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. 1990 Nov;172(11):6204–6216. doi: 10.1128/jb.172.11.6204-6216.1990

Genetic characterization of the stabilizing functions of a region of broad-host-range plasmid RK2.

R C Roberts 1, R Burioni 1, D R Helinski 1
PMCID: PMC526801  PMID: 2121707

Abstract

One of the regions responsible for the stable inheritance of the broad-host-range plasmid RK2 is contained within the PstI C fragment, located from coordinates 30.8 to 37.0 kb (P.N. Saurugger, O. Hrabak, H. Schwab, and R.M. Lafferty, J. Biotechnol. 4:333-343, 1986). Genetic analysis of this 6.2-kb region demonstrated that no function was present that stabilized by selectively killing plasmid-free segregants. The sequence from 36.0 to 37.0 kb mediated a twofold increase in plasmid copy number, but this region was not required for stabilization activity. The PstI C fragment was shown to encode a multimer resolution system from 33.1 to 35.3 kb. The resolution cis-acting site was mapped to 140 bp, sequenced, and observed to contain two directly repeated sequences of 6 and 7 bases and two perfect inverted repeats of 6 and 8 bases. The trans-acting factor(s) was mapped and functionally determined to encode a resolvase capable of catalyzing recombination at high frequency between cis-acting sites in either direct or inverted orientation. Multimer resolution alone did not account for complete plasmid stabilization by the PstI C fragment, since removal of regions adjacent to the 35.3-kb border of the minimal mrs locus dramatically reduced stabilization. The minimal region required for complete stabilization, from 32.8 to 35.9 kb, was capable of fully stabilizing plasmids independently of the replicon or the recA proficiency of the host. Stabilization activity was also fully expressed in several diverse gram-negative bacteria, whereas the F plasmid par locus functioned only in Escherichia coli. On the basis of these observations, we conclude that under the growth conditions used, the minimal stabilization locus encodes both an mrs activity and a stabilization activity that has the properties of a par locus.

