Table 1 Effects of mutations on promoter activity and CRP dependence

Column 1 lists the different constructions carrying mutations in the galP1 −10 element and extended −10 element. Column 2 shows the base sequence from position −20 to position +1 at each promoter, with the changes highlighted by underlining and non-bold type. The changes were originally constructed in the NM500 fragment, carrying one DNA site for CRP, but were then each transferred to the NM505 fragment carrying two DNA sites for CRP. To measure promoter activity, each fragment was cloned into plasmid pRW50 that carries the genes for resistance to tetracycline and the promoter-less lac operon [14,20]. Recombinants carrying galP1::lac fusions were then transformed into M182Δlac Δcrp cells or M182 Δlac crp+ cells. Promoter activities are expressed by β-galactosidase levels, measured as previously described [20], and are listed in columns 3–5 of the Table. Cells carrying the different pRW50 derivatives were grown aerobically at 37°C in LB (Luria–Bertani) medium supplemented with 35 μg/ml tetracycline. Values shown are the average of at least three independent assays and the values in parentheses indicate the fold stimulation by either one molecule of CRP bound at position −41.5, or a second molecule of CRP, bound upstream at position −90.5.