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.     

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.     

Introduction of the plasmid pKM101-associated muc genes into Saccharomyces cerevisiae

Bacteria-yeast shuttle plasmids containing the pKM101-associated muc genes were constructed by cloning an ARS TRP fragment into the plasmid pGW270 in both possible orientations. The insertion of Saccharomyces cerevisiae DNA into pGW270 had no effect on the mutator and protective phenotypes associated with the plasmid in Escherichia coli. Two such recombinant plasmids, pAA90 and pAA91 , were capable of efficient transformation of S. cerevisiae and were stably maintained in this organism. Hybridization experiments suggest that muc-specific mRNA was present in transformed yeast cells and a small amount was polyadenylated. The RNAs were not of a discrete size, all being smaller than the muc genes. The presence of the plasmid pAA91 , and to a lesser extent, pAA90 , in yeast resulted in a detectable increase in the reversion frequencies of three markers and in ultraviolet protection. These results are discussed in terms of studying the relationship of error-prone repair in bacteria and yeast and of developing improved yeast tester strains.     

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.     

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.     

Complementation of a pKM101 derivative that decreases resistance to UV killing but increases susceptibility to mutagenesis

The drug resistance plasmid pKM101 makes Escherichia coli resistant to the lethal effects of ultraviolet (UV) irradiation and more susceptible to mutagenesis by a variety of agents. The plasmid operon responsible for increasing mutagenesis has been termed mucAB (Mutagenesis, UV and chemical). We have isolated a derivative of pKM101 called pGW1975 which makes cells more sensitive to killing by UV but which retains the ability of pKM101 to increase susceptibility to methyl methanesulfonate (MMS) mutagenesis. pGW1975 increases UV mutagenesis less than pKM101 in a uvrA⁺ strain but more than pKM101 in a uvrA⁻ strain. muc⁻ point and insertion mutants of pKM101 and pGW1975 complement to restore the plasmid-mediated: (i) ability to reactivate UV-irradiated phage, (ii) resistance to killing by UV, and (iii) level of susceptibility to UV mutagenesis. We have identified a 2.0 kb region of pKM101 which is responsible for the complementation and which maps counterclockwise of mucAB.     

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.     

Identification and mapping of regions of the plasmid pKM101 which influence the growth rate and resistance to phleomycin E of Escherichia coli WP2.

The effects of deletion of various regions of the pKM101 genome on several phenotypes conferred by pKM101 in Escherichia coli WP2 cells were investigated. Differences in the response of cells carrying pKM101 or various pKM101 deletion derivatives to the mutagenic effects of phleomycin E can be attributed to differences in sensitivity to the lethal effects of phleomycin E. Resistance to phleomycin E is conferred by the pKM101 mucAB genes (or an adjacent gene) but observed only with pKM101 derivatives which have lost a 2.2-kilobase (BalI-KpnI-2) segment which completely includes the pKM101 endonuclease gene nuc. A pKM101 slow-growth determinant, distinct from the slo gene, has also been identified and localized in the 2.4-kilobase (BalI-KpnI-3) segment which is adjacent to the nuc gene. Loss of this region does not appear to substantially influence the toxic or mutagenic effects of phleomycin E.     

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.     

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.     

Molecular cloning of plasmid ColIb-P9 genes responsible for its mutator function

The localization of plasmid ColIb-P9 muc genes mediating the plasmids protective and mutagenesis-increasing activity has been determined. The increase of muc genes dose by cloning them within the multicopy vector has been shown to repress the mutator function of the plasmid. No essential homology has been revealed between ColIb-P9 muc gene nucleotide sequences, pKM101 muc genes with a similar function, and umuDC chromosome genes. It has been shown that the synthesis of 38 KD protein is essential for the manifestation of the mutator function of the plasmid.     

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.     

DNA sequence analysis of the imp UV protection and mutation operon of the plasmid TP110: identification of a third gene.

