Molecular logics in dual sensor regulation of enzyme activity – Phosphorylation OR blue-light activation of cyanobacterial diguanylate cyclases

Abstract Bacterial cells use multiple environmental cues to regulate levels of the second messenger cyclic dimeric GMP. This compound influences key lifestyle decisions such as motility, biofilm formation, or virulence. Although many diguanylate cyclases (DGCs) combined with various sensory domains have been studied previously, how distinct inputs are integrated within a single enzyme remains incompletely understood. Here, we investigate a cyanobacterial family of dual-sensor DGCs that combine an N-terminal receiver (Rec) domain followed by a light-oxygen-voltage (LOV) domain upstream of a diguanylate cyclase (GGDEF) domain. Using in vivo activity screening and in vitro characterisation, we determined how phosphorylation and blue light, individually and jointly, regulate enzyme activity. By measuring kinetic parameters across four defined functional states, unphosphorylated or phosphorylated, in combination with dark or light states, we reveal logic gate-like behaviours. One representative, LaRldC, integrates both signals with pronounced fold-changes in activity-, consistent with overall OR-type logic and with light acting as the dominant input. Our results demonstrate its function as a molecular gate coupling phosphorylation and illumination sensing to cyclic-di-GMP formation. These findings provide valuable insights into multi-signal decision-making in cyanobacteria and establish further understanding of how modular sensory domains are wired to control bacterial second-messenger signalling. Competing Interest Statement The authors have declared no competing interest.