scholarly journals Purification and characterization of glutathione reductase encoded by a cloned and over-expressed gene in Escherichia coli

1987 ◽  
Vol 245 (3) ◽  
pp. 875-880 ◽  
Author(s):  
N S Scrutton ◽  
A Berry ◽  
R N Perham
Keyword(s):  
Escherichia Coli ◽  
Glutathione Reductase ◽  
Reductase Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Fold Increase ◽  
Site Directed Mutagenesis ◽  
E Coli ◽  
Glutathione Reductase Activity ◽  
Rapid Purification ◽  
Sepharose 4B

An expression vector, pKGR, for the gor gene from Escherichia coli encoding glutathione reductase was constructed by subcloning of an AvaII fragment of the Clarke & Carbon bank plasmid pGR [Greer & Perham (1986) Biochemistry 25, 2736-2742] into the plasmid pKK223-3. The expression of glutathione reductase from the plasmid pKGR was found to have been successfully placed under the control of the tac promoter. Transformation of E. coli cells with this plasmid resulted in 100-200-fold increase in glutathione reductase activity in cell-free extracts. A rapid purification procedure for the enzyme, based on affinity chromatography on Procion Red HE-7B-CL-Sepharose 4B, was developed. The purified enzyme was homogeneous as judged by SDS/polyacrylamide-gel electrophoresis, and all its properties were consistent with the DNA sequence of the gene [Greer & Perham (1986) Biochemistry 25, 2736-2742] and with those previously reported for E. coli glutathione reductase [Mata, Pinto & Lopez-Barea (1984) Z. Naturforsch. C. Biosci. 39, 908-915]. These experiments have enabled an investigation of the protein chemical and mechanistic properties of the enzyme by site-directed mutagenesis.

scholarly journals Molecular Characterization of the Acid-Inducible asr Gene of Escherichia coli and Its Role in Acid Stress Response

2003 ◽  
Vol 185 (8) ◽  
pp. 2475-2484 ◽  
Author(s):  
Vaida Šeputienė ◽  
Domantas Motiejūnas ◽  
Kęstutis Sužiedėlis ◽  
Henrik Tomenius ◽  
Staffan Normark ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Signal Peptide ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Acid Stress ◽  
Acid Tolerance ◽  
Site Directed Mutagenesis ◽  
E Coli ◽  
Acid Shock

ABSTRACT Enterobacteria have developed numerous constitutive and inducible strategies to sense and adapt to an external acidity. These molecular responses require dozens of specific acid shock proteins (ASPs), as shown by genomic and proteomic analysis. Most of the ASPs remain poorly characterized, and their role in the acid response and survival is unknown. We recently identified an Escherichia coli gene, asr (acid shock RNA), encoding a protein of unknown function, which is strongly induced by high environmental acidity (pH < 5.0). We show here that Asr is required for growth at moderate acidity (pH 4.5) as well as for the induction of acid tolerance at moderate acidity, as shown by its ability to survive subsequent transfer to extreme acidity (pH 2.0). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western analysis of acid-shocked E. coli cells harboring a plasmid-borne asr gene demonstrated that the Asr protein is synthesized as a precursor with an apparent molecular mass of 18 kDa. Mutational studies of the asr gene also demonstrated the Asr preprotein contains 102 amino acids. This protein is subjected to an N-terminal cleavage of the signal peptide and a second processing event, yielding 15- and 8-kDa products, respectively. Only the 8-kDa polypeptide was detected in acid-shocked cells containing only the chromosomal copy of the asr gene. N-terminal sequencing and site-directed mutagenesis revealed the two processing sites in the Asr protein precursor. Deletion of amino acids encompassing the processing site required for release of the 8-kDa protein resulted in an acid-sensitive phenotype similar to that observed for the asr null mutant, suggesting that the 8-kDa product plays an important role in the adaptation to acid shock. Analysis of Asr:PhoA fusions demonstrated a periplasmic location for the Asr protein after removal of the signal peptide. Homologues of the asr gene from other Enterobacteriaceae were cloned and shown to be induced in E. coli under acid shock conditions.

scholarly journals Expression of the Corynebacterium glutamicum panD Gene Encoding l-Aspartate-α-Decarboxylase Leads to Pantothenate Overproduction in Escherichia coli

1999 ◽  
Vol 65 (4) ◽  
pp. 1530-1539 ◽  
Author(s):  
Nicole Dusch ◽  
Alfred Pühler ◽  
Jörn Kalinowski
Keyword(s):  
Escherichia Coli ◽  
Corynebacterium Glutamicum ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Fold Increase ◽  
Specific Productivity ◽  
Decarboxylase Activity ◽  
Coding Region ◽  
E Coli ◽  
Enhanced Expression

