Fig. 2 |. c-di-AMP levels increase in response to impaired cell wall synthesis.

a, Schematic of the c-di-AMP-binding riboswitch reporter used to monitor c-di-AMP levels. Cyclic-di-AMP binds the kimA RNA hairpin, generating a transcriptional terminator upstream of lacZ. b, Bar graph showing β-galactosidase activity from the riboswitch reporter in the indicated B. subtilis strains lacking the native cyclases and harbouring an IPTG-regulated allele of cdaAR. Cells were grown in LB with the indicated IPTG concentrations for >5 generations before assaying. c, Bar graph showing c-di-AMP levels quantified by ELISA and normalized to OD₆₀₀ in the same conditions as in b. d, Bar graph showing β-galactosidase activity of the riboswitch reporter in the presence (+) or absence (Δ) of PBP1 or treated with (+) 5 μg ml⁻¹ moenomycin (MOE). All cells lack disA, cdaS and cdaAR, and harbour an IPTG-regulated allele of cdaAR expressed with 50 μM IPTG. e, Representative immunoblots of B. subtilis cells expressing CdaA-His under IPTG-inducible control with 50 μM IPTG in the presence (+) or absence (Δ) of PBP1. SigA controls for loading. Molecular weight markers in kDa are indicated. f, Bar graph showing β-galactosidase activity from the riboswitch reporter in the indicated PBP1 depletion strain, with different concentrations of IPTG. Data are means ± s.d. of 3 biological replicates (b,c,d,f).