FIGURE 2.

Regulation of T6SS1 genes by AphA. The negative and positive numbers represent the nucleotide position upstream and downstream of each target gene, respectively. Lanes C, T, A and G represent the Sanger sequencing reactions. (A) qRT-PCR. The relative mRNA level of each target gene was compared between ΔaphA and WT. (B) Primer extension. An oligonucleotide primer was designed to be complementary to the RNA transcript of each target gene. The primer extension products were analyzed with an 8 M urea -6% acrylamide sequencing gel. The transcription start sites were indicated by the arrow with nucleotide and position. (C) LacZ fusion assay. The entire promoter-proximal region of each target gene was cloned into pHRP309, and then transformed into WT or ΔaphA to determine the β-galactosidase activity (miller units) in cellular extracts. (D) EMSA. The entire promoter-proximal region of each target gene was incubated with increasing amounts of purified His-AphA protein, and then subjected to 6% (w/v) polyacrylamide gel electrophoresis. Shown below the binding was the schematic representation of the EMSA design.