Skip to main content
PMC home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1995 Nov;177(22):6338–6345. doi: 10.1128/jb.177.22.6338-6345.1995

A DNA segment conferring stable maintenance on R6K gamma-origin core replicons.

F Wu 1, I Levchenko 1, M Filutowicz 1
PMCID: PMC177482  PMID: 7592407

Abstract

The plasmid R6K gamma origin consists of two adjacent modules, the enhancer and the core, and requires R6K initiator protein pi for replication. While the core alone can replicate at a low level of wild-type pi protein, we show here that host cells do not stably maintain core plasmids. The presence of the enhancer segment confers stable inheritance on core plasmids without a significant change in average plasmid copy number. Deletions and site-directed mutagenesis indicated that the stability of core plasmids is not mediated by binding sites or consensus sequences in the enhancer for DnaA, pi protein, gyrase, Fis, or Dcm methylase. Proper segregation of core plasmids requires only the R6K stb or stability-related region, which includes the 20-bp segment of the 100-bp enhancer adjacent to the core. The use of the pi 116 mutant protein, which increases plasmid copy number fourfold, does not stabilize core plasmids lacking the enhancer. We also show that at an elevated level of wild-type pi, the gamma-origin plasmid is unstable, even in the presence of the enhancer. We discuss the differences and similarities between the R6K stability system and those found in other plasmids.

Full Text

The Full Text of this article is available as a PDF (438.9 KB).

