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D Touati

Publications and source records attributed to D Touati.

48 records · Page 3Linked to original sources
PubMed1989

Exonuclease III and the catalase hydroperoxidase II in Escherichia coli are both regulated by the katF gene product.

The levels of both exonuclease III (exo III, product of xthA) and hydroperoxidase II (HP-II, product of katE) activity in Escherichia coli were influenced by a functional katF gene. The katF gene product is also necessary for synthesis of HP-II. Mutations in either katF or xthA, but not katE, result in sensitivity to H2O2 and near-UV (300-400 nm) radiation. Exo III, encoded by the xthA locus, recognizes and removes nucleoside 5'-monophosphates near apurinic and apyrimidinic sites in damaged DNA. Extracts of katF mutant strains had little detectable exo III activity. When a katF+ plasmid was introduced into the katF mutant, exo III activity exceeded wild-type levels. We propose that the katF gene is a trans-acting positive regulator of exo III and HP-II enzymes, both of which are involved in cellular recovery from oxidative damage.

Apurinic Acid↗
PubMed1989

The molecular genetics of superoxide dismutase in E. coli. An approach to understanding the biological role and regulation of SODS in relation to other elements of the defence system against oxygen toxicity.

Superoxide-mediated oxidative stress initiates a chain of events resulting in numerous cellular injuries. We have used genetics and E. coli to investigate the role and regulation of superoxide dismutase (SOD) and its relationship with the other constituents of the oxygen toxicity defence system. Structural SOD genes have been cloned and sequenced, permitting us to refine structural analysis and to isolate SOD-deficient mutants. The conditional oxygen sensitivity of these mutants, together with their increased mutation rate, demonstrated the essential biological role of SOD. Furthermore the complementation of SOD-lacking E. coli deficiencies by introducing a plasmid containing the gene encoding the human SOD supported the proposal that superoxide dismutation is the physiological function of SOD. Regulation of the MnSOD, through which the global SOD level in E. coli is modulated, has been studied using operon and protein fusions with the lactose operon, and led to the conclusion of a multicontrol of MnSOD. Isolation and characterization of regulation mutants are in progress, with the aim of identifying effectors and targets involved in the response to superoxide-mediated oxidative stress.

Escherichia coli↗
PubMed1988

Iron superoxide dismutase. Nucleotide sequence of the gene from Escherichia coli K12 and correlations with crystal structures.

The nucleotide sequence of the iron superoxide dismutase gene from Escherichia coli K12 has been determined. Analysis of the DNA sequence and mapping of the mRNA start reveal a unique promoter and a putative rho-independent terminator, and suggest that the Fe dismutase gene constitutes a monocistronic operon. The gene encodes a polypeptide product consisting of 192 amino acid residues with a calculated Mr of 21,111. The published N-terminal amino acid sequence of E. coli B Fe dismutase (Steinman, H. M., and Hill, R. L. (1973) Proc. Natl. Acad. Sci. U.S.A. 70, 3725-3729), along with the sequences of seven other peptides reported here, was located in the primary structure deduced from the K12 E. coli gene sequence. A new molecular model for iron dismutase from E. coli, based on the DNA sequence and x-ray data for the E. coli B enzyme at 3.1 A resolution, allows detailed comparison of the structure of the iron enzyme with manganese superoxide dismutase from Thermus thermophilus HB8. The structural similarities are more extensive than indicated by earlier studies and are particularly striking in the vicinity of the metal-ligand cluster, which is surrounded by conserved aromatic residues. The combined structural and sequence information now available for a series of Mn and Fe superoxide dismutases identifies variable regions in these otherwise very similar molecules; the principal variable site occurs in a surface region between the two long helices which dominate the N-terminal domain.

Amino Acid Sequence↗
PubMed1988

Molecular genetics of superoxide dismutases.

