Mutagenesis by neocarzinostatin in Escherichia coli and Salmonella typhimurium: requirement for umuC+ or plasmid pKM101.

Neocarzinostatin, a protein with antibiotic activity, is a bacterial mutagen. We have investigated the mutagenicity of neocarzinostatin towards Salmonella typhimurium and discovered that, unlike the situation in Escherichia coli, neocarzinostatin will revert base pair substitution mutations (missense or nonsense). However, when the R46 factor derivative, plasmid pKM101, was introduced, the mutagenicity of neocarzinostatin towards base pair substitution-carrying mutants of S. typhimurium was readily detected. Neocarzinostatin had only modest activity in reverting a frameshift mutation in S. typhimurium, but that activity, too, required the presence of pKM101. Mutant pKM101 plasmids which no longer enhanced mutagenesis also lost their ability to promote neocarzinostatin-induced mutations. Finally, the umuC36 mutation, which renders E. coli nonmutable by ultraviolet light, also rendered the bacteria nonmutable by neocarzinostatin. The effect of the umuC36 mutation was suppressed by plasmid pKM101.     

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.     

The role of the gene umuC and plasmid muc genes in mutagenesis induced by dioxidine in E. coli K12

The mutability induced by dioxidine in E. coli cells has been shown to be stringently dependent on a function of chromosomal umuC+ gene. Suppression of an umuC mutation by plasmids pKM101 or ColIb, restoring the dioxidine induced mutability, proves the possibility of umuC gene functional complementation by the plasmid muc+ genes.     

Isolation and characterization of mutants of the plasmid pKM101 deficient in their ability to enhance mutagenesis and repair.

A screening procedure was developed for identifying mutants of the plasmid pKM101 no longer capable of enhancing mutagenesis. The test was based on the large pKM101-mediated increase in the number of Gal+ papillae observed on colonies of Salmonella typhimurium gal mutants plated on tetrazolium-galactose plates in the presence of a mutagen. The pKM101 mutant plasmids transferred normally, were stably maintained in cells, caused normal levels of ampicillin resistance, and still imparted sensitivity to phage Ike to their hosts. However, the pKM101 mutants had lost the ability to (i) enhance the reversion of both point and frameshift mutations, (ii) protect the cells against killing by UV irradiation, (iii) increase the spontaneous reversion rates of point mutations, (iv) enhance plasmid-mediated reactivation of UV-irradiated phage P22, (v) enhance Weigle reactivation. One pKM101 mutant with different properties from the others was identified by its increased spontaneous mutator effect. It is suggested that pKM101 amplifies the activity of the inducible error-prone repair systems in bacteria and that this is the function of pKM101 in the Ames Salmonella tester strains used for detection of carcinogens as mutagens.     

Weakening of bacteriophage lambda EcoK DNA restriction in the presence of plasmid pKM101 ard+. I. General characteristics and genetic localization

The host-controlled K-restriction of unmodified phage lambda is ten to hundred-fold alleviated in the E. coli K12 strain, carring plasmid pKM101 of N-incompatibility group. By restriction mapping Tn5 insertion in pKM101, which reduced pKM101-mediated alleviation of K-restriction, was shown to by located within BglII-B-fragment approximately 9 kb anticlockwise from the EcoRI-site of pKM101. We have termed the gene(s) promoting the alleviation of K-restriction ARD (Alleviation of Restriction of DNA). It was shown that (i) plasmid pKM101-mediated alleviation of K-restriction did not depend on bacterial genes LexA, RecBC, umuC and plasmid gene muc; (ii) ard gene did not mediate EcoK type modification of DNA and did not enhance the modification activity of EcoK system in a way similar to that observed with RAL gene of phage lambda. Action of Ard gene of plasmid pKM101 is highly specific: alleviation of restriction of DNA lambda takes place only in K-strains of E. coli and is practically absent in B-strains and also in E. coli strains which have restricting enzymes of 11 type, EcoRI and EcoRIII.     

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.     

