Figure 3.

S. aureus dacA suppressor strains do not produce c-di-AMP but acquire mutations in genes coding for transport or respiration-related proteins. A, determination of cellular c-di-AMP levels by LC-MS/MS. Cell extracts (three biological replicates) were prepared from the suppressor strains dacA-S1, dacA-S2, and dacA-S4, and the production of c-di-AMP was assessed by LC-MS/MS. The extracts were prepared and analyzed at the same time as WT LAC* and the low-level c-di-AMP dacA_G206S strain as reported in a previous study (20). No c-di-AMP could be detected in extracts derived from strains dacA-S1, dacA-S2, and dacA-S4, and c-di-AMP levels determined for the WT and dacA_G206S mutant as part of the previous study (20) are shown as controls. B and C, schematic representation of proteins whose genes were found to be mutated in dacA suppressor strains and their predicted cellular functions. B, schematic representation and function of the OpuD and AlsT transporters. OpuD is a predicted (and as part of this study experimentally confirmed) glycine betaine and a reported weak proline transporter (43); AlsT is a predicted l-alanine/sodium symporter, but this substrate specificity could not be confirmed as part of this study. C, simplified view of the aerobic respiratory chain in S. aureus. The NADH dehydrogenase oxidizes NADH to NAD⁺, and electrons are transferred onto MQ to yield MQH₂. The electrons are subsequently moved onto the heme A component of the cytochrome aa₃ complex, composed of the QoxA, -B, -C, and -D proteins. Upon reduction of ½O₂ to H₂O by the cytochrome complex, protons (H⁺) are extruded and utilized by the F₁F₀-ATPase for the production of ATP. Suppressor mutations were identified in genes coding for HepS, HemB, CtaA (a membrane protein but shown for simplicity as a soluble protein), and QoxB, required for MQ, HemA, and cytochrome aa₃ complex formation, respectively. The proteins for which gene variations were found in suppressor strains are indicated in purple in the schematics.