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J. Bacteriol. doi:10.1128/JB.00437-07
Copyright (c) 2007, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

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

Department of Chemistry and Biochemistry University of Oklahoma, Norman, OK 73072; Robert-Koch Institut, Wernigerode Branch, National Reference Centre for Salmonellae and other Enterics, Burgstrasse 37, D-38855 Wernigerode, Germany

	Abstract

H8 derived from a collection of Salmonella enterica serotype Enteritidis bacteriophage. Its morphology and genomic structure closely resembled those of bacteriophage T5 in the Family Siphoviridae. H8 infected Salmonella serotypes Enteritidis and Typhimurium, and E. coli by initial adsorption to the outer membrane protein FepA. Ferric enterobactin inhibited H8 binding to E. coli FepA (IC₅₀ = 98 nM), and other ferric catecholate receptors (Fiu, Cir, IroN) did not participate in phage adsorption. H8 infection was TonB-dependent, but exbB mutations in Salmonella or E. coli did not prevent infection; only exbB tolQ/R double mutants were resistant to H8. Experiments with deletion and substitution mutants showed that the receptor-phage interaction first involves residues distributed over the protein's outer surface, and then narrows to the same charged (R316) or aromatic (Y260) residues that participate in the binding and transport of ferric enterobactin and colicins B and D. These data rationalize the multifunctionality of FepA: toxic ligands like bacteriocins and phage penetrate the OM by parasitizing residues in FepA that are adapted to the transport of the natural ligand, ferric enterobactin. DNA sequence determinations revealed the complete H8 genome, of 104.4 kb. 120 of its 143 predicted Orfs were homologous to Orfs in T5, at a level of 84% identity and 89% similarity. As in T5, the H8 structural genes clustered on the chromosome according to their function in the phage life cycle. The T5 genome contains a large section of deletable DNA that is absent in H8: compared to T5, H8 contains a 9000 bp deletion in the early region of its chromosome, and 9 potentially unique gene products. Sequence analyses of the tail proteins of phages in the same family showed that relative to pb5 (Oad) of T5 and Hrs of BF23, the FepA-binding protein (Rbp) of H8 contains unique acidic and aromatic residues. These side chains may promote binding to basic and aromatic residues in FepA, that normally function in the adsorption of FeEnt. Furthermore, a predicted H8 tail protein showed extensive identity/similarity to pb2 of T5, suggesting that it also functions in pore formation through the cell envelope. The variable region of this protein contains a potential TonB-box, intimating that it participates in the TonB-dependent stage of the phage infection process.

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