Methods in Molecular Biology Volume 954, 2013, pp 183-195

Induction of Competence for Natural Transformation in Legionella pneumophila and Exploitation for Mutant Construction

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Abstract

Many Gram-positive and Gram-negative bacteria possess natural competence mechanisms for DNA ­capture and internalization that play an important role in diversifying adaptation of bacteria through horizontal gene transfer. Natural transformation and other mechanisms of horizontal gene transfer are dependent on DNA recombination. Natural competence can be exploited both for studying adaptation and horizontal gene transfer as well as for genetic engineering of a strain. We report here different approaches to measure competence on solid and in liquid media by using a reporter plasmid where GFP is fused to the comEA gene or by inducing competence and measuring transformability induced by DNA-damaging stress. Finally we describe a method where competence is induced through a combined temperature and aeration shift, which may be exploited for the construction of mutants in Legionella pneumophila. This approach seems to be less prone to the appearance of secondary mutations during mutant construction as compared to procedures using electroporation.