Screening for membrane-permeabilizing mutants of the poliovirus protein 3AB.
Synthesis of the poliovirus polypeptide 3AB in bacterial cells results in an increase in membrane permeability. The alterations observed resemble those elicited by bacteriophage lytic proteins, which are presumed to cause pore formation in biological membranes. This property has been exploited in the development of an in vivo screening system that allows morphological differentiation of Escherichia coli clones expressing either wild-type 3AB or variant 3AB proteins lacking the ability to permeabilize bacteria. Expression of the wild-type 3AB gene in the presence of a chromogenic beta-galactosidase substrate causes E. coli clones to stain dark blue. In contrast, bacterial mutants that synthesize 3AB proteins with alterations in the hydrophobic domain lack pore-forming activity and stain to a light blue colour, allowing differentiation from wild-type clones. This phenotypic property correlates with the rate of entry of the beta-galactosidase substrate into the bacteria. The method developed here was used to screen more than 8000 E. coli clones after random PCR mutagenesis of the poliovirus 3AB gene. Our results show the existence of three different domains involved in the permeabilizing activity of 3AB protein. Twenty individual amino acid substitutions were identified in clones that showed the mutant phenotype and such bacteria displayed different reduced levels of permeability towards ONPG, hygromycin B, lysozyme and uridine. The procedure reported here may be of general interest to understand structure-function relationships in other eukaryotic proteins known to form pores.
Lama
J
J
Centro de Biología Molecular 'Severo Ochoa' (CSIC-UAM), Universidad Autónoma de Madrid, Canto Blanco, Spain. Lama@AXP1.salk.edu
Carrasco
L
L
D001665:Binding Sites; D002463:Cell Membrane Permeability; D004926:Escherichia coli; D016296:Mutagenesis; D017955:Poliovirus / Q000235:genetics / Q000378:metabolism*; D019105:Random Amplified Polymorphic DNA Technique; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D014758:Viral Core Proteins / Q000235:genetics / Q000378:metabolism*
D011993:Recombinant Fusion Proteins; D014758:Viral Core Proteins