Characterization of the sodF gene region of Frankia sp. strain ACN14a and complementation of Escherichia coli sod mutant

2003 ◽  
Vol 49 (4) ◽  
pp. 294-300 ◽  
Author(s):  
Joëlle Maréchal ◽  
Renata Santos ◽  
Yasser Hammad ◽  
Nicole Alloisio ◽  
Anne-Marie Domenach ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Superoxide Dismutase ◽  
Root Exudates ◽  
Alnus Glutinosa ◽  
Triple Mutant ◽  
Iron Superoxide Dismutase ◽  
E Coli ◽  
Its Gene ◽  
Tac Promoter ◽  
The One

The Frankia sp. strain ACN14a superoxide dismutase SodF was previously shown to be induced in response to Alnus glutinosa root exudates, and its gene was sequenced. We report here the sequence of the 9-kb genomic segment surrounding the sodF gene and further characterize this gene and its product. Nine ORFs coding for various proteins, such as regulators, acetyl-CoA transferases, and a bacterioferritin A next to the sodF gene, were found. Northern blot analysis showed that the sodF gene was expressed as a major 1-kb transcript, which indicates that it has its own promoter. The sodF gene strongly complemented an Escherichia coli triple mutant (sodA sodB recA), restoring aerobic growth when the gene was expressed from the synthetic tac promoter but when expressed from its own promoter showed only slight rescue, suggesting that it was poorly recognized by the E. coli RNA polymerase. It is noteworthy that this is the first time that a Frankia gene has been reported to complement an E. coli mutant. The superoxide dismutase activity of the protein was inactivated by hydrogen peroxide, indicating that the metal ligand is iron, which is supported by analysis of the protein sequence. Thus, the SodF protein induced in Frankia by root exudates is an iron-containing enzyme similar to the one present in the nodules.Key words: Frankia, iron superoxide dismutase, sodF, E. coli complementation.

scholarly journals Vaccination with Live Escherichia coli ExpressingBrucella abortus Cu/Zn Superoxide Dismutase Protects Mice against Virulent B. abortus

1999 ◽  
Vol 67 (2) ◽  
pp. 986-988 ◽  
Author(s):  
Angel A. Oñate ◽  
Ramesh Vemulapalli ◽  
Edilia Andrews ◽  
Gerhardt G. Schurig ◽  
Stephen Boyle ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Superoxide Dismutase ◽  
Fusion Protein ◽  
Vaccine Strain ◽  
Brucella Abortus ◽  
Proliferative Response ◽  
E Coli ◽  
The One

ABSTRACT Vaccination of mice with Escherichia coli expressingBrucella Cu/Zn superoxide dismutase (SOD) [E. coli(pBSSOD)] induced a significant level of protection against virulent Brucella abortus challenge, although this level was not as high as the one reached with B. abortusvaccine strain RB51. In addition, vaccination with E. coli(pBSSOD) induced antibodies to Cu/Zn SOD and a strong proliferative response of splenocytes when stimulated in vitro with a thioredoxin-Cu/Zn SOD fusion protein.

Peroxynitrite-induced nitration of tyrosine-34 does not inhibit Escherichia coli iron superoxide dismutase

2001 ◽  
Vol 360 (3) ◽  
pp. 563-567 ◽  
Author(s):  
Laurent SOULÈRE ◽  
Catherine CLAPAROLS ◽  
Jacques PÉRIÉ ◽  
Pascal HOFFMANN
Keyword(s):  
Escherichia Coli ◽  
Superoxide Dismutase ◽  
Protonation State ◽  
Iron Superoxide Dismutase ◽  
E Coli ◽  
Tyrosine Residues ◽  
Physiological Conditions ◽  
Spectral Changes ◽  
Diffusion Limited ◽  
Peroxynitrite Anion

The peroxynitrite anion is a potent oxidizing agent, formed by the diffusion-limited combination of nitric oxide and superoxide, and its production under physiological conditions is associated with the pathologies of a number of inflammatory and neurodegenerative diseases. Nitration of Escherichia coli iron superoxide dismutase (Fe-SOD) by peroxynitrite was investigated, and demonstrated by spectral changes and electrospray mass spectroscopic analysis. HPLC and mass studies of the tryptic digests of the mono-nitrated Fe-SOD indicated that tyrosine-34 was the residue most susceptible to nitration by peroxynitrite. Exclusive nitration of this residue occurred when Fe-SOD was exposed to a cumulative dose of 0.4mM peroxynitrite. Unlike with human Mn-SOD, this single modification did not inactivate E. coli Fe-SOD at pH7.4. When Fe-SOD was exposed to higher concentrations of peroxynitrite (7mM), eight tyrosine residues per subunit of the protein, of the nine available, were nitrated without loss of catalytic activity of the enzyme. The pKa of nitrated tyrosine-34 was determined to be 7.95±0.15, indicating that the peroxynitrite-modified enzyme appreciably maintains its protonation state under physiological conditions.

