1887

Abstract

Excision of the transposon Tn from sites of insertion in plasmid DNA was shown to occur at high frequency in . Plasmids with Tn insertions conjugated poorly into and adversely affected growth compared to the respective parental plasmids. The kanamycin-resistance phenotype of Tn5 was expressed poorly in and kanamycin-sensitive strains were common during the manipulation of the transconjugants. Examination of plasmid DNA isolated from kanamycin-sensitive transconjugants revealed excision of Tn5 sequences. A plasmid containing a selectable marker (mercury resistance) inactivated by a Tn insertion was constructed, and Tn5 excised precisely, permitting the expression of the mercury-resistance marker at high frequency (10) in and at the expected low frequency (10) in . The recombinational mechanism that promotes frequent Tn excision in operated in the absence of the gene product. Fragments of Tn were also examined for excision and instability in . A plasmid containing the terminal 485 bp of inverted repeat sequences from Tn5, but lacking the transposase or kanamycin-resistance genes, also showed precise excision of Tn DNA at high frequency (10) in . Unlike plasmids containing a complete Tn insertion, this plasmid transferred to at normal frequencies and growth of the host was not severely impaired. In contrast, plasmids containing either IS50 element transferred to at greatly reduced frequencies, and transconjugants containing the IS50R element (which contains the active transposase gene) were small and especially difficult to maintain. transconjugants harbouring a plasmid containing only the DNA between the IS elements (which included the kanamycin-resistance gene) were of normal size and stably maintained. These observations suggest that frequent and precise excision of Tn in required the long inverted repeat sequences at the termini of Tn5. The adverse effects conferred by IS50 on the transconjugant formation and growth of were apparently not required to promote Tn excision.

Loading

Article metrics loading...

/content/journal/micro/10.1099/00221287-136-5-789
1990-05-01
2024-11-08
Loading full text...

Full text loading...

/deliver/fulltext/micro/136/5/mic-136-5-789.html?itemId=/content/journal/micro/10.1099/00221287-136-5-789&mimeType=html&fmt=ahah

