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

2001 ◽  
Vol 47 (1) ◽  
pp. 63-71 ◽  
Author(s):  
Barbara A Qurollo ◽  
Paul E Bishop ◽  
Hosni M Hassan
Keyword(s):  
Stationary Phase ◽  
Azotobacter Vinelandii ◽  
Cosmid Library ◽  
Insertion Mutation ◽  
Upstream Sequence ◽  
Nitrogen Fixing ◽  
Coding Region ◽  
Gene Encoding ◽  
Growth And Survival ◽  
Superoxide Dismutase Gene

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.

scholarly journals Molecular Cloning and Nucleotide Sequence of the Superoxide Dismutase Gene and Characterization of Its Product fromBacillus subtilis

1998 ◽  
Vol 180 (14) ◽  
pp. 3697-3703 ◽  
Author(s):  
Takashi Inaoka ◽  
Yoshinobu Matsumura ◽  
Tetsuaki Tsuchido
Keyword(s):  
Superoxide Dismutase ◽  
Stationary Phase ◽  
Specific Activity ◽  
Upstream Region ◽  
Gene Encoding ◽  
Sod Activity ◽  
Vegetative Cells ◽  
Reading Frame ◽  
Superoxide Dismutase Gene

ABSTRACT Bacillus subtilis was found to possess one detectable superoxide dismutase (Sod) in both vegetative cells and spores. The Sod activity in vegetative cells was maximal at stationary phase. Manganese was necessary to sustain Sod activity at stationary phase, but paraquat, a superoxide generator, did not induce the expression of Sod. The specific activity of purified Sod was approximately 2,600 U/mg of protein, and the enzyme was a homodimer protein with a molecular mass of approximately 25,000 per monomer. The gene encoding Sod, designatedsodA, was cloned by the combination of several PCR methods and the Southern hybridization method. DNA sequence analysis revealed the presence of one open reading frame consisting of 606 bp. Several putative promoter sites were located in the upstream region ofsodA. The deduced amino acid sequence showed high homology with other bacterial manganese Sods. Conserved regions in bacterial manganese Sod could also be seen. The phenotype of double mutantEscherichia coli sodA sodB, which could not grow in minimal medium without supplemental amino acids, was complemented by the expression of B. subtilis sodA.

scholarly journals Transcriptional Analysis of an Ammonium-Excreting Strain of Azotobacter vinelandii Deregulated for Nitrogen Fixation

2017 ◽  
Vol 83 (20) ◽  
Author(s):  
Brett M. Barney ◽  
Mary H. Plunkett ◽  
Velmurugan Natarajan ◽  
Florence Mus ◽  
Carolann M. Knutson ◽  
...  
Keyword(s):  
Nitrogen Fixation ◽  
Stationary Phase ◽  
Gene Transcription ◽  
Biological Nitrogen Fixation ◽  
Azotobacter Vinelandii ◽  
Nitrogen Fixing ◽  
Wild Type ◽  
Content Type ◽  
Ammonium Production ◽  
Biological Nitrogen

ABSTRACT 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 nifL result in the deregulation of the nif transcriptional activator NifA in the nitrogen-fixing bacterium Azotobacter vinelandii, 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 rnf1 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. IMPORTANCE The transcriptional differences found during stationary-phase ammonium accumulation show a strong contrast between the deregulated (nifL-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 A. vinelandii 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.

Clostridium pasteurianum W5 synthesizes two NifH-related polypeptides under nitrogen-fixing conditions

Microbiology ◽  
2005 ◽  
Vol 151 (7) ◽  
pp. 2353-2362 ◽  
Author(s):  
Murat Kasap ◽  
Jiann-Shin Chen
Keyword(s):  
Mass Spectrometry ◽  
Dna Sequences ◽  
Blot Analysis ◽  
Azotobacter Vinelandii ◽  
Upstream Region ◽  
Clostridium Pasteurianum ◽  
Nitrogen Fixing ◽  
Coding Region ◽  
Flight Mass Spectrometry ◽  
Separation Of Proteins

