Isolation and Characterization of FecA- and FeoB-Mediated Iron Acquisition Systems of the SpirocheteLeptospira biflexaby Random Insertional Mutagenesis

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Abstract
The specific mechanisms by whichLeptospiraspp. acquire iron from their ecological niches are unknown. A major factor contributing to our ignorance of spirochetal biology is the lack of methods for genetic analysis of these organisms. In this study, we have developed a system for random transposon mutagenesis ofLeptospira biflexausing amarinertransposon,Himar1. To demonstrate the validity ofHimar1in vivo transposon mutagenesis inL. biflexa, a screen of mutants for clones impaired in amino acid biosynthesis was first performed, enabling the identification of tryptophan and glutamate auxotrophs. To investigate iron transporters, 2,000L. biflexatransposon mutants were screened onto media with and without hemin, thus allowing the identification of five hemin-requiring mutants, and the putative genes responsible for this phenotype were identified. Three mutants had distinct insertions in a gene encoding a protein which shares homology with the TonB-dependent receptor FecA, involved in ferric citrate transport. We also identified two mutants with aHimar1insertion into afeoB-like gene, the product of which is required for ferrous iron uptake in many bacterial organisms. Interestingly, the growth inhibition exhibited by thefecAandfeoBmutants was relieved by deferoxamine, suggesting the presence of a ferric hydroxamate transporter. These results confirm the importance of iron for the growth ofLeptospiraand its ability to use multiple iron sources.