-
Applied and Environmental Microbiology Oct 2017Biological nitrogen fixation is accomplished by a diverse group of organisms known as diazotrophs and requires the function of the complex metalloenzyme nitrogenase....
Biological nitrogen fixation is accomplished by a diverse group of organisms known as diazotrophs and requires the function of the complex metalloenzyme nitrogenase. Nitrogenase and many of the accessory proteins required for proper cofactor biosynthesis and incorporation into the enzyme have been characterized, but a complete picture of the reaction mechanism and key cellular changes that accompany biological nitrogen fixation remain to be fully elucidated. Studies have revealed that specific disruptions of the antiactivator-encoding gene result in the deregulation of the transcriptional activator NifA in the nitrogen-fixing bacterium , triggering the production of extracellular ammonium levels approaching 30 mM during the stationary phase of growth. In this work, we have characterized the global patterns of gene expression of this high-ammonium-releasing phenotype. The findings reported here indicated that cultures of this high-ammonium-accumulating strain may experience metal limitation when grown using standard Burk's medium, which could be amended by increasing the molybdenum levels to further increase the ammonium yield. In addition, elevated levels of nitrogenase gene transcription are not accompanied by a corresponding dramatic increase in hydrogenase gene transcription levels or hydrogen uptake rates. Of the three potential electron donor systems for nitrogenase, only the gene cluster showed a transcriptional correlation to the increased yield of ammonium. Our results also highlight several additional genes that may play a role in supporting elevated ammonium production in this aerobic nitrogen-fixing model bacterium. The transcriptional differences found during stationary-phase ammonium accumulation show a strong contrast between the deregulated (-disrupted) and wild-type strains and what was previously reported for the wild-type strain under exponential-phase growth conditions. These results demonstrate that further improvement of the ammonium yield in this nitrogenase-deregulated strain can be obtained by increasing the amount of available molybdenum in the medium. These results also indicate a potential preference for one of two ATP synthases present in as well as a prominent role for the membrane-bound hydrogenase over the soluble hydrogenase in hydrogen gas recycling. These results should inform future studies aimed at elucidating the important features of this phenotype and at maximizing ammonium production by this strain.
Topics: Ammonium Compounds; Azotobacter vinelandii; Bacterial Proteins; Gene Expression Regulation, Bacterial; Hydrogenase; Multigene Family; Nitrogen; Nitrogen Fixation; Nitrogenase
PubMed: 28802272
DOI: 10.1128/AEM.01534-17 -
Journal of Bacteriology Jun 1979The properties of Azotobacter vinelandii deoxyribonucleic acid (DNA) and folded chromosomes were studied and compared to those of Escherichia coli as a standard. Based...
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.
Topics: Azotobacter; Base Sequence; Chromosomes, Bacterial; Cytosine; DNA, Bacterial; Escherichia coli; Guanosine; Hot Temperature; Nucleic Acid Denaturation; Nucleic Acid Renaturation; Species Specificity
PubMed: 378943
DOI: 10.1128/jb.138.3.871-877.1979 -
Molecular Microbiology Mar 2011The major part of biological nitrogen fixation is catalysed by the molybdenum nitrogenase that carries at its active site the iron and molybdenum cofactor (FeMo-co). The...
The major part of biological nitrogen fixation is catalysed by the molybdenum nitrogenase that carries at its active site the iron and molybdenum cofactor (FeMo-co). The nitrogen fixation (nif) genes required for the biosynthesis of FeMo-co are derepressed in the absence of a source of fixed nitrogen. The nifB gene product is remarkable because it assembles NifB-co, a complex cluster proposed to comprise a [6Fe-9S-X] cluster, from simpler [Fe-S] clusters common to other metabolic pathways. NifB-co is a common intermediate of the biosyntheses of the cofactors present in the molybdenum, vanadium and iron nitrogenases. In this work, the expression of the Azotobacter vinelandii nifB gene was uncoupled from its natural nif regulation to show that NifB protein levels are lower in cells growing diazotrophically than in cells growing at the expense of ammonium. A. vinelandii carries a duplicated copy of the ATPase component of the ubiquitous ClpXP protease (ClpX2), which is induced under nitrogen fixing conditions. Inactivation of clpX2 resulted in the accumulation of NifB and NifEN and a defect in diazotrophic growth, especially when iron was in short supply. Mutations in nifE, nifN and nifX or in nifA also affected NifB accumulation, suggesting that NifB susceptibility to degradation might vary during its catalytic cycle.
