Plasmid pKM101-dependent repair and mutagenesis in Escherichia coli cells with mutations lexB30 tif and zab-53 in the recA gene

Bacterial survival after UV irradiation was increased in E. coli K12 lexB30 and tif zab-53 mutants harboring plasmid pKM101. Mutagenesis in response to UV was observed in these bacteria which, in absence of pKM101, are not UV-mutable. The mutator effect observed in unirradiated wild-type cells containing pKM101 was higher after incubation at 30°C with adenine than at 37°C. This effect was still enhanced by tif mutation, even in the tif zab-53 strain, but it was abolished by lexB30 mutation. In the tif zab-53 (pKM101) strain, repair and mutagenesis of UV-irradiated phage λ was observed, but not in the lexB30 mutant carrying pKM101. The pKM101 mutant, pGW1, was unable to protect tif zab-53 bacteria against killing by UV, whereas the protection of lexB30 was intermediate; moreover, it did not promote the mutator effect at 30°C or enhance phage repair and mutagenesis in tif zab-53 cells. All UV-induced bacterial mutations in lexB30 (pKM101) strain were suppressors; in contrast, true revertants were found after UV irradiation of the tif zab-53 (pKM101) cells.We suggest that the constitutive activity of RecA protein is enough for the production of UV-promoted suppressor mutations, whereas true reversions require a more active form of this protein which could exert its effects directly or by acting at a regulatory level on other cellular functions.     

Bacterial mutagenicity tests on 4-chloromethylbiphenyl and 2 structural analogues

4CMB, 4HMB and BC were tested in 5 strains of S. typhimurium and 2 strains of E. coli without S9. 4HMB was negative in all strains. 4CMB was a strong positive mutagen in TA1535, TA1537, TA1538, TA98, TA100 and WP2uvrA⁻(pKM101), and BC was a weak mutagen in TA100 and WP2uvrA⁻(pKM101). Positivity was determined as a dose response over 3 or more points, in repeat experiments, giving a significant correlation coefficient.     

Comparative bacterial mutagenicity studies with 8-methoxypsoralen and 4,5′,8-trimethylpsoralen in the presence of near-ultraviolet light and in the dark

2 strains of S. typhimurium, TA98 and TA100, and 2 strains of E. coli, WP2(pKM101) and WP2uvrA⁻(pKM101) were used to study mutagenesis by 8-methoxypsoralen (8-MOP) and 4,5′,8-trimethylpsoralen (4,5′,8-TMP) in the dark and in the presence of near-ultraviolet (NUV) light both without metabolic activation and with rat-liver S9 at 3 levels (4, 10 and 30% in standard cofactors).The S9-independent base substitution mutagenic activity of 8-MOP plus NUV light was confirmed in WP2(pKM101), and a similar activity was seen for 4,5′,8-TMP, although neither substance was active in TA100. The frameshift mutagenic activity of 8-MOP in the dark in TA98 was not confirmed despite histidine levels which would ensure DNA replication, but this may be due to the lower concentrations of 8-MOP achieved in the common solvent system adopted.Both 8-MOP and 4,5′,8-TMP were mutagenic in WP2uvrA⁻(pKM101) after microsomal activation, and the responses were similar whether experiments were conducted in the dark or in NUV light. In view of the oral administration of 8-MOP to psoriasis patients, this finding may be of relevance in risk assessment, and tends to suggest that topical application of 4,5′,8-TMP to psoriatic patients may present reduced risk of malignant disease.     

Localization of the plasmid (pKM101) gene(s) involved in recA+lexA+-dependent mutagenesis

Summary Twenty Tn5 insertion mutants of the drug resistance plasmid pKM101 have been isolated that are unable to enhance mutagenesis with ultraviolet (UV) irradiation or methyl methanesulfonate. By restriction mapping, the Tn5 insertion in each of these pKM101 mutants was shown to be within a 1.9 kb region of the plasmid genome. We have termed this segment of the pKM101 map the muc (mutagenesis: UV and chemical) gene(s). Characterization of these mutants indicated that any Tn5 insertion within the muc gene(s) abolished the ability of pKM101 to: (a) enhance spontaneous, UV and chemical mutagenesis, (b) increase host survival following UV-irradiation, (c) increase the survival of UV-irradiated phage plated on irradiated or unirradiated cells, and (d) suppress the repair and mutagenesis deficiencies of a umuC ^– mutant. Possible models to explain the role of the pKM101 muc gene(s) in mutagenesis and repair are discussed.     

Plasmid (pKM101)-mediated enhancement of repair and mutagenesis: Dependence on chromosomal genes in Escherichia coli K-12

Summary The drug resistance plasmid pKM101 plays a major role in the Ames Salmonella/microsome carcinogen detecting system by enhancing chemical mutagenesis. It is shown that in Escherichia coli K-12 the plasmid pKM101 enhances both spontaneous and methyl methanesulfonate-caused reversion of an ochre mutation, bacterial survival after ultraviolet irradiation, and reactivation of ultraviolet-irradiated in unirradiated cells. All these effects are shown to be dependent on the recA ⁺ lexA⁺ genotype but not on the recB ⁺ recC⁺ or recF ⁺ genotypes. The recA lexA-dependence of the plasmid-mediated repair and mutagenesis suggests an interaction with the cell's inducible error-prone repair system. The presence of pKM101 is shown to cause an additional increase in methyl methanesulfonate mutagenesis in a tif mutant beyond that caused by growth at 42°. The presence of the plasmid raises the level of the Weigle-reactivation curve for the reactivation of ultraviolet-irradiated in E. coli and causes a shift of the maximum to a higher UV fluence. These observations suggest that pKM101 does not exert its effects by altering the regulation of the cell's error-prone repair system but rather by supplying a mechanistic component or components.     

Frameshift mutagenesis by acridines in wild-type, uvrB and polA strains of Salmonella typhimurium with and without plasmid pKM101

A large range of acridines, including several anilinoacridines which are active as antitumour agents, have been studied for their ability to revert derivatives of Salmonella typhimurium strains carrying the frameshift marker hisC3076. The strains used all carried deep-rough (rfa) mutations, and were either wild-type with respect to DNA-repair capacity or carried uvrB, polA1 or polA3 (amber) mutations. Derivatives with and without the mutation-enhancing N group plasmid pKM101 were also used. 9-Aminoacridine and other acridines appeared similar to the anilinoacridines for the most part, in that frameshift mutagenesis and toxicity appeared to be unaffected by the uvrB mutation or by the presence of plasmid pKM101. Exceptions were ICR191, 3-NO2-acridine and 1- or 3-NO2-anilinoacridine derivatives in which mutagenesis was increased in uvrB strains and also when pKM101 was present. These compounds were slightly more toxic in the uvrB background, but less toxic when pKM101 was present in either the uvrB or wild-type backgrounds. Mutagenesis by most compounds was reduced by the polA1 mutation and virtually eliminated (except in the case of ICR191) by the polA3 mutation. Plasmid pKM101 occasionally enhanced mutagenesis in the polA1 strain, whereas in the polA3 it appeared to have no effect whatsoever. Again, there were no obvious differences in toxicity between Pol+ and Pol- strains.     

Frameshift mutagenesis by nitracrine analogues in wild-type, uvrB, polA and recA strains of Salmonella typhimurium, with and without plasmid pKM101

The mutagenic potential of 9-[(3-dimethylaminopropyl)amino]-acridine and its 1-, 2-, 3- and 4-nitro derivatives was studied in several strains of Salmonella typhimurium carrying the frameshift marker hisC3076. The strains all carried deep rough (rfa) mutations, and were either wild-type with respect to DNA repair capacity or carried recA, uvrB, polA1 or polA3 (amber) mutations. Derivatives with and without plasmid pKM101 were also studied. The des-nitro compound resembled 9 aminoacridine and other simple intercalating compounds. Both toxicity and mutagenesis were apparently unaffected by the uvrB and recA mutations or by the presence of plasmid pKM101. However, mutagenicity was reduced by the polA1 mutation, and virtually eliminated by the polA3 mutation. The drug was substantially more toxic in the latter, slightly more toxic in the former, of these polA- strains. Plasmid pKM101 enhanced mutagenesis and protected from toxicity in both polA1- and polA3- strains, although it did not restore either of these parameters to the level in the wild-type strain. The 2-nitro compound was generally similar to the des-nitro compound, except that it was considerably more toxic and apparently non-mutagenic in the recA-bearing strain. By contrast, mutagenicity of the 3- and 4-nitro compounds was enhanced by the uvrB mutation and by the presence of the plasmid. These compounds were highly toxic but non-mutagenic in the recA- strain, and showed some increased toxicity in polA1- and polA3- strains. The 1-nitro compound has been previously found to cross-link DNA. Unlike well-characterised cross-linkers such as mitomycin C it was highly mutagenic in the uvrB- strain, and this mutagenesis was enhanced by plasmid pKM101, but eliminated by the recA mutation. At high doses, where the drug was completely toxic towards uvrB- or recA-carrying strains, it became mutagenic in the DNA-repair-proficient strains. This 'high-dose' mutagenesis was enhanced by plasmid pKM101, but reduced by the polA1 mutation and almost eliminated by the polA3 mutation. Although there are several possible interpretations of these data, they are compatible with the suggestion that the lesion induced by high doses (but not by low doses) of nitracrine is a cross-link, but that this is not the major mutagenic lesion.     

Simultaneous mutations up to six distal sites using a phosphorylation-free and ligase-free polymerase chain reaction-based mutagenesis

In this study, we present an efficient phosphorylation-free and ligase-free PCR-based multiple site-directed mutagenesis that allows simultaneous mutations up to six distal sites. This method could be extended to any plasmid DNA that is isolated from dam⁺Escherichia coli strains, and the results showed that the simultaneously mutagenic efficiencies of quadruple mutation and sextuple mutation were up to 80% and 40%, respectively.     

The role of the excision and error-prone repair systems in mutagenesis by fluorinated quinolones in Salmonella typhimurium

Patterns of reversion produced by ciprofioxacin, enoxacin and ofloxacin in Salmonella typhimurium strains carrying the hisG428 ochre mutation have been studied. These fluorinated quinolones produce a significant increase in reversion of this mutation, even when it is located on the chromosome. Nevertheless, reversion is higher when the hisG428 mutation is on the multicopy plasmid pAQ1 than when it is on the chromosome. Reversion of hisG428 induced by fluorinated quinolones is abolished both in a uvrB genetic background and in the absence of the plasmid pKM101. Therefore, mutagenesis produced by fluorinated quinolones in the Salmonella mutagenicity assay is significantly affected by both the excision repair and the error-prone repair systems. Furthermore, fluorinated quinolones are also detected as moderate mutagens with the base substitution hisG46 mutation when both repair systems are functional in the tester strain.     

Effect of plasmids col I and pKM101, alone or in combination, on spontaneous and induced mutability of salmonellae

Comparative studies of plasmids col I and pKM101 effect on lethal and mutagenic response to UV-light and chemical agents (4NQ0, EMS, agent N012074) has been carried out in Salmonella strains used for screening of mutagens (potential carcinogens). It has been found that the plasmid pKM101 has more pronounced effect as compared with coll plasmid. Contrary to plasmid pKM101-mediated ability to form UV-induced frameshift mutation, colI factor lacks this ability and very slightly enhances the rate of frameshift mutagenesis induced by chemical agents under study. The colicinogenic factor is found to enhance only the rate of base-pair substitutions, whereas plasmid pKM101 enhances the rate of both base-pair substitutions and frameshift mutations. We were unable to demonstrate combined effect of these two plasmids on the rate of either spontaneous or induced mutations. Possible mechanisms of plasmid-mediated bacterial mutagenesis and repair are discussed.     

Mutagenicity and cytotoxicity of the carcinogen-mutagen aflatoxin B1 in Streptococcus pneumoniae (Pneumococcus) and Salmonella typhimurium: dependence on DNA repair functions

Strains R6, R6x and R6uvr-1 of Streptococcus pneumoniae (Pneumococcus) are sensitive to the cytotoxic effects of the mutagen/carcinogen aflatoxin B₁ (AFB₁). R6uvr-1 is more prone to the cytotoxic effects of AFB₁ than the repair-proficient parental strain, R6. The same differential susceptibility of strains R6, R6x and R6uvr-1 was observed when UV light replaced metabolically activated AFB₁. All pneumococcal strains were immutable by AFB₁. AFB₁ mutagenesis in Salmonella typhimurium strains was dependent on a functional RecA gene product. The enhancing effects of ΔuvrB and plasmid pKM101 were found to be additive. Data presented are consistent with the following: (i) AFB₁ toxic effects are due mainly to DNA binding of AFB₁; (ii) AFB₁ mutagenesis is dependent on error-prone DNA repair; (iii) Pneumococcus lacks an active error-prone (SOS) DNA-repair system.     

A new strain of Salmonella typhimurium reverted by mitomycin C and N-methyl-N′-nitro-N-nitrosoguanidine—a possible universal tester for mutagenic compounds

A strain of Salmonella typhimurium, SO1007, which carries the amber mutation trpD28 plus the plasmid pKM101 was reverted very efficiently by two mutagens with different mutagenic specificities and modes of action: mitomycin C (MC) and N-methyl-N′-nitro-N-nitrosoguanidine (NG). By selecting revertants on minimal agar supplemented with anthranilic acid (AA), two distinct phenotypic classes of TrpD28 revertants can be recovered: prototrophs (MM⁺) and anthranilate utilizers (AA⁺). Since each phenotypic class is known to be caused by a variety of mutational events, reversion of trpD28 on minimal-anthranilate medium may be useful for detecting mutagenic agents regardless of the types of mutations they may cause. Thus, strains like SO 1007 may be useful as ‘universal’ detectors of mutagenic compounds. In the course of these experiments we also observed that pKM101 does not protect but, on the contrary, sensitizes the host bacteria slightly to the toxic effects of MC.     

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 decifient 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 ΔoxyR30 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 ΔoxyR2). 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 f-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.     

Synergistic effect of an Escherichia coli mutator gene on mutagenesis by ultraviolet radiation and by alkylating agents

E. coli strains differing in a gene responsible for high spontaneous mutability (mut HI) were compared for their mutability by UV radiation and by the alkylating agents ethyl methanesulfonate and methyl methanesulfonate. All three exogenous mutagenic agents induced significantly higher frequencies of mutants with impaired carbohydrate-fermenting ability when the mutator allele rather than the wild-type allele was present. Thus the mut HI gene product possibly increases the probability of replication error due to alterations in the structure of the template strand of DNA. An attempt to detect an synergistic effect for UV-induced suppressor mutations was unsuccessful. The failure may have been due to the particular method used for scoring this type of mutation.     

Plasmid pKM101 sensitization of Escherichia coli strains to the action of ionizing radiation: the effect of the plasmid on survivability and induced mutagenesis

The wild type Escherichia coli K-12 has been shown to be sensitized to inactivation by gamma-irradiation by the plasmid pKM101. The dnaA strains of E. coli are more sensitive to gamma-rays killing effect, as compared with the wild type E. coli, pKM101 plasmid showing only slight sensitizing effect. "Cis" or "trans" position of the plasmid in relation to the chromosome plays no role in sensitization, while the plasmid effect on UV-induced killing and mutability depends on "trans" position of the plasmid before irradiation. gamma-Rays induced mutability to prototrophy is completely dependent on the presence of pKM101 in "trans" in wild type and dnaA strains before irradiation.     

LexA-independent expression of a mutant mucAB operon.

pKM101 is a naturally occurring plasmid that carries mucAB, an analog of the umuDC operon, the gene products of which are required for the SOS-dependent processing of damaged DNA necessary for most mutagenesis. Genetic studies have indicated that mucAB expression is controlled by the SOS regulatory circuit, with LexA acting as a direct repressor. pGW16 is a pKM101 derivative obtained by N-methyl-N'-nitro-N-nitrosoguanidine mutagenesis that was originally identified on the basis of its ability to cause a modest increase in spontaneous mutation rate. In this report, we show that pGW16 differs from pKM101 in being able to enhance methyl methanesulfonate mutagenesis and to confer substantial resistance to UV killing in a lexA3 host. The mutation carried by pGW16 is dominant and was localized to a 2.4-kb region of pGW16 that includes the mucAB coding region and approximately 0.6 kb of the 5'-flanking region. We determined the sequence of a 119-bp fragment containing the region upstream of mucAB and identified a single-base-pair change in that region, a G.C-to-A.T transition that alters a sequence homologous to known LexA-binding sites. DNA gel shift experiments indicate that LexA protein binds poorly to a 125-bp fragment containing this mutation, whereas a fragment containing the wild-type sequence is efficiently bound by LexA. This mutation also alters an overlapping sequence that is homologous to the -10 region of Escherichia coli promoters, moving it closer to the consensus sequence. The observation that the synthesis of pGW16-encoded mucAB proteins in maxicells is increased relative to that of pKM101-encoded mucAB proteins even in the absence of a lexA+ plasmid suggests that this mutation also increases the activity of the mucAB promoter.     

