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PubMed1979

Studies on the aerobic non-symbiotic nitrogen-fixing bacteria, other than Azotobacter, in Egyptian soils.

Twenty isolates of micro-organisms capable of growing on nitrogen-deficient medium and found as contaminants in Azotobacter cultures were isolated from Egyptian soils and studied for their morphological, cultural, and physiological properties. These micro-organisms s are members of Rhizobiaceae, Pseudomonadaceae, Achromobacteriaceae, Enterobacteriaceae, Micrococcaceae, Bacillaceae, and Streptomycetaceae as well as some yeasts. In nitrogen-free medium the micro-organism fixed only small amounts of atmospheric nitrogen, hardly exceeding 3 ppm and because of their low sugar consumption rates, efficiences of N2-fixation sometimes reaching 18 mg nitrogen fixed/g carbon oxidized were recorded. Addition of 15 ppm combined nitrogen to the medium increased the amounts of nitrogen fixed to 3--9 ppm.

Aerobiosis↗

PubMed1985

Azotobacter vinelandii nifD- and nifE-encoded polypeptides share structural homology.

The Azotobacter vinelandii nifE gene was isolated and its complete nucleotide sequence was determined. The amino acid sequences deduced from the A. vinelandii nifE and nifD gene sequences were compared and found to share striking primary sequence homology. This homology implies a functional and possibly an evolutionary relationship between these two gene products. The structural homology is discussed with regard to the potential FeMo cofactor binding properties of these polypeptides and the possible role of a nifEN product complex as a surrogate MoFe protein.

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PubMed1987

Site-directed mutagenesis of the nitrogenase MoFe protein of Azotobacter vinelandii.

A strategy has been formulated for the site-directed mutagenesis of the Azotobacter vinelandii nifDK genes. These genes encode the alpha and beta subunits of the MoFe protein of nitrogenase, respectively. Six mutant strains, which produce MoFe proteins altered in their alpha subunit by known single amino acid substitutions, have been produced. Three of these transversion mutations involve cysteine-to-serine changes (at residues 154, 183, and 275), two involve glutamine-to-glutamic acid changes (at residues 151 and 191), and one involves an aspartic acid-to-glutamic acid change (at residue 161). All three possible phenotypic responses are observed within this group- i.e., normal, slow, and no growth in the absence of a fixed-nitrogen source. Two-dimensional gel electrophoresis indicates that all mutants accumulate normal levels of the subunits of both nitrogenase component proteins. Whole-cell and crude-extract acetylene-reduction activities indicate substantial levels of Fe protein activity in all strains. In contrast, MoFe protein activities do not parallel the diazotrophic growth capability for all strains. Two strains appear to exhibit altered substrate discrimination. Such analyses should aid in the identification of metallocluster-binding sites and subunit-subunit interaction domains of the MoFe protein and also provide insight into the mechanistic roles of the various prosthetic groups in catalysis.

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PubMed1993

Structure at pH 6.5 of ferredoxin I from Azotobacter vinelandii at 2.3 A resolution.

Ferredoxin I from Azotobacter vinelandii (AvFdI) is an iron-sulfur protein composed of 106 amino acids, seven Fe atoms and eight inorganic S* atoms. A crystallographic redetermination of its structure showed the originally reported structure to be incorrect. We report here the crystal structure of AvFdI at pH 6.5. Extensive refinement has led to a final R value of 0.170 for all 6986 non-extinct reflections in the range 10-2.3 A using a solvent model which includes 98 discrete solvent atoms with occupancies between 0.3 and 1.0 and an average B value of 22.5 A(2). The first half of the peptide chain closely resembles that of the 55-residue ferredoxin from Peptococcus aerogenes (PaFd), while the remainder consists of three turns of helix and a series of loops which form a cap over part of the molecular core. Despite the similarities in structure and surroundings, the corresponding 4Fe4S* clusters in PaFd and AvFdI have strikingly different redox potentials; a possible explanation has been sought in the differing hydration models for the two molecules.

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PubMed1999

Adaptation and population dynamics of Azotobacter vinelandii during aerobic biological treatment of olive-mill wastewater.

Olive-mill wastewater (OMW) has a high organic and polyphenol content and is resistant to biodegradation. Its disposal leads to a major environmental pollution problem in the Mediterranean basin. The detoxification of OMW following inoculation with Azotobacter vinelandii (strain A) was performed for two successive 5-day-period cycles in an aerobic, biowheel-type reactor, under non-sterile conditions. The phytotoxicity of the processed product was reduced by over 90% at the end of both cycles. To exclusively monitor the A. vinelandii population in the reactor a most probable number-PCR approach was employed and applied daily to serial dilutions of total DNA extracted from reactor samples. PCR sensitivity was independent of the presence of OMW or non-target DNA. The A. vinelandii population dynamics were successfully monitored, showing an initial adaptation period, followed by a sharp population maximum on the fourth day of both cycles (1.6x10(8) and 9.6x10(7) cells ml(-1) respectively), after a major phytotoxicity decline. N(2) fixation rates were estimated using the acetylene reduction assay and reached a peak during the first 1-2 days of each cycle (36 and 29 nmol C(2)H(2) ml(-1) h(-1) respectively). The data are consistent with an initial physiological adaptation phase, where the presence of phenolic compounds limits A. vinelandii growth but stimulates N(2) fixation, followed by a rapid growth phase as phytotoxicity declines.