scholarly journals Identification and Function of the pdxY Gene, Which Encodes a Novel Pyridoxal Kinase Involved in the Salvage Pathway of Pyridoxal 5′-Phosphate Biosynthesis in Escherichia coli K-12

1998 ◽  
Vol 180 (7) ◽  
pp. 1814-1821 ◽  
Author(s):  
Yong Yang ◽  
Ho-Ching Tiffany Tsui ◽  
Tsz-Kwong Man ◽  
Malcolm E. Winkler
Keyword(s):  
Escherichia Coli ◽  
De Novo ◽  
Specific Activity ◽  
Salvage Pathway ◽  
Pyridoxal Kinase ◽  
Triple Mutant ◽  
Reading Frame ◽  
E Coli ◽  
Specific Activities ◽  
K 12

ABSTRACT pdxK encodes a pyridoxine (PN)/pyridoxal (PL)/pyridoxamine (PM) kinase thought to function in the salvage pathway of pyridoxal 5′-phosphate (PLP) coenzyme biosynthesis. The observation that pdxK null mutants still contain PL kinase activity led to the hypothesis that Escherichia coli K-12 contains at least one other B6-vitamer kinase. Here we support this hypothesis by identifying the pdxY gene (formally, open reading frame f287b) at 36.92 min, which encodes a novel PL kinase. PdxY was first identified by its homology to PdxK in searches of the complete E. coli genome. Minimal clones of pdxY + overexpressed PL kinase specific activity about 10-fold. We inserted an omega cassette intopdxY and crossed the resultingpdxY::ΩKanr mutation into the bacterial chromosome of a pdxB mutant, in which de novo PLP biosynthesis is blocked. We then determined the growth characteristics and PL and PN kinase specific activities in extracts ofpdxK and pdxY single and double mutants. Significantly, the requirement of the pdxB pdxK pdxY triple mutant for PLP was not satisfied by PL and PN, and the triple mutant had negligible PL and PN kinase specific activities. Our combined results suggest that the PL kinase PdxY and the PN/PL/PM kinase PdxK are the only physiologically important B6vitamer kinases in E. coli and that their function is confined to the PLP salvage pathway. Last, we show thatpdxY is located downstream from pdxH (encoding PNP/PMP oxidase) and essential tyrS (encoding aminoacyl-tRNATyr synthetase) in a multifunctional operon.pdxY is completely cotranscribed with tyrS, but about 92% of tyrS transcripts terminate at a putative Rho-factor-dependent attenuator located in thetyrS-pdxY intercistronic region.

Response of hydroperoxidase and superoxide dismutase deficient mutants of Escherichia coli K-12 to oxidative stress

1988 ◽  
Vol 34 (10) ◽  
pp. 1171-1176 ◽  
Author(s):  
Herb E. Schellhorn ◽  
Hosni M. Hassan
Keyword(s):  
Escherichia Coli ◽  
Superoxide Dismutase ◽  
Oxidant Stress ◽  
Relative Importance ◽  
E Coli ◽  
Redox Active ◽  
Chemical Oxidants ◽  
Dependent Growth ◽  
K 12 ◽  
Generalized Transduction

In Escherichia coli, the coordinate action of two antioxidant enzymes, superoxide dismutase and hydroperoxidase (catalase), protect the cell from the deleterious effects of oxyradicals generated during normal aerobic respiration. To evaluate the relative importance of these two classes of enzymes, strains of E. coli deficient in superoxide dismutase and (or) hydroperoxidase were constructed by generalized transduction and their physiological responses to oxygen and oxidant stress examined. Superoxide dismutase was found to be more important than hydroperoxidase in preventing oxygen-dependent growth inhibition and mutagenesis, and in reducing sensitivity to redox-active compounds known to generate the superoxide anion. However, both types of enzymes were required for an effective defense against chemical oxidants that generate superoxide radicals and hydrogen peroxide.

scholarly journals Properties of human testis-specific lactate dehydrogenase expressed from Escherichia coli

