1887

Abstract

XerCD-mediated recombination at converts multimers of plasmid ColE1 to monomers, maximizing the number of independently segregating molecules and minimizing the frequency of plasmid loss. In addition to XerCD, recombination requires the accessory factors ArgR and PepA. The promoter P, located centrally within , is also required for stable plasmid maintenance. P is active in plasmid multimers and directs transcription of a short RNA, Rcd, which appears to inhibit cell division. It has been proposed that Rcd is part of a checkpoint which ensures that multimer resolution is complete before the cell divides. This study has shown that ArgR does not act as a transcriptional repressor of P in plasmid monomers. P is unusual in that the −35 and −10 hexamers are separated by only 15 bp and this study has demonstrated that increasing this to a more conventional spacing results in elevated activity. An increase to 17 bp resulted in a 10- to 20-fold increase in activity, while smaller effects were seen when the spacer was increased to 16 bp or 18 bp. These observations are consistent with the hypothesis that P activation involves realignment of the −35 and −10 sequences within a recombinational synaptic complex. This predicts that a 17 bp spacer promoter derivative should be down-regulated by plasmid multimerization, and this is confirmed experimentally.

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2001-11-01
2024-12-07
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References

  1. Ackerson, J. W. & Gralla, J. D. (1982).In vivo expression of lac promoter variants with altered −10, −35, and spacer sequences. Cold Spring Harbor Symp Quant Biol 47, 473-476. [Google Scholar]
  2. Ansari, A. Z., Chael, M. L. & O’Halloran, T. V. (1992). Allosteric underwinding of DNA is a critical step in positive control of transcription by Hg-MerR. Nature 355, 87-89.[CrossRef] [Google Scholar]
  3. Aoyama, T., Takanami, M., Ohtsuka, E., Taniyama, Y, Marumoto, R., Sato, H. & Ikehara, M. (1983). Essential structure of E. coli promoter: effect of spacer length between the two consensus sequences on promoter function. Nucleic Acids Res 17, 5855-5864. [Google Scholar]
  4. Bachmann, B. J. (1972). Pedigrees of some mutant strains of Escherichia coli K-12. Bacteriol Rev 36, 525-557. [Google Scholar]
  5. Berman, M. L. & Landy, A (1979). Promoter mutations in the transfer RNA gene tyrT of Escherichia coli. Proc Natl Acad Sci USA 76, 4303-4307.[CrossRef] [Google Scholar]
  6. Blakely, G., May, G., McCulloch, R., Arciszewska, L. K., Burke, M., Lovett, S. T. & Sherratt, D. J. (1993). Two related recombinases are required for site-specific recombination at dif and cer in E. coli K12. Cell 75, 351-361.[CrossRef] [Google Scholar]
  7. Cohen, S. N., Chang, A. C. Y. & Hsu, L. (1972). Non-chromosomal antibiotic resistance in bacteria: genetic transformation of Escherichia coli by R-factor DNA. Proc Natl Acad Sci USA 69, 2110-2114.[CrossRef] [Google Scholar]
  8. Collis, C. M., Molloy, P. L., Both, G. W. & Drew, H. R. (1989). Influence of the sequence-dependent flexure of DNA on transcription in E. coli. Nucleic Acids Res 17, 9447-9468.[CrossRef] [Google Scholar]
  9. Colloms, S. D., Sykora, P., Szatmari, G. & Sherratt, D. J. (1990). Recombination at ColE1 cer requires the Escherichia coli xerC gene product, a member of the lambda integrase family of site-specific recombinases. J Bacteriol 172, 6973-6980. [Google Scholar]
  10. Guhathakurta, A. & Summers, D. K. (1995). Involvement of ArgR and PepA in the pairing of ColE1 dimer resolution sites. Microbiology 141, 1163-1171.[CrossRef] [Google Scholar]
  11. Guhathakurta, A., Viney, I. & Summers, D. K. (1996). Accessory proteins impose site selectivity during ColE1 dimer resolution. Mol Microbiol 20, 613-620.[CrossRef] [Google Scholar]
  12. Hagerman, P. J. (1990). Sequence-directed curvature of DNA. Annu Rev Biochem 59, 755-781.[CrossRef] [Google Scholar]
  13. Harley, C. B. & Reynolds, R. P. (1987). Analysis of E. coli promoter sequences. Nucleic Acids Res 15, 2343-2361.[CrossRef] [Google Scholar]
  14. Hawley, D. K. & McClure, W. R. (1983). Compilation and analysis of Escherichia coli promoter DNA sequences. Nucleic Acids Res 11, 2237-2255.[CrossRef] [Google Scholar]
  15. Hodgman, T. C., Griffiths, H. & Summers, D. K. (1998). Nucleoprotein architecture and ColE1 dimer resolution: a hypothesis. Mol Microbiol 29, 545-558.[CrossRef] [Google Scholar]
  16. Jordi, B. J. A. M., Owen-Hughes, T. A., Hulton, C. S. J. & Higgins, C. F. (1995). DNA twist, flexibility and transcription of the osmoregulated proU promoter of Salmonella typhimurium. EMBO J 14, 5690-5700. [Google Scholar]
  17. Kennedy, C. K. (1971). Induction of colicin production by high temperature or inhibition of protein synthesis. J Bacteriol 108, 10-19. [Google Scholar]
  18. Koo, H.-S., Wu, H.-M. & Crothers, D. M. (1986). DNA bending at adenine thymine tracts. Nature 320, 501-506.[CrossRef] [Google Scholar]
