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EMBO J. Mar 15, 1999; 18(6): 1712–1721.
PMCID: PMC1171257

Distribution of minichromosomes in individual Escherichia coli cells: implications for replication control.

Abstract

A novel method was devised to measure the number of plasmids in individual Escherichia coli cells. With this method, involving measurement of plasmid-driven expression of the green fluorescent protein gene by flow cytometry, the copy number distribution of a number of different plasmids was measured. Whereas natural plasmids had fairly narrow distributions, minichromosomes, which are plasmids replicating only from a cloned oriC copy, have a wide distribution, suggesting that there is no copy number control for minichromosomes. When the selection pressure (kanamycin concentration) for minichromosomes was increased, the copy number of minichromosomes was also increased. At up to 30 minichromosomes per host chromosome, replication and growth of the host cell was unaffected. This is evidence that there is no negative element for initiation control in oriC and that there is no incompatibility between oriC located on the chromosome and minichromosome. However, higher copy numbers led to integration of the minichromosomes at the chromosomal oriC and to initiation asynchrony of the host chromosome. At a minichromosome copy number of approximately 30, the cell's capacity for synchronous initiation is exceeded and free minichromosomes will compete out the chromosome to yield inviable cells, unless the minichromosomes are incorporated into the chromosome.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Asai T, Bates DB, Boye E, Kogoma T. Are minichromosomes valid model systems for DNA replication control? Lessons learned from Escherichia coli. Mol Microbiol. 1998 Aug;29(3):671–675. [PMC free article] [PubMed]
  • Atlung T, Løbner-Olesen A, Hansen FG. Overproduction of DnaA protein stimulates initiation of chromosome and minichromosome replication in Escherichia coli. Mol Gen Genet. 1987 Jan;206(1):51–59. [PubMed]
  • Berg DE, Davies J, Allet B, Rochaix JD. Transposition of R factor genes to bacteriophage lambda. Proc Natl Acad Sci U S A. 1975 Sep;72(9):3628–3632. [PMC free article] [PubMed]
  • BERTANI G. Studies on lysogenesis. I. The mode of phage liberation by lysogenic Escherichia coli. J Bacteriol. 1951 Sep;62(3):293–300. [PMC free article] [PubMed]
  • Bipatnath M, Dennis PP, Bremer H. Initiation and velocity of chromosome replication in Escherichia coli B/r and K-12. J Bacteriol. 1998 Jan;180(2):265–273. [PMC free article] [PubMed]
  • Blattner FR, Plunkett G, 3rd, Bloch CA, Perna NT, Burland V, Riley M, Collado-Vides J, Glasner JD, Rode CK, Mayhew GF, et al. The complete genome sequence of Escherichia coli K-12. Science. 1997 Sep 5;277(5331):1453–1462. [PubMed]
  • Bolivar F, Rodriguez RL, Greene PJ, Betlach MC, Heyneker HL, Boyer HW, Crosa JH, Falkow S. Construction and characterization of new cloning vehicles. II. A multipurpose cloning system. Gene. 1977;2(2):95–113. [PubMed]
  • Boye E, Løbner-Olesen A. The role of dam methyltransferase in the control of DNA replication in E. coli. Cell. 1990 Sep 7;62(5):981–989. [PubMed]
  • Boye E, Løbner-Olesen A, Skarstad K. Timing of chromosomal replication in Escherichia coli. Biochim Biophys Acta. 1988 Dec 20;951(2-3):359–364. [PubMed]
  • Bramhill D, Kornberg A. Duplex opening by dnaA protein at novel sequences in initiation of replication at the origin of the E. coli chromosome. Cell. 1988 Mar 11;52(5):743–755. [PubMed]
  • Braun RE, O'Day K, Wright A. Autoregulation of the DNA replication gene dnaA in E. coli K-12. Cell. 1985 Jan;40(1):159–169. [PubMed]
  • Campbell JL, Kleckner N. E. coli oriC and the dnaA gene promoter are sequestered from dam methyltransferase following the passage of the chromosomal replication fork. Cell. 1990 Sep 7;62(5):967–979. [PubMed]
  • Casadaban MJ, Cohen SN. Analysis of gene control signals by DNA fusion and cloning in Escherichia coli. J Mol Biol. 1980 Apr;138(2):179–207. [PubMed]
