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 specifi c 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.