Heat shock inhibits the induced expression of the SOS genes and SoxRS regulons in Escherichia coli

Oxidative stress formed in Escherichia coli cells is known to bring about a complex induction of alternative DNA repair processes, including SOS, SoxRS, and heat-shock response (HSR). The modification by heat shock of the expression of sfiA and soxS genes induced by oxidative agents H2O2, menadione and 4-nitroquinoline-1-oxide (4NQO) was studied for the first time. Quantitative parameters of gene expression were examined in E. coli strains with fused genes (promoters) sfiA::lacZ and soxS::lacZ. The expression of these genes induced by cell treatment with H2O2, but not menadione or 4NQO, was shown to decrease selectively after exposure to heat shock. Since genetic activity of menadione and 4NQO depends mainly on the formation of superoxide anion O2-, it is assumed that the effect of selective inhibition by heat-shock of sfiA and soxS gene expression in experiments with H2O2 is connected with activity of DnaK heat shock protein, which, unlike other heat-shock proteins, cannot be induced by superoxide anion O2-.     

Induction of the SOS response by hydrogen peroxide in various Escherichia coli mutants with altered protection against oxidative DNA damage.

The induction of the SOS response by H2O2 was measured in Escherichia coli by means of a sfiA::lacZ operon fusion. The effects of mutations in genes involved in DNA repair or DNA metabolism on the SOS response were investigated. We found that in an uvrA mutant, H2O2 induced the SOS response at lower concentrations than in the uvr+ parent strain, indicating that some lesions induced by H2O2 may be repaired by the uvrABC-dependent excision repair system. A nth mutation, yielding deficiency in thymine glycol DNA glycosylase, had no detectable effect on SOS induction, indicating that thymine glycol, a DNA lesion expected to be induced by H2O2, does not participate detectably in the induction of the SOS response by this chemical under our conditions. H2O2 still induced the SOS response in a dnaC(Ts) uvrA double mutant under conditions in which no DNA replication proceeds, suggesting that this chemical induces DNA strand breaks. Induction of the SOS response by H2O2 was also assayed in various mutants affected in genes suspected to be important for protection against oxidative stress. Mutations in the catalase genes, katE and katG, had only minor effects. However, in an oxyR deletion mutant, in which the adaptative response to H2O2 does not occur, SOS induction occurred at much lower H2O2 concentrations than in the oxyR+ parent strain. These results indicate that some enzymes regulated by the oxyR gene are, under our conditions, more important than catalase for protection against the H2O2-induced DNA damages which trigger the SOS response.     

Interplay of SOS induction,recombinant gene expression,and multimerization of plasmid vectors in Escherichia coli

Using pBR322- and pUC-derived plasmid vectors, a homologous (Escherichia coli native esterase) and three heterologous proteins (human interleukin-2, human interleukin-6, and Zymomonas levansucrase) were synthesized in E. coli IC2015(recA::lacZ) and GY4786 (sfiA::lacZ) strains. Via time-course measurement of beta-galactosidase activity in each recombinant culture, the SOS induction was estimated in detail and the results were systematically compared. In recombinant E. coli, the SOS response did not happen either with the recombinant insert-negative plasmid backbone alone or the expression vectors containing the homologous gene. Irrespective of gene expression level and toxic activity of synthesized foreign proteins, the SOS response was induced only when the heterologous genes were expressed using a particular plasmid vector, indicating strong dependence on the recombinant gene clone and the selection of a plasmid vector system. It is suggested that in recombinant E. coli the SOS response (i.e., activation of recA expression and initial sfiA expression) may be related neither to metabolic burden nor toxic cellular event(s) by synthesized heterologous protein, but may be provoked by foreign gene-specific interaction between a foreign gene and a plasmid vector. Unlike in E. coli XL1-blue(recA(-)) strains used, all expression vectors encoding each of the three heterologous proteins were multimerized in E. coli IC2015 strains in the course of cultivation, whereas the expression vectors containing the homologous gene never formed the plasmid multimers. The extent of multimerization was also dependent on a foreign gene insert in the expression vector. As a dominant effect of the SOS induction, recombinant plasmid vectors used for heterologous protein expression appear to significantly form various multimers in the recA(+) E. coli host.     

Induction of SOS and adaptive responses by alkylating agents in Escherichia coli mutants deficient in 3-methyladenine-DNA glycosylase activities

The induction of SOS and adaptive responses by alkylating agents was studied in Escherichia coli mutants tagA and alkA deficient in 3-methyladenine-DNA glycosylase activities. The SOS response was measured using an sfiA::lacZ operon fusion. The sfiA operon, in the double mutant tagA alkA, is induced at 5-50-fold lower concentrations of all tested methylating and ethylating compounds, as compared to the wild-type strain. In all cases, the tagA mutation, which inactivates the constitutive and specific 3-alkyladenine-DNA glycosylase I (TagI), sensitizes the strain to the SOS response. The sensitization effect of alkA mutation, which inactivates the inducible 3-alkyladenine-DNA glycosylase II (TagII), is observed under conditions which allow the induction of the adaptive response. We conclude that the persistence of 3-methyladenine and 3-ethyladenine residues in DNA most likely leads to the induction of the SOS functions. In contrast, the adaptive response, evaluated by O6-methylguanine-DNA methyltransferase activity in cell extracts, was not affected by either tagA or alkA mutations. The results suggest that the SOS and adaptive responses use different alkylation products as an inducing "signal". However, adaptation protein TagII inhibits the induction of the SOS response to some extent, due to its action at the level of signal production. Finally, we provide conditions to improve short-term bacterial tests for the detection of genotoxic alkylating agents.     

Mutagenesis and stress responses induced in Escherichia coli by hydrogen peroxide

Killing of Escherichia coli by hydrogen peroxide proceeds by two modes. Mode one killing appears to be due to DNA damage, has a maximum near 1 to 3 mM H2O2, and requires active metabolism during exposure. Mode two killing is due to uncharacterized damage, occurs in the absence of metabolism, and exhibits a classical multiple-order dose-response curve up to at least 50 mM H2O2 (J. A. Imlay and S. Linn, J. Bacteriol. 166:519-527, 1986). H2O2 induces the SOS response in proportion to the degree of killing by the mode one pathway, i.e., induction is maximal after exposure to 1 to 3 mM H2O2. Mutant strains that cannot induce the SOS regulon are hypersensitive to peroxide. Analysis of the sensitivities of mutants that are deficient in individual SOS-regulated functions suggested that the SOS-mediated protection is due to the enhanced synthesis of recA protein, which is rate limiting for recombinational DNA repair. Specifically, strains wholly blocked in both SOS induction and DNA recombination were no more sensitive than mutants that are blocked in only one of these two functions, and strains carrying mutations in uvrA, -B, -C, or -D, sfiA, umuC or -D, ssb, or dinA, -B, -D, -F, -G, -H, -I, or -J were not abnormally sensitive to killing by H2O2. After exposure to H2O2, mutagenesis and filamentation also occurred with the dose response characteristic of SOS induction and mode one killing, but these responses were not dependent on the lexA-regulated umuC mutagenesis or sfiA filamentation functions, respectively. Exposure of E. coli to H2O2 also resulted in the induction of functions under control of the oxyR regulon that enhance the scavenging of active oxygen species, thereby reducing the sensitivity to H2O2. Catalase levels increased 10-fold during this induction, and katE katG mutants, which totally lack catalase, while not abnormally sensitive to killing by H2O2 in the naive state, did not exhibit the induced protective response. Protection equal to that observed during oxyR induction could be achieved by the addition of catalase to cultures of naive cells in an amount equivalent to that induced by the oxyR response. Thus, the induction of catalase is necessary and sufficient for the observed oxyR-directed resistance to killing by H2O2. Although superoxide dismutase appeared to be uninvolved in this enhanced protective response, sodA sodB mutants, which totally lack superoxide dismutase, were especially sensitive to mode one killing by H2O2 in the naive state. gshB mutants, which lack glutathione, were not abnormally sensitive to killing by H2O2.     

