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PubMed1979

Covalently bound non-coenzyme phosphorus residues in flavoproteins: 31P nuclear magnetic resonance studies of Azotobacter flavodoxin.

In addition to the 5'-phosphate ester on its flavin mononucleotide (FMN) moiety, flavodoxin from Azotobacter vinelandii contains 2 moles of tightly bound phosphate. One non-coenzyme phosphate group is covalently bound to the protein, as it remains with the protein on acid precipitation, whereas the other phosphate is released. The invariance of the (31)P nuclear magnetic resonance chemical shift of the covalently bound phosphate (-0.8 ppm relative to 85% phosphoric acid) with pH, even in the presence of protein denaturants, implies it is in a diester linkage to the protein. Because no evidence could be found for the presence of covalently bound sugars, nucleotides, or phospholipids, it is suggested that the phosphate residue forms a diester linkage with two hydroxyl amino acids in the protein. The only other suggestion of a phosphodiester linkage in proteins is from previous studies on pepsin and pepsinogen [Perlmann, G. E. (1955) Adv. Prot. Chem. 10, 1-30]. The observed changes in (31)P chemical shift with pH show that the covalent phosphorus in pepsinogen has ionization properties of a monoester rather than a diester. The (31)P resonance of the FMN phosphate occurs at -5.6 ppm in native Azotobacter flavodoxin. No ionization of the protein-bound FMN phosphate is observed since the chemical shift does not change appreciably in the pH range of 5.5-9.5. The chemical shift data suggest, but do not prove, that the coenzyme phosphate in its protein-bound form is dianionic. Chemical analysis of several other flavoenzymes from a variety of sources shows the presence of covalently bound phosphorus in quantities stoichiometric with the flavin content in most of the enzymes tested. Thus, the presence of covalent phosphorus in flavoenzymes may be a general phenomenon with currently unknown catalytic significance.

Apoproteins↗

PubMed1990

Molecular cloning and expression of a novel catechol 2,3-dioxygenase gene from the benzoate meta-cleavage pathway in Azotobacter vinelandii.

Azotobacter vinelandii strain 206 degrades benzoate via the meta-cleavage pathway. In a genomic library derived from this organism a clone was obtained which carried and expressed the gene for the third enzyme in this pathway, catechol 2,3-dioxygenase (EC 1.13.11.2), on a 5.9 kb SalI restriction fragment. The structural gene was more precisely mapped on an internal 1.6 kb EcoRI fragment which, after insertion into expression vectors, directed the synthesis of a 33 kDa polypeptide. The gene showed very little or no homology with isofunctional genes derived from Pseudomonas. Comprehensive substrate specificity analysis showed significant differences between the specific activities obtained from the cloned gene product and extracts derived from Azotobacter itself.

Azotobacter↗

PubMed1994

The FeSII protein of Azotobacter vinelandii is not essential for aerobic nitrogen fixation, but confers significant protection to oxygen-mediated inactivation of nitrogenase in vitro and in vivo.

