FIG. 10.

Analysis of the dctA transcript. (A) Northern blotting. Total RNA was extracted from MC1000 (wild type [wt]), JRG1999 (crp), and JRG1999 transformed with pGS279 (crp⁺). Strains were grown aerobically in L broth with or without 1% glucose (Glu). Following electrophoresis and capillary transfer, RNA was hybridized with a labeled dctA fragment. The arrow on the right indicates the specifically hybridizing dctA transcripts. The size of the dctA mRNA transcript (in kilonucleotides) is indicated. (B) Determination of the dctA transcriptional start site by reverse transcriptase-mediated primer extension. RNA was isolated from MC1000 grown aerobically in L broth. Lane P indicates the primer extension product with primer P2_dctA. Similar results were obtained with primer P1_dctA (results not shown). The sequencing ladder (lanes A, C, G, and T) was generated by using primer P2_dctA and pGS753 as template. The corresponding nucleotide sequence, its complement, and the transcriptional start site (indicated by an arrow) are shown. (C) Nucleotide sequence of the dctA promoter region. Coordinates are from reference 51. The experimentally determined +1 site is boxed and labeled, as are the deduced −35 and −10 sites and the predicted CRP site. Residues matching the corresponding consensus sequence for the CRP, −35, −10, +1, and Shine-Dalgarno (S-D) sites are in bold and underlined. The positions of the predicted start codon of dctA and termination codon of f651 are in bold.