Summary
We have analysed as a function of cell doubling times the control of R6K plasmid replication in rep + and rep strains of Escherichia coli. The rep mutation results in an alteration or loss of an enzyme that unwinds helical DNA. We found in rep + bacteria that R6K relative dosage (plasmids per genome equivalent) remained nearly constant as growth rates increased. From this we concluded that the average plasmid concentration (plasmids per unit cell mass or volume) fell relative to the average concentration of chromosome origins when growth rates increased. In this context, the control of R6K replication is similar to that of other plasmids as seen by different workers. We also found that the relative dosage of R6K in rep mutants is greater than in rep + bacteria when both strains were grown at fast growth rates. This finding was expected since at fast growth rates the number of genome equivalents per unit mass is expected to be lower in rep mutants. Unexpectedly, however, we found the effect of the rep mutation on R6K relative dosage had occurred in a step-like manner at a slow growth rate of about 120 min per generation. This implies that both the relative dosage and concentration of R6K had increased in a step-like manner. We also found that the effect of the rep mutation on R6K concentration was lost at fast growth rates while the effect of the mutation on R6K relative dosage was not lost.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Bachman, B.J., Low, K.B., Taylor, A.L.: Recalibrated linkage map of Escherichia coli K-12. Bacteriol. Rev. 40, 116–167 (1976)
Chandler, M.G., Pritchard, R.H.: The effect of gene concentration and relative gene dosage on gene output in Escherichia coli. Mol. Gen. Genet. 138, 127–141 (1975)
Clewell, D.B., Helinski, D.R.: Effect of growth conditions on the formation of the relaxation complex of supercoiled ColE1 deoxyribonucleic acid and protein in Escherichia coli. J. Bacteriol. 110, 1135–1146 (1972)
Clewell, D.B., Helinski, D.R.: Supercoiled circular DNA-protein complex in Escherichia coli: purification and induced conversion to an open circular DNA form. Proc. Natl. Acad. Sci. U.S.A. 62, 1159–1166 (1969)
Collins, J., Pritchard, R.H.: The relationship between chromosome replication and Flac episome replication in Escherichia coli. J. Mol. Biol. 78, 143–155 (1973)
Cress, D.E., Kline, B.C.: Isolation and characterization of Escherichia coli chromosomal mutants affecting plasmid copy number. J. Bacteriol. 125, 635–642 (1976)
Denhardt, D.T., Iwaya, M., Larison, L.L.: The rep mutation. II. Its effect on Escherichia coli and on the replication of bacteriophage ϕX174. Virology 49, 486–496 (1972)
Donachie, W.D.: Relationship between cell size and time of initiation of DNA replication. Nature 219, 1077–1079 (1968)
Engberg, B., Nordström, K.: Replication of the R-factor R1 in K-12 at different growth rates. J. Bacteriol. 123, 179–186 (1975)
Friesen, J.D.: Measurement of DNA synthesis in bacterial cells. In: Methods in enzymology (L. Grossman and K. Moldave, eds.), Vol. 12, Part B, pp. 625–640. New York: Academic Press Inc. 1968
Hershfield, V., Boyer, H.W., Chow, L., Helinski, D.R.: Characterization of a mini-ColE1 plasmid. J. Bacteriol. 126, 447–453 (1976)
Kline, B.C., Helinski, D.R.: F sex factor of Escherichia coli. Size and purification in form of a strand specific relaxation complex of supercoiled deoxyribonucleic acid and protein. Biochemistry 10, 4975–4980 (1971)
Kline, B.C., Miller, J.R., Cress, D.E., Wlodarczyk, M., Manis, J.J., Otten, M.R.: Nonintegrated plasmid-chromosome complexes in Escherichia coli. J. Bacteriol. 127, 881–889 (1976)
Kontomichalou, P., Mitani, M., Clowes, R.C.: Circular R-factor molecules controlling penicillinase synthesis, replicating in E. coli under either relaxed or stringent control. J. Bacteriol. 104, 34–44 (1970)
Kool, A.J., NiJkamp, H.J.: Isolation and characterization of a copy mutant of the bacteriocinogenic plasmid CloDF13. J. Bacteriol. 120, 569–578 (1974)
Kupersztoch-Portnoy, Y.M., Miklos, G.L.G., Helinski, D.R.: Properties of the relaxation complexes of supercoiled deoxyribonucleic acid and protein of the R plasmids R64, R28K and R6K. J. Bacteriol. 120, 545–548 (1974)
Lane, H.E.D., Denhardt, D.T.: The rep mutation. III. Altered structure of the replicating Escherichia coli chromosome. J. Bacteriol. 120, 805–814 (1974)
Lane, H.E.D., Denhardt, D.T.: The rep mutation. IV. Slower movement of replication forks in Escherichia coli rep strains. J. Mol. Biol. 97, 99–112 (1975)
Lennox, E.S.: Transduction of linked genetic characters of the host by bacteriophage P1. Virology 1, 190–206 (1955)
Macrina, F.L., Weatherly, G.C., Curtiss, R. III: R6K plasmid replication: influence of chromosomal genotype in minicell-producing strains of Escherichia coli K-12. J. Bacteriol. 120, 1387–1400 (1974)
Manis, J.J., Kline, B.C.: F plasmid incompatibility and copy number genes: Their map locations and interactions. Plasmid 1, 492–507 (1978)
Morris, C.F., Hashimoto, H., Mickel, S., Rownd, R.: Round of replication mutant of a drug resistance factor. J. Bacteriol. 118, 855–866 (1974)
Novick, R.P., Clowes, R.C., Cohen, S.N., Curtiss, R. III, Datta, N., Falkow, S.: A uniform nomenclature for bacterial plasmids: a proposal. Bacteriol. Rev. 40, 168–189 (1976)
Prentki, P., Chandler, M.G., Caro, L.: Replication of prophage P1 during the cell cycle of Escherichia coli. Mol. Gen. Genet. 152, 71–76 (1977)
Pritchard, R.H.: On the growth and form of a bacterial cell. Philos. Trans. R. Soc. Lond. (Biol.) 267, 303–336 (1974)
Pritchard, R.H., Chandler, M.G., Collins, H.: Independence of F replication and chromosome replication in Escherichia coli. Mol. Gen. Genet. 138, 143–155 (1975)
Rush, M., Novick, R., DeLap, R.: Detection and quantitation of Staphylococcus aureus penicillinase plasmid deoxyribonucleic acid by reassociation kinetics. J. Bacteriol. 124, 1417–1423 (1975)
Scott, J.F., Eisenberg, S., Bertoch, L., Kornberg, A.: A mechanism of duplex DNA replication revealed by enzymatic studies of phage ϕX174: catalytic strand separation in advance of replication. Proc. Natl. Acad. Sci. U.S.A. 74, 193–197 (1977)
Uhlin, B.E., Nordström, K.: Plasmid incompatibility and control of replication: copy mutants of the R-factor R1 in Escherichia coli K-12. J. Bacteriol. 124, 641–649 (1975)
Author information
Authors and Affiliations
Additional information
Communicated by B.A. Bridges
Rights and permissions
About this article
Cite this article
Otten, M.R., Wlodarcyzk, M., Kline, B.C. et al. Control of plasmid R6K copy numbers in isogenic rep + and rep Escherichia coli strains. Molec. Gen. Genet. 177, 493–499 (1980). https://doi.org/10.1007/BF00271489
Received:
Issue Date:
DOI: https://doi.org/10.1007/BF00271489