RpoE, AphA, and LuxR bind to the <i>luxR</i> promoter.

Abstract

<p>(<b>A</b>) EMSA and DNase I footprinting analysis were used to analyze whether LuxR and AphA specifically bind to the <i>luxR</i> promoter. In EMSA assays, various concentrations of LuxR and AphA proteins were added to mixtures of poly(dI:dC) and Cy5-labeled <i>luxR</i> promoter DNA. In the DNase I footprinting assays, electropherograms of a DNase I digest of the P<i>luxR</i> promoter probe after it was incubated with or without the LuxR and AphA proteins. The level at which the respective nucleotide sequences were protected by the proteins is indicated below. (<b>B</b>) Diagram the showing promoter region of the <i>luxR</i> gene. The LuxR and AphA binding sites and the ribosome binding site (RBS) are underlined. The region protected by RpoE (RpoEB) is shadow-boxed. The well-conserved nucleotides that were bound to specific proteins are indicated with asterisks. The transcription start site (TSS) is labeled as +1. (<b>C-D</b>) EMSA assays of LuxR and RpoE mixtures binding to <i>luxR</i> promoter or mutant <i>luxR</i> promoter regions in the absence of the LBSI (P_<i>luxR</i>ΔLBSI) or LBSII binding site (P_<i>luxR</i>ΔLBSII). The amounts of LuxR or RpoE protein (nM) that were used are as indicated, and 20 ng of each Cy5-labelled probe was added to the EMSA reactions. (<b>E</b>) The promoter activity is shown for P_<i>luxR</i> and its variants that were fused to <i>lacZ</i>. The strains were grown at 22°C, 30°C, 37°C and 42°C for 9 h, and <i>β</i>-galactosidase activity was then assayed. The results are shown as the mean ± S.D. (<i>n</i> = 3).</p