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D Touati

Publications and source records attributed to D Touati.

At least 37 records · Page 2Linked to original sources
PubMed1997

Recovery of culturability of an HOCl-stressed population of Escherichia coli after incubation in phosphate buffer: resuscitation or regrowth?

An Escherichia coli population harvested in exponential phase at about 10(8) cells/ml was treated in phosphate buffer with HOCl at concentrations ranging from 0.4 to 1 mg/liter (7.7 to 19 microM). The HOCl stress resulted in the appearance of three cell subpopulations: a majority of dead (nonrespiring) cells, a few culturable cells (10(2) to 10(4)), and about 10(7) viable but nonculturable cells. In the absence of any added exogenous nutrient, a culturable population could be recovered after 1 day of incubation in phosphate buffer, and such a population would reach a cell density close to 10% of the initial density of the stressed population, whatever the initial number of survivors. When a small number of untreated cells were mixed with the stressed population, growth of the untreated cells was observed, demonstrating that damaged cells provided nutrients. Similarly, a filtrate and a disrupted-cell filtrate of the stressed population supported growth of untreated cells with the same efficiency. The number of CFU (untreated or stressed) at plateau phase depended on the initial density of the stressed cells. Taken together, these results suggest that recovery in phosphate buffer of an HOCl-stressed population is in large part due to growth of a few culturable cells at the expense of damaged cells. However, comparison of the growth rates of the stressed culturable population and of untreated bacteria growing in filtrate showed significantly faster growth of the stressed cells, a fact not fully compatible with the hypothesis that recovery is only the simple growth of survivors. We suggest, therefore, that in addition to growth of the few culturable stressed cells, there is repair and growth of some mildly injured viable but nonculturable cells.

Buffers↗
PubMed1997

Survival of Escherichia coli exposed to visible light in seawater: analysis of rpoS-dependent effects.

We investigated the effect of visible light on Escherichia coli in seawater microcosms. Escherichia coli lost its ability to form colonies in marine environments when exposed to artificial continuous visible light. Survival of illuminated bacteria during the stationary phase was drastically reduced in the absence of the sigma factor (RpoS or KatF) that regulates numerous genes induced in this phase. In the stationary phase, double catalase mutants katE katG and mutants defective in the protein Dps (both catalase and Dps are involved in resistance to hydrogen peroxide (H2O2)), were more sensitive to light. In the exponenital phase, a mutation in oxyR, the regulatory gene of the adaptive response to H2O2, increased sensitivity to light, further suggesting that deleterious effects might be associated with H2O2 production. However, in the stationary phase, the katE katG dps mutant was considerably more resistant to visible light than the rpoS mutant, suggesting rpoS-dependent protection against deleterious effects other than those related to H2O2. The deleterious action of visible light was less important when the salinity decreased. In freshwater, rpoS and katE katG dps mutants did not show a drastic difference in sensitivity to light suggesting that osmolarity sensitizes E. coli to those deleterious effects of visible light that are unrelated to H2O2.

Bacterial Proteins↗
PubMed1996

Hypochlorous acid stress in Escherichia coli: resistance, DNA damage, and comparison with hydrogen peroxide stress.

We have investigated the mechanisms of killing of Escherichia coli by HOCl by identifying protective functions. HOCl challenges were performed on cultures arrested in stationary phase and in exponential phase. Resistance to HOCl in both cases was largely mediated by genes involved in resistance to hydrogen peroxide (H2O2). In stationary phase, a mutation in rpoS, which controls the expression of starvation genes including those which protect against oxidative stress, renders the cells hypersensitive to killing by HOCl. RpoS-regulated genes responsible for this sensitivity were dps, which encodes a DNA-binding protein, and, to a lesser extent, katE and katG, encoding catalases; all three are involved in resistance to H2O2. In exponential phase, induction of the oxyR regulon, an adaptive response to H2O2, protected against HOCl exposure, and the oxyR2 constitutive mutant is more resistant than the wild-type strain. The genes involved in this oxyR-dependent resistance have not yet been identified, but they differ from those primarily involved in resistance to H2O2, including katG, ahp, and dps. Pretreatment with HOCl conferred resistance to H2O2 in an OxyR-independent manner, suggesting a specific adaptive response to HOCl. fur mutants, which have an intracellular iron overload, were more sensitive to HOCl, supporting the generation of hydroxyl radicals upon HOCl exposure via a Fenton-type reaction. Mutations in recombinational repair genes (recA or recB) increased sensitivity to HOCl, indicative of DNA strand breaks. Sensitivity was visible in the wild type only at concentrations above 0.6 mg/liter, but it was observed at much lower concentrations in dps recA mutants.

