Fig. 1
Regulation of toxR by QsvR. V. parahaemolyticus strains were grown in HI broth at 37 °C, and bacterial cells were harvested at an OD600 value of 0.8. Negative and positive numbers indicate the nucleotide positions upstream and downstream of toxR, respectively. a qPCR. Relative mRNA levels of toxR were tested in WT and ΔqsvR strains. b Primer extension. An oligonucleotide primer complementary to the toxR RNA transcript was designed. The primer extension products were analyzed with an 8 M urea-6% acrylamide sequencing gel. c LacZ fusion. The promoter-proximal DNA region of toxR was cloned into the pHRP309 plasmid and then transferred into WT and ΔqsvR strains to determine promoter activity (Miller units) in the cellular extracts. d EMSA. The radioactively-labelled promoter-proximal DNA fragments of toxR were incubated with increasing amounts of His-QsvR and analyzed using 4% (w/v) polyacrylamide gel electrophoresis. e DNase I footprinting. Labelled coding or noncoding DNA probes were incubated with increasing amounts of His-QsvR and analyzed using DNase I footprinting. The footprint regions are indicated by vertical bars at the corresponding sequence positions. Lanes C, T, A and G represent Sanger sequencing reactions