ABSTRACT The Corynebacterium glutamicum panD gene was identified by functional complementation of an Escherichia coli panDmutant strain. Sequence analysis revealed that the coding region ofpanD comprises 411 bp and specifies a protein of 136 amino acid residues with a deduced molecular mass of 14.1 kDa. A definedC. glutamicum panD mutant completely lackedl-aspartate-α-decarboxylase activity and exhibited β-alanine auxotrophy. The C. glutamicum panD(panDC.g. ) as well as the E. coli panD (panDE.c. ) genes were cloned into a bifunctional expression plasmid to allow gene analysis in C. glutamicum as well as in E. coli. The enhanced expression of panDC.g. in C. glutamicum resulted in the formation of two distinct proteins in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, leading to the assumption that the panDC.g. gene product is proteolytically processed into two subunits. By increased expression of panDC.g. in C. glutamicum, the activity of l-aspartate-α-decarboxylase was 288-fold increased, whereas the panDE.c. gene resulted only in a 4-fold enhancement. The similar experiment performed inE. coli revealed that panDC.g. achieved a 41-fold increase and that panDE.c. achieved a 3-fold increase of enzyme activity. The effect of thepanDC.g. and panDE.c. gene expression in E. coli was studied with a view to pantothenate accumulation. Only by expression of thepanDC.g. gene was sufficient β-alanine produced to abolish its limiting effect on pantothenate production. In cultures expressing the panDE.c. gene, the maximal pantothenate production was still dependent on external β-alanine supplementation. The enhanced expression ofpanDC.g. in E. coli yielded the highest amount of pantothenate in the culture medium, with a specific productivity of 140 ng of pantothenate mg (dry weight)−1h−1.

Functional Analysis of the Escherichia coli Molybdopterin Cofactor Biosynthesis Protein MoeA by Site-Directed Mutagenesis

2002 ◽  
Vol 383 (2) ◽  
pp. 319-323 ◽  
Author(s):  
C. Sandu ◽  
R. Brandsch
Keyword(s):  
Escherichia Coli ◽  
Nitrate Reductase ◽  
Nitrate Reductase Activity ◽  
Reductase Activity ◽  
Site Directed Mutagenesis ◽  
E Coli ◽  
Sitedirected Mutagenesis ◽  
Cofactor Biosynthesis

AbstractFive moeA mutants were generated by replacing some conserved amino acids of MoeA by sitedirected mutagenesis. The mutants were assayed for the ability to restore in vivo nitrate reductase activity of the moeA mutant Escherichia coli JRG97 and in vitro Neurospora crassa nit-1 nitrate reductase activity. The replacements Asp59AlaGly60Ala, Asp259Ala, Pro298AlaPro301Ala abolished the function of MoeA in Momolybdopterin formation and stabilization, reflected in the inability to restore nitrate reductase activity. The replacements Gly251AlaGly252Ala reduced, and that of Pro283Ala had no effect, on nitrate reductase activity. E. coli JRG97 cells transformed with mutants that failed to restore nitrate reductase activity showed by HPLC analysis a decreased level of molybdopterinderived dephospho FormA as compared to bacteria transformed with wildtype moeA. The effects of the amino acid replacements on MoeA function may be explained in correlation with the MoeA crystal structure.

scholarly journals A genetically detoxified derivative of heat-labile Escherichia coli enterotoxin induces neutralizing antibodies against the A subunit.

1994 ◽  
Vol 180 (6) ◽  
pp. 2147-2153 ◽  
Author(s):  
M Pizza ◽  
M R Fontana ◽  
M M Giuliani ◽  
M Domenighini ◽  
C Magagnoli ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Cholera Toxin ◽  
Neutralizing Antibodies ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
E Coli ◽  
Heat Labile ◽  
A Subunit ◽  
Adp Ribosyltransferase ◽  
Derivatives Of

Escherichia coli enterotoxin (LT) and the homologous cholera toxin (CT) are A-B toxins that cause travelers' diarrhea and cholera, respectively. So far, experimental live and killed vaccines against these diseases have been developed using only the nontoxic B portion of these toxins. The enzymatically active A subunit has not been used because it is responsible for the toxicity and it is reported to induce a negligible titer of toxin neutralizing antibodies. We used site-directed mutagenesis to inactivate the ADP-ribosyltransferase activity of the A subunit and obtained nontoxic derivatives of LT that elicited a good titer of neutralizing antibodies recognizing the A subunit. These LT mutants and equivalent mutants of CT may be used to improve live and killed vaccines against cholera and enterotoxinogenic E. coli.