The sequence of the imp operon of the plasmid TP110 (which belongs to the Incl1 incompatibility group) has been determined, and is shown to contain three open reading frames. This operon, involved in UV protection and mutation, is functionally analogous to the umuDC operon of E. coli and the mucAB operon of the plasmid pKM101, which belongs to the quite unrelated IncN incompatibility group. The umu and muc operons however contain only two open reading frames, coding for proteins of approximately 16kD and 46kD. The high degree of homology between the two 16kD proteins (UmuD and MucA) and between the two 46kD proteins (UmuC and MucB) clearly shows their relatedness. This is shown also to extend to the imp gene products, with ImpA sharing homology with UmuD and MucA, and ImpB sharing homology with UmuC and MucB. However, the two imp genes are preceded in the operon by a third gene, impC, which encodes a small protein of 9.5kD and which has no equivalent in the umu and muc operons.     

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.     

Identification of pKM101-encoded loci specifying potentially lethal gene products.

Two pKM101-encoded loci (designated kilA and kilB) have been identified which elaborate products that are potentially lethal to the bacterial cell. The lethal effects of each of these products is inhibited by two other plasmid-encoded loci, designated korA and korB (for kil override). Both korA and korB are required to control the lethality of either kil gene. In the presence of korA and korB both kil genes have other phenotypes: kilB is necessary for conjugal transfer, whereas kilA is responsible for the small-colony morphology on defined media that is characteristic of pKM101-containing strains (the Slo phenotype).     

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     

Physical mapping of the plasmid R205 and identification of a site responsible for increased UV-induced mutability of the bacterial host

A physical map of the conjugative IncN plasmid R205 (56.1 kb) was constructed. The distribution of cleavage sites for investigated restriction enzymes is asymmetric. It was found that R205 suppresses the mutant phenotype of E. coli K12 umuC or umuD strains deficient in UV-induced mutagenesis. A mini-derivative of R205, designated pMU4 (15.1 kb) preserves the ability of the parent plasmid to increase the survival and induced mutagenesis of UV-irradiated host cells. A region of R205 located between 0 and 2.0 kb-on the plasmid map seems to contain information necessary for complementation of mutation in the host genes umuD/C, Hybridization between this region of pMU4 and plasmid pGW1700 bearing mucAB genes of pKM101 was observed.     

Entry exclusion determinant(s) of IncN plasmid pKM101.

pKM101 renders its host a poor recipient in conjugal matings with genetically distinguishable derivatives of itself. The gene(s) primarily responsible for this, denoted eex, is located in between genes required for both conjugal transfer and sensitivity to donor-specific bacteriophage, although it itself is not necessary for transfer. A gene linked to, or coincident with, the region needed for vegetative plasmid replication also inhibited establishment of related plasmids under certain conditions. Construction of an operon fusion between eex and the Escherichia coli lac promoter has shown that this gene is transcribed in a clockwise fashion on the circular map of pKM101. To date, we have not been able to visualize a protein product(s) of the eex gene(s).     

Bacteriophage PSP3 and phiR73 activator proteins: analysis of promoter specificities.

The effect of plasmid pKM101 on the survival of Escherichia coli AB1157, growing in minimal medium, in the presence of a 4-quinolone DNA gyrase inhibitor was investigated. The presence of this plasmid decreased susceptibility to the quinolone ciprofloxacin, whereas mucAB genes present in a multicopy plasmid did not. The same effect of pKM101 was detected in a recA430 mutant, confirming that it was not really related to the SOS response. In contrast, when survival assays were performed under amino acid starvation conditions, pKM101 did not confer protection against ciprofloxacin. All of these results indicated that the synthesis of a product(s), different from MucAB, which was encoded by the plasmid pKM101 increased the rate of survival of the AB1157 strain in the presence of quinolone. To identify the gene(s) responsible for this phenotype, several plasmid derivatives carrying different portions of pKM101 were constructed. The 2.2-kb region containing korB, traL, korA, and traM genes was sufficient to decrease susceptibility to quinolone. This plasmidic fragment also made the AB1157 host strain grow more slowly (the Slo phenotype). Moreover, the suppression of the Slo phenotype by addition of adenine to the cultures abolished the decreased susceptibility to quinolone. These results are evidence that the protection against quinolone conferred by this region of pKM101 in strain AB1157 is a direct consequence of the slow growth rate.     

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.