Figure 6.

ATP generated by RocG-mediated glutamate catabolism is stored in spores for future germination

(A and B) Spores of LR33 (Parental), LR38 (ΔrocR ΔahrC), LR207 (ΔrocR ΔahrC, amyE::rocR-ahrC) strains, lacking gudB, were disrupted for ATP extraction. ATP levels were determined using an ATP bioluminescence assay kit (Promega), referring to a standard curve of known ATP concentrations (A). The ATP levels in spores are the total ATP in extracts (nmol)/dry weight of spores (g) (B). Shown are average values and SD obtained from three independent biological repeats.

(C) Upper panel: shows a schematic description of ATP detection in vivo from the forespore compartment during sporulation by luciferase-catalyzed luminescence reaction (Parrell and Kroos, 2020). Lower panel: LR209 [Parental (P_spoIIQ-luc)], LR227 [Mut (PspoIIQ-luc, P_IPTG-rocG, ΔrocR ΔahrC)] strains were induced to sporulate in DSM medium. At t₀ of sporulation, IPTG was added to induce rocG expression, and samples were subjected to luminescence assay at the indicated time points. Shown are luminescence values in arbitrary units (AU) normalized to total luciferase levels measured by Western blot analysis.

(D) Spores derived from the cultures described in (C) were purified, induced to germinate by L-Ala (10 mM) (t = 0), and germination was followed by monitoring the relative fluorescence units (RFU) of Tb³⁺-DPA. Shown is a representative experiment out of three independent biological repeats.