Oxidative and SOS-response in Escherichia coli and their interference]

Interference between the oxidative and SOS responses in Escherichia coli was studied. The oxidative response involves both reactive oxygen scavenging system and DNA repair systems which are distinct from either the SOS or adaptive response to alkylating agents. The oxyR gene is a positive regulatory gene for the oxidative response and controls at least 9 proteins which are induced by treatment with H2O2. This gene is not a portion of the SOS regulon that involves at least 17 different genes in E. coli and controls the SOS response--another inducible and nonspecific repair activity. The SOS response was measured in E. coli PQ37 by means of a sfiA: :lacZ operon fusion according to "SOS Chromotest" in a completely automated system "Bioscreen C" (Labsystems, Finland). Our data have shown that: 1) H2O2 was a potent inducer of sfiA gene--one of the SOS genes; 2) there was strong negative effect of the oxidative response on the subsequent induction of the SOS response. In common with our previous findings it should be concluded that there is an interference between the SOS response--on the one hand, and the adaptive and oxidative responses--on the other. The nonspecific heat shock response is proposed to be a main key in these interferences.     

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.     

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.     

Alkylating Agents Induce Uvm, a Reca-Independent Inducible Mutagenic Phenomenon in Escherichia Coli

Noninstructive DNA damage in Escherichia coli induces SOS functions hypothesized to be required for mutagenesis and translesion DNA synthesis at noncoding DNA lesions. We have recently demonstrated that in E. coli cells incapable of SOS induction, prior UV-irradiation nevertheless strongly enhances mutagenesis at a noninstructive lesion borne on M13 DNA. Here, we address the question whether this effect, named UVM for UV modulation of mutagenesis, can be induced by other DNA damaging agents. Exponentially growing δrecA cells were pretreated with alkylating agents before transfection with M13 single-stranded DNA bearing a site-specific ethenocytosine residue. Effect of cell pretreatment on survival of the transfected DNA was determined as transfection efficiency. Mutagenesis at the ethenocytosine site in pretreated or untreated cells was analyzed by multiplex DNA sequencing, a phenotype-independent technology. Our data show that 1-methyl-3-nitro-1-nitrosoguanidine, N-nitroso-N-methylurea and dimethylsulfate, but not methyl iodide, are potent inducers of UVM. Because alkylating agents induce the adaptive response to defend against DNA alkylation, we asked if the genes constituting the adaptive response are required for UVM. Our data show that MNNG induction of UVM is independent of ada, alkA and alkB genes and define UVM as an inducible mutagenic phenomenon distinct from the E. coli adaptive and SOS responses.     

Role of mismatch repair in the Escherichia coli UVM response.

Mutagenesis at 3,N4-ethenocytosine (epsilonC), a nonpairing mutagenic lesion, is significantly enhanced in Escherichia coli cells pretreated with UV, alkylating agents, or H2O2. This effect, termed UVM (for UV modulation of mutagenesis), is distinct from known DNA damage-inducible responses, such as the SOS response, the adaptive response to alkylating agents, or the oxyR-mediated response to oxidative agents. Here, we have addressed the hypothesis that UVM results from transient depletion of a mismatch repair activity that normally acts to reduce mutagenesis. To test whether the loss of mismatch repair activities results in the predicted constitutive UVM phenotype, E. coli cells defective for methyl-directed mismatch repair, for very-short-patch repair, or for the N-glycosylase activities MutY and MutM were treated with the UVM-inducing agent 1-methyl-3-nitro-1-nitrosoguanidine, with subsequent transfection of M13 viral single-stranded DNA bearing a site-specific epsilonC lesion. Survival of the M13 DNA was measured as transfection efficiency, and mutation fixation at the lesion was characterized by multiplex sequencing technology. The results showed normal UVM induction patterns in all the repair-defective strains tested. In addition, normal UVM induction was observed in cells overexpressing MutH, MutL, or MutS. All strains displayed UVM reactivation, the term used to describe the increased survival of epsilonC-containing DNA in UVM-induced cells. Taken together, these results indicate that the UVM response is independent of known mismatch repair systems in E. coli and may thus represent a previously unrecognized misrepair or misreplication pathway.     

The Escherichia coli K-12 SOS chromotest agar spot test for simple, rapid detection of genotoxic agents

The Escherichia coli K-12 SOS chromotest is a colorimetric (beta-galactosidase induction) system for detecting genotoxic chemicals as agents which induce filamentation in response to DNA damage. The chromotest was modified from a liquid suspension assay to a simple, convenient agar spot test, which was performed in the manner of a related colorimetric prophage induction assay (BIA). Chromotest agar dishes yielded optimal results after 16-18 h incubation, presumably because of the agar growth characteristics of tester strain PQ37. Of 44 tested chemicals, nitro aromatics, cytotoxic/antitumor agents, polycyclic hydrocarbons and aflatoxins showed good activity. Alkylating agents such as MNNG and MMS were active only at high concentrations. Compounds active in both the chromotest and BIA were active at 10-100-fold lower concentrations in the chromotest. The chromotest appeared to be less effective than the Salmonella Ames mutagenicity test in the detection of diverse classes of chemical carcinogens. The chromotest may be a useful alternative to the BIA in the study of particular classes of genotoxic compounds.     

A comparison of the relative activities of 8 radiosensitizers in the SOS chromotest

Misonidazole, and RSU 1069 and 6 of its analogues are all reported to show increased cytotoxicity towards hypoxic cells compared to oxic cells. DNA is considered to be the target through which these drugs exert their cytotoxic activity. Therefore we monitored induction of the SOS response in uvrABC excinuclease proficient and deficient strains of E. coli, under oxic and hypoxic conditions, as an indirect method of assessing the activity of these drugs towards DNA in a biological system. This was done using the SOS chromotest which utilizes E. coli strains which possess a sfiA::lacZ fusion allowing induction of the SOS response to be monitored by assaying beta-galactosidase activity. All of the drugs tested here show some induction of the SOS response in both uvrABC excinuclease proficient and deficient strains. Data shown here suggests that the uvrABC excinuclease is important in the production of a SOS induction signal from RSU 1069-induced DNA lesions and that RSU 1069 may act as a crosslinking agent. The data also shows that SOS induction activity and toxicity do not necessarily correlate and that production of a SOS induction signal may occur via a different pathway for RSU 1069 than for its analogues.     

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.     

Structural requirement for the induction of the adaptive response in Escherichia coli among N-(substituted alkyl)-N-nitrosoureas

Using E. coli CSH26 transformed with a plasmid carrying an alkA'-lacZ' fused gene, a series of N-(substituted alkyl)-N-nitrosoureas were subjected to a colorimetric assay to evaluate their capacity to induce the adaptive response, an inducible DNA-repair network in E. coli. Some of these derivatives induced the response in greater or lesser degrees, while others did not. Several structural requirements for the induction were disclosed. The capacity of these derivatives to induce the SOS response, which is another inducible DNA-repair network, was also evaluated using E. coli transformed with a plasmid carrying a umuC'-lacZ' fused gene. Since all the derivatives induced the SOS response, the structural requirements for the adaptive response disclosed in this study are substantially related to the molecular mechanism involved in the adaptive response.     

Induction of the SOS response in Escherichia coli cells under osmotic stress and in the presence of N-methyl-N'-nitro-N-nitrosoguanidine

We investigated the dynamics of the SOS response induction and the frequency of reversions induced by the monofunctional alkylating compound N-methyl-N'-nitro-N-nitrosoguanidine in Escherichia coli cells exposed to osmotic stress for 1 h. During the stress treatment of the wild-type cultures adapted and not adapted to the alkylating agent, the maximum SOS response values and induced reversion frequencies were recorded twice. The SOS response values and induced reversion frequencies remained unchanged during the whole period after attaining the maximum values in adapted and nonadapted cells carrying a mutation in the excision repair gene. Presumably, the SOS mutagenesis mechanisms are turned on in the cells with an inactivated excision repair system earlier than in wild-type cells.     

Functional recA, lexA, umuD, umuC, polA, and polB genes are not required for the Escherichia coli UVM response.

The Escherichia coli UVM response is a recently described phenomenon in which pretreatment of cells with DNA-damaging agents such as UV or alkylating agents significantly enhances mutation fixation at a model mutagenic lesion (3,N4-ethenocytosine; epsilon C) borne on a transfected M13 single-stranded DNA genome. Since UVM is observed in delta recA cells in which SOS induction should not occur, UVM may represent a novel, SOS-independent, inducible response. Here, we have addressed two specific hypothetical mechanisms for UVM: (i) UVM results from a recA-independent pathway for the induction of SOS genes thought to play a role in induced mutagenesis, and (ii) UVM results from a polymerase switch in which M13 replication in treated cells is carried out by DNA polymerase I (or DNA polymerase II) instead of DNA polymerase III. To address these hypotheses, E. coli cells with known defects in recA, lexA, umuDC, polA, or polB were treated with UV or 1-methyl-3-nitro-1-nitrosoguanidine before transfection of M13 single-stranded DNA bearing a site-specific ethenocytosine lesion. Survival of the transfected DNA was measured as transfection efficiency, and mutagenesis at the epsilon C residue was analyzed by a quantitative multiplex DNA sequencing technology. Our results show that UVM is observable in delta recA cells, in lexA3 (noninducible SOS repressor) cells, in LexA-overproducing cells, and in delta umuDC cells. Furthermore, our data show that UVM induction occurs in the absence of detectable induction of dinD, an SOS gene. These results make it unlikely that UVM results from a recA-independent alternative induction pathway for SOS gene.     

Influence of dam and mismatch repair system on mutagenic and SOS-inducing activity of methyl methanesulfonate in Escherichia coli

In contrast to earlier reports (Mohn et al., 1980; Glickman, 1982), we show that E. coli dam- cells are able to mutate following MMS treatment. Since the mutagenicity of MMS has been regarded as largely dependent on induction of the SOS functions, E. coli strains bearing the recA::lacZ or umuC::lacZ fusions were used to determine the ability of MMS to induce the SOS functions in the various dam+ and dam- strains. The mutagenicity of MMS was also tested in several of these strains. The results show that (i) there is no direct correlation between SOS-inducing ability and mutagenicity potency of MMS; and (ii) most of the premutagenic lesions induced by MMS are removed from DNA of dam+ or dam- cells by the mismatch repair system. The role of strand breaks in repair of mismatches induced by alkylating agents is discussed.     

Anti-genotoxic and free-radical scavenging activities of extracts from (Tunisian) Myrtus communis

The effect of extracts from leaves of Myrtus communis on the SOS reponse induced by Aflatoxin B1 (AFB1) and Nifuroxazide was investigated in a bacterial assay system, i.e. the SOS chromotest with Escherichia coli PQ37. Aqueous extract, the total flavonoids oligomer fraction (TOF), hexane, chloroform, ethyl acetate and methanol extracts and essential oil obtained from M. communis significantly decreased the SOS response induced by AFB1 (10 microg/assay) and Nifuroxazide (20 microg/assay). Ethyl acetate and methanol extracts showed the strongest inhibition of the induction of the SOS response by the indirectly genotoxic AFB1. The methanol and aqueous extracts exhibited the highest level of protection towards the SOS-induced response by the directly genotoxic Nifuroxazide. In addition to anti-genotoxic activity, the aqueous extract, the TOF, and the ethyl acetate and methanol extracts showed an important free-radical scavenging activity towards the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical. These results suggest the future utilization of these extracts as additives in chemoprevention studies.     

Para-aminobenzoic acid inhibits a set of SOS functions in Escherichia coli K12

PABA - Vitamin H1 of group B, has obtained increasing fundamental interest as a very potent natural antimutagen after a series of our publications since 1979. In the first set of our experiments, we studied PABA in the assays with the alkylating agent N-methyl-N-nitrosourea (MNU). Mutagenic efficiency of this agent was suppressed up to 10-fold when PABA was administered into Escherichia coli cells concurrently with the mutagen or prior to the mutagenic treatment. NMR spectrometric and UV-spectrophotometric measurements did not reveal an interaction between the direct acting MNU and PABA, typical for some N-nitroso compounds and phenolics. PABA suppressed the error-prone DNA repair pathway induced by UV-irradiation. PABA decreased MNU-induced phage lambda lysogenic induction more than two orders of magnitude. PABA inhibited the thermal shift up to 400-fold in phage lambda from the permissive to non-permissive temperature in E. coli mutant tif-1 and decreased about two-fold W-reactivation of UV-damaged phage lambda. Chloramphenicol treatment of the cells just after the mutagenic treatment prevented the occurrence of PABA specific activity. The results suggest that PABA affects the SOS DNA repair pathway and the mutagenic response of E. coli. PABA appears to be an effective bioantimutagen reducing mutagenesis by modulating the error-prone DNA repair (SOS) response.     

