Skip to main content
SpringerLink
Log in

Construction of high-density transposon mutant library of Staphylococcus aureus using bacteriophage ϕ11

  • Protocol
  • Published:
Journal of Microbiology Aims and scope Submit manuscript

Abstract

Transposon mutant libraries are an important resource to study bacterial metabolism and pathogenesis. The fitness analysis of mutants in the libraries under various growth conditions provides important clues to study the physiology and biogenesis of structural components of a bacterial cell. A transposon library in conjunction with next-generation sequencing techniques, collectively named transposon sequencing (Tn-seq), enables high-throughput genome profiling and synthetic lethality analysis. Tn-seq has also been used to identify essential genes and to study the mode of action of antibacterials. To construct a high-density transposon mutant library, an efficient delivery system for transposition in a model bacterium is essential. Here, I describe a detailed protocol for generating a high-density phage-based transposon mutant library in a Staphylococcus aureus strain, and this protocol is readily applicable to other S. aureus strains including USA300 and MW2.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  • Badarinarayana, V., Estep, P.W.3rd, Shendure, J., Edwards, J., Tavazoie, S., Lam, F., and Church, G.M. 2001. Selection analyses of insertional mutants using subgenic-resolution arrays. Nat. Biotechnol. 19, 1060–1065.

    Article  CAS  PubMed  Google Scholar 

  • Carver, T., Harris, S.R., Berriman, M., Parkhill, J., and McQuillan, J.A. 2012. Artemis: an integrated platform for visualization and analysis of high-throughput sequence-based experimental data. Bioinformatics 28, 464–469.

    Article  CAS  PubMed  Google Scholar 

  • Chao, M.C., Abel, S., Davis, B.M., and Waldor, M.K. 2016. The design and analysis of transposon insertion sequencing experiments. Nat. Rev. Microbiol. 14, 119–128.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Chao, M.C., Pritchard, J.R., Zhang, Y.J., Rubin, E.J., Livny, J., Davis, B.M., and Waldor, M.K. 2013. High-resolution definition of the Vibrio cholerae essential gene set with hidden Markov model-based analyses of transposon-insertion sequencing data. Nucleic Acids Res. 41, 9033–9048.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Cho, H. 2021. Transposon insertion site sequencing (TIS) of Pseudomonas aeruginosa. J. Microbiol. 59, 1067–1074.

    Article  CAS  PubMed  Google Scholar 

  • Coe, K.A., Lee, W., Stone, M.C., Komazin-Meredith, G., Meredith, T.C., Grad, Y.H., and Walker, S. 2019. Multi-strain Tn-Seq reveals common daptomycin resistance determinants in Staphylococcus aureus. PLoS Pathog. 15, e1007862.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Fey, P.D., Endres, J.L., Yajjala, V.K., Widhelm, T.J., Boissy, R.J., Bose, J.L., and Bayles, K.W. 2013. A genetic resource for rapid and comprehensive phenotype screening of nonessential Staphylococcus aureus genes. mBio 4, e00537–12.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Goodman, A.L., McNulty, N.P., Zhao, Y., Leip, D., Mitra, R.D., Lozupone, C.A., Knight, R., and Gordon, J.I. 2009. Identifying genetic determinants needed to establish a human gut symbiont in its habitat. Cell Host Microbe 6, 279–289.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Karash, S. and Yahr, T.L. 2022. Genome-wide identification of Pseudomonas aeruginosa genes important for desiccation tolerance on inanimate surfaces. mSystems 7, e0011422.

    Article  PubMed  Google Scholar 

  • Koo, B.M., Kritikos, G., Farelli, J.D., Todor, H., Tong, K., Kimsey, H., Wapinski, I., Galardini, M., Cabal, A., Peters, J.M., et al. 2017. Construction and analysis of two genome-scale deletion libraries for Bacillus subtilis. Cell Syst. 4, 291–305.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Lampe, D.J., Akerley, B.J., Rubin, E.J., Mekalanos, J.J., and Robertson, H.M. 1999. Hyperactive transposase mutants of the Himar1 mariner transposon. Proc. Natl. Acad. Sci. USA 96, 11428–11433.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Langridge, G.C., Phan, M.D., Turner, D.J., Perkins, T.T., Parts, L., Haase, J., Charles, I., Maskell, D.J., Peters, S.E., Dougan, G., et al. 2009. Simultaneous assay of every Salmonella Typhi gene using one million transposon mutants. Genome Res. 19, 2308–2316.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Lee, W., Do, T., Zhang, G., Kahne, D., Meredith, T.C., and Walker, S. 2018. Antibiotic combinations that enable one-step, targeted mutagenesis of chromosomal genes. ACS Infect. Dis. 4, 1007–1018.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Lee, W., VanderVen, B.C., Fahey, R.J., and Russell, D.G. 2013. Intracellular Mycobacterium tuberculosis exploits host-derived fatty acids to limit metabolic stress. J. Biol. Chem. 288, 6788–6800.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Murry, J.P., Sassetti, C.M., Lane, J.M., Xie, Z., and Rubin, E.J. 2008. Transposon site hybridization in Mycobacterium tuberculosis. In Osterman, A.L. and Gerdes, S.Y. (eds) Microbial Gene Essentiality: Protocols and Bioinformatics. Methods in Molecular Biology, vol. 416, pp. 45–59. Humana Press, Totowa, New Jersey, USA.

    Chapter  Google Scholar 

  • Nazarova, E.V., Montague, C.R., La, T., Wilburn, K.M., Sukumar, N., Lee, W., Caldwell, S., Russell, D.G., and VanderVen, B.C. 2017. Rv3723/LucA coordinates fatty acid and cholesterol uptake in Mycobacterium tuberculosis. eLife 6, e26969.

    Article  PubMed  PubMed Central  Google Scholar 

  • Pasquina, L., Santa Maria, J.P.Jr., McKay Wood, B., Moussa, S.H., Matano, L.M., Santiago, M., Martin, S.E.S., Lee, W., Meredith, T.C., and Walker, S. 2016. A synthetic lethal approach for compound and target identification in Staphylococcus aureus. Nat. Chem. Biol. 12, 40–45.

    Article  CAS  PubMed  Google Scholar 

  • Price, M.N., Wetmore, K.M., Waters, R.J., Callaghan, M., Ray, J., Liu, H., Kuehl, J.V., Melnyk, R.A., Lamson, J.S., Suh, Y., et al. 2018. Mutant phenotypes for thousands of bacterial genes of unknown function. Nature 557, 503–509.

    Article  CAS  PubMed  Google Scholar 

  • Reznikoff, W.S. 2008. Transposon Tn5. Annu. Rev. Genet. 42, 269–286.

    Article  CAS  PubMed  Google Scholar 

  • Rubin, E.J., Akerley, B.J., Novik, V.N., Lampe, D.J., Husson, R.N., and Mekalanos, J.J. 1999. In vivo transposition of mariner-based elements in enteric bacteria and mycobacteria. Proc. Natl. Acad. Sci. USA 96, 1645–1650.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Salama, N.R., Shepherd, B., and Falkow, S. 2004. Global transposon mutagenesis and essential gene analysis of Helicobacter pylori. J. Bacteriol. 186, 7926–7935.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Santa Maria, J.P.Jr., Sadaka, A., Moussa, S.H., Brown, S., Zhang, Y.J., Rubin, E.J., Gilmore, M.S., and Walker, S. 2014. Compound-gene interaction mapping reveals distinct roles for Staphylococcus aureus teichoic acids. Proc. Natl. Acad. Sci. USA 111, 12510–12515.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Santiago, M., Lee, W., Fayad, A.A., Coe, K.A., Rajagopal, M., Do, T., Hennessen, F., Srisuknimit, V., Müller, R., Meredith, T.C., et al. 2018. Genome-wide mutant profiling predicts the mechanism of a Lipid II binding antibiotic. Nat. Chem. Biol. 14, 601–608.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Santiago, M., Matano, L.M., Moussa, S.H., Gilmore, M.S., Walker, S., and Meredith, T.C. 2015. A new platform for ultra-high density Staphylococcus aureus transposon libraries. BMC Genomics 16, 252.

    Article  PubMed  PubMed Central  Google Scholar 

  • Sassetti, C.M., Boyd, D.H., and Rubin, E.J. 2001. Comprehensive identification of conditionally essential genes in mycobacteria. Proc. Natl. Acad. Sci. USA 98, 12712–12717.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Siegrist, M.S. and Rubin, E.J. 2009. Phage transposon mutagenesis. In Parish, T. and Brown, A. (eds) Mycobacteria Protocols. Methods in Molecular Biology, vol 465, pp. 311–323. Humana Press, Totowa, New Jersey, USA.

    Chapter  Google Scholar 

  • Stryjewski, M.E. and Corey, G.R. 2014. Methicillin-resistant Staphylococcus aureus: an evolving pathogen. Clin. Infect. Dis. 58, S10–S19.

    Article  CAS  PubMed  Google Scholar 

  • van Opijnen, T., Bodi, K.L., and Camilli, A. 2009. Tn-seq: high-throughput parallel sequencing for fitness and genetic interaction studies in microorganisms. Nat. Methods 6, 767–772.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • van Opijnen, T., Lazinski, D.W., and Camilli, A. 2014. Genome-wide fitness and genetic interactions determined by Tn-seq, a high-throughput massively parallel sequencing method for microorganisms. Curr. Protoc. Mol. Biol. 106, 7.16.1–7.16.24.

    Article  PubMed  Google Scholar 

  • Wang, H., Claveau, D., Vaillancourt, J.P., Roemer, T., and Meredith, T.C. 2011. High-frequency transposition for determining antibacterial mode of action. Nat. Chem. Biol. 7, 720–729.

    Article  CAS  PubMed  Google Scholar 

  • Zhang, Y.J., Ioerger, T.R., Huttenhower, C., Long, J.E., Sassetti, C.M., Sacchettini, J.C., and Rubin, E.J. 2012. Global assessment of genomic regions required for growth in Mycobacterium tuberculosis. PLoS Pathog. 8, e1002946.

    Article  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

We thank all Wonsik Lee’s lab members for discussion and comments. This work was supported by a grant from the National Research Foundation (NRF) of Korea, funded by the Korean government (MSIT) (grant numbers NRF-2020M3A9H5104234 and NRF-2020R1C1C1014695).

Author information

Authors and Affiliations

  1. Department of Pharmacy, School of Pharmacy, Sungkyunkwan University, Suwon, 16419, Republic of Korea

    Wonsik Lee

Authors
  1. Wonsik Lee
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Wonsik Lee.

Additional information

Conflict of Interest

The authors have no conflict of interest to report.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Lee, W. Construction of high-density transposon mutant library of Staphylococcus aureus using bacteriophage ϕ11. J Microbiol. 60, 1123–1129 (2022). https://doi.org/10.1007/s12275-022-2476-2

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12275-022-2476-2

Keywords

Search

Navigation