Manuel Blanco

Mutagenicity of 80 chemicals in Escherichia coli tester strains IC203, deficient in OxyR, and its oxyR(+) parent WP2 uvrA/pKM101: detection of 31 oxidative mutagens.

Strain IC203, deficient in OxyR, and its oxyR(+) parent WP2 uvrA/pKM101 (denoted IC188) are the basis of a new bacterial reversion assay, the WP2 Mutoxitest, which has been used in the evaluation of 80 chemicals for oxidative mutagenicity. The following 31 oxidative mutagens were recognized by their greater mutagenic response in IC203 than in IC188: (1) peroxides: hydrogen peroxide (HP), t-butyl hydroperoxide (BOOH) and cumene hydroperoxide (COOH); (2) benzoquinones (BQ): 2-methyl-1,4-BQ, 2,6-dimethyl-1,4-BQ and 2,3, 5,6-tetramethyl-1,4-BQ; (3) naphthoquinones (NQ): 1,4-NQ, 2-methyl-1, 4-NQ and 2-hydroxy-1,4-NQ; (4) phenol derivatives: catechol, hydroquinone, pyrogallol, 1,2,4-benzenetriol, t-butylhydroquinone, gallic acid and 4-aminophenol; (5) catecholamines: DL- and L-dopa, DL- and L-epinephrine, dopamine and L-norepinephrine; (6) thiols: L-cysteine methyl ester, L-cysteine ethyl ester, L-penicillamine and dithiothreitol; (7) diverse: 3,4-dihydroxyphenylacetic acid, hypoxanthine and xanthine, both in the presence of xanthine oxidase, L-ascorbic acid plus copper (II) and phenazine methosulfate. Among these oxidative mutagens, 25 were found to be uniquely positive in IC203. With the exception of BOOH and COOH, mutagenesis by all oxidative mutagens was inhibited by catalase present in rat liver S9, indicating that it is mediated by HP generation, probably in autoxidation reactions. These catalase-sensitive oxidative mutagens were poor inducers of mutations derived from 8-oxoguanine lesions, whereas such mutations were efficiently induced by organic hydroperoxides. The results support the usefulness of incorporating IC203 in the bacterial battery for testing of chemicals. The well-characterized oxidative mutagens available with the use of the WP2 Mutoxitest may serve as a reference in studies on the genotoxicity of oxidative stress.

Repair mechanisms involved in prophage reactivation and UV reactivation of UV-irradiated phage λ

Abstract Survival of UV-irradiated phage λ is increased when the host is lysogenic for a homologous heteroimmune prophage such as λ imm434 (prophage reactivation). Survival can also be increased by UV-irradiating slightly the non-lysogenic host (UV reactivation). Experiments on prophage reactivation were aimed at evaluating, in this recombination process, the respective roles of phage and bacterial genes as well as that of the extent of homology between phage and prophage. To test whether UV reactivation was dependent upon recombination between the UV-damaged phage and cellular DNAs, lysogenic host cells were employed. Such hosts had thus as much DNA homologous to the infecting phage as can be attained. Therefore, if recombination between phage and host DNAs was involved in this repair process, it could clearly be evidenced. By using unexposed or UV-exposed host cells of the same type, prophage reactivation and UV reactivation could be compared in the same genetic background. The following results were obtained: (1) Prophage reactivation is strongly decreased in a host carrying recA mutations but quite unaffected by mutation lex-I known to prevent UV reactivation; (2) In the absence of the recA + function, the red + but not the int + function can substitute for recA + to produce prophage reactivation, although less efficiently; (3) Prophage reactivation is dependent upon the number of prophages in the cell and upon their degree of homology to the infecting phage. The presence in a recA host of two prophages either in cis (on the chromosome) or in trans (on the chromosome and on an episome) increases the efficiency of prophage reactivation; (4) Upon prophage reactivation there is a high rate of recombination between phage and prophage but no phage mutagenesis; (5) The rate of recombination between phage and prophage decreases if the host has been UV-irradiated whereas the overall efficiency of repair is increased. Under these conditions UV reactivation of the phage occurs as in a non-lysogen, as attested by the high rate of mutagenesis of the restored phage. These results demonstrate that UV reactivation is certainty not dependent upon recombination between two pre-existing DNA duplexes. The hypothesis is offered that UV reactivation involves a repair mechanism different from excision and recombination repair processes.

New Escherichia coli WP2 tester strains highly sensitive to reversion by oxidative mutagens.

New Escherichia coli strains have been added to the WP2 mutagenicity test for the specific detection of oxidative mutagens. Strain IC203 derives from WP2 uvrA/pKM101 and is highly sensitive to oxidative stress due to a deficiency in the OxyR function. Following exposure to t-butyl hydroperoxide (BuOOH) or menadione (MD), but not to 4-nitroquinoline 1-oxide (4NQO), strain IC203 (oxyR) shows increased mutability with respect to the oxyR+ parent. The advantage that the OxyR deficiency confers on IC203 strain in detecting oxidative mutagens is not obtained with strains deficient in either katG or ahpCF, two OxyR-regulated genes. Strain IC206, a derivative of WP2 uvrA carrying a deletion of the umuDC genes and deficient in the MutY glycosylase, has also been added to the WP2 test for the detection of SOS-independent mutations promoted by 8-oxoguanine lesions. Induction of these mutations was observed after treatment with BuOOH, but not after MD or 4NQO exposure. The two new strains, IC203 and IC206, can be useful for the screening of mutations resulting from oxidative stress as well as in studies on antioxidants preventing mutagenesis.

Different efficiency of UmuDC and MucAB proteins in UV light induced mutagenesis in Escherichia coli

SummaryTwo multicopy plasmids carrying either the umuDC or the mucAB operon were used to compare the efficiency of UmuDC and MucAB proteins in UV mutagenesis of Escherichia coli K12. It was found that in recA+uvr+bacteria, plasmid pIC80, mucAB+mediated UV mutagenesis more efficiently than did plasmid pSE117, umuDC+. A similar result was obtained in lexA51(Def) cells, excluding the possibility that this was due to a differential regulation by LexA of the umuDC and mucAB operons. We conclude that some structural characteristic of the UmuDC and MucAB proteins determines their different efficiency in UV mutagenesis. This characteristic could be also responsible for the observation that in the recA430 mutant, pIC80 but no pSE117 can mediate UV mutagenesis. In the recA142 mutant, pIC80 also promoted UV mutagenesis more efficiently than pSE117. In this mutant, the recombination proficiency, the protease activity toward LexA and the mutation frequency were increased by the presence of adenine in the medium. In recA+uvrB5 bacteria, plasmid pSE117,umuDC caused both an increase in UV sensitivity as well as a reduction in the mutation frequency. These nagative effects resulting from the overproduction of UmuDC proteins were higher in recA142 uvrB5 than in recA+uvrB5 cells. In contrast, overproduction of MucAB proteins in excision-deficient bacteria containing pIC80 led to a large increase in the mutation frequency. We suggest that the functional differences between UmuDC and MucAB proteins might be due to their different dependence on the direct role of RecA protease in UV mutagenesis.

Mutants of Escherichia coli K12 (?)+ non-inducible by thymine deprivation: I. Method of isolation and classes of mutants obtained

A selective enrichment method based upon differential killing by thymine deprivation of inducible as compared to non-inducible lysogens was employed to isolate mutants of Escherichia coli K 12(λ)+ deficient in lysogenic induction. The efficiency of the method is such that about 1% of the surviving colonies are resistant to thymineless induction.SummaryA selective enrichment method based upon differential killing by thymine deprivation of inducible as compared to non-inducible lysogens was employed to isolate mutants of Escherichia coli K 12(λ)+ deficient in lysogenic induction. The efficiency of the method is such that about 1% of the surviving colonies are resistant to thymineless induction.About half of the mutants are recA recombination-deficient. Two other classes of non-inducible Rec+ mutants can be distinguished. No temperature conditional bacterial mutations could be obtained.Our results demonstrate that deficiency in the recA gene product is not the only bacterial factor which prevents lysogenic induction.

E. coli K12 inf: A mutant deficient in prophage λ induction and cell filamentation

SummaryThe bacterial mutant inf-3 (λ) is not inducible and does not form filaments following thymine starvation. Lysogenic induction is neither produced by ultraviolet light (UV) nor promoted by tif-1. This phenotype is due to a mutation infA3 located between 60 and 73 min on the E. coli K12 map.The inf mutant is resistant to X-ray and UV irradiation, in contrast to all other known non-inducible bacterial mutants. It is Rec+ and able to perform host cell reactivation as well as UV-reactivation of phage λ. After exposure to UV light, its DNA is degraded more than that of the parent and the resumption of DNA synthesis is delayed by 30 min; nevertheless, the cell survival is analogous to that of the parent. The inf mutant is also resistant to thymine starvation, for at least 3 hours.Wild type phage λ forms clear plaques on a lawn of non-lysogenic inf bacteria; a corresponding low level of lysogenization is found. The capacity of inf bacteria to reproduce phages λ, T4 or T6 is impaired.No gross defect in DNA transcription has been detected. Nevertheless, this mutant might have a slight alteration in the transcription process or in any other process involved in gene expression. This alteration might affect the regulation of DNA replication and cell division as well as prophage λ induction.

Specificity of spontaneous and t-butyl hydroperoxide-induced mutations in ΔoxyR strains of Escherichia coli differing with respect to the SOS mutagenesis proficiency and to the MutY and MutM functions

Mutations induced by oxidative DNA damage appear to occur by two pathways, differing in their dependence on SOS mutagenesis. We have analysed the specificity of mutations produced by each pathway. Base substitutions generating extragenic suppressors were characterized in Trp+ revertants of Escherichia coli strains carrying the trpE65 ochre mutation, which were hypersensitive to oxidative mutagenesis due to a deletion of the oxyR gene. In strain IC3821, containing MucA/B proteins and therefore proficient for SOS mutagenesis, the more frequently scored base substitutions, either spontaneous or induced by t-butyl hydroperoxide (BuOOH), were T:A-A:T transversions, followed by G:C-A:T transitions, while the frequency of G:C-T:A transversions was lower. This SOS-dependent mutability could be promoted by abasic sites. In strains IC3894 (mutY) and IC3981 (mutY mutM), lacking mutagenesis proteins, SOS-independent revertants arose almost exclusively via G:C-T:A transversions probably derived from oxidatively damaged 8-oxoguanine/adenine mispairs. Formation of these mispairs in IC3894 and IC3981 would be enhanced by BuOOH treatment since it caused a significant increase in the revertant number. Strains IC3894 and IC3981 could have a complementary role to that of IC3821 to analyse the mutagenicity and the mutational specificity of oxidants.

Influence of the recF143 mutation of Escherichia coli K12 on prophage λ induction

Prophage λ induction in a recF143 mutant of E. coli K12 was studied. The recF143 (λ) lysogen was inducible by UV irradiation or treatment with mitomycin C. However, the time required for the onset of derepression brought about by these treatments was longer in the recF143 mutant than in rec + strains, suggesting that the induction pathway was altered in the recF143 mutant. The rec143 (λ) lysogen was induced at very low doses of UV irradiation or mitomycin C treatment. Moreover, the presence of the recF143 mutation increased the sensitivity to thermal induction of a tif strain.

Screening for metabolites from Penicillium novae-zeelandiae displaying radical-scavenging activity and oxidative mutagenicity: isolation of gentisyl alcohol

In the search for new natural products with anti-oxidant activity, we have combined the cell-free assay based on the scavenging of the stable radical 2,2-diphenyl-1-picrylhydrazyl (DPPH), with a bioassay that detects oxidative mutagens. This bioassay uses a new Escherichia coli tester strain, IC203, specifically sensitive to oxidative stress due to a deficiency in the OxyR function. OxyR is a redox-sensitive transcriptional activator of genes encoding anti-oxidant enzymes such as catalase and peroxiredoxin alkyl hydroperoxide reductase. The positive response observed in E. coli IC203 with several known anti-oxidants, including cysteine, catechol and ascorbic acid, suggested to us the usefulness of the mutagenicity assay for a rapid screening of anti-oxidant compounds. The extract from Penicillium novae-zeelandiae was found to scavenge the DPPH radical. Subsequently, guided by the DPPH-scavenging assay and the oxidative mutagenesis assay, we isolated and identified three compounds in fractions from that active extract: patulin (1). 3-hydroxybenzyl alcohol (2). and gentisyl alcohol (2,5-dihydroxybenzyl alcohol) (3). Of these, gentisyl alcohol showed both DPPH-scavenging activity and oxidative mutagenicity. This compound also gave rise to intracellular formation of superoxide, evaluated by monitoring the oxidation of dihydroethidium, and was able to inhibit mutagenesis induced by the model oxidant t-butyl hydroperoxide (t-BuOOH).

Increased mutability by oxidative stress in OxyR-deficient Escherichia coli and Salmonella typhimurium cells: clonal occurrence of the mutants during growth on nonselective media

Escherichia coli and Salmonella typhimurium strains deficient in the OxyR-regulated adaptive response to oxidative stress were used to study the mode in which spontaneous SOS-dependent mutations are generated in a distressed bacterial population. When assayed on supplemented selective medium, the E. coli strain IC3821 (trpE65), carrying the delta oxyR30 mutation and containing the plasmid pRW144 (mucA/B), showed a frequency of spontaneous Trp+ revertants similar to that of the oxyR+ control. Instead, the IC3821 strain exhibited an enhancement in the clonal occurrence of spontaneous revertants arising at random during growth on a nonselective medium. A similar enhancement was observed for the S. typhimurium strain TA4125 (hisG428 delta oxyR2). The mutator effect observed in oxyR- cells would be induced by an increased background of reactive oxygen species; it provides a model for studying the mutability of a cell population constantly exposed to mutation-inducing agents. In the IC3821 strain, revertants were induced by t-butyl hydroperoxide with higher efficiency than in oxyR+. We suggest that strain IC3821 could be useful for the detection of SOS-dependent mutagenesis induced by chemical oxidants.