Generating a Collection of Insertion Mutations in the Staphylococcus aureus Genome Using bursa aurealis

  • Taeok Bae
  • Elizabeth M. Glass
  • Olaf Schneewind
  • Dominique Missiakas
Part of the Methods in Molecular Biology™ book series (MIMB, volume 416)


Staphylococcus aureus is the leading cause of wound and hospital-acquired infections. The emergence of strains with resistance to all antibiotics has created a serious public health problem. Transposon-based mutagenesis can be used to generate libraries of mutants and to query genomes for factors involved in nonessential pathways, such as virulence and antibiotic resistance. Ideally, such studies should employ defined and complete sets of isogenic mutants and should be conducted so as to permit acquisition and comparison of the complete data sets. Such systematic knowledge can reveal entire pathways and can be exploited for the rational design of therapies. The mariner-based transposon, bursa aurealis, can be used to generate random libraries of mutants in laboratory strains and clinical isolates of S. aureus. This chapter describes a procedure for isolating mutants and mapping the insertion sites on the chromosome.

Key Words

bursa aurealis Himar 1 transposase mariner mutagenesis Staphylococcus aureus transposon library temperature sensitive 


  1. 1.
    Fitzgerald, J. R., Sturdevant, D. E., Mackie, S. M., Gill, S. R., and Musser, J. M. (2001) Evolutionary genomics of Staphylococcus aureus: insights into the origin of methicillin-resistant strains and the toxic shock syndrome epidemic. Proc. Natl. Acad. Set U.S.A. 98, 8821–8826.CrossRefGoogle Scholar
  2. 2.
    Baba, T., Takeuchi, F., Kuroda, M., Yuzawa, H., Aoki, K., Oguchi, A., et al. (2002) Genome and virulence determinants of high virulence community-acquired MRSA. Lancet 359, 1819–1827.CrossRefPubMedGoogle Scholar
  3. 3.
    Kuroda, M., Ohta, T., Uchiyama, I., Baba, T., Yuzawa, H., Kobayashi, L., et al. (2001) Whole genome sequencing of methicillin-resistant Staphylococcus aureus. Lancet 357, 1225–1240.CrossRefPubMedGoogle Scholar
  4. 4.
    Archer, G. L. (1998) Staphylococcus aureus: a well-armed pathogen. Clin. Infect. Dis. 26, 1179–1181.CrossRefPubMedGoogle Scholar
  5. 5.
    Ní Eidhin, D., Perkins, S., Francois, P., Vaudaux, P., Höök, M., and Foster, T. J. (1998) Clumping factor B (ClfB), a new surface-located fibrinogen-binding adhesin of Staphylococcus aureus. Mol. Microbiol. 30, 245–257.CrossRefPubMedGoogle Scholar
  6. 6.
    Ton-That, H., Mazmanian, S. K., and Schneewind, O. (2001) The role of sortase enzymes in Gram-positive bacteria. Trends Microbiol. 9, 101–102.CrossRefGoogle Scholar
  7. 7.
    Ruvkun, G. B., and Ausubel, F. M. (1981) A general method for site-directed mutagenesis in prokaryotes. Nature 289, 85–88.CrossRefPubMedGoogle Scholar
  8. 8.
    Foster, T. J. (1998) Molecular genetic analysis of staphylococcal virulence. Methods Microbiol. 27, 432–454.Google Scholar
  9. 9.
    Bae, T., and Schneewind, O. (2006) Allelic replacement in Staphylococcus aureus with inducible counter-selection. Plasmid 55, 58–63.CrossRefPubMedGoogle Scholar
  10. 10.
    Schwan, W. R., Coulter, S. N., Ng, E. Y., Lnghorne, M. H., Ritchie, H. D., Brody, L. L., et al. (1998) Identification and characterization of the PutP proline permease that contributes to in vivo survival of Staphylococcus aureus in animal models. Infect. Immun. 66, 567–572.Google Scholar
  11. 11.
    Mei, J. M., Nourbakhsh, F., Ford, C. W., and Holden, D. W. (1997) Identification of Staphylococcus aureus virulence genes in a murine model of bacteraemia using signature-tagged mutagenesis. Mol. Microbiol. 26, 399–407.CrossRefPubMedGoogle Scholar
  12. 12.
    Bae, T., Banger, A. K., Wallace, A., Glass, E. M., Aslund, F., Schneewind, O., and Missiakas, D. M. (2004) Staphylococcus aureus virulence genes identified by bursa aurealis mutagenesis and nematode killing. Proc. Natl. Acad. Sci. U.S.A. 101, 12312–12317.CrossRefPubMedGoogle Scholar
  13. 13.
    Ji, Y., Zhang, B., Van, S. F., Horn, W. P., Woodnutt, G., Burnham, M. K., and Rosenberg, M. (2001) Identification of critical staphylococcal genes using conditional phenotypes generated by antisense RNA. Science 293, 2266–2269.CrossRefPubMedGoogle Scholar
  14. 14.
    Forsyth, R. A., Haselbeck, R. J., Gohlsen, K. L., Yamamoto, R. T., Xu, H., Trawick, J. D., et al. (2002) A genome-wide strategy for the identification of essential genes in Staphylococcus aureus. Mol. Microbiol. 43, 1387–1400.CrossRefPubMedGoogle Scholar
  15. 15.
