Phosphorylation of a conserved intrinsically disordered region is necessary for activation of a bacterial Hanks-type Ser/Thr kinase signaling pathway

Intrinsically disordered regions (IDRs) are found throughout all domains of life, yet their contribution to bacterial signaling has remained unclear. Here we show that phosphorylation of a conserved juxtamembrane IDR is necessary for activation of the bacterial Hanks-type Ser/Thr kinase-phosphatase PrkC/PrpC signaling pathway in Bacillus subtilis. Phosphoablative mutation of a conserved IDR phosphosite has strong effect on kinase-activity-dependent phenotypes, including intrinsic {beta}-lactam resistance and stationary phase survival. Using a synthetic quantitative reporter for kinase activity, mutational analysis, and mathematical modeling, we show that phosphorylation of the IDR promotes trans autoactivation of the kinase, and that this modification is essential for amplifying kinase activity in response to a signal. Phylogenetic analysis demonstrates that this IDR and associated putative phosphosites proximal to the kinase domain are highly conserved across prokaryotic species that diverged at the last universal common ancestor. Together these findings suggest that kinase-domain proximal IDR phosphorylation has a critical role in bacterial Ser/Thr signaling, with direct implications for understanding antibiotic resistance mechanisms and developing kinase-targeting antimicrobials. Author SummaryBacteria use protein kinases to sense and respond to their environment. Despite decades of research, the activation process of an evolutionarily ancient group of bacterial Ser/Thr kinases that serve as master regulators of responses to antibiotics remains poorly understood. In this work we show that phosphorylation of an unstructured protein region, an intrinsically disordered region (IDR), is required for activating the prototypical Ser/Thr bacterial kinase in Bacillus subtilis. Without this phosphorylation, bacteria are more sensitive to {beta}-lactam antibiotics and show severe survival defects in stationary phase. Using genetic analysis and mathematical modeling, we show that IDR phosphorylation allows kinases to activate each other, generating signal amplification. This kinase IDR is conserved across bacteria and archaea that diverged billions of years ago, suggesting it arose early in evolution. Because closely related kinases in clinically important pathogens share these features, our findings suggest this IDR as a target for developing antimicrobials that could disrupt bacterial responses to antibiotic treatment.