Rhythmic-phasic c-di-AMP signaling: a new paradigm for bacterial growth synchronization governed by a...

Cyclic di-adenosine monophosphate (c-di-AMP) is an essential bacterial second messenger that regulates bacterial physiology, survival, and pathogenesis. While current models describe c-di-AMP regulation as a linear synthesis-degradation paradigm through opposing activities of diadenylate cyclases (DACs) and c-di-AMP-specific phosphodiesterases (PDEs), they fail to capture the temporal complexity of c-di-AMP signaling dynamics in coordination with discrete growth-phase transitions, metabolic state, and redox homeostasis. In Porphyromonas gingivalis, a key Gram-negative proteolytic oral pathobiont relevant to oral and systemic disease, measurable canonical c-di-AMP degradation is absent, suggesting that c-di-AMP signaling operates through an atypical regulatory architecture mediated by the atypical phosphodiesterase PDE_Pg. Here, we identify rhythmic-phasic c-di-AMP signaling scheme in which PDE_Pg plays a critical role by coordinating with bacterial growth phase, metabolism, and cell cycle progression.

We employed an integrated experimental and structural modeling framework that combined bacterial genetics, growth-phase-resolved nucleotide profiling, whole membrane fraction biochemical assays, untargeted metabolomics, transcriptomics, proteomics, post-translational modification profiling, ultrastructural transmission electron microscopy, structural modeling, and molecular dynamics simulations to dissect the pseudoenzymatic-controlled c-di-AMP regulatory module in P. gingivalis.

This study demonstrates that growth phase synchronization in P. gingivalis requires rhythmic-phasic c-di-AMP signaling, characterized by coordinated depletion-replenishment episodes of intracellular c-di-AMP during growth-phase transitions. This regulatory network is orchestrated by two key components: the atypical HD-domain PDE homolog, PDE_Pg, and the conserved serine residue (Ser-226) within the DAC_Pg SEE loop. PDE_Pg maintains c-di-AMP homeostasis through a noncanonical regulatory mechanism rather than through measurable PDE-mediated hydrolysis. Disruption of PDE_Pg profoundly remodels bacterial physiology, resulting in altered c-di-AMP dynamics, impaired peptide-dependent metabolic adaptation, disrupted ATP homeostasis, and widespread post-translational remodeling, including condition-specific phosphorylation of the c-di-AMP cyclase DAC_Pg at the conserved Ser226 residue. Functional genetic analyses demonstrate that Ser226 is required for proper coordination of rhythmic-phasic c-di-AMP signaling and metabolic adaptation, while pantothenate-mediated rescue further supports the contribution of metabolic homeostasis to these processes. Along with structural modeling and molecular dynamics simulations, these findings support a model in which noncanonical regulatory protein (pseudoenzymatic function) PDE_Pg coordinates rhythmic-phasic c-di-AMP signaling and physiological adaptation in P. gingivalis.

Together, our findings establish a new paradigm in bacterial second messenger signaling, showing that pseudoenzymatic scaffolds coordinate rhythmic c-di-AMP dynamics that synchronize nutrient acquisition, redox metabolism, and cell-cycle progression, with broad implications for microbial physiology and pathogenesis.