Journal: Journal of Bacteriology
Article Title: The Transcriptional Regulator HlyU Positively Regulates Expression of exsA, Leading to Type III Secretion System 1 Activation in Vibrio parahaemolyticus
doi: 10.1128/JB.00653-17
Figure Lengend Snippet: Electrophoresis mobility shift assay (EMSA) and DNA footprinting assays indicate HlyU binding to a region within the exsA promoter. (A) Six percent TBE-polyacrylamide gels were loaded with reaction mixtures containing exsA promoter DNA and increasing amounts of purified HlyU-His or bovine serum albumin (BSA). An exsA promoter DNA-only control was included as indicated. The white box indicates slow-migrating, concentrated DNA species. (B) A 6% TBE-polyacrylamide gel was loaded with reaction mixtures containing nleH1 DNA and increasing amounts of purified HlyU-His. Following electrophoresis, gels were stained with SYBR green to specifically stain DNA. (C) A DNase I footprinting assay using purified HlyU-His was used to identify a DNA binding region for HlyU-His within the exsA promoter. The approximate base pair numbers of the HlyU-His footprint region are indicated (259 to 315 bp) and are based on capillary electrophoresis internal size standards. The DNA sequence associated with the identified HlyU protected region is displayed below the chromatogram. A 7-bp inverted repeat (labeled in blue) and separated by 14 bp is centrally located within the HlyU-His protected region.
Article Snippet: Purified HlyU-His or BSA (B9000S; New England BioLabs) was mixed with a PCR-amplified exsA promoter DNA fragment in binding buffer (10 mM Tris [pH 7.5 at 20°C], 1 mM EDTA, 0.1 M KCl, 0.1 mM dithiothreitol, 5%, vol/vol, glycerol, 0.01 mg ml−1 BSA).
Techniques: Electrophoresis, Mobility Shift, DNA Footprinting, Binding Assay, Purification, Staining, SYBR Green Assay, Footprinting, Sequencing, Labeling