  • Chang AC, Cohen SN. Construction and characterization of amplifiable multicopy DNA cloning vehicles derived from the P15A cryptic miniplasmid. J Bacteriol. 1978 Jun;134(3):1141–1156. [PMC free article] [PubMed]
  • Cooper S, Keasling JD. Cycle-specific replication of chromosomal and F-plasmid origins. FEMS Microbiol Lett. 1998 Jun 15;163(2):217–222. [PubMed]
  • Cormack BP, Valdivia RH, Falkow S. FACS-optimized mutants of the green fluorescent protein (GFP). Gene. 1996;173(1 Spec No):33–38. [PubMed]
  • Davies J, Smith DI. Plasmid-determined resistance to antimicrobial agents. Annu Rev Microbiol. 1978;32:469–518. [PubMed]
  • del Solar G, Giraldo R, Ruiz-Echevarría MJ, Espinosa M, Díaz-Orejas R. Replication and control of circular bacterial plasmids. Microbiol Mol Biol Rev. 1998 Jun;62(2):434–464. [PMC free article] [PubMed]
  • Donachie WD. Relationship between cell size and time of initiation of DNA replication. Nature. 1968 Sep 7;219(5158):1077–1079. [PubMed]
  • Eliasson A, Nordström K. Replication of minichromosomes in a host in which chromosome replication is random. Mol Microbiol. 1997 Mar;23(6):1215–1220. [PubMed]
  • Fuller RS, Funnell BE, Kornberg A. The dnaA protein complex with the E. coli chromosomal replication origin (oriC) and other DNA sites. Cell. 1984 Oct;38(3):889–900. [PubMed]
  • Guzman LM, Belin D, Carson MJ, Beckwith J. Tight regulation, modulation, and high-level expression by vectors containing the arabinose PBAD promoter. J Bacteriol. 1995 Jul;177(14):4121–4130. [PMC free article] [PubMed]
  • Hansen FG, Christensen BB, Atlung T. The initiator titration model: computer simulation of chromosome and minichromosome control. Res Microbiol. 1991 Feb-Apr;142(2-3):161–167. [PubMed]
  • Hansen FG, Atlung T, Braun RE, Wright A, Hughes P, Kohiyama M. Initiator (DnaA) protein concentration as a function of growth rate in Escherichia coli and Salmonella typhimurium. J Bacteriol. 1991 Aug;173(16):5194–5199. [PMC free article] [PubMed]
  • Helmstetter CE, Thornton M, Zhou P, Bogan JA, Leonard AC, Grimwade JE. Replication and segregation of a miniF plasmid during the division cycle of Escherichia coli. J Bacteriol. 1997 Feb;179(4):1393–1399. [PMC free article] [PubMed]
  • Hiraga S. Novel F prime factors able to replicate in Escherichia coli Hfr strains. Proc Natl Acad Sci U S A. 1976 Jan;73(1):198–202. [PMC free article] [PubMed]
  • Jensen MR, Løbner-Olesen A, Rasmussen KV. Escherichia coli minichromosomes: random segregation and absence of copy number control. J Mol Biol. 1990 Sep 20;215(2):257–265. [PubMed]
  • Keasling JD, Palsson BO, Cooper S. Cell-cycle-specific F plasmid replication: regulation by cell size control of initiation. J Bacteriol. 1991 Apr;173(8):2673–2680. [PMC free article] [PubMed]
  • Kitagawa R, Mitsuki H, Okazaki T, Ogawa T. A novel DnaA protein-binding site at 94.7 min on the Escherichia coli chromosome. Mol Microbiol. 1996 Mar;19(5):1137–1147. [PubMed]
  • Kitagawa R, Ozaki T, Moriya S, Ogawa T. Negative control of replication initiation by a novel chromosomal locus exhibiting exceptional affinity for Escherichia coli DnaA protein. Genes Dev. 1998 Oct 1;12(19):3032–3043. [PMC free article] [PubMed]
  • Koppes LJ. Nonrandom F-plasmid replication in Escherichia coli K-12. J Bacteriol. 1992 Apr;174(7):2121–2123. [PMC free article] [PubMed]
  • Koppes LJ, von Meyenburg K. Nonrandom minichromosome replication in Escherichia coli K-12. J Bacteriol. 1987 Jan;169(1):430–433. [PMC free article] [PubMed]
  • Landoulsi A, Malki A, Kern R, Kohiyama M, Hughes P. The E. coli cell surface specifically prevents the initiation of DNA replication at oriC on hemimethylated DNA templates. Cell. 1990 Nov 30;63(5):1053–1060. [PubMed]
  • Leonard AC, Helmstetter CE. Cell cycle-specific replication of Escherichia coli minichromosomes. Proc Natl Acad Sci U S A. 1986 Jul;83(14):5101–5105. [PMC free article] [PubMed]
