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. 2018 Aug 3;9:1799. doi: 10.3389/fmicb.2018.01799

FIGURE 4.

FIGURE 4

Site-directed mutational analysis of the promoter region of sptA. (A) Sequences of the promoter regions of the construct D215 and its mutants. The BRE motif, TATA box, and WW motif are boxed. The position of the transcriptional start site (+1) is indicated. The inverted repeat sequences (IR1f/IR1r, IR2f/IR2r, and IR3f/IR3r) are indicated by arrows. The region from bp –51 to bp –43 is shaded. The regions of three semi-palindromic sequences (SPS1, SPS2, and SPS3) are shown. In the sequences of the mutants (M1 to M3-6), the mutated and unaltered nucleotides are shown in letters and dashes, respectively. (B) Promoter activity assay. The ΔsptA2 mutants harboring the recombinant plasmids were grown in 23% MGM with 5 μg ml-1 mevinolin at 37°C. The mid-log and stationary phase culture supernatants were subjected to azocaseinolytic activity assay. The levels of sptA transcripts of mid-log and stationary phase cells were determined by qRT-PCR analysis using 16S rRNA as an internal control, and the transcript levels of the mutants were determined relative to that of construct D215 at mid-log phase. Values are expressed as the means and standard deviations (error bars) of three independent experiments.