Functional role of the diadenylate cyclase gene cdaA in stress responses of the dairy starter Streptococcus thermophilus.

Streptococcus thermophilus is a key dairy starter culture widely used in the production of yogurt and cheese, where its robustness in stress adaptation and growth performance is critical for industrial fermentation efficiency. Cyclic di-AMP (c-di-AMP) is a key second messenger involved in regulating osmotic homeostasis and stress adaptation in bacteria. However, the role of c-di-AMP in S. thermophilus remains unexplored. Here, cdaA, encoding a diadenylate cyclase containing a conserved DisA_N domain responsible for c-di-AMP synthesis, was identified in S. thermophilus S-3 through bioinformatic analysis and validated by LC-MS/MS. The cdaA gene knockout strain, S-3ΔcdaA, was unable to synthesize c-di-AMP and exhibited a markedly shortened lag phase. Stress response assays revealed that S-3ΔcdaA was sensitive to osmotic and ethanol stress, indicating that cdaA is essential for stress tolerance. Specifically, S-3ΔcdaA showed almost no growth in chemically defined medium (CDM) supplemented with 300 mM potassium or sodium chloride, whereas the wild-type S-3 reached OD600 values of 0.72 and 0.52, respectively. Under 5% ethanol stress, the specific growth rate of S-3ΔcdaA dropped significantly to 0.35 relative to S-3 compared to 1.10 in CDM medium alone. Interestingly, S-3ΔcdaA retained the ability to grow under 0.01% oxgall stress, reaching an OD600 of 0.83, while growth of S-3 was completely abolished. It suggested that absence of cdaA confers a survival advantage under bile salt stress. Taken together, these results showed that cdaA is critical for osmotic and ethanol tolerance but negatively modulates bile salt resistance in S. thermophilus.