title: Generation and use of site-directed chromosomal cyaA' translational fusions in Salmonella enterica.
abstract: CyaA from Bordetella pertussis is a calmodulin-dependent adenylate cyclase. Fusions to the catalytic domain of CyaA (CyaA') are useful tools to detect translocation of type III secretion system effectors from gram-negative pathogens like Salmonella enterica. These fusions are usually generated using plasmids with strong promoters. Here, we describe a protocol to insert the CyaA'-encoding sequence in a specific site in the bacterial chromosome in order to get a monocopy fusion whose expression is driven by the native promoter. We also describe the procedure to detect translocation of a CyaA' fusion into mammalian cells.
authors.affiliation: Departamento de Genética, Facultad de Biología, Universidad de Sevilla, Avda Reina Mercedes, 6, 41012, Sevilla, Spain, framos@us.es.
authors.forename: Francisco
authors.initials: F
authors.lastname: Ramos-Morales
authors.forename: Elena
authors.initials: E
authors.lastname: Cardenal-Muñoz
authors.forename: Mar
authors.initials: M
authors.lastname: Cordero-Alba
authors.forename: Fernando
authors.initials: F
authors.lastname: Baisón-Olmo
mesh_terms: D000262:Adenylyl Cyclases / Q000235:genetics* / Q000378:metabolism*; D000818:Animals; D019677:Artificial Gene Fusion / Q000379:methods*; D010582:Bacteriophage lambda / Q000235:genetics; D002876:Chromosomes, Bacterial / Q000235:genetics*; D006367:HeLa Cells; D006801:Humans; D051379:Mice; D014176:Protein Biosynthesis; D019779:Salmonella enterica / Q000201:enzymology* / Q000235:genetics*
chemical_list: D000262:Adenylyl Cyclases