FIGURE 6.
Pro2 and Cys35 are required for formaldehyde sensing by FrmR. A, LC-MS chromatogram following multiple-reaction monitoring of purified FrmR. Ion transition 405.19/488.24 is for analyte PHSPEDK. B, β-galactosidase activity in SL1344 containing PfrmRA-frmR (circles), PfrmRA-frmRC35A (triangles), or PfrmRA-frmRP2S (squares) fused to lacZ grown to mid-exponential phase in M9 minimal medium in the presence of formaldehyde (MNIC = 50 μm). Values are means of three biological replicates (each performed in triplicate) with S.D. C and D, anisotropy change upon titration of a limiting concentration of frmRAPro (10 nm) with FrmRC35A (C) or FrmRP2S (D) in the presence of 5 mm EDTA (closed symbols) and with the addition of 20 μm formaldehyde (open symbols). Data were fit to a model describing a 2:1 protein tetramer (nondissociable)/DNA stoichiometry (binding with equal affinity) (50, 86), and lines represent simulated curves produced from the average (apparent) KDNA determined across the experimental replicates shown. Symbol shapes represent individual experiments. E, apparent KDNA values of FrmR (black symbols), FrmRP2S (gray symbols), and FrmRC35A (open symbols) with increasing formaldehyde concentration. Values are means of three replicates with S.D. (error bars).