The FeSII protein of Azotobacter vinelandii has been proposed to mediate the 'conformational protection' of the molybdenum-dependent nitrogenase components against oxygen inactivation. We have cloned and characterized the structural gene for the FeSII protein (the fesII locus). Hybridization studies did not reveal the presence of fesII-like genes in a number of diverse species of well-studied nitrogen-fixing bacteria, with the exception of Azotobacter chroococcum. The fesII locus is transcriptionally expressed during both nitrogen fixing and non-nitrogen fixing conditions, although the level of its message is upregulated by approximately 2.5-fold during nitrogen fixation. The promoter region was identified by primer extension analysis, and is similar to other sigma 70-type promoters. Mutants devoid of the FeSII protein were constructed. These mutants possessed growth characteristics on a variety of carbon substrates during non-diazotrophic as well as diazotrophic growth that were essentially indistinguishable from the wild-type strain. Nevertheless, the nitrogenase activity in cell-free extracts is significantly more sensitive to irreversible oxygen inactivation in the mutants as compared with the wild type. When treated with 250 mM NaCl (a condition known to dissociate FeSII from nitrogenase components), the wild-type and mutant extracts were equally hypersensitive to oxygen inactivation. Upon energy starvation, conditions in which 'respiratory protection' is inoperable, the MoFe and Fe proteins of nitrogenase are degraded much more rapidly in vivo in the deletion mutants, compared to the wild type. Strains relying on either the vanadium or the 'iron-only' alternative nitrogenases exhibited similar growth rates irrespective of the presence or absence of the FeSII protein, and the in vitro inactivation of the vanadium nitrogenase components was not affected by the lack of the FeSII protein. All in all, these results are consistent with a model whereby 'respiratory protection' is the major physiological mechanism responsible for the protection of all three nitrogenases during energy-supplemented growth. Upon energy starvation, however, 'conformational protection', mediated by the FeSII protein is capable of temporarily protecting the conventional molybdenum nitrogenase components from inactivation and subsequent degradation.

Aerobiosis↗

PubMed1978

Effect of high pressure, detergents and phospholipase on the break in the Arrhenius plot of Azotobacter nitrogenase.

It is shown that lipids are responsible for the breaks in the Arrhenius plots of Azotobacter nitrogenase. The physical evidence is that temperature at which the break occurs increases with increasing pressure by 20 K/1000 atm. This is in agreement with the pressure dependence of the transition temperature of several synthetic phospholipids. We also find the same pressure dependence for the broad transitions observed in Escherichia coli phosphatidylethanolamine and in the membrane lipids from Azotobacter itself. Detergents and phospholipase remove the break. Reconstruction can be performed only with specific phospholipids.

Azotobacter↗

PubMed1986

Characterization of three different flavodoxins from Azotobacter vinelandii.

The flavodoxins from Azotobacter vinelandii cells grown N2-fixing and from cells grown on NH4OAc have been purified and characterized. The purified flavodoxins from these cells are a mixture of three different flavodoxins (Fld I, II, III) with different primary structures. The three proteins were separated by fast protein liquid chromatography; Fld I eluted at 0.38 M KCl, Fld II at 0.43 M KCl and Fld III at 0.45 M KCl. The most striking difference between the three flavodoxins was the midpoint potential (pH 7.0, 25 degrees C) of the semiquinone/hydroquinone couple, which was -320 mV for Fld I and -500 mV for the other two flavodoxins (Fld II and Fld III). All three flavodoxins were present in cells grown on NH4OAc. In cells grown on N2 as N source only Fld I and Fld II were found. The concentration of Fld II was 10-fold higher in N2-fixing cells than in cells grown on NH4OAc. Evidence has been obtained that Fld II is involved in electron transport to nitrogenase. As will be discussed, our observation that preparations of Azotobacter flavodoxin are heterogeneous, has consequences for the published data.

Azotobacter↗

PubMed1988

Lipoamide dehydrogenase from Azotobacter vinelandii. Molecular cloning, organization and sequence analysis of the gene.

The gene encoding lipoamide dehydrogenase from Azotobacter vinelandii has been cloned in Escherichia coli. Fragments of 9-23 kb from Azotobacter vinelandii chromosomal DNA obtained by partial digestion with Sau3A were ligated into the BamHI site of plasmid pUC9. E. coli TG2 cells were transformed with the resulting recombinant plasmids. Screening for clones which produced A. vinelandii lipoamide dehydrogenase was performed with antibodies raised against the purified enzyme. A positive colony was found which produced complete chains of lipoamide dehydrogenase as concluded form SDS gel electrophoresis of the cell-free extract, stained for protein or used for Western blotting. After subcloning of the 14.7-kb insert of this plasmid the structural gene could be located on a 3.2-kb DNA fragment. The nucleotide sequence of this subcloned fragment (3134 bp) has been determined. The protein-coding sequence of the gene consists of 1434 bp (478 codons, including the AUG start codon and the UAA stop codon). It is preceded by an intracistronic region of 85 bp and the structural gene for succinyltransferase. A putative ribosome-binding site and promoter sequence are given. The derived amino acid composition is in excellent agreement with that previously published for the isolated enzyme. The predicted relative molecular mass is 50223, including the FAD. The overall homology with the E. coli enzyme is high with 40% conserved amino acid residues. From a comparison with the three-dimensional structure of the related enzyme glutathione reductase [Rice, D. W., Schultz, G. E. & Guest, J. R. (1984) J. Mol. Biol. 174, 483-496], it appears that essential residues in all four domains have been conserved. The enzyme is strongly expressed, although expression does not depend on the vector-encoded lacZ promoter. The cloned enzyme is, in all the respects tested, identical with the native enzyme.

