Effect of pKM101 plasmid on lethal and mutagenic damage in UV-irradiated E. coli strains

Introduction of the R-factor plasmid pKM101 increased resistance to UV-killing in uvr lexA(Ind-) recA+ strains of E. coli K12 as well as B, while their UV mutability was not affected. Similar effects were also observed in those strains when the 18-B plasmid (a pBR322 derivative carrying the region (about 5 kb) of the 35.4 kb pKM101 plasmid) was introduced. The muc genes which are considered to be involved in error-prone repair are contained in 18-B. These results suggest the possibility that the pKM101 effect requires the host recA gene and a common genetic region, including the muc genes, in both plasmids and is associated with some unmutable repair systems.     

Plasmid pGW16, a derivative of pKM101, increases post-UV DNA synthesis, but sensitises some strains of Escherichia coli to UV

Plasmid pKM101, which carries muc genes that are analogous in function to chromosomal umu genes, protected Escherichia coli strains AB1157 uvrB+ umuC+, JC3890 uvrB umuC+, TK702 uvrB+ umuC and TK501 uvrB umuC against ultraviolet irradiation (UV). Plasmid pGW16, a derivative of pKM101 selected for its increased spontaneous mutator effect, also gave some protection to the UmuC-deficient strains, TK702 and TK501. However, it sensitised the wild-type strain AB1157 to low, but protected against high doses of UV, whilst sensitising strain JC3890 to all UV doses tested. Even though its UV-protecting effects varied, pGW16 was shown to increase both spontaneous and UV-induced mutation in all strains. Another derivative of pKM101, plasmid pGW12, was shown to have lost all spontaneous and UV-induced mutator effects and did not affect post-UV survival. Plasmids pKM101 and pGW16 increased post-UV DNA synthesis in strains AB1157 and TK702, whereas pGW12 had no effect. Similarly, the wild-type UV-protecting plasmids R46, R446b and R124 increased post-UV DNA synthesis in strain TK501, but the non-UV-protecting plasmids R1, RP4 and R6K had no effect. These results accord with the model for error-prone DNA repair that requires umu or muc gene products for chain elongation after base insertion opposite non-coding lesions. They also suggest that the UV-sensitizing effects of pGW16 on umu+ strains can be explained in terms of overactive DNA repair resulting in lethal, rather than repaired UV-induced lesions.     

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.     

Lowered induction of genetic tandem duplications in Salmonella by the pKM101 plasmid

Summary Selection for 3-amino-1,2,4-triazole (AT) resistance in certain strains of Salmonella typhimurium has been previously shown to select for genetic tandem duplications of the histidine operon. We show here that agents which induce tandem duplications are less effective in such induction in the presence of the pKM101 plasmid. The presence of the plasmid also produces an increase in AT-resistance due to mechanisms other than duplication, presumably because pKM101 produces high levels of error-prone repair. We suggest that high levels of error-prone repair may cause decreases in tandem duplication induction and propose that error-prone repair and tandem duplication may be alternative cellular responses to certain DNA lesions.     

Postreplication DNA repair in Escherichia coli cells. III. Repair independent of the presence of pKM101 and COLIb-P9 plasmids

The presence of pKM101 or ColIb-P9 plasmids in E. coli leads to the increase in the survival of UV-irradiated cells of wild type and of polAI, recB21 recC22 and dnaGts mutants; it does not change the survival of recA13 and lex3 mutants and does not influence kinetics and efficiency of postreplication repair (PRR) of DNA in cells of all the strains examined (with the exception of PG3 dnaGts mutant whose PRR of DNA in the presence of pKM101 plasmid is somewhat lower). The survival of both plasmid-containing and plasmid-free bacteria treated with chloramphenicol decreases in the same degree, but the survival of chloramphenicol-treated recA13, lex3 recB21 rec C22 mutants does not change. The pKM101 plasmid does not lend the dnaGts mutant a new capacity of repairing postreplication gaps with the participation of inducible component of PRR; the chloramphenicol-sensitive component of PRR is absent in this mutant. Plasmid and plasmid-free E. coli strains of wild type and of the polA1 mutant do not differ by the kinetics and level of inducible chloramphenicol-sensitive component of PRR of DNA.     

