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Journal of Bacteriology Jan 1991Strains of aerobic, microaerobic, nonsymbiotic, and symbiotic dinitrogen-fixing bacteria were screened for the presence of alternative nitrogenase (N2ase) genes by DNA...
Strains of aerobic, microaerobic, nonsymbiotic, and symbiotic dinitrogen-fixing bacteria were screened for the presence of alternative nitrogenase (N2ase) genes by DNA hybridization between genomic DNA and DNA encoding structural genes for components 1 of three different enzymes. A nifDK gene probe was used as a control to test for the presence of the commonly occurring Mo-Fe N2ase, a vnfDGK gene probe was used to show the presence of V-Fe N2ase, and an anfDGK probe was used to detect Fe N2ase. Hitherto, all three enzymes have been identified in Azotobacter vinelandii OP, and all but the Fe N2ase are present in Azotobacter chroococcum ATCC 4412 (MCD1). Mo-Fe N2ase and V-Fe N2ase structural genes only were confirmed in this strain and in two other strains of A. chroococcum (ATCC 480 and ATCC 9043). A similar pattern was observed with Azotobacter beijerinckii ATCC 19360 and Azotobacter nigricans ATCC 35009. Genes for all three systems are apparently present in two strains of Azotobacter paspali (ATCC 23367 and ATCC 23833) and also in Azomonas agilis ATCC 7494. There was no good evidence for the existence of any genes other than Mo-Fe N2ase structural genes in several Rhizobium meliloti strains, cowpea Rhizobium strain 32H1, or Bradyrhizobium japonicum. Nitrogenase and nitrogenase genes in Azorhizobium caulinodans behaved in an intermediate fashion, showing (i) the formation of ethane from acetylene under Mo starvation, a characteristic of alternative nitrogenases, and (ii) a surprising degree of cross-hybridization to the vnfDGK, but not the anfDGK, probe. vnfDGK- and anfDGK-like sequences were not detected in two saccharolytic Pseudomonas species or Azospirillum brasilense Sp7. The occurrence of alternative N2ases seems restricted to members of the family Azotobacteraceae among the aerobic and microaerobic diazotrophs tested, suggesting that an ability to cope with O2 when fixing N2 may be an important factor influencing the distribution of alternative nitrogenases.
Topics: Azotobacter; DNA Probes; DNA, Bacterial; Escherichia coli; Genes, Bacterial; Gram-Negative Aerobic Bacteria; Nitrogen Fixation; Nitrogenase; Nucleic Acid Hybridization; Pseudomonas; Rhizobium; Species Specificity
PubMed: 1987127
DOI: 10.1128/jb.173.1.365-371.1991 -
ELife Feb 2023The planetary biosphere is powered by a suite of key metabolic innovations that emerged early in the history of life. However, it is unknown whether life has always...
The planetary biosphere is powered by a suite of key metabolic innovations that emerged early in the history of life. However, it is unknown whether life has always followed the same set of strategies for performing these critical tasks. Today, microbes access atmospheric sources of bioessential nitrogen through the activities of just one family of enzymes, nitrogenases. Here, we show that the only dinitrogen reduction mechanism known to date is an ancient feature conserved from nitrogenase ancestors. We designed a paleomolecular engineering approach wherein ancestral nitrogenase genes were phylogenetically reconstructed and inserted into the genome of the diazotrophic bacterial model, enabling an integrated assessment of both in vivo functionality and purified nitrogenase biochemistry. Nitrogenase ancestors are active and robust to variable incorporation of one or more ancestral protein subunits. Further, we find that all ancestors exhibit the reversible enzymatic mechanism for dinitrogen reduction, specifically evidenced by hydrogen inhibition, which is also exhibited by extant nitrogenase isozymes. Our results suggest that life may have been constrained in its sampling of protein sequence space to catalyze one of the most energetically challenging biochemical reactions in nature. The experimental framework established here is essential for probing how nitrogenase functionality has been shaped within a dynamic, cellular context to sustain a globally consequential metabolism.
