FIGURE 8.
The relationship between glutathione and formaldehyde sensing by FrmR. A, survival of wild type Salmonella SL1344 (solid circles) or ΔgshA (open circles) grown to mid-exponential phase in M9 minimal medium in the presence of formaldehyde. Values are means of three biological replicates (each performed in triplicate) with S.D. (error bars). B, β-galactosidase activity of SL1344 (solid symbols) or ΔgshA (open symbols) containing PfrmRA-frmR fused to lacZ grown to mid-exponential phase in M9 minimal medium in the presence of formaldehyde (MNIC = 50 and 20 μm for wild type and ΔgshA, respectively; see supplemental Fig. S2 for corresponding growth data). Values are means of at least three biological replicates (each performed in triplicate) with S.D. C, anisotropy change upon titration of a limiting concentration of frmRAPro (10 nm) with FrmR in the presence of 5 mm EDTA and 800 μm GSH in the absence (gray symbols) or presence (open symbols) of 20 μm formaldehyde. Symbol shapes represent individual experiments. 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. D, intracellular glutathione concentration in Salmonella cells following growth to exponential phase in M9 minimal medium aerobically (O2) or anaerobically with TMAO as an alternative electron acceptor. Values are means of three biological replicates with S.D. E, representative (n = 3) LC-MS chromatograms of ion transitions detected in mid-logarithmic Salmonella SL1344 cells under aerobic growth conditions. Transitions are for analyte GQVEALER (solid black line) or labeled internal standard (IS) (GQVEALER[13C6,15N4], where R[13C6,15N4] represents 13C,15N-labeled arginine) (dashed gray line). F, fractional modification by formaldehyde of FrmR (solid black line), GSH (solid gray line), or FrmR (dashed red line) and RcnR (dashed blue line; tighter limit as indicated by the blue arrow) in the presence of GSH in Salmonella cells grown anaerobically with TMAO. Formaldehyde affinities of 10−5, 10−4 (tighter limit), and 1.77 × 10−3 m (73) were used for FrmR, RcnR, and GSH, respectively. Intracellular abundance was determined for FrmR (16.1 ± 0.2 nm) and GSH (1.2 ± 0.4 mm) and estimated for RcnR, as described under “Experimental Procedures.” G, the role of glutathione in formaldehyde detoxification and sensing in Salmonella. In the absence of effector, Salmonella FrmR represses the frm promoter. Formaldehyde directly modifies FrmR (reaction 1 in Fig. 1) via a deduced intersubunit methylene bridge between Pro2 and Cys35 (Fig. 5, up to four per tetramer) derepressing frm expression. GSH inhibits formaldehyde detection (reaction 3 in Fig. 1), and despite the high [glutathione], the affinity of FrmR for formaldehyde is sufficiently tight relative to GSH to enable expression of FrmA to coincide with the appearance of its substrate. The Salmonella frm operon lacks frmB, and YeiG may catalyze the final detoxification step. S-HMG, S-(hydroxymethyl)glutathione.