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At least 775 records · Page 43Linked to original sources

PubMed1975

Nitrogenase in synchronized Azotobacter vinelandii OP.

Azotobacter vinelandii OP was synchronized by the continuous phased culture technique. The nitrogenase (nitrogen:(acceptor)oxidoreductase)(EC 1.7.99.2) activity of the culture was determined continuously within the fermentor by acetylene reduction. Addition of NH4+ in excess of 5 x 10(-3)M to the culture lowered nitrogenase activity immediately. Other sources of fixed nitrogen had no immediate effect on nitrogenase activity, but nitrogenase synthesis decreased in the cell cycle following the one in which the fixed nitrogen was added.

Acetates↗

PubMed1976

The growth of nitrogen-fixing Azotobacter chroococcum in continuous culture under intense aeration.

Azotobacter chroococcum (ATCC 7493) was grown in continuous culture with intense vortex aeration (stirring rate 1750 rpm) with up to 50% O2 in the gas phase. Under these conditions the dissolved O2 generally remained at zero while the cell growth rose to about twice the normally accepted value. The meaning of the term "O2-limitation" in N2-fixing A. chroococcum cultures is critically examined.

Azotobacter↗

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↗

PubMed1979

Effects of oxygen tension on the lipid composition of Azotobacter chroococcum.

Lipid content and composition were determined in Azotobacter chroococcum grown, under nitrogen-fixing conditions, in continuous culture with intense aeration under atmospheres containing between 5 and 40% O2. Total lipid content remained almost constant at approximately 9% of dry weight. Phospholipid content was maximal at 20% O2 where it accounted for 92% of total lipid, and was minimal at 40% O2. Phosphatidylethanolamine was the only species of phosphatide detected in this fraction. Neutral lipid content was minimal at 20% O2 and maximal at 40% O2 where it represented approximately 30% of the total lipid. Glycolipid remained between 2 and 13% of total lipid throughout. The principal fatty acids of A. chroococcum were hexadecanoic (C16:0), hexadecenoic (C16:1), and octadecenoic acid (C18:1) at all O2 tensions, but C18:1 increased at the expense of C16:1 at higher O2 tensions, particularly in free fatty acid and phospholipid fractions. [U-14C]acetate was readily incorporated into lipid at both 20 and 40% O2, but total incorporation was much greater at 20% O2.

Azotobacter↗

PubMed1980

Superoxide dismutase from nitrogen-fixing Azotobacter chroococcum: purification, characterization, and intracellular location.

Superoxide dismutase (SOD, EC 1.15.1.1) from nitrogen-fixing Azotobacter chroococcum was purified and identified as being similar to the manganese SOD of other procaryotes. The enzyme was relatively thermostable and insensitive to cyanide. A molecular weight of approximately 33 000 was estimated. Superoxide dismutase was found to be cytoplasmic (not bound to cell membranes) in A. chroococcum, but some enzyme was released by sonication of membrane vesicles.

Azotobacter↗

PubMed1984

Ubiquinone in hydrogen metabolism by Azotobacter vinelandii.

Extraction with n-heptane abolished over 95% of the NADH oxidase and the hydrogenase activity in membrane preparations from Azotobacter vinelandii. Incorporation of ubiquinone-8 or plastoquinone restored each reaction to about 55% of its original activity.

Azotobacter↗

PubMed1987

Cellular incorporation of poly-beta-hydroxybutyrate into plasma membranes of Escherichia coli and Azotobacter vinelandii alters native membrane structure.

Under growth-limiting conditions or conditions which mediate genetic transformation, Escherichia coli and Azotobacter vinelandii incorporate poly-beta-hydroxybutyrate into their plasma membranes. Genetic transformation competence of both bacteria increased in proportion to the concentration of membrane poly-beta-hydroxybutyrate. The effects of this lipid polymer on membrane structure were investigated by freeze-fracture electron microscopy. Before poly-beta-hydroxybutyrate incorporation, freeze-fracture revealed a typical mosaic of particles and pits on both concave and convex surfaces of the plasma membrane. As the cells incorporated the lipid polymer into the membrane, transformability developed and small semiregular plaques which possessed shallow particles were seen. These plaques grew in size and frequency as the membrane poly-beta-hydroxybutyrate concentrations and transformability increased.