References

  1. Berg D. E. 1977; Insertion and excision of the transposable kanamycin resistance determinant Tn5 . In DNA Insertion Elements, Plasmids and Episomes pp. 205–212 Bukhari A. I., Shapiro J. A., Adhya S. L. Edited by Cold Spring Harbor, NY: Cold Spring Harbor Laboratory;
    [Google Scholar]
  2. Berg D. E., Berg C. M. 1983; The procaryotic transposable element Tn5 . Biol/Technology 1:417–435
    [Google Scholar]
  3. Berg D. E., Egner C., Hirschel B. J., Howard J., Johnsrud L., Jorgensen R. A., Tlsty T. D. 1980; Insertion, excision, and inversion of Tn5 . Cold Spring Harbor Symposia on Quantitative Biology 45:115–123
    [Google Scholar]
  4. Birnboim H. C., Doly J. 1979; A rapid extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Research 7:1513–1525
    [Google Scholar]
  5. Bolivar R., Rodriguez R. L., Greene J. G., Betlach M., Heyneker H. L., Boyer H. W. 1977; Construction and characterization of new cloning vehicles. II. A multipurpose cloning system. Gene 2:95–113
    [Google Scholar]
  6. Boyer H. W., Roulland-Dussoix D. 1969; A complementation analysis of the restriction and modification of DNA in Escherichia coli . Journal of Molecular Biology 41:459–472
    [Google Scholar]
  7. Bryan L. E., Nicas T., Holloway B. W., Crowther C. 1980; Aminoglycoside-resistant mutation of Pseudomonas aeruginosa defective in cytochrome c552 and nitrate reductase. Antimicrobial Agents and Chemotherapy 17:71–79
    [Google Scholar]
  8. Close T. J., Rodriguez R. L. 1982; Construction and characterization of the chloramphenicol-resistance gene cartridge: a new approach to the transcriptional mapping of extrachromosomal elements. Gene 20:305–316
    [Google Scholar]
  9. Cohen S. N., Chang A. C. Y., Hsu C. L. 1972; Non-chromosomal antibiotic resistance in bacteria: genetic transformation of Escheri-chia coli by R factor DNA. Proceedings of the National Academy of Sciences of the United States of America 69:2110–2114
    [Google Scholar]
  10. Deretic V., Gill J. F., Chakrabarty A. M. 1987; Alginate biosynthesis: a model system for gene regulation and function in Pseudomonas . Bio/Technology 5:469–477
    [Google Scholar]
  11. Dykstra C. C., Prasher D., Kushner S. R. 1984; Physical and biochemical analysis of the cloned recB and recC genes of Escherichia coli K-12. Journal of Bacteriology 157:21–27
    [Google Scholar]
  12. Egner C., Berg D. E. 1981; Excision of transposon Tn5 is dependent on the inverted repeats but not on the transposase function of Tn5 . Proceedings of the National Academy of Sciences of the United States of America 78:459–463
    [Google Scholar]
  13. Figurski D., Helinski D. R. 1979; Replication of an origin- containing derivative of plasmid RK2 dependent on a plasmid function provided in trans. Proceedings of the National Academy of Sciences of the United States of America 76:1648–1652
    [Google Scholar]
  14. Flynn J. L., Ohman D. E. 1988a; Cloning of genes from mucoid Pseudomonas aeruginosa which control spontaneous conversion to the alginate production phenotype. Journal of Bacteriology 170:1452–1460
    [Google Scholar]
  15. Flynn J. L., Ohman D. E. 1988b; Use of gene replacement cosmid vector for cloning alginate conversion genes from mucoid and nonmucoid Pseudomonas aeruginosa strains: algS controls expression of algT . Journal of Bacteriology 170:3228–3236
    [Google Scholar]
  16. Friedman A. M., Long S. R., Brown S. E., Buikema W. J., Ausubel F. M. 1982; Construction of a broad host range cosmid cloning vector and its use in the genetic analysis of Rhizobium mutants. Gene 18:289–296
    [Google Scholar]
  17. Gendel S. M. 1987; Instability of Tn5 inserts in cyanobacterial cloning vectors. Journal of Bacteriology 169:4426–4430
    [Google Scholar]
  18. Goldberg J. B., Ohman D. E. 1984; Cloning and expression in Pseudomonas aeruginosa of a gene involved in the production of alginate. Journal of Bacteriology 158:1115–1121
    [Google Scholar]
  19. Goldberg J. B., Ohman D. E. 1987; Construction and characterization of Pseudomonas aeruginosa algB mutants: role of algB in high level production of alginate. Journal of Bacteriology 169:1593–1602
    [Google Scholar]
  20. Gordenin D. A., Trofimova M. V., Shaburova O. N., Pavlov Y. I., Chernoff Y. O., Chekuolene Y. V., Proscyavichus Y. Y., Sasnauskas K. V., Janulaitis A. A. 1988; Precise excision of bacterial transposon Tn5 in yeast. Molecular and General Genetics 213:388–393
    [Google Scholar]