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Selected References

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

  1. Abeles A. L., Friedman S. A., Austin S. J. Partition of unit-copy miniplasmids to daughter cells. III. The DNA sequence and functional organization of the P1 partition region. J Mol Biol. 1985 Sep 20;185(2):261–272. doi: 10.1016/0022-2836(85)90402-4. [DOI] [PubMed] [Google Scholar]
  2. Abremski K., Hoess R., Sternberg N. Studies on the properties of P1 site-specific recombination: evidence for topologically unlinked products following recombination. Cell. 1983 Apr;32(4):1301–1311. doi: 10.1016/0092-8674(83)90311-2. [DOI] [PubMed] [Google Scholar]
  3. Altenbuchner J., Schmitt R. Transposon Tn1721: site-specific recombination generates deletions and inversions. Mol Gen Genet. 1983;190(2):300–308. doi: 10.1007/BF00330655. [DOI] [PubMed] [Google Scholar]
  4. Austin S. J. Plasmid partition. Plasmid. 1988 Jul;20(1):1–9. doi: 10.1016/0147-619x(88)90001-7. [DOI] [PubMed] [Google Scholar]
  5. Austin S., Wierzbicki A. Two mini-F-encoded proteins are essential for equipartition. Plasmid. 1983 Jul;10(1):73–81. doi: 10.1016/0147-619x(83)90059-8. [DOI] [PubMed] [Google Scholar]
  6. Austin S., Ziese M., Sternberg N. A novel role for site-specific recombination in maintenance of bacterial replicons. Cell. 1981 Sep;25(3):729–736. doi: 10.1016/0092-8674(81)90180-x. [DOI] [PubMed] [Google Scholar]
  7. Baldwin T. O., Berends T., Bunch T. A., Holzman T. F., Rausch S. K., Shamansky L., Treat M. L., Ziegler M. M. Cloning of the luciferase structural genes from Vibrio harveyi and expression of bioluminescence in Escherichia coli. Biochemistry. 1984 Jul 31;23(16):3663–3667. doi: 10.1021/bi00311a014. [DOI] [PubMed] [Google Scholar]
  8. Birnboim H. C., Doly J. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 1979 Nov 24;7(6):1513–1523. doi: 10.1093/nar/7.6.1513. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Bishop P. E., Brill W. J. Genetic analysis of Azotobacter vinelandii mutant strains unable to fix nitrogen. J Bacteriol. 1977 May;130(2):954–956. doi: 10.1128/jb.130.2.954-956.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Boyer H. W., Roulland-Dussoix D. A complementation analysis of the restriction and modification of DNA in Escherichia coli. J Mol Biol. 1969 May 14;41(3):459–472. doi: 10.1016/0022-2836(69)90288-5. [DOI] [PubMed] [Google Scholar]
  11. 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]
  12. Carter P., Bedouelle H., Winter G. Improved oligonucleotide site-directed mutagenesis using M13 vectors. Nucleic Acids Res. 1985 Jun 25;13(12):4431–4443. doi: 10.1093/nar/13.12.4431. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Depicker A., De Block M., Inzé D., Van Montagu M., Schell J. IS-like element IS8 in RP4 plasmid and its involvement in cointegration. Gene. 1980 Sep;10(4):329–338. doi: 10.1016/0378-1119(80)90153-5. [DOI] [PubMed] [Google Scholar]
  14. Ditta G., Stanfield S., Corbin D., Helinski D. R. Broad host range DNA cloning system for gram-negative bacteria: construction of a gene bank of Rhizobium meliloti. Proc Natl Acad Sci U S A. 1980 Dec;77(12):7347–7351. doi: 10.1073/pnas.77.12.7347. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Dodd H. M., Bennett P. M. Location of the site-specific recombination system of R46: a function necessary for plasmid maintenance. J Gen Microbiol. 1986 Apr;132(4):1009–1020. doi: 10.1099/00221287-132-4-1009. [DOI] [PubMed] [Google Scholar]
  16. Durland R. H., Toukdarian A., Fang F., Helinski D. R. Mutations in the trfA replication gene of the broad-host-range plasmid RK2 result in elevated plasmid copy numbers. J Bacteriol. 1990 Jul;172(7):3859–3867. doi: 10.1128/jb.172.7.3859-3867.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Fong S. T., Stanisich V. A. Location and characterization of two functions on RP1 that inhibit the fertility of the IncW plasmid R388. J Gen Microbiol. 1989 Mar;135(3):499–502. doi: 10.1099/00221287-135-3-499. [DOI] [PubMed] [Google Scholar]
