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Legionella pp 145-157 | Cite as

Genetic Manipulation of Non-pneumophila Legionella: Protocols Developed for Legionella longbeachae

  • Elizabeth Korevaar
  • Chen Ai Khoo
  • Hayley J. NewtonEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1921)

Abstract

Current biomedical research into Legionnaires’ disease is dominated by studies of Legionella pneumophila, largely because this pathogen is responsible for approximately 90% of clinical disease worldwide. However, in certain geographical regions, infections with non-pneumophila species are responsible for a significant proportion of diagnosed Legionnaires’ disease. Understanding the pathogenesis of these non-pneumophila species of Legionella is an important step toward clinical intervention. The capacity to genetically manipulate these pathogens is essential in order to understand the genetic factors that contribute to infection and the environmental life cycle of these bacteria. The capacity to delete, mutate, and relocate genetic regions of interest allows molecular research into gene function and importance. In this chapter, methods are outlined to introduce plasmids into Legionella by electroporation. This technique is particularly useful as it is often the essential preliminary step to experiments that observe the behavior of the bacterium under altered conditions, for example, the transformation of bacteria with reporter plasmids to monitor Dot/Icm effector translocation. Electroporation is a well-established method for transformation of competent bacteria, and here specific protocols are provided, suiting a range of materials and conditions that have been successfully applied to L. longbeachae and L. dumoffii. Additionally, a homologous recombination approach to delete genetic regions of interest in L. longbeachae is outlined. The application of these techniques allows for identification of the genetic determinants of non-pneumophila Legionella virulence and for important comparative studies with other Legionella species.

Key words

Legionella longbeachae Legionella dumoffii Electroporation Transformation pSR47s Homologous recombination 

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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Elizabeth Korevaar
    • 1
  • Chen Ai Khoo
    • 1
  • Hayley J. Newton
    • 1
    Email author
  1. 1.Department of Microbiology and ImmunologyUniversity of Melbourne at the Peter Doherty Institute for Infection and ImmunityMelbourneAustralia

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