TrkA of Streptococcus mitis CCUG31611 binds cyclic di-adenosine monophosphate and is required for growth in low potassium conditions

Cyclic di-adenosine monophosphate (c-di-AMP) is a bacterial second messenger regulating many physiological processes in bacteria. In the oral commensal species <i>Streptococcus mitis</i>, c-di-AMP is involved in regulating metabolism, growth, colony morphology, chain length, biofilm formation and DNA stress tolerance. However, no c-di-AMP-regulated effector proteins have yet been characterized in <i>S. mitis</i>. In this study, we first show that a Δ<i>cdaA</i> mutant, unable to produce c-di-AMP, grows slowly under low environmental potassium conditions. Growth of the <i>cdaA</i> mutant was not restored by reintroducing <i>cdaA</i> in the original locus (KB<i>cdaA</i>). Whole-genome sequencing of multiple KB<i>cdaA</i> isolates revealed secondary mutations in a putative potassium transporter. The mutations were predicted to result in the truncation of the protein or the alteration of a conserved glycine residue essential for selective potassium uptake, disrupting protein function. A Δ<i>pde2</i> mutant overproducing c-di-AMP survived poorly under high environmental sodium concentrations. We then characterized the potassium transporter regulator protein TrkA. Biochemical analyses of the purified recombinant TrkA protein revealed that it specifically binds c-di-AMP with high affinity <i>in vitro</i>. Using deletion mutants of <i>trkA</i>, we demonstrate that TrkA is essential for growth under low environmental potassium conditions. Ultra-high-performance liquid chromatography coupled to tandem mass spectrometry revealed lower c-di-AMP concentration in the Δ<i>trkA</i> mutant compared to the WT. This was not due to transcriptional regulation of the expression of the c-di-AMP turnover proteins CdaA, Pde1 or Pde2. C-di-AMP production is not affected by the extracellular potassium concentrations under the conditions tested. We also demonstrate a potential role of TrkA in UV stress tolerance but do not characterize the mechanism in this study.