Counter-Selection Method for Markerless Allelic Exchange in Bordetella bronchiseptica Based on sacB Gene From...

Bordetella bronchiseptica is a gram-negative bacterium that causes respiratory tract infections. It is a natural pathogen of a wide variety of mammals, including some used as laboratory models. This makes B. bronchiseptica an ideal organism to study pathogen-host interactions in order to unveil molecular mechanisms behind pathogenic processes. Even though genetic engineering is an essential tool in this area, there are just a few reports about genome manipulation techniques in this organism. In this article we describe an allelic exchange protocol based on double crossover recombination facilitated by the Bacillus subtilis sacB gene that can be applied for partial or complete gene knockouts, single-nucleotide mutations, or even introduction of coding sequences for transcriptional fusions. In contrast to previously employed techniques, this protocol renders genetically manipulated chromosomes without foreign DNA and enables the construction of successive genome manipulation using the same vector backbone. The entire procedure has been developed for fast and reliable manipulations with a total duration of 2 weeks. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Setting up strains Basic Protocol 2: Homologous recombination (first crossing-over) Alternate Protocol: B. bronchiseptica electroporation Basic Protocol 3: Screening for sucrose-sensitive clones Basic Protocol 4: Homologous recombination (second crossing-over) Basic Protocol 5: PCR screening of putative marker-exchange mutants Support Protocol: Electrocompetent cell preparation.