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PubMed1977

Purification and Some Biological Properties of Asparaginase from Azotobacter vinelandii.

Asparaginase was found in the soluble fraction of cells of Azotobacter vinelandii, and its activity remained the same during growth of the organism in a nitrogen-free medium. The specific activity and the yield of A. vinelandii increased twofold in the presence of ammonium sulfate. Within limits, the temperature (30 to 37 degrees C) and pH (6.5 to 8.0) of the medium showed little effect on the levels of enzyme activity. The enzyme was purified to near homogeneity by standard methods of enzyme purification, including affinity chromatography, and had optimum activity at pH 8.6 and 48 degrees C. The approximate molecular weight was 84,000. The apparent K(m) value for the substrate was 1.1 x 10 M. Metal ions or sulfhydryl reagents were not required for enzyme activity. Cu, Zn, and Hg showed concentration-dependent inhibition, whereas amino and keto acids had no effect on the enzyme activity. Asparaginase was stable when incubated with rat serum and ascites fluid. The enzyme had no effect on the membrane of sheep erythrocytes and did not inhibit the incorporation of radioactive precursors into deoxyribonucleic acid, ribonucleic acid, and protein in Yoshida ascites sarcoma cells. Asparaginase activity was not detected in the tumor cells.

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PubMed1981

Effects of salinity on acetylene reduction (nitrogen fixation) and respiration in a marine azotobacter.

An acetylene-reducing (nitrogen-fixing) bacterium, identified as Azotobacter sp., was isolated from a site in the Canary Creek Marsh, Del. Acetylene reduction activity of the isolate was maximal at 15 to 25 per thousand NaCl, with no activity observed at 0 or 60 per thousand. Respiration studies showed similar results, with maximal activity occurring at a slightly lower salinity (10 to 20 per thousand NaCl). The salinities over which peak activity occurred fell within the normal range of in situ salinity (20 to 28 per thousand total salinity).

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PubMed1983

Siderophores Produced by Nitrogen-Fixing Azotobacter vinelandii OP in Iron-Limited Continuous Culture.

Azotobacter vinelandii requires a high complement of iron and an efficient iron acquisition system to support nitrogen fixation. To circumvent problems inherent in batch culture trace metal studies, continuous cultures were used to measure the response of A. vinelandii to iron stress. Iron was found to be growth limiting for nitrogen-fixing A. vinelandii at a concentration as high as 12.5 muM; iron was growth sufficient at 25 muM. Iron-stressed A. vinelandii in continuous culture formed 2,3-hydroxybenzoic acid (DHB), 2-N,6-N-di-(2,3-dihydroxybenzoyl)-l-lysine (DHBL), and a chromophoric yellow-green fluorescent peptide (YGFP). At a fixed dilution rate of 0.1 h, steady-state growth occurred at growth-limiting iron concentrations. DHB and DHBL were quantitatively measured during iron-limited steady states and iron-sufficient states by Arnow colorimetric assays. YGFP was determined by absorbance measurements taken at 380 nm, and the concentration was calculated from the reported specific absorption coefficient. Biomass increased and DHBL, DHB, and YGFP concentrations decreased as the concentration of growth-limiting iron was increased in the culture vessel and medium reservoirs. DHBL was the major siderophore and YGFP was the minor siderophore species produced during iron-limited equilibrium growth. A low level of DHB and YGFP, but no DHBL, was formed under iron-sufficient conditions. These results provide further physiological evidence that DHB, YGFP, and especially DHBL may function as siderophores in nitrogen-fixing A. vinelandii.

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PubMed1985

Bacterial Alginate Produced by a Mutant of Azotobacter vinelandii.

The optimum conditions in shaken flasks for production of bacterial alginate by mutant C-14 of Azotobacter vinelandii NCIB 9068 and a comparison of the properties of bacterial and algal alginates were investigated. The largest amount of bacterial alginate was obtained in about 110 h by a culture grown on optimum medium at 34 degrees C and 170-rpm shaking speed. The viscosity of the culture broth was 18,400 cps and the alginate concentration reached 6.22 g/liter. The viscosity of the purified bacterial alginate was as high as 11,200 cps at a low concentration (0.6%). A greater than fivefold concentration of algal alginate was required to reach the same viscosity at a low shear rate. A solution of bacterial alginate was more pseudoplastic than that of algal alginate was. No significant differences were observed in other properties of bacterial and algal alginates such as gel formation with calcium ion, thermostability, and effect of temperature, pH, and sodium chloride on viscosity.