Amino Acid Sequence↗

PubMed1992

A reinvestigation of the pre-steady-state ATPase activity of the nitrogenase from Azotobacter vinelandii.

The pre-steady-state ATPase activity of nitrogenase has been reinvestigated. The exceptionally high burst in the hydrolysis of MgATP by the nitrogenase from Azotobacter vinelandii communicated by Cordewener et al. (1987) [Cordewener J., ten Asbroek A., Wassink H., Eady R. R., Haaker H. & Veeger C. (1987) Eur. J. Biochem. 162, 265-270] was found to be caused by an apparatus artefact. A second possible artefact in the determination of the stoichiometry of the pre-steady-state ATPase activity of nitrogenase was observed. Acid-quenched mixtures of dithionite-reduced MoFe or Fe protein of Azotobacter vinelandii nitrogenase and MgATP contained phosphate above the background level. It is proposed that due to this reaction, quenched reaction mixtures of nitrogenase and MgATP may contain phosphate in addition to the phosphate released by the ATPase activity of the nitrogenase complex. It was feasible to monitor MgATP-dependent pre-steady-state proton production by the absorbance change at 572 nm of the pH indicator o-cresolsulfonaphthalein in a weakly buffered solution. At 5.6 degrees C, a pre-steady-state phase of H+ production was observed, with a first-order rate constant of 2.2 s-1, whereas electron transfer occurred with a first-order rate constant of 4.9 s-1. At 20.0 degrees C, MgATP-dependent H+ production and electron transfer in the pre-steady-state phase were characterized by observed rate constants of 9.4 s-1 and 104 s-1, respectively. The stopped-flow technique failed to detect a burst in the release of protons by the dye-oxidized nitrogenase complex. It is concluded that the hydrolysis rate of MgATP, as judged by proton release, is lower than the rate of electron transfer from the Fe protein to the MoFe protein.

Adenosine Triphosphatases↗

PubMed2005

Utilization of the metal-cyano complex tetracyanonickelate (II) by Azotobacter vinelandii.

AIMS: The ability of Azotobacter vinelandii, a N(2)-fixing bacterium, to biodegrade tetracyanonickelate (TCN) was evaluated. METHODS AND RESULTS: The amounts of TCN were measured spectrophotometrically. Ammonia was determined colorimetrically by the indophenol method. The produced methane from TCN conversion by A. vinelandii was detected by gas chromatography. Results showed that A. vinelandii was able to biodegrade 1 mmol l(-1) of TCN. Ammonia and methane were detected during the process of TCN degradation. Effects of exogenous nitrogen sources on TCN degradation were addressed in this study. Results revealed that the addition of ammonia (1, 5 and 10 mmol l(-1)) into the reaction mixtures caused decrease of TCN degradation rate during a 24-h incubation period. This inhibition was also observed when nitrite (5 and 10 mmol l(-1)) was added, whereas TCN degradation still proceeded after the addition of nitrate at the same concentrations. Furthermore, the rate of TCN utilization was strikingly enhanced when 0.8% of glucose was added. CONCLUSIONS: Azotobacter vinelandii can degrade 1 mmol l(-1) of TCN into ammonia and methane. However, the inhibitory effects of exogenous ammonia and nitrite on TCN degradation by this bacterium were found in this study. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report defining the capability of A. vinelandii to degrade TCN. This bacterium might have potential value in applied strategies for removing metal-cyano wastes. Furthermore, these findings would be helpful in designing a practical system inoculated with A. vinelandii for the treatment of TCN.