    Robertson, H. M., and Lampe, D. J. (1995) Recent horizontal transfer of a mariner transposable element among and between Diptera and Neuroptera. Mol. Biol. Evol. 12, 850–862.PubMedGoogle Scholar
  16. 16.
    Lampe, D. J., Churchill, M. E., and Robertson, H. M. (1996) A purified mariner transposase is sufficient to mediate transposition in vitro. EMBO J. 15, 5470–5479.PubMedGoogle Scholar
  17. 17.
    Trieu-Cuot, P., Poyart-Salmeron, C., Carlier, C., and Courvalin, P. (1990) Nucleotide sequence of the erythromycin resistance gene of the conjugative transposon Tn1545. Nucl. Acids Res. 18, 3660.CrossRefPubMedGoogle Scholar
  18. 18.
    Fitzgerald, S. N., and Foster, T. J. (2000) Molecular analysis of the tagF gene, encoding CDP-glycerol:poly(gycerophosphate) glycero-phosphotransferase of Staphylococcus epidermidis ATCC14990. J. Bacteriol. 182, 1046–1052.CrossRefPubMedGoogle Scholar
  19. 19.
    Villafane, R., Bechhofer, D. H., Narayanan, C. S., and Dubnau, D. (1987) Replication control genes of plasmid pE194. J. Bacteriol. 169, 4822–4829.PubMedGoogle Scholar
  20. 20.
    Iordanescu, S. (1975) Recombinant plasmid obtained from two different, compatible staphylococcal plasmids. J. Bacteriol. 124, 597–601.PubMedGoogle Scholar
  21. 21.
    Peschel, A., Ottenwalder, B., and Gotz, F. (1996) Inducible production and cellular location of the epidermin biosynthetic enzyme EpiB using an improved staphylococcal expression system. FEMS Microbiol. Lett. 137, 279–284.CrossRefPubMedGoogle Scholar
  22. 22.
    Janzon, L., and Arvidson, S. (1990) The role of the delta-lysin gene (hld) in the regulation of virulence genes by the accessory gene regulator (agr) in Staphylococcus aureus. EMBO J. 9, 1391–1399.PubMedGoogle Scholar
  23. 23.
    Kullik, I., Giachino, P., and Fuchs, T. (1998) Deletion of the alternative sigma factor sigmaB in Staphylococcus aureus reveals its function as a global regulator of virulence genes. J. Bacteriol. 180, 4814–4820.PubMedGoogle Scholar
  24. 24.
    Melton, D. A., Krieg, P. A., Rebagliati, M. R., Maniatis, T., Zinn, K., and Green, M. R. (1984) Efficient in vitro synthesis of biologically active RNA and RNA hybridization probes from plasmids containing a bacteriophage SP6 promoter. Nucleic Acids Res. 12, 7035–7056.CrossRefPubMedGoogle Scholar
  25. 25.
    Novick, R. P., Edelman, I., and Lofdahl, S. (1986) Small Staphylococcus aureus plasmids are transduced as linear multimers that are formed and resolved by replicative processes. J. Mol. Biol. 192, 209–220.CrossRefPubMedGoogle Scholar
  26. 26.
    Iordanescu, S., Surdeanu, M., Della Latta, P., and Novick, R. (1978) Incompatibility and molecular relationships between small Staphylococcal plasmids carrying the same resistance marker. Plasmid 1, 468–479.CrossRefPubMedGoogle Scholar
  27. 27.
    Duthie, E. S., and Lorenz, L. L. (1952) Staphylococcal coagulase: mode of action and antigenicity. J. Gen. Microbiol. 6, 95–107.PubMedGoogle Scholar
  28. 28.
    Peng, H. L., Novick, R. P., Kreiswirth, B., Kornblum, J., and Schlievert, P. (1988) Cloning, characterization, and sequencing of an accessory gene regulator (agr) in Staphylococcus aureus. J. Bacteriol. 170, 4365–4372.Google Scholar
  29. 29.
    Waldron, D. E., and Lindsay, J. A. (2006) Sau1: a novel lineage-specific type I restriction-modification system that blocks horizontal gene transfer into Staphylococcus aureus and between S. aureus isolates of different lineages. J. Bacteriol. 188, 5578–5585.CrossRefPubMedGoogle Scholar
  30. 30.
    Altschul, S. F., Gish, W., Miller, W., Myers, E. W., and Lipman, D. J. (1990) Basic local alignment search tool. J. Mol. Biol. 215, 403–410.PubMedGoogle Scholar
  31. 31.
    Kobayashi, K., Ehrlich, S. D., Albertini, A., Amati, G., Andersen, K. K., Arnaud, M., et al. (2003) Essential Bacillus subtilis genes. Proc. Natl. Acad. Sci. U.S.A. 100, 4678–4683.CrossRefPubMedGoogle Scholar
  32. 32.
    Tam, C., Glass, E. M., Anderson, D. M., and Missiakas, D. (2006) Transposon mutagenesis of Bacillus anthracis strain Sterne using Bursa aurealis. Plasmid 56, 74–77.CrossRefPubMedGoogle Scholar

Copyright information

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

Authors and Affiliations

  • Taeok Bae
    • 1
    • 2
  • Elizabeth M. Glass
    • 3
  • Olaf Schneewind
    • 1
  • Dominique Missiakas
    • 1
  1. 1.Department of MicrobiologyThe University of ChicagoChicago
  2. 2.Indiana University NorthwestCary
  3. 3.Argonne National LaboratoryMathematics and Computer Sciences DivisionArgonne

Personalised recommendations