Molecular genetics of SOD has been recently developed primarily due to the new biotechnologies. Different types of isoenzymes have now been cloned and sequenced from several species ranging from bacteria to human and plants. Knowledge of the nucleotide sequences permitted refinement of structural models and provided information on subcellular locations. Cloned genes allowed the production of large amounts of SOD. They have been used for physiological and regulation studies, structural and enzymatic analyses, and are vital tools for the isolation of mutants. Isolation of mutants is generally essential to the understanding of the biological function of the gene in question. Indeed, SOD deficient mutants have now been isolated in bacteria and yeast. Their properties support, at numerous levels, a major role of SOD in cellular defense against oxygen toxicity. Few data are presently available on the molecular basis of mechanisms that regulate the expression of SOD.

Animals↗
PubMed1988

Effects of oxygen stress on membrane functions in Escherichia coli: role of HPI catalase.

Different conditions of oxidative stress were used to study their effects on membrane transport in Escherichia coli K-12. The oxidizing conditions included H2O2, plumbagin (a redox cycling compound that generates superoxide radicals [O2-]), and increased partial pressure of oxygen. Both superoxide radical-generating conditions and H2O2 treatments were found to cause a rapid decrease in proton motive force-dependent and -independent transport. H2O2-pretreated cells had the ability to rapidly recover both proton motive force-dependent and -independent transport. The induction required transcription and translation and was dependent on oxyR+ and katG+, providing evidence that these genes play crucial roles in the rapid recovery of transport. The effects of oxidatively induced loss of proton motive force on cell growth and macromolecular synthesis were also investigated.

Biological Transport, Active↗
PubMed1988

Transcriptional and posttranscriptional regulation of manganese superoxide dismutase biosynthesis in Escherichia coli, studied with operon and protein fusions.

Protein and operon fusions between the manganese superoxide dismutase (MnSOD) gene, sodA, and genes of the lactose operon were constructed in an attempt to explore the effects of various factors on MnSOD expression and the level at which they operate. In sodA-lacZ protein fusions, induction of beta-galactosidase perfectly mimicked MnSOD induction (i.e., beta-galactosidase was not expressed in anaerobiosis and was induced by oxygen, redox-cycling compounds in aerobiosis, and iron chelators in anaerobiosis). In tac-sodA operon fusions, MnSOD induction was monitored only by the lactose operon inducer isopropyl-beta-D-thiogalactopyranoside. Various plasmids carrying part or all of the sodA regulatory and structural region inhibited aerobic beta-galactosidase induction in sodA-lacZ fusions. This included plasmids carrying only the transcription start and upstream region and also plasmids which did not contain this region and in which MnSOD was under foreign transcriptional control. The role of metal ions was also investigated. Addition of Mn(II) enhanced MnSOD activity but did not affect induction. The anaerobic expression of MnSOD from the oxygen-insensitive tac promoter was enhanced threefold by iron-chelating agents, implying a posttranscriptional or most likely a posttranslational modulation of enzyme activity via metal ions. To accommodate all these data, multiregulation of MnSOD is proposed.

Base Sequence↗
PubMed1987

Human copper-zinc superoxide dismutase complements superoxide dismutase-deficient Escherichia coli mutants.

An Escherichia coli double mutant, sodAsodB, that is deficient in both bacterial superoxide dismutases (Mn superoxide dismutase and iron superoxide dismutase) is unable to grow on minimal medium in the presence of oxygen and exhibits increased sensitivity to paraquat and hydrogen peroxide. Expression of the evolutionarily unrelated eukaryotic CuZn superoxide dismutase in the sodAsodB E. coli mutant results in a wild-type phenotype with respect to aerobic growth on minimal medium and in resistance to paraquat and hydrogen peroxide. This supports the hypothesis that superoxide dismutation is the in vivo function of these proteins. Analysis of the growth of sodAsodB cells containing plasmids encoding partially active CuZn superoxide dismutases, produced by in vitro mutagenesis, shows a correlation between cell growth and enzyme activity. Thus, the sodAsodB strain provides a controlled selection for varying levels of superoxide dismutase activity.