Topics: Nitrogenase; Nitrogen Fixation; Azotobacter vinelandii; Amino Acid Sequence; Nitrogen
PubMed: 36799917
DOI: 10.7554/eLife.85003 -
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 -
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 -
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 -
FEMS Microbiology Reviews Oct 2000The hypothesis of respiratory protection, originally formulated on the basis of results obtained with Azotobacter species, postulates that consumption of O(2) at the... (Review)
Review
The hypothesis of respiratory protection, originally formulated on the basis of results obtained with Azotobacter species, postulates that consumption of O(2) at the surface of diazotrophic prokaryotes protects nitrogenase from inactivation by O(2). Accordingly, it is assumed that, at increased ambient O(2) concentrations, nitrogenase activity depends on increased activities of a largely uncoupled respiratory electron transport system. The present review compiles evidence indicating that cellular O(2) consumption as well as both the activity and the formation of the respiratory system of Azotobacter vinelandii are controlled by the C/N ratio, that is to say the ratio at which the organism consumes the substrate (i.e. the source of carbon, reducing equivalents and ATP) per source of compound nitrogen. The maximal respiratory capacity which can be attained at increased C/N ratios, however, is controlled, within limits, by the ambient O(2) concentration. When growth becomes N-limited at increased C/N ratios, cells synthesize nitrogenase and fix N(2). Under these diazotrophic conditions, cellular O(2) consumption remains constant at a level controlled by the O(2) concentration. Control by O(2) has been studied on the basis of both whole cell respiration and defined segments of the respiratory electron transport chain. The results demonstrate that the effect of O(2) on the respiratory system is restricted to the lower range of O(2) concentrations up to about 70 microM. Nevertheless, azotobacters are able to grow diazotrophically at dissolved O(2) concentrations of up to about 230 microM indicating that respiratory protection is not warranted at increased ambient O(2) concentrations. This conclusion is supported and extended by a number of results largely excluding an obvious relationship between nitrogenase activity and the actual rate of cellular O(2) consumption. On the basis of theoretical calculations, it is assumed that the rate of O(2) diffusion into the cells is not significantly affected by respiration. All of these results lead to the conclusion that, in the protection of nitrogenase from O(2) damage, O(2) consumption at the cell surface is less effective than generally assumed. It is proposed that alternative factors like the supply of ATP and reducing equivalents are more important.
Topics: Azotobacter; Carbon; Electron Transport; Nitrogen; Nitrogen Fixation; Nitrogenase; Oxygen; Oxygen Consumption; Substrate Specificity
PubMed: 10978541
DOI: 10.1111/j.1574-6976.2000.tb00545.x -
Journal of Bacteriology Aug 1964Cohen, Gary H. (University of Vermont, Burlington), and Donald B. Johnstone. Extracellular polysaccharides of Azotobacter vinelandii. J. Bacteriol. 88:329-338....
Cohen, Gary H. (University of Vermont, Burlington), and Donald B. Johnstone. Extracellular polysaccharides of Azotobacter vinelandii. J. Bacteriol. 88:329-338. 1964.-Extracellular polysaccharides synthetized by Azotobacter vinelandii strains 155, 102, and 3A were shown to be carboxylic acid heteropolysaccharides of apparent high molecular weight. Cells were grown in a nitrogen-free, mineral broth medium with 2% sucrose. Extracellular slime was recovered by centrifugation and purified by repeated alcohol precipitation and Sevag deproteinization. Capsular polysaccharide was recovered from washed cells by mild alkaline digestion. Methods of isolation and purification appeared to provide polysaccharide showing no evidence of heterogeneity when examined by chemical and physical methods. Infrared analysis of purified slime from the three strains suggested fundamental structural similarities. Colorimetric, paper chromatographic, and enzymatic analyses on both intact and acid-hydrolyzed slime polysaccharide indicated that the polymers contained in common galacturonic acid, [alpha] d-glucose, and rhamnose at a ratio of approximately 43:2:1, as well as a hexuronic acid lactone, probably mannurono-lactone. However, as shown by chemical and infrared analysis, minor differences did exist; namely, slime from strain 155 and 102 contained o-acetyl groups, whereas slime from strain 3A contained none. A sialic acid-like component (1.5% of dry weight of the polysaccharide, calculated as N-acetyl neuraminic acid), was found only in the slime of strain 155. Capsular polysaccharide composition closely resembled that for slime. It is of interest that the major slime components were identical whether the energy source provided for the cells was sucrose, glucose, fructose, or ethanol.
