Regulation of Potassium Homeostasis in Mycoplasma bovis by the Cyclic Adenylate Synthetase MbovP496

Cyclic di-AMP (c-di-AMP) is a critical and universally present second messenger that helps regulate potassium (K⁺) concentrations, ensuring osmotic balance. Despite its significance, 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. First,we found that MbovP496, a typical member of the DisA_N family, possesses the ability to synthesize c-di-AMP, facilitated by the conserved DGA and RHR motifs. Subsequently,we verified that the mutant strain lacking functional MbovP496, designated T5.415, displayed inhibited growthwhen co-culturedwith host cells or grown in a PPLO medium supplemented with exogenous K+. This growth defect was associated with increased expression of the K+ uptake transporters MbovP421 and MbovP415.Further analysis revealed thatMbovP421 serves as a direct receptor for c-di-AMP, and the amino acid residues R174, V180, and P192 of MbovP421 are critical for the interaction. In addition, disruption of the interaction in the T5.415 mutant resulted in significantly higher intracellular K⁺ concentration, highlighting the regulatory impact of c-di-AMP on K⁺ homeostasis. RNA sequencinganalysis further revealed that the differentially expressed genes between WT and T5.415 were enriched in pathways related totransmembrane transportand pyruvate metabolism. In conclusion, this study identified MbovP496 as an active c-di-AMP synthetase in M. bovisand elucidated its product’s interaction with the K+uptake transporter MbovP421. These findings provide valuable insights into the regulationof intracellular K+ concentrationby c-di-AMP synthetase in mycoplasmas.