On the involvement of singlet oxygen in mutation induction by 8-methoxypsoralen and UVA irradiation in Escherichia coli K-12

The possible mutagenic effects induced by singlet oxygen, which is formed during UVA irradiation of bacterial cells pretreated with 8-methoxypsoralen (8-MOP), were investigated. As genetic endpoint, back mutation from arg₅₆^? to arg⁺ was assayed in strain Escherichia coli K-12/343/113/uvrB; this system, in preliminary experiments, was rather sensitive to 8-MOP-induced photodynamic effects. To assess the involvement of singlet oxygen (¹O₂) in the mutation induction process, 2 tests were applied, namely, comparative mutation induction in D₂O and in H₂O media (pH 7.0) and quenching of ¹O₂ with 1,4-diazabicyclo[2.2.2]octane (DABCO).When photodynamy was performed with the indicator cells suspended in D₂O buffer, the mutagenic effect was substantially higher than that obtained with cells suspended in H₂O buffer; this increase was even more pronounced when the incubation mixtures were thoroughly oxygenated before irradiation. D₂O itself was not mutagenic under the present experimental conditions. Addition of DABCO in concentrations of 0.1–10 mM to the irradiation mixtures effectively reduced the number of 8-MOP-induced mutant yields by about 40%. DABCO itself had no effect on cell viability or on spontaneous mutation frequency under our experimental conditions.From these 2 sets of results, and from the preliminary findings that the photomutagenic effect of 8-MOP is higher in the uvrB derivative than in the corresponding excision-repair-proficient parent strain, which is in concordance with previous observations in other E coli strains, it can be concluded that ¹O₂ generated upon UVA irradiation of 8-MOP solutions is probably responsible for part of the observed genetic effects.     

UV protection and mutagenesis in uvrD, uvrE and recL strains of Escherichia coli carrying the pKM101 plasmid.

The effect of the pKM101 plasmid on UV mutagenesis and survival was examined in DNA-repair-deficient strains of E. coli carrying the uvrD, uvrE and recL mutations. Although enhancement of UV mutagenesis by pKM101 was found in all 3 strains, UV protection was only observed in the uvrD strain. We conclude that the plasmid not only requires lexA+ recA+ functions of the cell, but also those of uvrE+ recL+ for its UV-protective effect.     

Modulation of allele leakiness and adaptive mutability inEscherichia coli

It is shown that partial phenotypic suppression of two ochre mutations (argE3 andlacZU118) and an amber mutation (inargE) by sublethal concentrations of streptomycin in anrpsL ⁺ (streptomycin-sensitive) derivative of theEscherichia coli strain AB1157 greatly enhances their adaptive mutability under selection. Streptomycin also increases adaptive mutability brought about by theppm mutation described earlier. Inactivation ofrecA affects neither phenotypic suppression by streptomycin nor replication-associated mutagenesis but abolishes adaptive mutagenesis. These results indicate a causal relationship between allele leakiness and adaptive mutability.     

Inhibition of Homologous Recombination by the Plasmid MucA′B Complex

By its functional interaction with a RecA polymer, the mutagenic UmuD′C complex possesses an antirecombination activity. We show here that MucA′B, a functional homolog of the UmuD′C complex, inhibits homologous recombination as well. In F⁻ recipients expressing MucA′B from a P_tac promoter, Hfr × F⁻ recombination decreased with increasing MucA′B concentrations down to 50-fold. In damage-induced pKM101-containing cells expressing MucA′B from the native promoter, recombination between a UV-damaged F lac plasmid and homologous chromosomal DNA decreased 10-fold. Overexpression of MucA′B together with UmuD′C resulted in a synergistic inhibition of recombination. RecA[UmuR] proteins, which are resistant to UmuD′C inhibition of recombination, are inhibited by MucA′B while promoting MucA′B-promoted mutagenesis efficiently. The data suggest that MucA′B and UmuD′C contact a RecA polymer at distinct sites. The MucA′B complex was more active than UmuD′C in promoting UV mutagenesis, yet it did not inhibit recombination more than UmuD′C does. The enhanced mutagenic potential of MucA′B may result from its inherent superior capacity to assist DNA polymerase in trans-lesion synthesis. In the course of this work, we found that the natural plasmid pKM101 expresses around 45,000 MucA and 13,000 MucB molecules per lexA(Def) cell devoid of LexA. These molecular Muc concentrations are far above those of the chromosomally encoded Umu counterparts. Plasmid pKM101 belongs to a family of broad-host-range conjugative plasmids. The elevated levels of the Muc proteins might be required for successful installation of pKM101-like plasmids into a variety of host cells.