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The Effectors and Sensory Sites of Formaldehyde-responsive Regulator FrmR and Metal-sensing Variant *

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ABSTRACT

The DUF156 family of DNA-binding transcriptional regulators includes metal sensors that respond to cobalt and/or nickel (RcnR, InrS) or copper (CsoR) plus CstR, which responds to persulfide, and formaldehyde-responsive FrmR. Unexpectedly, the allosteric mechanism of FrmR from Salmonella enterica serovar Typhimurium is triggered by metals in vitro, and variant FrmRE64H gains responsiveness to Zn(II) and cobalt in vivo. Here we establish that the allosteric mechanism of FrmR is triggered directly by formaldehyde in vitro. Sensitivity to formaldehyde requires a cysteine (Cys35 in FrmR) conserved in all DUF156 proteins. A crystal structure of metal- and formaldehyde-sensing FrmRE64H reveals that an FrmR-specific amino-terminal Pro2 is proximal to Cys35, and these residues form the deduced formaldehyde-sensing site. Evidence is presented that implies that residues spatially close to the conserved cysteine tune the sensitivities of DUF156 proteins above or below critical thresholds for different effectors, generating the semblance of specificity within cells. Relative to FrmR, RcnR is less responsive to formaldehyde in vitro, and RcnR does not sense formaldehyde in vivo, but reciprocal mutations FrmRP2S and RcnRS2P, respectively, impair and enhance formaldehyde reactivity in vitro. Formaldehyde detoxification by FrmA requires S-(hydroxymethyl)glutathione, yet glutathione inhibits formaldehyde detection by FrmR in vivo and in vitro. Quantifying the number of FrmR molecules per cell and modeling formaldehyde modification as a function of [formaldehyde] demonstrates that FrmR reactivity is optimized such that FrmR is modified and frmRA is derepressed at lower [formaldehyde] than required to generate S-(hydroxymethyl)glutathione. Expression of FrmA is thereby coordinated with the accumulation of its substrate.

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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 × 10−3m (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.
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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 × 10−3m (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.

Mentions: The substrates of the FrmR-regulated alcohol dehydrogenase from Salmonella (FrmA) are predicted to be the formaldehyde and nitrosylated adducts of GSH, S-(hydroxymethyl)glutathione and S-nitrosoglutathione, respectively, by analogy to E. coli (Fig. 2B) (47, 69). Despite the evidence that DNA binding by FrmR is directly weakened by formaldehyde in vitro (Fig. 3A), glutathione adducts of formaldehyde might represent the predominant available species during formaldehyde stress conditions. Notably, glutathione has been shown to act positively on metal detection by FrmRE64Hin vivo, suggesting that the protein may interact with glutathione adducts (Fig. 1) (50). Deletion of gshA, encoding γ-glutamate-cysteine ligase (70), required for the first step in glutathione biosynthesis, renders Salmonella more sensitive to exogenous formaldehyde compared with the wild type strain (Fig. 8A), as expected if (as in E. coli) glutathione is required for formaldehyde detoxification in Salmonella by formation of S-(hydroxymethyl)glutathione. However, formaldehyde-mediated derepression of PfrmRA-frmR was not impaired in ΔgshA cells (Fig. 8B), indicating that formation of formaldehyde-glutathione adducts is not an absolute requirement for FrmR responsiveness to formaldehyde in vivo. Indeed, expression levels from PfrmRA-frmR were higher in ΔgshA than in wild type, at equivalent exogenous formaldehyde concentrations (Fig. 8B), consistent with FrmR detecting increased formaldehyde accumulation in the cytosol of ΔgshA cells, due to reduced FrmA activity and/or due to glutathione acting negatively on the modification of FrmR by formaldehyde.


The Effectors and Sensory Sites of Formaldehyde-responsive Regulator FrmR and Metal-sensing Variant *
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 × 10−3m (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.
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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 × 10−3m (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.
Mentions: The substrates of the FrmR-regulated alcohol dehydrogenase from Salmonella (FrmA) are predicted to be the formaldehyde and nitrosylated adducts of GSH, S-(hydroxymethyl)glutathione and S-nitrosoglutathione, respectively, by analogy to E. coli (Fig. 2B) (47, 69). Despite the evidence that DNA binding by FrmR is directly weakened by formaldehyde in vitro (Fig. 3A), glutathione adducts of formaldehyde might represent the predominant available species during formaldehyde stress conditions. Notably, glutathione has been shown to act positively on metal detection by FrmRE64Hin vivo, suggesting that the protein may interact with glutathione adducts (Fig. 1) (50). Deletion of gshA, encoding γ-glutamate-cysteine ligase (70), required for the first step in glutathione biosynthesis, renders Salmonella more sensitive to exogenous formaldehyde compared with the wild type strain (Fig. 8A), as expected if (as in E. coli) glutathione is required for formaldehyde detoxification in Salmonella by formation of S-(hydroxymethyl)glutathione. However, formaldehyde-mediated derepression of PfrmRA-frmR was not impaired in ΔgshA cells (Fig. 8B), indicating that formation of formaldehyde-glutathione adducts is not an absolute requirement for FrmR responsiveness to formaldehyde in vivo. Indeed, expression levels from PfrmRA-frmR were higher in ΔgshA than in wild type, at equivalent exogenous formaldehyde concentrations (Fig. 8B), consistent with FrmR detecting increased formaldehyde accumulation in the cytosol of ΔgshA cells, due to reduced FrmA activity and/or due to glutathione acting negatively on the modification of FrmR by formaldehyde.

View Article: PubMed Central - PubMed

ABSTRACT

The DUF156 family of DNA-binding transcriptional regulators includes metal sensors that respond to cobalt and/or nickel (RcnR, InrS) or copper (CsoR) plus CstR, which responds to persulfide, and formaldehyde-responsive FrmR. Unexpectedly, the allosteric mechanism of FrmR from Salmonella enterica serovar Typhimurium is triggered by metals in vitro, and variant FrmRE64H gains responsiveness to Zn(II) and cobalt in vivo. Here we establish that the allosteric mechanism of FrmR is triggered directly by formaldehyde in vitro. Sensitivity to formaldehyde requires a cysteine (Cys35 in FrmR) conserved in all DUF156 proteins. A crystal structure of metal- and formaldehyde-sensing FrmRE64H reveals that an FrmR-specific amino-terminal Pro2 is proximal to Cys35, and these residues form the deduced formaldehyde-sensing site. Evidence is presented that implies that residues spatially close to the conserved cysteine tune the sensitivities of DUF156 proteins above or below critical thresholds for different effectors, generating the semblance of specificity within cells. Relative to FrmR, RcnR is less responsive to formaldehyde in vitro, and RcnR does not sense formaldehyde in vivo, but reciprocal mutations FrmRP2S and RcnRS2P, respectively, impair and enhance formaldehyde reactivity in vitro. Formaldehyde detoxification by FrmA requires S-(hydroxymethyl)glutathione, yet glutathione inhibits formaldehyde detection by FrmR in vivo and in vitro. Quantifying the number of FrmR molecules per cell and modeling formaldehyde modification as a function of [formaldehyde] demonstrates that FrmR reactivity is optimized such that FrmR is modified and frmRA is derepressed at lower [formaldehyde] than required to generate S-(hydroxymethyl)glutathione. Expression of FrmA is thereby coordinated with the accumulation of its substrate.

No MeSH data available.


Related in: MedlinePlus