Decoding the interactome for Cyclic-di-AMP–producing enzyme diadenylate cyclase (DacA)

Abstract Diadenylate cyclase (DacA) synthesizes the second messenger cyclic di-AMP (c-di-AMP), which regulates essential cellular processes across many Gram-positive and select Gram-negative bacteria/archaeal lineages. Although DacA is known to interact with regulators such as GlmM and CdaR, the breadth and functional relevance of its interactome remains poorly defined. Our study seeks to identify novel protein-protein interactions to further elucidate their unknown regulatory mechanisms and cellular roles. Using Streptococcus mutans as a model, we engineered a Flag-tagged strain (DacA-FLAG) and then performed co-immunoprecipitation under non-crosslinked and crosslinked conditions followed by mass spectrometry. We identified 22 candidates interacting proteins in non-crosslinked samples, 18 in crosslinked samples, and 6 shared between conditions. Selected partners were validated in vivo using split luciferase complementation. Notably, SMU_723 emerged as a key binding partner. AlphaFold-guided modeling predicated a direct DacA and SMU_723 interaction interface involving threonine 147, glutamine 148, and threonine 149 in DacA . Site-directed mutagenesis of these residues impaired binding, confirming their critical role. An SMU_723 deletion phenocopied a dacA deletion strain, sharing prolonged lag phase, aberrant cell morphology, reduced acid production and acid tolerance, impaired sorbitol metabolism, decreased colonization in a Drosophila model, and delayed growth upon calcium stimulation. These shared phenotypes suggest a functional and possibly regulatory link between DacA and SMU_723. Given SMU_723 sequence homology consistent with a calcium transporter, these data suggest that the DacA–723 interaction contributes to calcium homeostasis and/or calcium-responsive signaling. Collectively, our findings expand the DacA interaction network and implicate calcium transport in c-di-AMP-mediated regulation in S. mutans . Importance Mapping the DacA interactome reveals how environmental and intracellular cues tune c-di-AMP signaling to control stress adaptation, ion balance, and virulence traits in S. mutans . By identifying new DacA-associated proteins and validating SMU_723 as a previously unrecognized interactor with genetic and phenotypic linkage to DacA, this study provides new insights into the mechanistic framework for c-di-AMP regulation in S. mutans . The connection between DacA and a putative calcium transporter highlights a plausible axis that couples second-messenger signaling to calcium homeostasis, with implications for biofilm physiology and pathogenesis. These insights open new avenues to therapeutically modulate c-di-AMP pathways.