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At least 199 records · Page 11Linked to original sources
PubMed1987

The nucleotide sequence of the sigma factor gene ntrA (rpoN) of Azotobacter vinelandii: analysis of conserved sequences in NtrA proteins.

The nucleotide sequence of the Azotobacter vinelandii ntrA gene has been determined. It encodes a 56916 Dalton acidic polypeptide (AvNtrA) with substantial homology to NtrA from Klebsiella pneumoniae (KpNtrA) and Rhizobium meliloti (RmNtrA). NtrA has been shown to act as a novel RNA polymerase sigma factor but the predicted sequence of AvNtrA substantiates our previous analysis of KpNtrA in showing no substantial homology to other known sigma factors. Alignment of the predicted amino acid sequences of AvNtrA, KpNtrA and RmNtrA identified three regions; two showing greater than 50% homology and an intervening sequence of less than 10% homology. The predicted protein contains a short sequence near the centre with homology to a conserved region in other sigma factors. The C-terminal region contains a region of homology to the beta' subunit of RNA polymerase (RpoC) and two highly conserved regions one of which is significantly homologous to known DNA-binding motifs. In A. vinelandii, ntrA is followed by another open reading frame (ORF) which is highly homologous to a comparable ORF downstream of ntrA in K. pneumoniae and R. meliloti.

Amino Acid Sequence↗
PubMed1987

Tn10 mutagenesis in Azotobacter vinelandii.

The tetracycline-resistant transposon Tn10 and its "high-hopper" derivative Tn10HH104 were introduced into the Azotobacter vinelandii genome using suicide conjugative plasmids derived from pRK2013. Several types of mutants induced by either of these elements are described. Nif- mutants (deficient in nitrogen fixation) were easily isolated, whereas the isolation of other mutant types (auxotrophs, sugar non-users) required special selection conditions. The characterization of the mutations as transposon insertions was often complicated and sometimes required a combination of genetic and physical tests. A common source of complication, the existence of double inserts, was found among the mutants induced by Tn10HH104 but not among those induced by Tn10. Both the high-hopper and the wild-type element proved to undergo secondary transpositions, albeit at different frequencies. Another type of complication, the existence of heterozygotes, occurred because of the high level of redundancy of the A. vinelandii genome.

Azotobacter vinelandii↗
PubMed1984

Cloning and organisation of some genes for nitrogen fixation from Azotobacter chroococcum and their expression in Klebsiella pneumoniae.

By DNA hybridisation, restriction fragments of genomic DNA from Azotobacter chroococcum and A. vinelandii bearing sequences homologous to Klebsiella pneumoniae nitrogenase structural genes were detected. These were different in the two species and inconsistent with the arrangement of the homologous sequences as a contiguous cluster of unique genes. The use of a DNA probe specific for nifH showed that in A. chroococcum two nifH-like sequences were present in the genome. From gene libraries for A. chroococcum, several recombinant cosmid clones bearing nif genes were identified and physically mapped. One copy of the nifH-like sequences was closely linked to nifD and K, the order of genes being as for K. pneumoniae. This cluster was sub-cloned into the broad host-range vector pKT230. The resultant plasmid complemented for C2H2-reduction but not growth in N2 several Nif- mutants of A. vinelandii and K. pneumoniae and also abolished growth in N2 in Nif+ parents. The inhibition was ascribed to a short region adjacent to nifH, which probably corresponds to the promoter as its inhibitory affects were alleviated by provision of K. pneumoniae nifA in multiple copies. 3 sizes of transcripts are produced from the region containing nifH and nifD of A. chroococcum in cultures derepressing for nif. A region bearing homology to a fragment of the K. pneumoniae nif cluster bearing nifV was identified 15 Kb away from nifHDK in A. chroococcum however the order of genes is probably similar to that of K. pneumoniae.

Azotobacter↗
PubMed1988

Characterization of the gene for the Fe-protein of the vanadium dependent alternative nitrogenase of Azotobacter vinelandii and construction of a Tn5 mutant.