3-Methyladenine residues in DNA induce the SOS function sfiA in Escherichia coli.

The induction by methylating agents of the SOS function sfiA was measured by means of a sfiA::lac operon fusion in Escherichia coli mutants defective in alkylation repair. The sfiA operon was turned on at a 10-fold lower concentration of methylmethane sulfonate or dimethyl sulfate in tagA strains, lacking specific 3-methyladenine-DNA glycosylase, than in wild-type strains. In contrast, the induction of sfiA by u.v. light was not affected by a tagA mutation. We confirm that tagA strains specifically accumulate 3-methyladenine in their DNA. We conclude that the persistence of 3-methyladenine in E. coli DNA most likely induces the SOS functions. Results on in vitro DNA synthesis further suggest that this induction is due to an unscheduled arrest of DNA synthesis at this lesion.     

The induction of SOS function in Escherichia coli K-12/PQ37 by 4-nitroquinoline oxide (4-NQO) and fecapentaenes-12 and -14 is bile salt sensitive: implications for colon carcinogenesis

The response of Escherichia coli to genotoxic agents involves the triggering of a complex system of genes known as the SOS response. In E. coli PQ37, a test organism used for the assessment of genotoxicity, lacZ, the beta-galactosidase gene is placed under the control of sfiA, one of the SOS genes through an operon fusion. The induction of beta-galactosidase activity, when the organism is exposed to genotoxic agents, is an indirect measure of the genotoxic activity of the test compound. Incubation of E. coli PQ37 with either 4-nitroquinoline oxide (4-NQO) or one of the fecal mutagens, fecapentaene-12 or -14 (F-12 or F-14) in the presence of sodium taurocholate or sodium deoxycholate resulted in a significant enhancement of induction of beta-galactosidase activity. The molecular mechanisms of 4-NQO-induced mutagenesis in E. coli are similar to those of the effects of UV light in which both replication-dependent and repair-dependent pathways of mutagenesis exist. Since E. coli PQ37 is excision-repair-deficient, alternate pathways are involved in this system. Bile salts by themselves do not trigger the SOS response, and hence their role in enhancing the SOS-inducing potency of mutagens may involve the potentiation of the cleavage-inactivation of lexA (repressor of SOS) by the protein product of the SOS-controlled gene, recA. The potentiating effect of bile salts on the fecal mutagens, F-12 and F-14, has implications in their suspected role in colon carcinogenesis associated with high-fat, low-fiber diets.     

The role of putrescine in of oxidative stress defense genes expression regulation in Escherichia coli

The role of putrescine in the adaptive response of Escherichia coli grown aerobically in synthetic M9 medium with glucose to the H2O2-induced oxidative stress was studied. Under oxidative stress, the expression of the single-copy reporter gene fusions oxyR::lacZ and katG::lacZ was found to undergo biphasic changes, which were most pronounced in glucose-starved E. coli cells. The concentration-dependent activating effect of putrescine on the expression of the oxyR regulon genes was maximum when the oxyR gene was inhibited by high concentrations of hydrogen peroxide.     

Induction of the SOS response by hydroxyurea in Escherichia coli K12

Hydroxyurea at concentrations higher than 10(-2) M induced the recA and sfiA genes of E. coli as well as the lambda prophage by a pathway independent of the recBC genes. In addition, the hydroxyurea-mediated induction of the SOS response is accompanied by a recA-dependent decrease on the cellular ATP pool. The presence of the multicopy plasmid pPS2, harboring the nrdAB genes (encoding the ribonucleoside reductase enzyme), abolished the hydroxyurea-induced expression of the recA gene. These data lead us to suggest that induction of the SOS response by hydroxyurea is due to the blocking of DNA replication by the inhibition of the ribonucleoside reductase complex activity.     

