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At least 19 recordsLinked to original sources
PubMed1976

Physiological factors affecting transformation of Azotobacter vinelandii.

Cells of Azotobacter vinelandii (ATCC 12837) can be transformed by exogenous deoxyribonucleic acid towards the end of exponential growth. Transformation occurs at very low frequencies when the deoxyribonucleic acid is purified or when the transformation is carried out in liquid medium. Optimal transformation occurs on plates of Burk nitrogen-free glucose medium containing either high phosphate (10 mM) or low calcium (0 to 0.29 mM) content. Higher levels of calcium are inhibitory, whereas magnesium ions are essential for transformation and growth. Extracellular polymer and capsule are increasingly inhibitory to transformation and are most abundant when the calcium content of the medium is high. Transformation is optimal at pH 7.0 to 7.1 and at 30 C, conditions which also coincide with minimal extracellular polymer production. Nonencapsulated strains are excellent transformation recipients. Glycine-induced pleomorphism reduces the transformation frequency and the degree of inhibition is dependent on the phosphate concentration of the medium. Rifampin resistance and shifts from adenine, hypoxanthine, uracil, and nitrogenase auxotrophy to prototrophy can be achieved. Although single marker transfer is always greater than double marker transfer, the data suggest that rifampin resistance is linked to hypoxanthine, adenine and uracil protorophy at intervals of increasing distance. Rifampin resistance did not appear to be linked to nitrogenase.

Adenine↗
PubMed1975

Relationship between calcium and uroinic acids in the encystment of Azotobacter vinelandii.

Encystment of Azotobacter vinelandii (ATCC 12837) in modified Burk nitrogen-free medium (pH 7.0) containing 0.2 percent beta-hydroxybutyrate occurs optimally in 0.37 to 0.44 mM solutions of calcium ions. Suspension of cells in media deficient in calcium results in abortive encystment characterized by the release of viscous cyst coat material. Mature cysts rupture in ethylene glycol-bis-(beta-aminoethyl ether)-N,N'-tetraacetic acid, suggesting that calcium is a structural component of the cyst coat. Maximal stimulation of encystment by calcium ions occurs prior to the completion of the cyst exine or outer coat. The uronic acid composition of cyst components is dependent on calcium levels in the medium. Uronic acids account for 31.7 percent of the intine (inner coat) and 13 percent of the exine dry weight, and only mannuronic and guluronic acids are present in these fractions. These can be extracted as homo- and heteropolymeric sequence "blocks" characteristic of alginic acids. The polyuronic acid fraction of both the cyst coats contain approximately equal amounts of heteropolymeric (mannuronic acid/guluronic acid) blocks. The exine, however, is richer in polyguluronic acid and the intine is richer in polymannuronic acid. As a result, the mannuronic acid/guluronic acid ratio of the exine is lower than that of the intine. Slimes that form in abortive encystment are rich in polymannuronic acid and have a high mannuronic acid/guluronic acid ratio. A polymannuronic acid 5-epimerase is active in the mature cyst central body and the encystment culture fluid.

Azotobacter↗
PubMed1979

Optimal conditions for transformation of Azotobacter vinelandii.

Optimal transformation of Azotobacter vinelandii OP required a 20-min incubation of the competent cells with deoxyribonucleic acid at 30 degrees C in buffer (pH 6.0 to 8.0) containing 8 mM magnesium sulfate. Nitrogen-fixing transformants of nitrogen fixation-deficient recipients could be plated immediately on selective medium, but transformants acquiring rifampin and streptomycin resistance required preincubation in nonselective medium. The three phenotypes achieved an approximately equal and stable frequency after 17 h (six generations) of growth in nonselective medium.

Azotobacter↗
PubMed1979

Phosphate-limited culture of Azotobacter vinelandii.

Batch cultures of Azotobacter vinelandii grown in phosphate-deficient media were compared with control cultures grown in phosphate-sufficient media. Phosphate limitation was assessed by total cell yield and by growth kinetics. Although cell protein, nucleic acids, and early growth rate were unaffected by phosphate deficiency, cell wall structure, oxygen uptake, and cell viability were significantly affected. Also, phosphate-limited cells contained much larger amounts of poly-beta-hydroxybutyric acid but lower adenylate nucleotide energy charge than did control cells. The ratio of adenosine 5'-triphosphate to adenosine 5'-diphosphate was much lower in phosphate-deficient cells. The data indicate a substrate saving choice of three metabolic pathways available to this organism under different growth conditions.

Adenine Nucleotides↗
PubMed1976

ATP-dependent calcium transport in isolated membrane vesicles from Azotobacter vinelandii.