Previous studies identified five nifH-like genes (nifH2 through nifH6) in Clostridium pasteurianum (strain W5), where the nifH1 gene encodes the nitrogenase iron protein. Transcripts of these nifH genes, with the exception of nifH3, were detected in molybdenum-sufficient nitrogen-fixing cells. However, the size of the transcripts, the level of transcription and the presence of polypeptides encoded by the nifH-like genes were not reported. The nifH2 and nifH6 genes were extremely similar, as they seemed to differ by only two bases in a span of 2481 bp, one in the coding region and another in the upstream region. Re-examination of the DNA sequences revealed that the coding region of nifH2 and nifH6 was identical, whereas the difference in the upstream region was confirmed. Results from the authors' ongoing study of the nif genes of single-colony isolates of C. pasteurianum suggest that the nifH6 designation should be eliminated. Here the size of mRNA from nifH2 and the detection of the NifH2 polypeptide in nitrogen-fixing cells of C. pasteurianum are reported. Northern blot analysis of periodically collected nitrogen-fixing cells showed that the nifH1 and nifH2 mRNAs were present throughout growth. Addition of ammonium acetate repressed the transcription of both these genes similarly. Using an antiserum raised against NifH of Azotobacter vinelandii, two NifH-related bands were detected by Western blot analysis after electrophoretic separation of proteins in extracts of nitrogen-fixing C. pasteurianum cells. After separation of proteins by preparative SDS-PAGE, the NifH polypeptides were characterized by MALDI-TOF-MS (matrix-assisted laser desorption/ionization time-of-flight mass spectrometry) and by ES-MS/MS (electrospray tandem mass spectrometry) analyses. The results confirmed the presence of NifH2, in addition to NifH1, in nitrogen-fixing C. pasteurianum cells.

scholarly journals The Bacillus subtilis ydjL (bdhA) Gene Encodes Acetoin Reductase/2,3-Butanediol Dehydrogenase

2008 ◽  
Vol 74 (22) ◽  
pp. 6832-6838 ◽  
Author(s):  
Wayne L. Nicholson
Keyword(s):  
Bacillus Subtilis ◽  
Stationary Phase ◽  
Amino Acid Sequences ◽  
Insertion Mutation ◽  
Early Stationary Phase ◽  
Gene Encoding ◽  
Genome Database ◽  
A Minor ◽  
Butanediol Dehydrogenase

ABSTRACT Bacillus subtilis is capable of producing 2,3-butanediol from acetoin by fermentation, but to date, the gene encoding the enzyme responsible, acetoin reductase/2,3-butanediol dehydrogenase (AR/BDH), has remained unknown. A search of the B. subtilis genome database with the amino acid sequences of functional AR/BDHs from Saccharomyces cerevisiae and Bacillus cereus resulted in the identification of a highly similar protein encoded by the B. subtilis ydjL gene. A knockout strain carrying a ydjL::cat insertion mutation was constructed, which (i) abolished 2,3-butanediol production in early stationary phase, (ii) produced no detectable AR or BDH activity in vitro, and (iii) accumulated the precursor acetoin in early stationary phase. The ydjL::cat mutation also affected the kinetics of lactate but not acetate production during stationary-phase cultivation with glucose under oxygen limitation. A very small amount of 2,3-butanediol was detected in very-late-stationary-phase (96-hour) cultures of the ydjL::cat mutant, suggesting the existence of a second gene encoding a minor AR activity. From the data, it is proposed that the major AR/BDH-encoding gene ydjL be renamed bdhA.

scholarly journals Effects of A6E Mutation on Protein Expression and Supramolecular Assembly of Yeast Asparagine Synthetase

Biology ◽  
2021 ◽  
Vol 10 (4) ◽  
pp. 294
Author(s):  
Thunyarat Surasiang ◽  
Chalongrat Noree
Keyword(s):  
Protein Expression ◽  
Stationary Phase ◽  
Lymphoblastic Leukemia ◽  
Energy Levels ◽  
Supramolecular Assembly ◽  
Asparagine Synthetase ◽  
Gene Encoding ◽  
Synthetase Deficiency ◽  
Asparagine Synthetase Deficiency ◽  
Intracellular Energy

Asparagine synthetase deficiency (ASD) has been found to be caused by certain mutations in the gene encoding human asparagine synthetase (ASNS). Among reported mutations, A6E mutation showed the greatest reduction in ASNS abundance. However, the effect of A6E mutation has not yet been tested with yeast asparagine synthetase (Asn1/2p). Here, we constructed a yeast strain by deleting ASN2 from its genome, introducing the A6E mutation codon to ASN1, along with GFP downstream of ASN1. Our mutant yeast construct showed a noticeable decrease of Asn1p(A6E)-GFP levels as compared to the control yeast expressing Asn1p(WT)-GFP. At the stationary phase, the A6E mutation also markedly lowered the assembly frequency of the enzyme. In contrast to Asn1p(WT)-GFP, Asn1p(A6E)-GFP was insensitive to changes in the intracellular energy levels upon treatment with sodium azide during the log phase or fresh glucose at the stationary phase. Our study has confirmed that the effect of A6E mutation on protein expression levels of asparagine synthetase is common in both unicellular and multicellular eukaryotes, suggesting that yeast could be a model of ASD. Furthermore, A6E mutation could be introduced to the ASNS gene of acute lymphoblastic leukemia patients to inhibit the upregulation of ASNS by cancer cells, reducing the risk of developing resistance to the asparaginase treatment.