  19. Lozinski, T., Adrych-Rozek, K., Markiewicz, W. T. & Wierzchowski, K. L. (1991). Effect of DNA bending in various regions of a consensus-like Escherichia coli promoter on its strength in vivo and structure of the open complex in vitro. Nucleic Acids Res 19, 2947-2953.[CrossRef] [Google Scholar]
  20. McKenney, K., Shimatake, H., Court, D., Schmeisser, U., Brady, C. & Rosenberg, M. (1981). A system to study promoter and terminator signals recognized by Escherichia coli RNA polymerase. In Gene Amplification and Analysis , pp. 383-415. Edited by J. G. Chirikjian & T. S. Papa. Amsterdam:Elsevier.
  21. Mandecki, W. & Reznikoff, W. S. (1982). A lac promoter with a changed distance between −10 and −35 regions. Nucleic Acids Res 10, 903-912.[CrossRef] [Google Scholar]
  22. Mandecki, W., Goldman, R. A., Powell, B. S. & Caruthers, M. H. (1985).lac up-promoter mutants with increased homology to the consensus promoter sequence. J Bacteriol 164, 1353-1355. [Google Scholar]
  23. Messing, J. (1983). New M13 vectors for cloning. Methods Enzymol 101, 20-78. [Google Scholar]
  24. Norrander, J., Kempe, T. & Messing, J. (1983). Construction of improved M13 vectors using oligodeoxynucleotide-directed mutagenesis. Gene 26, 101-106.[CrossRef] [Google Scholar]
  25. O’Halloran, T. V., Frantz, B., Shin, M. K., Ralston, D. M. & Wright, J. G. (1989). The MerR heavy metal receptor mediates positive activation in a topologically novel transcription complex. Cell 56, 119-129.[CrossRef] [Google Scholar]
  26. Parkhill, J. & Brown, N. L. (1990). Site-specific insertion and deletion mutants in the mer promoter-operator region of Tn501; the nineteen base-pair spacer is essential for normal induction of the promoter by MerR. Nucleic Acids Res 18, 5157-5162.[CrossRef] [Google Scholar]
  27. Patient, M. E. & Summers, D. K. (1993). ColE1 multimer formation triggers inhibition of Escherichia coli cell division. Mol Microbiol 9, 1089-1095.[CrossRef] [Google Scholar]
  28. Rowe, C. D. & Summers, D. K. (1999). The quiescent-cell expression system for protein synthesis in Escherichia coli. Appl Environ Microbiol 65, 2710-2715. [Google Scholar]
  29. Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989).Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
  30. Sharpe, M. E., Chatwin, H. M., Macpherson, C., Withers, H. L. & Summers, D. K. (1999). Analysis of the ColE1 stability determinant Rcd. Microbiology 145, 2135-2144.[CrossRef] [Google Scholar]
  31. Stefano, J. E. & Gralla, J. D. (1982). Spacer mutations in the lac ps promoter. Proc Natl Acad Sci USA 79, 1069-1072.[CrossRef] [Google Scholar]
  32. Stirling, C. J., Stewart, G. & Sherratt, D. J. (1988a). Multicopy plasmid stability in Escherichia coli requires host-encoded functions that lead to plasmid site-specific recombination. Mol Gen Genet 214, 80-84.[CrossRef] [Google Scholar]
  33. Stirling, C. J., Szatmari, G., Stewart, G., Smith, M. C. M. & Sherratt, D. J. (1988b). The arginine repressor is essential for plasmid stabilizing site-specific recombination at the ColE1 cer locus. EMBO J 7, 4389-4395. [Google Scholar]
  34. Stirling, C. J., Colloms, S. D., Collins, J. F., Szatmari, G. & Sherratt, D. J. (1989).xerB, an Escherichia coli gene required for plasmid ColE1 site-specific recombination, is identical to pepA, encoding aminopeptidase A, a protein with substantial similarity to bovine lens leucine aminopeptidase. EMBO J 8, 1623-1627. [Google Scholar]
  35. Sträter, N., Sherratt, D. J. & Colloms, S. D. (1999). X-ray structure of aminopeptidase A from Escherichia coli and a model for the nucleoprotein complex in Xer site-specific recombination. EMBO J 18, 4513-4522.[CrossRef] [Google Scholar]
  36. Summers, D. K. (1991). The kinetics of plasmid loss. Trends Biotechnol 9, 273-278.[CrossRef] [Google Scholar]
  37. Summers, D. K. (1998). Timing, self-control and a sense of direction are the secrets of multicopy plasmid stability. Mol Microbiol 29, 1137-1145.[CrossRef] [Google Scholar]
  38. Summers, D. K. & Sherratt, D. J. (1984). Multimerization of high copy number plasmids causes instability: ColE1 encodes a determinant essential for plasmid monomerization and stability. Cell 36, 1097-1103.[CrossRef] [Google Scholar]
  39. Summers, D. K. & Sherratt, D. J. (1988). Resolution of ColE1 dimers requires a DNA sequence implicated in the three-dimensional organization of the cer site. EMBO J 7, 851-858. [Google Scholar]
  40. Summers, D. K., Beton, C. W. H. & Withers, H. L. (1993). Multicopy plasmid instability: the dimer catastrophe hypothesis. Mol Microbiol 8, 1031-1038.[CrossRef] [Google Scholar]
  41. Vieira, J. & Messing, J. (1982). The pUC plasmids, an M13mp7-derived system for insertion mutagenesis and sequencing with synthetic universal primers. Gene 19, 259-268.[CrossRef] [Google Scholar]
  42. Yanisch-Perron, C., Vieira, J. & Messing, J. (1985). Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene 33, 103-119.[CrossRef] [Google Scholar]
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