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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

Effect of growth rate on plasmid maintenance by Escherichia coli HB101(pAT153)

The effects of changing the composition of the growth medium, the dilution rate and the source of the bacterial host on maintenance of the plasmid pAT153 in Escherichia coli HB101 have been studied. In a medium supplemented with Casamino acids, the plasmid was maintained longer during phosphate-limited growth at a dilution rate of 0.3 h-1 than at 0.15 h-1. In contrast, phosphate-limited growth was not achieved when the Casamino acids were replaced by proline, leucine and thiamin to satisfy the auxotrophic requirements of the host. Although 100% of the bacteria were still ampicillin resistant after 72 generations of growth at a dilution rate of 0.15 h-1, the original plasmid had almost totally been replaced by a structurally modified plasmid which lacked a functional tet gene. Further experiments confirmed that neither the host nor the plasmid was retained unchanged in the minimal medium. The changes were highly reproducible and reflected periodic selection of sub-populations which were either plasmid-free or carried a structurally modified plasmid, which had reverted to Leu+ or Pro+, or had acquired other chromosomal mutations which gave them a selective advantage. We conclude that in complex media the plasmid is maintained longer by E. coli HB101 at a high than at a low growth rate and that different results reported from different laboratories are largely due to differences in analytical techniques and the growth medium rather than to differences in the bacterial host or the plasmid used. A fermenter-adapted strain was isolated which reproducibly maintained the plasmid longer during phosphate-limited continuous growth than the original strain which had been cultured on laboratory media.     

Plasmid pKM101 sensitization of Escherichia coli strains to the action of ionizing radiation: the effect of the plasmid on survivability and induced mutagenesis

The wild type Escherichia coli K-12 has been shown to be sensitized to inactivation by gamma-irradiation by the plasmid pKM101. The dnaA strains of E. coli are more sensitive to gamma-rays killing effect, as compared with the wild type E. coli, pKM101 plasmid showing only slight sensitizing effect. "Cis" or "trans" position of the plasmid in relation to the chromosome plays no role in sensitization, while the plasmid effect on UV-induced killing and mutability depends on "trans" position of the plasmid before irradiation. gamma-Rays induced mutability to prototrophy is completely dependent on the presence of pKM101 in "trans" in wild type and dnaA strains before irradiation.     

Physical mapping of hemolysin plasmid pCW2, which codes for virulence of a nephropathogenic Escherichia coli strain.

The hemolysin plasmid pCW2, which enhances the virulence of Escherichia coli, has been mapped. Comparison of the region coding for hemolysin with other hemolysin determinants reveals differences that could explain differences in the ability to confer virulence.     

The influence of the growth environment on the stability of a drug resistance plasmid in Escherichia coli K12.

Populations of Escherichi coli K12 containing the plasmid TP120 which coded for resistance to ampicillin, streptomycin, sulphonamide and tetracycline were grown in a chemostat under carbon-limited and phosphorus-limited conditions. With time resistance to one or more of the drugs was lost, resulting in the production of mutant populations which were more competitive than the parent population. The resistance to tetracycline was always lost under both carbon and phosphorus limitations, but resistance to the other three drugs was lost only during phosphate-limited growth. Strains of E. coli which had lost resistance to one or more of the drugs were capable of higher maximum specific growth rates than the parent strain.     

An alteration leading to loss of ability to support phleomycin mutagenesis in the pKM101-derived plasmid pGW16 is located in or close to the mucAB genes

The differences between the plasmid pKM101 and its derivative pGW16, which has lost the ability to support muc-dependent phleomycin mutagenesis, while retaining other muc-dependent phenotypes, have been further investigated. Deletion derivatives which retain only 10.8 kb (approximately one third) of the pKM101 genome but retain the muc genes have been constructed from both pKM101 and pGW16. The deletion derivatives confer protection and mutagenesis-enhancing properties similar to those of their respective parents, indicating that the lesion in the mutant plasmid pGW16 lies in or close to the muc genes. Differences in the muc-dependent phenotypes of strains containing pKM101 or pGW16 suggest that the pGW16 lesion results in either differential loss of function in the muc gene products, or constitutive expression of the muc gene products.