Extended Data Fig. 7 |. An acute increase in c-di-AMP levels causes membrane invaginations independent of phospholipid biosynthesis.

Representative images of B. subtilis cells harboring Phy-cdaS(L44F). Cells were induced with 500 μM IPTG for 10 min and then treated with cerulenin (12.5 μg/mL) to block phospholipid synthesis for 5 min prior to harvest. Images were acquired ~ 5 minutes later due to cell concentration, mounting, and imaging. The delay in cerulenin addition (10 min after IPTG-induction) was chosen based on control experiments showing that the drug rapidly inhibits transcription from an IPTG-regulated promoter (Supplementary Fig. 6). Importantly, no membrane invaginations are detectable at 10 min after IPTG addition (Extended Data Fig. 6). Thus, cerulenin treatment at 10 minutes enabled cdaS(L44F) expression and inhibition of phospholipid biosynthesis prior to the generation of membrane invaginations. Membrane accumulation did not increase over the next 75 minutes in cerulenin treated cells, suggesting that the severity of the membrane invaginations is partially due to uncoupled phospholipid synthesis from growth. Cells constitutively expressed cytoplasmic mCherry and were stained with TMA-DPH to label membranes. Sites of membrane invagination in the presence or absence of cerulenin are highlighted (yellow carets). Scale bar indicates 5 μm.