Article Figures & Data
- FIG 1
Model of the general stress response (GSR) system of alphaproteobacteria, including defined molecular components of C. crescentus GSR. (A) Under normal growth conditions, the ECF σ factor, σT, is bound and inhibited by the anti-σ factor NepR. Upon stress encounter, the sensor histidine kinase, PhyK, is proposed to phosphorylate PhyR, thereby increasing its affinity for NepR and releasing σT to bind RNAP. IM, inner membrane. (B) Surface representation of the structure of the σ-like domain of PhyR (PhyR-SL) (in white; M1 to E138) bound to NepR (in dark pink; R30 to E62) (PDB code 3T0Y) (5). (C) Amino acid sequence and secondary structure of C. crescentus NepR: N-terminal flanking region (FR1; M1 to Q32), α-helix 1 (α1; A33 to N47), linker (L; E48 to P51), α-helix 2 (α2; D52 to A61), and C-terminal flanking region (FR2; E62 to E68). Three putative NepR start codons are highlighted in yellow.
- FIG 2
Interaction of full-length and mutant NepR alleles with PhyR, PhyR σ-like domain (PhyR-SL), and σT assessed by bacterial two-hybrid (BTH) assay. (Top right) Cartoon representation of the full-length (FL), truncated, and site-directed NepR mutant alleles assayed for interaction with σT, PhyR, and PhyR-SL. (Middle) Protein interaction results reported as β-galactosidase activity, measured in BTH reporter strains transformed with pUT18c expressing nepR alleles pictured on top right: full length (FL; M1 to E68), start codon 2 (SC2; M8 to E68), start codon 3 (SC3; M14 to E68), flanking region 1 deleted (ΔFR1; Q31 to E68), flanking region 2 deleted (ΔFR2; M1 to E62), short version (SV; Q31 to E62), and polyalanine linker (poly-A); each of these NepR alleles was measured for interaction with PhyR, PhyR-SL, or σT expressed from pKT25. (Bottom left) Positive (leucine zipper vectors), negative (empty vectors, EV), and vector controls. All assays were performed in triplicate; error bars represent standard deviations. (Bottom right) Stability of NepR alleles expressed in the BTH reporter strain was assessed by Western blotting using T18 antiserum.
- FIG 3
Functional analysis of full-length and truncated NepR alleles as regulators of GSR transcription in C. crescentus. (A) Measured β-galactosidase activity from the σT-dependent PsigU-lacZ reporter plasmid. β-Galactosidase activities were measured in WT and ΔnepR ΔsigT backgrounds containing a plasmid expressing sigT from a xylose-inducible promoter (Pxyl-sigT), in the presence (+) or absence (−) of sigT inducer (0.2% xylose) and the presence (+) or absence (−) of osmotic upshock stress (150 mM sucrose). Empty vector (EV) controls (Pxyl and Pvan) are also included. (B) σT-dependent transcription measured in a ΔnepR ΔsigT strain expressing sigT from Pxyl-sigT. Transcription was assayed as a function of full-length (nepRFL) and terminally truncated (nepRSV) nepR alleles expressed from a vanillate-inducible promoter (Pvan-nepRFL or Pvan-nepRSV). Boundaries of expressed nepR alleles are as follows: nepRFL, M1 to E68; nepRSV, Q31 to E62. β-Galactosidase activities were assayed in the presence (+) and absence (−) of nepR induction (0.5 mM vanillate) and in the presence (+) and absence (−) of osmotic upshock (150 mM sucrose). Transcription was compared to an empty vector (EV) control strain. Stability and function of HA-tagged nepR alleles were further evaluated by dot blotting and β-galactosidase transcriptional assays described in Fig. S4 in the supplemental material. All assays were performed in triplicate; error bars represent standard deviations.
- FIG 4
Measuring the effect of NepR termini on σ/anti-σ complex folded stability. (A) Expression and affinity purification of His-σT in presence or absence of NepRFL or NepRSV. When overexpressed in isolation, His-σT is in the insoluble cell fraction (pellet [P]); no protein was retrieved from the soluble fraction (SF) after Ni2+ affinity chromatography (lane 2). When coexpressed with untagged NepRFL or NepRSV, no His-σT is evident in the insoluble pellet fraction (P; lanes 3 and 4). The protein purifies as a σT/NepR (σ/anti-σ) complex in the soluble fraction (SF; lanes 5 and 6). (B) His-σT was coexpressed with MBP-NepR alleles. The complexes were sequentially purified by nickel and amylose affinity chromatographies and vice versa. Resolved proteins were visualized by Coomassie blue staining and support 1:1 binding for σT/NepRFL and σT/NepRSV. (C) Circular dichroism (CD) spectra of σT/NepRFL and σT/NepRSV complexes at 26°C. (D) (Left) Representative normalized CD thermal denaturation trace of the σT/NepRFL and σT/NepRSV complexes; molar ellipticity at 222 nm was normalized by setting the value at 26°C to 0% and the value at 72°C to 100%. Change in ellipticity at 222 nm (3 acquisitions every 2°C) was plotted and fitted according to a Boltzmann model. (Right) Melting temperatures were measured four times on independent protein preparations. The horizontal bar represents the mean of all four measures. His-σT/MBP control and gel filtration elution profiles of the different complexes used for CD Tm are presented in Fig. S6 in the supplemental material.
- FIG 5
Acetyl phosphate (AcP)-dependent phosphorylation of PhyR requires NepR and PhyR residue D192. (A) Radiograph showing phosphorylation of PhyR (left) and PhyRD192A (right) by [32P]AcP in the presence or absence of equivalent concentrations of MBP, MBP-NepRFL, or MBP-NepRSV after a 120-min incubation. (B) Kinetics of AcP-dependent phosphorylation of PhyR alone or incubated with equimolar amounts (10 µM) of MBP, MBP-NepRFL, or MBP-NepRSV. Time is shown in minutes; the maximum level of PhyR phosphorylation observed was set to 100%. All experiments were performed in triplicate; error bars represent standard deviations.
- FIG 6
NepR anti-σ function requires its unconserved, disordered termini. A NepRSV allele missing the termini does not bind phospho-PhyR and forms a less stably folded complex with σT. Notably, phosphorylation of PhyR in vitro is strongly enhanced by addition of NepR, suggesting an additional layer of GSR regulation in which direct NepR interaction can influence activation of PhyR as an anti-anti-σ factor.
- TABLE 1
Microscopic association (Ka) and dissociation (Kd) and calculated equilibrium (KD) affinities of PhyR binding to full-length NepR (NepRFL) and the short version of NepR missing FR1 and FR2 (NepRSV)
PhyR MBP-NepRFL MBP-NepRSV Ka, (1/M ⋅ s) × 105 Kd, (1/s) × 10−1 KD, nM Ka, (1/M ⋅ s) × 105 Kd, (1/s) × 10−1 KD, nM His-PhyR-SL 5.08 ± 1.78 1.1 ± 0.16 191 ± 7 4.65 ± 0.98 2.08 ± 0.26 455 ± 54 His-PhyR~P 1.34 ± 0.15 0.97 ± 0.31 744 ± 289 NDa ND ND ↵a ND, not determined.
- TABLE 2
Calculated phospho-PhyR (PhyR~P) half-life in the presence of buffer, MBP, MBP-NepRFL or MBP-NepRSVa
Protein PhyR~P half-life (h) PhyR~P + buffer 45.7 ± 6.8 PhyR~P + MBP 44.6 ± 8.5 PhyR~P + MBP-NepRFL 49.9 ± 2.2 PhyR~P + MBP-NepRSV 47.8 ± 3.8 ↵a Values presented are the averages of results from three independent experiments ± standard deviations.
Supplemental Material
Figure S1
Conservation and disorder prediction of the different regions of NepR. (A) Amino acid sequence alignment of C. crescentus (Cc) NepR with orthologous sequences from other alphaproteobacteria (Me, Methylobacterium extorquens; Bj, Bradyrhizobium japonicum USDA110B; Rp, Rhodopseudomonas palustris CGA009; Ml, Mesorhizobium loti MAFF303099; Sm, Sinorhizobium meliloti 1021; and Ba, Brucella abortus 2308). Residues are highlighted according to degree of conservation. (B) Intrinsic NepR protein disorder as a function of amino acid position predicted by the PONDR-FIT algorithm (B. Xue, R. L. Dunbrack, R. W. Williams, A. K. Dunker, V. N. Uversky, Biochim Biophys Acta 1804:996–1010, 2010, doi:10.1016/j.bbapap.2010.01.011). Graph plots calculated disorder disposition versus amino acid position (residue index). Amino acids with a predicted disorder disposition greater than 0.5 (red line) are classified as being intrinsically disordered. The secondary structure of NepR is shown above the graph. Download Figure S1, PDF file, 0.8 MB.
Copyright © 2015 Herrou et al.
This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-ShareAlike 3.0 Unported license, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original author and source are credited.
Figure S2
NepR translation start sites. (A) Ribosome footprinting data of the translated C. crescentus genome (J. M. Schrader et al., PLoS Genet 10:e1004463, 2014, doi:10.1371/journal.pgen.1004463) in the context of three possible NepR translation start sites at positions 1, 8, and 14 (1 = start codon at C. crescentus NA1000 nucleotide coordinate 3744767). Orange peaks correspond to the relative abundance of ribosomes that occupy the 5′ region of nepR mRNA. (B) Translational fusion constructs containing the nepR promoter with start codons PnepR1, PnepR2, and PnepR3 fused to lacZ; start codons were isolated in these constructs by mutation of adjacent starts to alanine codon GCG. Translation from each start (1, 2, and 3) was assessed relative to a fusion in which all three putative start codons were mutated to GCG (Ala). Quantification of translation demonstrates the highest expression from start codon 2 compared to the control. The first and third start sites are expressed at lower levels. (C) lacZ translational fusion constructs containing the nepR promoter onto which each start codon was added sequentially (PnepR1, PnepR12, and PnepR123). β-Galactosidase activities for each fusion are compared to empty vector control (EV). All experiments were conducted in triplicate; error bars represent standard deviations. Download Figure S2, PDF file, 0.4 MB.
Copyright © 2015 Herrou et al.
This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-ShareAlike 3.0 Unported license, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original author and source are credited.
Figure S3
Additional bacterial two-hybrid (BTH) protein interaction assay controls. (A) BTH assays confirm the absence of interaction between T25-PhyRD>A and T18c-NepR. Positive, negative, and empty vector controls are displayed below the graph. The stability of T25-PhyRD>A was assessed by Western blotting using PhyR antiserum and compared to wild-type PhyR. (B) (1) “Reverse” control experiment. NepR alleles were cloned into pKT25, and interactions with T18c-PhyR, T18c-PhyR-SL, and T18c-σT were assessed. (2) A 30-amino-acid linker connecting the T18 adenylate cyclase subunit to NepRSV was introduced to confirm that steric clashes do not impair any possible interaction between T18c-NepRSV and T25-PhyR. Positive, negative, and empty vector controls (EV) for both graphs are displayed. All experiments were conducted in triplicate; error bars represent standard deviations. Download Figure S3, PDF file, 0.7 MB.
Copyright © 2015 Herrou et al.
This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-ShareAlike 3.0 Unported license, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original author and source are credited.
Figure S4
Assessing stability of NepR alleles in C. crescentus. (A) Steady-state levels of N- or C-terminal HA-tagged NepR alleles expressed in C. crescentus evaluated by dot blot assay using HA antiserum. Spot intensities for independent replicate experiments were integrated and are plotted below the blots. The empty vector (EV) strain carrying plasmid pMT806 was used as a negative control. Error bars represent standard deviations. (B) σT-dependent transcription measured in a ΔnepR ΔsigT strain expressing sigT from Pxyl-sigT. Transcription was assayed as a function of full-length (nepRFL) and terminally truncated (nepRSV) HA-tagged nepR alleles expressed from a vanillate-inducible promoter (Pvan-nepRFL or Pvan-nepRSV). Boundaries of expressed HA-tagged nepR alleles are as follows: nepRFL, M1 to E68; nepRSV, Q31 to E62. β-Galactosidase activities were assayed in the presence (+) and absence (−) of nepR induction (0.5 mM vanillate) and in the presence (+) and absence (−) of osmotic upshock (150 mM sucrose). We note that N- and C-terminally tagged versions of NepRFL appear to be less effective anti-σ factors than untagged NepR, though they do retain anti-σT activity. Download Figure S4, PDF file, 1 MB.
Copyright © 2015 Herrou et al.
This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-ShareAlike 3.0 Unported license, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original author and source are credited.
Figure S5
Surface plasmon resonance (SPR) studies of PhyR-NepR interaction. Raw SPR binding data are presented in black; fits to the data are in red. Residuals to the fits are plotted below the sensorgrams. Boundaries of assayed NepR alleles are as follows: NepRFL, M1 to E68; NepRSV, Q31 to E62. (A) Association and dissociation of immobilized His-PhyR-SL with MBP-NepRFL made to flow at concentrations of 1,000, 500, 250, 125, and 62.5 nM (black). (B) Association and dissociation of immobilized His-PhyR-SL with MBP-NepRSV made to flow under the same conditions and concentrations as described in panel A. (C) Association and dissociation of immobilized His-PhyR with MBP-NepRFL made to flow at concentrations of 1,000, 500, 250, 125, and 62.5 nM in the presence of 5 mM acetyl phosphate (AcP) and 5 mM Mg2+. (D) Association and dissociation of immobilized His-PhyR with MBP-NepRSV made to flow under the same conditions and concentrations as described in panel C. (E) SPR protein-protein interaction controls. Interaction between His-PhyR and MBP was assessed (at 1,000, 500, 250, 125, and 62.5 nM) in the presence of AcP (5 mM) and Mg2+ (5 mM). (F) SPR protein-protein interaction controls. Interaction between His-PhyR-SL and MBP was assessed (at 1,000, 500, 250, 125, and 62.5 nM). RU, resonance units. Color code: PhyR sigma-like (SL) domain, black; PhyR receiver (R) domain, green; phosphoryl group (P), red; NepR, dark pink; maltose-binding protein (MBP), blue. All binding assays were performed in triplicate. Download Figure S5, PDF file, 2.3 MB.
Copyright © 2015 Herrou et al.
This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-ShareAlike 3.0 Unported license, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original author and source are credited.
Figure S6
(A) Purification of His-σT/NepRFL (top) and His-σT/NepRSV (bottom) σ/anti-σ complexes by size exclusion chromatography. Elution of protein complexes from Superdex 200 (void volume, 11.9 ml) was monitored spectrophotometrically at 280 nm; elution volumes and the corresponding estimated molecular weights are shown on top of each peak. To ensure that purified complexes (σT/NepRFL and σT/NepRSV) had comparable oligomeric states, fractions from the second, higher-volume peaks were used for CD-thermal denaturation experiments (resolved by 16% SDS-PAGE) (highlighted with black stars). The volume of this peak is consistent with σ/anti-σ heterotetramer (red triangle) as previously described for Brucella (H. S. Kim, C. C. Caswell, R. Foreman, R. M. Roop II, and S. Crosson, J Biol Chem 288:13906–13916, 2013, doi:10.1074/jbc.M113.459305); heterotetramerization was also described for the closely related Caulobacter PhyR-SL/NepR complex (J. Herrou, G. Rotskoff, Y. Luo, B. Roux, and S. Crosson, Proc Natl Acad Sci U S A 109:E1415–E1423, 2012, doi:10.1073/pnas.1116887109). The lower-volume peak corresponds to a larger oligomeric complex and was not used for CD-thermal denaturation. (B) MBP coexpression control confirms that σT solubility is determined by NepR and not MBP. MBP was coexpressed with His-σT. Lane 1, resuspended pellet showing His-σT in the insoluble inclusion fraction; lanes 2 and 3, a small amount of unstable His-σT is present in the soluble fraction after Ni2+ affinity purification; no bands corresponding to MBP are visualized in this fraction; if soluble His-σT fraction is then loaded into an amylose resin column, no bands corresponding to MBP or His-σT can be visualized, confirming that MBP and His-σT do not interact in this assay; lanes 4 and 5, MBP is present in the soluble fraction after amylose resin affinity purification; no bands corresponding to His-σT are visible. After the soluble MBP fraction is loaded onto an Ni2+ resin column and washed, no MBP or His-σT is visible, again confirming an absence of interaction. Download Figure S6, PDF file, 0.7 MB.
Copyright © 2015 Herrou et al.
This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-ShareAlike 3.0 Unported license, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original author and source are credited.
Table S1
Primers used in this study. Table S1, PDF file, 0.1 MB.
Copyright © 2015 Herrou et al.
This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-ShareAlike 3.0 Unported license, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original author and source are credited.
Table S2
Strains used in this study. Table S2, PDF file, 0.1 MB.
Copyright © 2015 Herrou et al.
This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-ShareAlike 3.0 Unported license, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original author and source are credited.
Additional Files
Supplementary Data
Supplementary Data
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- Figure sf6, PDF - Figure sf6, PDF
- Table st1, PDF - Table st1, PDF
- Table st2, PDF - Table st2, PDF
