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Background <p>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 <i>Porphyromonas gingivalis</i>, 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<sub><i>Pg</i></sub>. Here, we identify rhythmic-phasic c-di-AMP signaling scheme in which PDE<sub><i>Pg</i></sub> plays a critical role by coordinating with bacterial growth phase, metabolism, and cell cycle progression.</p> Methods <p>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 <i>P. gingivalis</i>.</p> Results <p>This study demonstrates that growth phase synchronization in <i>P. gingivalis</i> 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<sub><i>Pg</i></sub>, and the conserved serine residue (Ser-226) within the DAC<sub><i>Pg</i></sub> SEE loop. PDE<sub><i>Pg</i></sub> maintains c-di-AMP homeostasis through a noncanonical regulatory mechanism rather than through measurable PDE-mediated hydrolysis. Disruption of PDE<sub><i>Pg</i></sub> 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<sub><i>Pg</i></sub> 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<sub><i>Pg</i></sub> coordinates rhythmic-phasic c-di-AMP signaling and physiological adaptation in <i>P. gingivalis</i>.</p> Conclusion <p>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.</p>

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Rhythmic-phasic c-di-AMP signaling: a new paradigm for bacterial growth synchronization governed by a pseudo-phosphodiesterase and modifiable diadenylate cyclase

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2026年7月29日

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  • Suraj Adhikari,
  • Shirin Ghods,
  • Ratnam S. Seelan,
  • Masoud Hamidi,
  • Michael Sekula,
  • Pritha Bagchi,
  • David A. Scott,
  • Richard J. Lamont,
  • Fata Moradali

摘要

Background

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 PDEPg. Here, we identify rhythmic-phasic c-di-AMP signaling scheme in which PDEPg plays a critical role by coordinating with bacterial growth phase, metabolism, and cell cycle progression.

Methods

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.

Results

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, PDEPg, and the conserved serine residue (Ser-226) within the DACPg SEE loop. PDEPg maintains c-di-AMP homeostasis through a noncanonical regulatory mechanism rather than through measurable PDE-mediated hydrolysis. Disruption of PDEPg 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 DACPg 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) PDEPg coordinates rhythmic-phasic c-di-AMP signaling and physiological adaptation in P. gingivalis.

Conclusion

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.