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Scientific Reports Jul 2020Bacterial alginate initially consists of 1-4-linked β-D-mannuronic acid residues (M) which can be later epimerized to α-L-guluronic acid (G). The family of AlgE...
Bacterial alginate initially consists of 1-4-linked β-D-mannuronic acid residues (M) which can be later epimerized to α-L-guluronic acid (G). The family of AlgE mannuronan C-5-epimerases from Azotobacter vinelandii has been extensively studied, and three genes putatively encoding AlgE-type epimerases have recently been identified in the genome of Azotobacter chroococcum. The three A. chroococcum genes, here designated AcalgE1, AcalgE2 and AcalgE3, were recombinantly expressed in Escherichia coli and the gene products were partially purified. The catalytic activities of the enzymes were stimulated by the addition of calcium ions in vitro. AcAlgE1 displayed epimerase activity and was able to introduce long G-blocks in the alginate substrate, preferentially by attacking M residues next to pre-existing G residues. AcAlgE2 and AcAlgE3 were found to display lyase activities with a substrate preference toward M-alginate. AcAlgE2 solely accepted M residues in the positions - 1 and + 2 relative to the cleavage site, while AcAlgE3 could accept either M or G residues in these two positions. Both AcAlgE2 and AcAlgE3 were bifunctional and could also catalyze epimerization of M to G. Together, we demonstrate that A. chroococcum encodes three different AlgE-like alginate-modifying enzymes and the biotechnological and biological impact of these findings are discussed.
Topics: Alginates; Amino Acid Sequence; Azotobacter; Azotobacter vinelandii; Bacterial Proteins; Biocatalysis; Carbohydrate Epimerases; Genes, Bacterial; Multigene Family; Sequence Alignment; Substrate Specificity
PubMed: 32719381
DOI: 10.1038/s41598-020-68789-3 -
Journal of Bacteriology May 1953
Topics: Azotobacter; Enzymes; Hydrogenase; Nitrogenase
PubMed: 13069414
DOI: 10.1128/jb.65.5.511-517.1953 -
Journal of Bacteriology Aug 1978Dormant 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...
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.
Topics: Azotobacter; Cell Membrane; Cytoplasmic Granules; Hydroxybutyrates; Vacuoles
PubMed: 681284
DOI: 10.1128/jb.135.2.641-646.1978 -
Journal of Bacteriology Jan 1986Azotobacter vinelandii mutants defective for acetate utilization that were resistant to fluoroacetate (FA) were isolated. FA-resistant mutant AM6 failed to transport...
Azotobacter vinelandii mutants defective for acetate utilization that were resistant to fluoroacetate (FA) were isolated. FA-resistant mutant AM6 failed to transport [14C]acetate and lacked enzymatic activity for both acetate kinase and phosphotransacetylase. Growth of wild-type A. vinelandii was sensitive to 10 mM glycine; however, all FA-resistant strains were resistant to glycine toxicity. Isolated mutants that were spontaneously resistant to glycine were also resistant to FA and lacked both acetate kinase and phosphotransacetylase activity. The glycine-resistant mutant AM3, unlike mutant AM6, was capable of growth on acetate. The mutant strain AM6 was unable to growth under acetate-glucose diauxie conditions. Glucose utilization in this mutant, unlike that in wild-type A. vinelandii, was permanently arrested in the presence of acetate. Revertants of strain AM6 were selected on plates with acetate or acetate-glucose. Two classes of revertants were isolated. Class I revertant mutants AM31 and AM35 were positive for both acetate kinase and phosphotransacetylase activities. These revertants were also sensitive to both FA and glycine. Class II revertant strains AM32 and AM34 still lacked acetate kinase and phophotransacetylase activity. Both of these revertants remained resistant to FA and glycine.
Topics: Acetate Kinase; Acetates; Acetic Acid; Acetyltransferases; Azotobacter; Fluoroacetates; Glucose; Mutation; Oxygen Consumption; Phosphate Acetyltransferase; Phosphotransferases
PubMed: 3001033
DOI: 10.1128/jb.165.1.6-12.1986 -
PloS One 2014Melanins, the ubiquitous hetero-polymer pigments found widely dispersed among various life forms, are usually dark brown/black in colour. Although melanins have variety...