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PubMed1986

Survival of Azotobacter spp. in Dry Soils.

Dry soils stored in glass containers in the laboratory and protected from contamination for periods of 22 to 24 years yielded numerous colonies of Azotobacter chroococcum and other members of the family Azotobacteraceae. These results were compared with those reported in 1974, and the findings are uniformly consistent in terms of surviving populations. The data prove that these bacteria remain viable after prolonged periods of dormancy in much the same way as do the endospores of gram-positive bacteria.

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PubMed1986

Sodium-Dependent Growth of Azotobacter chroococcum.

The majority of Azotobacter chroococcum strains in a collection obtained from Alberta soils were absolutely dependent on Na for growth. Two strains from the American Type Culture Collection also were either Na dependent or were stimulated by Na. Optimal growth required 0.8 mM Na and was limited at 0.2 to 0.35 mM Na. Growth promoted by 0.8 mM Na was inhibited by Rb > K >> NH(4) but was not affected by Li. Growth inhibition by Rb and K was overcome by increasing the Na concentration present in the medium. Excellent growth in media containing limiting Na was obtained when 25 mM Li or 25 mM Mg was added. Li was significantly more effective in replacing Na than was Mg. Na was required for growth on all C sources (glucose, sucrose, melibiose, and mannitol) and N sources (N(2), NH(4), and NO(3)) and was required throughout the pH range of growth. Cells suspended in Na -deficient medium or in distilled water did not appear to lyse or lose viability.

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PubMed1987

Growth of Azotobacter vinelandii on Soil Nutrients.

Azotobacter vinelandii cells grew well in a medium made from soil and distilled water which contained little or no carbohydrate. They utilized p-hydroxybenzoic acid and other phenolic acids, soil nitrogen, and water-soluble mineral substances. Seventeen soils which supported excellent growth of A. vinelandii contained 11 to 18 different phenolic acids each, including p-hydroxybenzoic, m-hydroxybenzoic, vanillic, p-coumeric, syringic, cis- and trans-ferrulic, and other unidentified aromatic acids. Three white, chalky "caliche" soils which were taken from areas where no plants grew failed to support the growth of A. vinelandii, and these contained no, two, and three phenolic acids, respectively. A. vinelandii did not fix nitrogen when growing in dialysates of soils which contained numerous phenolic acids. Growth was ample and rapid in most of the soils tested, but cell morphology was different from that usually seen in chemically defined, nitrogen-free media which contain glucose.

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PubMed1987

Iron-Dependent Production of Hydroxamate by Sodium-Dependent Azotobacter chroococcum.

The sodium-dependent strain 184 of Azotobacter chroococcum was unable to grow significantly in iron-limited medium, but did produce iron-repressible outer membrane proteins. Siderophores were not produced under these conditions. Citric acid was excreted, but not in response to iron limitation. This strain, however, was able to grow in insoluble mineral iron sources, and under these conditions the cells produced a hydroxamate. Growth on minerals and hydroxamate production was dependent on a low level of freely exchangeable iron. Optimal hydroxamate production was observed with 0.75 muM ferric citrate, and hydroxamate production was repressed by >5 muM iron. Despite this iron requirement, hyroxamate was only formed during internal iron limitation of the cells. Iron-containing cells were able to grow in iron-limited medium but only produced hydroxamate when their iron-per-cellular-protein content was low. These results, the spectral changes observed upon Fe addition, and iron-uptake coincident with hydroxamate production suggested that the hydroxamate was a siderophore.

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PubMed1988

Zn Increases Siderophore Production in Azotobacter vinelandii.

When Azotobacter vinelandii was grown in the presence of low levels of iron, the addition of 20 or 40 muM ZnSO(4) caused earlier production of the catechol siderophores and a dramatic increase in the amount of azotobactin. The level of cellular iron was not significantly lowered in Zn -grown cells, which suggested that Zn was not causing more severe, or earlier, iron limitation. Also, Zn did not appear to affect production of the high-molecular-weight outer membrane iron-repressible proteins that presumably function as ferrisiderophore receptors. Spectrophotometric examination of ion binding to the siderophores revealed that while the siderophores appeared to bind Zn, only in the case of azotochelin was iron unable to completely overcome any Zn -induced changes in the absorption spectra. This appeared to rule out direct competition of Zn with iron for binding to the siderophores. Fe uptake was depressed both in Zn -grown cells and in Zn -free cells to which Zn was added during the uptake assay, except with azotobactin, with which the level of Fe uptake by Zn -grown cells was close to control levels. These results suggested two possible sites where Zn could be acting, one involving the biosynthesis of siderophores and possibly the genetic regulation of the iron assimilation system and the other involving an internal point common to iron assimilation by both high- and low-affinity iron uptake.