Selected References

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

  1. Beaucage S. L., Miller C. A., Cohen S. N. Gyrase-dependent stabilization of pSC101 plasmid inheritance by transcriptionally active promoters. EMBO J. 1991 Sep;10(9):2583–2588. doi: 10.1002/j.1460-2075.1991.tb07799.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Biek D. P., Cohen S. N. Propagation of pSC101 plasmids defective in binding of integration host factor. J Bacteriol. 1992 Feb;174(3):785–792. doi: 10.1128/jb.174.3.785-792.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. 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]
  4. Conley D. L., Cohen S. N. Effects of the pSC101 partition (par) locus on in vivo DNA supercoiling near the plasmid replication origin. Nucleic Acids Res. 1995 Feb 25;23(4):701–707. doi: 10.1093/nar/23.4.701. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Conley D. L., Cohen S. N. Isolation and characterization of plasmid mutations that enable partitioning of pSC101 replicons lacking the partition (par) locus. J Bacteriol. 1995 Feb;177(4):1086–1089. doi: 10.1128/jb.177.4.1086-1089.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Crosa J. H. Three origins of replication are active in vivo in the R plasmid RSF1040. J Biol Chem. 1980 Dec 10;255(23):11075–11077. [PubMed] [Google Scholar]
  7. De Lucia P., Cairns J. Isolation of an E. coli strain with a mutation affecting DNA polymerase. Nature. 1969 Dec 20;224(5225):1164–1166. doi: 10.1038/2241164a0. [DOI] [PubMed] [Google Scholar]
  8. Dellis S., Filutowicz M. Integration host factor of Escherichia coli reverses the inhibition of R6K plasmid replication by pi initiator protein. J Bacteriol. 1991 Feb;173(3):1279–1286. doi: 10.1128/jb.173.3.1279-1286.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Dellis S., Schatz T., Rutlin K., Inman R. B., Filutowicz M. Two alternative structures can be formed by IHF protein binding to the plasmid R6K gamma origin. J Biol Chem. 1992 Dec 5;267(34):24426–24432. [PubMed] [Google Scholar]
  10. Deng W. P., Nickoloff J. A. Site-directed mutagenesis of virtually any plasmid by eliminating a unique site. Anal Biochem. 1992 Jan;200(1):81–88. doi: 10.1016/0003-2697(92)90280-k. [DOI] [PubMed] [Google Scholar]
  11. Filutowicz M., Appelt K. The integration host factor of Escherichia coli binds to multiple sites at plasmid R6K gamma origin and is essential for replication. Nucleic Acids Res. 1988 May 11;16(9):3829–3843. doi: 10.1093/nar/16.9.3829. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Filutowicz M., Davis G., Greener A., Helinski D. R. Autorepressor properties of the pi-initiation protein encoded by plasmid R6K. Nucleic Acids Res. 1985 Jan 11;13(1):103–114. doi: 10.1093/nar/13.1.103. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Filutowicz M., Dellis S., Levchenko I., Urh M., Wu F., York D. Regulation of replication of an iteron-containing DNA molecule. Prog Nucleic Acid Res Mol Biol. 1994;48:239–273. doi: 10.1016/s0079-6603(08)60857-0. [DOI] [PubMed] [Google Scholar]
  14. Filutowicz M., McEachern M. J., Helinski D. R. Positive and negative roles of an initiator protein at an origin of replication. Proc Natl Acad Sci U S A. 1986 Dec;83(24):9645–9649. doi: 10.1073/pnas.83.24.9645. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Filutowicz M., McEachern M., Greener A., Mukhopadhyay P., Uhlenhopp E., Durland R., Helinski D. Role of the pi initiation protein and direct nucleotide sequence repeats in the regulation of plasmid R6K replication. Basic Life Sci. 1985;30:125–140. doi: 10.1007/978-1-4613-2447-8_13. [DOI] [PubMed] [Google Scholar]
  16. Filutowicz M., Uhlenhopp E., Helinski D. R. Binding of purified wild-type and mutant pi initiation proteins to a replication origin region of plasmid R6K. J Mol Biol. 1986 Jan 20;187(2):225–239. doi: 10.1016/0022-2836(86)90230-5. [DOI] [PubMed] [Google Scholar]
  17. Fuller R. S., Funnell B. E., 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. doi: 10.1016/0092-8674(84)90284-8. [DOI] [PubMed] [Google Scholar]
  18. Germino J., Bastia D. Interaction of the plasmid R6K-encoded replication initiator protein with its binding sites on DNA. Cell. 1983 Aug;34(1):125–134. doi: 10.1016/0092-8674(83)90142-3. [DOI] [PubMed] [Google Scholar]
  19. Germino J., Bastia D. Primary structure of the replication initiation protein of plasmid R6K. Proc Natl Acad Sci U S A. 1982 Sep;79(18):5475–5479. doi: 10.1073/pnas.79.18.5475. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Hattman S. Partial purification of the Escherichia coli K-12 mec+ deoxyribonucleic acid-cytosine methylase: in vitro methylation completely protects bacteriophage lambda deoxyribonucleic acid against cleavage by R-EcoRII. J Bacteriol. 1977 Mar;129(3):1330–1334. doi: 10.1128/jb.129.3.1330-1334.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Hedges R. W., Datta N., Coetzee J. N., Dennison S. R factors from Proteus morganii. J Gen Microbiol. 1973 Aug;77(2):249–259. doi: 10.1099/00221287-77-2-249. [DOI] [PubMed] [Google Scholar]
