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

## TL;DR

This study explores how Mycoplasma bovis regulates potassium levels using the enzyme CdaM, which produces a signaling molecule called c-di-AMP.

## Contribution

The study identifies CdaM as a key player in potassium homeostasis in M. bovis and reveals its interaction with the K+ transporter TrkA.

## Key findings

- CdaM is a functional diadenylate cyclase that synthesizes c-di-AMP in Mycoplasma bovis.
- Loss of CdaM disrupts potassium homeostasis and causes growth defects in M. bovis.
- TrkA interacts with c-di-AMP, and specific residues are critical for this interaction.

## Abstract

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.