Transposon Sequencing in Leptospira interrogans.

The development of random transposon mutagenesis in L. interrogans has enabled the creation of large libraries of mutants, facilitating the identification of genes involved in key phenotypes such as virulence. However, phenotypic screening of individual mutants from these libraries remains time-consuming and labor-intensive. Here, we describe a transposon-sequencing protocol (Tn-Seq) that combines random transposon mutagenesis with high-throughput sequencing to simultaneously assess fitness changes across a large collection of L. interrogans mutants under defined conditions. Inoculating individual animals with a pooled set of mutants enables rapid identification of candidate virulence genes based on reduced fitness. This approach significantly reduces time and resource requirements compared with traditional screening techniques.