Abstract
In comparison to other bacterial species, genetics of leptospires are in their infancy. Recently, we developed a system for random transposon mutagenesis in the saprophyte Leptospira biflexa and then applied this approach to the pathogen L. interrogans. Thousands of random mutants can be readily obtained in L. biflexa by random insertion of Himar1 in the genome, thereby generating extensive libraries of mutants that could be screened for phenotypes affecting diverse aspects of the biology of the bacterium. This system should be particularly useful for the identification of new genes of unknown function in Leptospira spp. This chapter describes a procedure for transposition in L. biflexa via conjugation of a plasmid delivering Himar1, isolation of mutants, and mapping of the insertion sites on the chromosome.
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References
Louvel, H., and Picardeau, M. (2007) Genetic Manipulation of Leptospira biflexa, J. Wiley and Sons, Hoboken, NJ
Picardeau, M., et al. (2008) Genome sequence of the saprophyte Leptospira biflexa provides insights into the evolution of Leptospira and the pathogenesis of leptospirosis, PLoS ONE 3, e1607
Louvel, H., Saint Girons, I., and Picardeau, M. (2005) Isolation and characterization of FecA- and FeoB-mediated iron acquisition systems of the spirochete Leptospira biflexa by random insertional mutagenesis, J. Bacteriol. 187, 3249–3254
Picardeau, M. (2008) Conjugative transfer between Escherichia coli and Leptospira spp. as a new genetic tool, Appl. Environ. Microbiol. 74, 319–322
Louvel, H., et al. (2006) Comparative and functional genomic analyses of iron transport and regulation in Leptospira spp., J. Bacteriol. 188, 7893–7904
Ellinghausen, H. C., and McCullough, W. G. (1965) Nutrition of Leptospira pomona and growth of 13 other serotypes: fractionation of oleic albumin complex and a medium of bovine albumin and polysorbate 80, Am. J. Vet. Res. 26, 45–51
Johnson, R. C., and Harris, V. G. (1967) Differentiation of pathogenic and saprophytic leptospires, J. Bacteriol. 94, 27–31
Vallenet, D., et al. (2009) MicroScope: a platform for microbial genome annotation and comparative genomics, Database (Oxford) bap021
Poggi, D., et al. (2010) Antibiotic resistance markers for genetic manipulations of Leptospira spp., Appl. Environ. Microbiol. 76, 4882–4885
Murray, G. L., et al. (2009) Genome-wide transposon mutagenesis in pathogenic Leptospira spp., Infect. Immun. 77, 810–816
Lampe, D. J., et al. (1999) Hyperactive transposase mutants of the Himar1 mariner transposon, Proc Natl Acad Sci U S A. 96, 11428–11433
Acknowledgments
We thank Gerald Murray (Monash University, Australia) for sharing the protocol of semirandom PCR.
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Slamti, L., Picardeau, M. (2012). Construction of a Library of Random Mutants in the Spirochete Leptospira Biflexa Using a Mariner Transposon. In: Bigot, Y. (eds) Mobile Genetic Elements. Methods in Molecular Biology, vol 859. Humana Press. https://doi.org/10.1007/978-1-61779-603-6_9
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DOI: https://doi.org/10.1007/978-1-61779-603-6_9
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