Membrane vesicles from Azotobacter vinelandii O prepared by osmotic lysis of spheroplasts in tris (hydroxymethyl) aminomethane/acetate buffer (pH 7.8) contain a latent adenosine triphosphatase (ATPase). The ATPase can be activated when the vesicles are incubated in the presence of an electron donor (D-lactate) and a mixture of adenosine diphosphate and inorganic phosphate or by controlled treatment with trypsin. After the ATPase is activated, the membrane vesicles in the presence of adenosine triphosphate accumulate calcium but not glucose or rubidium (in the presence of valinomycin). ATP-dependent calcium uptake follows Michaelis-Menten kinetics with a Km of 48 muM and a Vmax of 20 nmol/min/mg of membrane protein and is highly specific for calcium over cations magnesium, barium, lanthanum, sodium, potassium, and lithium. The calcium accumulated in the presence of ATP is freely exchangeable with external calcium and is rapidly released in the presenceof uncouplers or ATPase inhibitors. Calcium uptake in the presenceof ATP is blocked by dicyclohexylcarbodiimide, ADP, p-chloromercuriphenylsulfonate, by the proton-conducting ionophores m-chlorophenylcarbonylcyanide hydrazone, nigericin, monensin, and gramicidin D, but not by potassium cyanide, anoxia, or valinomycin (in the presence of potassium). Measurements of the external pH of vesicle suspensions reveal that protons are actively taken up by the membranes during hydrolysis of ATP. These results suggest that vesicles prepared under these conditions have a topology which is inverted with respect to the intact cell and that calcium is accumulated by means of proton antiport.

Adenosine Diphosphate↗
PubMed1978

Kinetic studies on electron transfer and interaction between nitrogenase components from Azotobacter vinelandii.

Kinetic properties of electron transfer by nitrogenase of Azotobacter vinelandii are dependent on the concentration of the two components of nitrogenase. An excess of the MoFe protein inhibits electron transfer in a distinctive manner, and the inhibition is reversed by increasing levels of reductant. The saturation curve for Fe protein is hyperbolic, indicating that only one Fe protein molecule per MoFe protein is required for full activity in ATP hydrolysis and electron transfer. These results can be interpreted on the basis of a complex between the Fe protein and the MoFe protein that dissociates rapidly during turnover. Both 2:1 and 1:1 complexes (Fe-MoFe) are active. Dithionite appears to be a relatively poor reductant for nitrogenase from Azotobacter vinelandii, whereas azotobacter flavodoxin is much better. In the presence of the flavodoxin it is possible to increase the specific activity of the Fe protein more than 50% relative to its activity with dithionite alone as a reductant; specific activities greater than 3000 nmol of C2H4 formed min(-1) (mg of Fe protein)(-1) have been observed.

Adenosine Triphosphate↗
PubMed1978

Induction of transformation competence in Azotobacter vinelandii iron-limited cultures.

Azotobacter vinelandii strains UW (Nif+) or UW1 (Nif-) were induced to form competent cells by growing cultures in Burk medium without added iron. Competent cells were generated in either liquid or solid medium. The competent culture was highly colored by the characteristic fluorescent green pigment of A. vinelandii but all pigmented cultures were not competent. In liquid culture, competent cells required a fixed nitrogen source (ammonia, nitrate, or urea) that was also a nitrogenase repressor. Glucose, sucrose, glycerol, and mannitol (1% w/v) were excellent C-sources for competence induction while acetate (1% w/v) and beta-hydroxybutyrate (0.2% w/v) were ineffective. Cells induced in liquid culture remained competent for a 12-h period before the end of the exponential growth phase, then competence decreased rapidly upon the initiation of the stationary phase of growth.

Azotobacter↗
PubMed1978

Ultrastructural and physiological changes occurring upon germination and outgrowth of Azotobacter vinelandii cysts.

Dormant cysts of Azotobacter vinelandii germinated at 30 degrees C in Burk nitrogen-free media containing 1% glucose. Samples taken at intervals and examined by electron microscopy revealed that as germination progressed, vesicle-like and fibrillar structures became visible in the intine region. Lamellae associated with the cell membrane appeared in the central body at 6 h post-initiation of germination. Both electron micrographic and chemical analysis showed that the poly-beta-hydroxybutyrate content of cysts decreased significantly after 4 h of germination. Dormant cysts were resistant to sonic oscillation, but this property was lost during their conversion to metabolically active vegetative cells.

