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Journal of Bacteriology Jul 1989Ferric reductase activity was examined in Azotobacter vinelandii and was found to be located in the cytoplasm. The specific activities of soluble cell extracts were not...
Ferric reductase activity was examined in Azotobacter vinelandii and was found to be located in the cytoplasm. The specific activities of soluble cell extracts were not affected by the iron concentration of the growth medium; however, activity was inhibited by the presence of Zn2+ during cell growth and also by the addition of Zn2+ to the enzyme assays. Intracellular Fe2+ levels were lower and siderophore production was increased in Zn2+-grown cells. The ferric reductase was active under aerobic conditions, had an optimal pH of approximately 7.5, and required flavin mononucleotide and Mg2+ for maximum activity. The enzyme utilized NADH to reduce iron supplied as a variety of iron chelates, including the ferrisiderophores of A. vinelandii. The enzyme was purified by conventional protein purification techniques, and the final preparation consisted of two major proteins with molecular weights of 44,600 and 69,000. The apparent Km values of the ferric reductase for Fe3+ (supplied as ferric citrate) and NADH were 10 and 15.8 microM, respectively, and the data for the enzyme reaction were consistent with Ping Pong Bi Bi kinetics. The approximate Ki values resulting from inhibition of the enzyme by Zn2+, which was a hyperbolic (partial) mixed-type inhibitor, were 25 microM with respect to iron and 1.7 microM with respect to NADH. These results suggested that ferric reductase activity may have a regulatory role in the processes of iron assimilation in A. vinelandii.
Topics: Azotobacter; Bacterial Proteins; Enzyme Activation; FMN Reductase; Iron Chelating Agents; Kinetics; Molecular Weight; Oxidoreductases; Siderophores; Zinc
PubMed: 2525550
DOI: 10.1128/jb.171.7.4031-4037.1989 -
Journal of Bacteriology Jan 1966Parker, Laura T. (Louisiana State University, Baton Rouge), and M. D. Socolofsky. Central body of the Azotobacter cyst. J. Bacteriol. 91:297-303. 1966.-Sodium citrate...
Parker, Laura T. (Louisiana State University, Baton Rouge), and M. D. Socolofsky. Central body of the Azotobacter cyst. J. Bacteriol. 91:297-303. 1966.-Sodium citrate was found to effect extensive rupture of cyst coats of Azotobacter vinelandii. By filtering a citrate-ruptured cyst suspension through a Millipore microfiber glass prefilter, a preparation of viable central bodies was obtained that contained less than 1% residual cysts and vegetative cells. Electron micrographs showed the central bodies to have a cell wall and cell membrane. Free central bodies germinated into typical vegetative cells. Central bodies exhibited approximately the same resistance to ultraviolet radiation, sonic treatment, and elevated temperatures as did vegetative cells; cysts were much more resistant. Manometric experiments indicated that central bodies and cysts have almost the same oxidative capabilities. Results of resistance studies indicated that the central body is a contracted vegetative cell encased in a protective coat. The cyst coat appears to account for the resistance of the cyst.
Topics: Azotobacter; Citrates; Edetic Acid; In Vitro Techniques; Microscopy, Electron
PubMed: 4955249
DOI: 10.1128/jb.91.1.297-303.1966 -
The ISME Journal Jan 2017Nitrogen fixation is advantageous in microbial competition when bioavailable nitrogen is scarce, but has substantial costs for growth rate and growth efficiency. To...
Nitrogen fixation is advantageous in microbial competition when bioavailable nitrogen is scarce, but has substantial costs for growth rate and growth efficiency. To quantify these costs, we have developed a model of a nitrogen-fixing bacterium that constrains mass, electron and energy flow at the scale of the individual. When tested and calibrated with laboratory data for the soil bacterium Azotobacter vinelandii, the model reveals that the direct energetic cost of nitrogen fixation is small relative to the cost of managing intracellular oxygen. It quantifies the costs and benefits of several potential oxygen protection mechanisms present in nature including enhanced respiration (respiratory protection) as well as the production of extracellular polymers as a barrier to O diffusion, and increasing cell size. The latter mechanisms lead to higher growth efficiencies relative to respiratory protection alone. This simple, yet mechanistic framework provides a quantitative model of nitrogen fixation, which can be applied in ecological simulations.
Topics: Azotobacter vinelandii; Models, Biological; Nitrogen; Nitrogen Fixation; Oxygen
PubMed: 27740611
DOI: 10.1038/ismej.2016.97 -
Journal of Bacteriology Jul 1962Socolofsky, M. D. (University of Texas, Austin) and Orville Wyss. Resistance of the Azotobacter cyst. J. Bacteriol. 84:119-124. 1962-The Azotobacter cysts were found to...
Socolofsky, M. D. (University of Texas, Austin) and Orville Wyss. Resistance of the Azotobacter cyst. J. Bacteriol. 84:119-124. 1962-The Azotobacter cysts were found to be more resistant than the vegetative cells to various harmful agents. Studies involving ultraviolet irradiation indicated that cysts required twice as great a dosage, as correspondingly treated vegetative cells, to be 90% inactivated. The acquisition of ultraviolet resistance during the encystment process was gradual and appeared to be related to the formation of exine and intine. A slow loss of ultraviolet resistance during germination was also noted. The cysts exhibited no marked resistance to heat, although they were extremely resistant to gamma radiation, sonic treatment, and desiccation. Evidence was presented indicating that the cyst is not a bacterial endospore. The encystment process may confer a survival advantage upon the organism by coupling the low endogenous respiration rate with the ability to withstand desiccation.
Topics: Azotobacter; Cysts; Hot Temperature; Spores, Bacterial
PubMed: 13914732
DOI: 10.1128/jb.84.1.119-124.1962 -
The Biochemical Journal Oct 1967
Topics: Azotobacter; Chromatography, Ion Exchange; Cold Temperature; Freezing; Nitrogen Fixation; Oxidation-Reduction; Oxidoreductases
PubMed: 6056637
DOI: 10.1042/bj1050003c -
Journal of Bacteriology Jan 1970A nitrogenase system that remains in the supernatant fluid after centrifuging for 3 hr at 180,000 x g can be extracted from Azotobacter vinelandii by osmotic lysis of...
A nitrogenase system that remains in the supernatant fluid after centrifuging for 3 hr at 180,000 x g can be extracted from Azotobacter vinelandii by osmotic lysis of the bacteria. This nitrogenase preparation is oxygen-labile and appears to be similar, though not identical, to that obtained from Clostridium pasteurianum. The particulate characteristic and oxygen stability of previously described preparations are likely due to the method of cell disruption, e.g., in the French pressure cell. The data support a nitrogenase model system in the intact cell in which oxygen-labile enzymes are protected from oxygen by the extensive internal membranous system which Azotobacter synthesize only when they fix nitrogen.
Topics: Acetylene; Azotobacter; Cell Membrane; Centrifugation; Microscopy, Electron; NAD; Nitrogen Fixation; Osmosis; Oxidation-Reduction; Oxidoreductases; Oxygen
PubMed: 4312544
DOI: 10.1128/jb.101.1.292-296.1970 -
Journal of Bacteriology Feb 2008The general stress response mediated by the sigma factor RpoS is important for survival of bacteria in adverse environments. A mutant unable to produce RpoS was...
The general stress response mediated by the sigma factor RpoS is important for survival of bacteria in adverse environments. A mutant unable to produce RpoS was constructed using the diazotrophic bacterium Azotobacter vinelandii strain UW. Under nondesiccating, solid-medium growth conditions the wild type was culturable for 16.5 years, while the rpoS mutant remained viable for only 10 months. The rpoS mutant exhibited reduced survival compared to the wild type following hydrogen peroxide stress, and stationary phase cells were killed rapidly by 15 mM H2O2. Three catalases (Kat1, Kat2, and Kat3) were expressed in the wild type under the conditions used. Kat2 was expressed in exponential phase during shake flask growth and could be induced under highly aerated conditions in all growth phases, suggesting that there was induction by reactive oxygen intermediates. Kat3 was possibly an isoform of Kat2. In contrast, Kat1 was expressed in an RpoS-dependent manner during the mid-exponential to late stationary phases. RpoS expression did not occur exclusively in stationary phase but was influenced by changes in carbon and nitrogen source availability. There was 26- to 28-fold induction of the RpoS protein during acetate-to-glucose and ammonium-to-N2 diauxic shifts. Following recovery of growth on the alternative carbon or nitrogen source, RpoS protein concentrations declined rapidly to a basal level. However, rpoS mRNA levels did not correlate directly to RpoS levels, suggesting that there was posttranscriptional regulation. Evidence obtained using the RpoS-dependent reporter Kat1 suggested that there is regulation of the RNAP:RpoS holoenzyme at the level of complex formation or activity.
