Cleavage cascade of the sigma regulator FecR orchestrates TonB-dependent signal transduction

Abstract

TonB-dependent signal transduction is a versatile mechanism observed in gram-negative bacteria that integrates energy-dependent substrate transport with signal relay. In Escherichia coli, the TonB-ExbBD motor complex energizes the TonB-dependent outer membrane transporter FecA, facilitating ferric citrate import. FecA also acts as a sensor, transmitting signals to the cytoplasmic membrane protein FecR, which eventually activates the cytoplasmic sigma factor FecI, driving transcription of the fec operon. Building on our previous finding that FecR undergoes functional maturation through a three-step cleavage process [T. Yokoyama et al., J. Biol. Chem. 296, 100673 (2021)], we here describe the complete mechanism of FecR-mediated ferric citrate signaling involving FecA and TonB. The cleavage cascade begins with FecR autoproteolysis prior to membrane integration. The soluble C-terminal domain (CTD) fragment of FecR is cotranslocated with the N-terminal domain (NTD) fragment through a twin-arginine translocation (Tat) system-mediated process. In the periplasm, the interaction between the CTD and NTD fragments prevents further cleavage. Binding of ferric citrate induces a conformational change in FecA, exposing its TonB box to the periplasmic space. This structural alteration is transmitted to the interacting FecR CTD via the motor function of TonB, resulting in the release of the CTD blockage from the NTD. Consequently, the successive cleavage of FecR's NTD is initiated, culminating in the ferric citrate signal-induced activation of fec gene expression. Our findings reveal that the regulation of FecR cleavage, controlled by the TonB-FecA axis, plays a central role in the bacterial response to ferric citrate signals.

Keywords: Fec system; Tat system; Ton system; iron transport; signal transduction.

Fig. 1.

The FecR CTD fragment is degraded depending on the ferric citrate signal. (A) Schematic representation of the FecR processing cascade, adapted with modifications from (15). FecR undergoes an initial autocleavage, generating CL(a) and the CTD fragment (1st cleavage). CL(a) is then truncated at its C-terminal region to produce CL(b) in response to the ferric citrate signal (2nd cleavage). Subsequently, CL(b) is cleaved by the intramembrane protease RseP, yielding CL(c) (3rd cleavage). The released CL(c) interacts with the alternative sigma factor FecI to activate transcription of the fec operon. IM and Cyto represent the inner membrane and the cytoplasm, respectively. (B) Schematic representation of the domain organization of FecR and its derivatives used in this study. The transmembrane region (TM) of FecR is shown as a cylinder. (C and D) Cleavage profiles of 3xFLAG-FecR-PA (C) and 3xFLAG-MBP-FecR-PA (D) in response to Na₃-citrate (citrate). E. coli strains, YK167 (rseP⁺, +) or YK191 (ΔrseP, Δ), harboring pYK212 (3xFLAG-FecR-PA) or pYK214 (3xFLAG-MBP-FecR-PA), were grown at 30 °C in M9-based medium containing 1 mM isopropyl-β-D-thiogalactopyranoside (IPTG) and 10 μM FeCl₃, with or without 1 mM Na₃-citrate until mid-log phase. Total cellular proteins were acid-precipitated, dissolved in SDS sample buffer containing 10% 2-mercaptoethanol (ME), and analyzed by SDS-PAGE followed by immunoblotting with the indicated antibodies. (C and D, Left). FL, CL(a), CL(b), CL(c), and CTD indicate the full-length protein, N-terminal cleavage product CL(a), CL(b), CL(c), and the C-terminal cleavage product CTD fragment, respectively. The band intensities of fragments derived from 3xFLAG-MBP-FecR-PA in indicated strains were quantified (D, Middle and Right). Band intensities are presented as a percentage of the total intensity of all fragments (D, Middle) or normalized to the levels in the citrate-depleted condition (D, Right). Data represent the means ± SD from two biologically independent experiments. Student’s t test was carried out to compare the values between the groups. *P < 0.05. (E) Schematic representation of ferric citrate signal–dependent processing of the FecR CTD and CL(a).

