The ESX-5 system from Mycobacterium xenopi was expressed in Mycobacterium smegmatis on a constituatively expressed plasmid. This expression plasmid, pMX-Mxen-ESX-5 enocodes the seceretion complex and 3 secreted substrate pairs : EsxM+EsxN; PE19.1+PPE26.1 ; and PE19.2+PPE26.2. Several mutants in the secretion apparatus were generated and the secretion of the plasmid encoded substrates monitored.