Topics: Amino Acid Sequence; Azotobacter vinelandii; Bacterial Proteins; Endopeptidase Clp; Gene Expression Regulation, Bacterial; Molecular Sequence Data; Nitrogen; Nitrogen Fixation; Sequence Alignment
PubMed: 21231969
DOI: 10.1111/j.1365-2958.2011.07540.x -
Journal of Bacteriology Sep 1966Sobek, J. M. (University of Southwestern Louisiana, Lafayette), J. F. Charba, and W. N. Foust. Endogenous metabolism of Azotobacter agilis. J. Bacteriol. 92:687-695....
Sobek, J. M. (University of Southwestern Louisiana, Lafayette), J. F. Charba, and W. N. Foust. Endogenous metabolism of Azotobacter agilis. J. Bacteriol. 92:687-695. 1966-Ribonucleic acid, deoxyribonucleic acid, cellular carbohydrate, and the cold trichloroacetic acid and acidic alcohol fractions of the cell do not appear to function as endogenous reserves for Azotobacter agilis. The immediate endogenous reserve of cells grown on glucose, acetate, or succinate was poly-beta-hydroxybutyric acid (PHB). Viability of the cells during starvation was dependent upon the initial levels of PHB and the growth substrate. Cells with high initial PHB levels survived longer than cells with lower levels. Cells from succinate-grown cultures had lower PHB levels than cells from glucose-grown cultures, but were capable of maintaining their viability longer. Cellular protein may also serve as a secondary endogenous reserve substrate for this organism.
Topics: Acetates; Azotobacter; Bacterial Proteins; Butyrates; Carbohydrate Metabolism; Carbon Isotopes; DNA, Bacterial; Glucose; Manometry; RNA, Bacterial; Radiometry; Succinates
PubMed: 5922542
DOI: 10.1128/jb.92.3.687-695.1966 -
Journal of Bacteriology May 1968Methylamine and 2-methylalanine appeared to act as co-repressors of nitrogenase in Azotobacter vinelandii OP. They inhibited the growth of this organism on molecular...
Methylamine and 2-methylalanine appeared to act as co-repressors of nitrogenase in Azotobacter vinelandii OP. They inhibited the growth of this organism on molecular nitrogen but not on nitrate, ammonia, or Casamino Acids; they prevented the formation of nitrogenase by cells transferred from repression to induction conditions; and they did not inhibit the activity of nitrogenase in vitro. A mutant of strain OP, selected on the basis of its relative resistance to methylalanine, appeared partially constitutive because nitrogenase in this strain was less sensitive to repressors than was the enzyme in the wild-type strain.
Topics: Alanine; Amines; Ammonia; Azotobacter; Enzyme Repression; Glucosephosphate Dehydrogenase; Mutation; Nitrogen Fixation; Oxidoreductases
PubMed: 5650079
DOI: 10.1128/jb.95.5.1721-1726.1968 -
Journal of Bacteriology Oct 1969When young cells of Azotobacter vinelandii are impinged on membrane filters, washed free of carbon substrate, and placed on a mineral salts basal medium, the culture...
When young cells of Azotobacter vinelandii are impinged on membrane filters, washed free of carbon substrate, and placed on a mineral salts basal medium, the culture will proceed to encyst although at a slower rate than if n-butanol were supplied as a substrate. The endogenous cysts are depleted in polybeta-hydroxybutyrate and have a narrower intine but show an increased resistance to desiccation and are susceptible to lysis by chelating agents. Membrane-supported cells reveal details of the encystment process such as the formation of a zone within the capsule prior to exine formation and the early deposition of exine structures.