Melanins, the ubiquitous hetero-polymer pigments found widely dispersed among various life forms, are usually dark brown/black in colour. Although melanins have variety of biological functions, including protection against ultraviolet radiation of sunlight and are used in medicine, cosmetics, extraction of melanin from the animal and plant kingdoms is not an easy task. Using complementary physicochemical techniques (i.e. MALDI-TOF, FTIR absorption and cross-polarization magic angle spinning solid-state (13)C NMR), we report here the characterization of melanins extracted from the nitrogen-fixing non-virulent bacterium Azotobacter chroococcum, a safe viable source. Moreover, considering dihydroxyindole moiety as the main constituent, an effort is made to propose the putative molecular structure of the melanin hetero-polymer extracted from the bacterium. Characterization of the melanin obtained from Azotobacter chroococcum would provide an inspiration in extending research activities on these hetero-polymers and their use as protective agent against UV radiation.
Topics: Azotobacter; Melanins; Nitrogen Fixation; Spectrum Analysis
PubMed: 24416247
DOI: 10.1371/journal.pone.0084574 -
Molecular Microbiology Mar 2011Nitrogen fixation by the free-living organism Azotobacter vinelandii can occur through the activity of three different systems that are genetically distinct but...
Nitrogen fixation by the free-living organism Azotobacter vinelandii can occur through the activity of three different systems that are genetically distinct but mechanistically related. A combination of bioinformatic and biochemical-genetic studies has revealed that at least 82 different genes are likely to be associated with the formation and regulation of these systems. Studies performed over many years have established that cross-talk occurs between the various nitrogen fixation systems, and that expression and fine-tuning of their activities are integrated with overall cellular physiology. Martinez-Noel and co-workers now report another newly discovered aspect of the process. Evidence is presented to suggest that a nitrogen fixation-specific paralogue of ClpX is used to control the accumulation of proteins involved in formation of a metal-sulphur cluster that provides a nitrogenase active site. The intriguing aspect of this work is that it indicates that the nitrogen fixation-associated ClpX must recruit ClpP, for which a paralogue is not duplicated within any of the nitrogen fixation regions of the genome, to achieve its function related to nitrogen fixation. Inspection of the A. vinelandii genome indicates that such recruitment of cellular housekeeping components is a common feature used to integrate nitrogen fixation with global cellular physiology.
Topics: Azotobacter vinelandii; Bacterial Proteins; Gene Expression Regulation, Bacterial; Nitrogen Fixation; Nitrogenase
PubMed: 21338415
DOI: 10.1111/j.1365-2958.2011.07541.x -
Proceedings of the National Academy of... Jun 1979Circular dichroism (CD) and magnetic circular dichroism (MCD) spectra of nitrogenase components (MoFe protein and Fe protein) from Azotobacter vinelandii (Av) and...
Circular dichroism (CD) and magnetic circular dichroism (MCD) spectra of nitrogenase components (MoFe protein and Fe protein) from Azotobacter vinelandii (Av) and Klebsiella pneumoniae (Kp) have been obtained in the near infrared-visible-near ultraviolet spectral region. Previously, visible CD was reported to be absent or barely detectable in nitrogenase proteins; MCD spectra have not been reported. The chiroptical spectra can be measured in solution at room temperature, an advantage relative to spectroscopic methods requiring cryogenic sample temperatures. Absorption spectra were also obtained. The CD and MCD are markedly more structured, and thus interpretively more useful, than the corresponding absorption spectra. The dithionite-reduced MoFe proteins (Av1, Kp1) have nearly identical CD and MCD, demonstrating identical numbers and types of metal centers in similar protein environments. The CD and MCD cannot be explained solely in terms of contributions from known 4-Fe or 2-Fe clusters; the near-infrared MCD is inconsistent with the presence of known 4-Fe clusters. CD and MCD spectra of Lauth's violet-oxidized Kp1 are also reported. The reduced Fe proteins (Av2, Kp2) have similar CD and MCD, again indicating significant conservation of chromophore environment. The spectra clearly demonstrate the presence of a reduced bacterial ferredoxin-like (C(3-)) 4-Fe cluster. No obvious evidence of additional chromophores is observed. CD, MCD, and absorption spectra of Av1-oxidized Av2 are reported. The absorption spectrum shows the expected shoulder near 390 nm. The CD and MCD are characteristic of a C(2-) 4-Fe cluster; in particular, the diagnostic near-infrared MCD peak is observed at approximately 8300 cm(-1). The CD of Av2 oxidized in the presence and absence of MgATP are radically different, providing the first direct evidence for MgATP interaction with Fe protein in this oxidation state.