Inactivation or amplification of the spa2 gene, encoding a potential stationary-phase regulator, affects differentiation in Streptomyces ambofaciens

1997 ◽  
Vol 43 (12) ◽  
pp. 1118-1125 ◽  
Author(s):  
Martine Aubert ◽  
Elisabeth Weber ◽  
Brigitte Gintz ◽  
Bernard Decaris ◽  
Keith F. Chater
Keyword(s):  
Stationary Phase ◽  
Antibiotic Production ◽  
Morphological Differentiation ◽  
Aerial Mycelium ◽  
Detectable Effect ◽  
Multicopy Plasmid ◽  
Gene Encoding ◽  
Wild Type Phenotype ◽  
Streptomyces Ambofaciens ◽  
Mycelial Development

The deduced product of the spa2 gene of Streptomyces ambofaciens is a homologue of RspA, involved in stationary-phase σs factor regulation in Escherichia coli. This suggests that Spa2 could play a part in stationary-phase-associated differentiation in S. ambofaciens. The disruption of spa2 led to reductions in aerial mycelial development and associated spore pigmentation. The mutant phenotype reverted to the wild-type phenotype when the disruption construct spontaneously excised. The spa2 disruption had no detectable effect on growth rates in different media or antibiotic production and resistance. When spa2 was placed on a multicopy plasmid, a severe defect in formation and pigmentation of aerial mycelium resulted. These results strongly suggest that Spa2 is involved in a complex manner in the morphological differentiation process.Key words: Streptomyces, differentiation, stationary-phase regulator.

An essential yeast gene encoding a TTAGGG repeat-binding protein

1993 ◽  
Vol 13 (2) ◽  
pp. 1306-1314
Author(s):  
C Brigati ◽  
S Kurtz ◽  
D Balderes ◽  
G Vidali ◽  
D Shore
Keyword(s):  
Dna Binding ◽  
Binding Protein ◽  
Binding Activity ◽  
Insertion Mutation ◽  
Yeast Gene ◽  
Gene Encoding ◽  
Cloned Gene ◽  
Diploid Cells ◽  
Ttaggg Repeat

A yeast gene encoding a DNA-binding protein that recognizes the telomeric repeat sequence TTAGGG found in multicellular eukaryotes was identified by screening a lambda gt11 expression library with a radiolabeled TTAGGG multimer. This gene, which we refer to as TBF1 (TTAGGG repeat-binding factor 1), encodes a polypeptide with a predicted molecular mass of 63 kDa. The TBF1 protein, produced in vitro by transcription and translation of the cloned gene, binds to (TTAGGG)n probes and to a yeast telomeric junction sequence that contains two copies of the sequence TTAGGG separated by 5 bp. TBF1 appears to be identical to a previously described yeast TTAGGG-repeat binding activity called TBF alpha. TBF1 produced in vitro yields protein-DNA complexes with (TTAGGG)n probes that have mobilities on native polyacrylamide gels identical to those produced by partially purified TBF alpha from yeast cells. Furthermore, when extracts are prepared from a strain containing a TBF1 gene with an antigen tag, we find that the antigen copurifies with the predominant (TTAGGG)n-binding activity in the extracts. The DNA sequence of TBF1 was determined. The predicted protein sequence suggests that TBF1 may contain a nucleotide-binding domain, but no significant similarities to any other known proteins were identified, nor was an obvious DNA-binding motif apparent. Diploid cells heterozygous for a tbf1::URA3 insertion mutation are viable but upon sporulation give rise to tetrads with only two viable spores, both of which are Ura-, indicating that the TBF1 gene is essential for growth. Possible functions of TBF1 (TFB alpha) are discussed in light of these new results.

Optimal conditions for induction of competence in nitrogen-fixing Azotobacter vinelandii

1982 ◽  
Vol 28 (4) ◽  
pp. 389-397 ◽  
Author(s):  
William J. Page
Keyword(s):  
Dissolved Oxygen ◽  
Sodium Acetate ◽  
Azotobacter Vinelandii ◽  
Nitrogenase Activity ◽  
Cell Mass ◽  
Apparent Competition ◽  
Competence Development ◽  
Optimal Conditions ◽  
Nitrogen Fixing ◽  
Ph Range

Competence development in nitrogen-fixing Azotobacter vinelandii cells was optimal at pH 7.2–7.4 which necessitated additional buffering of the iron-limited nitrogen-free competence medium or the addition of a suitable organic acid salt, e.g., sodium acetate. An autolysin was active in this pH range and competent cells were more susceptible to autolysis than the general cell population. Competence development also required restricted aeration of the culture, and only those cultures that attained zero dissolved oxygen became competent. Restricted aeration served to protect the iron-limited cell nitrogenase from oxygen inactivation thus allowing the culture to reach zero dissolved oxygen. The inclusion of additional sources of reductant, e.g., malate, in buffered competence medium resulted in increased respiration and protection of nitrogenase, increased cell mass, and poly-β-hydroxybutyrate synthesis, but decreased competence. A possible explanation for the apparent competition between competence development and nitrogenase activity is discussed.

Sign in / Sign up

Export Citation Format

Share Document