Construction of a library of random mutants in the spirochete Leptospira biflexa using a mariner transposon

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.