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.     

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.     

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.     

Elimination of plasmid pKM101 fromSalmonella typhimurium by monoammonium salts

The frequency of elimination of plasmid pKM101 fromSalmonella typhimurium TA92 exposed to the action of 1-alkyl-1-ethylpiperidinium bromides and N-alkyl-N-[5-(benzoyloxy)-3-oxapentyl]-N,N-dimethylammonium bromides was nonlinear in the homologous series. Change in the length of the alkyl chain markedly affected the elimination properties of the piperidine derivatives but had no effect on the elimination of benzoyl derivatives. Piperidines exhibited a weaker elimination capacity than the benzoyl derivatives. The most potent eliminator was the octylbenzoyl derivative, which causes the elimination of the plasmid in 80–85% cells.     

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.     

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.     

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.     

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.     

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.     

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.     

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).     

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.     

Restriction endonuclease cleavage map of pKM101: Relationship to parental plasmid R46

Summary A detailed restriction endonuclease cleavage map of the plasmid pKM101 has been constructed. pKM101 plasmids containing individual Tn5 insertions were used to facilitate the ordering of restriction fragments generated by enzymes cleaving pKM101 at multiple sites. By restriction enzyme analysis, pKM101 (35.4 kilobases) appears to have arisen from its clinically-isolated parent by deletion of a single DNA region which codes for three of the four drug resistances carried by R46.     

Genetic and biochemical analysis of an endonuclease encoded by the IncN plasmid pKM101.

The IncN plasmid pKM101 nuc gene encodes a periplasmically localized endonuclease. DNA sequence analysis indicates that this gene encodes a hydrophilic protein of about 19.5 kDa containing a hydrophobic signal sequence. nuc is homologous to a partially sequenced open reading frame adjacent to the sog gene of the plasmid CollB-P9, a plasmid known to encode an endonuclease similar to that of pKM101. A partially sequenced tra gene directly upstream of nuc is homologous to the virB11 gene of Agrobacterium tumefaciens. We have partially purified the pKM101 nuclease by osmotic shock and cation exchange chromatography, and used this enzyme preparation to sequence the protein's amino terminus. The first 13 amino acids of the mature protein match amino acids 23 to 35 of the predicted sequence, indicating that the protein is proteolytically processed to a molecular mass of approximately 17 kDa, probably during export to the periplasmic space. The enzyme was able to attack many sites along an end labelled duplex DNA substrate, but showed clearly preferred cleavage sites, and may cleave preferentially at purine-rich regions.     

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.     

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.     

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.     

IncN plasmid pKM101 and IncI1 plasmid ColIb-P9 encode homologous antirestriction proteins in their leading regions.

The IncN plasmid pKM101 (a derivative of R46), like the IncI1 plasmid ColIb-P9, carries a gene (ardA, for alleviation of restriction of DNA) encoding an antirestriction function. ardA was located about 4 kb from the origin of transfer, in the region transferred early during bacterial conjugation. The nucleotide sequence of ardA was determined, and an appropriate polypeptide with the predicted molecular weight of about 19,500 was identified in maxicells of Escherichia coli. Comparison of the deduced amino acid sequences of the antirestriction proteins of the unrelated plasmids pKM101 and ColIb (ArdA and Ard, respectively) revealed that these proteins have about 60% identity. Like ColIb Ard, pKM101 ArdA specifically inhibits both the restriction and modification activities of five type I systems of E. coli tested and does not influence type III (EcoP1) restriction or the 5-methylcytosine-specific restriction systems McrA and McrB. However, in contrast to ColIb Ard, pKM101 ArdA is effective against the type II enzyme EcoRI. The Ard proteins are believed to overcome the host restriction barrier during bacterial conjugation. We have also identified two other genes of pKM101, ardR and ardK, which seem to control ardA activity and ardA-mediated lethality, respectively. Our findings suggest that ardR may serve as a genetic switch that determines whether the ardA-encoded antirestriction function is induced during mating.