Ionizing and ultraviolet radiation-induced reversion of sequenced frameshift mutations in Escherichia coli: a new role for umuDC suggested by delayed photoreactivation

The ultraviolet (UV) and gamma radiation-induced reversion of the trpA21, trpA9813, and trpE9777 sequenced-frameshift mutations were studied in Escherichia coli K-12 with or without the plasmid pKM101. Radiation induced the reversion of all 3 frameshifts, and pKM101 enhanced this reversion 10-50-fold. Factors influencing the differential radiation revertability of frameshifts are discussed. The two most revertable frameshifts, trpE9777 and trpA9813, were used as probes to understand the role of the umuDC genes in radiation-induced frameshift reversion. Unlike the UV radiation-induced reversion of base-substitution mutations, the reversion of these frameshifts was not enhanced in a uvrA umuC strain by photoreactivation after a post-UV-irradiation incubation. The UmuDC proteins are suggested to have functions in the radiation induction of frameshifts that are more complex than are their functions in the induction of base substitutions.     

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.     

Lack of umuDC gene functions in Vibrio cholerae cells

Attempts to identify an umuDC analog, using interspecific complementation of Escherichia coli mutants with plasmids containing a gene bank of Vibrio cholerae, were not successful. The DNA from none of the vibrio species examined including marine vibrios hybridized to E. coli umuC and umuD gene sequences. These cells are not mutable by ultraviolet (UV) light and cannot Weigle-reactivate UV-irradiated choleraphages, suggesting that vibrios are deficient in the umuDC operon. This possibility is supported by the fact that when the plasmid pKM101 carrying the mucAB genes is introduced into V. cholerae cells, they acquire the UV-mutable phenotype and UV-irradiated choleraphages can be Weigle-reactivated.     

Frameshift mutagenesis by chloroquine in Escherichia coli and Salmonella typhimurium

Chloroquine can be detected as a direct-acting mutagen in plate-incorporation assays using the excision-deficient Salmonella typhimurium strain TA97, but very much more effectively using the repair-proficient Escherichia coli strain DG1669 which carries the lacZ19124 marker. When tested at concentrations of 200-1000 micrograms/plate with strain DG1669, the mutagenicity of chloroquine is enhanced by the addition of Aroclor-induced rat-liver S9. Further experiments indicated that chloroquine-induced reversion frequencies were essentially identical in wild-type, recA, umuC and uvrC derivatives of DG1669, as well as in strains carrying the mutation enhancing plasmid pKM101, over a wide range of doses (0-1200 micrograms/plate). These results suggest that neither excision repair nor SOS-type repair are important in chloroquine-induced frameshift mutagenesis.     

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.     

The ColIb-CA53 plasmid suppresses umuC- and umuD-mutations in Escherichia coli K-12

The presence of the ColIa-CA53 plasmid in umuC and umuD mutant Escherichia coli K-12 cells restores their mutability under UV irradiation to a level that even exceeds that of the isogenic umuC+umuD+ strains, as well as increases their resistance to the lethal effects of UV irradiation. The ColIb-P9 plasmid which suppresses the umuC mutant phenotype, as we have shown earlier, acts in the same manner with respect to the umuD mutant cells. The results of the study demonstrate that both plasmids encode products that are functionally similar to those of the chromosomal genes umuC and umuD. The plasmids ColIa-CA53, ColIb-P9 and pKM101 are shown to have practically the same effect upon the mutagenesis and survival of the umuC, umuD mutant cells.     

Effect of plasmid pKM101 on the expression of bacterial genes not related to DNa metabolism]

An experimental system ensuring fusion of bacterial genes to the lac operon of the Mu dl(Aplac) phage was used. Fusion operons in which the lac operon was under the control of promoters of the elt gene, responsible for synthesis of the LT toxin, of the tetracyclin-resistance tet gene, and sfiA gene encoding filament production, was studied. Using this experimental system, plasmid pKM101 was shown to be capable of activating the expression of the above Escherichia coli and Salmonella typhimurium genes, which is manifested as the activation of beta-galactosidase synthesis. The activation of the elt gene expression by the pKM101 plasmid was also confirmed in experiments on detecting the LT toxin synthesized by bacteria carrying this plasmid. Effect of the plasmid on the activation of elt operon expression, unlike the effect of this plasmid on mutability, does not depend on the functioning of the lexA and recA genes, i.e., this is not a SOS-regulated process. The mutant plasmid pGW12, a derivative of pKM101, deficient in the mucAB genes responsible for mutagenesis, causes a more pronounced activation of the elt gene than plasmid pKM101.     

