Cyclic-di-adenosine monophosphate (c-di-AMP) is required for osmotic regulation in Staphylococcus aureus but dispensable for viability in anaerobic conditions. Growth of Staphylococcus in the absence of c-di-AMP

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.

环二腺苷单磷酸（cyclic di-adenosine monophosphate，c-di-AMP）是近年发现的信号分子，对厚壁菌门（Firmicutes）的存活至关重要——该细菌类群包含金黄色葡萄球菌（Staphylococcus aureus）等多种重要致病菌。然而，该分子的确切功能尚未明确。dacA作为金黄色葡萄球菌中编码合成c-di-AMP所需二腺苷酸环化酶（diadenylate cyclase）的基因，在丰富培养基中培养细菌时无法被敲除，表明该培养条件下c-di-AMP对细菌生长不可或缺。本研究证实，可在化学成分限定培养基（chemically defined medium）中构建金黄色葡萄球菌dacA缺失突变株。与既往研究结果一致，该突变株在丰富培养基中呈现严重生长缺陷。基于这一生长缺陷表型，我们筛选得到生长恢复的抑制突变株（suppressor strains），以解析c-di-AMP依赖的通路。研究在alsT与opuD中发现了可绕过dacA必需性的突变：二者分别编码预测的氨基酸转运蛋白与渗透溶质转运蛋白，其中opuD被本研究证实为金黄色葡萄球菌主要的甘氨酸甜菜碱摄取系统。灭活此类转运蛋白或可阻止细胞内过量积累渗透溶质与氨基酸，进一步佐证了c-di-AMP在渗透调节中的关键作用。此外，我们还在hepS、hemB、ctaA与qoxB中获得了抑制突变，这些基因编码呼吸作用所需的蛋白。进一步实验表明，厌氧条件下dacA并非细菌生长所必需。综上，本研究揭示了c-di-AMP信号网络在金黄色葡萄球菌有氧呼吸（而非无氧呼吸）中的必需功能。