Topics: Azotobacter; Bacteriolysis; Culture Media; Filtration; Freeze Drying; Membranes, Artificial; Microscopy, Electron
PubMed: 5344107
DOI: 10.1128/jb.100.1.475-479.1969 -
European Journal of Biochemistry Apr 1989The gene encoding the dihydrolipoyl transacetylase (E2) component from Azotobacter vinelandii has been cloned in Escherichia coli. High expression of the gene was found...
The gene encoding the dihydrolipoyl transacetylase (E2) component from Azotobacter vinelandii has been cloned in Escherichia coli. High expression of the gene was found when the cells were grown for more than 14 h. The E2 produced was partially active, varying 10 and 90% in different experiments. By limited proteolysis of the protein it was shown that the catalytic domain was incorrectly folded, caused by formation of intermolecular or intramolecular S-S bridges. The enzyme was fully activated after unfolding in 2.5 M guanidine hydrochloride containing 2 mM dithiothreitol, followed by refolding by dialysis. Active E2 was isolated in a simple three-step procedure. It possessed a specific activity in the same order as that found after isolation of E2 from purified pyruvate dehydrogenase complex from A. vinelandii. Active E2 comprises about 7% of the total soluble cellular protein in the E. coli clone. By genetic manipulation, deletion mutants of E2 were created, one encoding the lipoyl domain and the N-terminal half of the pyruvate-dehydrogenase (E1)- and lipoamide-dehydrogenase (E3)-binding domain, the other encoding the catalytic domain and the C-terminal half of the E1- and E3-binding domain. In E. coli expression of both mutants was observed.
Topics: Acetyltransferases; Azotobacter; Chromatography, Gel; Chromatography, Ion Exchange; Dihydrolipoyllysine-Residue Acetyltransferase; Enzyme Activation; Escherichia coli; Genes; Genes, Bacterial; Kinetics; Molecular Weight; Plasmids; Polyethylene Glycols; Pyruvate Dehydrogenase Complex; Recombinant Proteins
PubMed: 2653824
DOI: 10.1111/j.1432-1033.1989.tb14692.x -
Journal of Bacteriology Apr 1988A 3.8-kilobase-pair EcoRI fragment which corrects the mutations carried by the NifB- Azotobacter vinelandii strains CA30 and UW45 was cloned, and its nucleotide sequence...
A 3.8-kilobase-pair EcoRI fragment which corrects the mutations carried by the NifB- Azotobacter vinelandii strains CA30 and UW45 was cloned, and its nucleotide sequence was determined. Four complete open reading frames (ORFs) and two partial ORFs were found. The translation product of the first partial ORF is the carboxy-terminal end of a protein homologous to the nifA gene product from Klebsiella pneumoniae. A 285-base-pair sequence containing a potential nif promoter and nif regulatory sites separates this nifA gene from the first complete ORF which encodes a protein homologous to nifB gene products from K. pneumoniae and Rhizobium species. The Tn5 insertion in strain CA30 and the nif-45 mutation of strain UW45 are located within this nifB gene. The ORF downstream from nifB predicts an amino acid sequence with a cysteine residue pattern that is characteristic of ferredoxins. No similarities were found between the translation product of the third complete ORF and those of nif genes from other organisms. At the carboxy-terminal end of the predicted translation product of the fourth complete ORF, 30 of 60 amino acid residues were identical with the sequence of the nifQ gene product from K. pneumoniae. The partial ORF located at the end of the fragment encodes the N-terminal part of a potential protein with an unknown function. Northern (RNA) blot analysis indicated that transcripts from the region containing the four complete ORFs were NH4+ repressible and that the transcription products were identical in cells derepressed under conditions of Mo sufficiency or Mo deficiency or in the presence of vanadium. In contrast to the NifB- strain CA30, which is Nif- under all conditions, mutants that carry mutations affecting the C-terminal end of nifB or genes located immediately downstream from nifB, grew under all N2-fixing conditions. However, in the presence of Mo, most of the strains required 1,000 times the amount of molybdate that is sufficient for maximal growth of the wild-type strain CA under N2-fixing conditions. Growth data from strain CA37, which carries a Kanr insertion in nifQ, indicate that nifQ in A. vinelandii is not required for N2 fixation in the presence of V2O5 or under Mo-deficient conditions. Growth studies and acetylene reduction assays performed on two nifEN deletion strains showed that nifE and nifN are required for N2 fixation under Mo sufficiency, as previously observed (K. E. Brigle, M. C. Weiss, W. E. Newton, and D. R. Dean, J. Bacteriol. 169:1547-1553, 1987), but not under conditions of Mo deficiency or in the presence of 50 nM V2O5.
