The interaction between a bacteriophage and its host is mediated by the phage's receptor binding protein (RBP). Despite its fundamental role in governing phage activity and host range, the molecular rules of RBP function remain a mystery. Here, we systematically dissect the functional role of every residue in the tip domain of T7 phage RBP using a novel phage genome engineering method called ORACLE (Optimized Recombination, Accumulation and Library Expression). ORACLE is a high-throughput, locus-specific, sequence-programmable method to create a large, unbiased library of phage variants at a targeted gene locus. Using ORACLE, we generated all single amino acid substitutions at every site (1660 variants) of the tip domain to quantify the functional role of all variants on multiple bacterial hosts. This rich dataset allowed us to cross compare functional profiles of each host to precisely identify regions of functional importance, many which were previously unknown. Host-specific substitution patterns displayed differences in site specificity and physicochemical properties of mutations indicating exquisite adaptation to individual hosts. Comparison of enriched variants across hosts also revealed a tradeoff between activity and host range. We discovered gain-of-function variants effective against resistant hosts and host-constricting variants that selectively eliminated certain hosts. We demonstrate therapeutic utility against uropathogenic E. coli by engineering a highly active T7 variants to avert emergence of spontaneous resistance of the pathogen. Our approach presents a generalized framework for systematic and comprehensive characterization of sequence-function relationships in phages on an unprecedented scale.

中文翻译：

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通过深突变扫描绘制的T7噬菌体受体结合域的功能格局。

噬菌体与其宿主之间的相互作用由噬菌体的受体结合蛋白（RBP）介导。尽管RBP在控制噬菌体活性和宿主范围方面起着基本作用，但其RBP功能的分子规则仍然是一个谜。在这里，我们使用一种称为ORACLE（优化的重组，积累和文库表达）的新型噬菌体基因组工程方法，系统地分析了T7噬菌体RBP末端结构域中每个残基的功能。ORACLE是一种高通量，基因座特异性，序列可编程的方法，可在目标基因基因座处创建大型，无偏见的噬菌体变体文库。使用ORACLE，我们在末端结构域的每个位点（1660个变体）生成了所有单个氨基酸取代，以量化所有变体在多个细菌宿主上的功能作用。这个丰富的数据集使我们可以交叉比较每个主机的功能配置文件，以精确地识别具有功能重要性的区域，其中许多以前是未知的。宿主特异性替代模式显示出位点特异性和突变的理化特性差异，表明它们对个体宿主的适应性强。跨宿主的富集变体的比较还揭示了活性和宿主范围之间的权衡。我们发现了有效抵抗抗性宿主的功能获得型变体和选择性抑制某些宿主的抑制宿主的变体。我们通过工程化一个高活性的T7变体来避免病原体的自发抵抗，证明了对尿路致病性大肠杆菌的治疗效用。