Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

High-throughput, quantitative analyses of genetic interactions in E. coli

Nature Methods volume 5, pages 781–787 (2008)Cite this article

Abstract

Large-scale genetic interaction studies provide the basis for defining gene function and pathway architecture. Recent advances in the ability to generate double mutants en masse in Saccharomyces cerevisiae have dramatically accelerated the acquisition of genetic interaction information and the biological inferences that follow. Here we describe a method based on F factor–driven conjugation, which allows for high-throughput generation of double mutants in Escherichia coli. This method, termed genetic interaction analysis technology for E. coli (GIANT-coli), permits us to systematically generate and array double-mutant cells on solid media in high-density arrays. We show that colony size provides a robust and quantitative output of cellular fitness and that GIANT-coli can recapitulate known synthetic interactions and identify previously unidentified negative (synthetic sickness or lethality) and positive (suppressive or epistatic) relationships. Finally, we describe a complementary strategy for genome-wide suppressor-mutant identification. Together, these methods permit rapid, large-scale genetic interaction studies in E. coli.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on SpringerLink
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: A flowchart depicting the different steps used in GIANT-coli.
Figure 2: A 12 × 12 genetic interaction matrix to validate GIANT-coli.
Figure 3: A toolkit that facilitates the use of GIANT-coli in genome-wide analyses.
Figure 4: Genome-wide screens using GIANT-coli.

Similar content being viewed by others

References

  1. Boone, C., Bussey, H. & Andrews, B.J. Exploring genetic interactions and networks with yeast. Nat. Rev. Genet. 8, 437–449 (2007).

    Article  CAS  Google Scholar 

  2. Tong, A.H. et al. Systematic genetic analysis with ordered arrays of yeast deletion mutants. Science 294, 2364–2368 (2001).

    Article  CAS  Google Scholar 

  3. Tong, A.H. et al. Global mapping of the yeast genetic interaction network. Science 303, 808–813 (2004).

    Article  CAS  Google Scholar 

  4. Pan, X. et al. A robust toolkit for functional profiling of the yeast genome. Mol. Cell 16, 487–496 (2004).

    Article  CAS  Google Scholar 

  5. Collins, S.R. et al. Functional dissection of protein complexes involved in yeast chromosome biology using a genetic interaction map. Nature 466, 806–810 (2007).

    Article  Google Scholar 

  6. Schuldiner, M. et al. Exploration of the function and organization of the yeast early secretory pathway through an epistatic miniarray profile. Cell 123, 507–519 (2005).

    Article  CAS  Google Scholar 

  7. Roguev, A., Wiren, M., Weissman, J.S. & Krogan, N.J. High-throughput genetic interaction mapping in the fission yeast Schizosaccharomyces pombe. Nat. Methods 4, 861–866 (2007).

    Article  CAS  Google Scholar 

  8. Byrne, A.B. et al. A global analysis of genetic interactions in Caenorhabditis elegans. J. Biol. 6, 8 (2007).

    Article  Google Scholar 

  9. Breitkreutz, B.J. et al. The BioGRID Interaction Database: 2008 update. Nucleic Acids Res. 36, D637–D640 (2008).

    Article  CAS  Google Scholar 

  10. Ara, K. et al. Bacillus minimum genome factory—effective utilization of microbial genome information. Biotechnol. Appl. Biochem 46, 1169–1178 (2006).

    Google Scholar 

  11. Ito, M., Baba, T. & Mori, H. Functional analysis of 1440 Escherichia coli genes using the combination of knock-out library and phenotype microarrays. Metab. Eng. 7, 318–327 (2005).

    Article  CAS  Google Scholar 

  12. Low, K.B. Hfr strains of Escherichia coli K-12. In Escherichia coli and Salmonella: Cellular and Molecular Biology Vol. 2 (ed. Neidhardt, F.C. et al.) 2402–2405 (ASM Press, Washington, DC, 1996).

    Google Scholar 

  13. Francois, V., Conter, A. & Louarn, J.M. Properties of new Escherichia coli Hfr strains constructed by integration of pSC101-derived conjugative plasmids. J. Bacteriol. 172, 1436–1440 (1990).

    Article  CAS  Google Scholar 

  14. Bachmann, B.J. Derivations and genotypes of some mutant derivatives of Escherichia Coli K-12. In Escherichia coli and Salmonella: Cellular and Molecular Biology Vol. 2 (ed. Neidhardt, F.C. et al.) 2460–2488 (ASM Press, Washington, DC, 1996).

    Google Scholar 

  15. Baba, T. et al. Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection. Mol. Syst. Biol. 2, 0008 (2006).

    Article  Google Scholar 

  16. Nielsen, H.J., Youngren, B., Hansen, F.G. & Austin, S. Dynamics of Escherichia coli chromosome segregation during multifork replication. J. Bacteriol. 189, 8660–8666 (2007).

