characteristic of eukaryotic plant LEA proteins. In this study, we found that disruption of the lea1 gene caused a loss of the cysts' viability after 3 months of desiccation, whereas at 6 months, wild-type or lea2 mutant strain cysts remained viable. Vegetative cells of the lea1 mutant were more sensitive to osmotic stress; cysts developed by this mutant were also more sensitive to high temperatures than cysts or vegetative cells of the wild type or of the lea2 mutant. Expression of lea1 was induced several fold during encystment. In addition, the protective effects of these proteins were assessed in Escherichia coli cells. We found that E. coli cells overexpressing avLEA1 were more tolerant to salt stress than control cells; finally, in vitro analysis showed that avLEA1 protein was able to prevent the freeze thaw-induced inactivation of lactate dehydrogenase. In conclusion, avLEA1 is essential for the survival of A. vinelandii in dry conditions and for protection against hyper-osmolarity, two major stress factors that bacteria must cope with for survival in the environment. This is the first report on the role of bacterial LEA proteins on the resistance of cysts to desiccation.
Topics: Amino Acid Sequence; Arabidopsis; Azotobacter vinelandii; Bacterial Proteins; Databases, Genetic; Escherichia coli; L-Lactate Dehydrogenase; Mutagenesis; Osmotic Pressure; Plant Proteins; Recombinant Proteins; Sequence Alignment; Stress, Physiological; Temperature; Thermotolerance
PubMed: 28258486
DOI: 10.1007/s12192-017-0781-1