Role of nitric oxide in the SOS response in Escherichia coli

Having one electron with unpaired spin, nitric oxide (NO) shows high reactivity and activates or inhibits free radical chain reactions. NO toxic and genotoxic effects appear to be the result of intracellular formation of peroxinitrite that can induce some cellular damages, including DNA strand breaks, DNA base oxidation, destruction of the key enzymes, etc. Taking into account the character of DNA damages being formed under NO activity, we proposed a formation of the SOS signal and induction the SOS DNA repair response in E. coli cells treated with NO physiological donors--DNIC and GSNO. The ability of NO donor compounds to induce the SOS DNA response in E. coli PQ37 with sfiA::lacZ operon fusion is reported here at the first time. So, the SOS DNA repair response induction is one of the function of nitric oxide.     

Toxicity and mutagenicity of plumbagin and the induction of a possible new DNA repair pathway in Escherichia coli.

Actively growing Escherichia coli cells exposed to plumbagin, a redox cycling quinone that increases the flux of O2- radicals in the cell, were mutagenized or killed by this treatment. The toxicity of plumbagin was not found to be mediated by membrane damage. Cells pretreated with plumbagin could partially reactivate lambda phage damaged by exposure to riboflavin plus light, a treatment that produces active oxygen species. The result suggested the induction of a DNA repair response. Lambda phage damaged by H2O2 treatment were not reactivated in plumbagin-pretreated cells, nor did H2O2-pretreated cells reactivate lambda damaged by treatment with riboflavin plus light. Plumbagin treatment did not induce lambda phage in a lysogen, nor did it cause an increase in beta-galactosidase production in a dinD::Mu d(lac Ap) promoter fusion strain. Cells pretreated with nonlethal doses of plumbagin showed enhanced survival upon exposure to high concentrations of plumbagin, but were unchanged in their susceptibility to far-UV irradiation. polA and recA mutants were not significantly more sensitive than wild type to killing by plumbagin. However, xth-1 mutants were partially resistant to plumbagin toxicity. It is proposed that E. coli has an inducible DNA repair response specific for the type of oxidative damage generated during incubation with plumbagin. Furthermore, this response appears to be qualitatively distinct from the SOS response and the repair response induced by H2O2.     

Diesel emissions revisited: is the carcinogenicity due to a genotoxic mechanism?

The induction of recA, umuC and sfiA genes by quercetin was studied in the presence and in the absence of S9 mix. The inducing activity of quercetin is higher for sfiA than for recA and umuC genes in the absence of S9 mix. The putative genotoxic metabolites of quercetin produced by S9 mix display different inducing activities of the three SOS genes as compared to quercetin. The induction of sfiA gene is decreased by the presence of S9 mix, whereas an opposite effect was observed concerning umuC and recA. These data suggest that the error-prone repair pathway participates in mutagenesis by quercetin and its metabolites. Moreover, the type of DNA damage exerted by quercetin seems to be determined by its metabolic fate. The importance of testing for the induction of other SOS genes, together with sfiA, in the study of SOS functions as a genotoxic index is emphasized.     

Inhibition of the SOS response of Escherichia coli by the Ada protein.

Induction of the adaptive response by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) caused a decrease in the UV-mediated expression of both recA and sfiA genes but not of the umuDC gene. On the other hand, the adaptive response did not affect the temperature-promoted induction of SOS response in a RecA441 mutant. The inhibitory effect on the UV-triggered expression of the recA and sfiA genes was not dependent on either the alkA gene or the basal level of RecA protein, but rather required the ada gene. Furthermore, an increase in the level of the Ada protein, caused by the runaway plasmid pYN3059 in which the ada gene is regulated by the lac promoter, inhibited UV-mediated recA gene expression even in cells to which the MNNG-adaptive treatment had not been applied. This inhibitory effect of the adaptive pretreatment was not observed either in RecBC- strains or in RecBC mutants lacking exonuclease V-related nuclease activity. However, RecF- mutants showed an adaptive response-mediated decrease in UV-promoted induction of the recA gene.     