Azotobacter↗

PubMed1981

Oxidation-reduction titration of cytochrome components in the electron transport chaim of Azotobacter vinelandii.

The multiple cytochrome components in the electron transport particle of Azotobacter vinelandii were resolved and their oxidation-reduction midpoint potentials were determined by a simultaneous potentiometric and absorption measurements under anaerobic condition with or without CO. The midpoints of the individual cytochrome component corresponding to the membrane-bound types were also determined in the solubilized fractions prepared by a differential detergent solubilization of the membrane particles of A vinelandii. Two cytochromes of b type, one with an absorption maximum measured at 559 nm and another at 561 nm in the membrane particle, were resolved and their Em, 7.4 values determined to be - 30 mV and +122mV, respectively. Cytochrome b 559 reacted in both membrane-bound and solubilized forms, however, cytochrome b561 was inert to CO treatment. Only one cytochrome of c type (c4) measured at 575-551 nm was resolved, its midpoint potential at pH 7.4 was +322mV in the membrane-bound form and +278mV in the solubilized form. This c-type cytochrome had no CO reactivity. Cytochrome d, a CO-reactive component, had a midpoint of +270mV in the membrane fraction. The midpoint of cytochrome a1 in its membrane-bound form could not be measured accurately because of its low concentration. However, in the solubilized preparations, cytochrome a1 apparently had a red shift with an absorption maximum at 613 nm, with an estimated Em, 7.4 of - 45 mV, while cytochrome d was no longer detected, possibly because of denaturation.

Azotobacter↗

PubMedDate not supplied

Simultaneous uptake of galactose and glucose by Azotobacter vinelandii.

Azotobacter vinelandii growing on galactosides induced two distinct permeases for glucose and galactose. The apparent Vmax and Km of the galactose permease were 16 nmol galactose/min per 10(10) cells and 0.5 mM, respectively. The apparent Vmax and Km of the glucose permease were 7.8 nmol glucose/min per 10(10) cells and 0.04 mM, respectively. Excess glucose had no effect on the galactose uptake. However, excess galactose inhibited glucose transport. The galactosides-induced glucose permease also exhibited different uptake kinetics from that induced by glucose.

Azotobacter vinelandii↗

PubMed2001

Characterization of the iron superoxide dismutase gene of Azotobacter vinelandii: sodB may be essential for viability.

Azotobacter vinelandii contains two superoxide dismutases (SODs), a cytoplasmic iron-containing enzyme (FeSOD), and a periplasmic copper/zinc-containing enzyme (CuZnSOD). In this study, the FeSOD was found to be constitutive, while the activity of CuZnSOD increased as the culture entered the stationary phase. Total SOD (units/mg protein) in stationary phase cells grown under nitrogen-fixing conditions was not significantly different from those grown under non-nitrogen-fixing conditions. The gene encoding FeSOD (sodB) was isolated from an A. vinelandii cosmid library. A 1-kb fragment containing the coding region and 400 base pairs of upstream sequence was cloned and sequenced. The nucleotide sequence and the deduced amino acid sequence had a high degree of homology with other bacterial FeSODs, particularly with P. aeruginosa. Attempts to construct a sodB mutant by recombination of a sodB::kan insertion mutation into the multicopy chromosome of A. vinelandii were unsuccessful even in the presence of SOD mimics or nutritional supplements. These results suggest that FeSOD may be essential for the growth and survival of A. vinelandii, and that the periplasmic CuZnSOD cannot replace the function of FeSOD.

Amino Acid Sequence↗

PubMed2002

Cloning, sequencing, and expression of a gene encoding the monomeric isocitrate dehydrogenase of the nitrogen-fixing bacterium, Azotobacter vinelandii.

