Regulation of potassium homeostasis in Mycoplasma bovis by the diadenylate cyclase CdaM

Cyclic di-AMP (c-di-AMP) is a critical second messenger in many Gram-positive bacteria and archaea that regulates intracellular potassium (K + ) concentrations, ensuring osmotic balance. However, the precise mechanisms of K + regulation by c-di-AMP in Mycoplasma species remain largely unexplored. In this study, we used the ruminant pathogen Mycoplasma bovis ( M. bovis ) as a model to investigate this mechanism. We identified CdaM (MbovP496), a member of the DisA_N family, a member of the DisA_N family, as a functional diadenylate cyclase capable of synthesizing c-di-AMP, and demonstrated that its enzymatic activity depends on the conserved DGA and RHR motifs. Deletion of cdaM ( cdaM* ) abolished c-di-AMP production and resulted in pronounced growth defects when M. bovis was co-cultured with host cells or grown in PPLO medium supplemented with exogenous K + . These phenotypes were accompanied by increased expression of the K + uptake transporter TrkA (MbovP421). We further demonstrated that TrkA acts as a direct receptor for c-di-AMP and identified the residues R174, V180, and P192 as critical for this interaction. Loss of CdaM resulted in significantly elevated intracellular K + levels, underscoring the essential role of c-di-AMP in maintaining K + homeostasis. Transcriptomic analysis further revealed that genes differentially expressed between the wild type strain and cdaM* mutant were enriched in pathways related to transmembrane transport and pyruvate metabolism, indicating broader metabolic reprogramming associated with disrupted c-di-AMP signaling. In conclusion, this study identifies CdaM as a key determinant of K + adaptation in M. bovis and elucidates the molecular interaction between its product and the K + uptake transporter TrkA. Together, these findings provide important insights into c-di-AMP-mediated regulation of intracellular K + concentration in mycoplasmas and highlight DisA_N family proteins as potential targets for antimicrobial intervention.