Figure 9.

Mutations in the FapR malonyl-CoA binding pocket are deleterious for cell growth. (A) Fluorescence anisotropy changes upon addition of FapR_R106A to 9.5 nM 34 bp F-dsDNA (black circles); on addition of FapR_G107V,L128W to 9.5 nM 34 bp F-dsDNA (white circles); on addition of malonyl-CoA to the previously formed F-dsDNA/FapR_WT complex (gray triangles); on addition of malonyl-CoA to the previously formed F-dsDNA/FapR_R106A complex (black triangles); and on addition of malonyl-CoA to the previously formed F-dsDNA/FapR_G107V,L128W complex (white triangles). (B) Plasmids were constructed in which fapR_WT, fapR_R106A or fapR_G107V,L128W were under the control of a xylose-inducible promoter (Pxyl) and introduced into a strain, GS364, that was a mutant for fapR and contained a PfabHA-lacZ fusion. The strains were grown on LB solid medium in the absence and in the presence of xylose (1%) (C) β-galactosidase activities from PfabHA-lacZ in strain GS364 expressing fapR_WT,fapR_R106A or fapR_G107V,L128W. To induce expression of the fapR alleles, the different strains previously grown on LB agar solid medium, were resuspended in LB medium supplemented with xylose 1% to an OD₅₂₅ of 0.1. After two hours of growth at 37°C, samples were collected and β-galactosidase activities determined.