The transcriptional start site of comEA and the putative CRP-S site. (A) Sequence of the upstream region of comEA. Numbers in black below the nucleotide sequence indicate the position relative to the start codon (ATG, as indicated); numbers in red above the nucleotide sequence refer to the position relative to the transcription start site at position −24 (highlighted by the arrow and +1). The putative −10 and −35 regions are also highlighted in bold and underlined. The newly identified putative CRP-S site is centered at position −44.5 (sequence surrounded by the gray box). (B) Putative CRP-S sites identified in the upstream region of comEA and qstR and their site-directed mutagenesis. The conserved bases of the putative CRP-S site are indicated in bold (compared to the motif in silico predicted by Cameron and Redfield for other competence genes of Vibrionaceae). The putative CRP-S site upstream of comEA and qstR was entirely deleted in the constructs ΔCRP-S and ΔCRP-S_qstR, respectively. Changes made to generate the indicated CRP-S motif variants are underlined.