Fig 9. A GerSD106R mutation affects the stability of GerS protein and leads to slower germination.
(A) Spore germination as measured by the change in OD600 in response to germinant in gerSD106R and cwlDR169D mutant spores relative to wild-type spores. Purified spores were resuspended in BHIS, and germination was induced by adding taurocholate (1% final concentration). The ratio of the OD600 of each strain at a given time point relative to the OD600 at time zero is plotted. The mean of three assays from 3 independent spore preparations are shown. Shading represents the standard deviation as the area between error bars for each time point measured. Statistical significance relative to wild-type was determined using two-way ANOVA and Tukey’s test. ***, p< 0.0005; **** p < 0.0001. (B) Germination efficiency of gerSD106R and cwlDR169D point mutant spores. The number of colony forming units (CFUs) produced by germinating spores is shown. The mean OD600 determined from three independent spore preparations is shown along with the associated standard deviations. Statistical analyses relative to the wild-type were performed using one-way ANOVA and Tukey’s test. **, p < 0.005; ****, p < 0.0001. (C) Western blot analysis of CwlD and GerS levels in sporulating cells from the wild-type (WT), ΔgerS, ΔgerS complemented with either gerS, or gerS encoding either wild-type or D106R FLAG-tagged GerS variants as well as ΔcwlD, ΔcwlD complemented with either cwlD encoding either wild-type or R169D GerS variants. CwlD-c and GerS-c indicate cleaved form of both wild-type proteins; asterisks indicate the cleaved form of FLAG-tagged. Δspo0A (Δ0A) was used as negative control for sporulating cells, and SpoIVA was used as a loading control. GerS and CwlD levels were analyzed using GerS, CwlD and FLAG antibodies. (B) Western blot analysis of CwlD and GerS levels in spores from the strains used in the experiments described in (C). SleC was used as a loading control.