High-throughput suppressor screens unveil functional convergence of single-gene lysis proteins

In contrast to canonical dsDNA phages, lysis in ssRNA Fiersviridae and ssDNA Microviridae phages is encoded by a minimal single gene (sgl), to meet the size constraints of some of the smallest genomes on earth. To achieve lysis, Sgl proteins exploit evolutionary "weak spots" in the bacterial cell wall by inhibiting specific steps in cell wall synthesis. Although a handful of these proteins have been characterized, the potentially diverse "weak spots" targeted by most Sgls remains enigmatic. Here, we repurpose Dub-seq for genome-wide assessment of host suppressors of Sgl activity and apply to eight diverse Sgls awaiting molecular target characterization. In addition to known molecular mechanisms, we discover a complex network of suppressors across the spectrum of genes involved in cell wall biogenesis. We highlight the first case of Sgl convergent evolution between unrelated proteins by determining the molecular target of SglPP7 as MurJ, suggesting the existence of universal "weak spots" in the bacterial cell wall.

与典型双链DNA（dsDNA）噬菌体不同，单链RNA菲尔斯噬菌体科（ssRNA Fiersviridae）与单链DNA微小噬菌体科（ssDNA Microviridae）的裂解过程由最小单基因（sgl）编码，以适配地球上部分极小基因组的尺寸限制。为实现细菌裂解，Sgl蛋白通过抑制细胞壁合成的特定步骤，利用细菌细胞壁演化形成的“薄弱位点”。尽管已有少量这类蛋白得到表征，但大多数Sgl所靶向的潜在多样“薄弱位点”仍不明朗。本研究将Dub-seq技术重新改造，用于全基因组水平评估Sgl活性的宿主抑制因子，并将其应用于8种尚未明确分子靶点的不同Sgl蛋白。除已知的分子机制外，我们还发现了一张覆盖细胞壁生物发生相关全部基因的复杂抑制因子网络。我们通过确定SglPP7的分子靶点为MurJ，首次揭示了无关联蛋白间Sgl趋同演化的案例，这表明细菌细胞壁中存在通用的“薄弱位点”。