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:

eSGA: E. coli synthetic genetic array analysis

Nature Methods volume 5, pages 789–795 (2008)Cite this article

This article has been updated

Abstract

Physical and functional interactions define the molecular organization of the cell. Genetic interactions, or epistasis, tend to occur between gene products involved in parallel pathways or interlinked biological processes. High-throughput experimental systems to examine genetic interactions on a genome-wide scale have been devised for Saccharomyces cerevisiae, Schizosaccharomyces pombe, Caenorhabditis elegans and Drosophila melanogaster, but have not been reported previously for prokaryotes. Here we describe the development of a quantitative screening procedure for monitoring bacterial genetic interactions based on conjugation of Escherichia coli deletion or hypomorphic strains to create double mutants on a genome-wide scale. The patterns of synthetic sickness and synthetic lethality (aggravating genetic interactions) we observed for certain double mutant combinations provided information about functional relationships and redundancy between pathways and enabled us to group bacterial gene products into functional modules.

NOTE: In the version of this article initially published online two author names (Gabriel Moreno-Hagelseib and Constantine Christopolous) were spelled incorrectly. The correct author names are Gabriel Moreno-Hagelsieb and Constantine Christopoulos. The error has been corrected for the print, PDF and HTML versions of this article.

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

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

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

Figure 1: eSGA outline.
Figure 2: Map of the query mutations and detection of aggravating genetic interactions by eSGA.
Figure 3: Evaluation of data quality and statistical significance of interaction (S) scores from genome-wide screens.
Figure 4: Clustering of genetic interaction profiles from genome-wide screens.

Similar content being viewed by others

Change history

  • 17 August 2008

    NOTE: In the version of this article initially published online two author names (Gabriel Moreno-Hagelseib and Constantine Christopolous) were spelled incorrectly. The correct author names are Gabriel Moreno-Hagelsieb and Constantine Christopoulos. The error has been corrected for the print, PDF and HTML versions of this article.

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. Costanzo, M., Giaever, G., Nislow, C. & Andrews, B. Experimental approaches to identify genetic networks. Curr. Opin. Biotechnol. 17, 472–480 (2006).

    Article  CAS  Google Scholar 

  3. Beyer, A., Bandyopadhyay, S. & Ideker, T. Integrating physical and genetic maps: from genomes to interaction networks. Nat. Rev. Genet. 8, 699–710 (2007).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  6. 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 

  7. Collins, S.R. et al. Functional dissection of protein complexes involved in yeast chromosome biology using a genetic interaction map. Nature 446, 806–810 (2007).

    Article  CAS  Google Scholar 

  8. Baba, T. et al. Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection. Mol. Syst. Biol. 2 2006.0008 (2006).

  9. Kogoma, T. Stable DNA replication: interplay between DNA replication, homologous recombination and transcription. Microbiol. Mol. Biol. Rev. 61, 212–238 (1997).

    CAS  PubMed  PubMed Central  Google Scholar 

  10. Ippen-Ihler, K.A. & Minkley, E.G. Jr. The conjugation system of F, the fertility factor of Escherichia coli. Annu. Rev. Genet. 20, 593–624 (1986).

    Article  CAS  Google Scholar 

  11. Kowalczykowski, S.C., Dixon, D.A., Eggleston, A.K., Lauder, S.D. & Rehrauer, W.M. Biochemistry of homologous recombination in Escherichia coli. Microbiol. Mol. Biol. Rev. 58, 401–465 (1994).

    CAS  Google Scholar 

  12. Yu, D. et al. An efficient recombination system for chromosome engineering in Escherichia coli. Proc. Natl. Acad. Sci. USA 97, 5978–5983 (2000).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  14. Susman, M. General bacterial genetics. Annu. Rev. Genet. 4, 135–176 (1970).

    Article  CAS  Google Scholar 

  15. Bachmann, B.J. Pedigrees of some mutant strains of Escherichia coli K-12. Bacteriol. Rev. 36, 525–557 (1972).

    CAS  PubMed  PubMed Central  Google Scholar 

  16. Fontecave, M., Ollagnier de Choudens, S. & Barras, B. Py, F. Mechanisms of iron-sulfur cluster assembly: the SUF machinery. J. Biol. Inorg. Chem. 10, 713–721 (2005).

    Article  CAS  Google Scholar 

  17. Tokumoto, U., Kitamura, S., Fukuyama, K. & Takahashi, Y. Interchangeability and distinct properties of bacterial Fe-S cluster assembly systems: functional replacement of the isc and suf operons in Escherichia coli with the nifSU-like operon from Helicobacter pylori. J. Biochem. 136, 199–209 (2004).

    Article  CAS  Google Scholar 

  18. Tokumoto, U. & Takahashi, Y. Genetic analysis of the isc operon in Escherichia coli involved in the biogenesis of cellular iron-sulfur proteins. J. Biochem. 130, 63–71 (2001).

    Article  CAS  Google Scholar 

  19. Outten, F.W., Djaman, O. & Storz, G. A suf operon requirement for Fe–S cluster assembly during iron starvation in Escherichia coli. Mol. Microbiol. 52, 861–872 (2004).

    Article  CAS  Google Scholar 

  20. Lu, J., Yang, J., Tan, G. & Ding, H. Complementary roles of SufA and IscA in the biogenesis of iron-sulfur clusters in Escherichia coli. J. Biochem. 409, 535–543 (2008).

    Article  CAS  Google Scholar 

  21. Mani, R., St. Onge, R.P., Hartman, J.L., Giaever, G. & Roth, F.P. Defining genetic interaction. Proc. Natl. Acad. Sci. USA 105, 3461–3466 (2008).

    Article  CAS  Google Scholar 

  22. Fox, M.S. Some features of genetic recombination in prokaryotes. Annu. Rev. Genet. 12, 47–68 (1978).

    Article  CAS  Google Scholar 

  23. St. Onge, R.P. et al. Systematic pathway analysis using high-resolution fitness profiling of combinatorial gene deletions. Nat. Genet. 39, 199–206 (2007).

    Article  CAS  Google Scholar 

  24. Von Mering, C. et al. STRING 7-recent developments in the integration and prediction of protein interactions. Nucleic Acids Res. 35, D358–D362 (2007).

    Article  CAS  Google Scholar 

  25. Lauhon, C.T. Requirement for IscS in biosynthesis of all thionucleosides in Escherichia coli. J. Bacteriol. 184, 6820–6829 (2002).

    Article  CAS  Google Scholar 

  26. Davierwala, A.P. et al. The synthetic genetic interaction spectrum of essential genes. Nat. Genet. 37, 1147–1152 (2005).

    Article  CAS  Google Scholar 

  27. Ding, H., Harrison, K. & Lu, J. Thioredoxin reducatse system mediates iron binding in iscA and iron delivery for the iron-sulfur cluster assembly in IscU. J. Biol. Chem. 280, 30432–30437 (2005).

    Article  CAS  Google Scholar 

  28. Fernandes, A.P. et al. A novel monothiol glutaredoxin (Grx4) from Escherichia coli can serve as a substrate for thioredoxin reductase. J. Biol. Chem. 280, 24544–24552 (2005).

    Article  CAS  Google Scholar 

  29. Mizoguchi, H., Mori, H. & Fujio, T. Escherichia coli minimum genome factory. Biotechnol. Appl. Biochem. 46, 157–167 (2007).

    Article  CAS  Google Scholar 

  30. Eisen, M.B., Spellman, P.T., Brown, P.O. & Botstein, D. Cluster analysis and display of genome-wide expression patterns. Proc. Natl. Acad. Sci. USA 95, 14863–14868 (1998).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank M. Costanzo for comments on the manuscript. This work was supported by a Canadian Institute of Health Research (CIHR) grant (82852) to J.F.G. and A.E., by a US National Institutes of Health grant to B.L.W. (GM62662), by Grant-in-Aid for Scientific Research on Priority Areas from the Ministry of Education, Sports, Science and Technology of Japan Core Research for Evolutional Science and Technology, and Japan Science and Technology grants to H.M., by a CIHR grant (GSP-41567) to B.J.A. and C.B., by a partial postdoctoral fellowship from the Mexican Science and Technology Research Council to J.J.D.-M., and by a Laboratory Directed Research and Development grant to G.B. The phage-λ Red system was kindly provided by D.L. Court (National Cancer Institute).

Author information

Author notes

  1. Gareth Butland

    Present address: Present address: Life Sciences Division, Lawrence Berkeley National Laboratory, 1 Cyclotron Road, Berkeley, California 94720, USA.,

  2. Gareth Butland and Mohan Babu: These authors contributed equally to this work.

Authors and Affiliations

  1. Banting and Best Department of Medical Research, Terrence Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, 160 College Street, Toronto, M5S 3E1, Canada

    Gareth Butland, Mohan Babu, J Javier Díaz-Mejía, Fedyshyn Bohdana, Sadhna Phanse, Joyce Li, Oxana Pogoutse, Henry Lo, Jas Wasniewski, Constantine Christopoulos, Pascal Venn, Anahita Safavi-Naini, Natalie Sourour, Simone Caron, Ja-Yeon Choi, Ludovic Laigle, Anaies Nazarians-Armavil, Avnish Deshpande, Sarah Joe, Brenda J Andrews, Charles Boone, Huiming Ding, Bilal Sheikh, Jack F Greenblatt & Andrew Emili

  2. Life Sciences Division, Lawrence Berkeley National Laboratory, 1 Cyclotron Road, Berkeley, 94720, California, USA

    Gareth Butland, Barbara Gold & Wenhong Yang

  3. Department of Biology, Wilfrid Laurier University, 75 University Avenue West, N2L 3C5, Waterloo, Canada

    J Javier Díaz-Mejía & Gabriel Moreno-Hagelsieb

  4. Department of Molecular Genetics, University of Toronto, 1 King's College Circle, Toronto, M5S 1A8, Canada

    Alla G Gagarinova, Mehrab Ali, Brenda J Andrews, Jack F Greenblatt & Andrew Emili

