The genus Leptospira belongs to the order Spirochaetales and is composed of both saprophytic and pathogenic members, such as Leptospira biflexa and L. interrogans, respectively. A major factor contributing to our ignorance of spirochetal biology is the lack of methods for genetic analysis of these organisms. We have developed a system for transposon mutagenesis of L. biflexa using a mariner transposon, Himar1. This mutagenesis approach yields a randomly distributed set of insertion mutations throughout the genome, which can be screened for specific phenotypes. An analysis of transposon mutants has allowed the identification of genes required for diverse biological activities such as amino acid biosynthesis and metal transport. The development of numerous genetic tools for saprophytic species of Leptospira enables the use of these bacteria such as L. biflexa as a model bacterium We therefore sequenced the genome of the saprophyte L. biflexa consisting of a 3.6-megabase large chromosome and a 278-kilobase small chromosome. Comparative genomics, in combination with gene inactivation, give us significant functional information on iron homeostasis in Leptospira.