Construction of Long Chromosomal Deletion Mutants of Escherichia coli and Minimization of the Genome

  • Jun-ichi Kato
  • Masayuki Hashimoto
Part of the Methods in Molecular Biology™ book series (MIMB, volume 416)


Genetic information consists of protein- and RNA-coding genes that exist in a range of sizes and noncoding cis- and trans-acting sequence elements. The use of long chromosomal deletion mutations is a powerful method for identifying essential genetic information through experimental reduction of the genome to its minimal gene set. Taking advantage of recent technical advances, we constructed sequence-specific long deletion mutations of the Escherichia coli chromosome. In a recent report (1), we described a set of E. coli medium-scale deletions (MDs) and large-scale deletions (LDs). Several LD mutations were combined to generate an engineered strain lacking ∼30% of the parental chromosome. We then constructed another set of deletion mutations, MDs and small-scale deletions (SDs), and identified additional essential genetic regions using complementation analysis. To delete the remaining essential chromosomal regions, we developed an Flp recombinase target (FRT)-based system of site-specific recombination to move chromosomal regions onto mini-F plasmids in vivo. In this report, we describe the details of the construction of several of these types of large chromosomal deletion mutants.

Key Words

chromosome deletions Escherichia coli essential genes FLP recombinase red 


  1. 1.
    Hashimoto, M., Ichimura, T., Mizoguchi, H., Tanaka, K., Fujimitsu, K., Keyamura, K., et al. (2005) Cell size and nucleoid organization of engineered Escherichia coli cells with a reduced genome. Mol. Microbiol. 55, 137–149.CrossRefPubMedGoogle Scholar
  2. 2.
    Hashimoto-Gotoh, T., Kume, A., Masahashi, W., Takeshita, S., and Fukuda, A. (1986) Improved vector, pHSG664, for direct streptomycin-resistance selection: cDNA cloning with G:C-tailing procedure and subcloning of double-digest DNA fragments. Gene 41, 125–128.CrossRefPubMedGoogle Scholar
  3. 3.
    Datsenko, K. A., and Wanner, B. L. (2000) One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc. Natl. Acad. Sci. U.S.A. 97, 6640–6645.CrossRefPubMedGoogle Scholar
  4. 4.
    Murphy, K. C., Campellone, K. G., and Poteete, A. R. (2000) PCR-mediated gene replacement in Escherichia coli. Gene 246, 321–330.CrossRefPubMedGoogle Scholar
  5. 5.
    Yu, D., Ellis, H. M., Lee, E. C., Jenkins, N. A., Copeland, N. G., and Court, D. L. (2000) An efficient recombination system for chromosome engineering in Escherichia coli. Proc. Natl. Acad. Sci. U.S.A. 97, 5978–5983.CrossRefPubMedGoogle Scholar
  6. 6.
    Murphy, K. C. (1998) Use of bacteriophage λ recombination functions to promote gene replacement in Escherichia coli. J. Bacteriol. 180, 2063–2071.PubMedGoogle Scholar
  7. 7.
    Ayres, E. K., Thomson, V. J., Merino, G., Balderes, D., and Figurski, D. H. (1993) Precise deletions in large bacterial genomes by vector-mediated excision (VEX). The trfA gene of promiscuous plasmid RK2 is essential for replication in several gram-negative hosts. J. Mol. Biol. 230, 174–185.CrossRefPubMedGoogle Scholar
  8. 8.
    Posfai, G., Koob, M., Hradecna, Z., Hasan, N., Filutowicz, M., and Szybalski, W. (1994) In vivo excision and amplification of large segments of the Escherichia coli genome. Nucleic Acids Res. 22, 2392–2398.CrossRefPubMedGoogle Scholar
  9. 9.
    Wild, J., Hradecna, Z., Posfai, G., and Szybalski, W. (1996) A broad-host-range in vivo pop-out and amplification system for generating large quantities of 50-to 100-kb genomic fragments for direct DNA sequencing. Gene 179, 181–188.CrossRefPubMedGoogle Scholar
  10. 10.
    Posfai, G., Koob, M. D., Kirkpatrick, H. A., Blattner, F. R. (1997) Versatile insertion plas-mids for targeted genome manipulations in bacteria: isolation, deletion, and rescue of the pathogenicity island LEE of the Escherichia coli O157:H7 genome. J. Bacteriol. 179, 4426–4428.PubMedGoogle Scholar
  11. 11.
    Yoon, Y. G., Cho, J. H., and Kim, S. C. (1998) Cre/loxP-mediated excision and amplification of large segments of the Escherichia coli genome. Genet. Anal. 14, 89–95.PubMedGoogle Scholar
  12. 12.
    Murayama, N., Shimizu, H., Takiguchi, S., Baba, Y., Amino, H., Horiuchi, T., et al. (1996) Evidence for involvement of Escherichia coli genes pmbA, csrA and a previously unrecognized gene tldD, in the control of DNA gyrase by letD (ccdB) of sex factor F. J. Mol. Biol. 256, 483–502.CrossRefPubMedGoogle Scholar

Copyright information

© Humana Press Inc., a part of Springer Science+Business Media, LLC 2008

Authors and Affiliations

  • Jun-ichi Kato
    • 1
  • Masayuki Hashimoto
    • 2
  1. 1.Department of Biological Sciences, Graduate School of Science and EngineeringTokyo Metropolitan UniversityTokyoJapan
  2. 2.Division of Gene Research, Department of Life Science, Research Center for Human and Environmental ScienceShinshu UniversityNaganoJapan

Personalised recommendations