Mutagenesis and repair of DNA damage caused by nitrogen mustard, N,N'-bis(2-chloroethyl)-N-nitrosourea (BCNU), streptozotocin, and mitomycin C in E. coli

Cytotoxicity and mutagenesis by streptozotocin, BCNU, nitrogen mustard, and mitomycin C were evaluated in E. coli mutants deficient in SOS repair, SOS-mediated mutagenesis, the adaptive response, and mutants that engage in aberrant mismatch repair. The results demonstrate that premutagenic lesions are caused by nitrogen mustard, BCNU and streptozotocin that are not repaired by ada or recognized by umuDC. Further, recA mutants were hypomutable after exposure to nitrogen mustard, BCNU, and streptozotocin compared to wild type. With the exception of the monofunctional nitrosourea, streptozotocin, both recA and uvrA gene products contribute to the repair of DNA damage caused by the alkylating agents tested. In the case of streptozotocin, although recA mutants were more sensitive than wild type, uvrA mutants were not. Moreover, while ada and alkA E. coli mutants showed increased sensitivity to streptozotocin, they were not more sensitive to the other alkylating agents evaluated.     

Evaluation of the SOS chromotest

In the present investigation, the SOS chromotest with E. coli PQ37 was evaluated. The potential to identify different kinds of bacterial mutagens was examined. 124 chemicals of different chemical classes were tested. Their responses in the SOS chromotests were compared to reported test results obtained with the Ames test.     

Mutagenic effect on Escherichia coli bacteria of 8-hydroxy-2'-deoxyguanosine--a DNA base damage product induced by oxygen radicals and ionizing radiation]

The effect of 8-oxo-2'-deoxyguanosine (8-oxo-dG) (8-hydroxydeoxyguanosine)--a DNA base damage product induced by oxygen radicals and irradiation on survival and mutagenesis in Escherichia coli strains C-600 and P-687 was investigated. Survival and mutagenesis curves, in dependence of 8-oxo-dG concentrations in the medium, ranging from 0.2 through 10 mM, were obtained. Bacterial survival at all 8-oxo-dG concentrations tested was shown to be no lesser than in the control. The mutagenic effect of 8-oxo-dG was tested by frequency of reversions in the absence of leucine and threonine. A non-linear dependence of mutagenesis on the concentration was observed. Linear increase in the amount of revertants took place at concentrations of 8-oxo-dG lower than 1 mM, and being kept constant at higher concentrations. Induction of SOS repair under the action of 8-oxo-dG in E. coli PQ37 strain was estimated according to alteration of activity of beta-galactosidase in the SOS chromotest. Weak induction of the SOS response was observed within the wide range of 8-oxo-dG concentration values, which points to a lack of genotoxicity and independence of mutagenesis on SOS repair.     

Expression of DNA damage-inducible genes of Escherichia coli upon treatment with methylating, ethylating and propylating agents

Several alkylation-inducible genes have been identified by construction of Mu-d1 (Apr lac) fusions to genes whose expression is increased in response to alkylation treatment, but not UV treatment. We have examined the induction of 4 different alkylation-inducible genes by treatment with a variety of methylating and ethylating agents, and a propylating agent. We have compared the induction of the alkylation-inducible genes with the induction of the sulA gene, which is a component of the SOS response to DNA damage. We find that the Ada-regulated adaptive response genes (ada-alkB, alkA and aidB) are induced primarily in response to methylation treatment. The ada-independent aidC gene is induced upon treatment with agents that alkylate predominantly by SN1 nucleophilic attack. aidC induction occurs only when cells are not aerated during treatment. The SOS response, as indicated by sulA induction, is strongly induced by all types of alkylating agents used.