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.     

Plasmid-dosage effects on ultraviolet, visible light and methyl methanesulfonate mutagenesis in Salmonella typhimurium

Salmonella typhimurium LT2 strains bearing plasmids pKM101, R64 or pColIb-P9 demonstrated enhanced UV survival when compared with strains not bearing plasmids. A strain of S. typhimurium bearing both pKM101 and pColIb-P9 survived UV irradiation slightly better than either of the single-plasmid strains. Spontaneous reversion of the hisG46 and trpE8 missense alleles was enhanced in each single-plasmid strain, and for the dual-plasmid strain containing pKM101 and pColIb-P9 enhancement represented a near additivity of the response seen for the single-plasmid strains. Following exposure to UV or visible-light irradiation, reversion of hisG46 and trpE8 was also enhanced in each single-plasmid strain, but quantitatively greater in the dual-plasmid strain and was equal to or slightly greater than additive the responses of the single-plasmid strains. In contrast to visible-light irradiation, UV exposure resulted in two phenotypic Trp+-revertant classes. One Trp+ class, having normal colony size (2.0 mm) and similar in number to His+ revertants, was comprised of intragenic revertants of trpE8, while the predominant Trp+ class, having smaller colony size (0.8 mm), represented intergenic suppressor revertants, illuminating the differences in mutation and/or repair specificity for UV and visible-light exposure. Methyl methane-sulfonate (MMS)-induced reversion of hisG46 was similar in effect to that seen with UV or visible-light irradiation. Plasmids pKM101 or pColIb-P9 enhanced the frequency of hisG46 reversion, while a more than additive response was seen in a strain with both plasmids. Furthermore, MMS-induced reversion of hisG46 was also observed to be greatest in a strain bearing plasmid R64 (incompatibility group I alpha) and pKM101, when compared with single-plasmid strains bearing either R64 or pKM101.     

R46-derived recombinant plasmids affecting DNA repair and mutation in E. coli

Summary Recombinant plasmids which render their host less mutable and more sensitive to some DNA-damaging agents have been isolated from the N-group plasmid R46. These plasmids have been physically mapped and found to originate from the region of R46 that has been deleted in pKM101. This deleted region is well removed from the muc region of R46 and pKM101 which is responsible for the mutator effects of these plasmids.The effect of these anti-mutagenic plasmids on the ability of pKM101 to complement umuC mutations has been examined, and they have been found to inhibit the complementation of such strains. We propose that these plasmids may code for a negative control function acting on the muc gene.     

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.     

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.     

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

Patterns of reversion produced by ciprofloxacin, 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.     

Isolation of plasmid pKM101 in the Stocker laboratory

pKM101 is a mutagenesis-enhancing resistance transfer plasmid (R plasmid) that was introduced into several tester strains used in the Salmonella/microsome mutation assay (Ames test). Plasmid pKM101 has contributed substantially to the effectiveness of the Ames assay, which is used on a world-wide basis to detect mutagens and is required by many government regulatory agencies for approval to market new drugs and other chemical agents. Widely used since 1975, the Ames test is still regarded as one of the most sensitive genetic toxicity assays and a useful short-term test for predicting carcinogenicity in animals. Plasmid pKM101, which is a deletion derivative of plasmid R46 (also referred to as R-Brighton after its origin of isolation in Brighton, England), has also been used to elucidate molecular mechanisms of mutagenesis. It was isolated in the laboratory of Professor Bruce A.D. Stocker at Stanford University as part of my doctoral research with 20 R plasmids. Professor Stocker's phenomenal insight into the genetics of Salmonella typhimurium and plasmid behavior was a major factor that led to the isolation of pKM101. This paper includes a tribute to Bruce Stocker, together with a summary of my research with mutagenesis-enhancing R plasmids and a brief discussion of the molecular mechanisms involved in pKM101 plasmid-mediated bacterial mutagenesis.     

The muc+ gene of plasmid pKM101 prevents respiration shutoff in far ultraviolet-irradiated Salmonella typhimurium

Summary The plasmid pKM101 is known to protect Escherichia coli and Salmonella typhimurium against killing by far UV irradiation and to enhance UV-induced mutagenesis. The muc ⁺ gene of the plasmid is responsible for both of these effects. This paper shows that respiration of S. typhimurium shuts off about an hour after UV irradiation and that pKM101 prevents the shutoff. Plasmids which contained Tn5 translocatable elements, either in (and having produced a muc mutation) or flanking the muc ⁺ gene, have been introduced into S. typhimurium. The muc mutant plasmid, which does not protect its host against UV killing and does not enhance UV induced mutagenesis, also does not protect against UV induced respiration shutoff. Like-wise, plasmids in which the Tn5 translocatable elements flank the nuc ⁺ gene protect against shutoff of respiration. Thus the muc ⁺ gene of pKM101 is responsible for protection against UV induced shutoff of respiration in S. typhimurium.Research sponsored by the Office of Health and Environmental Research, U.S. Department of Energy, under contract W-7405-eng-26 with the Union Carbide Corporation and by the National Science Foundation under grant No. PCM 7908647 with the University of Tennessee, Knoxville     

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.     