Topics: Amino Acid Sequence; Azotobacter; Base Sequence; Cloning, Molecular; DNA Restriction Enzymes; Deoxyribonuclease EcoRI; Genes, Bacterial; Molecular Sequence Data; Mutation; Nitrogen Fixation; Nitrogenase; Nucleic Acid Hybridization; Plasmids; Protein Biosynthesis; RNA, Bacterial; Sequence Homology, Nucleic Acid; Transcription, Genetic
PubMed: 2450865
DOI: 10.1128/jb.170.4.1475-1487.1988 -
Journal of Bacteriology Dec 1991The nucleotide sequence (3,600 bp) of a second copy of nifENX-like genes in Azotobacter vinelandii has been determined. These genes are located immediately downstream... (Comparative Study)
Comparative Study
The nucleotide sequence (3,600 bp) of a second copy of nifENX-like genes in Azotobacter vinelandii has been determined. These genes are located immediately downstream from vnfA and have been designated vnfENX. The vnfENX genes appear to be organized as a single transcriptional unit that is preceded by a potential RpoN-dependent promoter. While the nifEN genes are thought to be evolutionarily related to nifDK, the vnfEN genes appear to be more closely related to nifEN than to either nifDK, vnfDK, or anfDK. Mutant strains (CA47 and CA48) carrying insertions in vnfE and vnfN, respectively, are able to grow diazotrophically in molybdenum (Mo)-deficient medium containing vanadium (V) (Vnf+) and in medium lacking both Mo and V (Anf+). However, a double mutant (strain DJ42.48) which contains a nifEN deletion and an insertion in vnfE is unable to grow diazotrophically in Mo-sufficient medium or in Mo-deficient medium with or without V. This suggests that NifE and NifN substitute for VnfE and VnfN when the vnfEN genes are mutationally inactivated. AnfA is not required for the expression of a vnfN-lacZ transcriptional fusion, even though this fusion is expressed under Mo- and V-deficient diazotrophic growth conditions.
Topics: Amino Acid Sequence; Azotobacter vinelandii; Base Sequence; Cloning, Molecular; Escherichia coli; Genes, Bacterial; Genotype; Molecular Sequence Data; Mutagenesis, Insertional; Nitrogen Fixation; Nitrogenase; Plasmids; Protein Biosynthesis; Sequence Homology, Nucleic Acid; Transcription, Genetic
PubMed: 1938952
DOI: 10.1128/jb.173.23.7565-7572.1991 -
European Journal of Biochemistry Nov 1981
A 13C nuclear-magnetic-resonance study on free flavins and Megasphaera elsdenii and Azotobacter vinelandii flavodoxin. 13C-enriched flavins as probes for the study of flavoprotein active sites.
Topics: Azotobacter; Binding Sites; Flavin Mononucleotide; Flavins; Flavodoxin; Flavoproteins; Hydrogen-Ion Concentration; Magnetic Resonance Spectroscopy; Oxidation-Reduction; Riboflavin; Veillonellaceae
PubMed: 7308219
DOI: 10.1111/j.1432-1033.1981.tb05666.x