Azotobacter↗
PubMed1976

Control of transformation competence in Azotobacter vinelandii by nitrogen catabolite derepression.

Azotobacter vinelandii (ATCC 12837) became competent to be transformed by exogenous deoxyribonucleic acid towards the end of exponential growth. Competence in wild-type and nitrogenase auxotrophic (nif-) strains was repressed by the addition of ammonium salts or urea to the transformation medium. Transformation of wild-type cells and nif- strains was optimal on nitrogen-free or nitrogen-limiting medium, respectively. Transformation of wild-type cells also was enhanced when the transformation medium had low molydbate content. During the development of competence, nitrogen was growth limiting, whereas carbon (glucose) was in excess. Carbon source shift-down was not effective in inducing competence. Shifting glucose-grown wild-type cells to medium containing 0.2% beta-hydroxybutyrate initiated encystment and also induced competence. The addition of glucose to this medium blocked encystment and early competence induction and reduced the transformation frequency to the basal level. Cyclic adenosine 3',5'-monophosphate induced competence in wild-type nitrogen-fixing cells and increased the transformation frequency 1,000-fold over the basal level. Exogenous cyclic adenosine 3',5'-monophosphate however, did not reverse nitrogen repression of competence in ammonia-grown wild-type or nif- strains.

Ammonia↗
PubMed1979

Structure of pyridine nucleotide transhydrogenase from Azotobacter vinelandii.

1. Pyridine nucleotide transhydrogenase of Azotobacter vinelandii purified by affinity chromatography consists of a mixture of polydisperse rods at neutral pH. No other structures are seen by electron microscopy. 2. At high pH (8.5--9.0) the rods depolymerize. Complete depolymerization can be achieved in 0.1 M Tris-Cl pH 9.0. The depolymerized enzyme has a molecular weight of 421000 (sedimentation equilibrium), its sedimentation coefficient s20, w = 15 S and its Stokes' radius Rs = 7 nm. Since gel electrophoresis in the presence of sodium dodecyl sulphate shows that transhydrogenase consists of a single polypeptide chain of molecular weight (54 +/- 2) X 10(3) it follows that the depolymerized enzyme has an octameric quaternary structure. We propose that this octamer serves as the functional monomeric unit ('unimer') from which the polymeric form of transhydrogenase is constructed. 3. Gel filtration and sucrose gradient centrifugation studies of cell-free extracts from A. vinelandii show the unimer to be the predominant active species.

Azotobacter↗
PubMed1979

The oxidation-reduction potentials of cytochrome o + c4 and cytochrome o purified from Azotobacter vinelandii.

Oxidation-reduction titrations of Azotobacter vinelandii cytochrome o + c4 and cytochrome o were performed with simultaneous potential and absorbance measurements under anaerobic conditions. Cytochrome c4 has a midpoint potential (Em, 7.4) of 260mV and purified cytochrome o has an Em, 7.4 of -18mV. Little change in the midpoint potential of cytochrome o was observed when titrated in the pH range 6.2--9.8.

Azotobacter↗
PubMed1977

Isolation of membrane vesicles with inverted topology by osmotic lysis of Azotobacter vinelandii spheroplasts.

Membrane vesicles were prepared from Azotobacter vinelandii spheroplasts by lysis in either potassium phosphate (pH 7.0) or Tris1-acetate (pH 7.8) buffers. These 2 types of preparations differ considerably in their properties: 1) Examination by scanning electron microscopy reveals that the Pi vesicles consist primarily of closed structures 0.6-0.8 micrometer in diameter with a rough or particulate surface similar to that of spheroplasts. The Tris vesicles are significantly smaller, 0.1-0.3 micrometer in diameter, and have a much smoother surface structure. 2) Antisera from rabbits immunized with A. vinelandii lipopolysaccharide antigen will agglutinate Pi vesicles but not Tris vesicles. 3) Tris vesicles have a fourfold higher specific activity of latent H+-ATPase than Pi vesicles. After exposure to Triton X-100 similar ATPase activities are observed for both types of vesicles. 4) Pi vesicles transport calcium in the presence of ATP or lactate at less than 30% of the rats observed for Tris vesicles. 5) Tris vesicles have less than 22% of the transport capacity of Pi vesicles for accumulation of labeled sucrose and less than 3% of the capacity for valinomycin-induced uptake of rubidium observed during respiration. 6) Quinacrine fluorescence intensity is reduced by 30% during lactate oxidation and 20% during ATP hydrolysis by Tris vesicles. Under similar conditions, fluorescence in Pi vesicles is quenched by only 7% and less than 2%, respectively. These findings suggest that Pi vesicles have the normal orientation of the intact cell whereas Tris vesicles have an inverted topology.

