Colicin A hybrids: a genetic tool for selection of type II secretion-proficient Pseudomonas strains

The Gram-negative bacterium Pseudomonas aeruginosa secretes the majority of its extracellular proteins by the type II secretion mechanism, a two-step process initiated by translocation of signal peptide-bearing exoproteins ac ross the inner membrane. The periplasmic forms are transferred across the o uter membrane by a machinery consisting of 12 rep gene products. Although t he type II secretion machinery is conserved among Gram-negative bacteria, i nteractions between the secreted proteins and the machinery are specific. T he lack of a selectable phenotype has hampered the development of genetic s trategies for studying type II secretion. We report a novel strategy to ide ntify rare events, such as those that allow heterologous secretion or ident ification of extragenic suppressors correcting xcp defects. This is based o n creating a host-vector system where the non-secretory phenotype is lethal . The original tool we designed is a hybrid protein containing elastase and the pore-forming domain of colicin A.