Summary
A two-step mutant plasmid, originating from the resistance plasmid R1drd-19, was shown to replicate without control of the plasmid copy number when the host bacteria were grown at temperatures above 35°C. The uncontrolled plasmid replication, so called runaway-replication, eventually inhibits the host cell growth and results in a conditionally lethal phenotype. As much as 75% of the total DNA in the cells was found to be covalently closed circular plasmid DNA at the stage when the cell growth stopped. At 30°C the mutant plasmid had a strictly controlled copy number and the onset and kinetics of runaway-replication was studied by temperatureshift experiments. The synthesis of plasmid DNA after a temperature shift from 30°C to 40°C (or 37°C) was found to be exponential with a doubling time of about half the doubling time of the host bacteria, in broth medium as well as in minimal salt-glucose medium. In broth medium the plasmid population had a doubling time of about 12 min which is longer than expected if it is assumed that the rate of elongation during the DNA polymerization is the only limitation on the uncontrolled replication. The results indicate that the minimum time required between successive replications of a plasmid copy is determined by some rate-limiting step(s) presumably after the duplication process. The uncontrolled plasmid replication was shown to be inhibited by the protein synthesis inhibitor chloramphenicol. The plasmid synthesis stopped within 20 min after the addition of the drug and the plasmid DNA increment was less than 50%, suggesting that the uncontrolled plasmid replication requires de novo protein synthesis. Similarly, the RNA polymerase inhibitor rifampicin did inhibit the plasmid replication. The mutational alteration could be reversed, and the copy number control restored, by lowering the temperature before the host cell growth was inhibited. This type of plasmid mutant offers new possibilities in studies of host cell-plasmid interactions.
Similar content being viewed by others
References
Adams, M.H.: Bacteriophages. New York: Interscience 1959
Andreoli, P.M.: Ph.D. Thesis. Free University, Amsterdam (1978)
Bachman, B.J.: Pedigrees of some mutant strains of Escherichia coli K-12. Bact. Rev. 36, 525–557 (1972)
Berg, D.E.: Genes of phage λ essential for λdv plasmids. Virology 62, 224–233 (1974)
Bertani, G.: Studies on lysogenesis. I. The mode of phage liberation by lysogenic Escherichia coli. J. Bact. 62, 293–300 (1951)
Boman, H.G., Eriksson-Grennberg, K.G., Normark, S., Matsson, E.: Resistance of Escherichia coli to penicillins. IV. Genetic study of mutants resistant to DL-ampicillin concentrations of 100 μg/ml. Genet. Res. (Camb.) 12, 169–185 (1968)
Carter, B.J., Smith, M.G.: Intracellular pools of bacteriophage λ deoxyribonucleic acid. J. molec. Biol. 50, 713–718 (1970)
Clewell, D.B.: Nature of ColEI plasmid replication in Escherichia coli in the presence of chloramphenicol. J. Bact. 110, 667–676 (1972)
Cohen, S.N., Chang, A.C.Y., Hsu, L.: Nonchromosomal antibiotic resistance in bacteria: Genetic transformation of Escherichia coli by R-factor DNA. Proc. nat. Acad. Sci. (Wash.) 69, 2110–2114 (1972)
Cooper, S., Helmstetter, C.E.: Chromosomal replication and the division cycle of Escherichia coli B/r. J. molec. Biol. 31, 319–340 (1968)
Cress, D.E., Kline, B.C.: Isolation and characterization of Escherichia coli chromosomal mutants affecting plasmid copy number. J. Bact. 125, 635–642 (1976)
Davis, R., Vapnek, D.: In vivo transcription of R-plasmid deoxyribonucleic acid in Escherichia coli strains with altered antibiotic resistance levels and/or conjugal proficiency. J. Bact. 125, 1148–1155 (1976)
