Figure 3.

Adaptive mutant LiaR^D191N that confers increased daptomycin resistance in E. faecalis S613 dramatically increases LiaR affinity for target DNA sequences. E. faecalis response regulator LiaR–DNA interactions were measured with MST. To determine the K_d, increasing concentrations of LiaR^D191N was added to 40 nM of fluorescently labeled DNAs (Supplementary Information). F_norm (normalized fluorescence) was plotted on the y-axis in per mil [‰] unit (meaning every 1000) against the total concentration of the titrated partner on a log₁₀ scale on the x-axis (21). The resulting K_d values based on average from six independent MST measurements. Note, when the markers were increased in size for readability the error bars became covered in some cases. (A) The binding of LiaR^D191N (magenta diamonds), LiaR^D50E (purple circles), LiaR^D50A (blue triangles), LiaR^D50E/D191N (green triangles) and LiaR^DBD(D191N) (red squares) to the consensus sequence within the liaXYZ operon or LiaR^D191N with a random sequence (black circles). (B) The binding of LiaR^D191N (magenta diamonds), LiaR^D50E (purple circles), LiaR^D50A (blue triangles), LiaR^DBD(D191N) (red squares), LiaR^D50E/D191N (green triangles) to the extended site of the liaFSR operon. (C) The binding of LiaR^D191N to the entire contiguous protected regions (Figure 2B, D) of liaXYZ (cyan diamonds) and liaFSR (magenta circles).