Isocitrate dehydrogenase (IDH: EC 1.1.1.42) of Azotobacter vinelandii was purified to an electrophoretically homogeneous state, and a gene (icd) encoding this enzyme was cloned and sequenced. The N-terminal amino acid sequence of the purified enzyme was consistent with that deduced from the nucleotide sequence of the icd gene. The deduced amino acid sequence of this gene showed high identity (62-66%) to those of the other bacterial monomeric IDHs. Expression of the icd gene in Escherichia coli was examined by measuring the enzyme activity and mRNA level. Primer extension analyses revealed that two species of mRNAs with different lengths of 5'-untranslated regions (TS-1 and TS-2) were present, of which the 5'-terminals (TS-1 and TS-2 sites) were cytosines located at 244 bp and 101 bp upstream of translational initiation codon, respectively. Conserved promoter elements were present at -35 and -10 regions from the TS-1 site, whereas no such a common motif was found in the upstream region of the TS-2 site. Deletion of the promoter elements upstream of the TS-1 site resulted in complete loss of IDH activity in the E. coli transformant. When the promoter elements upstream of the TS-1 site were intact, the levels of TS-1 and TS-2 were varied greatly by altering exogenous nutrients for growth. The cells grown in a nutrient-rich medium produced large amounts of TS-1 and had a low level of IDH activity. In a nutrient-poor medium, the cells contained large amounts of TS-2 and high levels of IDH activity.

5' Untranslated Regions↗

PubMedDate not supplied

Aerobic and anaerobic microbial degradation of poly-beta-hydroxybutyrate produced by Azotobacter chroococcum.

Food industry wastewater served as a carbon source for the synthesis of poly-beta-hydroxybutyrate (PHB) by Azotobacter chroococcum. The content of polymer in bacterial cells grown on the raw materials reached 75%. PHB films were degraded under aerobic, microaerobic, and anaerobic conditions in the presence and absence of nitrate by microbial populations of soil, sludges from anaerobic and nitrifying/denitrifying reactors, and sediment from a sludge deposit site. Changes in molecular mass, crystallinity, and mechanical properties of PHB were studied. Anaerobic degradation was accompanied by acetate formation, which was the main intermediate utilized by denitrifying bacteria or methanogenic archaea. On a decrease in temperature from 20 to 5 degrees C in the presence of nitrate, the rate of PHB degradation was 7.3 times lower. Under anaerobic conditions and in the absence of nitrate, no PHB degradation was observed, even at 11 degrees C. The enrichment cultures of denitrifying bacteria obtained from soil and anaerobic sludge degraded PHB films for a short time (3-7 d). The dominant species in the enrichment culture from soil were Pseudomonas fluorescens and Pseudomonas stutzeri. The rate of PHB degradation by the enrichment cultures depended on the polymer molecular weight, which reduced with time during biodegradation.

Aerobiosis↗

PubMed2001

A persulfurated cysteine promotes active site reactivity in Azotobacter vinelandii Rhodanese.

Active site reactivity and specificity of RhdA, a thiosulfate:cyanide sulfurtransferase (rhodanese) from Azotobacter vinelandii, have been investigated through ligand binding, site-directed mutagenesis, and X-ray crystallographic techniques, in a combined approach. In native RhdA the active site Cys230 is found persulfurated; fluorescence and sulfurtransferase activity measurements show that phosphate anions interact with Cys230 persulfide sulfur atom and modulate activity. Crystallographic analyses confirm that phosphate and hypophosphite anions react with native RhdA, removing the persulfide sulfur atom from the active site pocket. Considering that RhdA and the catalytic subunit of Cdc25 phosphatases share a common three-dimensional fold as well as active site Cys (catalytic) and Arg residues, two RhdA mutants carrying a single amino acid insertion at the active site loop were designed and their phosphatase activity tested. The crystallographic and functional results reported here show that specific sulfurtransferase or phosphatase activities are strictly related to precise tailoring of the catalytic loop structure in RhdA and Cdc25 phosphatase, respectively.

Azotobacter vinelandii↗

PubMedDate not supplied

Molecular recognition between Azotobacter vinelandii rhodanese and a sulfur acceptor protein.

