Skip to main content
PMC home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1990 Nov;172(11):6568–6572. doi: 10.1128/jb.172.11.6568-6572.1990

Mini-Tn5 transposon derivatives for insertion mutagenesis, promoter probing, and chromosomal insertion of cloned DNA in gram-negative eubacteria.

V de Lorenzo 1, M Herrero 1, U Jakubzik 1, K N Timmis 1
PMCID: PMC526846  PMID: 2172217

Abstract

A collection of Tn5-derived minitransposons has been constructed that simplifies substantially the generation of insertion mutants, in vivo fusions with reporter genes, and the introduction of foreign DNA fragments into the chromosome of a variety of gram-negative bacteria, including the enteric bacteria and typical soil bacteria like Pseudomonas species. The minitransposons consist of genes specifying resistance to kanamycin, chloramphenicol, streptomycin-spectinomycin, and tetracycline as selection markers and a unique NotI cloning site flanked by 19-base-pair terminal repeat sequences of Tn5. Further derivatives also contain lacZ, phoA, luxAB, or xylE genes devoid of their native promoters located next to the terminal repeats in an orientation that affords the generation of gene-operon fusions. The transposons are located on a R6K-based suicide delivery plasmid that provides the IS50R transposase tnp gene in cis but external to the mobile element and whose conjugal transfer to recipients is mediated by RP4 mobilization functions in the donor.