  22. Hübner P., Arber W. Mutational analysis of a prokaryotic recombinational enhancer element with two functions. EMBO J. 1989 Feb;8(2):577–585. doi: 10.1002/j.1460-2075.1989.tb03412.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Ingmer H., Cohen S. N. Excess intracellular concentration of the pSC101 RepA protein interferes with both plasmid DNA replication and partitioning. J Bacteriol. 1993 Dec;175(24):7834–7841. doi: 10.1128/jb.175.24.7834-7841.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Inuzuka M., Helinski D. R. Requirement of a plasmid-encoded protein for replication in vitro of plasmid R6K. Proc Natl Acad Sci U S A. 1978 Nov;75(11):5381–5385. doi: 10.1073/pnas.75.11.5381. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Jones C. S., Osborne D. J., Stanley J. Molecular comparison of the IncX plasmids allows division into IncX1 and IncX2 subgroups. J Gen Microbiol. 1993 Apr;139(4):735–741. doi: 10.1099/00221287-139-4-735. [DOI] [PubMed] [Google Scholar]
  26. Kelley W., Bastia D. Replication initiator protein of plasmid R6K autoregulates its own synthesis at the transcriptional step. Proc Natl Acad Sci U S A. 1985 May;82(9):2574–2578. doi: 10.1073/pnas.82.9.2574. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Koch C., Vandekerckhove J., Kahmann R. Escherichia coli host factor for site-specific DNA inversion: cloning and characterization of the fis gene. Proc Natl Acad Sci U S A. 1988 Jun;85(12):4237–4241. doi: 10.1073/pnas.85.12.4237. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Kolter R., Helinski D. R. Plasmid R6K DNA replication. II. Direct nucleotide sequence repeats are required for an active gamma-origin. J Mol Biol. 1982 Oct 15;161(1):45–56. doi: 10.1016/0022-2836(82)90277-7. [DOI] [PubMed] [Google Scholar]
  29. Kolter R., Inuzuka M., Helinski D. R. Trans-complementation-dependent replication of a low molecular weight origin fragment from plasmid R6K. Cell. 1978 Dec;15(4):1199–1208. doi: 10.1016/0092-8674(78)90046-6. [DOI] [PubMed] [Google Scholar]
  30. Kontomichalou P., Mitani M., Clowes R. C. Circular R-factor molecules controlling penicillinase synthesis, replicating in Escherichia coli under either relaxed or stringent control. J Bacteriol. 1970 Oct;104(1):34–44. doi: 10.1128/jb.104.1.34-44.1970. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Kostyal D. A., Farrell M., McCabe A., Mei Z., Firshein W. Replication of an RK2 miniplasmid derivative in vitro by a DNA/membrane complex extracted from Escherichia coli: involvement of the dnaA but not dnaK host proteins and association of these and plasmid-encoded proteins with the inner membrane. Plasmid. 1989 May;21(3):226–237. doi: 10.1016/0147-619x(89)90046-2. [DOI] [PubMed] [Google Scholar]
  32. Lockshon D., Morris D. R. Sites of reaction of Escherichia coli DNA gyrase on pBR322 in vivo as revealed by oxolinic acid-induced plasmid linearization. J Mol Biol. 1985 Jan 5;181(1):63–74. doi: 10.1016/0022-2836(85)90324-9. [DOI] [PubMed] [Google Scholar]
  33. Manen D., Goebel T., Caro L. The par region of pSC101 affects plasmid copy number as well as stability. Mol Microbiol. 1990 Nov;4(11):1839–1846. doi: 10.1111/j.1365-2958.1990.tb02032.x. [DOI] [PubMed] [Google Scholar]
  34. Marinus M. G., Carraway M., Frey A. Z., Brown L., Arraj J. A. Insertion mutations in the dam gene of Escherichia coli K-12. Mol Gen Genet. 1983;192(1-2):288–289. doi: 10.1007/BF00327681. [DOI] [PubMed] [Google Scholar]
  35. Maxam A. M., Gilbert W. Sequencing end-labeled DNA with base-specific chemical cleavages. Methods Enzymol. 1980;65(1):499–560. doi: 10.1016/s0076-6879(80)65059-9. [DOI] [PubMed] [Google Scholar]
  36. McEachern M. J., Bott M. A., Tooker P. A., Helinski D. R. Negative control of plasmid R6K replication: possible role of intermolecular coupling of replication origins. Proc Natl Acad Sci U S A. 1989 Oct;86(20):7942–7946. doi: 10.1073/pnas.86.20.7942. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. McEachern M. J., Filutowicz M., Helinski D. R. Mutations in direct repeat sequences and in a conserved sequence adjacent to the repeats result in a defective replication origin in plasmid R6K. Proc Natl Acad Sci U S A. 1985 Mar;82(5):1480–1484. doi: 10.1073/pnas.82.5.1480. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Meacock P. A., Cohen S. N. Partitioning of bacterial plasmids during cell division: a cis-acting locus that accomplishes stable plasmid inheritance. Cell. 1980 Jun;20(2):529–542. doi: 10.1016/0092-8674(80)90639-x. [DOI] [PubMed] [Google Scholar]
