Abstract
Cyclic di-adenosine monophosphate (c-di-AMP) is a recently discovered signaling molecule important for the survival of Firmicutes, a large bacterial group that includes notable pathogens such as Staphylococcus aureus. However, the exact role of this molecule has not been identified. dacA, the S. aureus gene encoding the diadenylate cyclase enzyme required for c-di-AMP production, cannot be deleted when bacterial cells are grown in rich medium, indicating that c-di-AMP is required for growth in this condition. Here, we report that an S. aureus dacA mutant can be generated in chemically defined medium. Consistent with previous findings, this mutant had a severe growth defect when cultured in rich medium. Using this growth defect in rich medium, we selected for suppressor strains with improved growth to identify c-di-AMP–requiring pathways. Mutations bypassing the essentiality of dacA were identified in alsT and opuD, encoding a predicted amino acid and osmolyte transporter, the latter of which we show here to be the main glycine betaine–uptake system in S. aureus. Inactivation of these transporters likely prevents the excessive osmolyte and amino acid accumulation in the cell, providing further evidence for a key role of c-di-AMP in osmotic regulation. Suppressor mutations were also obtained in hepS, hemB, ctaA, and qoxB, coding proteins required for respiration. Furthermore, we show that dacA is dispensable for growth in anaerobic conditions. Together, these findings reveal an essential role for the c-di-AMP signaling network in aerobic, but not anaerobic, respiration in S. aureus.
Highlights
Cyclic di-adenosine monophosphate (c-di-AMP) is a recently discovered signaling molecule important for the survival of Firmicutes, a large bacterial group that includes notable pathogens such as Staphylococcus aureus
We show that c-di-AMP production is dispensable for the growth of S. aureus in chemically defined medium and in rich medium supplemented with additional sodium or potassium chloride
We have investigated the requirement of c-diAMP for the growth of S. aureus. c-di-AMP was previously shown to be required for the growth of S. aureus in rich medium [23]; here we show that inactivation of the glycine betaine transporter OpuD, the predicted amino acid transporter AlsT, or several proteins required for respiration can bypass the requirement of this signaling molecule for the growth of S. aureus. c-di-AMP is important for the growth of several other Gram-positive bacteria, and bypass mutations that allow L. monocytogenes and B. subtilis to grow in the absence of c-di-AMP have been described [34, 35, 37]
Summary
Cyclic di-adenosine monophosphate (c-di-AMP) is a recently discovered signaling molecule important for the survival of Firmicutes, a large bacterial group that includes notable pathogens such as Staphylococcus aureus. We found that inactivating mutations in a glycine betaine– uptake system and in a predicted amino acid transporter bypass the requirement of c-di-AMP for the growth of S. aureus in rich medium, further highlighting a key role of c-di-AMP in osmotic regulation. We found that mutations in genes encoding central aerobic respiration enzymes can bypass the requirement for c-di-AMP and that c-di-AMP is dispensable for the growth of S. aureus under anaerobic conditions. Taken together, these results highlight an important function for c-diAMP during aerobic but not anaerobic growth
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