  18. Garfinkel D. J., Simpson R. B., Ream L. W., White F. F., Gordon M. P., Nester E. W. Genetic analysis of crown gall: fine structure map of the T-DNA by site-directed mutagenesis. Cell. 1981 Nov;27(1 Pt 2):143–153. doi: 10.1016/0092-8674(81)90368-8. [DOI] [PubMed] [Google Scholar]
  19. Garnier T., Saurin W., Cole S. T. Molecular characterization of the resolvase gene, res, carried by a multicopy plasmid from Clostridium perfringens: common evolutionary origin for prokaryotic site-specific recombinases. Mol Microbiol. 1987 Nov;1(3):371–376. doi: 10.1111/j.1365-2958.1987.tb01944.x. [DOI] [PubMed] [Google Scholar]
  20. Gerdes K., Molin S. Partitioning of plasmid R1. Structural and functional analysis of the parA locus. J Mol Biol. 1986 Aug 5;190(3):269–279. doi: 10.1016/0022-2836(86)90001-x. [DOI] [PubMed] [Google Scholar]
  21. Gerlitz M., Hrabak O., Schwab H. Partitioning of broad-host-range plasmid RP4 is a complex system involving site-specific recombination. J Bacteriol. 1990 Nov;172(11):6194–6203. doi: 10.1128/jb.172.11.6194-6203.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Grinter N. J., Brewster G., Barth P. T. Two mechanisms necessary for the stable inheritance of plasmid RP4. Plasmid. 1989 Nov;22(3):203–214. doi: 10.1016/0147-619x(89)90003-6. [DOI] [PubMed] [Google Scholar]
  23. Hakkaart M. J., van den Elzen P. J., Veltkamp E., Nijkamp H. J. Maintenance of multicopy plasmid Clo DF13 in E. coli cells: evidence for site-specific recombination at parB. Cell. 1984 Jan;36(1):203–209. doi: 10.1016/0092-8674(84)90090-4. [DOI] [PubMed] [Google Scholar]
  24. Hanahan D. Studies on transformation of Escherichia coli with plasmids. J Mol Biol. 1983 Jun 5;166(4):557–580. doi: 10.1016/s0022-2836(83)80284-8. [DOI] [PubMed] [Google Scholar]
  25. 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]
  26. Hogan J. E., Kline B. C., Levy S. B. Regions on the F plasmid which affect plasmid maintenance and the ability to segregate into Escherichia coli minicells. Plasmid. 1982 Jul;8(1):36–44. doi: 10.1016/0147-619x(82)90039-7. [DOI] [PubMed] [Google Scholar]
  27. Holmes D. S., Quigley M. A rapid boiling method for the preparation of bacterial plasmids. Anal Biochem. 1981 Jun;114(1):193–197. doi: 10.1016/0003-2697(81)90473-5. [DOI] [PubMed] [Google Scholar]
  28. Jaffé A., Ogura T., Hiraga S. Effects of the ccd function of the F plasmid on bacterial growth. J Bacteriol. 1985 Sep;163(3):841–849. doi: 10.1128/jb.163.3.841-849.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Kitts P. A., Symington L. S., Dyson P., Sherratt D. J. Transposon-encoded site-specific recombination: nature of the Tn3 DNA sequences which constitute the recombination site res. EMBO J. 1983;2(7):1055–1060. doi: 10.1002/j.1460-2075.1983.tb01545.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Kolter R., Helinski D. R. Construction of plasmid R6K derivatives in vitro: characterization of the R6K replication region. Plasmid. 1978 Sep;1(4):571–580. doi: 10.1016/0147-619x(78)90014-8. [DOI] [PubMed] [Google Scholar]
  31. Lane D., de Feyter R., Kennedy M., Phua S. H., Semon D. D protein of miniF plasmid acts as a repressor of transcription and as a site-specific resolvase. Nucleic Acids Res. 1986 Dec 22;14(24):9713–9728. [PMC free article] [PubMed] [Google Scholar]
  32. Lanka E., Barth P. T. Plasmid RP4 specifies a deoxyribonucleic acid primase involved in its conjugal transfer and maintenance. J Bacteriol. 1981 Dec;148(3):769–781. doi: 10.1128/jb.148.3.769-781.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Ludtke D. N., Austin S. J. The plasmid-maintenance functions of the P7 prophage. Plasmid. 1987 Jul;18(1):93–98. doi: 10.1016/0147-619x(87)90083-7. [DOI] [PubMed] [Google Scholar]
  34. Martin K. A., Friedman S. A., Austin S. J. Partition site of the P1 plasmid. Proc Natl Acad Sci U S A. 1987 Dec;84(23):8544–8547. doi: 10.1073/pnas.84.23.8544. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Merryweather A., Barth P. T., Wilkins B. M. Role and specificity of plasmid RP4-encoded DNA primase in bacterial conjugation. J Bacteriol. 1986 Jul;167(1):12–17. doi: 10.1128/jb.167.1.12-17.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. NEWTON J. W., WILSON P. W., BURRIS R. H. Direct demonstration of ammonia as an intermediate in nitrogen fixation by Azotobacter. J Biol Chem. 1953 Sep;204(1):445–451. [PubMed] [Google Scholar]