    Article  CAS  Google Scholar 

  17. Nielsen, H.J., Li, Y., Youngren, B., Hansen, F.G. & Austin, S. Progressive segregation of the Escherichia coli chromosome. Mol. Microbiol. 61, 383–393 (2006).

    Article  CAS  Google Scholar 

  18. Collins, S.R., Schuldiner, M., Krogan, N.J. & Weissman, J.S. A strategy for extracting and analyzing large-scale quantitative epistatic interaction data. Genome Biol. 7, R63 (2006).

    Article  Google Scholar 

  19. Hoekstra, W.P. & Havekes, A.M. On the role of the recipient cell during conjugation in Escherichia coli. Antonie Van Leeuwenhoek 45, 13–18 (1979).

    Article  CAS  Google Scholar 

  20. Rizzitello, A.E., Harper, J.R. & Silhavy, T.J. Genetic evidence for parallel pathways of chaperone activity in the periplasm of Escherichia coli. J. Bacteriol. 183, 6794–6800 (2001).

    Article  CAS  Google Scholar 

  21. Charlson, E.S., Werner, J.N. & Misra, R. Differential effects of yfgL mutation on Escherichia coli outer membrane proteins and lipopolysaccharide. J. Bacteriol. 188, 7186–7194 (2006).

    Article  CAS  Google Scholar 

  22. Onufryk, C., Crouch, M.L., Fang, F.C. & Gross, C.A. Characterization of six lipoproteins in the σE regulon. J. Bacteriol. 187, 4552–4561 (2005).

    Article  CAS  Google Scholar 

  23. Rouviere, P.E. & Gross, C.A. SurA, a periplasmic protein with peptidyl-prolyl isomerase activity, participates in the assembly of outer membrane porins. Genes Dev. 10, 3170–3182 (1996).

    Article  CAS  Google Scholar 

  24. Sklar, J.G., Wu, T., Kahne, D. & Silhavy, T.J. Defining the roles of the periplasmic chaperones SurA, Skp, and DegP in Escherichia coli. Genes Dev. 21, 2473–2484 (2007).

    Article  CAS  Google Scholar 

  25. Danese, P.N., Snyder, W.B., Cosma, C.L., Davis, L.J. & Silhavy, T.J. The Cpx two-component signal transduction pathway of Escherichia coli regulates transcription of the gene specifying the stress-inducible periplasmic protease, DegP. Genes Dev. 9, 387–398 (1995).

    Article  CAS  Google Scholar 

  26. Pogliano, J., Lynch, A.S., Belin, D., Lin, E.C. & Beckwith, J. Regulation of Escherichia coli cell envelope proteins involved in protein folding and degradation by the Cpx two-component system. Genes Dev. 11, 1169–1182 (1997).

    Article  CAS  Google Scholar 

  27. Bardwell, J.C., McGovern, K. & Beckwith, J. Identification of a protein required for disulfide bond formation in vivo. Cell 67, 581–589 (1991).

    Article  CAS  Google Scholar 

  28. Leichert, L.I. & Jakob, U. Protein thiol modifications visualized in vivo. PLoS Biol. 2, e333 (2004).

    Article  Google Scholar 

  29. Qu, J., Mayer, C., Behrens, S., Holst, O. & Kleinschmidt, J.H. The trimeric periplasmic chaperone Skp of Escherichia coli forms 1:1 complexes with outer membrane proteins via hydrophobic and electrostatic interactions. J. Mol. Biol. 374, 91–105 (2007).

    Article  CAS  Google Scholar 

  30. Parsons, L.M., Lin, F. & Orban, J. Peptidoglycan recognition by Pal, an outer membrane lipoprotein. Biochemistry 45, 2122–2128 (2006).

    Article  CAS  Google Scholar 

  31. Cascales, E. & Lloubes, R. Deletion analyses of the peptidoglycan-associated lipoprotein Pal reveals three independent binding sequences including a TolA box. Mol. Microbiol. 51, 873–885 (2004).

    Article  CAS  Google Scholar 

  32. Cascales, E., Lloubes, R. & Sturgis, J.N. The TolQ-TolR proteins energize TolA and share homologies with the flagellar motor proteins MotA-MotB. Mol. Microbiol. 42, 795–807 (2001).

    Article  CAS  Google Scholar 

  33. Gerding, M.A., Ogata, Y., Pecora, N.D., Niki, H. & de Boer, P.A. The trans-envelope Tol-Pal complex is part of the cell division machinery and required for proper outer-membrane invagination during cell constriction in E. coli. Mol. Microbiol. 63, 1008–1025 (2007).

