c-di-AMP-Regulated K⁺ Importer KtrAB Affects Biofilm Formation, Stress Response, and SpeB Expression in Streptococcus...

The second messenger cyclic di-AMP (c-di-AMP) controls biofilm formation, stress response, and virulence in Streptococcus pyogenes The deletion of the c-di-AMP synthase gene, dacA, results in pleiotropic effects including reduced expression of the secreted protease SpeB. Here, we report a role for K⁺ transport in c-di-AMP-mediated SpeB expression. The deletion of ktrB in the ΔdacA mutant restores SpeB expression. KtrB is a subunit of the K⁺ transport system KtrAB that forms a putative high-affinity K⁺ importer. KtrB forms a membrane K⁺ channel, and KtrA acts as a cytosolic gating protein that controls the transport capacity of the system by binding ligands including c-di-AMP. SpeB induction in the ΔdacA mutant by K⁺ specific ionophore treatment also supports the importance of cellular K⁺ balance in SpeB production. The ΔdacA ΔktrB double deletion mutant not only produces wild-type levels of SpeB but also partially or fully reverts the defective ΔdacA phenotypes of biofilm formation and stress responses, suggesting that many ΔdacA phenotypes are due to cellular K⁺ imbalance. However, the null pathogenicity of the ΔdacA mutant in a murine subcutaneous infection model is not restored by ktrB deletion, suggesting that c-di-AMP controls not only cellular K⁺ balance but also other metabolic and/or virulence pathways. The deletion of other putative K⁺ importer genes, kup and kimA, does not phenocopy the deletion of ktrB regarding SpeB induction in the ΔdacA mutant, suggesting that KtrAB is the primary K⁺ importer that is responsible for controlling cellular K⁺ levels under laboratory growth conditions.