FIG. 4.
In vitro transcription of the ctaA promoter with RNAP from stage T0, T3, T4, or T5 cells and with ResD or ResD∼P suggests two promoters. (A) In vitro transcription of the ctaA promoter (224-bp EcoRI fragment from pXH43) with B. subtilis RNAP isolated at stage T0 (lanes 1 to 7) or T3 (lanes 8 to 12) from cells cultured in SSG. Phosphorylation of ResD and in vitro transcription reactions were carried out as described in Materials and Methods. Samples were separated electrophoretically on 8 M urea–6% polyacrylamide gels. WT, wild type; M, 100-nt RNA marker; −, absent; +, present. ResD concentrations are given above the sample lanes. (B) Enhancement of ctaA promoter expression from RNAP isolated from cells at the T4 (lanes 2 to 4) or T5 (lanes 5 and 6) stage of growth in SSG medium and with ResD or ResD∼P. Arrows at the right indicate the ctaA upstream (≈ 100-nt) and downstream (≈ 80-nt) promoter transcripts. In vitro expression of the upstream ctaA promoter (lane 7) using RNAP isolated from a Spo0A mutant strain is shown. All the reaction procedures were the same as those mentioned above.