Molecular and General Genetics MGG

, Volume 204, Issue 2, pp 273–280 | Cite as

Replicon fusions promoted by insertion sequences on Pseudomonas cepacia plasmid pTGL6

  • Gary Barsomian
  • T. G. Lessie
Article

Summary

Plasmid pMR5 (pRP1ts) failed to replicate in Pseudomonas cepacia at 47° C. Selection at this temperature for maintenance of tetracycline resistance associated with this plasmid allowed isolation of cointegrate plasmids formed by fusion of pMR5 with pTGL6, a 170 kb plasmid harbored by P. cepacia 249. In the cointegrate plasmids pTGL100, pTGL101, and pTGL102, different regions of pTGL6 were involved in fusion with the same tra-2-containing region of pMR5. Formation of all three plasmids was promoted by insertion sequences on pTGL6, which were also represented in the chromosome.

Two different copies of a 1.3 kb element, IS401, were involved in formation of pTGL100 and pTGL101. Another insertion sequence, IS402 (1 kb), promoted the fusion which formed pTGL102. Southern hybridization experiments indicated that each of the cointegrate plasmids contained an additional copy of the fusion mediating element. Plasmid pTGL100 was observed to resolve into two independent replicons: pTGL6 and pTGL105 (pMR5::IS401), a novel derivative of pMR5 containing a copy of IS401.

The third cointegrate plasmid, pTGL102, evolved in two steps: fusion of pTGL6 and pMR5 mediated by IS402, and transposition of IS411 (1.9 kb) to a region of pMR5 distinct from that involved in the fusion. Plasmid pTGL6 contained one copy of IS402 and IS411 while pTGL102 contained two copies of each of these elements.

Key words

Pseudomonas cepacia Insertion sequences Replicaon fusions Plasmid pTGL6 Plasmid pMR5 

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. Ballard RW, Palleroni NJ, Doudoroff M, Stanier RY, Mandel M (1970) Taxonomy of the aerobic pseudomonads: Pseudomonas cepacia, P. marginata, P. alliicola, and P. caryophilli. J Gen Microbiol 60:199–214Google Scholar
  2. Beckman W, Lessie TG (1979) Response of Pseudomonas cepacia to β-lactam antibiotics: utilization of penicillin G as the carbon source. J Bacteriol 140:1126–1128Google Scholar
  3. Beckman W, Lessie TG (1981) Characterization of plasmids in mutant strains of Pseudomonas cepacia unable to utilize penicillin or octopine. Abstr Annu Meeting Am Soc Microbiol H112, p 132Google Scholar
  4. Berka TR, Lessie TG (1984) Enzymes related to galactose utilization in Pseudomonas cepacia. Curr Microbiol 11:43–48Google Scholar
  5. Birnboim HC, Doly J (1979) A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res 7:1513–1523Google Scholar
  6. Chang ACY, Cohen SN (1978) Construction and characterization of amplifiable multicopy DNA cloning vehicles derived from P15A cryptic mini-plasmid. J Bacteriol 134:1141–1156Google Scholar
  7. Cihlar RL, Lessie TG, Holt SC (1979) Characterization of bacteriophage CP1, an organic solvent sensitive phage associated with Pseudomonas cepacia. Can J Microbiol 24:1404–1412Google Scholar
  8. Currier TC, Nester EW (1976) Isolation of covalently closed circular DNA of high molecular weight from bacteria. Anal Biochem 76:431–441Google Scholar
  9. Depicker A, De Block M, Inze D, Van Montagu M, Schell J (1980) IS-like element IS8 and its involvement in cointegration. Gene 10:329–338Google Scholar
  10. Ederer GM, Matsen JM (1972) Colonization and infection with Pseudomonas cepacia. J Infect Dis 125:613–618Google Scholar
  11. Gaffney TD (1985) Characterization of plasmid PC1 of Pseudomonas cepacia: analysis of insertion sequence mediated rearrangements and isolation of cured strains. Ph.D. thesis University of Massachusetts, Amherst, 124 ppGoogle Scholar
  12. Grindley NDF, Reed RR (1985) Transpositional recombination in procaryotes. Annu Rev Biochem 54:863–896Google Scholar
  13. Kado CI, Liu ST (1981) Rapid procedure for detection and isolation of large and small plasmids. J Bacteriol 145:1365–1373Google Scholar
  14. Kilbane JJ, Chatterjee DK, Karns JS, Kellogg ST, Chakrabarty AM (1982) Biodegradation of 2,4,5-trichlorophenoxyacetic acid by a pure culture of Pseudomonas cepacia. Appl Environ Microbiol 44:72–78Google Scholar
  15. Kushner SR (1978) An improved method for transformation of Escherichia coli with ColE1 derived plasmids. In: Boyer HW, Nicosia S (eds) Genetic engineering. Elsevier/North Holland Biomedical Press, Amsterdam, p 17Google Scholar
  16. Lessie TG, Gaffney T (1986) Catabolic potential of Pseudomonas cepacia. In: Sokatch JR, Ornston LN (eds) The bacteria, the biology of Pseudomonas, vol 10 Academic Press, Orlando, in pressGoogle Scholar
  17. Levy SB (1984) Resistance to the tetracyclines. In: Bryan LE (ed) Antimicrobial, drug resistance. Academic Press, Orlando, pp 191–240Google Scholar
  18. Maniatis T, Fritsch ED, Sambrook J (eds) (1982) Molecular cloning. Cold Spring Harbor Laboratory, Cold Spring Harbor, NYGoogle Scholar
  19. Marmur J (1961) A procedure for the isolation of deoxyribonucleic acid from micro-organisms. J Mol Biol 3:208–218Google Scholar
  20. Norskey G, Gaffney T, Lessie TG (1984) Insertional activation of β-lactamase and β-galactosidase genes of plasmids RP1 and GC91.14. Abstr Annu Meeting Am Soc Microbiol H42, p 98Google Scholar
  21. Otsubo E, Zenilman M, Ohtsubo H, McCormick M, Machida C, Machida Y (1980) Mechanisms of insertion and cointegration mediated by IS1 and Tn3. Cold Spring Harbor Symp Quant Biol XLV:283–295Google Scholar
  22. Palleroni NJ, Doudoroff M (1972) Taxonomic subdivisions of the genus Pseudomonas. Ann Rev Phytopathol 10:73–100Google Scholar
  23. Palleroni NJ, Holmes B (1981) Pseudomonas cepacia sp. nov., nom. rev. Int J System Biol 31:479–481Google Scholar
  24. Rigby PJW, Dieckmann M, Rhodes C, Berg P (1977) Labelling deoxyribonucleic acid to high specific activity in vitro by nick translation with DNA polymerase 1. J Mol Biol 113:237–251Google Scholar
  25. Robinson MK, Bennett PM, Falkow S, Dodd HM (1980) Isolation of a temperature sensitive derivative of RP1 Plasmid 3:343–347Google Scholar
  26. Southern EM (1975) Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol 98:503–517Google Scholar
  27. Shapiro JA (1979) Molecular model for the transposition and replication of bacteriohage Mu and other transposable elements. Proc Natl Acad Sci 76:1933–1937Google Scholar
  28. Stanier RY, Palleroni NJ, Doudoroff M (1966) The acrobic pseudomonads: a taxonomic study. J Gen Microbiol 43:159–271Google Scholar

Copyright information

© Springer-Verlag 1986

Authors and Affiliations

  • Gary Barsomian
    • 1
  • T. G. Lessie
    • 1
  1. 1.Department of MicrobiologyUniversity of MassachusettsAmherstUSA

Personalised recommendations