Legionella pp 183-195 | Cite as

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

Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 954)

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.

Key words

Legionella Natural competence Transformation Mutant construction Horizontal gene transfer 

Notes

Acknowledgments

X. Charpentier is supported by the young investigator program ATIP-Avenir from the CNRS and INSERM and by a grant from Sanofi. C.  Buchrieser receives support from the Institut Pasteur, the Centre national de la recherché scientifique (CNRS), and the Institut Carnot-Pasteur MI and from the ANR-10-PATH-004 project, in the frame of ERA-Net PathoGenoMics.

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Copyright information

© Springer Science+Business Media New York 2013

Authors and Affiliations

  1. 1.Biology of Intracellular BacteriaInstitut PasteurParisFrance
  2. 2.CNRS UMR 3525ParisFrance
  3. 3.Unité de Microbiologie, Adaptation et PathogénieVillerubanneFrance

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