Fig. 2.

The CTD fragment is exported to the periplasm concurrently with the membrane insertion of CL(a) via the Tat pathway. (A) Cleavage profiles of 3xFLAG-FecR-PA in response to Na₃-citrate in the presence or absence of TatABC. YK167 (tatABC⁺) or YK1196 (ΔtatABC) cells harboring pYK212 (3xFLAG-FecR-PA) were transformed with either pSTD689 (vector control, vec) or pYK345 (pTatABC). Cells were grown, and the total cellular proteins were analyzed as described in Fig. 1C. (B) A schematic representation of FecR cleavage in a TatABC-deficient strain. The first cleavage occurs in the cytoplasm, producing CL(a) and the CTD fragment. Peri, IM, and Cyto indicate the periplasm, the inner membrane, and the cytoplasm, respectively. (C) A schematic representation of the mal-PEG-2k modification assay. The localization of the CTD fragment is assessed by its modifiability with membrane-impermeable mal-PEG-2k. (D) Cellular localization of the CTD fragment assessed by the mal-PEG-2k modification assay in the presence or absence of TatABC. HM1742 (tatABC⁺) or YK2238 (ΔtatABC) cells harboring pYK1001 (3xFLAG-FecR-PA C271A, pFecR Cys-less) or pYK1025 (3xFLAG-FecR-PA C271A/A218C, pFecR A218C) were grown in Na₃-citrate-free medium as described in Fig. 1C. Spheroplasts were prepared from these cells and treated with 2 mM mal-PEG-2k (mal-PEG) in the presence or absence of 1% Triton X-100 (Triton). Total proteins were analyzed as described in Fig. 1C. An asterisk indicates a lysozyme band (D, Left). The levels of mal-PEG-2k-modification of the CTD fragment were quantified and presented as a percentage of the total levels of the mal-PEG-2k-modified and -unmodified bands. Data represent the means ± SD from two biologically independent experiments. Student’s t test was carried out to compare the values between the groups. **P < 0.01; ns, P > 0.05 (D, Right). (E) Tat pathway dependence of the CTD fragment export assessed by the mal-PEG-2k modification assay. YK2238 (ΔtatABC) cells harboring pYK1025 (3xFLAG-FecR-PA C271A/A218C) were transformed with pYK1000 encoding TatABC under the control of the araBAD promoter. A schematic workflow of the experiment is shown (E, Upper Left). Cells were grown at 30 °C in M9-based medium containing 10 μM FeCl₃ and 0.05% fucose, which inhibits the expression from the araBAD promoter, until mid-log phase. FecR expression was induced with 1 mM IPTG for 30 min. Cells were then washed, resuspended in M9-based IPTG-free medium without or with 0.2% arabinose to induce TatABC expression (indicated as 0 min), and further incubated at 30 °C. At the indicated time point, the cells were collected, and the mal-PEG-2k modification assay was performed as described in (D). The immunoblot image (E, Lower Left) and quantification of mal-PEG-2k-modification (E, Right) are shown. The modification level is plotted against the induction time of TatABC expression. Data represent the means ± SD from two biologically independent experiments. Student’s t test was carried out to compare the values between the groups. **P < 0.01; ns, P > 0.05. (F) Schematic representation of the model in which the CTD fragment is exported to the periplasm via the Tat pathway. After the first cleavage, the CTD fragment remains associated with CL(a) and is transported to the periplasm together with the membrane insertion of CL(a). (G) Coimmunoprecipitation assay demonstrating the in vivo (G, Left) and in vitro (G, Right) interaction between the CTD fragment and CL(a). Crude membranes were prepared from YK167 cells harboring pYK2054 (pFecR-PA) or pYK212 (p3xFLAG-FecR-PA), solubilized with Triton X-100, and subjected to immunoprecipitation using anti-FLAG antibody (G, Left). For the in vitro assay, FecR-PA and 3xFLAG-FecR-PA were synthesized using the PURE system in the presence of n-Dodecyl-β-D-maltoside (DDM) and subjected to immunoprecipitation with anti-FLAG antibody (G, Right). Compared to the 3×FLAG-FecR-PA construct, three times the amount of membrane lysate or reaction solution of the PURE system was used for immunoprecipitation of the FecR-PA construct. Proteins in the input, flow-through, wash, and eluate fractions were analyzed as described in Fig. 1C. Asterisks indicate nonspecific bands.

