A genetically encoded biosensor reveals heterogenous cAMP dynamics coordinating growth and resuscitation in Mycobacterium tuberculosis

3',5'- Cyclic adenosine monophosphate (cAMP) serves as a global regulatory hub in Mycobacterium tuberculosis (Mtb), which dedicates approximately 1% of its genome to the synthesis, degradation and sensing of this secondary messenger. Despite cAMPs central role in Mtb virulence and metabolic adaptation, we currently do not have any tools to monitor cAMP dynamics in real time at single-cell resolution. Here, we report the development and validation of rmgCarvi, a genetically encoded biosensor adapted from eukaryotic systems for use in mycobacteria. By utilizing cAMP-responsive cpGFP and constitutive mCherry fluorescence, rmgCarvi provides a high-fidelity ratiometric readout of intracellular cAMP levels, revealing that carbon source and growth state act as primary determinants of cAMP dynamics. Real-time single-cell imaging using rmgCarvi demonstrated robust correlations between cAMP and growth rate, with pronounced cAMP spikes coinciding with cell division. In starved Mtb populations, a bimodal distribution in cAMP levels dictates a distinct hysteresis during regrowth upon nutrient supplementation. Low-cAMP subpopulations are associated with strong induction in cAMP levels and a longer lag before initiation of growth, while high cAMP cells do not exhibit significant induction in cAMP and have recovery times uncorrelated with prior cAMP levels. Finally, use of rmgCarvi in infection studies captured host species-specific responses, including a strong cAMP induction upon internalization followed by a decline in murine, but not human, macrophages. By resolving cAMP dynamics with unprecedented cellular and temporal resolution, rmgCarvi provides a framework for understanding how Mtb tunes this crucial metabolite to navigate the complex microenvironments in the host. Significance StatementWe report the evaluation of a ratiometric genetic biosensor, rmgCarvi, for real-time single-cell monitoring of cAMP dynamics in mycobacteria. This biosensor reveals how Mycobacterium tuberculosis precisely tunes cAMP within a narrow range ("Goldilocks zone") to coordinate metabolic adaptation, and growth across complex nutrient environments. In nutrient-starved populations, we identify a bimodal cAMP distribution that functions as a phenotypic memory, dictating resuscitation kinetics upon nutrient restoration. As the first cAMP biosensor validated in any mycobacterial species, rmgCarvi facilitates the discovery of host-specific signaling dynamics during infection and provides a platform for screening of compounds that perturb cAMP homeostasis. This study provides a framework to explore how Mtb utilizes this secondary messenger molecule to survive and adapt within complex host environments.