Comparative lethal effects of uv and ionizing radiation in Ames tester strains of Salmonella

In the three (parent-daughter) pairs of Ames Salmonella tester strains TA1535-TA100, TA1537-TA2637, and TA1538-TA98 in which the daughter strains carry the pKM101 plasmid but the parent strains do not, the pKM101 plasmid uniformly confers resistance of the host to uv radiation which indicates that the muc genes of the plasmid are present and function correctly in all three daughter strains. This uniform protection against killing by uv contrasts with the lethality responses of the same parent-daughter pairs to ionizing radiation (ir) where pKM101 again confers lethality protection to TA100 and TA2637 but sensitizes TA98 toward the lethal effects of ir. From these results we conclude that the pathways for error-prone repair of lethal lesions induced by uv and by ionizing radiation are not the same and that the muc genes of the plasmid alone are not sufficient to carry out error-prone repair of lethal lesions induced by ionizing radiation. We infer that a segment of plasmid DNA that is present in TA100 and TA2637 and is required to repair potentially lethal damage induced by ir is deleted in TA98.     

Lethal effect of formaldehyde on Escherichia coli strains carrying or lacking the plasmid pKM101

The presence of plasmid pKM101 in Escherichia coli cells results in a slight increase in their sensitivity of lethal effect of formaldehyde. Plasmid ability to sensitize bacterial cells to formaldehyde inactivation is controlled by some chromosomal (uvrE, uvrA, recA) and plasmid-borne (mucAB) genes and depends on SOS-DNA repair activity. Plasmid pKM101 is capable of decreasing the level of repair reliability of DNA damaged by formaldehyde thus causing increased bacterial sensitivity to this agent.     

Photosensitized inactivation of plasmid and bacteriophage lambda DNA: relative contribution to the lethal effect of monoadducts and diadducts of 8-methoxypsoralen and their repair in SOS-induced Escherichia coli cells

The curves of UV (254 nm)-inactivation and inactivation by furocoumarin derivatives + UVA radiation (PUVA) of bacteriophage lambda and biologically active plasmid pBR322 were measured using Escherichia coli K12 bacteria with different defects of DNA repair system as a ghost. The ratio of mono- and diadducts (interstrand cross-links) of 8-methoxypsoralen was determined that are formed after treating the DNA of pBR322 and bacteriophage lambda with PUVA. It is shown that, on the average, about five monoadducts per one diadduct are formed in DNA of pBR322, and about 0.9 monoadducts per one diadduct are formed in lambda phage DNA. An increased (up to 50%) efficiency of SOS-repair of monoadducts of 8-methoxypsoralen in DNA of pBR322 and lambda in the presence of plasmid pKM101 muc+ (incN) was found.     

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.     

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.     

Paradoxical behaviour of pKM101; inhibition of uvr-independent crosslink repair in Escherichia coli by muc gene products

In strains of Escherichia coli deficient in excision repair (uvrA or uvrB), plasmid pKM101 muc+ but not pGW219 mucB::Tn5 enhanced resistance to angelicin monoadducts but reduced resistance to 8-methoxy-psoralen interstrand DNA crosslinks. Thermally induced recA-441 (= tif-1) bacteria showed an additional resistance to crosslinks that was blocked by pKM101. Plasmid-borne muc+ genes also conferred some additional sensitivity to gamma-radiation and it is suggested that a repair step susceptible to inhibition by muc+ gene products and possibly involving double-strand breaks may be involved after both ionizing radiation damage and psoralen crosslinks.     

Mutational Specificity of Ultraviolet Light in ESCHERICHIA COLI with and without the R Plasmid Pkm101

Plasmid pKM101 provides UV protection and increases the frequency of spontaneous and UV-induced mutations in Escherichia coli. By analyzing reversion patterns of defined trpA alleles, we showed that pKM101 altered the mutational specificity of UV-induced mutations. Certain UV-induced base-pair substitutions were strongly enhanced, while others were decreased in frequency in the presence of pKM101. This result suggests an interaction between cellular misrepair and an error-prone repair function(s) provided by pKM101. We have also examined UV mutational specificity in the absence of pKM101 and found the following: (1) UV preferentially enhances missense, as well as nonsense, intergenic suppressor mutations; (2) UV causes all possible base-pair substitutions as well as frameshift mutations; (3) G·C base pairs are more susceptible to UV mutagenesis than A·T base pairs at the same nucleotide positions; and (4) UV-induced mutations can occur at nucleotide positions that are not part of pyrimidine-pyrimidine sequences.     

Inducible reactivation and mutagenesis of UV-irradiated bacteriophage P22 in Salmonella typhimurium LT2 containing the plasmid pKM101.

