Figure 5.

Glutamate supplementation at the onset of sporulation suppresses ΔrocR ΔahrC germination deficiency

(A) LR33 (Parental), LR38 (ΔrocR ΔahrC), LR137 (ΔrocG) strains, lacking gudB, were grown in DSM sporulation medium with or without 2.5 mM glutamate. After 20 hrs of incubation, spores were purified, induced to germinate on an agarose pad supplemented with L-Ala (10 mM) (t = 0), and followed by time-lapse microscopy. Shown are phase contrast images captured at the indicated time points from a representative experiment, out of three independent biological repeats. Graphs on the right show the quantification of the percentages of the initial number of the phase bright spores (n ≥ 300 for each strain). Scale bar, 1 μm.

(B) LR33 (Parental), LR38 (ΔrocR ΔahrC), LR137 (ΔrocG) strains, lacking gudB, were grown in DSM sporulation medium with or without 2.5 mM glutamate. After 20 hrs of incubation, spores were purified, induced to germinate by L-Ala (10 mM) (t = 0), and germination was followed by DPA release, monitoring the relative fluorescence units (RFU) of Tb³⁺-DPA. Shown is a representative experiment out of three independent biological repeats.

(C) LR33 (Parental), LR38 (ΔrocR ΔahrC) strains, lacking gudB, were grown in DSM sporulation medium with or without 2.5 mM glutamate. After 20 hrs of incubation, spores were purified and analyzed for ATP level using ATP Bioluminescence Detection Kit (Promega). Shown are average values and SD obtained from three independent biological repeats.