FIGURE 6.

Bacterial-one-hybrid assays showing interaction between expR promoter (R1, R4) and QsrR which was potentiated by WSHM. (A) Schematic diagram of bacterial one-hybrid system. The HIS3/URA3 cistron is in the pH3U3 prey vector. The target DNA sequences were ligated to pH3U3. The binding of DNA-binding domain (DBD, ligated to pB1H1) with the target DNA sequence enables the transcription of HIS3 and URA3. The expression of HIS3 enabled E. coli USO grow on NM selective medium containing the desired concentration of 3-amino-triazole (3-AT), a competitive inhibitor of HIS3. The expression of URA3 prevented E. coli USO growth on medium containing 5- fluoroorotic acid (5-FOA). This figure was adopted from Meng et al. (2005). (B) Schematic diagram of the promoter region of gene expR which was cloned into pH3U3. “+1” in red was marked as the transcription start site of expR which was located in 87 bp upstream of ATG of expR (Charoenpanich et al., 2013). Regions R1, R2, and R4 were ligated to pH3U3 prey vector to detect the binding region of QsrR. Region R1 is the entire intergenic region between expR and SMc03900. Region R2 is the middle region of R1 which lost the 87bp right flank and the 76bp left flank of R1. Region R4 is the left flank of R1 (142 bp). The blue arrows indicate transcriptional direction of expR and SMc03900. Region R2 was excluded from further analyses because of the high level self-activation of (E. coli USO: pB1H1/pH3U3-R2) (see Materials and Methods for details). (C) The interaction of QsrR and pexpR was assayed by the growth of bacteria on selective medium (NM lacking histidine) supplemented with 2, 3, or 5 mM of 3-AT. The genetic information of test strains is labeled on the left. “+/–” indicated that qsrR gene was cloned into pB1H1 (+) or the plasmid was left empty (–), and similar information for pH3U3. Images were assembled and the marked lines were eliminated without modifying the observed data in Adobe Photoshop CS5.