Figure 5.

Biotin-regulated repressor activity of SaBPL and mutants. (A) In vivo expression assays were performed in an E. coli reporter strain containing chromosomally integrated repressor and promoter constructs [23]. LacZ units were calculated by subtracting the value generated by the control strain containing no integrated promoter construct (LacZ unit ≤10). Expression of the β-galactosidse reporter gene, under the control of promoters (B) bioO or (C) bioY, was measured in media containing varying concentrations of biotin. Transcription factors SaBPL (orange), SaBPL D200E (green) and SaBPL F123G (black) were analyzed alongside a control strain that harbored no repressor protein (blue). Error bars represent SEM from independent biological replicates (n = 6). The amount of biotin to reach half-maximum repression (K_{R biotin}) was calculated from the curves using GraphPad Prism. The K_{R biotin} for wildtype SaBPL repressing bioO and bioY was 4.3 ± 1.9 nM and 8.2 ± 0.7 nM, respectively. The K_{R biotin} for SaBPL-D200E repressing bioO and bioY was 13.9 ± 3.4 nM and >500 nM, respectively. The K_{R biotin} for SaBPL-F123G repressing bioO and bioY was 15.3 ± 3.5 nM and >500 nM.