Characterization of an endonuclease associated with the drug resistance plasmid pKM101.

An endonuclease was detected in strains of Salmonella typhimurium containing the drug resistance plasmid pKM101. The enzyme was not detectable in strains lacking this plasmid, but it was present in strains containing mutants of pKM101 that were no longer able to enhance host cell mutagenesis. The endonuclease had a molecular weight of roughly 75,000 and, at pH 7.0, was equally active on single-stranded and duplex deoxyribonucleic acid (DNA). The reaction with single-stranded DNA was optimal at pH 5.5, whereas with duplex DNA the optimum was pH 6.8. The enzyme required a divalent cation for activity, and it had no detectable exonuclease activity with single-stranded or duplex DNA. The endonuclease extensively degraded DNA with no apparent base specificity, forming 5'-phosphomonoester termini. Although characterization of the endonuclease has not revealed its function, the enzyme does not appear to be a restriction endonuclease.     

Effect of plasmid pKM101 in ultraviolet irradiated uvr+ and uvr- Escherichia coli.

The effect of plasmid pKM101 on UV irradiated excision proficient and excision deficient cells was investigated. The plasmid increased the survival of excision proficient cells while partially inhibiting thymine dimer excision. The frequency of mutations was almost unchanged. In excision deficient cells the effect of the plasmid on survival was less pronounced while cell mutability was increased. Our data indicate that the mucAB genes (carried by the plasmid) influence the two types of cells in a different way.     

R46 and pKM101 plasmid-mediated resistance to ionizing radiation in Escherichia coli

The ability of the R46 R factor and its derivative pKM101 to modify sensitivity to 60Co gamma radiation was studied. In Escherichia coli K12 both plasmids enhanced bacterial survival after 60Co gamma irradiation. This effect was dependent on recA+ genotype but not on recB+, recB+ recC+, and recF+ genotypes. 5-Fluorouracil eliminated the R46 R factor from the parent and its rec- mutant strains. These strains lost not only the antibiotic resistance coded for R46 R factor but their radioresistance as well.     

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.     

A plasmid carrying mucA and mucB genes from pKM101 in Haemophilus influenzae and Escherichia coli.

The plasmid pMucAMucB, constructed from the Haemophilus influenzae vector pDM2, and a similar plasmid, constructed from pBR322, increased the survival after UV irradiation of Escherichia coli AB1157 with the umu-36 mutation and also caused UV-induced mutation in the E. coli strain. In H. influenzae, pMucAMucB caused a small but reproducible increase in survival after UV irradiation in wild-type cells and in a rec-1 mutant, but there was no increase in spontaneous mutation in the wild type or in the rec-1 mutant and no UV-induced mutation.     

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.     

Temperature-sensitive replication of plasmid pKM101

A mutant of Escherichia coli K-12, IB10 carrying the ts10 mutation has been isolated. The mutation affects replication and inheritance of pKM101 plasmid. Incubation of the mutant under non-selective conditions of 42 degrees C resulted in the formation of R-cell population. The frequency of temperature-independent clones was 2,1 X 10(-5). The defect of pKM101 replication was shown to result in growth inhibition of host cells at a non-permissive temperature. The host growth only started after elimination of the plasmid. The mechanisms are likely to exist governing the participation of plasmid gene products in processes related to host growth. The influence of ts10 mutation on replication of other plasmids was studied. It was established that ts10 did not affect replication of R6K, RP4 and Flac+ plasmids. However, replication of R15, R205 as well as of pKM101 plasmid stopped under conditions of non-permissive temperature in IB10 mutant. Obviously, ts10 mutation results in defective replication of plasmids only belonging to the N-incompatibility group (IncPN). It is shown that R6K, RP4, Flac+ plasmids are not able to correct pKM101 replication in the mutant at 42 degrees C.     

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.     

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.     

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.     

Effects of plasmid pKM101 on the expression of Escherichia coli and Salmonella typhimurium genes under ultraviolet irradiation

The study focused on plasmid pKM101, which is a necessary component of the short-term test of Eim's system (Salmonella-microsome test), to detect the potential carcinogens through their mutagen activity. We found a previously unknown feature of the plasmid to enhance the expression of certain plasmid and chromosome genes. The purpose of the present study was to examine and specify the role of operon mucAB responsible for the mutation properties of the plasmid in activating the expression of bacterial genes. An ultraviolet-induction examination of bacterial genes, with the mutants of plasmid pKM101 affecting operon mucAB being used, showed that the function of genes mucAB did activate, but, on the contrary, suppressed the induction of genes elt (i.e. of genes controlling the formation of LT-toxin of Escherichia coli) and of sfiA (SOS-regulated gen E. col controlling the cell division.     

Conjugal transfer system of the IncN plasmid pKM101.

The conjugal transfer system of the broad-host range IncN plasmid pKM101 was analyzed genetically. Its organization differed significantly from that of the F plasmid. The tra genes are located in three regions, each between 3 and 4 kilobases in length. All of the genes in the first two regions are required for sensitivity to "donor-specific" phage which bind to the plasmid-mediated sex pilus, and these genes therefore are involved in the synthesis, and possibly retraction, of the sex pilus. The plasmid's origin of transfer was localized to a 1.2-kilobase region at an extreme end of the transfer region. Using two different methods, we have identified 11 complementation groups required for transfer. One of these, traC, is of special interest in that mutations at this locus can be partially suppressed if, prior to mating, cells carrying a traC mutant plasmid are incubated with cells which elaborate sex pili but are unable to transfer their plasmids. One possible explanation for this is that pilus-elaborating cells can donate traC gene product to a traC mutant in a form that can be reused.     

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.     

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.     

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.     

Fertility inhibition of RP1 by IncN plasmid pKM101.

IncN plasmids, including pKM101, strongly inhibit the conjugal transfer of cohabiting IncP plasmids. We localized the pKM101 DNA sufficient for this phenomenon to a 1.1-kilobase region (denoted fip). Two fip-deficient Tn5 insertion derivatives of pKM101 were isolated; neither affected other pKM101-mediated functions. fip did not inhibit either the synthesis of the IncP plasmid's sex pilus or its ability to mediate entry exclusion against other IncP plasmids.     

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).