Advertisement

High-Throughput Insertion Tracking by Deep Sequencing for the Analysis of Bacterial Pathogens

  • Sandy M. S. Wong
  • Jeffrey D. Gawronski
  • David Lapointe
  • Brian J. AkerleyEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 733)

Abstract

Whole-genome techniques toward identification of microbial genes required for their survival and growth during infection have been useful for studies of bacterial pathogenesis. The advent of massively parallel sequencing platforms has created the opportunity to markedly accelerate such genome-scale analyses and achieve unprecedented sensitivity, resolution, and quantification. This chapter provides an overview of a genome-scale methodology that combines high-density transposon mutagenesis with a mariner transposon and deep sequencing to identify genes that are needed for survival in experimental models of pathogenesis. Application of this approach to a model pathogen, Haemophilus influenzae, has provided a comprehensive analysis of the relative role of each gene of this human respiratory pathogen in a murine pulmonary model. The method is readily adaptable to nearly any organism amenable to transposon mutagenesis.

Key words

HITS Himar1 Mariner Transposon mutagenesis Bacteria Haemophilus influenzae Genetic footprinting Deep sequencing 

Notes

Acknowledgments

This work was supported in part by grant from the NIH (AI49437) to B.J.A. Initial development of the method was conducted in collaboration with Georgia Giannoukos and Doyle Ward of the Broad Institute.

References

  1. 1.
    Hensel, M., Shea, J. E., Gleeson, C., Jones, M. D., Dalton, E., and Holden, D. W. (1995) Simultaneous identification of bacterial virulence genes by negative selection, Science 269, 400  –  403.PubMedCrossRefGoogle Scholar
  2. 2.
    Sassetti, C. M., Boyd, D. H., and Rubin, E. J. (2001) Comprehensive identification of conditionally essential genes in mycobacteria, Proc Natl Acad Sci U S A 98, 12712–12717.PubMedCrossRefGoogle Scholar
  3. 3.
    Salama, N. R., Shepherd, B., and Falkow, S. (2004) Global transposon mutagenesis and essential gene analysis of Helicobacter pylori, J Bacteriol 186, 7926  –7935.PubMedCrossRefGoogle Scholar
  4. 4.
    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.PubMedCrossRefGoogle Scholar
  5. 5.
    Gawronski, J. D., Wong, S. M., Giannoukos, G., Ward, D. V., and Akerley, B. J. (2009) Tracking insertion mutants within libraries by deep sequencing and a genome-wide screen for Haemophilus genes required in the lung, Proc Natl Acad Sci U S A 106, 16422–16427.PubMedCrossRefGoogle Scholar
  6. 6.
    Vila-Corcoles, A., Ochoa-Gondar, O., Rodriguez-Blanco, T., Raga-Luria, X., and Gomez-Bertomeu, F. (2009) Epidemiology of community-acquired pneumonia in older adults: a population-based study, Respir Med 103, 309–316.PubMedCrossRefGoogle Scholar
  7. 7.
    Sethi, S., and Murphy, T. F. (2001) Bacterial infection in chronic obstructive pulmonary disease in 2000: a state-of-the-art review, Clin Microbiol Rev 14, 336  –363.PubMedCrossRefGoogle Scholar
  8. 8.
    Akerley, B. J., Rubin, E. J., Novick, V. L., Amaya, K., Judson, N., and Mekalanos, J. J. (2002) A genome-scale analysis for identification of genes required for growth or survival of Haemophilus influenzae, Proc Natl Acad Sci U S A 99, 966  –971.PubMedCrossRefGoogle Scholar
  9. 9.
    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 U S A 96, 1645  –1650.PubMedCrossRefGoogle Scholar
  10. 10.
    Lampe, D. J., Grant, T. E., and Robertson, H. M. (1998) Factors affecting transposition of the Himar1 mariner transposon in vitro, Genetics 149, 179–187.PubMedGoogle Scholar
  11. 11.
    Wong, S. M., and Akerley, B. J. (2008) Identification and analysis of essential genes in Haemophilus influenzae, Methods Mol Biol 416, 27–  44.PubMedCrossRefGoogle Scholar
  12. 12.
    Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A., and Struhl, K. (1995), John Wiley and Sons, Inc.Google Scholar
  13. 13.
    Quail, M. A., Kozarewa, I., Smith, F., Scally, A., Stephens, P. J., Durbin, R., Swerdlow, H., and Turner, D. J. (2008) A large genome center’s improvements to the Illumina sequencing system, Nat Methods 5, 1005  –1010.PubMedCrossRefGoogle Scholar
  14. 14.
    Bentley, D. R., Balasubramanian, S., Swerdlow, H. P., Smith, G. P., Milton, J., Brown, C. G., et al. (2008) Accurate whole human genome sequencing using reversible terminator chemistry, Nature 456, 53  –  59.PubMedCrossRefGoogle Scholar
  15. 15.
    Li, R., Li, Y., Kristiansen, K., and Wang, J. (2008) SOAP: short oligonucleotide alignment program, Bioinformatics 24, 713  –714.PubMedCrossRefGoogle Scholar
  16. 16.
    Singh, I. R., Crowley, R. A., and Brown, P. O. (1997) High-resolution functional mapping of a cloned gene by genetic footprinting, Proc Natl Acad Sci U S A 94, 1304  –1309.PubMedCrossRefGoogle Scholar
  17. 17.
    Akerley, B. J., Rubin, E. J., Camilli, A., Lampe, D. J., Robertson, H. M., and Mekalanos, J. J. (1998) Systematic identification of essential genes by in vitro mariner mutagenesis, Proc Natl Acad Sci U S A 95, 8927–  8932.PubMedCrossRefGoogle Scholar
  18. 18.
    Hood, D. W., Deadman, M. E., Allen, T., Masoud, H., Martin, A., Brisson, J. R., Fleischmann, R., Venter, J. C., Richards, J. C., and Moxon, E. R. (1996) Use of the complete genome sequence information of Haemophilus influenzae strain Rd to investigate lipopolysaccharide biosynthesis, Mol Microbiol 22, 951–  965.PubMedCrossRefGoogle Scholar
  19. 19.
    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.PubMedCrossRefGoogle Scholar
  20. 20.
    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., Wain, J., Parkhill, J., and Turner, A. K. (2009) Simultaneous assay of every Salmonella Typhi gene using one million transposon mutants, Genome Res 19, 2308  –2316.PubMedCrossRefGoogle Scholar
  21. 21.
    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.PubMedCrossRefGoogle Scholar
  22. 22.
    Skerra, A. (1992) Phosphorothioate primers improve the amplification of DNA sequences by DNA polymerases with proofreading activity, Nucleic Acids Res 20, 3551–  3554.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  • Sandy M. S. Wong
  • Jeffrey D. Gawronski
  • David Lapointe
  • Brian J. Akerley
    • 1
    Email author
  1. 1.Department of Molecular Genetics and MicrobiologyUniversity of Massachusetts Medical SchoolWorcesterUSA

Personalised recommendations