The inducible (Weigle) reactivation of UV-irradiated bacteriophage P22 has been examined on strains of Salmonella typhimurium with and without the mutagenesis-enhancing plasmid pKM101. A large inducible reactivation was observed in the plasmid-containing strain, but only a small response was observed in the strain lacking the plasmid. An increased frequency of clear-plaque mutants was detected among the survivors. The efficiencies of the plasmid-mediated and cellular repair processes have been determined. The kinetics of induction of the phage reactivation have been investigated. The relationship of the observed results to the inducible reactivation of UV-irradiated lambda in Escherichia coli and to error-prone repair is discussed.     

Segregation of the mutator property of plasmid R46 from its ultraviolet-protecting property

Summary Plasmid R46 (an R factor conferring resistance to ampicillin, sulfonamides, streptomycin and tetracycline) reduces the bactericidal effect of UV irradiation but increases its mutagenic effect (reversion of hisG46), and raises the frequency of spontaneous reversion (mutator effect). Putative deletion mutants of R46 were obtained by transduction of the plasmid, then two successive conjugal transfers. Plasmids of five of six deletion classes, each with a different combination of drug resistance traits, retained conjugative ability and the UV-protecting, mutagenesis-enhancing and mutator effects of R46. (pKM101, used in the Ames system to enhance responsiveness to chemical mutagens, is one such mutant of R46.) Plasmids of a sixth class, represented by pKM115, conferred resistance only to streptomycin and were non-conjugative. All of several such plasmids (of independent origin) had a much stronger mutator effect than did R46, but lacked UV-protecting ability and did not enhance the mutagenic effect of UV irradiation. We infer that R46 possesses: (i) a gene, uvp, which increases capacity for error-prone repair of UV-damaged DNA, and thus causes both UV protection and enhancement of UV mutagenesis; (ii) gene(s) whose action in the absence of gene uvp greatly increases the frequency of spontaneous reversion of hisG46. A plasmid of another incompatibility group, pLS51, has UV-protecting and mutagenesis-enhancing effect but lacks the mutator property; introduction of pLS51 into a clone of hisG46 carrying a pKM115-type plasmid greatly reduced its spontaneous reversion rate, as expected if pLS51 also has a uvp gene able to modulate the mutator effect of R46-derived gene(s) in the pKM115-type plasmid.     

Effects of DNA-repair processes on the induction of genetic duplications in bacteria by ultraviolet light

A straightforward positive selection for genetic duplication is possible in strains of Salmonella typhimurium that carry the aroC321 allele. Strains with a single copy of this allele require phenylalanine, tyrosine and tryptophan for growth. Such strains give rise to tryptophan prototrophs, which still require phenylalanine and tyrosine, through the formation of a duplication that includes about 30% of the chromosome. We have constructed strains that permit the simultaneous study of duplications and mutations and have used these strains to explore the effects of DNA repair processes on the induction of duplications by ultraviolet light (UV). UV causes dose-dependent increases in the frequency of duplications in bacteria. The exposure required to induce duplications is much less in a delta uvrB strain than in repair-proficient strains, suggesting that duplications result from DNA lesions that are subject to excision repair. The photoreversibility of UV-induced preduplication lesions implicates pyrimidine dimers in the induction of duplications. Unlike its effect on the induction of mutations, the error-prone repair process associated with plasmid pKM101 does not enhance the induction of duplications. The prevention of duplication-formation by a recA mutation suggests that the formation of duplications involves recombinational events. Taken together, the data indicate that the same DNA lesions can be mutagenic and recombinagenic in bacteria, but that the two effects involve different pathways of processing DNA damage.     

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.     

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.     

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.     

Induction of presumed somatic gene mutations in mice by 2-naphthylamine

Plasmid pKM101, whose mucA and B genes endow cells with enhanced mutation frequency and enhanced resistance to far-ultraviolet radiation (FUV) (254 nm), had no influence on these properties when cells were damaged by near-ultraviolet radiation (NUV) (300-400 nm). Thus, NUV lesions did not lead to induction of SOS repair and subsequent expression of mucA and B genes on plasmid pKM101. Further, when cells were pre-irradiated with NUV and subsequently irradiated with FUV, there was a blockage of SOS repair, including the repair normally controlled by genes on pKM101.     

Reduced restriction of EcoK in the presence of the plasmid pKM ard+. II. Cloning of the gene ard

Plasmid pKM101 affects the I type restriction EcoK in E. coli. The gene ard responsible for alleviation of EcoK restriction was shown to be located within the BglIIB fragment of pKM101. Here we have cloned ard gene into high copy vectors pBR322 and pUC12. Plasmid pD12 was constructed by introducing into pUC12 a 1.87 kb HindIII-Pst fragment, carrying ard gene. Tn5 and Tn1000 insertions were obtained in the ard gene region.