Chapter 9
Transposon Sequencing in Leptospira interrogans
Kristel Lourdault and James Matsunaga
Abstract
Our limited understanding of the relationship of genotype to phenotype in the spirochete Leptospira
interrogans stems from the inefficiency of the genetic tools available to manipulate the pathogen. The
recent development of random transposon mutagenesis in L. interrogans has allowed the creation of large
libraries of mutants, permitting the identification of several genes involved in certain functions such as
virulence. However, the process of phenotypically screening individual mutants in the library remains timeand labor-intensive. Here, we describe a transposon sequencing technique (Tn-Seq), which combines
random transposon mutagenesis with high-throughput sequencing for screening L. interrogans mutants
more rapidly with fewer resources than traditional methods.
Key words Leptospira, High-throughput sequencing, Virulence factors, Transposon, Genomic
library, Fitness
1 Introduction
The contribution of most leptospiral genes to the characteristics of
Leptospira interrogans remains unknown due to the poor performance of the tools currently used to genetically manipulate the
spirochete [1]. The development of random transposon mutagenesis in L. interrogans a decade ago opened the door to the identification of gene function at a genome-wide scale [2]. However, the
process of individually screening mutants for altered phenotypes is
laborious, particularly for identification of virulence genes, which
requires a large number of animals.
To facilitate the identification of virulence factors, we recently
applied transposon sequencing (Tn-Seq) to L. interrogans [3–
6]. Tn-Seq, which combines random transposon mutagenesis
with high-throughput sequencing, is the perfect tool to screen
the fitness of a large number of mutants under a given condition.
Briefly, mutants are pooled (input pool) and grown under a condition of interest to study in vitro fitness. To examine the fitness of the
mutants in vivo, the pools are inoculated into animals and later
Nobuo Koizumi and Mathieu Picardeau (eds.), Leptospira spp.: Methods and Protocols, Methods in Molecular Biology, vol. 2134,
https://doi.org/10.1007/978-1-0716-0459-5_9, © Springer Science+Business Media, LLC, part of Springer Nature 2020
97
Transposon Sequencing in Leptospira interrogans
Kristel Lourdault and James Matsunaga
Abstract
Our limited understanding of the relationship of genotype to phenotype in the spirochete Leptospira
interrogans stems from the inefficiency of the genetic tools available to manipulate the pathogen. The
recent development of random transposon mutagenesis in L. interrogans has allowed the creation of large
libraries of mutants, permitting the identification of several genes involved in certain functions such as
virulence. However, the process of phenotypically screening individual mutants in the library remains timeand labor-intensive. Here, we describe a transposon sequencing technique (Tn-Seq), which combines
random transposon mutagenesis with high-throughput sequencing for screening L. interrogans mutants
more rapidly with fewer resources than traditional methods.
Key words Leptospira, High-throughput sequencing, Virulence factors, Transposon, Genomic
library, Fitness
1 Introduction
The contribution of most leptospiral genes to the characteristics of
Leptospira interrogans remains unknown due to the poor performance of the tools currently used to genetically manipulate the
spirochete [1]. The development of random transposon mutagenesis in L. interrogans a decade ago opened the door to the identification of gene function at a genome-wide scale [2]. However, the
process of individually screening mutants for altered phenotypes is
laborious, particularly for identification of virulence genes, which
requires a large number of animals.
To facilitate the identification of virulence factors, we recently
applied transposon sequencing (Tn-Seq) to L. interrogans [3–
6]. Tn-Seq, which combines random transposon mutagenesis
with high-throughput sequencing, is the perfect tool to screen
the fitness of a large number of mutants under a given condition.
Briefly, mutants are pooled (input pool) and grown under a condition of interest to study in vitro fitness. To examine the fitness of the
mutants in vivo, the pools are inoculated into animals and later
Nobuo Koizumi and Mathieu Picardeau (eds.), Leptospira spp.: Methods and Protocols, Methods in Molecular Biology, vol. 2134,
https://doi.org/10.1007/978-1-0716-0459-5_9, © Springer Science+Business Media, LLC, part of Springer Nature 2020
97