Skip to main content
PMC home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1981 Jan;78(1):425–429. doi: 10.1073/pnas.78.1.425

Introduction of transposon Tn5 into Myxococcus for analysis of developmental and other nonselectable mutants

Jerry M Kuner 1,*, Dale Kaiser 1
PMCID: PMC319066  PMID: 16592958

Abstract

The transposon Tn5, which carries a gene for kanamycin resistance, can be introduced into Myxococcus xanthus, an organism that undergoes a primitive cycle of development, from Escherichia coli by the specialized transducing phage P1::Tn5. Tn5 DNA sequences, but no P1 sequences, are found in the stable kanamycin-resistant transductants. Tn5 transposes from P1 to many different chromosomal sites in Myxococcus. In each independent transductant of Myxococcus examined, the Tn5 element is found in a different DNA fragment produced by cleaving cell DNA with a restriction endonuclease. Moreover, different Tn5 insertions have been found linked to the first 20 different genetic sites tested. Once inserted into the Myxococcus chromosome, Tn5 remains fixed in position during growth and when transferred to another strain by generalized transduction. To analyze developmental or other mutants that have no selectable phenotype themselves, a general method has been devised, and tested, for the systematic isolation of a Tn5 insertion near any arbitrary locus.

Keywords: transduction, transposition, stability, linked insertion, bacteriophage P1

Full text

PDF
426

Images in this article

426Image
on p.426
427Image
on p.427
427Image
on p.427
427Image
on p.427
428Image
on p.428

