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Transposon sequencing (Tn-seq) has greatly accelerated the rate at which gene function can be profiled in microbial organisms. This technique has been applied to the study of the dental caries pathogen Streptococcus mutans where it has been used to generate large transposon mutant libraries. Coupled with high-throughput sequencing and bioinformatics tools, culture of these transposon mutant libraries has facilitated the identification of essential and conditional essential genes. In this chapter, we describe a procedure for performing Tn-seq studies in S. mutans that covers pooled transposon mutant construction, in vitro culture, and DNA library sequencing and data analysis., (© 2022. Springer Science+Business Media, LLC, part of Springer Nature.)

Generation of a random transposon mutant library is advantageous in Leptospira as site-directed mutagenesis remains a challenge, especially in pathogenic species. This procedure is typically completed by transformation of Leptospira with a Himar1 containing plasmid via conjugation with Escherichia coli as a donor cell. Here we describe the methodology to generate random transposon mutants in the saprophyte Leptospira biflexa via conjugation of plasmid pSW29T-TKS2 harbored in E. coli β2163. Determination of transposon insertion site by semi-random nested PCR will also be described. A similar methodology may be employed to generate Tn mutants of pathogenic Leptospira species.