Transposon Mutagenesis of the Lyme Disease Agent Borrelia burgdorferi - Bacterial Pathogenesis: Methods and Protocols - page 82

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Transposon Mutagenesis of the Lyme Disease Agent
Borrelia burgdorferi
Philip E. Stewart and Patricia A. Rosa
Summary
Borrelia burgdorferi , the causative agent of Lyme disease, is an obligate parasite
that cycles between vertebrate hosts and tick vectors. Attempts to understand the genetic
factors that allow B. burgdorferi to sense, adapt to, and survive in different environments
have been limited by a relatively low transformation rate. Here, we describe a mariner -
based transposon system that achieves saturating levels of random mutagenesis in B.
burgdorferi . In comparison with allelic exchange, which targets a single locus, transposon
mutagenesis can create libraries of mutants encompassing disruptions of all genes. Suitably
designed screens or selections of such a library permit the recovery of mutants exhibiting
a desired phenotype. The system described here allows rapid identification of the genetic
locus responsible for the mutant phenotype. With appropriate modifications, this mariner -
based transposon can be adapted to other spirochetes and bacteria with inefficient genetic
transformation methods.
Key Words: Borrelia burgdorferi ; spirochete; DNA transposable element; Himar1 ;
insertional mutagenesis.
1. Introduction
Transposons are DNA elements with the ability to move, or transpose, to
new locations within a genome. Several modified transposon systems have
proven useful as genetic tools for insertional inactivation of genes, identifying
conditionally regulated genes, and genome sequencing (reviewed in ref. 1 ).
These techniques have identified microbial genes essential for growth and
virulence, as well as loci affecting physiology and morphology (2,3,4,5,6,7,8,9) .