Adenosine Triphosphatases↗
PubMed1977

Generation of a transmembrane electric potential during respiration by Azotobacter vinelandii membrand vesicles.

Membrane vesicles isolated from Azotobacter vinelandii strain O by lysis of spheroplasts in potassium of sodium phosphate buffer develop a transmembrane electric potential during respiration. The magnitude of this potential was determined by three independent methods: (i) fluorescence of 3,3'-dipropylthiodicarbocyanine and 3,3'-dihexyloxacarbocyanine; (ii) uptake of 86Rb+ in the presence of valinomycin; and (iii) uptake of [3H]triphenylmethyl phosphonium. In method (i), the relative fluorescence of these cyanine dyes in the presence of intact cells or derived vesicles is quenched during oxication of electron donors. A linear relationship between this quenching and a potassium diffusion potential was employed to calibrate the probe response. In method (ii), the steady-state concentration ratio of rubidium across the vesicle membrane during oxidation of L-malate was converted to potential by the Nernst equation. In method (iii), the steady-state concentration ratio of this lipophilic cation was likewise converted to a potential. With the exception of 3,3'-dihexyloxacarbocyanine fluorescence, these methods gave good agreement for the potential developed during L-malate oxidation by membrane vesicles. A value of 75 to 80 mV (inside negative) was obtained for vesicles prepared in potassium phosphate, and 104 mV (inside negative) was obtained for vesicles prepared in sodium phosphate. Electrogenic expulsion of hydrogen ion was observed during L-malate oxidation, and the amount of proton exodus was greater in potassium rather than the sodium-containing vesicles. This indicates the presence of a sodium-proton antiport mechanism. In addition, D-glucose uptake was observed during development of a potassium diffusion potential that was artificially imposed across the vesicle membrane. These observations suggest the presence of a glucose-proton symport mechanism in accordance with the principles of Mitchell.

Azotobacter↗
PubMed1975

Characterization studies on the membrane-bound adenosine triphosphatase (ATPase) of Azotobacter vinelandii.

The adenosinetriphosphatase (ATPase) (EC 3.6.1.3) activity in Azotobacter vinelandii concentrates in the membranous R3 fraction that is directly associated with Azotobacter electron transport function. Sonically disrupted Azotobacter cells were examined for distribution of ATPase activity and the highest specific activity (and activity units) was consistently found in the particulate R3 membranous fraction which sediments on ultracentrifugation at 144 000 X g for 2 h. When the sonication time interval was increased, the membrane-bound ATPase activity could neither be solubilized nor released into the supernatant fraction. Optimal ATPase activty occurred at pH 8.0; Mg2+ ion when added to the assay was stimulatory. Maximal activity always occurred when the Mg2+:ATP stoichiometry was 1:1 on a molar ratio at the 5 mM concentration level. Sodium and potassium ions had no stimulatory effect. The reaction kinetics were linear for the time intervals studied (0-60 min). The membrane-bound ATPase in the R3 fraction was stimulated 12-fold by treatment wiTH TRypsin, and fractionation studies showed that trypsin treatment did not solubilize ATPase activity off the membranous R3 electron transport fraction. The ATPase was not cold labile and the temperature during the preparation of the R3 fraction had no effect on activity; overnight refrigeration at 4 degrees C, however, resulted in a 25% loss of activity as compared with a 14% loss when the R3 fraction was stored overnight at 25 degrees C. A marked inactivation (although variable, usually about 60%) did occur by overnight freezing (-20 degrees C), and subsequent sonication failed to restore ATPase activity. This indicates that membrane reaggregation (by freezing) was not responsible for ATPase inactivation. The addition of azide, ouabain, 2,4-dinitrophenol, or oligomycin to the assay system resulted in neither inhibition nor stimulation of the ATPase activity. The property of trypsin activation and that ATPase activity is highest in the R3 electron transport fraction suggests that its probable functional role is in coupling of electron transport to oxidative phosphorylation.

Adenosine Triphosphatases↗
PubMed1978

Respiration-coupled calcium transport by membrane vesicles from Azotobacter vinelandii.