The occurrence of rhodanese-like proteins in the major evolutionary phyla, together with the observed abundance of these proteins also within the same genome, suggests that their function cannot be limited to cyanide scavenging. The aim of the present study was to investigate whether Azotobacter vinelandii RhdA, an enzyme possessing unique biochemical and structural features with respect to other members of rhodanese homology superfamily, could recognize a suitable protein as a potential acceptor of the sulfane sulfur held on its catalytic Cys residue. Both the potential sulfur-delivery RhdA-S and the sulfur-deprived RhdA were found to interact with either holo- or apo-adrenodoxin, the 'substrate' protein used in this work. Interaction of RhdA-S with apo-adrenodoxin led to mobilization of RhdA-S sulfane sulfur. Under appropriate conditions, the sulfur released from RhdA-S was productively used for 2Fe-2S cluster reconstitution to yield holo-adrenodoxin from apo-adrenodoxin in the absence of any other sulfur source. A comparison of the reactivity of RhdA-S with protein and non-protein thiols allowed also some insights into the accessibility of the sulfane sulfur carried by RhdA.

Adrenodoxin↗

PubMedDate not supplied

Structure elucidation of azotobactin 87, isolated from Azotobacter vinelandii ATCC 12837.

Chromopeptide siderophores (azotobactin 87-I and -II) were isolated from an iron deficient culture medium of Azotobacter vinelandii ATCC 12837 (= DSM 87). Their structures were elucidated by chemical degradation studies and spectroscopic methods, especially 2D-NMR-techniques. Total assignments of 1H-, 13C-, and 15N-resonances based on 2D-HOHAHA-, 1H/13C-HMQC-, 1H/13C-HMBC-, 1H/15N-HMQC/TOCSY-, and 1H/15N-HMBC-experiments are given as well as sequential information derived from 1H/1H-NOESY-, 1H/13C-HMBC- and 1H/15N-HMBC-experiments. Both Az 87-I and Az 87-II consist of a tetracyclic chromophore-- (1S)8,9-dihydroxy-4-oxo-2,3,4,5-tetrahydro-1H,10cH-3a,5,10b- triazaacephenantrylene-1-carboxylic acid--and a decapeptide chain linked with the N-terminus to the carboxy group of the chromophore containing also modified, non-proteinogenic amino acids. The sequence L-Ser-D-Ser-L-Hse-Gly-D-threo-OHAsp-Hse-Hse-Hse-D-N5OH-N5-R- Hbu-Orn-L-Hse was determined for Az 87-I, while Az 87-II contains a C-terminal L-Hse-lactone instead. Iron is chelated by the catecholic group of the chromophore, the beta-hydroxy aspartic acid, and the hydroxamate function formed by N5-hydroxyornithine and R-beta-hydroxybutyric acid.

Amino Acid Sequence↗

PubMed1990

Dissimilation of 2,4-dichlorophenoxyacetic acid by Azotobacter chroococcum.

1. A strain of Azotobacter chroococcum which could use 2,4-dichlorophenoxyacetic acid (2,4-D) as sole carbon source was isolated. 2. The strain metabolized 2,4-D via p-chlorophenoxyacetic acid, p-chlorophenol and 4-chlorocatechol; the last metabolite was cleaved by catechol 1,2-dioxygenase. 3. The enzyme exhibited broad substrate specificity.

2,4-Dichlorophenoxyacetic Acid↗

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↗

PubMed1975

Transcription of bacteriophage deoxyribonucleic acid. Comparison of Escherichia coli and Azotobacter vinelandii sigma subunits.

The effect of the sigma subunit of RNA polymerase on the rate and asymmetry of the in vitro transcription of Escherichia coli and Azotobacter vinelandii phage DNAs has been studied with purified E. coli and A. vinelandii RNA polymerases and hybrid enzymes containing the core subunits of one enzyme and sigma from the other. The effect of sigma on the rate of transcription is characteristic of the template and not of the enzyme and depends on ionic strength. The rate of transcription of A. vinelandii phage A21 DNA is decreased by sigma at high ionic strength, but shows the more characteristic stimulation at KCl concentrations below 0.05 M. In contrast, the stimulation by sigma of T4 DNA transcription increased with an increase in the KCl concentrations. All combinations of core and sigma subunits behaved similarly with respect to stimulation or inhibition by sigma and with respect to asymmetric transcription of S13 replicative form (RF)DNA. However, the heterologous, but not the homologous combinations of core and sigma transcribed A21 symmetrically. S13 RF DNA in the superhelical, but not in the relaxed configuration, is transcribed asymmetrically by the A. vinelandii core enzyme. A role for the core subunits in specific site recognition is indicated by this observation.

Animals↗