Induction of NhaR, GadE, and GadW by YdeO in E. coli.

YY5002 (gadE-3xflag) (a), YY5003 (gadW-3xflag) (b), and BW25113, (c) harboring pTrc99A (−) or pYdeO (+), were grown in LB medium until logarithmic phase. After centrifugation the lysate solution was prepared in lysis buffer containing 8 M urea by sonication. The lysates were subjected to western blotting as described in Materials and Methods. Anti-FLAG (SIGMA) and anti-NhaR (Lab preparation) were used for detection of GadE-3xFLAG/GadW-3xFLAG, and NhaR, respectively [A]. The amounts of GadE-3xFLAG, GadW-3xFLAg, and NhaR were represented as the ratio of level of RNA polymerase-α subunit, detected by anti-α (Neoclone) [B].