Fig. 3.

The CTD fragment regulates the signal-dependent cleavage of CL(a). (A) Schematic representation of the domain organization of 3xFLAG-MBP-FecR and its CL(a)-mimic mutant lacking the CTD fragment. (B) Cleavage profiles of the CL(a)-mimic mutant in response to Na₃-citrate in the presence or absence of RseP. YK167 (rseP⁺, +) or YK191 (ΔrseP, Δ) cells harboring either pYK147 (p3xFLAG-MBP-FecR WT) or pYK172 [p3xFLAG-MBP-FecR CL(a)-mimic] were grown and total cellular proteins were analyzed. The immunoblot image (B, Upper) and the quantified band intensities (B, Lower) are presented as described in Fig. 1D. Student’s t test was carried out to compare the values between the groups. *P < 0.05; **P < 0.01; ns, P > 0.05. (C) Ability of the FecR CL(a)-mimic mutant to transmit the ferric citrate signal. YK627 (ΔfecR) cells harboring pYK149 (P_fecA-lacZ) and pSTD343 (lacI) were transformed with either pSTD1060 (vector), pYK188 (pFecR WT), or pYK341 [pFecR CL(a)-mimic]. Cells were grown at 30 °C in M9-based medium containing 1 mM IPTG and 0.1 μM FeCl₃, with or without 1 mM Na₃-citrate, until mid-log phase, and their LacZ activities were measured. Relative LacZ activities, normalized to that of the cells harboring pYK188 grown in the same medium with 1 mM Na₃-citrate, are shown. Data represent the means ± SD from two biologically independent experiments. Student’s t test was carried out to compare the values between the groups. ***P < 0.001; ns, P > 0.05. (D) Schematic representation of the role of the CTD. The CL(a)-mimic mutant undergoes a constitutive second cleavage, independent of the ferric citrate signal.

Fig. 4.

Dissociation of the CTD fragment from CL(a) is essential for ferric citrate signal–dependent CL(a) cleavage and CTD degradation. (A) AlphaFold3-predicted structure of the FecR CL(a)-CTD complex. CL(a) and the CTD fragment are shown in pink and red, respectively. The C-terminal residue of CL(a) (G181) and the N-terminal residue of the CTD fragment (T182) are depicted as sphere models. The side chains of the cysteine-introduced residues (K115 and N266) are presented as stick models. (B) Effect of the disulfide cross-linking between CL(a) and the CTD fragment on CL(a) cleavage and CTD degradation. YK167 cells harboring pYK369 (pFecA) were transformed with plasmids encoding the indicated 3xFLAG-MBP-FecR-PA Cys-less derivatives. FecA is constitutively overproduced from pYK369 to provide sufficient signal transduction to FecR. A schematic workflow of the experiment is shown (B, Upper). Cells were grown at 30 °C in M9-based medium containing 1 mM IPTG and 10 μM FeCl₃ until mid-log phase. The cells were then washed and resuspended in IPTG-free M9-based medium containing 10 μM FeCl₃ and 1 mM diamide. After 1 h of cultivation at 30 °C to allow disulfide bond formation, the cells were washed again, resuspended in IPTG-free M9-based medium containing 10 μM FeCl₃ with or without 1 mM Na₃-citrate, incubated for additional 1 h at 30 °C, and harvested. Total cellular proteins were acid-precipitated, dissolved in SDS sample buffer containing 25 mM N-ethylmaleimide (NEM; to bock free thiol groups), with or without 10% ME, and analyzed by immunoblotting as described in Fig. 1D (B, Middle). “CL(a)xCTD” indicates the disulfide cross-linked product. Band intensities of CL(a) and CL(c) (B, Lower Left) and the CTD fragment (B, Lower Right) under reducing conditions (+ME) were quantified and shown as described in Fig. 1D. Student’s t test was carried out to compare the values between the groups. *P < 0.05; ***P < 0.001; ns, P > 0.05. (C) Effect of reducing the disulfide bond between CL(a) and the CTD fragment on CL(a) cleavage and CTD degradation. Cells were grown and treated as described in (B). After treatment with diamide, the cells were washed, resuspended in IPTG-free M9-based medium containing 10 μM FeCl₃ and the indicated concentration of tris(2-carboxyethyl)phosphine (TCEP), and incubated at 30 °C for 30 min. The cells were then washed again, resuspended in IPTG-free M9-based medium containing 10 μM FeCl₃ with or without 1 mM Na₃-citrate, incubated for 1 h at 30 °C, and harvested. Proteins were analyzed as described in (B), and the results are shown in the same format. Student’s t test was carried out to compare the values between the groups. *P < 0.05; **P < 0.01; ns, P > 0.05.

