Rabsch Wolfgang, Ma Li, Wiley Graham, Najar Fares Z, Kaserer Wallace, Schuerch Daniel W, Klebba Joseph E, Roe Bruce A, Laverde Gomez Jenny A, Schallmey Marcus, Newton Salete M C, Klebba Phillip E
Department of Chemistry and Biochemistry, University of Oklahoma, 620 Parrington Oval, Norman, OK 73019, USA.
J Bacteriol. 2007 Aug;189(15):5658-74. doi: 10.1128/JB.00437-07. Epub 2007 May 25.
H8 is derived from a collection of Salmonella enterica serotype Enteritidis bacteriophage. Its morphology and genomic structure closely resemble those of bacteriophage T5 in the family Siphoviridae. H8 infected S. enterica serotypes Enteritidis and Typhimurium and Escherichia coli by initial adsorption to the outer membrane protein FepA. Ferric enterobactin inhibited H8 binding to E. coli FepA (50% inhibition concentration, 98 nM), and other ferric catecholate receptors (Fiu, Cir, and 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 or exbB tolR 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 outer membrane 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. A total of 120 of its 143 predicted open reading frames (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 DNA that can be deleted and that is absent in H8: compared to T5, H8 contains a 9,000-bp deletion in the early region of its chromosome, and nine 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 ferric enterobactin. Furthermore, a predicted H8 tail protein showed extensive identity and 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.
H8源自一组肠炎沙门氏菌血清型肠炎噬菌体。其形态和基因组结构与长尾噬菌体科的噬菌体T5极为相似。H8通过最初吸附在外膜蛋白FepA上感染肠炎沙门氏菌血清型肠炎和鼠伤寒以及大肠杆菌。肠铁载体抑制H8与大肠杆菌FepA的结合（50%抑制浓度为98 nM），其他儿茶酚铁受体（Fiu、Cir和IroN）不参与噬菌体吸附。H8感染依赖TonB，但沙门氏菌或大肠杆菌中的exbB突变并不阻止感染；只有exbB tolQ或exbB tolR双突变体对H8具有抗性。缺失和替代突变体实验表明，受体与噬菌体的相互作用首先涉及分布在蛋白质外表面的残基，然后缩小到参与肠铁载体以及大肠杆菌素B和D的结合与转运的相同带电（R316）或芳香族（Y260）残基。这些数据解释了FepA的多功能性：诸如细菌素和噬菌体之类的有毒配体通过寄生FepA中适应天然配体肠铁载体转运的残基穿透外膜。DNA序列测定揭示了104.4 kb的完整H8基因组。其143个预测开放阅读框（ORF）中的120个与T5中的ORF同源，同一性水平为84%，相似性水平为89%。与T5一样，H8结构基因根据其在噬菌体生命周期中的功能聚集在染色体上。T5基因组包含一大段可删除的DNA，而H8中不存在该段DNA：与T5相比，H8在其染色体早期区域有一个9000 bp的缺失，以及九个潜在的独特基因产物。同一家族中噬菌体尾蛋白的序列分析表明，相对于T5的pb5（Oad）和BF23的Hrs，H8的FepA结合蛋白（Rbp）包含独特的酸性和芳香族残基。这些侧链可能促进与FepA中通常在肠铁载体吸附中起作用的碱性和芳香族残基的结合。此外，预测的H8尾蛋白与T5的pb2具有广泛的同一性和相似性，表明它也在通过细胞包膜形成孔道中发挥作用。该蛋白的可变区包含一个潜在的TonB框，表明它参与噬菌体感染过程中依赖TonB的阶段。