Topics: Azotobacter vinelandii; Bacterial Proteins; Carbon; Catalase; DNA, Bacterial; Gene Expression Regulation, Bacterial; Heat-Shock Response; Hydrogen Peroxide; Molecular Sequence Data; Nitrogen; Oxidative Stress; RNA Processing, Post-Transcriptional; RNA, Bacterial; RNA, Messenger; Sequence Analysis, DNA; Sigma Factor
PubMed: 18055600
DOI: 10.1128/JB.01571-06 -
Applied and Environmental Microbiology Apr 2012Concerns regarding the depletion of the world's reserves of oil and global climate change have promoted an intensification of research and development toward the...
Concerns regarding the depletion of the world's reserves of oil and global climate change have promoted an intensification of research and development toward the production of biofuels and other alternative sources of energy during the last years. There is currently much interest in developing the technology for third-generation biofuels from microalgal biomass mainly because of its potential for high yields and reduced land use changes in comparison with biofuels derived from plant feedstocks. Regardless of the nature of the feedstock, the use of fertilizers, especially nitrogen, entails a potential economic and environmental drawback for the sustainability of biofuel production. In this work, we have studied the possibility of nitrogen biofertilization by diazotrophic bacteria applied to cultured microalgae as a promising feedstock for next-generation biofuels. We have obtained an Azotobacter vinelandii mutant strain that accumulates several times more ammonium in culture medium than wild-type cells. The ammonium excreted by the mutant cells is bioavailable to promote the growth of nondiazotrophic microalgae. Moreover, this synthetic symbiosis was able to produce an oil-rich microalgal biomass using both carbon and nitrogen from the air. This work provides a proof of concept that artificial symbiosis may be considered an alternative strategy for the low-N-intensive cultivation of microalgae for the sustainable production of next-generation biofuels and other bioproducts.
Topics: Azotobacter; Biofuels; Biomass; Biotechnology; Chlorella; Culture Media; Fresh Water; Gene Deletion; Microalgae; Mutation; Nitrogen Fixation; Nitrogenase; Quaternary Ammonium Compounds; Scenedesmus; Symbiosis
PubMed: 22267660
DOI: 10.1128/AEM.06260-11 -
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 -
Encystment and polymer production by Azotobacter vinelandii in the presence of beta-hydroxybutyrate.Journal of Bacteriology Jun 1968Cells of Azotobacter vinelandii encysted in Burk's nitrogen-free liquid media which had been supplemented with n-butyl alcohol, beta-hydroxybutyrate, or crotonate....
Cells of Azotobacter vinelandii encysted in Burk's nitrogen-free liquid media which had been supplemented with n-butyl alcohol, beta-hydroxybutyrate, or crotonate. Butyraldehyde and butyrate did not influence the extent of encystment. In the absence of glucose, beta-hydroxybutyrate enhanced the rate and extent of encystment. In the presence of glucose, it promoted abortive encystment, which was manifested by the disorganization of the exine and the release of a highly viscous material into the medium. The soluble, viscous polymer was separated from the medium by a series of ethyl alcohol precipitations and identified as a mucopeptide. It was cleaved by treatment with lysozyme and lysostaphin with a concomitant increase in reducing power. It contained 13.9% N; 56% amino acids, as alanine (alanine, lysine, and glutamic acids); and 42% hexosamines. The polymer appeared to be similar to a noncross-linked peptidoglycan.
Topics: Alcohols; Azotobacter; Carbon Isotopes; Culture Media; Glucose; Hydroxybutyrates; Microscopy, Electron; Morphogenesis; Peptides; Polymers
PubMed: 5669905
DOI: 10.1128/jb.95.6.2336-2343.1968