Figure 3

Role of AcsR in acsA gene expression. A - Expression of a transcription reporter fusion of acsA. Wild-type and ΔacsR strains carrying an acsA::luxAB transcription fusion were grown in AB medium supplemented with 3 μg/ml tetracycline, and luminescence produced by each strain was determined using a luminometer during the whole growth period. The specific luciferase activities were presented by plotting the normalized values as relative light units (RLU) per biomass (OD₅₉₅); B - Gel-shift assay for binding of rAcsR to the regulatory region of acsA. A labeled 284-bp DNA fragment including an upstream region of acsA was incubated with various concentrations of rAcsR (0.5 ~ 5 μM). The reaction mixtures were separated on a 6% non-denaturing polyacrylamide gel. Phosphorylated rAcsR was used in another set of gel-shift assays. rAcsR (60 μg/ml) was incubated with for 1 h at 30°C in a buffer containing 100 mM Tris-HCl (pH 7.0), 10 mM MgCl₂, 125 mM KCl, and 50 mM dilithium acetyl-phosphate (Sigma); C – Competition assay for AcsR-acsA promoter DNA. For competition analysis, the identical but unlabeled DNA fragment (competitor DNA) was included in the reactions with 1 μM rAcsR at concentrations at 760 nM. As a nonspecific control, the gap DNA was added into the binding reaction at 760 nM.