Ammonia↗

PubMed1993

Differences in sensitivity to NADH of purified pyruvate dehydrogenase complexes of Enterococcus faecalis, Lactococcus lactis, Azotobacter vinelandii and Escherichia coli: implications for their activity in vivo.

The effect of NADH on the activity of the purified pyruvate dehydrogenase complexes (PDHc) of Enterococcus (Ec.) faecalis, Lactococcus lactis, Azotobacter vinelandii and Escherichia coli was determined in vitro. It was found that the PDHc of E. coli and L. lactis was active only at relatively low NADH/NAD ratios, whereas the PDHc of Ec. faecalis was inhibited only at high NADH/NAD ratios. The PDHc of Azotobacter vinelandii showed an intermediate sensitivity. The organisms were grown in chemostat culture under conditions that led to different intracellular NADH/NAD ratios and the PDHc activities in vivo could be calculated from the specific rates of product formation. Under anaerobic growth conditions, only Ec. faecalis expressed PDHc activity in vivo. The activities in vivo of the complexes of the different organisms were in good agreement with their properties determined in vitro. The physiological consequences of these results are discussed.

Aerobiosis↗

PubMed2003

Iron-regulated phenylalanyl-tRNA synthetase activity in Azotobacter vinelandii.

Azotobacter vinelandii strain UA22 was produced by pTn5luxAB mutagenesis, such that the promoterless luxAB genes were transcribed in an iron-repressible manner. Tn5luxAB was localized to a fragment of chromosomal DNA encoding the thrS, infC, rpmI, rplT, pheS and pheT genes, with Tn5 inserted in the 3'-end of pheS. The isolation of this mutation in an essential gene was possible because of polyploidy in Azotobacter, such that strain UA22 carried both wild-type and mutant alleles of pheS. Phenylalanyl-tRNA synthetase activity and PHES::luxAB reporter activity was partially repressed under iron-sufficient conditions and fully derepressed under iron-limited conditions. The ferric uptake regulator (Fur) bound to a DNA sequence immediately upstream of luxAB, within the pheS gene, but PHES::luxAB reporter activity was not affected by phenylalanine availability. This suggests there is novel regulation of pheST in A. vinelandii by iron availability.

Azotobacter vinelandii↗

PubMed1968

Effect of ion concentration on the rupture and survival of Azotobacter cysts.

The ability of ethylenediaminetetraacetic acid (EDTA) and dipicolinic acid (DPA) to rupture Azotobacter cysts was directly related to the molarity of the reaction mixture. Cysts were not ruptured at concentrations above 0.15 m (NaCl or phosphate). These higher salt concentrations prevented the formation of metallo:EDTA (or DPA) chelate complexes. Sudden shifts in the tonicity of NaCl or phosphate mixtures induced cyst rupture in the absence of EDTA. The effect of these mechanisms on the survival and germination of Azotobacter cysts in the soil is discussed.

Azotobacter↗

PubMed1975

Tetramethyl-p-phenylenediamine oxidase reaction in Azotobacter vinelandii.