  39. Miller C. A., Beaucage S. L., Cohen S. N. Role of DNA superhelicity in partitioning of the pSC101 plasmid. Cell. 1990 Jul 13;62(1):127–133. doi: 10.1016/0092-8674(90)90246-b. [DOI] [PubMed] [Google Scholar]
  40. Miller C. A., Tucker W. T., Meacock P. A., Gustafsson P., Cohen S. N. Nucleotide sequence of the partition locus of Escherichia coli plasmid pSC101. Gene. 1983 Oct;24(2-3):309–315. doi: 10.1016/0378-1119(83)90091-4. [DOI] [PubMed] [Google Scholar]
  41. Mukhopadhyay P., Filutowicz M., Helinski D. R. Replication from one of the three origins of the plasmid R6K requires coupled expression of two plasmid-encoded proteins. J Biol Chem. 1986 Jul 15;261(20):9534–9539. [PubMed] [Google Scholar]
  42. Nordström K., Austin S. J. Mechanisms that contribute to the stable segregation of plasmids. Annu Rev Genet. 1989;23:37–69. doi: 10.1146/annurev.ge.23.120189.000345. [DOI] [PubMed] [Google Scholar]
  43. Nordström K., Molin S., Aagaard-Hansen H. Partitioning of plasmid R1 in Escherichia coli. I. Kinetics of loss of plasmid derivatives deleted of the par region. Plasmid. 1980 Sep;4(2):215–227. doi: 10.1016/0147-619x(80)90011-6. [DOI] [PubMed] [Google Scholar]
  44. Ogden G. B., Pratt M. J., 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. doi: 10.1016/0092-8674(88)90186-9. [DOI] [PubMed] [Google Scholar]
  45. Palmer B. R., Marinus M. G. The dam and dcm strains of Escherichia coli--a review. Gene. 1994 May 27;143(1):1–12. doi: 10.1016/0378-1119(94)90597-5. [DOI] [PubMed] [Google Scholar]
  46. Ross W., Thompson J. F., Newlands J. T., Gourse R. L. E.coli Fis protein activates ribosomal RNA transcription in vitro and in vivo. EMBO J. 1990 Nov;9(11):3733–3742. doi: 10.1002/j.1460-2075.1990.tb07586.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Shafferman A., Flashner Y., Hertman I., Lion M. Identification and characterization of the functional alpha origin of DNA replication of the R6K plasmid and its relatedness to the R6K beta and gamma origins. Mol Gen Genet. 1987 Jun;208(1-2):263–270. doi: 10.1007/BF00330452. [DOI] [PubMed] [Google Scholar]
  48. Shafferman A., Helinski D. R. Structural properties of the beta origin of replication of plasmid R6K. J Biol Chem. 1983 Apr 10;258(7):4083–4090. [PubMed] [Google Scholar]
  49. Shafferman A., Kolter R., Stalker D., Helinski D. R. Plasmid R6K DNA replication. III. Regulatory properties of the pi initiation protein. J Mol Biol. 1982 Oct 15;161(1):57–76. doi: 10.1016/0022-2836(82)90278-9. [DOI] [PubMed] [Google Scholar]
  50. Shon M., Germino J., Bastia D. The nucleotide sequence of the replication origin beta of the plasmid R6K. J Biol Chem. 1982 Nov 25;257(22):13823–13827. [PubMed] [Google Scholar]
  51. Stalker D. M., Kolter R., Helinski D. R. Nucleotide sequence of the region of an origin of replication of the antibiotic resistance plasmid R6K. Proc Natl Acad Sci U S A. 1979 Mar;76(3):1150–1154. doi: 10.1073/pnas.76.3.1150. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Stalker D. M., Kolter R., Helinski D. R. Plasmid R6K DNA replication. I. Complete nucleotide sequence of an autonomously replicating segment. J Mol Biol. 1982 Oct 15;161(1):33–43. doi: 10.1016/0022-2836(82)90276-5. [DOI] [PubMed] [Google Scholar]
  53. Steck T. R., Drlica K. Bacterial chromosome segregation: evidence for DNA gyrase involvement in decatenation. Cell. 1984 Apr;36(4):1081–1088. doi: 10.1016/0092-8674(84)90058-8. [DOI] [PubMed] [Google Scholar]
  54. Tucker W. T., Miller C. A., Cohen S. N. Structural and functional analysis of the par region of the pSC 10 1 plasmid. Cell. 1984 Aug;38(1):191–201. doi: 10.1016/0092-8674(84)90540-3. [DOI] [PubMed] [Google Scholar]
  55. Wahle E., Kornberg A. The partition locus of plasmid pSC101 is a specific binding site for DNA gyrase. EMBO J. 1988 Jun;7(6):1889–1895. doi: 10.1002/j.1460-2075.1988.tb03022.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Wu F., Goldberg I., Filutowicz M. Roles of a 106-bp origin enhancer and Escherichia coli DnaA protein in replication of plasmid R6K. Nucleic Acids Res. 1992 Feb 25;20(4):811–817. doi: 10.1093/nar/20.4.811. [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. Wu F., Levchenko I., Filutowicz M. Binding of DnaA protein to a replication enhancer counteracts the inhibition of plasmid R6K gamma origin replication mediated by elevated levels of R6K pi protein. J Bacteriol. 1994 Nov;176(22):6795–6801. doi: 10.1128/jb.176.22.6795-6801.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. York D., Filutowicz M. Autoregulation-deficient mutant of the plasmid R6K-encoded pi protein distinguishes between palindromic and nonpalindromic binding sites. J Biol Chem. 1993 Oct 15;268(29):21854–21861. [PubMed] [Google Scholar]

Articles from Journal of Bacteriology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES