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Comparative Study
. 2016 Dec 9:6:38879.
doi: 10.1038/srep38879.

The mechanism of a formaldehyde-sensing transcriptional regulator

Katie J Denby  1 , Jeffrey Iwig  2 , Claudine Bisson  1 , Jodie Westwood  1 , Matthew D Rolfe  1 , Svetlana E Sedelnikova  1 , Khadine Higgins  3 , Michael J Maroney  3 , Patrick J Baker  1 , Peter T Chivers  2   4 , Jeffrey Green  1
Affiliations
Comparative Study

The mechanism of a formaldehyde-sensing transcriptional regulator

Katie J Denby et al. Sci Rep. .

Abstract

Most organisms are exposed to the genotoxic chemical formaldehyde, either from endogenous or environmental sources. Therefore, biology has evolved systems to perceive and detoxify formaldehyde. The frmRA(B) operon that is present in many bacteria represents one such system. The FrmR protein is a transcriptional repressor that is specifically inactivated in the presence of formaldehyde, permitting expression of the formaldehyde detoxification machinery (FrmA and FrmB, when the latter is present). The X-ray structure of the formaldehyde-treated Escherichia coli FrmR (EcFrmR) protein reveals the formation of methylene bridges that link adjacent Pro2 and Cys35 residues in the EcFrmR tetramer. Methylene bridge formation has profound effects on the pattern of surface charge of EcFrmR and combined with biochemical/biophysical data suggests a mechanistic model for formaldehyde-sensing and derepression of frmRA(B) expression in numerous bacterial species.

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Figures

Figure 1
Figure 1. Amino acid fingerprints associated with signal perception by members of the CsoR/RcnR family and amino acid sequences of EcFrmR and StyFrmR.
(a) Amino acid residues in the W-X-Y-Z fingerprint of CsoR/RcnR family proteins and the signals perceived by the indicated proteins. (b) Alignment of the E. coli (Ec) and S. enterica serovar Typhimurium (Sty) FrmR proteins. Identical (single letter code) and similar (+) residues, Pro2 (brown background), Cys35 and Cys70 (yellow background) are indicated. Residues of the W-X-Y-Z fingerprint (blue font) are indicated. Position W is shown to incorporate both Pro2 and Ser/His3 (as indicated by parentheses; see text for details). Residues on blue backgrounds have been implicated in DNA-binding in other CsoR/RcnR family members.
Figure 2
Figure 2. EcFrmR-mediated repression of frmRAB expression is relieved by formaldehyde.
(a) Cultures of E. coli PC677 carrying Pfrm-frmR-lacZ (open bars) or Pfrm-frmRstop-lacZ (gray bars) were grown as described in Methods in the absence and presence of the indicated concentrations of formaldehyde. β-Galactosidase activities (Miller units plotted on a log scale) were measured as a proxy for in vivo transcription from the frmRAB promoter. (b) β-Galactosidase activities (Miller units) of cultures of E. coli PC677 carrying Pfrm-frmR-lacZ were measured after anaerobic cultivation in the presence of the indicated aldehydes as described in Methods. Activities were normalized to that measured in the absence of formaldehyde. For both panels, the error bars represent the standard deviation from the mean (n = 3).
Figure 3
Figure 3. Formaldehyde enhances disassociation of the Pfrm-EcFrmR complex.
(a) Bio-Layer Interferometry (BLItz) assays. Reactions to evaluate the interaction of biotin-labeled Pfrm DNA, immobilized on a streptavidin probe, with EcFrmR were carried out with 10 different concentrations of EcFrmR (Table S4A). Representative traces for EcFrmR (6.16 μM tetramer, black line, 0.88 μM tetramer; red line), as well as EcFrmR pre-treated with 200-fold molar excess of formaldehyde (0.88 μM tetramer; blue line), and EcFrmR binding at a non-target DNA (PydhY, 0.88 μM EcFrmR tetramer; green line) are shown. (b) Pre-formed Pfrm-EcFrmR complexes were exposed to 10 different concentrations (Table S4C) of formaldehyde and disassociation curves were recorded. Traces for 0 (black); 0.05 mM (orange); 0.25 mM (gray); 0.62 mM (yellow); 1.25 mM (blue); 3.69 mM (green); 4.92 mM (dark blue); 7.38 mM (brown) are shown. (c) Single exponential fits to formaldehyde disassociation curves were used to obtain the observed rate constants (kobs) which were plotted against formaldehyde concentration to obtain the apparent second order rate constant. (d) Inhibition of frmRAB transcription by EcFrmR in vitro is relieved by formaldehyde. Reaction conditions are described in the Methods section. Left panel, Pfrm; right panel, Pndh. Lanes 1, RNA size markers, top to bottom: 600, 500, 400, 300, 200, 100 bases; Lanes 2, no EcFrmR; lanes 3, 1 nM EcFrmR tetramer; lane 4, 1 nM EcFrmR tetramer pre-treated with 200-molar excess formaldehyde. The locations of the frmR and ndh are indicated.
Figure 4
Figure 4. Identification of formaldehyde-insensitive EcFrmR protein variants.
(a) The Pfrm-frmR-lacZ reporter was modified to encode EcFrmR variants with the indicated amino acid substitutions. Cultures of E. coli PC677 carrying these reporters were grown under anaerobic conditions in the absence (open bars) of presence (gray bars) of formaldehyde (250 μM) and β-galactosidase activities (Miller units) were measured as described in Methods. The error bars represent the standard deviation from the mean (n = 3). (b) Maximum growth rates (μmax) of E. coli MG1655 frmRAB mutant transformed with plasmids expressing the frmRAB operon from Pfrm under the control of wild-type EcFrmR (closed bars), EcFrmR(P2A) (gray bars) or EcFrmR(C35A) (open bars) cultured in the presence of the indicated initial concentrations of formaldehyde. The mean and standard deviations (n = 3) and the p values for one-tailed t-tests are shown.
Figure 5
Figure 5. Structure of EcFrmR.
(a) Cartoon representations of uncross-linked (left) and cross-linked (right) EcFrmR monomers colored blue (N-terminal) to red (C-terminal). Secondary structure elements (α-helices, α1 to α3; loops, L1 and L2) are labeled and the amino acid residues (single letter code, P2, C35 and C70) involved in cross-linking and disulfide bond formation are shown as sticks. The disordered N-terminal region in the uncross-linked subunit is represented by the blue dashed line. (b) A comparison of the overall size and shape of the uncross-linked (left) and cross-linked (right) faces of the EcFrmR tetramer. The upper images show the arrangement of the helices on each face of the tetramer, the positions of the methylene bridges (P2′-C35) and the Cys70-Cys70′ disulfide bonds (S-S). The homodimer (A/B) on the uncross-linked face is drawn in shades of green and the cross-linked face (A′/B′) in shades of orange. The middle images show the expansion of the surface envelope upon cross-linking (black double headed arrow drawn between Arg14 Cα atoms, highlighted in pink). The lower images show the surface-charge on either side of the tetramer (red represents negative charge, blue positive charge and white neutral). (c) Section of the 2Fo-Fc map between chains A and A′ obtained when the coordinates for Pro2 and the methylene bridge were omitted from the refinement (black mesh, contoured at 1σ). Residues are indicated by their single letter codes.
Figure 6
Figure 6. Modeling the Pfrm–EcFrmR complex.
(a) The DNA sequence of the frmRAB promoter region (Pfrm) contains tandem EcFrmR binding sites consisting of ATAC/GTAT inverted repeats (bold) separated by G/C-rich tracts (italic) that form a larger inverted repeat (convergent red arrows). The size of EcFrmR (subunits colored in shades of green and orange as in Fig. 5b) suggests that two tetramers could bind to the frmRAB promoter region. One EcFrmR tetramer (side view) is shown on the top face of the DNA sequence and the other (top view) behind the DNA sequence, offset by approximately a quarter turn relative to the first tetramer. (b) Models of binary complexes formed from EcFrmR and A- or B-form DNA. One of the tandem EcFrmR binding sites of Pfrm (dark gray) is modeled as A- (left) and B-form (right) DNA. EcFrmR is shown as surface representation with subunits colored in shades of green (uncross-linked A/B face) and orange (cross-linked A′/B′ face), with the amino acid side-chains on the A/B face that are implicated in DNA-binding highlighted in blue.
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