FIGURE 4.

RacR binds to a specific region on the ggt promoter. (A) Nucleotide sequence and features of the ggt promoter. The start codon ATG is indicated in bold face, the putative -10 and -16 regions and ribosomal binding site (RBS) are underlined. A palindromic sequence is indicated with a horizontal bar. The previously identified RacR binding consensus sequence (van der Stel et al., 2015) is indicated above the predicted RacR binding site, vertical lines indicate matching nucleotides. Arrows indicate the 5′ termini and direction of the primers used to generate the ggt promoter elements for the luciferase reporter plasmids and EMSA bait DNA. The transcriptional start site of the ggt gene identified by (Dugar et al., 2013) is indicated with a hooked arrow. (B) Luciferase activities using different lengths of the region upstream of the ggt gene are determined in wt and racRS::Cm mutant bacteria. Cultures were grown until late-log phase at oxygen limiting conditions with the addition of nitrate. Data represents the mean and SE of three independent experiments. (C) EMSA experiments using the different ggt promoter elements. DIG-labeled PCR fragments (~50 fmol) were mixed with or without 50 pmol RacR and 25 pmol RacScyto in the presence of ATP. RacR-P phosphorylated RacR.