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

    Hirotada Mori, Barry L Wanner, Natsuko Yamamoto & Charles Boone

  6. Department of Biological Sciences, Purdue University, 915 W. State Street, West Lafayette, 47907, Indiana, USA

    Kirill A Datsenko

Authors
  1. Gareth Butland
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Mohan Babu
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. J Javier Díaz-Mejía
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Fedyshyn Bohdana
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Sadhna Phanse
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Barbara Gold
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Wenhong Yang
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Joyce Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Alla G Gagarinova
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Oxana Pogoutse
    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. Barry L Wanner
    View author publications

    You can also search for this author in PubMed Google Scholar

  13. Henry Lo
    View author publications

    You can also search for this author in PubMed Google Scholar

  14. Jas Wasniewski
    View author publications

    You can also search for this author in PubMed Google Scholar

  15. Constantine Christopoulos
    View author publications

    You can also search for this author in PubMed Google Scholar

  16. Mehrab Ali
    View author publications

    You can also search for this author in PubMed Google Scholar

  17. Pascal Venn
    View author publications

    You can also search for this author in PubMed Google Scholar

  18. Anahita Safavi-Naini
    View author publications

    You can also search for this author in PubMed Google Scholar

  19. Natalie Sourour
    View author publications

    You can also search for this author in PubMed Google Scholar

  20. Simone Caron
    View author publications

    You can also search for this author in PubMed Google Scholar

  21. Ja-Yeon Choi
    View author publications

    You can also search for this author in PubMed Google Scholar

  22. Ludovic Laigle
    View author publications

    You can also search for this author in PubMed Google Scholar

  23. Anaies Nazarians-Armavil
    View author publications

    You can also search for this author in PubMed Google Scholar

  24. Avnish Deshpande
    View author publications

    You can also search for this author in PubMed Google Scholar

  25. Sarah Joe
    View author publications

    You can also search for this author in PubMed Google Scholar

  26. Kirill A Datsenko
    View author publications

    You can also search for this author in PubMed Google Scholar

  27. Natsuko Yamamoto
    View author publications

    You can also search for this author in PubMed Google Scholar

  28. Brenda J Andrews
    View author publications

    You can also search for this author in PubMed Google Scholar

  29. Charles Boone
    View author publications

    You can also search for this author in PubMed Google Scholar

  30. Huiming Ding
    View author publications

    You can also search for this author in PubMed Google Scholar

  31. Bilal Sheikh
    View author publications

    You can also search for this author in PubMed Google Scholar

  32. Gabriel Moreno-Hagelsieb
    View author publications

    You can also search for this author in PubMed Google Scholar

  33. Jack F Greenblatt
    View author publications

    You can also search for this author in PubMed Google Scholar

  34. Andrew Emili
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

M.B. and G.B. coordinated experimental design and data analysis. M.B., G.B., H.L., J.W., P.V., C.C., L.L., M.A., K.A.D., N.Y., H.M. and B.L.W. performed optimization studies and constructed recipient arrays. J.L., A.S.-N., W.Y., A.G.G., O.P., G.B. and M.B. generated query deletion knockouts. M.B., F.B., N.S., S.C., J.-Y.C. and A.N.-A. performed genome-wide screens. M.B., S.P., A.D., S.J., B.S., H.D., B.J.A. and C.B. designed and performed quantitative image analysis. J.J.D.-M., M.B., S.P. and G.M.-H. performed informatics analyses. G.B., B.G. and W.Y. performed validation experiments. G.B., M.B., J.F.G. and A.E. drafted the manuscript. J.F.G. and A.E. conceived, designed and directed the project.

Corresponding authors

Correspondence to Jack F Greenblatt or Andrew Emili.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–5, Supplementary Methods, Supplementary Results, Supplementary Discussion (PDF 1941 kb)

Supplementary Table 1

List of recipient F- KEIO deletion mutant strains and SPA-tagged essential genes used in this study. (XLS 701 kb)

Supplementary Table 2

List of query deletion mutant strains used in the 39 genome-wide screens. (XLS 32 kb)

Supplementary Table 3

High-confidence genetic interaction filtered after accounting for linkage (30 kbp-window) using a stringent cut-off of |Z score| > = 4 (P < 0.0001). (XLS 284 kb)

Supplementary Table 4

Raw colony sizes (see Sheet 1), Normalized median colony sizes (see Sheet 2), |Z scores| (see Sheet 3) and interaction (S) scores (see Sheet 4) of each mutant gene pair from 39 genome-wide screens without any filtering parameters. (XLS 35759 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Butland, G., Babu, M., Díaz-Mejía, J. et al. eSGA: E. coli synthetic genetic array analysis. Nat Methods 5, 789–795 (2008). https://doi.org/10.1038/nmeth.1239

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nmeth.1239

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