Effect of pKM101 on cell killing and specificity of mutation induction by cis-diaminedichloroplatinum(II) in Escherichia coli K-12.

Cell killing and mutation induction in the lacI gene of Escherichia coli by cis-Pt(NH3)2Cl2 were studied in cells with different repair capacities, with and without pKM101. The presence of the plasmid pKM101 made repair-proficient cells more susceptible to killing by cis-Pt(NH3)2Cl2 and strongly enhanced mutation induction by that compound. Both effects were shown to be dependent upon excision repair. Characterization of the induced mutations in the lacI gene after cis-Pt(NH3)2Cl2 treatment of E. coli cells, by the LacI system, revealed that the mutagenic specificity of the Pt compound was strongly influenced by the presence of the pKM101 plasmid. With pKM101, 23% of the induced amber and ochre mutations resulted from substitutions at AT base pairs, whereas these mutations were hardly induced in cells without pKM101. These results suggest that pKM101-induced repair differs from normal SOS repair.     

Substitution of UmuD' for UmuD does not affect SOS mutagenesis

In order to study the role of UmuDC proteins in SOS mutagenesis, we have constructed new Escherichia coli K-12 strains to avoid i) over-production of Umu proteins, ii) the formation of unwanted mixed plasmid and chromosomal Umu proteins upon complementation. We inserted a mini-kan transposon into the umuD gene carried on a plasmid. The insertion at codon 24 ends protein translation and has a polar effect on the expression of the downstream umuC gene. We transferred umuD24 mutation to the E coli chromosome. In parallel, we subcloned umuD+ umuC+ or umuD' umuC+ genes into pSC101, a low copy number plasmid. In a host with the chromosomal umuD24 mutation, plasmids umuD+ umuC+ or umuD' umuC+ produced elevated resistance to UV light and increased SOS mutagenesis related to a gene dosage of about 3. UV mutagenesis was as high in umuD' umuC+ hosts devoid of UmuD+ protein as in umuD+ umuC+ hosts. UmuD' protein, the maturated form of UmuD, can substitute for UmuD in SOS mutagenesis.     

Repair promoted by plasmid pKM101 is different from SOS repair.

In E. coli K12 bacteria carrying plasmid pKM101, prophage lambda was induced at UV doses higher than in plasmid-less parental bacteria. UV-induced reactivation per se was less effective. Bacteria with pKM101 showed no alteration in their division cycle. Plasmid pKM101 coded for a constitutive error-prone repair different from the inducible error-prone repair called SOS repair. Plasmid pKM101 protected E. coli bacteria from UV damage but slightly sensitized them to X-ray lesions. Protection against UV damage was effective in mutant bacteria deficient in DNA excision-repair provided that the recA, lexA and uvrE genes were functional. Survival of phages lambda and S13 after UV irradiation was enhanced in bacteria carrying plasmid pKM101; phage lambda mutagenesis was also increased. Plasmid pKM101 repaired potentially lethal DNA lesions, although wild-type DNA sequences may not necessarily be restored; hence the mutations observed are the traces of the original DNA lesions.     

Conditions affecting the mutagenicity of sodium bisulfite in Salmonella typhimurium

Sodium bisulfite is a weak mutagen at pH 5 and 6 in S. typhimurium strains carrying the hisG46 and hisD6610 mutations, but is not mutagenic in strains with the hisC3076 or hisD3052 mutations. The bisulfite-induced base-pair substitution mutations were slightly enhanced by the presence of the plasmid, pKM101, but inhibited by the presence of the uvrB and rfa mutations. The hisO1242 mutation which causes constitutive expression of the histidine operon, produced a slight enhancement of frameshift (hisD6610), but not base-pair substitution (hisG46) mutations. Bisulfite-induced mutations appear to be the result of two different mechanisms which may be a function of the repair capacity of the strains. The data suggest that the deamination of cytosine may not be responsible for frameshift mutations, but may be responsible for base-pair substitution mutagenesis. Because the rate of bisulfite autooxidation appears to play a role in the mutagenic process, we are suggesting that the deamination of cytosine may be the result of oxidative damage rather than through the direct formation of a cytosine-bisulfite adduct. This is further supported by the much lower concentrations of bisulfite needed to cause mutagenicity than the 1 M concentrations cited to produce cytosine-bisulfite adducts.     