Selected References

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

  1. Abstracts of papers presented at the 1980 meetings of the Genetic Society of America. Boulder, Colorado August 18-20, 1980. Genetics. 1980;94(4 Pt 2 Suppl):1–16. [PMC free article] [PubMed] [Google Scholar]
  2. Berg D. E., Davies J., Allet B., Rochaix J. D. Transposition of R factor genes to bacteriophage lambda. Proc Natl Acad Sci U S A. 1975 Sep;72(9):3628–3632. doi: 10.1073/pnas.72.9.3628. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Burchard R. P., Dworkin M. Light-induced lysis and carotenogenesis in Myxococcus xanthus. J Bacteriol. 1966 Feb;91(2):535–545. doi: 10.1128/jb.91.2.535-545.1966. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Burchard R. P. Gliding motility mutants of Myxococcus xanthus. J Bacteriol. 1970 Nov;104(2):940–947. doi: 10.1128/jb.104.2.940-947.1970. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Campos J. M., Geisselsoder J., Zusman D. R. Isolation of bacteriophage MX4, a generalized transducing phage for Myxococcus xanthus. J Mol Biol. 1978 Feb 25;119(2):167–178. doi: 10.1016/0022-2836(78)90431-x. [DOI] [PubMed] [Google Scholar]
  6. Clewell D. B., Helinski D. R. Supercoiled circular DNA-protein complex in Escherichia coli: purification and induced conversion to an opern circular DNA form. Proc Natl Acad Sci U S A. 1969 Apr;62(4):1159–1166. doi: 10.1073/pnas.62.4.1159. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Davis R. W., Thomas M., Cameron J., St John T. P., Scherer S., Padgett R. A. Rapid DNA isolations for enzymatic and hybridization analysis. Methods Enzymol. 1980;65(1):404–411. doi: 10.1016/s0076-6879(80)65051-4. [DOI] [PubMed] [Google Scholar]
  8. Goldberg R. B., Bender R. A., Streicher S. L. Direct selection for P1-sensitive mutants of enteric bacteria. J Bacteriol. 1974 Jun;118(3):810–814. doi: 10.1128/jb.118.3.810-814.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Grunstein M., Hogness D. S. Colony hybridization: a method for the isolation of cloned DNAs that contain a specific gene. Proc Natl Acad Sci U S A. 1975 Oct;72(10):3961–3965. doi: 10.1073/pnas.72.10.3961. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Hagen D. C., Bretscher A. P., Kaiser D. Synergism between morphogenetic mutants of Myxococcus xanthus. Dev Biol. 1978 Jun;64(2):284–296. doi: 10.1016/0012-1606(78)90079-9. [DOI] [PubMed] [Google Scholar]
  11. Hershfield V., Boyer H. W., Yanofsky C., Lovett M. A., Helinski D. R. Plasmid ColEl as a molecular vehicle for cloning and amplification of DNA. Proc Natl Acad Sci U S A. 1974 Sep;71(9):3455–3459. doi: 10.1073/pnas.71.9.3455. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Hodgkin J., Kaiser D. Cell-to-cell stimulation of movement in nonmotile mutants of Myxococcus. Proc Natl Acad Sci U S A. 1977 Jul;74(7):2938–2942. doi: 10.1073/pnas.74.7.2938. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Jorgensen R. A., Rothstein S. J., Reznikoff W. S. A restriction enzyme cleavage map of Tn5 and location of a region encoding neomycin resistance. Mol Gen Genet. 1979;177(1):65–72. doi: 10.1007/BF00267254. [DOI] [PubMed] [Google Scholar]
  14. Kaiser D., Dworkin M. Gene transfer to myxobacterium by Escherichia coli phage P1. Science. 1975 Feb 21;187(4177):653–654. doi: 10.1126/science.803710. [DOI] [PubMed] [Google Scholar]
  15. Kaiser D., Manoil C., Dworkin M. Myxobacteria: cell interactions, genetics, and development. Annu Rev Microbiol. 1979;33:595–639. doi: 10.1146/annurev.mi.33.100179.003115. [DOI] [PubMed] [Google Scholar]
  16. Kleckner N., Roth J., Botstein D. Genetic engineering in vivo using translocatable drug-resistance elements. New methods in bacterial genetics. J Mol Biol. 1977 Oct 15;116(1):125–159. doi: 10.1016/0022-2836(77)90123-1. [DOI] [PubMed] [Google Scholar]
  17. Kleckner N., Steele D. A., Reichardt K., Botstein D. Specificity of insertion by the translocatable tetracycline-resistance element Tn10. Genetics. 1979 Aug;92(4):1023–1040. doi: 10.1093/genetics/92.4.1023. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. LENNOX E. S. Transduction of linked genetic characters of the host by bacteriophage P1. Virology. 1955 Jul;1(2):190–206. doi: 10.1016/0042-6822(55)90016-7. [DOI] [PubMed] [Google Scholar]
  19. Lindberg A. A. Bacteriophage receptors. Annu Rev Microbiol. 1973;27:205–241. doi: 10.1146/annurev.mi.27.100173.001225. [DOI] [PubMed] [Google Scholar]
  20. MCVITTIE A., MESSIK F., ZAHLER S. A. Developmental biology of Myxococcus. J Bacteriol. 1962 Sep;84:546–551. doi: 10.1128/jb.84.3.546-551.1962. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Martin S., Sodergren E., Masuda T., Kaiser D. Systematic isolation of transducing phages for Myxococcus xanthus. Virology. 1978 Jul 1;88(1):44–53. doi: 10.1016/0042-6822(78)90108-3. [DOI] [PubMed] [Google Scholar]
  22. Mayer H., Reichenbach H. Restriction endonucleases: general survey procedure and survey of gliding bacteria. J Bacteriol. 1978 Nov;136(2):708–713. doi: 10.1128/jb.136.2.708-713.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. McDonell M. W., Simon M. N., Studier F. W. Analysis of restriction fragments of T7 DNA and determination of molecular weights by electrophoresis in neutral and alkaline gels. J Mol Biol. 1977 Feb 15;110(1):119–146. doi: 10.1016/s0022-2836(77)80102-2. [DOI] [PubMed] [Google Scholar]
  24. Morris D. W., Parish J. H. Restriction in Myxococcus virescens. Arch Microbiol. 1976 Jun;108(2):227–230. doi: 10.1007/BF00428956. [DOI] [PubMed] [Google Scholar]
  25. Rigby P. W., Dieckmann M., Rhodes C., Berg P. Labeling deoxyribonucleic acid to high specific activity in vitro by nick translation with DNA polymerase I. J Mol Biol. 1977 Jun 15;113(1):237–251. doi: 10.1016/0022-2836(77)90052-3. [DOI] [PubMed] [Google Scholar]
  26. Rosner J. L. Formation, induction, and curing of bacteriophage P1 lysogens. Virology. 1972 Jun;48(3):679–689. doi: 10.1016/0042-6822(72)90152-3. [DOI] [PubMed] [Google Scholar]
  27. Shaw K. J., Berg C. M. Escherichia coli K-12 auxotrophs induced by insertion of the transposable element Tn5. Genetics. 1979 Jul;92(3):741–747. doi: 10.1093/genetics/92.3.741. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Southern E. M. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol. 1975 Nov 5;98(3):503–517. doi: 10.1016/s0022-2836(75)80083-0. [DOI] [PubMed] [Google Scholar]
  29. Zusman D. R., Krotoski D. M., Cumsky M. Chromosome replication in Myxococcus xanthus. J Bacteriol. 1978 Jan;133(1):122–129. doi: 10.1128/jb.133.1.122-129.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES