Mumbleau, M. M.; Hammond, M. C.

Cyclic dinucleotide (CDN) signaling molecules, such as cyclic di-GMP (c-di-GMP) and 3',3' cyclic GMP-AMP (cGAMP), are second messengers that play critical roles in phenotypic regulation, such as biofilm formation, host colonization, and bacterial virulence. Recently, hybrid promiscuous (Hypr) GGDEF proteins have been identified in certain bacteria to produce both cyclic dinucleotides. One such enzyme, Bd0367, from the predatory Bdellovibrio bacteriovorus, switches between synthesizing c-di-GMP and cGAMP to regulate the bacterial predation cycle and prey exit. However, the molecular mechanism controlling this switch remains unknown. Here, we introduce an RNA-based ratiometric, dual metabolite biosensor that enables simultaneous detection of c-di-GMP and cGAMP in live cells. This sensor integrates a Pepper-based biosensor for c-di-GMP detection and a Spinach2-based biosensor for cGAMP detection into a single transcript, producing distinct fluorescent outputs. In E. coli, the dual metabolite sensor reliably reported shifts in c-di-GMP/cGAMP production ratios from various CDN synthases, including Bd0367. Additionally, a histidine kinase was discovered as the probable regulatory partner of Bd0367. These findings demonstrate the sensor's capacity to assess relative CDN levels and to uncover complex signaling pathways. Together, this ratiometric dual metabolite biosensor provides a foundation for broader applications of fluorogenic RNA biosensors in dissecting bacterial signaling networks, microbial ecology, and host-pathogen interactions.