Effect of the plasmid ColIb-P9 on cellular processes related to DNA repair

Summary The plasmid ColIb-P9 introduced into Escherichia coli K12 umuC mutant cells suppresses the deficiencies in mutagenesis and repair of mutants after UV-irradiation. These data suggest that ColIb-P9 encodes a product with a function similar to that of the chromosomal gene umuC. Tn5 insertion mutants of ColIb-P9 were isolated with an altered ability to restore UV-mutagenesis in the umuC mutant. The same plasmid mutations were shown to eliminate the effects of ColIb-P9 on UV-mutagenesis, survival after UV and mitomycin C treatment, reactivation of UV-irradiated in unirradiated cells, Weigle-reactivation, induction of colicin E1 synthesis. The ColIb-P9 genes responsible for the enhancement of UV-mutagenesis were cloned within a 14 Md SalI fragment. Their location was established by restriction analysis of the mutant plasmid ColIb 6-13::Tn5.While the action of the plasmids ColIb-P9 and pKM101 is similar, these plasmids were shown to have opposite effects on cell survival and colicin E1 synthesis after mitomycin C treatment. A study of the mutant plasmids ColIb::Tn5 and pGW12 (muc ^- mutant of pKM101) has shown the difference in the effects of ColIb-P9 and pKM101 to be associated with the plasmid genes responsible for the protective and mutagenesis-enhancing effects of these plasmids in UV-irradiated cells.Abbreviations MC mitomycin C - ICS induction of colicin synthesis     

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.     

In vivo definition of the functional origin of leading strand replication on the lactococcal plasmid pFX2

The lactococcal plasmid pFX2 belongs to a family of plasmids, whose prototype is the streptococcal plasmid pMV158, that replicates by the rolling circle mechanism. Determination of the nucleotide sequence of the repX gene of pFX2 allowed us to make some minor corrections in the published sequence, and to show that the repX gene is identical to the rep gene of plasmid pWV01. We have established pFX2 in Escherichia coli and in Streptococcus pneumoniae. In the latter host, we have defined in vivo the nick site introduced by the RepX protein. Plasmid pFX2 and the pMV158 derivative pLS1 exhibit a moderate degree of incompatibility in S. pneumoniae. Cloning of the double strand origin (dso) of pFX2 into a high-copy-number plasmid that is compatible with the pMV158 replicon led to an increase in incompatibility toward pLS1. Plasmids pFX2 and pLS1 exhibit homologies in their Rep proteins and in their dso sequences, but not in their negative control elements. Thus, the observed incompatibility indicates that cross-recognition of Rep proteins and dso takes place. Received: 25 May 1998 / Accepted: 8 July 1998     

Spontaneous mutational specificity of drug resistance plasmid pKM101 in Escherichia coli.

Plasmid pKM101 enhances the frequency of spontaneous and ultraviolet light-induced mutations in Escherichia coli and protects the cells against the lethal effects of ultraviolet irradiation. By analyzing reversion patterns of defined trpA alleles, we showed that pKM101 caused all types of spontaneous base-pair substitution mutations with the possible exception of guanine . cytosine leads to adenine. thymine transitions. Neither insertion nor deletion frameshift mutations were enhanced. Transversions were more strongly enhanced than transitions, and adenine . thymine base pairs appeared more susceptible to pKM101 mutator activity than guanine . cytosine base pairs. In addition, there were effects from neighboring base pairs and genetic background that influenced the mutator activity of pKM101.     

Influence of uvrB and pKM101 on the spectrum of spontaneous, UV- and gamma-ray-induced base substitutions that revert hisG46 in Salmonella typhimurium

Oligonucleotide probes were used to identify base substitutions in 1089 revertants of hisG46 in Salmonella typhimurium that arose spontaneously or following irradiation with UV- or gamma-rays. The hisG46 allele, carrying a mutant CCC codon (Pro) in place of the wild-type codon CTC (Leu69) reverted via 6 distinguishable mutational events--C to T transitions at codon sites 1 or 2, C to A or C to G transversions at codon site 1, C to A at codon site 2, and an extragenic suppressor mutation. The distribution of hisG46 revertants differed among treatments and was influenced by the DNA-repair capacity of the bacteria. Plasmid pKM101 enhanced the frequencies of both spontaneous and induced mutations; transversion events were enhanced more efficiently by pKM101 than were transition events. Compared to Uvr+ bacteria, Uvr- bacteria had higher frequencies of spontaneous and induced mutations; transition mutations were enhanced more efficiently than were transversion mutations. The influence of DNA-repair activities on the mutational spectra provides some insights on the origins of spontaneous and UV-induced mutations.     

Functional organization of plasmid pKM101.