Bacterial Proteins↗
PubMed1996

Overproduction of the rbo gene product from Desulfovibrio species suppresses all deleterious effects of lack of superoxide dismutase in Escherichia coli.

In an attempt to isolate the superoxide dismutase (SOD) gene from the anaerobic sulfate-reducing bacterium Desulfoarculus baarsii, a DNA fragment was isolated which functionally complemented an Escherichia coli mutant (sodA sodB) deficient in cytoplasmic SODs. This region carries two open reading frames with sequences which are very similar to that of the rbo-rub operon from Desulfovibrio vulgaris. Independent expression of the rbo and rub genes from ptac showed that expression of rbo was responsible for the observed phenotype. rbo overexpression suppressed all deleterious effects of SOD deficiency in E. coli, including inactivation by superoxide of enzymes containing 4Fe-4S clusters and DNA damage produced via the superoxide-enhanced Fenton reaction. Thus, rbo restored to the sodA sodB mutant the ability to grow on minimal medium without the addition of branched amino acids, and growth on gluconate and succinate carbon sources was no longer impaired. The spontaneous mutation rate, which is elevated in SOD-deficient mutants, returned to the wild-type level in the presence of Rbo, which also restored aerobic viability of sodA sodB recA mutants. Rbo from Desulfovibrio vulgaris, but not Desulfovibrio gigas desulforedoxin, which corresponds to the NH2-terminal domain of Rbo, complemented sod mutants. The physiological role of Rbo in sulfate-reducing bacteria is unknown. In E. coli, Rbo may permit the bacterium to avoid superoxide stress by maintaining functional (reduced) superoxide sensitive 4Fe-4S clusters. It would thereby restore enzyme activities and prevent the release of iron that occurs after cluster degradation and presumably leads to DNA damage.

Amino Acid Sequence↗
PubMed1996

Cloning and characterization of the katA gene of Rhizobium meliloti encoding a hydrogen peroxide-inducible catalase.

To investigate the involvement of bacterial catalases of the symbiotic gram-negative bacterium Rhizobium meliloti in the development of Medicago-Rhizobium functional nodules, we cloned a putative kat gene by screening a cosmid library with a catalase-specific DNA probe amplified by PCR from the R. meliloti genome. Nucleotide sequence analysis of a 1.8-kb DNA fragment revealed an open reading frame, called katA, encoding a peptide of 562 amino acid residues with a calculated molecular mass of 62.9 kDa. The predicted amino acid sequence showed a high homology with the primary structure of monofunctional catalases from eucaryotes and procaryotes. The katA gene was localized on the chromosome, and the katA gene product was essentially found in the periplasmic space. A katA::Tn5 mutant was obtained and showed a drastic sensitivity to hydrogen peroxide, indicating an essential protective role of KatA. However, neither Nod nor Fix phenotypes were impaired in the mutant, suggesting that KatA is not essential for nodulation and establishment of nitrogen fixation. Exposure to a sublethal concentration of H2O2 enhanced KatA activity (100-fold) and also increased survival to subsequent H2O2 exposure at higher concentrations. No protection is observed in katA::Tn5, indicating that KatA is the major component of an adaptive response.

Adaptation, Physiological↗
PubMed1995

Lethal oxidative damage and mutagenesis are generated by iron in delta fur mutants of Escherichia coli: protective role of superoxide dismutase.

The Escherichia coli Fur protein, with its iron(II) cofactor, represses iron assimilation and manganese superoxide dismutase (MnSOD) genes, thus coupling iron metabolism to protection against oxygen toxicity. Iron assimilation is triggered by iron starvation in wild-type cells and is constitutive in fur mutants. We show that iron metabolism deregulation in fur mutants produces an iron overload, leading to oxidative stress and DNA damage including lethal and mutagenic lesions. fur recA mutants were not viable under aerobic conditions and died after a shift from anaerobiosis to aerobiosis. Reduction of the intracellular iron concentration by an iron chelator (ferrozine), by inhibition of ferric iron transport (tonB mutants), or by overexpression of the iron storage ferritin H-like (FTN) protein eliminated oxygen sensitivity. Hydroxyl radical scavengers dimethyl sulfoxide and thiourea also provided protection. Functional recombinational repair was necessary for protection, but SOS induction was not involved. Oxygen-dependent spontaneous mutagenesis was significantly increased in fur mutants. Similarly, SOD deficiency rendered sodA sodB recA mutants nonviable under aerobic conditions. Lethality was suppressed by tonB mutations but not by iron chelation or overexpression of FTN. Thus, superoxide-mediated iron reduction was responsible for oxygen sensitivity. Furthermore, overexpression of SOD partially protected fur recA mutants. We propose that a transient iron overload, which could potentially generate oxidative stress, occurs in wild-type cells on return to normal growth conditions following iron starvation, with the coupling between iron and MnSOD regulation helping the cells cope.

Aerobiosis↗
PubMed1994

The NAD(P)H:flavin oxidoreductase from Escherichia coli as a source of superoxide radicals.

The NAD(P)H:flavin oxidoreductase (encoded by the fre gene) of Escherichia coli is a soluble enzyme which, under aerobic conditions and together with NAD(P)H and flavins, generates superoxide radicals selectively. This was demonstrated from spin trapping experiments and from the ability of the flavin reductase to achieve a superoxide dismutase (SOD)-sensitive reduction of cytochrome c. The participation of the flavin reductase to O2-. generation in E. coli cells has been studied. Superoxide production in dialyzed cytosolic fraction of SOD-deficient E. coli was stimulated by the addition of flavins. There was no stimulation in soluble extracts of flavin reductase-deficient strains. Moreover, using fusions of sodA promoter to lacZ, we showed that sodA transcription was diminished in flavin reductase-deficient E. coli and that the induction of MnSOD by flavin reductase was SoxRS-independent. These results suggest that the flavin reductase might: (i) in vivo, be an important cytosolic site of O2-. generation; (ii) in vitro, serve as a simple, efficient, and selective O2-. generator.

Cyclic N-Oxides↗
PubMed1994

Anaerobic activation of arcA transcription in Escherichia coli: roles of Fnr and ArcA.

The ArcA and Fnr regulators of Escherichia coli, both of which are activated in anaerobic conditions, negatively regulate the sodA gene (coding for manganese superoxide dismutase), but Fnr has no effect on anaerobic sodA expression in a delta arcA delta fnr background (Compan and Touati, 1993). We show here that the sdh gene (coding for succinate dehydrogenase) is also negatively regulated by Fnr, but again Fnr exerts no control in a delta arcA background. One interpretation of these results is that Fnr activates arcA transcription. Using arcA-lac transcriptional and translational fusions, we show that arcA expression increases (about fourfold) in anaerobiosis and that both Fnr and ArcA are required for full expression. In a delta fnr background, there is no autoactivation, suggesting that ArcA enhances activation by Fnr. Transcript and sequence analyses reveal that the arcA upstream regulatory region lies within a 530 bp non-coding DNA fragment, which contains five putative promoter sequences and a putative Fnr-binding site. Identification of the transcription start sites indicates that transcription occurs in aerobiosis from three constitutive upstream promoters (Pe, Pd, Pc). In anaerobiosis an additional completely Fnr-dependent transcript starting at Pa is present; expression from Pa is reduced in the absence of ArcA, and Fnr activation at Pa blocks the weak anaerobic-dependent expression from Pb. Fnr activation of arcA transcription may play an important role in the co-ordination of expression of genes associated with aerobic and anaerobic metabolism during environmental changes.

Anaerobiosis↗
PubMed1994

Involvement of Escherichia coli DNA polymerase II in response to oxidative damage and adaptive mutation.

DNA polymerase II (Pol II) is regulated as part of the SOS response to DNA damage in Escherichia coli. We examined the participation of Pol II in the response to oxidative damage, adaptive mutation, and recombination. Cells lacking Pol II activity (polB delta 1 mutants) exhibited 5- to 10-fold-greater sensitivity to mode 1 killing by H2O2 compared with isogenic polB+ cells. Survival decreased by about 15-fold when polB mutants containing defective superoxide dismutase genes, sodA and sodB, were compared with polB+ sodA sodB mutants. Resistance to peroxide killing was restored following P1 transduction of polB cells to polB+ or by conjugation of polB cells with an F' plasmid carrying a copy of polB+. The rate at which Lac+ mutations arose in Lac- cells subjected to selection for lactose utilization, a phenomenon known as adaptive mutation, was increased threefold in polB backgrounds and returned to wild-type rates when polB cells were transduced to polB+. Following multiple passages of polB cells or prolonged starvation, a progressive loss of sensitivity to killing by peroxide was observed, suggesting that second-site suppressor mutations may be occurring with relatively high frequencies. The presence of suppressor mutations may account for the apparent lack of a mutant phenotype in earlier studies. A well-established polB strain, a dinA Mu d(Apr lac) fusion (GW1010), exhibited wild-type (Pol II+) sensitivity to killing by peroxide, consistent with the accumulation of second-site suppressor mutations. A high titer anti-Pol II polyclonal antibody was used to screen for the presence of Pol II in other bacteria and in the yeast Saccharomyces cerevisiae. Cross-reacting material was found in all gram-negative strains tested but was not detected in gram-positive strains or in S. cerevisiae. Induction of Pol II by nalidixic acid was observed in E. coli K-12, B, and C, in Shigella flexneri, and in Salmonella typhimurium.

Adaptation, Physiological↗
PubMed1993

Iron and oxygen regulation of Escherichia coli MnSOD expression: competition between the global regulators Fur and ArcA for binding to DNA.

Manganese superoxide dismutase (the sodA gene product) in Escherichia coli, is negatively regulated by two global regulators, ArcA (aerobic respiration control) and Fur (ferric uptake regulation), coupling its expression to aerobic metabolism and the intracellular iron pool. Footprinting analyses were carried out on the sodA promoter region with purified Fur protein and with ArcA protein overproduced in crude extracts. ArcA is able to bind in vitro in the absence of the in vivo triggering signal. The binding occurs in one step and study of contacts within the operator sequence reveals binding on one side of the double helix. The DNA protection extends to a much larger region (about 65 bp) than would be expected for a 27 kDa protein, suggesting polymerization. Both Fur and ArcA footprints encompass the -35 and -10 promoter region and there is considerable overlap of their binding sequences, in agreement with in vivo results suggesting that either regulator alone can block sodA transcription. Furthermore, competition experiments show that Fur and ArcA binding to the sodA promoter are mutually exclusive and that ArcA can easily displace Fur, but not vice versa. The biological significance of this in vitro behaviour is discussed.

Aerobiosis↗
PubMed1993

Interaction of six global transcription regulators in expression of manganese superoxide dismutase in Escherichia coli K-12.

Transcription of the sodA gene of Escherichia coli, which encodes manganese superoxide dismutase, is governed by six global regulators: the product of the soxRS locus (superoxide response) and mutated alleles of the soxQ locus (such as cfxB) act as activators; the products of the fur (ferric uptake regulation), arcA (aerobic regulation control), and fnr (fumarate nitrate reductase) genes and the integration host factor (IHF) negatively regulate sodA. The action of these effectors on the sodA promoter was investigated by using chromosomal sodA-lacZ operon fusions with intact or deleted promoters, different environmental conditions, and strains carrying different combinations of null mutations in the effector genes. The data allow us to assign target regions in the sodA promoter for activation by SoxRS and CfxB and for repression by Fur and ArcA. In aerobiosis, activation of sodA transcription by SoxRS was compatible with CfxB activation or Fur repression, whereas cfxB and fur controls were mutually exclusive. Repression by Fnr appeared, at least in part, to be ArcA dependent. IHF enhanced aerobic Fur repression, and in the absence of Fur, it enhanced anaerobic repression by ArcA. The DNA targets for Fur (encompassing the -35 region) and ArcA (from and downstream of the -35 region) appear to overlap, suggesting that Fur and ArcA repressions are mutually exclusive. Fur (in response to the iron pool) or ArcA, acting with Fnr and IHF (in response to the redox state of the cells), can block anaerobic sodA-lacZ expression with about equivalent efficiencies. The possible biological significance of this result is discussed.

Bacterial Outer Membrane Proteins↗
PubMed1991

Are appR and katF the same Escherichia coli gene encoding a new sigma transcription initiation factor?

The phenotype of Escherichia coli appR pleiotropic mutants has been compared with that of mutants in the katF gene, which lies in the same region and controls expression of catalase HPII (katE) and exonuclease III (xth). All the described characters of appR mutants--reduced pH 2.5 acid phosphatase level, overexpression of alkaline phosphatase and ability of crp or cya mutants to utilize some CAP + cAMP-dependent carbon sources--were reproduced by a katF:: Tn10 insertion. In all cases, the wild-type phenotype was restored by the presence of a plasmid-borne katF+ gene. Conversely, spontaneous appR mutants were hypersensitive to H2O2 to the same degree as katF mutants. We conclude that the appR gene is identical to katF, which encodes a putative new sigma factor (Mulvey and Loewen, 1989).

DNA Transposable Elements↗
PubMed1991

Two global regulators repress the anaerobic expression of MnSOD in Escherichia coli::Fur (ferric uptake regulation) and Arc (aerobic respiration control).

The expression of sodA, the Escherichia coli gene encoding manganese superoxide dismutase (MnSOD) is induced by aerobiosis and superoxide generators such as paraquat. Analysis of variants expressing sodA in the absence of oxygen has revealed that mutations in genes for two global regulatory systems, Fur (ferric uptake regulation) and Arc (aerobic respiration control), are simultaneously required for the expression of sodA in anaerobiosis. The Fur protein still represses sodA in an iron-dependent fashion in aerobiosis. Moreover, all mutants remain inducible by paraquat, indicating that the positive control of SoxR, which mediates the response to superoxide in E. coli, is still operative. Thus, in addition to the response to the superoxide-mediated oxidative stress which depends on SoxR, two global controls regulate MnSOD expression: ArcA couples MnSOD expression to respiration, and Fur couples it to the intracellular concentration of iron.

Aerobiosis↗
PubMed1991

The molecular genetics of superoxide dismutase in E. coli.

The biological role and the regulation of superoxide dismutase (SOD) in E. coli have been investigated using genetics. Cloning of both E. coli SOD genes permitted construction of mutants completely lacking SOD. The conditional oxygen sensitivity of those mutants, together with their increased mutation rate, demonstrated the essential biological role of SOD. SOD-deficient mutants constitute a powerful tool to assess a possible role of O-2 or SOD in biological processes. Complementation of their deficiencies by the expression of SOD originating from a different organism is used for screening libraries for SOD genes of other species. Regulation of MnSOD has been studied using protein and operon fusions with the lactose operon, and isolating regulation mutants. These studies reveal multiregulation of MnSOD including response to the superoxide mediated oxidative stress and response to variations of the intracellular redox state induced by metabolic changes.

Bacterial Proteins↗
PubMed1989

Mutations by near-ultraviolet radiation in Escherichia coli strains lacking superoxide dismutase.

In wild-type Escherichia coli, near-ultraviolet radiation (NUV) was only weakly mutagenic. However, in an allelic mutant strain (sodA sodB) that lacks both Mn- and Fe-superoxide dismutase (SOD) and assumed to have excess superoxide anion (O2-), NUV induced a 9-fold increase in mutation above the level that normally occurs in this double mutant. When a sodA sodB double mutant contained a plasmid carrying katG+ (excess HP-I catalase), mutation by NUV was reduced to wild-type (sodA+ sodB+) levels. Also, in the sodA sodB xthA triple mutant, which lacks exonuclease III (exoIII) in addition to SOD, the mutational frequency by NUV was reduced to wild-type levels. This synergistic action of NUV and O2- suggested that pre-mutational lesions occur, with exoIII converting these lesions to stable mutants. Exposure to H2O2 induced a 2.8-fold increase in mutations in sodA sodB double mutants, but was reduced to control levels when a plasmid carrying katG+ was introduced. These results suggest that NUV, in addition to its other effects on cells, increases mutations indirectly by increasing the flux of OH. radicals, possibly by generating excess H2O2.

DNA Damage↗