  21. Holloway B. W., Krishnapillai V., Morgan A. F. 1979; Chromosomal genetics of Pseudomonas . Microbiological Reviews 43:73–102
    [Google Scholar]
  22. Horn J. M., Ohman D. E. 1988; Transcriptional and translational analyses of recA mutant alleles in Pseudomonas aeruginosa . Journal of Bacteriology 170:1637–1650
    [Google Scholar]
  23. Isberg R. R., Lazaar A. L., Syvanen M. 1982; Regulation of Tni by the right-repeat proteins: control at the level of the transposition reaction?. Cell 30:883–892
    [Google Scholar]
  24. Johnson R. C., Yin J. C. P., Reznikoff W. S. 1982; Control of Tn5 transposition in Escherichia coli is mediated by protein from the right repeat. Cell 30:873–882
    [Google Scholar]
  25. Jorgensen R. A., Rothstein S. J., Reznikoff W. S. 1979; A restriction enzyme cleavage map of Tn5 and location of a region encoding neomycin resistance. Molecular and General Genetics 177:65–72
    [Google Scholar]
  26. Kleckner N. 1981; Transposable elements in prokaryotes. Annual Review of Genetics 15:341–404
    [Google Scholar]
  27. Lundblad V., Taylor A. F., Smith G. R., Kleckner N. 1984; Unusual alleles of recB and recC stimulate excision of inverted repeat transposons Tn10 and Tn5 . Proceedings of the National Academy of Sciences of the United States of America 81:824–828
    [Google Scholar]
  28. Meyer R., Laux R., Boch G., Hinds M., Bayly R., Shapiro J. A. 1982; Broad-host-range IncP-4 plasmid R1162: effects of deletions and insertions on plasmid maintenance and host range. Journal of Bacteriology 152:140–150
    [Google Scholar]
  29. Misra T. K., Brown N. L., Fritzinger D. C., Pridmore R. D., Barnes W. M., Haberstroh L., Silver S. 1984; Mercuric ion-resistance operons of plasmid R100 and transposon Tn501: the beginning of the operon including the regulatory region and the first two structural genes. Proceedings of the National Academy of Sciences of the United States of America 81:5975–5979
    [Google Scholar]
  30. Ohman D. E. 1986; Molecular genetics of exopolysaccharide production by mucoid Pseudomonas aeruginosa . European Journal of Clinical Microbiology 5:6–10
    [Google Scholar]
  31. Ohman D. E., Chakrabarty A. M. 1981; Genetic mapping of chromosomal determinants for the production of the exopolysaccharide alginate in a Pseudomonas aeruginosa cystic fibrosis isolate. Infection and Immunity 33:142–148
    [Google Scholar]
  32. Ohman D. E., Chakrabarty A. M. 1982; Utilization of human respiratory secretions by mucoid Pseudomonas aeruginosa of cystic fibrosis origin. Infection and Immunity 37:662–669
    [Google Scholar]
  33. Ohman D. E., West M. A., Flynn J. L., Goldberg J. B. 1985; Method for gene replacement in Pseudomonas aeruginosa used in construction of recA mutants: recA-independent instability of alginate production. Journal of Bacteriology 162:1068–1074
    [Google Scholar]
  34. O’Hoy K., Krishnapillai V. 1985; Transposon mutagenesis of the Pseudomonas aeruginosa PAO chromosome and the isolation of high frequency of recombination donors. FEMS Microbiology Letters 29:299–303
    [Google Scholar]
  35. Rothstein S. J., Reznikoff W. S. 1981; The functional differences in the inverted repeats of Tn5 are caused by a single base pair nonhomology. Cell 23:191–199
    [Google Scholar]
  36. Schilf W., Krishnapillai V. 1985; Genetic basis of non-transposition of Tn5 in Pseudomonas aeruginosa following mobilisation of RP4 Mob: :Tn5 from Escherichia coli . FEMS Microbiology Letters 28:351–354
    [Google Scholar]
  37. Sokol P. A. 1987; Tn5 insertion mutants of Pseudomonas aeruginosa deficient in surface expression of ferripyochelin-binding protein. Journal of Bacteriology 169:3365–3368
    [Google Scholar]
  38. Stapleton M. J., Jagger K. S., Warren R. L. 1984; Transposon mutagenesis of Pseudomonas aeruginosa exoprotease genes. Journal of Bacteriology 157:7–12
    [Google Scholar]
  39. Staskawicz B., Dahlbeck D., Keen N., Napoli C. 1987; Molecular characterization of cloned avirulence genes from race O and race 1 of Pseudomonas syringae pv. glycinea . Journal of Bacteriology 169:5789–5794
    [Google Scholar]
  40. Woodruff W. A., Hancock R. E. W. 1988; Construction and characterization of Pseudomonas aeruginosa protein F-deficient mutants after in vitro and in vivo insertion mutagenesis of the cloned gene. Journal of Bacteriology 170:2592–2598
    [Google Scholar]
/content/journal/micro/10.1099/00221287-136-5-789
Loading
/content/journal/micro/10.1099/00221287-136-5-789
Loading

Data & Media loading...

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error