A sequence homologous to the conventional nifH gene has been cloned from a different region of the Azotobacter vinelandii genome. Tn5 insertions were obtained in this clone and the mutagenized plasmid was used for marker exchange with A. vinelandii strain CA12 (delta nifHDK) to obtain Tn5 mutants. These mutants exhibited a Nif- phenotype in the presence of vanadium, unlike CA12 which was Nif+ on vanadium-containing medium. The gene in the cloned nifH-like region is therefore apparently involved in the vanadium dependent alternative pathway of nitrogen fixation. This gene, nifH2, has been sequenced and encodes a protein of 289 amino acids that is similar to nifH in nucleotide sequence, deduced amino acid sequence, predicted secondary structure and hydrophobicity profile. A second open reading frame downstream of nifH2 codes for a protein of 64 amino acids, similar to the ferredoxin (Fd)-like protein encoded downstream of nifH* in A. chroococum. Sequence analysis suggests that the nifH2 and Fd-like genes are in a single operon.

Amino Acid Sequence↗
PubMed1995

Analysis of a gene region required for dihydrogen oxidation in Azotobacter vinelandii.

The putative products of six Azotobacter vinelandii chromosomal open reading frames (ORFs) were suggested to be involved in dihydrogen (H2) metabolism [Chen and Mortenson (1992) Biochim Biophys Acta 1131, 199-202]. A promoterless lacZ-containing cassette was used to disrupt the ORFs. Qualitative analysis revealed that the lacZ genes were expressed only in those mutants where the directions of the inserted lacZ were identical to those of the ORFs, showing that the six ORFs were transcribed as predicted. Unlike wildtype (w.t.), none of the mutants could perform dioxygen (O2)-dependent H2-oxidation, even though Western immunoanalyses showed that the hydrogenase large subunit was present although in amounts less than in w.t. Only one of the mutants (a hypB mutant), grown in nickel-enriched media, showed meaningful restoration of the H2-oxidizing ability. From the above observations it is concluded that (a) the six-ORF region is transcriptionally active and involved in H2-oxidation, (b) the product of hypB is needed for nickel activation of hydrogenase, and (c) the six ORFs (genes) belong to two or more operons. Possible roles of the gene products for the assembly, modification, and processing of hydrogenase from its apoproteins and metal centers are discussed.

Azotobacter vinelandii↗
PubMed1980

Adenine nucleotide metabolism in Azotobacter vinelandii. Two metabolic pathways of AMP degradation.

AMP-degrading pathways in Azotobacter vinelandii cells were investigated. AMP nucleosidase (EC 3.2.2.4) was rapidly synthesized and reached a maximum at 24 h, while the activity of 5'-nucleotidase (EC 3.1.3.5) specific for AMP, which was negligible during the logarithmic phase of the growth, first appeared in 24 h-cultures, and reached a maximum after complete exhaustion of sucrose from the growth medium (70 h). Cell-free extracts of A. vinelandii of 48 h-cultures hydrolyzed AMP to ribose 5-phosphate and adenine in the presence of ATP, and adenine was deaminated to hypoxanthine. When ATP was excluded, AMP was dephosphorylated to adenosine, which was further metabolized to inosine, and finally to hypoxanthine. Hypoxanthine thus formed was reutilized for the salvage synthesis of IMP under the conditions where 5-phosphoribosyl 1-pyrophosphate was able to be supplied. These results suggest that the levels of ATP can determine the rate of AMP degradation by the AMP nucleosidase- and 5-'nucleotidase-pathways. The role of ATP in the AMP degradation was discussed in relation to the regulatory properties of AMP nucleosidase, inosine nucleosidase (EC. 3.2.2.2) and adenosine deaminase (EC 3.5.4.4).

Adenine Nucleotides↗
PubMed1980

Adenosine deaminase from Azotobacter vinelandii. Purification and properties.

Adenosine deaminase (EC 3.5.4.4) was found to occur in the extract of Azotobacter vinelandii, strain 0, and purified by heating at 65 degrees C, fractionation with ammonium sulfate, DEAE-cellulose chromatography and gel filtration on Sephadex G-150. Purified adenosine deaminase was effectively stabilized by the addition of ethylene glycol. The molecular weight of the enzyme was estimated to be 66,000 by gel filtration on Sephadex G-150. The enzyme specifically attacked adenosine and 2'-deoxyadenosine to the same extent, and formycin A to a lesser extent. The pH optimum of the enzyme was observed at pH 7.2. Double reciprocal plot of initial velocity versus adenosine concentration was concave upward, and Hill interaction coefficient was calculated to be 1.5, suggesting the allosteric binding of the substrate. ATP inhibited adenosine deaminase in an allosteric manner, whereas other nucleotides were without effect. The physiological significance of the enzyme was discussed in relation to salvage pathway of purine nucleotides.

Adenosine Deaminase↗
PubMed1978

Inosine nucleosidase from Azotobacter vinelandii. Purification and properties.

An enzyme catalyzing the hydrolysis of purine nucleosides was found to occur in the extract of Azotobacter vinelandii, strain O, and was highly purified by ammonium sulfate fractionation, DEAE-cellulose chromatography, hydroxylapatite chromatography and gel filtration on Sephadex G-150. A strict substrate specificity of the purified enzyme was shown with respect to the base components. The enzyme specifically attacked the nucleosides without amino groups in the purine moiety: inosine gave the maximum rate of hydrolysis and xanthosine was hydrolyzed to a lesser extent. The pH optimum of inosine hydrolysis was observed from pH 7 to 9, while xanthosine was hydrolyzed maximally at pH 7. The Km values of the enzyme for inosine were 0.65 and 0.85 mM at pH 7.1 and 9.0, respectively, and the value for xanthosine was 1.2 mM at pH 7.1. Several nucleotides inhibited the enzyme: the phosphate portions of the nucleotides were suggested to be responsible for the inhibition by nucleotides. Although the inhibition of the enzyme by nucleotides was apparently non-competitive type with respect to inosine, allosteric (cooperative) binding of the substrate was suggested in the presence of the inhibitor. The physiological significance of the enzyme was discussed in connection with the degradation and salvage pathways of purine nucleotides.

Azotobacter↗
PubMed1982

A reappraisal of antigenic determinants for nitrogenase from Azotobacter and nitrate reductase from Escherichia coli.

Previous work based on double immunodiffusion assays had shown that there are common antigenic determinants for nitrate reductase from Escherichia coli and component I of nitrogenase from Azotobacter vinelandii. Further work reported herein using a variety of immunoelectrophoretic techniques indicates that the cross-reaction between nitrate reductase and antiserum to component I of nitrogenase results from a contaminant antigen co-purified with nitrate reductase.

Antigens, Bacterial↗
PubMed1983

Localization and activities of nitrogenase, glutamine synthetase and glutamate synthase in Azotobacter vinelandii grown in oxygen-controlled continuous culture.

Azotobacter vinelandii was grown in oxygen-controlled continuous cultures under conditions of dinitrogen fixation. Different oxygen concentrations were adjusted with air. Cell-free extracts were employed to study the oxygen dependency of the intracellular distribution and activity of the following enzymes: nitrogenase, glutamine synthetase and glutamate synthase. Nitrogenase was localized exclusively in the soluble fraction. Its activity increased steeply when the oxygen concentration employed in growing the organism decreased from about 30% close to 0% air saturation. Glutamine synthetase was identified exclusively as a soluble enzyme. The degree of adenylylation of the enzyme increased from about one to about four parallel to nitrogenase activity when the oxygen concentration in the culture was lowered. Glutamate synthase was detected in both a soluble and a membrane-bound form. The sum of specific activities of both forms stayed constant irrespective of changes in the oxygen concentration. However, with increasing oxygen concentration, the proportion of the membrane-bound form increased up to two-thirds of the total activity.

