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

In contrast to dsDNA phages where multiple proteins are involved in programmed host lysis, lysis in ssRNA Fiersviridae and ssDNA Microviridae phages requires only a single gene (sgl for single gene lysis) to meet the size constraints of some of the smallest genomes in the biosphere. To achieve lysis, Sgl proteins exploit evolutionary "weak spots" in bacterial cell wall biogenesis. In several cases, this is done by inhibiting specific steps in Lipid II synthesis. Recently metatranscriptomics has revealed thousands of novel ssRNA phage genomes, each of which must carry at least one sgl gene. Determining the targets of these Sgl proteins could reveal novel vulnerabilities in bacterial envelope biogenesis and may lead to new antibiotics. Here, we employ a high-throughput genetic screen to uncover genome-wide host suppressors of Sgl activity and apply it to a set of diverse Sgls with unknown molecular targets. In addition to validating known molecular mechanisms, we determined that the Sgl of PP7, an ssRNA phage of P. aeruginosa, targets MurJ, the flippase responsible for Lipid II export which was previously shown to be the target of the Sgl of coliphage M. These two Sgls, which are unrelated and predicted to have opposite membrane topology, thus represent a case of convergent evolution. Another set of Sgls which are thought to cause lysis without inhibiting cell wall synthesis elicit a common set of multicopy suppressors, suggesting these Sgls act by the same or similar mechanism.