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

Elizabeth Korevaar, Chen Ai Khoo, Hayley J. Newton

Research output: Chapter in Book/Report/Conference proceedingChapter (Book)Other

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.
Original languageEnglish
Title of host publicationLegionella
Subtitle of host publicationMethods and Protocols
EditorsCarmen Buchrieser, Hubert Hilbi
Place of PublicationNew York NY USA
Chapter9
Pages145-157
Number of pages13
Edition2e
ISBN (Electronic)9781493990481
DOIs
Publication statusPublished - 2019
Externally publishedYes

Publication series

NameMethods in Molecular Biology
PublisherSpringer Science+Business Media, LLC, part of Springer Nature
Volume1921
ISSN (Print)1064-3745
ISSN (Electronic)1940-6029

Keywords

  • Legionella longbeachae
  • Legionella dumoffii
  • Electroporation
  • Transformation
  • pSR47s
  • Homologous recombination

Cite this