FIGURE 2.

Analysis of SraL sRNA regulation by RpoS. (A) SraL expression is dependent on RpoS. (Upper panel) SraL levels were detected by Northern blot using 15 μg of total RNA isolated at indicated time points during growth from S. Typhimurium wild-type (wt) and rpoS mutant strains and rpoS mutant strain carrying a constitutive plasmid expressing the wild-type rpoS allele (pISVA-002); 5S rRNA was used as loading control. (Lower panel) RpoS protein expression was monitored by Western blot analysis. Samples correspond to 5 × 10⁷ bacteria at the indicated time points. GroEL was used as a loading control. (B) Transcriptional activity of sraL promoter in late stationary phase. Samples from overnight grown cultures of wild-type SL1344 and the isogenic rpoS null mutant transformed with a plasmid expressing the transcriptional fusion of sraL promoter to lacZ reporter gene (pISVA-003) were used to assess β-galactosidase activity. (C) Transcriptional activity of sraL promoter upon osmotic shock. Bacterial cultures were grown to reach exponential growth phase (OD 0.3), and then NaCl was added to a final concentration of 0.5 M and let grow for one more hour. β-Galactosidase activity was measured before (t₀, white bar) and after the treatment (+NaCl, black bar). A control culture with no addition of NaCl was also carried in parallel (control, dashed-light gray bar). Bars correspond to the mean ± SD of three biological replicates. ***P < 0.001 by Student’s t-test; n.s. indicates nonsignificant.