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

Abstract

The receptor-binding protein pb5(T5) of bacteriophage T5, when expressed from the oad gene 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 of E. coli, where it blocks FhuA for subsequent adsorption of T5 (Mondigler et 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 within oad on the FhuA-binding properties of corresponding pb5 derivatives. Three classes of mutations were tested: (i) oad deletion derivatives, (ii) the oad mutation 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 the oad mutation. 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 pb5(T5). All other derivatives were inactive or almost inactive. Isolation and molecular characterization of phenotypic revertants of T5oad showed that all revertants were true genotypic revertants of the oad mutation. The oad mutation has been identified as a G to T exchange resulting in a substitution of Gly for Trp at position 166 of pb5(T5). DNA sequencing of the hrs gene of bacteriophage BF23 and comparing the deduced amino acid sequence of pb5(BF23) with that of pb5(T5) revealed distinct regions of similarity and nonsimilarity. We propose that the receptor-binding region of pb5(T5) (pb5(BF23)) is formed by the region of nonsimilarity extending from amino acid position 89 (88) to position 305 (283).

FepA- and TonB-Dependent Bacteriophage H8: Receptor Binding and Genomic Sequence

“…These genomic homologies identified the H8 receptor-binding tail protein (Rbp), and its comparison to the T5 and BF23 receptor binding proteins revealed regions that likely interact with the OM proteins FepA, FhuA, and BtuB, respectively. These data support and refine prior predictions of the T5 receptor binding domain in the oad structural gene (63). The analogous portion of the H8 Rbp, from residues 138 to 213, has a net charge of Ϫ8, consistent with the experimental evidence that H8 interacts with FepA in a similar way as the acidic siderophore FeEnt.…”

“…For H8, the Rbp is most homologous to Hrs and pb5 (Oad) of BF23 and T5, respectively, and Tpf is related to the pb2 (D18-19) of T5. Despite the fact that the products of oad and hrs may functionally replace each other in the tails of T5 and BF23, respectively (39, 48), Mondigler et al (62,63) reported that no homology exists between these genes. The CLUSTAL W alignment of the T5, BF23, and H8 receptor binding proteins (Fig.…”

“…Besides the genome of H8, reported herein, full or partial genomic sequences of other LGP-specific bacteriophage are known, including those of the TonB-dependent phage T1 and the TonB-independent phages T5 and BF23. Sequence data for BF23 is incomplete, but the genes that encode some of its tail proteins are known and sequenced (62,63). Blast analyses of H8 DNA identified ORFs at 94.7 kb and 82.8 kb that encode homologous proteins to the experimentally demonstrated receptor binding proteins of BF23 (Hrs) (48) and T5 (pb5, encoded by oad) (39, 63) and the pore-forming tail proteins of T5 (pb2) (7,28), respectively.…”

Cell Envelope Signaling in Escherichia coli

“…Alternatively, the large size of the phage particles relative to the other ligands may in some manner preclude the enhancement of FhuA-TonB interaction as detected in vitro. The identification and isolation of tail fiber proteins that constitute the minimal requirement for phage to participate in receptor recognition (54,55) will assist in the analysis of TonB coupling with TonB-dependent receptors. Future studies need to focus upon the ligand-induced conformational dynamics of FhuA which govern its physical association with TonB and upon those regions of FhuA which are required for the interaction with TonB.…”

Insights into Bacteriophage T5 Structure from Analysis of Its Morphogenesis Genes and Protein Components

“…However, this previous assignment must be revisited as pb2 is not long enough to extend from the bottom of the tail tube to the end of the central fiber (18). pb5, the receptor binding protein (RBP), ensures the binding of T5 to its receptor, the E. coli outer membrane protein FhuA (19,20). The FhuA-pb5 complex has been characterized in vitro (21)(22)(23), but the exact location of pb5 at the tail tip remains uncertain.…”