Fig. 2.

Dephosphorylation of the translational factor Tig is required for spore germination. (A) Spores of PY79 (WT), BZ98 (tig-R45A), BZ97 (tig-R45D), and LS38 (∆tig) strains were incubated on agarose supplemented with l-Ala (10 mM) and monitored by time lapse microscopy. Shown are phase contrast images from a representative experiment out of three independent biological repeats. (Scale bar: 1 μm.) (B) Spores of PY79 (WT), BZ98 (tig-R45A), BZ97 (tig-R45D), and LS38 (∆tig) strains were incubated on agarose supplemented with l-Ala (10 mM) and monitored by time lapse microscopy. Data are presented as percentages of the initial number of the phase-bright spores. Shown are average values and SDs obtained from three independent biological repeats (n ≥ 300 for each strain). (C) Spores of LS5 (ΔmetE), BZ103 (ΔmetE, tig-R45A), BZ102 (ΔmetE, tig-R45D), and LS80 (ΔmetE, ∆tig) strains were induced to germinate with l-Ala in the presence of AHA for 30 min. Shown is a dot blot analysis of protein samples (in duplicate) that were collected before (t = 0, dormant spores) and after (t = 30 min) germination induction. Samples (1:1 and 1:10 dilution) were spotted on a membrane that was subsequently probed with antibiotin antibodies. The obtained signal was compared with known amounts of biotinylated BSA. (D) Protein extracts from vegetative cells, dormant and germinating spores of strains: (i) AR71 (WT), BZ107 (tig-R45A), and BZ106 (tig-R45D) carrying malS-gfp; (ii) BZ118 (WT), BZ120 (tig-R45A), and BZ119 (tig-R45D) carrying rpsB-mCherry; and (iii) AR68 (WT), BZ144 (tig-R45A), and BZ145(tig-R45D) carrying pupG-gfp were subjected to Western blot analysis using an antibody against GFP or mCherry, respectively. Equal amounts of protein extracts were loaded. Germination was induced by suspending the spores in 10 mM l-Ala for 10 min.