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PubMed1988

Effects of Mannose on the Growth of N(2)-Fixing Azotobacter vinelandii.

Mannose is not a suitable substrate for N(2)-fixing Azotobacter vinelandii. However, when H(2) gas is provided, A. vinelandii can grow mixotrophically with H(2) as the energy source and mannose as the carbon source (T.-Y. Wong and R. J. Maier, J. Bacteriol. 163:528-533, 1985). In this report, seven sugars were used to determine whether A. vinelandii could derive energy from these sugars for mannose utilization. Supplementation of fructose- or galactose-limited medium with mannose did not influence the biomass produced by N(2)-fixing A. vinelandii. The presence of mannose in glucose- or maltose-limited cultures increased cell yield slightly. The addition of mannose decreased the total biomass in the melibiose-limited culture slightly. Mannose was a potent inhibitor of growth when sucrose or turanose was used as the primary sugar. The inhibitory effect of mannose on utilization of sucrose and turanose seems to be related to the energy requirement of the N(2)-fixing processes.

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PubMed1988

Sodium-Dependent Azotobacter chroococcum Strains Are Aeroadaptive, Microaerophilic, Nitrogen-Fixing Bacteria.

Na -dependent strains of Azotobacter chroococcum were observed to have very low reactivities with the H(2)O(2) spot test for catalase. The cell extract of the representative Na -dependent strain 184 contained a catalase specific activity that was 10-to 600-fold lower than those found in Na -independent strains of A. chroococcum. Peroxidase and superoxide dismutase activities existed in all strains, although only certain Na -dependent strains contained a peroxidase reactive with p-phenylenediamine. The activities of catalase and peroxidase in the Na -dependent strain 184 were dependent on iron availability, which helped to explain the iron-dependent growth characteristic of this strain. The activities of these enzymes were not increased by subjecting the cells to increased aeration, nitrogen-fixing conditions, or paraquat. Strain 184 was found to be very sensitive to H(2)O(2) or paraquat, even under iron-sufficient conditions, and was difficult to recover quantitatively on solid plating media. Strain 184 was more susceptible to H(2)O(2) when grown under low-aeration, nitrogen-fixing conditions than when it was grown in the presence of NH(4). Low population densities of strain 184 grew in nitrogen-free medium under microaerophilic conditions, while more dense populations were able to fix nitrogen under aerobic conditions. Therefore, these bacteria appeared to be aeroadaptive, microaerophilic, nitrogen-fixing bacteria.

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PubMed1989

Cytokinins in Azotobacter vinelandii Culture Medium.

Azotobacter vinelandii OP was grown to stationary phase in defined medium. The cell-free culture medium was analyzed for cytokinin content by XAD-2 and Sephadex LH-20 chromatography, thin-layer chromatography, tobacco callus bioassay, and enzyme immunoassay. Three cytokinin-active fractions were detected and tentatively identified as trans-zeatin, isopentenyladenosine, and isopentenyladenine. The total cytokinin activity was equivalent to 0.75 mug of kinetin per liter.

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PubMed1989

Isolation and Preliminary Characterization of Hydroxamic Acids Formed by Nitrogen-Fixing Azotobacter chroococcum B-8.

The free-living diazotroph Azotobacter chroococcum B-8 responded to iron-limited growth conditions by forming hydroxamic acids and an 85,000-dalton outer membrane protein. The Fe(III)-binding hydroxamate compounds stimulated the growth of Arthrobacter flavescens JG-9 and gave a positive Csaky reaction for bound hydroxylamines. The hydroxamates were isolated from liquid cultures by benzyl alcohol extraction and purified by size exclusion chromatography and high-performance liquid chromatography. Four high-performance liquid chromatography fractions, designated A, B, C, and D, had the characteristic hydroxamate absorption maximum at 420 to 423 nm, which did not shift over a pH range from 3.0 to 9.0. Cyclic voltammograms of the iron-hydroxamate complexes exhibited reduction potentials of -0.426 to -0.442 V for fractions A, B, and D and of -0.56 V for fraction C versus the normal hydrogen electrode at pH 8.0. Based on mass spectra, nominal molecular weights of 800 and 844 were assigned to ferrated compounds A and B, respectively. Reductive hydrolysis of compounds A and B in 57% hydriodic acid yielded ornithine as detected by gas chromatography-mass spectrometry. All of these physiological and chemical data strongly support the hypothesis that the high-affinity iron-binding compounds isolated from A. chroococcum B-8 are hydroxamic acids and probably function as siderophores for this diazotroph.

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