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. 1980 Dec;144(3):1126–1138. doi: 10.1128/jb.144.3.1126-1138.1980

Plasmid replication functions: two distinct segments of plasmid R1, RepA and RepD, express incompatibility and are capable of autonomous replication.

H Danbara, J K Timmis, R Lurz, K N Timmis
PMCID: PMC294779  PMID: 6254944

Abstract

The genetic determinants for replication and incompatibility of plasmid R1 were investigated by gene cloning methods, and three types of R1 miniplasmid derivatives were generated. The first, exemplified by plasmid pKT300, consisted of a single BglII endonuclease-generated deoxyribonucleic acid fragment derived from the R1 region that is located between the determinants for conjugal transfer and antibiotic resistance. Two types of miniplasmids could be formed from PstI endonuclease-generated fragments of pKT300. One of these, which is equivalent to miniplasmids previously generated from plasmids R1-19 and R1-19B2, consisted of two adjacent PstI fragments that encode the RepA replication system of plasmid R1. The other type contained a segment of R1, designated the RepD replication region, that is adjacent to the RepA region and that has not been identified previously as having the capacity for autonomous replication. Plasmid R1, therefore, contained two distinct deoxyribonucleic acid segments capable of autonomous replication. The RepA-RepD miniplasmid pKT300 had a copy number about eightfold higher than that of R1 and hence lacked a determinant for the regulation of plasmid copy number. Like R1, it was maintained stably in dividing bacteria. RepA miniplasmids had copy numbers which were two- to fourfold higher than that of R1 (i.e., which were lower than that of pKT300) and were maintained slightly less stably than those of pKT300 and R1. The RepD miniplasmid was not maintained stably in dividing bacteria. Previous experiments have shown that incompatibility of IncFII group plasmids is specified by a plasmid copy control gene. Despite the fact that RepA miniplasmids of R1 were defective in copy control, they nevertheless expressed incompatibility. This suggests that two genes are responsible for plasmid copy control, one that specifies incompatibility and is located on RepA miniplasmids and another that is located outside of, but adjacent to, the RepA replication region. Hybrid plasmids composed of pBR322 and one PstI fragment from the RepA region, P-8, exhibited incompatibility towards R2 and RepA miniplasmids but not the RepD miniplasmid, whereas hybrids composed of pBR322 and the PstI fragment of the RepD region, P-3, exhibited incompatibility towards R1 and the RepD miniplasmid but not RepA miniplasmids. These results indicate that the two replication systems are functionally distinct and that, although the RepA system is the principal replication system of R1, the RepD system also plays a role in the maintenance of this plasmid.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Andrés I., Slocombe P. M., Cabello F., Timmis J. K., Lurz R., Burkardt H. J., Timmis K. N. Plasmid replication functions. II. Cloning analysis of the repA replication region of antibiotic resistance plasmid R6-5. Mol Gen Genet. 1979 Jan 5;168(1):1–25. doi: 10.1007/BF00267929. [DOI] [PubMed] [Google Scholar]
  2. Bachmann B. J., Low K. B. Linkage map of Escherichia coli K-12, edition 6. Microbiol Rev. 1980 Mar;44(1):1–56. doi: 10.1128/mr.44.1.1-56.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Blohm D., Goebel W. Restriction map of the antibiotic resistance plasmid R1drd-19 and its derivatives pKN102 (R1drd-19B2) and R1drd-16 for the enzymes BamHI, HindIII, EcoRI and SalI. Mol Gen Genet. 1978 Nov 29;167(2):119–127. doi: 10.1007/BF00266905. [DOI] [PubMed] [Google Scholar]
  4. Bolivar F., Rodriguez R. L., Greene P. J., Betlach M. C., Heyneker H. L., Boyer H. W., Crosa J. H., Falkow S. Construction and characterization of new cloning vehicles. II. A multipurpose cloning system. Gene. 1977;2(2):95–113. [PubMed] [Google Scholar]
  5. Burkardt H. J., Riess G., Pühler A. Relationship of group P1 plasmids revealed by heteroduplex experiments: RP1, RP4, R68 and RK2 are identical. J Gen Microbiol. 1979 Oct;114(2):341–348. doi: 10.1099/00221287-114-2-341. [DOI] [PubMed] [Google Scholar]
  6. Chang A. C., Cohen S. N. Construction and characterization of amplifiable multicopy DNA cloning vehicles derived from the P15A cryptic miniplasmid. J Bacteriol. 1978 Jun;134(3):1141–1156. doi: 10.1128/jb.134.3.1141-1156.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Greene P. J., Heyneker H. L., Bolivar F., Rodriguez R. L., Betlach M. C., Covarrubias A. A., Backman K., Russel D. J., Tait R., Boyer H. W. A general method for the purification of restriction enzymes. Nucleic Acids Res. 1978 Jul;5(7):2373–2380. doi: 10.1093/nar/5.7.2373. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Heffron F., McCarthy B. J., Ohtsubo H., Ohtsubo E. DNA sequence analysis of the transposon Tn3: three genes and three sites involved in transposition of Tn3. Cell. 1979 Dec;18(4):1153–1163. doi: 10.1016/0092-8674(79)90228-9. [DOI] [PubMed] [Google Scholar]
  9. Ishii K., Hashimoto-Gotoh T., Matsubara K. Random replication and random assortment model for plasmid incompatibility in bacteria. Plasmid. 1978 Sep;1(4):435–445. doi: 10.1016/0147-619x(78)90002-1. [DOI] [PubMed] [Google Scholar]
  10. Kollek R., Oertel W., Goebel W. Isolation and characterization of the minimal fragment required for autonomous replication ("basic replicon") of a copy mutant (pKN102) of the antibiotic resistance factor R1. Mol Gen Genet. 1978 Jun 1;162(1):51–57. doi: 10.1007/BF00333850. [DOI] [PubMed] [Google Scholar]
  11. Kornberg A. Aspects of DNA replication. Cold Spring Harb Symp Quant Biol. 1979;43(Pt 1):1–9. doi: 10.1101/sqb.1979.043.01.003. [DOI] [PubMed] [Google Scholar]
  12. Lane D., Gardner R. C. Second EcoRI fragment of F capable of self-replication. J Bacteriol. 1979 Jul;139(1):141–151. doi: 10.1128/jb.139.1.141-151.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Meynell E., Datta N. Mutant drug resistant factors of high transmissibility. Nature. 1967 May 27;214(5091):885–887. doi: 10.1038/214885a0. [DOI] [PubMed] [Google Scholar]
  14. Meynell E., Datta N. The relation of resistance transfer factors to the F-factor (sex-factor) of Escherichia coli K12. Genet Res. 1966 Feb;7(1):134–140. doi: 10.1017/s0016672300009538. [DOI] [PubMed] [Google Scholar]
  15. Miki T., Easton A. M., Rownd R. H. Cloning of replication, incompatibility, and stability functions of R plasmid NR1. J Bacteriol. 1980 Jan;141(1):87–99. doi: 10.1128/jb.141.1.87-99.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Molin S., Nordström K. Control of plasmid R1 replication: functions involved in replication, copy number control, incompatibility, and switch-off of replication. J Bacteriol. 1980 Jan;141(1):111–120. doi: 10.1128/jb.141.1.111-120.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Molin S., Stougaard P., Uhlin B. E., Gustafsson P., Nordström K. Clustering of genes involved in replication, copy number control, incompatibility, and stable maintenance of the resistance plasmid R1drd-19. J Bacteriol. 1979 Apr;138(1):70–79. doi: 10.1128/jb.138.1.70-79.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Ohtsubo E., Feingold J., Ohtsubo H., Mickel S., Bauer W. Unidirectional replication in Escherichia coli of three small plasmids derived from R factor R12. Plasmid. 1977 Nov;1(1):8–18. doi: 10.1016/0147-619x(77)90004-x. [DOI] [PubMed] [Google Scholar]