Topics: Azotobacter; Azotobacter vinelandii; Centrifugation; Chemical Phenomena; Chemistry; Chromatography; Chromatography, Paper; Culture Media; Fructose; Glucose; Polysaccharides; Polysaccharides, Bacterial; Research; Rhamnose; Sucrose
PubMed: 14203348
DOI: 10.1128/jb.88.2.329-338.1964 -
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 -
Journal of Applied Microbiology Jan 2013To examine tannic acid (TA) utilization capacity by nitrogen-fixing bacteria, Azotobacter sp. SSB81, and identify the intermediate products during biotransformation....
AIMS
To examine tannic acid (TA) utilization capacity by nitrogen-fixing bacteria, Azotobacter sp. SSB81, and identify the intermediate products during biotransformation. Another aim of this work is to investigate the effects of TA on major biopolymers like extracellular polysaccharide (EPS) and polyhydroxybutyrate (PHB) synthesis.
METHODS AND RESULTS
Tannic acid utilization and tolerance capacity of the strain was determined according to CLSI method. Intermediate products were identified using high-performance liquid chromatography, LC-MS/MS and (1) H NMR analysis. Intermediates were quantified by multiple reactions monitoring using LC-MS/MS. The strain was able to tolerate a high level of TA and utilized through enzymatic system. Growth of Azotobacter in TA-supplemented medium was characterized by an extended lag phase and decreased growth rate. Presence of TA catalytic enzymes as tannase, polyphenol oxidase (PPO) and phenol decarboxylase was confirmed in cell lysate using their specific substrates. PPO activity was more prominent in TA-supplemented mineral medium after 48 h of growth when gallic to ellagic acid (EA) reversible reaction was remarkable. Phase contrast and scanning electron microscopic analysis revealed elongated and irregular size of Azotobacter cells in response to TA. (1) H NMR analysis indicated that TA was transformed into gallic acid (GA), EA and pyrogallol. Biopolymer (EPS and PHB) production was decreased several folds in the presence of TA compared with cells grown in only glucose medium.
CONCLUSIONS
This is the first evidence on the biotransformation of TA by Azotobacter and also elevated level of EA production from gallotannins. Azotobacter has developed the mechanism to utilize TA for their carbon and energy source.
SIGNIFICANCE AND IMPACT OF THE STUDY
The widespread occurrence and exploitation of Azotobacter sp. strain SSB81 in agricultural and forest soil have an additional advantage to utilize the soil-accumulated TA and detoxifies the allelopathic effect of constant accumulated TA in soil.
Topics: Azotobacter; Biotransformation; Carboxy-Lyases; Carboxylic Ester Hydrolases; Catechol Oxidase; Ellagic Acid; Gallic Acid; Hydroxybenzoates; Hydroxybutyrates; Nitrogen Fixation; Polysaccharides; Pyrogallol; Soil Microbiology; Tannins
PubMed: 23035941
DOI: 10.1111/jam.12030 -
The Journal of Biological Chemistry Jul 1967
Topics: Azotobacter; Chemical Phenomena; Chemistry; Flavin Mononucleotide; Fluorescence; Molecular Weight; Oxidation-Reduction; Photomicrography; Proteins; Spectrophotometry; Sulfhydryl Compounds; Ultracentrifugation
PubMed: 6029442
DOI: No ID Found