Interaction of the globular domains of pIII protein of filamentous bacteriophage fd with the F-pilus of Escherichia coli

Gene 3 protein (pIII), a minor coat protein at one end of the filamentous bacteriophage fd, is involved in initiating the infection by the virus of Escherichia coli cells that display an F-pilus. infection is thought to start with the adsorption of the D2 domain of pill to the tip of the pilus, retraction of the pilus, and penetration of the E. coli cell membrane mediated by an interaction between the D1 domain of pIII and the Tol protein complex in the membrane. A subgene encoding the pIII-D1D2 di-domain was created, and the subgene was successfully overexpressed in E. coli cells. Domains D1 and D2 were separated after limited proteolysis or a modified pIII-D1D2 (designated pIII-D1D2.trp) into which two tryptic cleavage sites were introduced at appropriate points. The purified pIII-D1D2 di-domain and pIII-D2 domain were able to bind to the F-pilus, competing with the wild-type pIII and delaying infection by the intact filamentous phage. The pIII-D1 domain was unable to bind to the F-pilus by this criterion. This provides conclusive evidence that the pIII-D2 domain is responsible for the adsorption to the tip of the F-pilus and can achieve this in the absence of domain D1, opening the way to identifying the molecular basis of the interaction of pIII-D2 with the pilus. (C) 1999 Academic Press.