  19. Paulson J. R., Laemmli U. K. The structure of histone-depleted metaphase chromosomes. Cell. 1977 Nov;12(3):817–828. doi: 10.1016/0092-8674(77)90280-x. [DOI] [PubMed] [Google Scholar]
  20. Sharp P. A., Cohen S. N., Davidson N. Electron microscope heteroduplex studies of sequence relations among plasmids of Escherichia coli. II. Structure of drug resistance (R) factors and F factors. J Mol Biol. 1973 Apr 5;75(2):235–255. doi: 10.1016/0022-2836(73)90018-1. [DOI] [PubMed] [Google Scholar]
  21. Silver L., Chandler M., de la Tour E. B., Caro L. Origin and direction of replication of the drug resistance plasmid R100.1 and of a resistance transfer factor derivative in synchronized cultures. J Bacteriol. 1977 Sep;131(3):929–942. doi: 10.1128/jb.131.3.929-942.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Silver R. P., Cohen S. N. Nonchromosomal antibiotic resistance in bacteria. V. Isolation and characterization of R factor mutants exhibiting temperature-sensitive repression of fertility. J Bacteriol. 1972 Jun;110(3):1082–1088. doi: 10.1128/jb.110.3.1082-1088.1972. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Sutcliffe J. G. Complete nucleotide sequence of the Escherichia coli plasmid pBR322. Cold Spring Harb Symp Quant Biol. 1979;43(Pt 1):77–90. doi: 10.1101/sqb.1979.043.01.013. [DOI] [PubMed] [Google Scholar]
  24. Synenki R. M., Nordheim A., Timmis K. N. Plasmid replication functions. III. Origin and direction of replication of a "mini" plasmid derived from R6-5. Mol Gen Genet. 1979 Jan 5;168(1):27–36. doi: 10.1007/BF00267930. [DOI] [PubMed] [Google Scholar]
  25. Taylor D. P., Cohen S. N. Structural and functional analysis of cloned DNA segments containing the replication and incompatibility regions of a miniplasmid derived from a copy number mutant of NR1. J Bacteriol. 1979 Jan;137(1):92–104. doi: 10.1128/jb.137.1.92-104.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Timmis K. N., Andrés I., Slocombe P. M. Plasmid incompatibility: cloning analysis of an incFII determinant of R6-5. Nature. 1978 May 4;273(5657):27–32. doi: 10.1038/273027a0. [DOI] [PubMed] [Google Scholar]
  27. Timmis K. N., Cabello F., Cohen S. N. Cloning and characterization of EcoRI and HindIII restriction endonuclease-generated fragments of antibiotic resistance plasmids R6-5 and R6. Mol Gen Genet. 1978 Jun 14;162(2):121–137. doi: 10.1007/BF00267869. [DOI] [PubMed] [Google Scholar]
  28. Timmis K., Cabello F., Cohen S. N. Cloning, isolation, and characterization of replication regions of complex plasmid genomes. Proc Natl Acad Sci U S A. 1975 Jun;72(6):2242–2246. doi: 10.1073/pnas.72.6.2242. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Timmis K., Cabello F., Cohen S. N. Utilization of two distinct modes of replication by a hybrid plasmid constructed in vitro from separate replicons. Proc Natl Acad Sci U S A. 1974 Nov;71(11):4556–4560. doi: 10.1073/pnas.71.11.4556. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. 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. 1975 Nov;124(2):641–649. doi: 10.1128/jb.124.2.641-649.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Yoshikawa M. Identification and mapping of the replication genes of an R factor, R100-1, integrated into the chromosome of Escherichia coli K-12. J Bacteriol. 1974 Jun;118(3):1123–1131. doi: 10.1128/jb.118.3.1123-1131.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]

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