Carcinogen-induced mutation spectrum in wild-type, uvrA and umuC strains of Escherichia coli. Strain specificity and mutation-prone sequences

Forward mutations induced by the ultimate carcinogen N-acetoxy-N-2-acetylaminofluorene (N-Aco-AAF) in the tetracycline resistance gene carried on plasmid pBR322 are shown to be dependent upon the induction of the host SOS functions in wild-type and umuC Escherichia coli cells. The mutation frequency in the umuC strain is equal to about 40% of the mutation frequency observed in the umu+ background. In the excision-repair-deficient uvrA mutant strain the mutagenic response is the same as in SOS-induced wild-type cells whether or not the uvrA bacteria are SOS-induced. Equal mutation frequencies are obtained in both the wild-type and the uvrA strains for equal modification levels although the survival of AAF-modified plasmid DNA is greatly reduced in the uvrA strain as compared to the wild-type strain. Sequence analysis of the mutations reveals that more than 90% of the N-Aco-AAF-induced mutations are frameshift mutations. Two types of mutational hotspots are observed occurring either at repetitive sequences or at non-repetitive sequences. Both types of mutants appear at similar locations and frequencies in both the wild-type and the uvrA strains. On the other hand, only the non-repetitive sequence mutants are obtained in the umuC background. These non-repetitive sequence mutants preferentially occur within the sequence 5' G-G-C-G-C-C 3' (the NarI restriction enzyme recognition sequence). The analysis of the -AAF binding spectrum to the same DNA fragment shows that there is no direct correlation between the modification spectrum and the mutation spectrum. We suggest that certain sequences are "mutation-prone" in the sense that only these sequences can be efficiently mutated as the result of an active processing mediated by specific proteins. When a sequence is said to be mutation-prone it probably corresponds to a particular structure that is induced within this sequence as a result of the binding to the DNA of the mutagen. This sequence-specific conformational change is the substrate for the protein(s) that fixes the mutation. The mutagenic processing pathway(s) is part of the cellular response to DNA-damaging agents (the so-called SOS response). Two pathways for frameshift mutagenesis are suggested by the data: an umuC-dependent pathway, which is involved in the mutagenic processing of lesions within repetitive sequences; an umuC-independent pathway responsible for the fixation of mutations within specific non-repetitive sequences.     

5-fluorouracil forward mutation assay in Salmonella: determination of mutational target and spontaneous mutational spectra

A forward mutation assay in Salmonella typhimurium that selects for 5-fluoruracil (FU) resistance has been developed. The two genes possibly involved in FU resistance, the uracil phosphoribosyl transferase gene (upp) and the uracil transport protein (uraA), have been cloned from S. typhimurium and sequenced. One hundred percent of FU-resistant clones display sequence changes in the upp gene, indicating that its loss is the major mechanism involved in FU resistance. The spontaneous mutational spectra at the upp locus were then determined in two S. typhimurium strains, FU100 and FU1535, that differ only in the presence of pKM101 plasmid. The pKM101 plasmid provides error-prone replicative bypass of DNA lesions and renders FU100 more susceptible to induced mutagenesis. Fluctuation analysis of FU-resistant clones demonstrated a 10-fold higher spontaneous mutation rate at the upp locus in FU100 relative to FU1535. Over 300 independent FU-resistant clones were then used to generate the spectra at the upp locus in both the strains. Approximately 40% of all the mutations were base substitutions, present at the same relative percentage in both the strains. Frameshift mutations also accounted for approximately 40% of the total; however, their incidence was slightly elevated in FU100. The remaining mutations were larger insertions and deletions, which were both slightly elevated in FU1535. pKM101 significantly elevated the rate of all classes of mutations at the upp locus, with profound effects on A:T to T:A transversions and -2-base frameshift mutations. These initial mutational spectra at the upp locus reveal 147 mutable sites, or 23% of the total 627-base coding sequence and suggest that the target can detect a diverse spectrum of mutagenic events.     

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.