It was possible to quantitate the tetramethyl-p-phenylenediamine (TMPD) oxidase reaction in Azotobacter vinelandii strain O using turbidimetrically standarized resting cell suspensions. The Q(O2) value obtained for whole cell oxidation of ascorbate-TMPD appeared to reflect the full measure of the high respiratory oxidative capability usually exhibited by this genera of organisms. The Q(O2) value for the TMPD oxidase reaction ranged from 1,700 to 2,000 and this value was equivalent to that obtained for the oxidation of the growth substrate, e.g., acetate. The kinetic analyses for TMPD oxidation by whole cells was similar to that obtained for the "particulate" A. vinelandii electron transport particle, that fraction which TMPD oxidase activity is exclusively associated with. Under the conditions used, there appeared to be no permeability problems; TMPD (reduced by ascorbate) readily penetrated the cell and oxidized at a rate comparable to that of the growth substrate. This, however, was not true for the oxidation of another electron donor, 2,6-dichloroindophenol, whose whole cell Q(O2) values, under comparable conditions, were twofold lower. The TMPD oxidase activity in A. vinelandii whole cells was found to be affected by the physiological growth conditions, and resting cells obtained from cells grown on sucrose, either under nitrogen-fixing conditions or on nitrate as the combined nitrogen source, exhibited low TMPD oxidase rates. Such low TMPD oxidase rates were also noted for chemically induced pleomorphic A. vinelandii cells, which suggests that modified growth conditions can (i) alter the nature of the intracellular terminal oxidase formed (or induced), or (ii) alter surface permeability, depending upon the growth conditions used. Preliminary studies on the quantitative TMPD oxidation reaction in mutant whole cells of both Azotobacter and a well-known Mucor bacilliformis strain AY1, deficient in cytochrome oxidase activity, showed this assay can be very useful for detecting respiratory deficiencies in the metabolism of whole cells.

2,6-Dichloroindophenol↗

PubMed1970

Correlation of ultrastructure in Azotobacter vinelandii with nitrogen source for growth.

Azotobacter synthesizes an extensive internal membranous nework when grown with air (N(2)), i.e., under conditions when these bacteria fix nitrogen. Very slight quantities of internal membrane, concentrated mainly about the cell periphery, are formed when Azotobacter grows with fixed nitrogen, i.e., ammonia and amino acids. Compared to cells growing with ammonia, cells utilizing atmospheric nitrogen as the sole nitrogen source are smaller in size and volume, grow one-third slower, and lack detectable poly-beta-hydroxybutyrate.

Air↗

PubMed1970

Properties of a soluble nitrogenase in Azotobacter.

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.

Acetylene↗

PubMed1985

Transformation of Azotobacter vinelandii with plasmid DNA.

Azotobacter vinelandii cells can be transformed at high frequencies with the broad-host-range plasmids pRK2501, RSF1010, and pGSS15, using a modification of the procedure developed by Page and von Tigerstrom (J. Bacteriol. 139:1058-1061, 1979) for chromosomal DNA-mediated transformation. The frequency of transformation per microgram of plasmid DNA per viable cell with pRK2501 and pGSS15 was about 5 X 10(-2) and 2 X 10(-2), respectively. With RSF1010, transformation frequencies ranged from 3 X 10(-4) to 4 X 10(-2). With each plasmid, the frequency of transformation was independent of the phase of the growth cycle. When concentrations of pRK2501 ranging from 0.1 to 51 micrograms of DNA were tested, the frequency of transformation was directly proportional to the amount of DNA. This linear response indicated that, although the uptake of plasmid DNA with this procedure may be inefficient, there is a high probability that once inside a cell the plasmid will be stably maintained. Cells that have been transformed with pRK2501 did not grow well on transforming medium which lacks iron and contains fixed nitrogen. However, on growth medium which contains iron and lacks fixed nitrogen, transformants produced distinctive colonies larger than those of nontransformed cells. Resistance to kanamycin due to transformation by pRK2501 was stably maintained for at least 10 successive generations in the absence of selective pressure. The present protocol should facilitate the molecular cloning of genes in Azotobacter spp.

Azotobacter↗

PubMed1985

Lesions in citrate synthase that affect aerobic nitrogen fixation by Azotobacter chroococcum.

