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.