High-throughput discovery of phage receptors using transposon insertion sequencing of bacteria

As the most abundant microbes on earth, novel bacteriophage (phage; bacteria-specific viruses) are readily isolated from environmental samples. However, it remains challenging to characterize phage-bacteria interactions, such as the host receptor(s) phage bind to initiate infection. Here, we tested whether transposon insertion sequencing, INSeq, could be used to identify bacterial genes involved in phage binding. As proof of concept, results showed that INSeq screens successfully identified genes encoding known receptors for previously characterized viruses of Escherichia coli (phage T6, T2, T4 and T7). INSeq screens were then used to identify genes involved during infection of six newly isolated coliphage. Results showed that candidate receptors could be successfully identified for the majority (five of six) of the phage; furthermore, genes encoding the phage receptor(s) were the top hit(s) in the analyses of the successful screens. INSeq screens provide a generally useful method for high-throughput discovery of phage receptors. We discuss limitations of our approach when examining uncharacterized phage, as well as usefulness of the method for exploring the evolution of broad versus narrow use of cellular receptors among phage in the biosphere.

作为地球上丰度最高的微生物类群，新型噬菌体（bacteriophage，phage；细菌特异性病毒）可便捷地从环境样本中分离获得。然而，解析噬菌体与细菌的互作机制仍颇具挑战，例如噬菌体结合以启动感染的宿主受体鉴定等问题。本研究验证了转座子插入测序（INSeq）能否用于鉴定参与噬菌体结合过程的细菌基因。作为概念验证实验，结果显示INSeq筛选可成功鉴定出此前已被表征的大肠杆菌（Escherichia coli）病毒——即T6、T2、T4及T7噬菌体——的已知受体编码基因。随后，研究利用INSeq筛选对6株新分离的大肠杆菌噬菌体（coliphage）感染过程中涉及的细菌基因进行了鉴定。结果表明，6株噬菌体中的多数（5株）可成功鉴定出候选受体；且在成功完成的筛选分析中，编码噬菌体受体的基因均为排名最高的命中结果。INSeq筛选为噬菌体受体的高通量发现提供了一种通用且实用的研究方法。本研究还讨论了该方法在分析未被表征的噬菌体时存在的局限性，以及其在探究生物圈中噬菌体对细胞受体使用谱（广谱vs窄谱）的进化规律方面的应用价值。