  37. Nash H. A. Integration and excision of bacteriophage lambda: the mechanism of conservation site specific recombination. Annu Rev Genet. 1981;15:143–167. doi: 10.1146/annurev.ge.15.120181.001043. [DOI] [PubMed] [Google Scholar]
  38. 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]
  39. 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. doi: 10.1016/0092-8674(83)90454-3. [DOI] [PubMed] [Google Scholar]
  40. Pansegrau W., Miele L., Lurz R., Lanka E. Nucleotide sequence of the kanamycin resistance determinant of plasmid RP4: homology to other aminoglycoside 3'-phosphotransferases. Plasmid. 1987 Nov;18(3):193–204. doi: 10.1016/0147-619x(87)90062-x. [DOI] [PubMed] [Google Scholar]
  41. Plasterk R. H., Ilmer T. A., Van de Putte P. Site-specific recombination by Gin of bacteriophage Mu: inversions and deletions. Virology. 1983 May;127(1):24–36. doi: 10.1016/0042-6822(83)90367-7. [DOI] [PubMed] [Google Scholar]
  42. Reed R. R., Grindley N. D. Transposon-mediated site-specific recombination in vitro: DNA cleavage and protein-DNA linkage at the recombination site. Cell. 1981 Sep;25(3):721–728. doi: 10.1016/0092-8674(81)90179-3. [DOI] [PubMed] [Google Scholar]
  43. Schurter W., Holloway B. W. Genetic analysis of promoters on the insertion sequence IS21 of plasmid R68.45. Plasmid. 1986 Jan;15(1):8–18. doi: 10.1016/0147-619x(86)90010-7. [DOI] [PubMed] [Google Scholar]
  44. Sherratt D., Dyson P., Boocock M., Brown L., Summers D., Stewart G., Chan P. Site-specific recombination in transposition and plasmid stability. Cold Spring Harb Symp Quant Biol. 1984;49:227–233. doi: 10.1101/sqb.1984.049.01.026. [DOI] [PubMed] [Google Scholar]
  45. Stokes H. W., Moore R. J., Krishnapillai V. Complementation analysis in Pseudomonas aeruginosa of the transfer genes of the wide host range R plasmid R18. Plasmid. 1981 Mar;5(2):202–212. doi: 10.1016/0147-619x(81)90021-4. [DOI] [PubMed] [Google Scholar]
  46. Summers D., Yaish S., Archer J., Sherratt D. Multimer resolution systems of ColE1 and ColK: localisation of the crossover site. Mol Gen Genet. 1985;201(2):334–338. doi: 10.1007/BF00425680. [DOI] [PubMed] [Google Scholar]
  47. Tabuchi A., Min Y. N., Kim C. K., Fan Y. L., Womble D. D., Rownd R. H. Genetic organization and nucleotide sequence of the stability locus of IncFII plasmid NR1. J Mol Biol. 1988 Aug 5;202(3):511–525. doi: 10.1016/0022-2836(88)90282-3. [DOI] [PubMed] [Google Scholar]
  48. Thomas C. M. Molecular genetics of broad host range plasmid RK2. Plasmid. 1981 Jan;5(1):10–19. doi: 10.1016/0147-619x(81)90074-3. [DOI] [PubMed] [Google Scholar]
  49. Thomas C. M. Recent studies on the control of plasmid replication. Biochim Biophys Acta. 1988 Mar 31;949(3):253–263. doi: 10.1016/0167-4781(88)90150-9. [DOI] [PubMed] [Google Scholar]
  50. Thomas C. M., Smith C. A. Incompatibility group P plasmids: genetics, evolution, and use in genetic manipulation. Annu Rev Microbiol. 1987;41:77–101. doi: 10.1146/annurev.mi.41.100187.000453. [DOI] [PubMed] [Google Scholar]
  51. Tsuchimoto S., Ohtsubo H., Ohtsubo E. Two genes, pemK and pemI, responsible for stable maintenance of resistance plasmid R100. J Bacteriol. 1988 Apr;170(4):1461–1466. doi: 10.1128/jb.170.4.1461-1466.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Uhlin B. E., Nordström K. R plasmid gene dosage effects in Escherichia coli K-12: copy mutants of the R plasmic R1drd-19. Plasmid. 1977 Nov;1(1):1–7. doi: 10.1016/0147-619x(77)90003-8. [DOI] [PubMed] [Google Scholar]
  53. Vieira J., Messing J. The pUC plasmids, an M13mp7-derived system for insertion mutagenesis and sequencing with synthetic universal primers. Gene. 1982 Oct;19(3):259–268. doi: 10.1016/0378-1119(82)90015-4. [DOI] [PubMed] [Google Scholar]
  54. Yanisch-Perron C., Vieira J., Messing J. Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene. 1985;33(1):103–119. doi: 10.1016/0378-1119(85)90120-9. [DOI] [PubMed] [Google Scholar]

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