Heterologous expression of B. subtilis yloC in E. coli disrupts RyhB sRNA regulation. (A) Schematic of the sodB translational reporter fusion for monitoring RyhB sRNA activity in E. coli (Chen et al. 2021). (B) Imaging colony fluorescence on an agar plate. (VA) Vector alone, (B.F.) bright field, (mCherry) fluorescence. (C) Quantitation of mCherry fluorescence; an average of four biological repeats. (****) P < 0.0001. (D) The same set of strains was subjected to northern blot analysis of RNA, with probing for RyhB RNA and the control SsrA RNA, as well as total protein extraction and Coomassie staining (Supplemental Fig. S5). (E) Relative levels of full-length RyhB were quantitated from two independent experiments, normalized to the SsrA loading control first, and then further compared with that of the vector control. Unpaired two-tailed Student's t-test was used to calculate statistical significance. (**) P < 0.01. (F) Bacterial two-hybrid test for YloC interaction with PNPase. The reporter strain BTH101 was cotransformed with plasmids T18 and T25 fused to PNPase, YicC, or YloC and assayed for β-galactosidase activity. Data are the mean and standard deviation of biological triplicates. Two-way ANOVA was used to calculate statistical significance. (ns) Not significant, P > 0.05; (****) P < 0.0001.