    Article  CAS  Google Scholar 

  34. Inoue, T. et al. Genome-wide screening of genes required for swarming motility in Escherichia coli K-12. J. Bacteriol. 189, 950–957 (2007).

    Article  CAS  Google Scholar 

  35. Bochner, B.R. New technologies to assess genotype-phenotype relationships. Nat. Rev. Genet. 4, 309–314 (2003).

    Article  CAS  Google Scholar 

  36. Butland, G. et al. Interaction network containing conserved and essential protein complexes in Escherichia coli. Nature 433, 531–537 (2005).

    Article  CAS  Google Scholar 

  37. Chen, C.S. et al. A proteome chip approach reveals new DNA damage recognition activities in Escherichia coli. Nat. Methods 5, 69–74 (2008).

    Article  CAS  Google Scholar 

  38. Mazurkiewicz, P., Tang, C.M., Boone, C. & Holden, D.W. Signature-tagged mutagenesis: barcoding mutants for genome-wide screens. Nat. Rev. Genet. 7, 929–939 (2006).

    Article  CAS  Google Scholar 

  39. Girgis, H.S., Liu, Y., Ryu, W.S. & Tavazoie, S. A comprehensive genetic characterization of bacterial motility. PLoS Genet. 3, 1644–1660 (2007).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank S. Lee, A. Wong and J. Lee for technical assistance, and C.J. Ingles, P.J. Kiley, C. Squires, S.A. Johnson, A. Hochschild & T.J. Silhavy for critically reading this manuscript and offering useful suggestions. This work was supported by Sandler Family Funding (to C.A.G. and to N.J.K.), US National Institutes of Health (GM036278 to C.A.G. and GM62662 to B.L.W.), Japanese Ministry of Education, Culture, Sports, Science and Technology (Core Research for Evolutional Science and Technology) Grant-in-Aid for Scientific Research and Grant-in-Aid for Scientific Research on Priority Areas (to H.M.). A.T. is a recipient of a European Molecular Biology Organization fellowship.

Author information

Authors and Affiliations

  1. Department of Microbiology and Immunology, University of California at San Francisco, 600 16th Street, San Francisco, 94158, California, USA

    Athanasios Typas, Robert J Nichols, Bentley Lim & Carol A Gross

  2. Department of Biology, Texas A&M University, 3258 TAMU College Station, 77843, Texas, USA

    Deborah A Siegele

  3. Department of Cellular and Molecular Pharmacology and The California Institute for Quantitative Biomedical Research, University of California at San Francisco, 1700 4th Street, San Francisco, 94158, California, USA

    Michael Shales, Sean R Collins, Hannes Braberg, Jonathan S Weissman & Nevan J Krogan

  4. Howard Hughes Medical Institute, University of California at San Francisco, 1700 4th Street, San Francisco, 94158, California, USA

    Sean R Collins & Jonathan S Weissman

  5. Graduate School of Biological Sciences, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma, 630-0101, Nara, Japan

    Natsuko Yamamoto, Rikiya Takeuchi & Hirotada Mori

  6. Department of Biological Sciences, Purdue University, LILY 1-228, West Lafayette, 47907, Indiana, USA

    Barry L Wanner

Authors
  1. Athanasios Typas
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Robert J Nichols
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Deborah A Siegele
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Michael Shales
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Sean R Collins
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Bentley Lim
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Hannes Braberg
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Natsuko Yamamoto
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Rikiya Takeuchi
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Barry L Wanner
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Hirotada Mori
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. Jonathan S Weissman
    View author publications

    You can also search for this author in PubMed Google Scholar

  13. Nevan J Krogan
    View author publications

    You can also search for this author in PubMed Google Scholar

  14. Carol A Gross
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

A.T., R.J.N., D.A.S., J.S.W., N.J.K. and C.A.G. designed research; A.T., R.J.N., D.A.S. and B.L. executed research; A.T., R.J.N., M.S., S.C. and H.B. analyzed data; N.Y., R.T., B.L.W. and H.M. contributed new reagents (single-gene knockout libraries and CIP plasmids); A.T., N.J.K. and C.A.G. wrote the paper; R.J.N., M.S., B.L.W. and J.S.W. edited the paper.

Corresponding authors

Correspondence to Nevan J Krogan or Carol A Gross.

Supplementary information

Supplementary Text and Figures

Supplementary Methods, Supplementary Figures 1–5, Supplementary Tables 1–3 (PDF 1706 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Typas, A., Nichols, R., Siegele, D. et al. High-throughput, quantitative analyses of genetic interactions in E. coli. Nat Methods 5, 781–787 (2008). https://doi.org/10.1038/nmeth.1240

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nmeth.1240

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing