ACS Chem. Biol. https://doi.org/10.1021/acschembio.7b01019 (2018)
Bacteria use 3′,5′-cyclic diguanosine monophosphate (c-di-GMP) as a signaling molecule to coordinate various processes including virulence and biofilm formation. Though a number of fluorescent biosensors exist to investigate c-di-GMP dynamics in cells, these tools are often not amenable to more complex environments. To address these issues, Dippel et al. developed chemiluminescent biosensors for c-di-GMP that avoid the need for external illumination. These protein-based biosensors consist of a PilZ domain inserted into a split luciferase fused to a fluorescent protein, such that binding of c-di-GMP to the PilZ domain enables luciferase activity and results in a fluorescence signal via bioluminescence resonance energy transfer. Because these biosensors are not hampered by autofluorescence, they can be used to assay c-di-GMP directly in lysates without further purification. Screening a library of diverse PilZ domains led to the identification of biosensor variants that are active and well-folded, exhibit high affinity and selectivity for c-di-GMP, and produce a large signal change upon binding. One of the resulting biosensors was used to develop an assay for diguanylate cyclase activity in Escherichia coli, and these biosensors could in the future be applied to the detection of c-di-GMP in complex biological systems or be adapted for the detection of other cyclic dinucleotides.