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

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.

Characterization of a pre-export enzyme-chaperone complex on the twin-arginine transport pathway.

The Tat (twin-arginine translocation) system is a protein targeting pathway utilized by prokaryotes and chloroplasts. Tat substrates are produced with distinctive N-terminal signal peptides and are translocated as fully folded proteins. In Escherichia coli, Tat-dependent proteins often contain redox cofactors that must be loaded before translocation. Trimethylamine N-oxide reductase (TorA) is a model bacterial Tat substrate and is a molybdenum cofactor-dependent enzyme. Co-ordination of cofactor loading and translocation of TorA is directed by the TorD protein, which is a cytoplasmic chaperone known to interact physically with the TorA signal peptide. In the present study, a pre-export TorAD complex has been characterized using biochemical and biophysical techniques, including SAXS (small-angle X-ray scattering). A stable, cofactor-free TorAD complex was isolated, which revealed a 1:1 binding stoichiometry. Surprisingly, a TorAD complex with similar architecture can be isolated in the complete absence of the 39-residue TorA signal peptide. The present study demonstrates that two high-affinity binding sites for TorD are present on TorA, and that a single TorD protein binds both of those simultaneously. Further characterization suggested that the C-terminal 'Domain IV' of TorA remained solvent-exposed in the cofactor-free pre-export TorAD complex. It is possible that correct folding of Domain IV upon cofactor loading is the trigger for TorD release and subsequent export of TorA.

Characterization of a periplasmic nitrate reductase in complex with its biosynthetic chaperone.

Escherichia coli is a Gram-negative bacterium that can use nitrate during anaerobic respiration. The catalytic subunit of the periplasmic nitrate reductase NapA contains two types of redox cofactor and is exported across the cytoplasmic membrane by the twin-arginine protein transport pathway. NapD is a small cytoplasmic protein that is essential for the activity of the periplasmic nitrate reductase and binds tightly to the twin-arginine signal peptide of NapA. Here we show, using spin labelling and EPR, that the isolated twin-arginine signal peptide of NapA is structured in its unbound form and undergoes a small but significant conformational change upon interaction with NapD. In addition, a complex comprising the full-length NapA protein and NapD could be isolated by engineering an affinity tag onto NapD only. Analytical ultracentrifugation demonstrated that the two proteins in the NapDA complex were present in a 1 : 1 molar ratio, and small angle X-ray scattering analysis of the complex indicated that NapA was at least partially folded when bound by its NapD partner. A NapDA complex could not be isolated in the absence of the NapA Tat signal peptide. Taken together, this work indicates that the NapD chaperone binds primarily at the NapA signal peptide in this system and points towards a role for NapD in the insertion of the molybdenum cofactor.