Demerec, M., Adelberg, E.A., Clark, A.J., Hartman, P.E.: A proposal for a uniform nomenclature in bacterial genetics. Genetics 54, 61–76 (1966)
Engberg, B., Hjalmarsson, K., Nordström, K.: Inhibition of cell division in Escherichia coli K-12 by the R-factor R1 and copy mutants of R1. J. Bact. 124, 633–640 (1975)
Engberg, B., Nordström, K.: Replication the R-factor R1 in Escherichia coli K-12 at different growth rates. J. Bact. 123, 179–186 (1975)
Folkmanis, A., Takeda, Y., Simuth, J., Gussin, G., Echols, H.: Purification and properties of a DNA-binding protein with characteristics expected for the Cro protein of bacteriophage λ, a repressor essential for lytic growth. Proc. nat. Acad. Sci. (Wash.) 73, 2249–2253 (1976)
Gaunce, A.P., D'Iorio, A.: Microdetermination of protein by an automated Lowry method. Biochemistry 37, 204–207 (1970)
Giles, K.W., Meyers, A.: An improved diphenylamine method for the estimation of deoxyribonucleic acid. Nature (Lond.) 206, 93 (1965)
Goebel, W.: Replication of plasmid DNA in Escherichia coli. Proc. Soc. gen. Microbiol. 1, 1–2 (1973)
Goebel, W.: Studies on the initiation of plasmid DNA replication. Europ. J. Biochem. 41, 51–62 (1974)
Goebel, W., Bonewald, R.: Class of small multicopy plasmids originating from the mutant antibiotic resistance factor R1drd-19B2. J. Bact. 123, 658–665 (1975)
Grindley, N.D.F., Grindley, J.N., Kelley, W.S.: Mutant plasmid with altered replication control. In: Microbiology — 1978 (Schlessinger, D., ed.). Washington D.C.: Am. Soc. Microbiol. 1978 71–73
Grindley, N.D.F., Kelley, W.S.: Effects of different alleles of the E. coli K 12 polA gene on the replication of non-transferring plasmids. Molec. gen. Genet. 143, 311–318 (1976)
Grinsted, J., Saunders, J.R., Ingram, L.C., Sykes, R.B., Richmond, M.H.: Properties of an R-factor which originated in Pseudomonas aeruginosa 1822. J. Bact. 110, 529–537 (1972)
Gustafsson, P.: Ph.D. Thesis. Umeå University, Umeå (1977)
Gustafsson, P., Nordström, K.: Temperature-dependent and amber copy mutants of plasmid R1drd-19 in Escherichia coli. Plasmid 1, 134–144 (1978)
Gustafsson, P., Nordström, K., Perram, J.W.: Selection and timing of replication of plasmids R1drd-19 and F′lac in Escherichia coli. Plasmid 1, 187–203 (1978)
Kool, A.J., Nijkamp, H.J.J.: Isolation and characterization of a copy mutant of the bacteriogenic plasmid Clo DF13. J. Bact. 120, 569–578 (1974)
Kupersztoch, Y.M., Helinski, D.R.: A catenated DNA molecule as an intermediate in the replication of the resistance transfer factor R6K in Escherichia coli. Biochem. biophys. Res. Commun. 54, 1451–1459 (1973)
Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J.: Protein measurement with the Folin phenol reagent. J. biol. Chem. 193, 265–275 (1951)
Macrina, F.L., Weatherly, G.G., Curtiss, R. III: R6K plasmid replication: Influence of chromosomal genotype in minicell-producing strains of Escherichia coli K-12. J. Bact. 120, 1387–1400 (1974)
Matsubara, K.: Genetic structure and regulation of a replicon of plasmid λdv. J. molec. Biol. 102, 427–439 (1976)
Matsubara, K., Takeda, Y.: Role of the tof gene in the production and perpetuation of the λdv plasmid. Molec. gen. Genet. 142, 225–230 (1975)
Meynell, E., Datta, N.: Mutant drug-resistant factors of high trans-missibility. Nature (Lond.) 214, 885–887 (1967)
Mickel, S., Bauer, W.: Isolation, by tetracycline selection, of small plasmids derived from R-factor R12 in Escherichia coli K-12. J. Bact. 127, 644–655 (1976)
Morris, C.F., Hashimoto, H., Mickel, S., Rownd, R.: Round of replication mutant of a drug resistance factor. J. Bact. 118, 855–866 (1974)