Azotobacter↗
PubMed1975

Molybdenum and iron as functional consitituents of the enzymes of the nitrate-reducing system of Azotobacter chroococcum.

The roles of molybdenum and iron in the enzymes of the assimilatory nitrate-reducing system from Azotobacter chroococcum have been investigated. 1. By adding 99 Mo-molybdate to a cell culture of A. chrocococcum with nitrate as the nitrogen source, it has been possible to incroporate the radioactive metal into a purified preparation of the enzyme nitrate reductase. 2. When 185 W-tungstate was supplied to a culture medium lacking added molybdate, a 185 W-labelled nitrate reductase preparation with negligible activity could be obtained. This in vivo incorporation of tungsten was competitively hindered by molybdenum. 3. The cellular level of nitrite reductase activity gradually increased in response to the addition of increasing amounts of iron to the culture medium. Under the same conditions, of the level of nitrate reductase activity was not affected.

Azotobacter↗
PubMed1985

Reversible and irreversible inactivation of cellular nitrogenase upon oxygen stress in Azotobacter vinelandii growing in oxygen controlled continuous culture.

Azotobacter vinelandii growing in oxygen controlled chemostat culture was subjected to sudden increases of ambient oxygen concentrations (oxygen stress) after adaptation to different oxygen concentrations adjustable with air (100% air saturation corresponds to 225 +/- 14 microM O2). Inactivations of cellular nitrogenase during stress (switch off) as well as after release of stress (switch on) were evaluated in vivo as depending on stress duration and stress height (delta pO2). Switch off was at its final extent within 1 min of stress. The extent of switch off, however, increased with stress height and was complete at delta pO2 between 8-10% air saturation irrespective of different oxygen concentrations the organisms were adapted to before stress, indicating that switch off is adaptable. Inactivation of nitrogenase measureable after switch on represents irreversible loss of activity. Irreversible inactivation was at its characteristic level within less than 3 min of stress and at a delta pO2 of less than 1% air saturation. The level of irreversible inactivation increased linearly with the oxygen concentration the organisms were adapted to before stress. Thus adaptation of cells to increased oxygen concentrations did not prevent increased susceptibility of nitrogenase to irreversible inhibition during oxygen stress. The fast response of irreversible inactivation at low stress heights suggests that it takes place already during stress. Thus switch off comprised both a reversible and an irreversible phase. The data showed that reversible inactivation of nitrogenase was less susceptible to oxygen stress than irreversible inactivation.(ABSTRACT TRUNCATED AT 250 WORDS)

Azotobacter↗
PubMed1975

Ammonium uptake by nitrogen fixing bacteria I. Azotobacter vinelandii.

Both the changes in the activities of nitrogenase, glutamine synthetase and glutamate dehydrogenase and in the extracellular and intracellular NH4+ concentrations were investigated during the transition from an NH4+ free medium to one containing NH4+ ions for a continuous culture of Azotobacter vinelandii. If added in amounts causing 80-100% repression of nitrogenase, ammonium acetate, lactate and phosphate are absorbed completely, whereas chloride, sulfate and citrate are only taken up to about 80%. After about 1-2 hrs the NH4+ remaining in the medium is absorbed too, indicating the induction or activation of a new NH4+ transport system. One of the new permeases allows the uptake of citrate in the presence of sucrose. Addition of inorganic NH4+ level leads to a reversible rise in the glutamine synthetase activity which is not prevented by chloramphenicol, and to a reversible decrease in nitrogenase activity. During these measurements glutamate dehydrogenase activity remains close to zero. The intracellular NH4+ level of about 0.6 mM does not change when extracellular NH4+ is taken up and repression of nitrogenase starts.

Acetates↗
PubMed1982

Effect of O2 limitation on growth and respiration of the wild type and an ascorbate-tetramethyl-p-phenylenediamine-oxidase-negative mutant strain of Azotobacter vinelandii.