Topics: Azotobacter; Circular Dichroism; Iron; Klebsiella pneumoniae; Magnetics; Molybdenum; Nitrogenase; Oxidation-Reduction; Protein Conformation
PubMed: 379860
DOI: 10.1073/pnas.76.6.2585 -
Journal of Bacteriology Apr 1988Four laboratory strains and two isolates of Azotobacter vinelandii were found to contain plasmids. Twenty-five laboratory strains which could fix nitrogen did not have...
Four laboratory strains and two isolates of Azotobacter vinelandii were found to contain plasmids. Twenty-five laboratory strains which could fix nitrogen did not have free, covalently closed circular plasmid DNA. The plasmids varied in size from 9 to 52 megadaltons, and each strain yielded only one plasmid. No discernible differences in ability to fix nitrogen were found between plasmid-bearing and cured cultures.
Topics: Azotobacter; DNA, Bacterial; DNA, Circular; Genes, Bacterial; Nitrogen Fixation; Plasmids
PubMed: 3350795
DOI: 10.1128/jb.170.4.1984-1985.1988 -
Journal of Bacteriology Sep 2011Most biological nitrogen (N(2)) fixation results from the activity of a molybdenum-dependent nitrogenase, a complex iron-sulfur enzyme found associated with a diversity...
Most biological nitrogen (N(2)) fixation results from the activity of a molybdenum-dependent nitrogenase, a complex iron-sulfur enzyme found associated with a diversity of bacteria and some methanogenic archaea. Azotobacter vinelandii, an obligate aerobe, fixes nitrogen via the oxygen-sensitive Mo nitrogenase but is also able to fix nitrogen through the activities of genetically distinct alternative forms of nitrogenase designated the Vnf and Anf systems when Mo is limiting. The Vnf system appears to replace Mo with V, and the Anf system is thought to contain Fe as the only transition metal within the respective active site metallocofactors. Prior genetic analyses suggest that a number of nif-encoded components are involved in the Vnf and Anf systems. Genome-wide transcription profiling of A. vinelandii cultured under nitrogen-fixing conditions under various metal amendments (e.g., Mo or V) revealed the discrete complement of genes associated with each nitrogenase system and the extent of cross talk between the systems. In addition, changes in transcript levels of genes not directly involved in N(2) fixation provided insight into the integration of central metabolic processes and the oxygen-sensitive process of N(2) fixation in this obligate aerobe. The results underscored significant differences between Mo-dependent and Mo-independent diazotrophic growth that highlight the significant advantages of diazotrophic growth in the presence of Mo.
Topics: Azotobacter vinelandii; DNA, Complementary; Evolution, Molecular; Gene Expression Profiling; Gene Expression Regulation, Bacterial; Genes, Bacterial; Genetic Association Studies; Molybdenum; Nitrogen Fixation; Reverse Transcriptase Polymerase Chain Reaction; Sequence Analysis, DNA
PubMed: 21724999
DOI: 10.1128/JB.05099-11 -
The EMBO Journal Feb 1986The ntrA, ntrB and ntrC products are responsible for regulating the transcription of many genes involved in the assimilation of poor nitrogen sources in enteric...
The ntrA, ntrB and ntrC products are responsible for regulating the transcription of many genes involved in the assimilation of poor nitrogen sources in enteric bacteria. The presence of a similar system in the non-enteric bacterium Azotobacter vinelandii is reported here. Genes analogous to ntrA and ntrC were isolated from an A. vinelandii gene library by complementation of Escherichia coli mutants. The gene encoding glutamine synthetase, glnA, was also isolated and found to be adjacent to ntrC but distant from ntrA, as it is in enteric organisms. The cloned Azotobacter genes also complemented Klebsiella pneumoniae mutants and hybridized to K. pneumoniae ntrA, ntrC and glnA gene probes. The role of ntrA and ntrC in A. vinelandii was established by using Tn5 insertions in the cloned genes to construct mutants by marker exchange. These mutants show that both ntrA and ntrC are required for the utilization of nitrate as a nitrogen source. However, ntrC is not required for nitrogen fixation by A. vinelandii, in contrast with K. pneumoniae where both ntrA and ntrC are essential.
Topics: Azotobacter; Cloning, Molecular; DNA Restriction Enzymes; Escherichia coli; Genes; Genes, Bacterial; Genetic Complementation Test; Genotype; Glutamate-Ammonia Ligase; Klebsiella pneumoniae; Mutation; Nitrogen; Nitrogenase
PubMed: 2872049
DOI: 10.1002/j.1460-2075.1986.tb04225.x