Fig. 5.

The FecA transporter, powered by the TonB motor, mediates the dissociation of the CTD fragment from CL(a), enabling ferric citrate signal–dependent CL(a) cleavage. (A) AlphaFold3-predicted structure of the CL(a)-CTD-FecA-TonB supercomplex. FecR CL(a), the CTD fragment, FecA, and TonB are shown in pink, red, green, and gray, respectively. Enlarged views of the interaction sites between TonB and FecA (Upper dashed box) and between FecA and the FecR CTD fragment (Middle and Lower dashed boxes) are shown. (B) Cleavage profiles of the FecR protein in the presence or absence of TonB. YK167 (tonB⁺) or YK2234 (ΔtonB) cells were transformed with pYK212 (p3xFLAG-FecR-PA). Cells were grown, and proteins were analyzed as described in Fig. 1C. (C) Effect of the depletion of PMF on CL(a) cleavage and CTD degradation. YK167 cells harboring pYK367 (pFecA) were transformed with pYK214 (p3xFLAG-MBP-FecR-PA). A schematic workflow of the experiment is shown (C, Upper). Cells were grown at 30 °C in M9-based medium containing 1 mM IPTG and 10 μM FeCl₃ until mid-log phase. The cells were then washed and resuspended in IPTG-free M9-based medium containing 10 μM FeCl₃ and 100 μM carbonyl cyanide m-chlorophenyl hydrazone (CCCP). After 15 min of incubation at 30 °C, the cells were washed again, resuspended in M9-based IPTG-free medium containing 10 μM FeCl₃ and 100 μM CCCP with or without 1 mM Na₃-citrate, incubated for additional 1 h at 30 °C, and the total cellular proteins were analyzed. The immunoblot image (C, Middle) and the quantified band intensities (C, Lower) are presented as described in Fig. 1D. Student’s t test was carried out to compare the values between the groups. *P < 0.05; ns, P > 0.05. (D) Effect of FecA mutations on the predicted FecA-FecR CTD interaction, assessed by FecR cleavage. YK167 (fecA⁺) or YK1602 (ΔfecA) cells harboring pYK214 (p3xFLAG-MBP-FecR-PA) were transformed with plasmids encoding the indicated FecA-His₁₀ derivatives. Cells were grown, and total cellular proteins were analyzed as described in Fig. 1D. Student’s t test was carried out to compare the values between the groups. *P < 0.05; **P < 0.01; ns, P > 0.05.

Fig. 6.

Model of the Fec signaling mechanism. The TonB-dependent transporter FecA mediates the progression of the FecR cleavage cascade in response to the ferric citrate signal. See the text for a detailed description.