Identification of the Receptor-Binding Regions of pb5 Proteins of Bacteriophages T5 and BF23

Abstract

The receptor-binding protein pb5T5of bacteriophage T5, when expressed from theoadgene cloned in pVK88 under the control of the phage T7 promoter/polymerase system, has been shown to bind to its FhuA receptor on the surface ofE. coli,where it blocks FhuA for subsequent adsorption of T5 (Mondigleret al., FEMS Microbiol. Lett.,130, 293–300, 1995). In the present study the blocking assay has been applied to analyze the effects of several mutations withinoadon the FhuA-binding properties of corresponding pb5 derivatives. Three classes of mutations were tested: (i)oaddeletion derivatives, (ii) theoadmutation known to interfere with FhuA-binding of T5 (Heller and Bryniok,J. Virol.,49, 20–25, 1984), and (iii) linker-insertion mutations at a site very close to theoadmutation. Of the corresponding pb5 derivatives only one, a deletion derivative lacking the 153 C-terminal amino acids, was as active in the blocking assay as wild-type pb5T5. All other derivatives were inactive or almost inactive. Isolation and molecular characterization of phenotypic revertants of T5oadshowed that all revertants were true genotypic revertants of theoadmutation. Theoadmutation has been identified as a G to T exchange resulting in a substitution of Gly for Trp at position 166 of pb5T5. DNA sequencing of thehrsgene of bacteriophage BF23 and comparing the deduced amino acid sequence of pb5BF23with that of pb5T5revealed distinct regions of similarity and nonsimilarity. We propose that the receptor-binding region of pb5T5(pb5BF23) is formed by the region of nonsimilarity extending from amino acid position 89 (88) to position 305 (283).