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 across the inner membrane. The periplasmic forms are transferred across the outer membrane by a machinery consisting of 12 xcp gene products. Although the type II secretion machinery is conserved among gram-negative bacteria, interactions between the secreted proteins and the machinery are specific. The lack of a selectable phenotype has hampered the development of genetic strategies for studying type II secretion. We report a novel strategy to identify rare events, such as those that allow heterologous secretion or identification of extragenic suppressors correcting xcp defects. This is based on 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.
Laboratoire d'Ingéniérie des Systémes Macromoléculaires, Marseille, France.
R
R
Voulhoux
A
A
Lazdunski
A
A
Filloux
D001426:Bacterial Proteins; D003087:Colicins / Q000737:chemistry / Q000235:genetics*; D003593:Cytoplasm / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D005020:Ethyl Methanesulfonate; D005822:Genetic Vectors; D015151:Immunoblotting; D008666:Metalloendopeptidases / Q000737:chemistry / Q000378:metabolism; D008954:Models, Biological; D016296:Mutagenesis; D009153:Mutagens; D010196:Pancreatic Elastase / Q000737:chemistry / Q000235:genetics; D010455:Peptides / Q000378:metabolism; D010641:Phenotype; D010957:Plasmids / Q000378:metabolism; D021382:Protein Sorting Signals; D017434:Protein Structure, Tertiary; D011550:Pseudomonas aeruginosa / Q000235:genetics* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics
D001426:Bacterial Proteins; D003087:Colicins; D009153:Mutagens; D010455:Peptides; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D005020:Ethyl Methanesulfonate; D010196:Pancreatic Elastase; D008666:Metalloendopeptidases; C043859:pseudolysin, Pseudomonas aeruginosa