1991 ◽  
Vol 273 (3) ◽  
pp. 587-592 ◽  
Author(s):  
K M LeVan ◽  
E Goldberg
Keyword(s):  
Escherichia Coli ◽  
Lactate Dehydrogenase ◽  
Prokaryotic Expression ◽  
Specific Activity ◽  
Human Testis ◽  
Reading Frame ◽  
E Coli ◽  
Heat Stable ◽  
Prokaryotic Expression Vector ◽  
Tac Promoter

The cDNA encoding the C4 isoenzyme of lactate dehydrogenase (LDH-C4) was engineered for expression in Escherichia coli. The Ldh-c open reading frame was constructed as a cassette for production of the native protein. The modified Ldh-c cDNA was subcloned into the prokaryotic expression vector pKK223-3. Transformed E. coli cells were grown to mid-exponential phase, and induced with isopropyl beta-D-thiogalactopyranoside for positive regulation of the tac promoter. Induced cells expressed the 35 kDa subunit, which spontaneously formed the enzymically active 140 kDa tetramer. Human LDH-C4 was purified over 200-fold from litre cultures of cells by AMP and oxamate affinity chromatography to a specific activity of 106 units/mg. The enzyme was inhibited by pyruvate concentrations above 0.3 mM, had a Km for pyruvate of 0.03 mM, a turnover number (nmol of NADH oxidized/mol of LDH-C4 per min at 25 degrees C) of 14,000 and was heat-stable.

scholarly journals Characterization of human and mouse granulocyte-macrophage-colony-stimulating factors derived from Escherichia coli

1987 ◽  
Vol 247 (1) ◽  
pp. 195-199 ◽  
Author(s):  
J L Schrimsher ◽  
K Rose ◽  
M G Simona ◽  
P Wingfield
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Amino Acid Sequences ◽  
Natural Material ◽  
Animal Cells ◽  
Colony Stimulating Factors ◽  
E Coli ◽  
Macrophage Colony ◽  
The One ◽  
Human And Mouse

Human and mouse granulocyte-macrophage-colony-stimulating factors (hGM-CSF and mGM-CSF, respectively), isolated from Escherichia coli cells expressing the corresponding human and mouse genes, have been characterized. The observed properties of the proteins have been compared with those properties which can be deduced from the DNA sequence alone and the published properties of natural GM-CSFs. The purified E. coli-derived proteins were found to have the expected molecular masses, amino acid compositions and N- and C-terminal amino acid sequences. The finding of 70-90% unprocessed N-terminal methionine for both proteins is discussed. The four Cys residues were found to be involved in two intramolecular disulphide bonds, linking the first and third, and second and fourth Cys residues. This disulphide bond arrangement is probably the one existing in natural material, since, although not glycosylated, both E. coli-derived proteins showed biological activity (colony stimulating assay for hGM-CSF, and cell proliferation assay for mGM-CSF) comparable with that reported for the respective proteins purified from animal cells.

Further investigation of the mechanism of Vitreoscilla hemoglobin (VHb) protection from oxidative stress in Escherichia coli

Biologia ◽  
2011 ◽  
Vol 66 (5) ◽  
Author(s):  
Meltem Akbas ◽  
Tugrul Doruk ◽  
Serhat Ozdemir ◽  
Benjamin Stark
Keyword(s):  
Oxidative Stress ◽  
Escherichia Coli ◽  
Hydrogen Peroxide ◽  
Superoxide Dismutase ◽  
Stationary Phase ◽  
Exponential Phase ◽  
Vitreoscilla Hemoglobin ◽  
Sod Activity ◽  
E Coli ◽  
Hydrogen Peroxide Treatment

AbstractIn Escherichia coli, Vitreoscilla hemoglobin (VHb) protects against oxidative stress, perhaps, in part, by oxidizing OxyR. Here this protection, specifically VHb-associated effects on superoxide dismutase (SOD) and catalase levels, was examined. Exponential or stationary phase cultures of SOD+ or SOD− E. coli strains with or without VHb and oxyR antisense were treated with 2 mM hydrogen peroxide without sublethal peroxide induction, and compared to untreated control cultures. The hydrogen peroxide treatment was toxic to both SOD+ and SOD− cells, but much more to SOD− cells; expression of VHb in SOD+ strains enhanced this toxicity. In contrast, the presence of VHb was generally associated in the SOD+ background with a modest increase in SOD activity that was not greatly affected by oxyR antisense or peroxide treatment. In both SOD+ and SOD− backgrounds, VHb was associated with higher catalase activity both in the presence and absence of peroxide. Contrary to its stimulatory effects in stationary phase, in exponential phase oxyR antisense generally decreased VHb levels.

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