Murotsu, T., Tanaka, H., Imaji, M., Koga, H., Matsubara, K., Horiuchi, T.: Purification and properties of a λ operator-binding protein which is expected to be autorepressor (tof protein) from E. coli carrying λdv plasmid. Molec. gen. Genet. 157, 139–147 (1977)
Nordström, K., Engberg, B., Gustafsson, P., Molin, S., Uhlin, B.E.: Copy mutants of the plasmid R1 as a tool in studies of control of plasmid replication. In: Topics in infectious diseases. Vol. 2. R-factors: Their properties and possible control (Drews, J., Högenauer, G., eds.), pp. 299–332 Wien: Springer 1977
Nordström, K., Ingram, L.C., Lundbäck, A.: Mutations in R-factors of Escherichia coli causing an increased number of R-factor copies per chromosome. J. Bact. 110, 562–569 (1972)
Nordström, U.M., Engberg, B., Nordström, K.: Competition for DNA polymerase III between the chromosome and the R-factor R1. Molec. gen. Genet. 135, 185–190 (1974)
Novick, R.P., Clowes, R.C., Cohen, S.N., Curtiss, R., III, Datta, N., Falkow, S.: Uniform nomenclature for bacterial plasmids: A proposal. Bact. Rev. 40, 168–189 (1976)
Novick, R.P., Smith, K., Sheehy, R.J., Murphy, E.: A catenated intermediate in plasmid replication. Biochem. biophys. Res. Commun. 54, 1460–1469 (1973)
Pritchard, R.H., Barth, P.T., Collins, J.: Control of DNA synthesis in bacteria. Symp. Soc. gen. Microbiol. 19, 263–297 (1969)
Sheehy, R.J., Novick, R.P.: Studies on plasmid replication V. Replicative intermidiates. J. molec. Biol. 93, 237–253 (1975)
Timmis, K., Winkler, U.: Gene dosage studies with pleiotropic mutants of Serratia marcescens superactive in the synthesis of marcescin A and certain other exocellular proteins. Molec. gen. Genet. 124, 207–217 (1973)
Uhlin, B.E., Gustafsson, P., Molin, S., Nordström, K., Blohm, D.: Replication and copy number control of the plasmid R1 in Escherichia coli. In: Microbial drug resistance. II. Microbial drug resitance and related plasmids (Hashimoto, H., Mitsuhashi, S., eds.). Tokyo: University of Tokyo Press 1978 (in press)
Uhlin, B.E., Nordström, K.: Plasmid incompatibility and control of replication: Copy mutants of the R-factor R1 in Escherichia coli K-12. J. Bact. 124, 641–649 (1975)
Uhlin, B.E., Nordström, K.: R plasmid gene dosage effects in Escherichia coli K-12: Copy mutants of the R plasmid R1drd-19. Plasmid 1, 1–7 (1977)
Veltkamp, E., Barendsen, W., Nijkamp, H.J.J.: Influence of protein and ribonucleic acid synthesis on the replication of the bacteriocinogenic factor Clo DF13 in Escherichia coli cells and minicells. J. Bact. 118, 165–174 (1974)
Veltkamp, E., Nijkamp, H.J.J.: Replication of the bacteriocinogenic plasmid Clo DF13 in thermosensitive Escherichia coli mutants defective in initiation of elongation of deoxyribonucleic acid replication. J. Bact. 120, 1227–1237 (1974)
Womble, D.D., Taylor, D.P., Rownd, R.H.: Method for obtaining more-accurate covalently closed circular plasmid-to-chromosome ratios from bacterial lysates by dye-buoyant density centrifugation. J. Bact. 130, 148–153 (1977)
Young, E.T., Sinsheimer, R.L.: Vegetative bacteriophage λ DNA II. Physical characterization and replication. J. molec. Biol. 30, 165–200 (1967)
Author information
Authors and Affiliations
Additional information
Communicated by W. Arber
Rights and permissions
About this article
Cite this article
Uhlin, B.E., Nordström, K. A runaway-replication mutant of plasmid R1drd-19: Temperature-dependent loss of copy number control. Molec. Gen. Genet. 165, 167–179 (1978). https://doi.org/10.1007/BF00269904
Received:
Issue Date:
DOI: https://doi.org/10.1007/BF00269904