Ferric Citrate Regulator FecR Is Translocated across the Bacterial Inner Membrane via a Unique Twin-Arginine Transport-Dependent Mechanism

Abstract

In Escherichia coli, citrate-mediated iron transport is a key nonheme pathway for the acquisition of iron. Binding of ferric citrate to the outer membrane protein FecA induces a signal cascade that ultimately activates the cytoplasmic sigma factor FecI, resulting in transcription of the fecABCDE ferric citrate transport genes. Central to this process is signal transduction mediated by the inner membrane protein FecR. FecR spans the inner membrane through a single transmembrane helix, which is flanked by cytoplasm- and periplasm-orientated moieties at the N and C termini. The transmembrane helix of FecR resembles a twin-arginine signal sequence, and the substitution of the paired arginine residues of the consensus motif decouples the FecR-FecI signal cascade, rendering the cells unable to activate transcription of the fec operon when grown on ferric citrate. Furthermore, the fusion of beta-lactamase C-terminal to the FecR transmembrane helix results in translocation of the C-terminal domain that is dependent on the twin-arginine translocation (Tat) system. Our findings demonstrate that FecR belongs to a select group of bitopic inner membrane proteins that contain an internal twin-arginine signal sequence.IMPORTANCE Iron is essential for nearly all living organisms due to its role in metabolic processes and as a cofactor for many enzymes. The FecRI signal transduction pathway regulates citrate-mediated iron import in many Gram-negative bacteria, including Escherichia coli The interactions of FecR with the outer membrane protein FecA and cytoplasmic anti-sigma factor FecI have been extensively studied. However, the mechanism by which FecR inserts into the membrane has not previously been reported. In this study, we demonstrate that the targeting of FecR to the cytoplasmic membrane is dependent on the Tat system. As such, FecR represents a new class of bitopic Tat-dependent membrane proteins with an internal twin-arginine signal sequence.

Keywords: Escherichia coli; iron acquisition; iron regulation; twin-arginine translocation.

FIG 1

(A) Schematic representation of FecR-mediated signal transduction. Binding of ferric citrate to the outer membrane protein FecA initiates a signal that is transmitted across the cytoplasmic membrane by FecR. (B) Sequence alignment of the putative internal Tat motif of FecR (residues 68 to 109) with the N-terminal Tat motif of TorA (residues 1 to 52). The Tat motif is boxed in red, and the TorA cleavage site is boxed in orange. The region of hydrophobicity/membrane-spanning region are boxed in green. Positively charged residues adjacent to the FecR c-region are indicated by arrows, and putative Sec avoidance (which corresponds to the WebLogo shown in panel D) is boxed in purple. (C) Consensus Tat sequence motif of predicted FecR eggNOG-derived orthologues, excluding the six sequences that do not contain a consensus twin-arginine motif. Amino acid positions relative to the twin-arginine motif are denoted below. (D) Sequence alignment consensus logo of the c-region of the predicted FecR eggNOG-derived orthologues. Charged amino acid residues are colored blue.

FIG 2

Mutation of twin-arginine residues inhibits periplasmic translocation of a FecR-BlaM fusion. (A) Domain architecture of the FecR-BlaM reporter. (B and C) The ampicillin (AMP) sensitivities of the FecR-BlaM reporter with RR-to-AA and RR-to-KK substitutions (E. coli strain, 10β) (B) and the FecR-BlaM reporter expressed in a Tat-deficient strain (E. coli strains HS3018-A and HS3018-A ΔtatABC) (C) were determined using M.I.C. Evaluator strips. Representative images of three biological replicates are shown.

FIG 3

(A to C) Cell localization of FecR-Bla-His reporter and FecR-His with RR-to-KK substitutions (10β) (A and B) or FecR-Bla-His expressed in a Tat-deficient strain (HS3018-A ΔtatABC) (C). Soluble and membrane fractions were resolved by SDS-PAGE and transferred to nitrocellulose membranes probed with an anti-6×His antibody. All gels were imaged prior to transfer to determine the total protein loaded in each well (shown in lower images). (A) Soluble fractions. Lane 1, FecR-Bla-His; lane 2, FecR-Bla-His R79/80A; lane 3, FecR-His; lane 4, FecR-His R79/80K. (B) Membrane fractions. Lane 1, FecR-Bla-His; lane 2, FecR-Bla-His R79/80A; lane 3, FecR-His; lane 4, FecR-His R79/80K. (C) Lane 1, HS3018-A FecR-Bla-His, soluble fraction; lane 2, HS3018-A Δtat FecR-Bla-His, soluble fraction; lane 3, HS3018-A FecR-Bla-His, membrane fraction; lane 4, HS3018-A Δtat FecR, FecR-Bla-His, membrane fraction. (D) Crude cell extracts were washed with either 0.2 M Na₂CO₃ or 4 M urea prior to membrane sedimentation. Lane 1, HS3018-A FecR-Bla-His, membrane fraction, with Na₂CO₃; lane 2, HS3018-A Δtat FecR-Bla-His, membrane fraction, with Na₂CO₃; lane 3, HS3018-A FecR-Bla-His, membrane fraction, with urea; lane 4, HS3018-A Δtat FecR-Bla-His, membrane fraction, with urea. wt, wild type.

FIG 4

Relative gene expression of the fecABCDE operon and fecR under iron-limiting conditions. Quantitative real-time PCR of a fecR mutant (strain BW25113) expressing either wild-type fecR or the fecR R79/80K substitution and an empty plasmid control was performed on RNA/cDNA extracted from cell cultures grown in medium supplemented with 2′,2′-dipyridyl and 1 mM sodium citrate. Error bars represent the standard deviation of mean values derived from three biological replicates.