Pervasive phosphorylation by phage T7 kinase disarms bacterial defenses

Bacteria and bacteriophages are in a constant arms race to develop bacterial defense and phage counter-defense systems. Currently known phage counter-defense systems are specific to (the activity of) the targeted bacterial defense system. Here, we uncover a mechanism by which the T7 bacteriophage broadly counteracts bacterial defenses using protein phosphorylation. We show that the T7 protein kinase (T7K), which was believed to specifically redirect the function of a few host proteins, is in fact a hyper-promiscuous, dual-specificity kinase enacting a massive wave of phosphorylation on virtually all host and phage proteins during infection. The scale of phosphorylation vastly exceeds the number of previously known phosphorylation events in E. coli, has no sequence motif specificity, and results in a higher proteome-wide phosphorylation density than that of mammalian cells which encode ~ 500 kinases. Stoichiometry analysis of phosphorylation sites revealed a strong bias of T7K activity towards nucleic acid-binding substrates, which we show is mediated by its C-terminal DNA-binding domain. This specificity for highly stoichiometric phosphorylation of nucleic acid-binding proteins enables the deactivation of DNA-targeting or -containing bacterial defense systems. We provide mechanistic insight into how T7K weakens two such defense systems, Retron-Eco9 and DarTG1, through specific phosphorylation events, with single phosphomimetic mutations in key sites of the toxins abolishing defense. Finally, by screening a large collection of E. coli strains, we provide evidence of broad counter-defense capacities for T7K in nature, as strains counteracted contain diverse bacterial defense systems.