Skip to main content
NCBI home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1991 Jan;173(2):896–899. doi: 10.1128/jb.173.2.896-899.1991

Transposon Tn5supF-based reverse genetic method for mutational analysis of Escherichia coli with DNAs cloned in lambda phage.

S H Phadnis 1, S Kulakauskas 1, B R Krishnan 1, J Hiemstra 1, D E Berg 1
PMCID: PMC207086  PMID: 1846153

Abstract

An efficient method for systematic mutational analysis of the Escherichia coli genome was developed. It entails Tn5supF transposition to lambda-E. coli hybrid phage clones (Kohara library) and then transduction of recipient cells to Sup+. Essential and nonessential genes are distinguished by the ability of insertion mutant phage to form haploid versus only heterozygous partial diploid bacterial recombinants.

Full text

PDF
896

Images in this article

898Image
on p.898

Selected References

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

  1. Anderson R. P., Roth J. R. Tandem genetic duplications in phage and bacteria. Annu Rev Microbiol. 1977;31:473–505. doi: 10.1146/annurev.mi.31.100177.002353. [DOI] [PubMed] [Google Scholar]
  2. Bachmann B. J. Linkage map of Escherichia coli K-12, edition 8. Microbiol Rev. 1990 Jun;54(2):130–197. doi: 10.1128/mr.54.2.130-197.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Berg D. E., Schmandt M. A., Lowe J. B. Specificity of transposon Tn5 insertion. Genetics. 1983 Dec;105(4):813–828. doi: 10.1093/genetics/105.4.813. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Berg D. E., Weiss A., Crossland L. Polarity of Tn5 insertion mutations in Escherichia coli. J Bacteriol. 1980 May;142(2):439–446. doi: 10.1128/jb.142.2.439-446.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Gutterson N. I., Koshland D. E., Jr Replacement and amplification of bacterial genes with sequences altered in vitro. Proc Natl Acad Sci U S A. 1983 Aug;80(16):4894–4898. doi: 10.1073/pnas.80.16.4894. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Guyer M. S. The gamma delta sequence of F is an insertion sequence. J Mol Biol. 1978 Dec 15;126(3):347–365. doi: 10.1016/0022-2836(78)90045-1. [DOI] [PubMed] [Google Scholar]
  7. HORIUCHI T., HORIUCHI S., NOVICK A. The genetic basis of hyper-synthesis of beta-galactosidase. Genetics. 1963 Feb;48:157–169. doi: 10.1093/genetics/48.2.157. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Hamilton C. M., Aldea M., Washburn B. K., Babitzke P., Kushner S. R. New method for generating deletions and gene replacements in Escherichia coli. J Bacteriol. 1989 Sep;171(9):4617–4622. doi: 10.1128/jb.171.9.4617-4622.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Jasin M., Schimmel P. Deletion of an essential gene in Escherichia coli by site-specific recombination with linear DNA fragments. J Bacteriol. 1984 Aug;159(2):783–786. doi: 10.1128/jb.159.2.783-786.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Joyce C. M., Grindley N. D. Method for determining whether a gene of Escherichia coli is essential: application to the polA gene. J Bacteriol. 1984 May;158(2):636–643. doi: 10.1128/jb.158.2.636-643.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Kalnins A., Otto K., Rüther U., Müller-Hill B. Sequence of the lacZ gene of Escherichia coli. EMBO J. 1983;2(4):593–597. doi: 10.1002/j.1460-2075.1983.tb01468.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Kohara Y., Akiyama K., Isono K. The physical map of the whole E. coli chromosome: application of a new strategy for rapid analysis and sorting of a large genomic library. Cell. 1987 Jul 31;50(3):495–508. doi: 10.1016/0092-8674(87)90503-4. [DOI] [PubMed] [Google Scholar]
  13. Kulakauskas S. T., Mitkaite G. V., Khmeliauskaite V. G. Izuchenie duplikatsii v raione genov phoA i lacZ na khromosome Escherichia coli K-12. Genetika. 1990 Nov;26(11):1926–1931. [PubMed] [Google Scholar]