Induction of the SOS system in a dam-3 mutant: a diagnostic strain for chemicals causing DNA mismatches

We have developed a strain of E. coli in which expression of the SOS function sfiA, monitored by means of a sfiA::lacZ operon fusion, is efficiently triggered by the two base analogues 2-aminopurine and 5-bromo-2'-deoxyuridine. This strain resulted from introduction of a dam-3 mutation into a Uvr+, Rfa+ derivative of strain PQ37 used in the SOS chromotest, a bacterial colorimetric assay for genotoxins (Quillardet et al., 1982). The dam-3 mutation affects the mismatch correction system in E. coli. We show that the SOS-inducing capacity of a weak SOS inducer such as the alkylating agent ethyl methanesulfonate was also increased in the dam-3 strain. We provide evidence that the increase in SOS inducibility due to the dam-3 mutation is specific for compounds causing DNA mismatches and propose the use of the dam-3 derivative of PQ37 as a diagnostic strain for such agents. This diagnostic strain can be a useful addition to the SOS chromotest.     

Inducible SOS response system of DNA repair and mutagenesis in Escherichia coli

Chromosomal DNA is exposed to continuous damage and repair. Cells contain a number of proteins and specific DNA repair systems that help maintain its correct structure. The SOS response was the first DNA repair system described in Escherichia coli induced upon treatment of bacteria with DNA damaging agents arrest DNA replication and cell division. Induction of the SOS response involves more than forty independent SOS genes, most of which encode proteins engaged in protection, repair, replication, mutagenesis and metabolism of DNA. Under normal growth conditions the SOS genes are expressed at a basal level, which increases distinctly upon induction of the SOS response. The SOS-response has been found in many bacterial species (e.g., Salmonella typhimurium, Caulobacter crescentus, Mycobacterium tuberculosis), but not in eukaryotic cells. However, species from all kingdoms contain some SOS-like proteins taking part in DNA repair that exhibit amino acid homology and enzymatic activities related to those found in E. coli. but are not organized in an SOS system. This paper presents a brief up-to-date review describing the discovery of the SOS system, the physiology of SOS induction, methods for its determination, and the role of some SOS-induced genes.     

Survival and SOS induction in cisplatin-treated Escherichia coli deficient in Pol II,RecBCD and RecFOR functions

Cisplatin is a potent anticancer agent forming intrastrand-crosslinks in DNA. The efficacy of cisplatin in chemotherapy can be limited by the development of tumor resistances such as elevated DNA repair or damage tolerance. In Escherichia coli, cisplatin treatment causes induction of the SOS regulon resulting in elevated levels of DNA Pol II, DNA Pol IV, DNA Pol V, the cell division inhibitor SfiA (SulA), homologous recombination (HR) and DNA repair. In this work, the roles of Pol II and HR in facilitating resistance of E. coli to cisplatin are studied. SOS induction levels were measured by beta-galactosidase assays in cisplatin-treated and untreated E. coli PQ30 that has the lacZ gene fused to the sfiA promoter. Comparative studies were carried out with derivatives of PQ30 constructed by P1 transduction that have transposon insertions in the polB gene, the recB gene blocking the RecBCD pathway of HR and genes of the RecFOR pathway of HR. Resistance of E. coli strains to cisplatin as determined by plating experiments decreased in the following order: parent PQ30 strain, polB > recO, recR, recF > recB. Both the RecBCD and RecFOR pathways of HR are important for survival when E. coli is exposed to cisplatin, because treatment of double mutants deficient in both pathways reduced colony forming ability to 37% in 6-9min in comparison to 39-120min for single mutants. Pol II and RecF appear to function in two distinct pathways to initiate replication blocked due to damage caused by cisplatin because function of Pol II was required for survival in mutants deficient in the RecFOR pathway after 2h of cisplatin treatment. In contrast, Pol II was not required for survival in recB mutants. SOS induction was delayed in RecFOR deficient mutants but occurred at high levels in the recB mutant soon after cisplatin treatment in a RecFOR-dependent way. An SfiA independent, DNA damage dependent pathway is apparently responsible for the filamentous cells observed after cisplatin or MMC treatments of these SfiA defective strains.