FIG 4.

SigS and SigS antisense Northern blot analysis in the context of SroAB activity. (A) Overnight cultures of SH1000 carrying pEPSA5, pEP-sigS, pEP-sroAB, pEP-sroA, or pEP-sroB were subcultured in 30 mL of TSB-Cm and grown to an OD₆₀₀ of 0.3. Cultures were induced with xylose to a final concentration of 2% for 30 min, and total RNA was isolated. Total RNA (3 μg) was analyzed by Northern blotting using biotin-labeled probes for 16S rRNA, SigS mRNA, SigSa (sigS antisense RNA resulting from the 3′ UTR of SAOUHSC_01899), SroA mRNA, and SroB mRNA. (B) Bacterial two-hybrid (BACTH) analysis of SroA and SroB. The sroA and sroB genes were cloned into BACTH plasmids pEB355 (T18) and pEB354 (T25) and analyzed on MacConkey-maltose agar plates at 30°C along with empty vector controls and positive controls pEB355-zip (T18-zip) and pEB354-zip (T25-zip).