Vevik Kristina, Sribasgaran Biramitha, Cai Kun, Naemi Ali-Oddin, Pedersen Kaspersen Håkon, Uhlig Silvio, Økstad Ole Andreas, Simm Roger
Institute of Oral Biology, University of Oslo, 0316 Oslo, Norway.
Norwegian Veterinary Institute, 1433 Ås, Norway.
Microbiology (Reading). 2025 Aug;171(8). doi: 10.1099/mic.0.001597.
Cyclic di-adenosine monophosphate (c-di-AMP) is a bacterial second messenger regulating many physiological processes in bacteria. In the oral commensal species , 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 . In this study, we first show that a Δ mutant, unable to produce c-di-AMP, grows slowly under low environmental potassium conditions. Growth of the mutant was not restored by reintroducing in the original locus (KB). Whole-genome sequencing of multiple KB 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 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 . Using deletion mutants of , 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 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.
环二磷酸腺苷（c-di-AMP）是一种细菌第二信使，可调节细菌中的许多生理过程。在口腔共生菌中，c-di-AMP参与调节代谢、生长、菌落形态、链长、生物膜形成和DNA应激耐受性。然而，尚未在[具体菌种未提及]中鉴定出c-di-AMP调节的效应蛋白。在本研究中，我们首先表明，无法产生c-di-AMP的Δ突变体在低环境钾条件下生长缓慢。通过在原始位点（KB）重新引入[相关基因未提及]，无法恢复该突变体的生长。对多个KB分离株进行全基因组测序，揭示了一个假定的钾转运蛋白中的二次突变。这些突变预计会导致蛋白质截短或改变选择性钾摄取所必需的保守甘氨酸残基，从而破坏蛋白质功能。过量产生c-di-AMP的Δ突变体在高环境钠浓度下存活不佳。然后，我们对钾转运蛋白调节蛋白TrkA进行了表征。对纯化的重组TrkA蛋白的生化分析表明，它以高亲和力特异性结合c-di-AMP。使用[相关基因未提及]的缺失突变体，我们证明TrkA在低环境钾条件下的生长中至关重要。超高效液相色谱-串联质谱分析表明，与野生型相比，Δ突变体中的c-di-AMP浓度较低。这不是由于c-di-AMP周转蛋白CdaA、Pde1或Pde2表达的转录调控。在所测试的条件下，c-di-AMP的产生不受细胞外钾浓度的影响。我们还证明了TrkA在紫外线应激耐受性中的潜在作用，但在本研究中未表征其机制。
