Bacterial dynamin homolog regulates growth phase-dependent c-di-GMP production

The bacterial second messenger bis-(3′-5′)-cyclic dimeric guanosine monophosphate (c-di-GMP) governs the transition from motile to sessile lifestyle by suppressing flagellar motility and promoting the synthesis of the extracellular biofilm matrix. In Escherichia coli , a main contributor to the global c-di-GMP pool is the diguanylate cyclase DgcE. It was recently shown that DgcE is activated by physical interaction with the dynamin-like GTPase RdcA and its accessory protein RdcB, but molecular details and physiological relevance of this regulation have not been fully revealed. Here, we used a combination of in vitro and in vivo experiments to characterize the complex formation between RdcA, RdcB, and DgcE. We corroborate that RdcA functions as a modular GTP sensor to control the activity of DgcE; while its C-terminal domain binds to and activates DgcE, the N-terminal GTPase domain confers regulation. We show that a drop in GTP levels triggers RdcA localization to the cytoplasmic membrane and its interaction with DgcE. RdcB coordinates the formation of larger RdcA-DgcE assemblies, and it is required for the DgcE activation by RdcA. Additionally, we demonstrate that the alarmone (p)ppGpp modulates the DgcE activation by counteracting the inhibitory effect of GTP on RdcA activity. Taken together, our findings further characterize a sophisticated regulatory mechanism of c-di-GMP signaling in E. coli , in which the ternary RdcA-RdcB-DgcE complex integrates GTP and (p)ppGpp signals to monitor amino acid starvation and/or cellular energy status, thus controlling transition to a multi-cellular, biofilm-associated lifestyle. IMPORTANCE As a central regulator of motile-to-sessile lifestyle switching, bis-(3′-5′)-cyclic dimeric guanosine monophosphate (c-di-GMP) needs to be dynamically modulated in response to growth phase transitions and environmental challenges. Our work characterizes a novel post-translational mechanism of c-di-GMP regulation, where a bacterial GTPase, RdcA, that is homologous to eukaryotic dynamins, couples the physiological state of cells to c-di-GMP production. This coupling includes a cell energy- or nutrient-dependent recruitment of RdcA to the cytoplasmic membrane, where it interacts with and activates a major c-di-GMP producing enzyme. In contrast to the established function of eukaryotic and bacterial dynamins in membrane remodeling, our study establishes an example of a dynamin-like protein functioning as a GTP-sensitive switch and trigger enzyme that controls second messenger signaling in bacteria.