Membrane vesicles, isolated from osmotic lysates of Azotobacter vinelandii spheroplasts in Tris-acetate buffer, rapidly accumulate calcium in the presence of an oxidizable substrate. The addition of D-lactate to vesicles increases the rate of calcium uptake by 34-fold; L-malate, NADH, NADPH, and reduced phenazine methosulfate are nearly as effective as lactate. The intravesicular calcium pool which accumulates under these conditions is rapidly discharged by isotopic exchange or in the presence of respiratory inhibitors, uncouplers, or EGTA. The uptake rates for calcium follow Michaelis-Menten kinetics yielding a Km of 48 microM and a V max of 45 nmoles/min/mg membrane protein. Initial rates of EGTA-induced calcium efflux also follow saturation kinetics, giving a V max identical to that for calcium entry; but the Km for exodus is 14 mM, assuming that free calcium accumulates in vesicles. The difference in the affinity of calcium for the entry and exit processes observed during respiration is sufficient to account for the estimated 150-fold calcium concentration gradient achieved under steady-state conditions. The uptake system is specific for calcium as opposed to other cations, but zinc and lanthanum are effective competitors. Calcium uptake is blocked when electron is inhibited by exposure of vesicles to p-chlormercuriphenylsulfonate, hydroxyquinoline-N-oxide, or cyanide, or under anoxic conditions. Divalent cation ionophores (A23187 and X537A) and proton ionophores (CCP and gramicidin D) also block calcium transport effectively. The electrogenic potassium ionophore valinomycin has no effect on lactate-dependent calcium uptake in the presence of potassium; but ionophores which induce electroneutral exchange of protons for sodium or potassium (monensin and nigericin, respectively) did block calcium transport in the presence of the appropriate cation. The fluorescence intensity of quinacrine (an amine probe) in the presence of A. vinelandii membrane vesicles is reduced by 25% on addition of lactate; the quenching is blocked by CCP. This indicates that a pH gradient (inside acid) is developed across the vesicle membrane during lactate oxidation. These results indicate that these membrane preparations contain vesicles of inverted topology (with respect to the intact cell) and suggest that calcium transport occurs by means of electroneutral calcium/proton antiport.

Azotobacter↗
PubMed1978

Isolation and partial characterization of two different subunits from the molybdenum-iron protein of Azotobacter vinelandii nitrogenase.

The molybdenum-iron protein of Azotobacter vinelandii nitrogenase was separated into two subunits of equal concentration by ion exchange chromatography on sulfopropyl (SP) Sephadex at pH 5.4 in 7 M urea. Better than 90% yield of each subunit was obtained on a preparative scale if the reduced carboxymethylated molybdenum-iron protein was incubated at 45 degrees C for 45 min prior to chromatography. Without the heating step low yields of the subunits were obtained. Although the amino acid compositions of the two subunits were very similar, the NH2-terminal sequences were completely different as determined by automated sequential Edman degradation. The sequence for the alpha subunit was NH2-Ser-Gln-Gln-Val-Asp-Lys-Ile-Lys-Ala-Ser-Tyr-Pro-Leu-Phe-Leu-Asp-Gln-Asp-Tyr- and for the beta subunit the sequence was NH2-Thr-Gly-Met-Ser-Arg-Glu-Glu-Val-Glu-Ser-Leu-Ile-Gln-Glu-Val-Leu-Glu-Val-Tyr-. Likewise the COOH-terminal sequences for the two subunits, as determined with carboxypeptidase Y, were tota-ly different. The sequence for the alpha subunit was -Leu-Arg-Val-COOH and that for the beta subunit was -Ile-(Phe, Glu)-Ala-Phe-COOH. Radioautographs of tryptic peptide maps were prepared for the molybdenum-iron protein and the two subunits which had been labeled at the cysteinyl residues with iodo[2-14C]acetic acid. These maps indicated that the two subunits had no cysteinyl peptides in common and that the cysteinyl residues were clustered in both subunits.

Amino Acid Sequence↗
PubMed1979

Characterization of Azotobacter vinelandii deoxyribonucleic acid and folded chromosomes.

The properties of Azotobacter vinelandii deoxyribonucleic acid (DNA) and folded chromosomes were studied and compared to those of Escherichia coli as a standard. Based on melting temperature and buoyant density measurements, the guanosine + cytosine content of purified A. vinelandii DNA was 65%, whereas that of E. coli DNA was 50%. The results of renaturation studies showed that the unique DNA sequence lengths of the two organisms were similar with Cot1/2 values of 7.3 +/- 0.4 mol.s/liter and 7.5 +/- 0.3 mol.s/liter, respectively, for A. vinelandii and E. coli. Folded chromosomes of A. vinelandii sedimented in a centrifugal field at a rate identical to those derived from E. coli, 1,600 to 1,700S. Based on the DNA content per cell and the mass of a single genome, A. vinelandii contains at least 40 chromosomes per cell.

Azotobacter↗