Quantifying c-di-GMP: A Call for Integration of Biological and Chemical Approaches

This is the authors' abstract. We don't add key points for this paper.

Bacteria sense, respond, and adapt to rapidly changing environments through highly sensitive intracellular networks. Among them, cyclic-di-GMP (c-di-GMP) is a well-characterized second messenger controlling the transition from planktonic to sessile lifestyles, making it a key determinant of bacterial adaptation and survival. Two main approaches have been developed to investigate c-di-GMP dynamics. The first uses biological biosensors for live-cell monitoring of intracellular c-di-GMP through transcriptional reporters, RNA-based sensors, or protein-based sensors. Their main advantage is real-time, spatiotemporal analysis in living cells, although they generally do not provide absolute quantification. The second approach relies on analytical chemistry, particularly liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS), which enables sensitive and accurate quantification of intracellular c-di-GMP across diverse bacterial species. However, the lack of standardized extraction protocols and the need for cell lysis prevent real-time measurements, providing only a snapshot of the total c-di-GMP pool. These approaches are complementary: biosensors reveal dynamic, single-cell responses, whereas LC-MS/MS provides precise global quantification. Nevertheless, most studies rely on only one method, limiting a comprehensive understanding of c-di-GMP biology. Combining both approaches would provide a more integrated view of c-di-GMP signaling and its role in bacterial physiology.