A class of Azotobacter chroococcum mutants induced by Tn1 that were defective in normal aerobic nitrogen fixation when grown on sugars (Fos-) were corrected by provision of alpha-ketoglutarate or glutamate. In a representative mutant, Fos252, rates of evolution of 14CO2 from [14C]acetate or [14C]glucose were 5% of the parental values, although uptake and incorporation were normal for both substrates. The results suggest that a lesion affects the entry of substrates into the tricarboxylic acid cycle. The activity of citrate synthase in Fos252 in vitro was 5% that of the parents. The citrate synthase (gltA) gene from Escherichia coli was cloned into broad-host-range vectors and mobilized into Fos252. The plasmids restored parental citrate synthase activities to Fos252 and complemented the inability to fix N2 in air. The data indicate that a mutation causing an intrinsic limitation in respiratory capacity abolishes normal aerobic N2 fixation, which is consistent with the hypothesis of respiratory protection for nitrogenase in Azotobacter species.

Azotobacter↗

PubMed1989

Nucleotide sequence and mutational analysis of the structural genes (anfHDGK) for the second alternative nitrogenase from Azotobacter vinelandii.

The nucleotide sequence of a region of the Azotobacter vinelandii genome exhibiting sequence similarity to nifH has been determined. The order of open reading frames within this 6.1-kilobase-pair region was found to be anfH (alternative nitrogen fixation, nifH-like gene), anfD (nifD-like gene), anfG (potentially encoding a protein similar to the product of vnfG from Azotobacter chroococcum), anfK (nifK-like gene), followed by two additional open reading frames. The 5'-flanking region of anfH contains a nif promoter similar to that found in the A. vinelandii nifHDK gene cluster. The presumed products of anfH, anfD, and anfK are similar in predicted Mr and pI to the previously described subunits of nitrogenase 3. Deletion plus insertion mutations introduced into the anfHDGK region of wild-type strain A. vinelandii CA resulted in mutant strains that were unable to grow in Mo-deficient, N-free medium but grew in the presence of 1 microM Na2MoO4 or V2O5. Introduction of the same mutations into the nifHDK deletion strain CA11 resulted in strains that grew under diazotrophic conditions only in the presence of vanadium. The lack of nitrogenase 3 subunits in these mutant strains was demonstrated through two-dimensional gel analysis of protein extracts from cells derepressed for nitrogenase under Mo and V deficiency. These results indicate that anfH, anfD, and anfK encode structural proteins for nitrogenase 3.

Amino Acid Sequence↗

PubMed1991

Nucleotide sequence and genetic analysis of the Azotobacter chroococcum nifUSVWZM gene cluster, including a new gene (nifP) which encodes a serine acetyltransferase.

Nucleotide sequence was obtained for a region of 7,099 bp spanning the nifU, nifS, nifV, nifW, nifZ, and nifM genes from Azotobacter chroococcum. Chromosomal mutations constructed at several sites within the locus confirmed a requirement for this region for expression of the molybdenum nitrogenase in this organism. The genes are tightly clustered and ordered as in Klebsiella pneumoniae except for two additional open reading frames (ORFs) between nifV and nifW. The arrangement of genes in A. chroococcum closely matches that described for Azotobacter vinelandii. The polypeptide encoded by ORF4 immediately downstream from nifV is 41% identical over 186 amino acids to the product of the cysE gene from Escherichia coli, which encodes serine acetyltransferase (SAT), a key enzyme in cysteine biosynthesis. Plasmids which potentially express ORF4 complemented E. coli JM39, a cysteine auxotroph which lacks SAT. SAT activity was detected in crude extracts of one such complemented strain. A strain of A. chroococcum carrying a chromosomal disruption of ORF4 grew normally with ammonium as the N source but more slowly than the parental strain when N2 was the sole N source. These data suggest that ORF4 encodes a nif-specific SAT required for optimizing expression of nitrogenase activity. ORF4 was assigned the name nifP. nifP may be required to boost rates of synthesis or intracellular concentrations of cysteine or methionine. Sequence identity between nifV and leuA gene products suggests that nifV may catalyze a condensation reaction analogous to that carried out by isopropylmalate synthase (LEUA) but in which acetyl coenzyme and alpha-ketoglutarate are substrates for the formation of homocitrate, the proposed product of NIFV activity.

Acetyltransferases↗