Tn5 insertion mutants and in vitro-generated deletion mutants of the mutagenesis-enhancing plasmid pKM101 have been used to identify several genetic regions on the pKM101 map. In clockwise order on the pKM101 map are: (i) the bla gene, coding for a beta-lactamase; (ii) the Slo region, responsible for retarding cell growth on minimal medium; (iii) the tra genes, enabling pKM101 to transfer conjugally; (iv) sensitivity to IKe phage (this function[s] maps within the tra region); (v) the muc gene(s), responsible for enhancing ultraviolet light and chemically induced mutagenesis in the cell; and (vi) the Rep region, essential for plasmid replication. The muc gene(s) and the Rep region are contained in a deoxyribonucleic acid region bounded by inverted repeated sequences.     

Some group N plasmids makeEscherichia coli K-12 strain AB1157 dependent on exogenous purine

Escherichia coli K-12 strain AB1157 given the conjugative group N plasmid R46 (or its derivative, pKM101, or plasmid R384N) grew only very slowly on defined medium containing the known growth-factor requirements of AB1157, which do not include any purine. Addition of adenine or hypoxanthine (or their nucleosides) restored normal growth; guanine and xanthine (and their nucleosides) were ineffective, because of thegpt defect caused by deletionproA2. Variants of AB1157(R46) able to grow rapidly on defined medium without purine were tetracycline-sensitive and/or transfer-defective; an, R46 gene,slo, causing purine auxotrophy, is inferred to be betweentet andtra.     

DNA repair in Proteus mirabilis. VI. Plasmid (R46-) mediated recovery and UV mutagenesis.

Summary The expression of plasmid R46-mediated recovery and mutagenic function (s) was studied in P. mirabilis, which is normally either weakly or nonmutable after UV exposure. The plasmid was found to confer on P. mirabilis enhanced UV resistance as well as UV-induced mutability for various types of forward mutations and reversion of the thr273 mutation. The plasmid enhanced survival of UV-irradiated phages in P. mirabilis both in unirradiated host cells and with increased efficiency after UV-exposure of host cells, as is characteristic of UV-inducible phage reactivation. Spontaneous mutability of P. mirabilis harboring R46 was about 2 to 7 times higher than that of cells without plasmid, depending on the marker, repair type, and plating density of the cells used. All of these R46-mediated rescue and mutagenic functions require the rec672⁺ gene function.It is assumed that the plasmid R46 adds functions to P. mirabilis comparable to those deficient in umuC and uvm mutants of E. coli (Kato and Shinoura, 1977; Steinborn, 1978) and that P. mirabilis possesses functions homologous to those controlled in E. coli by the recA ⁺ and lexA ⁺ genes.The significance of plasmid-mediated rescue and mutagenic functions for bacteria which lack the misrepair branch of mutagenesis, is 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.     

Plasmid pSK1002-mediated mutator effect and SOS response and SOS mutagenesis of 2,5-dichloronitrobenzol in Salmonella typhimurium

The plasmid pSK1002 (umuC'-'lacZ) could increase the number of revertants induced by methyl methanesulfonate (MMS) and 4-nitroquinoline-N-oxide (4NQO) in Salmonella typhimurium TA 1535 (his-). The values induced by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) were not different irrespective of the presence or absence of plasmid. However, the plasmid pKM101-mediated mutagenesis-enhancing effect was much greater than that mediated by pSK1002 as induced by the 3 mutagens mentioned above. Moreover, the plasmid pSK1002 could induced umu-mediated SOS response in the presence of any of these 3 mutagens or of mitomycin C, and a dose-response relationship was evident. It shows that pSK1002 (umuC'-'lacZ) has a dual biological effect, namely a mutator effect and the effect of inducing the SOS response. Besides, this study has proved SOS mutagenesis of 2,5-dichloronitrobenzol (2,5-DCNB) because of the dual indicator nature of pSK1002. Therefore, it is probable that pSK1002 could be further developed and applied in studying the relation between the SOS response and mutagenesis and in identifying environmental SOS mutagens.     

Indications that mutagenesis in Salmonella may be subject to catabolite repression

Spontaneous reversion of the base-pair substitution trpE8 marker in the LT2 sub-line of Salmonella typhimurium is significantly increased in the presence of the ultraviolet light-protecting and mutation-enhancing plasmid pKM101. The numbers of Trp⁺ revertants arising on plates of defined medium supplemented with trace amounts of nutrient broth have been found to depend upon the nature of the carbon source provided to support growth of both the background lawn and any revertants which may arise. For example, the yield of Trp⁺ revertants can be some 5–8 times greater when glycerol is the carbon source as compared to when glucose is the carbon source. S. typhimurium strain TA100, which carries the base-pair substitution hisG46 marker and pKM101, shows a similar response, although the difference is much smaller. Time-course experiments using both carbon sources indicate that the final trpE8 → Trp⁺ mutation yield is depressed by glucose rather than enhanced by a ‘mutagenic’ effect of glycerol. These results are consistent with the idea that a glucose-repressible function responsible for generating mutations can be switched on by growth on glycerol as sole carbon source. Evidence is also presented that many more mutational events occur in response to a mild temperature stress (42°) in populations growing on glycerol as carbon source than occur in populations growing on glucose.