Assessment of the phi6 lysis system using genetic complementation and heterologous expression in Escherichia coli and Pseudomonas syringae

The RNA cystovirus phi6 represents a unique evolutionary outlier, encoding a holin and endolysin similar to tailed phages, yet lacking an identified gene for outer membrane disruption. In this study, we investigated the phi6 lysis system using genetic complementation experiments and heterologous expression in <i>Escherichia coli</i> and <i>Pseudomonas syringae</i>. Our results demonstrate that the phi6 endolysin is functional in both bacterial systems, while the phi6 holin does not show activity in either bacterial system when expressed using moderate expression systems. Combinations of lambda lysis gene controls performed as expected in <i>P. syringae</i>. The phi6 holin requires overexpression or co-expression with other phi6 genes to exhibit lysis in <i>E. coli</i>. Overexpression of plasmids containing entire phi6 S-, M-, and L-segments cDNA in <i>E. coli</i> produced lysis profiles and cell morphology consistent with holin-endolysin expression, but outer membrane disruption was not observed. This suggests either phi6 has not evolved to carry an outer membrane disruptor or that the outer membrane disruptor is not active in our <i>E. coli</i> testbed. Our findings highlight the unusual nature of the phi6 lysis system.