  14. Kurnit D. M., Seed B. Improved genetic selection for screening bacteriophage libraries by homologous recombination in vivo. Proc Natl Acad Sci U S A. 1990 Apr;87(8):3166–3169. doi: 10.1073/pnas.87.8.3166. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Lodge J. K., Berg D. E. Mutations that affect Tn5 insertion into pBR322: importance of local DNA supercoiling. J Bacteriol. 1990 Oct;172(10):5956–5960. doi: 10.1128/jb.172.10.5956-5960.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Masters M. Location of purine genes on the physical map of Escherichia coli. J Bacteriol. 1990 Mar;172(3):1173–1173. doi: 10.1128/jb.172.3.1173.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Meek D. W., Hayward R. S. Nucleotide sequence of the rpoA-rplQ DNA of Escherichia coli: a second regulatory binding site for protein S4? Nucleic Acids Res. 1984 Jul 25;12(14):5813–5821. doi: 10.1093/nar/12.14.5813. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Ogawa T., Tomizawa J. Absortive lysogenization of bacteriophage lambda b2 and residual immunity of non-lysogenic segregants. J Mol Biol. 1967 Jan 28;23(2):225–245. doi: 10.1016/s0022-2836(67)80030-5. [DOI] [PubMed] [Google Scholar]
  19. Parker B., Marinus M. G. A simple and rapid method to obtain substitution mutations in Escherichia coli: isolation of a dam deletion/insertion mutation. Gene. 1988 Dec 20;73(2):531–535. doi: 10.1016/0378-1119(88)90517-3. [DOI] [PubMed] [Google Scholar]
  20. Parkinson J. S., Huskey R. J. Deletion mutants of bacteriophage lambda. I. Isolation and initial characterization. J Mol Biol. 1971 Mar 14;56(2):369–384. doi: 10.1016/0022-2836(71)90471-2. [DOI] [PubMed] [Google Scholar]
  21. Phadnis S. H., Huang H. V., Berg D. E. Tn5supF, a 264-base-pair transposon derived from Tn5 for insertion mutagenesis and sequencing DNAs cloned in phage lambda. Proc Natl Acad Sci U S A. 1989 Aug;86(15):5908–5912. doi: 10.1073/pnas.86.15.5908. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Russell C. B., Thaler D. S., Dahlquist F. W. Chromosomal transformation of Escherichia coli recD strains with linearized plasmids. J Bacteriol. 1989 May;171(5):2609–2613. doi: 10.1128/jb.171.5.2609-2613.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Shen P., Huang H. V. Homologous recombination in Escherichia coli: dependence on substrate length and homology. Genetics. 1986 Mar;112(3):441–457. doi: 10.1093/genetics/112.3.441. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Shevell D. E., Abou-Zamzam A. M., Demple B., Walker G. C. Construction of an Escherichia coli K-12 ada deletion by gene replacement in a recD strain reveals a second methyltransferase that repairs alkylated DNA. J Bacteriol. 1988 Jul;170(7):3294–3296. doi: 10.1128/jb.170.7.3294-3296.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Strausbaugh L. D., Bourke M. T., Sommer M. T., Coon M. E., Berg C. M. Probe mapping to facilitate transposon-based DNA sequencing. Proc Natl Acad Sci U S A. 1990 Aug;87(16):6213–6217. doi: 10.1073/pnas.87.16.6213. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Whoriskey S. K., Nghiem V. H., Leong P. M., Masson J. M., Miller J. H. Genetic rearrangements and gene amplification in Escherichia coli: DNA sequences at the junctures of amplified gene fusions. Genes Dev. 1987 May;1(3):227–237. doi: 10.1101/gad.1.3.227. [DOI] [PubMed] [Google Scholar]
  27. Winans S. C., Elledge S. J., Krueger J. H., Walker G. C. Site-directed insertion and deletion mutagenesis with cloned fragments in Escherichia coli. J Bacteriol. 1985 Mar;161(3):1219–1221. doi: 10.1128/jb.161.3.1219-1221.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES