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.

Insight into the Global Negative Regulation of Iron Scavenger 7-HT Biosynthesis by the SigW/RsiW System in Pseudomonas donghuensis HYS

“…P. aeruginosa PAO1 and P. putida KT2440 are widely distributed pathogenic microorganisms, and 19 ECF σ factors are found to be encoded within each of their genomes [ 36 , 37 ]. In contrast, the genome of P. donghuensis HYS encodes 20 ECF σ factors, 10 of which are significantly responsive to iron and most of which cooperate with FecR proteins, which is a relatively common transmembrane transport process involved in iron acquisition [ 48 ]. Two ECF σ factors of the σ 70 family, named sigW and σ16, attracted our attention.…”

Novel Tat-Dependent Protein Secretion

“…In their recent paper, Passmore et al (1) described the novel Tat (twin arginine translocation)dependent translocation of a bitopic cytoplasmic membrane protein with functional domains located in the periplasm and in the cytoplasm. The protein receives a transcriptional regulatory signal in the periplasm and transmits it across the cytoplasmic membrane to the cytoplasm, where it activates an extracytoplasmic function (ECF) sigma factor.…”

“…secretion by Tat was convincingly demonstrated in a straightforward experimental approach by Passmore et al(1). Residues 1-115 of FecR were fused to the Bla β-lactamase and the secretion of the construct was then tested based on the ampicillin resistance conferred by Bla in the periplasm.…”

“…(1). However, whereas most Tat proteins contain the Tat motif close to the N-terminus, the FecR Tat motif is located deeper inside the sequence (residues 68-109).…”

Identification of novel tail-anchored membrane proteins integrated by the bacterial twin-arginine translocase

“…Alongside the Sec pathway and YidC, the twin-arginine translocase (Tat) is the third system found in the cytoplasmic membrane of prokaryotes that is able to mediate membrane protein insertion. Although most Tat substrates identified to date are globular periplasmic proteins, the Tat pathway is able to integrate proteins that have a single hydrophobic helix located either at the N-or C-terminus of the substrate [6,7], or occasionally within the protein sequence [8,9].…”