FIG 5.
SigS mRNA turnover following overexpression of SroA. (A) Northern blot analysis of wild-type SH1000 carrying either pEPSA5 or pEP-sroA grown in 30 mL of TSB-Cm supplemented with MMS to a final concentration of 25 mM and an OD600 of 0.3. Cultures were then induced with xylose to a final concentration of 2% and incubated for an additional 30 min. Cells were harvested and resuspended in fresh TSB-Cm supplemented with rifampin to a final concentration of 500 μg/mL, and total RNA was isolated at 5- to 15-minute intervals. Total RNA (3 μg) was analyzed by Northern blotting using biotin-labeled probes for 16S rRNA and SigS mRNA. (B) Densitometry analysis of Northern blotting. (C) Calculation of the half-life (t1/2) of SigS mRNA based on densitometry analysis in panel B. Quantitative Northern blotting data from panel B were subjected to linear regression analysis using GraphPad Prism 9. Experiments were repeated at least three times, and data are presented as the mean plus or minus the standard error of the mean.