  • Leonard AC, Helmstetter CE. Replication patterns of multiple plasmids coexisting in Escherichia coli. J Bacteriol. 1988 Mar;170(3):1380–1383. [PMC free article] [PubMed]
  • Løbner-Olesen A, von Freiesleben U. Chromosomal replication incompatibility in Dam methyltransferase deficient Escherichia coli cells. EMBO J. 1996 Nov 1;15(21):5999–6008. [PMC free article] [PubMed]
  • Løbner-Olesen A, Atlung T, Rasmussen KV. Stability and replication control of Escherichia coli minichromosomes. J Bacteriol. 1987 Jun;169(6):2835–2842. [PMC free article] [PubMed]
  • Løbner-Olesen A, Skarstad K, Hansen FG, von Meyenburg K, Boye E. The DnaA protein determines the initiation mass of Escherichia coli K-12. Cell. 1989 Jun 2;57(5):881–889. [PubMed]
  • Løbner-Olesen A, Hansen FG, Rasmussen KV, Martin B, Kuempel PL. The initiation cascade for chromosome replication in wild-type and Dam methyltransferase deficient Escherichia coli cells. EMBO J. 1994 Apr 15;13(8):1856–1862. [PMC free article] [PubMed]
  • Lovett MA, Helinski DR. Method for the isolation of the replication region of a bacterial replicon: construction of a mini-F'kn plasmid. J Bacteriol. 1976 Aug;127(2):982–987. [PMC free article] [PubMed]
  • MacFerrin KD, Chen L, Terranova MP, Schreiber SL, Verdine GL. Overproduction of proteins using expression-cassette polymerase chain reaction. Methods Enzymol. 1993;217:79–102. [PubMed]
  • Marsh RC, Worcel A. A DNA fragment containing the origin of replication of the Escherichia coli chromosome. Proc Natl Acad Sci U S A. 1977 Jul;74(7):2720–2724. [PMC free article] [PubMed]
  • Messer W, Bergmans HE, Meijer M, Womack JE, Hansen FG, von Meyenburg K. Mini-chromosomes: plasmids which carry the E. coli replication origin. Mol Gen Genet. 1978 Jul 4;162(3):269–275. [PubMed]
  • Ogden GB, Pratt MJ, Schaechter M. The replicative origin of the E. coli chromosome binds to cell membranes only when hemimethylated. Cell. 1988 Jul 1;54(1):127–135. [PubMed]
  • Ogura T, Hiraga S. Partition mechanism of F plasmid: two plasmid gene-encoded products and a cis-acting region are involved in partition. Cell. 1983 Feb;32(2):351–360. [PubMed]
  • Pal SK, Mason RJ, Chattoraj DK. P1 plasmid replication. Role of initiator titration in copy number control. J Mol Biol. 1986 Nov 20;192(2):275–285. [PubMed]
  • Roth A, Messer W. High-affinity binding sites for the initiator protein DnaA on the chromosome of Escherichia coli. Mol Microbiol. 1998 Apr;28(2):395–401. [PubMed]
  • Schaper S, Messer W. Interaction of the initiator protein DnaA of Escherichia coli with its DNA target. J Biol Chem. 1995 Jul 21;270(29):17622–17626. [PubMed]
  • Siegele DA, Hu JC. Gene expression from plasmids containing the araBAD promoter at subsaturating inducer concentrations represents mixed populations. Proc Natl Acad Sci U S A. 1997 Jul 22;94(15):8168–8172. [PMC free article] [PubMed]
  • Skarstad K, Boye E, Steen HB. Timing of initiation of chromosome replication in individual Escherichia coli cells. EMBO J. 1986 Jul;5(7):1711–1717. [PMC free article] [PubMed]
  • Skarstad K, von Meyenburg K, Hansen FG, Boye E. Coordination of chromosome replication initiation in Escherichia coli: effects of different dnaA alleles. J Bacteriol. 1988 Feb;170(2):852–858. [PMC free article] [PubMed]
  • Sompayrac L, Maaloe O. Autorepressor model for control of DNA replication. Nat New Biol. 1973 Jan 31;241(109):133–135. [PubMed]
  • Stoker NG, Fairweather NF, Spratt BG. Versatile low-copy-number plasmid vectors for cloning in Escherichia coli. Gene. 1982 Jun;18(3):335–341. [PubMed]
  • von Meyenburg K, Hansen FG, Riise E, Bergmans HE, Meijer M, Messer W. Origin of replication, oriC, of the Escherichia coli K12 chromosome: genetic mapping and minichromosome replication. Cold Spring Harb Symp Quant Biol. 1979;43(Pt 1):121–128. [PubMed]
  • Wold S, Skarstad K, Steen HB, Stokke T, Boye E. The initiation mass for DNA replication in Escherichia coli K-12 is dependent on growth rate. EMBO J. 1994 May 1;13(9):2097–2102. [PMC free article] [PubMed]

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