Full text

PDF
6568

Selected References

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

  1. Boivin R., Chalifour F. P., Dion P. Construction of a Tn5 derivative encoding bioluminescence and its introduction in Pseudomonas, Agrobacterium and Rhizobium. Mol Gen Genet. 1988 Jul;213(1):50–55. doi: 10.1007/BF00333397. [DOI] [PubMed] [Google Scholar]
  2. Chang C. N., Kuang W. J., Chen E. Y. Nucleotide sequence of the alkaline phosphatase gene of Escherichia coli. Gene. 1986;44(1):121–125. doi: 10.1016/0378-1119(86)90050-8. [DOI] [PubMed] [Google Scholar]
  3. Elliott T., Geiduschek E. P. Defining a bacteriophage T4 late promoter: absence of a "-35" region. Cell. 1984 Jan;36(1):211–219. doi: 10.1016/0092-8674(84)90091-6. [DOI] [PubMed] [Google Scholar]
  4. Engebrecht J., Simon M., Silverman M. Measuring gene expression with light. Science. 1985 Mar 15;227(4692):1345–1347. doi: 10.1126/science.2983423. [DOI] [PubMed] [Google Scholar]
  5. Fellay R., Frey J., Krisch H. Interposon mutagenesis of soil and water bacteria: a family of DNA fragments designed for in vitro insertional mutagenesis of gram-negative bacteria. Gene. 1987;52(2-3):147–154. doi: 10.1016/0378-1119(87)90041-2. [DOI] [PubMed] [Google Scholar]
  6. Groisman E. A., Casadaban M. J. Mini-mu bacteriophage with plasmid replicons for in vivo cloning and lac gene fusing. J Bacteriol. 1986 Oct;168(1):357–364. doi: 10.1128/jb.168.1.357-364.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Gutierrez C., Devedjian J. C. A plasmid facilitating in vitro construction of phoA gene fusions in Escherichia coli. Nucleic Acids Res. 1989 May 25;17(10):3999–3999. doi: 10.1093/nar/17.10.3999. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Herrero M., de Lorenzo V., Timmis K. N. Transposon vectors containing non-antibiotic resistance selection markers for cloning and stable chromosomal insertion of foreign genes in gram-negative bacteria. J Bacteriol. 1990 Nov;172(11):6557–6567. doi: 10.1128/jb.172.11.6557-6567.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Joseph-Liauzun E., Fellay R., Chandler M. Transposable elements for efficient manipulation of a wide range of gram-negative bacteria: promoter probes and vectors for foreign genes. Gene. 1989 Dec 21;85(1):83–89. doi: 10.1016/0378-1119(89)90467-8. [DOI] [PubMed] [Google Scholar]
  10. 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]
  11. Krebs M. P., Reznikoff W. S. Transcriptional and translational initiation sites of IS50. Control of transposase and inhibitor expression. J Mol Biol. 1986 Dec 20;192(4):781–791. doi: 10.1016/0022-2836(86)90028-8. [DOI] [PubMed] [Google Scholar]
  12. Krebs M. P., Reznikoff W. S. Use of a Tn5 derivative that creates lacZ translational fusions to obtain a transposition mutant. Gene. 1988 Mar 31;63(2):277–285. doi: 10.1016/0378-1119(88)90531-8. [DOI] [PubMed] [Google Scholar]
  13. Kroos L., Kaiser D. Construction of Tn5 lac, a transposon that fuses lacZ expression to exogenous promoters, and its introduction into Myxococcus xanthus. Proc Natl Acad Sci U S A. 1984 Sep;81(18):5816–5820. doi: 10.1073/pnas.81.18.5816. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Legocki R. P., Legocki M., Baldwin T. O., Szalay A. A. Bioluminescence in soybean root nodules: Demonstration of a general approach to assay gene expression in vivo by using bacterial luciferase. Proc Natl Acad Sci U S A. 1986 Dec;83(23):9080–9084. doi: 10.1073/pnas.83.23.9080. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Mandecki W., Reznikoff W. S. A lac promoter with a changed distance between -10 and -35 regions. Nucleic Acids Res. 1982 Feb 11;10(3):903–912. doi: 10.1093/nar/10.3.903. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Manoil C. Analysis of protein localization by use of gene fusions with complementary properties. J Bacteriol. 1990 Feb;172(2):1035–1042. doi: 10.1128/jb.172.2.1035-1042.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Manoil C., Beckwith J. TnphoA: a transposon probe for protein export signals. Proc Natl Acad Sci U S A. 1985 Dec;82(23):8129–8133. doi: 10.1073/pnas.82.23.8129. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Manoil C., Mekalanos J. J., Beckwith J. Alkaline phosphatase fusions: sensors of subcellular location. J Bacteriol. 1990 Feb;172(2):515–518. doi: 10.1128/jb.172.2.515-518.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Miller V. L., Mekalanos J. J. A novel suicide vector and its use in construction of insertion mutations: osmoregulation of outer membrane proteins and virulence determinants in Vibrio cholerae requires toxR. J Bacteriol. 1988 Jun;170(6):2575–2583. doi: 10.1128/jb.170.6.2575-2583.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. O'Sullivan D. J., O'Gara F. Delivery system for creation of one-step in vivo lac gene fusions in Pseudomonas spp. involved in biological control. Appl Environ Microbiol. 1988 Nov;54(11):2877–2880. doi: 10.1128/aem.54.11.2877-2880.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Schneider K., Beck C. F. New expression vectors for identifying and testing signal structures for initiation and termination of transcription. Methods Enzymol. 1987;153:452–461. doi: 10.1016/0076-6879(87)53071-3. [DOI] [PubMed] [Google Scholar]
  22. Simon R., Quandt J., Klipp W. New derivatives of transposon Tn5 suitable for mobilization of replicons, generation of operon fusions and induction of genes in gram-negative bacteria. Gene. 1989 Aug 1;80(1):161–169. doi: 10.1016/0378-1119(89)90262-x. [DOI] [PubMed] [Google Scholar]
  23. Stachel S. E., An G., Flores C., Nester E. W. A Tn3 lacZ transposon for the random generation of beta-galactosidase gene fusions: application to the analysis of gene expression in Agrobacterium. EMBO J. 1985 Apr;4(4):891–898. doi: 10.1002/j.1460-2075.1985.tb03715.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Way J. C., Davis M. A., Morisato D., Roberts D. E., Kleckner N. New Tn10 derivatives for transposon mutagenesis and for construction of lacZ operon fusions by transposition. Gene. 1984 Dec;32(3):369–379. doi: 10.1016/0378-1119(84)90012-x. [DOI] [PubMed] [Google Scholar]
  25. Zukowski M. M., Gaffney D. F., Speck D., Kauffmann M., Findeli A., Wisecup A., Lecocq J. P. Chromogenic identification of genetic regulatory signals in Bacillus subtilis based on expression of a cloned Pseudomonas gene. Proc Natl Acad Sci U S A. 1983 Feb;80(4):1101–1105. doi: 10.1073/pnas.80.4.1101. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES