Additional file 12: Figure S6. of Phosphoproteome dynamics mediate revival of bacterial spores

Characterization of SspA-S6,S9,S58 phosphorylation mutants. (A) Spores of PY79 (wild type, WT), AR211 (sspA-S6A,S9A,S58A), and AR212 (sspA-S6D,S9D,S58D) strains were incubated in S7 defined medium supplemented with L-Ala (10 mM) at 37 °C, and optical density (OD600) was measured at the indicated time points. Data are presented as a fraction of the initial OD600 of the phase-bright spores. Decreasing OD600 signifies spore germination while increasing OD600 indicates spore outgrowth. The data points are averages of results obtained from three independent biological repeats. Error bars designate SD. (B) Spores of PY79 (wild type, WT), AR211 (sspA-S6A,S9A,S58A), and AR212 (sspA-S6D,S9D,S58D) strains were exposed to increasing UV (254 nm) doses (mj/cm2) and plated on LB for viable count. Percentage survival was calculated by dividing the viable spore titer at any given UV dose (mj/cm2) with the spore titer obtained from the non-irradiated spores. The data points are averages of results obtained from three independent biological repeats. Error bars designate SD. (C) Spores of PY79 (wild type, WT), AR211 (sspA-S6A,S9A,S58A), and AR212 (sspA-S6D,S9D,S58D) strains were germinated with L-Ala (10 mM). Samples were taken at the indicated time points, irradiated with 500 mj/cm2 UV (254 nm) and plated on LB. Percentage survival was calculated by dividing the viable spore titer at any given time point with the spore titer obtained from spores irradiated at the 0 time point. The data points are averages of results obtained from three independent biological repeats. Error bars designate SD. (D) Spores of AR186 (∆sspB), AR191 (sspA-S6A,S9A,S58A, ∆sspB), and AR192 (sspA-S6D,S9D,S58D ∆sspB) revival was followed as described in (A). (E) UV resistance of spores of AR186 (∆sspB), AR191 (sspA-S6A,S9A,S58A, ∆sspB), and AR192 (sspA-S6D,S9D,S58D, ∆sspB) was determined as described in (B). (F) UV resistance of AR186 (∆sspB), AR191 (sspA-S6A,S9A,S58A, ∆sspB), and AR192 (sspA S6D,S9D,S58D, ∆sspB) germinating spores was determined as described in (C). (PDF 112 kb)