scholarly journals In Escherichia coli Ammonia Inhibits Cytochrome bo3 But Activates Cytochrome bd-I

Antioxidants ◽  
2020 ◽  
Vol 10 (1) ◽  
pp. 13
Author(s):  
Elena Forte ◽  
Sergey A. Siletsky ◽  
Vitaliy B. Borisov
Keyword(s):  
Escherichia Coli ◽  
Molecular Mechanisms ◽  
Dissociation Constants ◽  
Reductase Activity ◽  
Ammonia Concentration ◽  
High Concentration ◽  
E Coli ◽  
Cytochrome Bd ◽  
Cytochrome Bo3 ◽  
Oxygen Reductase

Interaction of two redox enzymes of Escherichia coli, cytochrome bo3 and cytochrome bd-I, with ammonium sulfate/ammonia at pH 7.0 and 8.3 was studied using high-resolution respirometry and absorption spectroscopy. At pH 7.0, the oxygen reductase activity of none of the enzymes is affected by the ligand. At pH 8.3, cytochrome bo3 is inhibited by the ligand, with 40% maximum inhibition at 100 mM (NH4)2SO4. In contrast, the activity of cytochrome bd-I at pH 8.3 increases with increasing the ligand concentration, the largest increase (140%) is observed at 100 mM (NH4)2SO4. In both cases, the effector molecule is apparently not NH4+ but NH3. The ligand induces changes in absorption spectra of both oxidized cytochromes at pH 8.3. The magnitude of these changes increases as ammonia concentration is increased, yielding apparent dissociation constants Kdapp of 24.3 ± 2.7 mM (NH4)2SO4 (4.9 ± 0.5 mM NH3) for the Soret region in cytochrome bo3, and 35.9 ± 7.1 and 24.6 ± 12.4 mM (NH4)2SO4 (7.2 ± 1.4 and 4.9 ± 2.5 mM NH3) for the Soret and visible regions, respectively, in cytochrome bd-I. Consistently, addition of (NH4)2SO4 to cells of the E. coli mutant containing cytochrome bd-I as the only terminal oxidase at pH 8.3 accelerates the O2 consumption rate, the highest one (140%) being at 27 mM (NH4)2SO4. We discuss possible molecular mechanisms and physiological significance of modulation of the enzymatic activities by ammonia present at high concentration in the intestines, a niche occupied by E. coli.

Inhibition of Escherichia coli Trimethylamine-N-oxide Reductase by Food Preservatives1

1982 ◽  
Vol 45 (3) ◽  
pp. 241-243 ◽  
Author(s):  
M. KRUK ◽  
J. S. LEE
Keyword(s):  
Escherichia Coli ◽  
Benzoic Acid ◽  
Sorbic Acid ◽  
Reductase Activity ◽  
Resting Cells ◽  
E Coli

Trimethylamine-N-oxide (TMA-O) reductase activity of resting cells of Escherichia coli was inhibited by tetrasodium ethylenediaminetetraacetate (Na4EDTA), benzoic acid (BA and methylparaben (MP). The 50% inhibitory concentrations of Na4EDTA, BA and MP were 20.2, 1.2 and 32.4 mM, respectively. BA at pH 6.5 or below most effectively inhibited the TMA-O reductase. Sorbic acid (SA), up to 0.70 mM, had no effect on TMA-O reductase activity, but SA inhibited the growth and subsequent TMA production in E. coli at or above 0.3S mM.

scholarly journals Genome-Wide Screening Identifies Six Genes That Are Associated with Susceptibility to Escherichia coli Microcin PDI

2015 ◽  
Vol 81 (20) ◽  
pp. 6953-6963 ◽  
Author(s):  
Zhe Zhao ◽  
Lauren J. Eberhart ◽  
Lisa H. Orfe ◽  
Shao-Yeh Lu ◽  
Thomas E. Besser ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Atp Synthase ◽  
Disulfide Bonds ◽  
Gene Knockout ◽  
Single Gene ◽  
Site Directed Mutagenesis ◽  
Content Type ◽  
E Coli ◽  
Synthase Inhibitor ◽  
Loss Of Susceptibility

ABSTRACTThe microcin PDI inhibits a diverse group of pathogenicEscherichia colistrains. Coculture of a single-gene knockout library (BW25113;n= 3,985 mutants) against a microcin PDI-producing strain (E. coli25) identified six mutants that were not susceptible (ΔatpA, ΔatpF, ΔdsbA, ΔdsbB, ΔompF, and ΔompR). Complementation of these genes restored susceptibility in all cases, and the loss of susceptibility was confirmed through independent gene knockouts inE. coliO157:H7 Sakai. Heterologous expression ofE. coliompFconferred susceptibility toSalmonella entericaandYersinia enterocoliticastrains that are normally unaffected by microcin PDI. The expression of chimeric OmpF and site-directed mutagenesis revealed that the K47G48N49region within the first extracellular loop ofE. coliOmpF is a putative binding site for microcin PDI. OmpR is a transcriptional regulator forompF, and consequently loss of susceptibility by the ΔompRstrain most likely is related to this function. Deletion of AtpA and AtpF, as well as AtpE and AtpH (missed in the original library screen), resulted in the loss of susceptibility to microcin PDI and the loss of ATP synthase function. Coculture of a susceptible strain in the presence of an ATP synthase inhibitor resulted in a loss of susceptibility, confirming that a functional ATP synthase complex is required for microcin PDI activity. Intransexpression ofompFin the ΔdsbAand ΔdsbBstrains did not restore a susceptible phenotype, indicating that these proteins are probably involved with the formation of disulfide bonds for OmpF or microcin PDI.

scholarly journals In Vivo Bioavailability of Selenium in Selenium-Enriched Streptococcus thermophilus and Enterococcus faecium in CD IGS Rats

Antioxidants ◽  
2021 ◽  
Vol 10 (3) ◽  
pp. 463
Author(s):  
Gabriela Krausova ◽  
Antonin Kana ◽  
Marek Vecka ◽  
Ivana Hyrslova ◽  
Barbora Stankova ◽  
...  
Keyword(s):  
Glutathione Reductase ◽  
Enterococcus Faecium ◽  
Reductase Activity ◽  
Streptococcus Thermophilus ◽  
Heart Tissue ◽  
Sprague Dawley ◽  
Glutathione Reductase Activity ◽  
Novel Concept ◽  
The Individual

The selenium (Se) enrichment of yeasts and lactic acid bacteria (LAB) has recently emerged as a novel concept; the individual health effects of these beneficial microorganisms are combined by supplying the essential micronutrient Se in a more bioavailable and less toxic form. This study investigated the bioavailability of Se in the strains Enterococcus faecium CCDM 922A (EF) and Streptococcus thermophilus CCDM 144 (ST) and their respective Se-enriched forms, SeEF and SeST, in a CD (SD-Sprague Dawley) IGS rat model. Se-enriched LAB administration resulted in higher Se concentrations in the liver and kidneys of rats, where selenocystine was the prevalent Se species. The administration of both Se-enriched strains improved the antioxidant status of the animals. The effect of the diet was more pronounced in the heart tissue, where a lower glutathione reductase content was observed, irrespective of the Se fortification in LAB. Interestingly, rats fed diets with EF and SeEF had higher glutathione reductase activity. Reduced concentrations of serum malondialdehyde were noted following Se supplementation. Diets containing Se-enriched strains showed no macroscopic effects on the liver, kidneys, heart, and brain and had no apparent influence on the basic parameters of the lipid metabolism. Both the strains tested herein showed potential for further applications as promising sources of organically bound Se and Se nanoparticles.

Structure of the O-chain polysaccharide of the phenol-phase soluble lipopolysaccharide of Escherichia coli 0:157:H7

1986 ◽  
Vol 64 (1) ◽  
pp. 21-28 ◽  
Author(s):  
Malcolm B. Perry ◽  
Leann MacLean ◽  
Douglas W. Griffith
Keyword(s):  
Escherichia Coli ◽  
Brucella Abortus ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Extraction Procedure ◽  
Periodate Oxidation ◽  
Cross Reactivity ◽  
E Coli ◽  
Structure Formula ◽  
Phenol Water

The phenol-phase soluble lipopolysaccharide isolated from Escherichia coli 0:157 by the hot phenol–water extraction procedure was shown by sodium dodecyl sulfate–polyacrylamide gel electrophoresis, periodate oxidation, methylation, and 13C and 1H nuclear magnetic resonance studies to be an unbranched linear polysaccharide with a tetrasaccharide repeating unit having the structure:[Formula: see text]The serological cross-reactivity of E. coli 0:157 with Brucella abortus, Yersinia enterocolitica (serotype 0:9), group N Salmonella, and some other E. coli species can be related immunochemically to the presence of 1,2-glycosylated N-acylated 4-amino-4,6-dideoxy-α-D-mannopyranosyl residues in the O-chains of their respective lipopolysaccharides.

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