Azotobacter vinelandii strain AVOP (wild type) and an ascorbate-N,N,N',N'-tetramethylene-p-phenylenediamine oxidase-negative mutant (AV11) were each grown in O2-limited chemostat cultures. The results showed that the mutant strain grew and used O2 less efficiently than the wild-type strain. Respiration rates of membrane particles with NADH or malate as the substrate were similar for each strain. Succinate oxidase activity was about fourfold lower in membrane particles prepared from mutant than from wild-type strain. Cyanide at a concentration that completely inhibited ascorbate-TMPD oxidase activity resulted in a 50% inhibition of NADH oxidase activity in membrane particles of AVOP. These data suggest that the cytochrome o, a1, oxidase branch of the respiratory chain may be important in the physiology of A. vinelandii under O2-limiting growth conditions.

Azotobacter↗
PubMed1984

Activation studies by phospholipids on the purified cytochrome c4:o oxidase of Azotobacter vinelandii.

A modified procedure is described that was used to solubilize and purify the TMPD-dependent cytochrome c4:o oxidase from Azotobacter vinelandii. Two functional components (Fractions I and V) were obtained after DEAE-cellulose chromatography. Fraction V contained both cytochrome c4 (3.6 nmol/mg protein) and cytochrome o (1.6 nmol/mg protein). This cytochrome oxidase complex oxidized TMPD at "moderate" rates. Fraction I, a clear greenish-yellow fraction, contained primarily phosphatidylethanolamine with some phosphatidylglycerol. Fraction I itself could not oxidize TMPD, but when it was preincubated with Fraction V, a 2-4-fold stimulation in TMPD oxidase activity occurred. Other "authentic" micellar phospholipids also readily activated TMPD oxidase activity in Fraction V. The maximum activation effect obtained with Fraction I was in essence duplicated with purified phosphatidylethanolamine.

Azotobacter↗
PubMed1988

The DNA gyrase inhibitors, nalidixic acid and oxolinic acid, prevent iron-mediated repression of catechol siderophore synthesis in Azotobacter vinelandii.

Low concentrations of nalidixic acid and oxolinic acid that were just inhibitory to Azotobacter vinelandii growth promoted the production of the catechol siderophores azotochelin and aminochelin, in the presence of normally repressive concentrations of Fe3+. There was a limited effect on the pyoverdin siderophore, azotobactin, where low concentrations of Fe3+ were rendered less repressive, but the repression by higher concentrations of Fe3+ was normal. These drugs did not induce high-molecular-mass iron-repressible outer-membrane proteins and similar effects on the regulation of catechol siderophore synthesis were not produced by novobiocin, coumermycin, or ethidium bromide. The timing of nalidixic acid and Fe3+ addition to iron-limited cells was critical. Nalidixic acid had to be added before iron-repression of catechol siderophore synthesis and before the onset of iron-sufficient growth. Continued production of the catechol siderophores, however, was not due to interference with normal iron uptake. These data indicated that nalidixic acid prevented normal iron-repression of catechol siderophore synthesis but could not reverse iron repression once it had occurred. The possible roles of DNA gyrase activity in the regulation of catechol siderophore synthesis is discussed.

Azotobacter↗
PubMed1999

Effect of some herbicides on the production of lysine by Azotobacter chroococcum.

Production of lysine by Azotobacter chroococcum strain H23 was studied in chemically-defined media amended with different concentrations of alachlor, metolachlor, 2,4-D, 2,4,5-T and 2,3,6-TBA. The presence of 5, 10, and 50 micrograms/ml of alachlor or 2,3,6-TBA significantly decreased quantitative production of lysine. However, the presence 2,4-D or 2,4,5-T at concentrations of 10 and 50 micrograms/ml enhanced the production of lysine. Quantitative production of lysine was not affected as consequence of the addition of metolachlor to the culture medium, showing that the release lysine to the culture media by A. chroococcum was not affected by that herbicide.

2,4,5-Trichlorophenoxyacetic Acid↗