Chapter 8
Creating a Library of Random Transposon Mutants
in Leptospira
Christopher J. Pappas, Hui Xu, and Md A. Motaleb
Abstract
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.
Key words Spirochete, Leptospira, Himar1, Transposon library, Mutagenesis, Conjugation, Nested
PCR
1 Introduction
The genus Leptospira belongs to the phylum Spirochaetes, which
consists of saprophytic species (e.g., L. biflexa), intermediate species (e.g., L. licerasiae), and pathogenic species (e.g.,
L. interrogans), of which the latter are primarily responsible for
the disease leptospirosis [1–3]. Leptospirosis is an emerging zoonotic disease of global importance, with over 1.7 million cases of
severe disease occurring annually [4]. To facilitate our understanding of the biology of Leptospira, such as virulence factors, host
adaptation mechanisms, or its physiology, effective genetic manipulation tools are required [1, 5–9].
Studies of Leptospira genes completed in the 1990s were primarily focused on functional complementation of Escherichia coli
mutants. This led to the identification and characterization of
Leptospira genes recA, rfb, asd, and trpE [10–13]. Later, electroporation and conjugation were both successfully applied to introduce
DNA (by suicide or replicative plasmids) into saprophytic and
pathogenic Leptospira species with selective antibiotic markers of
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_8, © Springer Science+Business Media, LLC, part of Springer Nature 2020
77
Creating a Library of Random Transposon Mutants
in Leptospira
Christopher J. Pappas, Hui Xu, and Md A. Motaleb
Abstract
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.
Key words Spirochete, Leptospira, Himar1, Transposon library, Mutagenesis, Conjugation, Nested
PCR
1 Introduction
The genus Leptospira belongs to the phylum Spirochaetes, which
consists of saprophytic species (e.g., L. biflexa), intermediate species (e.g., L. licerasiae), and pathogenic species (e.g.,
L. interrogans), of which the latter are primarily responsible for
the disease leptospirosis [1–3]. Leptospirosis is an emerging zoonotic disease of global importance, with over 1.7 million cases of
severe disease occurring annually [4]. To facilitate our understanding of the biology of Leptospira, such as virulence factors, host
adaptation mechanisms, or its physiology, effective genetic manipulation tools are required [1, 5–9].
Studies of Leptospira genes completed in the 1990s were primarily focused on functional complementation of Escherichia coli
mutants. This led to the identification and characterization of
Leptospira genes recA, rfb, asd, and trpE [10–13]. Later, electroporation and conjugation were both successfully applied to introduce
DNA (by suicide or replicative plasmids) into saprophytic and
pathogenic Leptospira species with selective antibiotic markers of
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_8, © Springer Science+Business Media, LLC, part of Springer Nature 2020
77