Copper↗
PubMed1986

Isolation of superoxide dismutase mutants in Escherichia coli: is superoxide dismutase necessary for aerobic life?

Mu transposons carrying the chloramphenicol resistance marker have been inserted into the cloned Escherichia coli genes sodA and sodB coding for manganese superoxide dismutase (MnSOD) and iron superoxide dismutase (FeSOD) respectively, creating mutations and gene fusions. The mutated sodA or sodB genes were introduced into the bacterial chromosome by allelic exchange. The resulting mutants were shown to lack the corresponding SOD by activity measurements and immunoblot analysis. Aerobically, in rich medium, the absence of FeSOD or MnSOD had no major effect on growth or sensitivity to the superoxide generator, paraquat. In minimal medium aerobic growth was not affected, but the sensitivity to paraquat was increased, especially in the sodA mutant. A sodA sodB double mutant completely devoid of SOD was also obtained. It was able to grow aerobically in rich medium, its catalase level was unaffected and it was highly sensitive to paraquat and hydrogen peroxide; the double mutant was unable to grow aerobically on minimal glucose medium. Growth could be restored by removing oxygen, by providing an SOD-overproducing plasmid or by supplementing the medium with the 20 amino acids. It is concluded that the total absence of SOD in E. coli creates a conditional sensitivity to oxygen.

Aerobiosis↗
PubMed1986

Oxygen-dependent mutagenesis in Escherichia coli lacking superoxide dismutase.

Escherichia coli double mutants (sodA sodB) completely lacking superoxide dismutase (SOD) have greatly enhanced mutation rates during aerobic growth. Single mutants lacking manganese SOD (MnSOD) but possessing iron SOD (FeSOD) have a smaller increase, and single mutants lacking FeSOD but possessing MnSOD do not show such an increase. The enhancement of mutagenesis is completely dependent on the presence of oxygen, and treatments that increase the flux of superoxide radicals produce even higher levels of mutagenesis. The presence of a plasmid overproducing either form of SOD reduces the level of mutagenesis to that of wild type, showing that the O2-dependent enhancement results from a lack of SOD. The enhancement of mutagenesis is RecA-independent, and a complete lack of SOD does not induce the SOS response during aerobic growth. However, the enhanced mutagenesis in aerobically grown sodA sodB mutants is largely dependent on functional exonuclease III, suggesting that the increased flux of superoxide radicals results in DNA lesions that can be acted on by this enzyme, leading to mutations.

DNA Damage↗
PubMed1984

Cloning of the iron superoxide dismutase gene (sodB) in Escherichia coli K-12.

A clone overproducing iron superoxide dismutase has been isolated from an Escherichia coli cosmid bank. Subcloning located the gene responsible for iron superoxide dismutase overproduction on a 6.6-kilobase PstI restriction endonuclease fragment. Maxicell analysis, followed by immunological identification of iron superoxide dismutase protein, demonstrated that the structural gene, sodB, of iron superoxide dismutase has been cloned.

Base Composition↗
PubMed1983

Cloning and mapping of the manganese superoxide dismutase gene (sodA) of Escherichia coli K-12.

An Escherichia coli gene bank composed of large DNA fragments (about 40 kilobases) was constructed by using the small cosmid pHC79. From it, a clone was isolated for its ability to overproduce superoxide dismutase. The enzyme overproduced was manganese superoxide dismutase, as determined by electrophoresis and antibody precipitation. Maxicell analysis and two-dimensional O'Farrell polyacrylamide gel electrophoresis demonstrated that the structural gene, sodA, of manganese superoxide dismutase was cloned. Subcloning fragments from the original cosmid located the sodA gene within a 4.8-kilobase EcoRI-BamHI fragment. This fragment was inserted into a lambda phage which was deleted for the att region and consequently could only lysogenize by recombination between the cloned bacterial DNA insertion and the bacterial chromosome. Genetic mapping of the prophage in such lysogens indicated that the chromosomal sodA locus lies near 87 min on the E. coli map.

Chromosome Mapping↗