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).     

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.     

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.     

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.     

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.     

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.     

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.     

Genetic analysis of the mobilization and leading regions of the IncN plasmids pKM101 and pCU1

The conjugative IncN plasmids pKM101 and pCU1 have previously been shown to contain identical oriT sequences as well as conserved restriction endonuclease cleavage patterns within their tra regions. Complementation analysis and sequence data presented here indicate that these two plasmids encode essentially identical conjugal DNA-processing proteins. This region contains three genes, traI, traJ, and traK, transcribed in the same orientation from a promoter that probably lies within or near the conjugal transfer origin (oriT). Three corresponding proteins were visualized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and complementation analysis confirmed that this region contains three tra complementation groups. All three proteins resemble proteins of the IncW plasmid R388 and other plasmids thought to have roles in processing of plasmid DNA during conjugation. The hydropathy profile of TraJ suggests a transmembrane topology similar to that of several homologous proteins. Both traK and traI were required for efficient interplasmid site-specific recombination at oriT, while traJ was not required. The leading region of pKM101 contains three genes (stbA, stbB, and stbC), null mutations in which cause elevated levels of plasmid instability. Plasmid instability was observed only in hosts that are proficient in interplasmid recombination, suggesting that this recombination can potentially lead to plasmid loss and that Stb proteins somehow overcome this, possibly via site-specific multimer resolution.     

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.     

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.     

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.     

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.     

TraC of IncN Plasmid pKM101 Associates with Membranes and Extracellular High-Molecular-Weight Structures in Escherichia coli

Conjugative transfer of IncN plasmid pKM101 is mediated by the TraI-TraII region-encoded transfer machinery components. Similar to the case for the related Agrobacterium tumefaciens T-complex transfer apparatus, this machinery is needed for assembly of pili to initiate cell-to-cell contact preceding DNA transfer. Biochemical and cell biological experiments presented here show extracellular localization of TraC, as suggested by extracellular complementation of TraC-deficient bacteria by helper cells expressing a functional plasmid transfer machinery (S. C. Winans, and G. C. Walker, J. Bacteriol. 161:402-410, 1985). Overexpression of TraC and its export in large amounts into the periplasm of Escherichia coli allowed purification by periplasmic extraction, ammonium sulfate precipitation, and column chromatography. Whereas TraC was soluble in overexpressing strains, it partly associated with the membranes in pKM101-carrying cells, possibly due to protein-protein interactions with other components of the TraI-TraII region-encoded transfer machinery. Membrane association of TraC was reduced in strains carrying pKM101 derivatives with transposon insertions in genes coding for other essential components of the transfer machinery, traM, traB, traD, and traE but not eex, coding for an entry exclusion protein not required for DNA transfer. Cross-linking identified protein-protein interactions of TraC in E. coli carrying pKM101 but not derivatives with transposon insertions in essential tra genes. Interactions with membrane-bound Tra proteins may incorporate TraC into a surface structure, suggested by its removal from the cell by shearing as part of a high-molecular-weight complex. Heterologous expression of TraC in A. tumefaciens partly compensated for the pilus assembly defect in strains deficient for its homolog VirB5, which further supported its role in assembly of conjugative pili. In addition to its association with high-molecular-weight structures, TraC was secreted into the extracellular milieu. Conjugation experiments showed that secreted TraC does not compensate transfer deficiency of TraC-deficient cells, suggesting that extracellular complementation may rely on cell-to-cell transfer of TraC only as part of a bona fide transfer apparatus.     

Conjugal transfer system of plasmid RP4: analysis by transposon 7 insertion.

We have begun an analysis in Escherichia coli of the conjugal transfer functions of the broad-host-range plasmid RP4. We have isolated 19 tra mutants of RP4, generated by insertion of transposon 7, and mapped their insertion sites by restriction endonuclease analysis. These sites fall into two separate regions on either side of the kanamycin resistance determinant. The transfer rates of the mutants range from 10% of that of RP4 to an undetectable level. Spot tests with the P-1 pilus-specific phages PRR1, Pf3, and PR4 and electron microscopic examination for pili have classified the mutants into several phenotypes consistent with their having normal, retracted, or no pili. Analysis of transient plasmid heterozygotes, created by P1 transduction, divided the tra mutants into a minimum of five complementation groups. Some of these groups contain more than one phenotypic class and may represent more than one gene because of the possible polar and deletion effects of Tn7 insertion.     

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.     

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.     

The Hydrophobic TraM Protein of pKM101 Is Required for Conjugal Transfer and Sensitivity to Donor-Specific Bacteriophage

pKM101 is a self-transmissible plasmid of the IncN incompatibility group. Analysis of the DNA sequences of the genes required for conjugal transfer suggested the existence of a previously uncharacterized open reading frame, designatedtraM,that might be required for conjugation. Merodiploid strains containing transposon insertion mutations either intraMor in neighboringtragenes were used to demonstrate thattraMconstitutes a new complementation group essential for conjugation and donor phage sensitivity. The hydrophobicity profile of TraM suggests that it contains a signal sequence. The remainder of TraM is also composed predominantly of hydrophobic amino acids but contains one possible surface exposed loop. TraM–alkaline phosphatase and TraM–β-galactosidase fusion proteins supported the hypothesis that TraM has a small cytoplasmic loop. We were unable to detect heterologous complementation between anytramutation and its homolog from thevirBoperon ofAgrobacterium tumefaciens.