Influence of the TorD signal peptide chaperone on Tat-dependent protein translocation

The twin-arginine translocation (Tat) pathway transports folded proteins across energetic membranes. Numerous Tat substrates contain co-factors that are inserted before transport with the assistance of redox enzyme maturation proteins (REMPs), which bind to the signal peptide of precursor proteins. How signal peptides are transferred from a REMP to a binding site on the Tat receptor complex remains unknown. Since the signal peptide mediates both interactions, possibilities include: i) a coordinated hand-off mechanism; or ii) a diffusional search after REMP dissociation. We investigated the binding interaction between substrates containing the TorA signal peptide (spTorA) and its cognate REMP, TorD, and the effect of TorD on the in vitro transport of such substrates. We found that Escherichia coli TorD is predominantly a monomer at low micromolar concentrations (dimerization KD > 50 μM), and this monomer binds reversibly to spTorA (KD ≈ 1 μM). While TorD binds to membranes (KD ≈ 100 nM), it has no apparent affinity for Tat translocons and it inhibits binding of a precursor substrate to the membrane. TorD has a minimal effect on substrate transport by the Tat system, being mildly inhibitory at high concentrations. These data are consistent with a model in which the REMP-bound signal peptide is shielded from recognition by the Tat translocon, and spontaneous dissociation of the REMP allows the substrate to engage the Tat machinery. Thus, the REMP does not assist with targeting to the Tat translocon, but rather temporarily shields the signal peptide.

Influence of the TorD signal peptide chaperone on Tat-dependent protein translocation

The twin-arginine translocation (Tat) pathway transports folded proteins across energetic membranes. Numerous Tat substrates contain co-factors that are inserted before transport with the assistance of redox enzyme maturation proteins (REMPs), which bind to the signal peptide of precursor proteins. How signal peptides are transferred from a REMP to a binding site on the Tat receptor complex remains unknown. Since the signal peptide mediates both interactions, possibilities include: i) a coordinated hand-off mechanism; or ii) a diffusional search after REMP dissociation. We investigated the binding interaction between substrates containing the TorA signal peptide (spTorA) and its cognate REMP, TorD, and the effect of TorD on the in vitrotransport of such substrates. We found that Escherichia coli TorD is predominantly a monomer at low micromolar concentrations (dimerization KD > 50 M), and this monomer binds reversibly to spTorA (KD 1 M). While TorD binds to membranes (KD 100 nM), it has no apparent affinity for Tat translocons and it inhibits binding of a precursor substrate to the membrane. TorD has a minimal effect on substrate transport by the Tat system, being mildly inhibitory at high concentrations. These data are consistent with a model in which the REMP-bound signal peptide is shielded from recognition by the Tat translocon, and spontaneous dissociation of the REMP allows the substrate to engage the Tat machinery. Thus, the REMP does not assist with targeting to the Tat translocon, but rather temporarily shields the signal peptide.

Disruption of the Pseudomonas aeruginosa Tat system perturbs PQS-dependent quorum sensing and biofilm maturation through loss of the Rieske cytochrome bc1 sub-unit

Extracellular DNA (eDNA) is a major constituent of the extracellular matrix of P seudomonas aeruginosa biofilms and its release is regulated via the pseudomonas quinolone signal (PQS) dependent quorum sensing (QS). By screening a P. aeruginosa transposon library to identify factors required for DNA release, mutants with insertions in the twin-arginine translocation (Tat) pathway were identified as exhibiting reduced eDNA release, and defective biofilm architecture with enhanced susceptibility to tobramycin. P. aeruginosa tat mutants showed substantial reductions in pyocyanin, rhamnolipid and membrane vesicle (MV) production consistent with perturbation of 2-heptyl-3-hydroxy-4-quinolone (PQS) dependent QS as demonstrated by changes in pqsA expression and 2-alkyl-4-quinolone (AQ) production. Provision of exogenous PQS to the tat mutants did not return pqsA , rhlA or phzA1 expression or pyocyanin production to wild type levels. However, transformation of the tat mutants with the AQ-independent pqs effector pqsE restored phzA1 expression and pyocyanin production. Since mutation or inhibition of Tat prevented PQS-driven auto-induction, we sought to identify the Tat secretion substrate responsible. A pqsA::lux fusion was introduced into each of 34 validated P. aeruginosa Tat substrate deletion mutants. Analysis of each mutant for reduced bioluminescence revealed that the signalling defect was associated with the Rieske iron-sulfur subunit of the cytochrome bc 1 complex. In common with the parent strain, a Rieske mutant exhibited defective PQS signalling, AQ production, rhlA expression and eDNA release that could be restored by genetic complementation. Thus, lack of the Rieske sub-unit export is clearly responsible for the Tat-mediated perturbation of PQS-dependent QS, the loss of virulence factor production, biofilm eDNA and the tobramycin tolerance of P. aeruginosa biofilms.

Twin-arginine translocation (Tat) mutants in Salmonella enterica serovar Typhimurium have increased susceptibility to cell wall targeting antibiotics

ABSTRACT The twin-arginine translocation (Tat) system is a protein secretion system that is conserved in bacteria, archaea and plants. In Gram-negative bacteria, it is required for the export of folded proteins from the cytoplasm to the periplasm. There are 30 experimentally verified Tat substrates in Salmonella, including hydrogenase subunits, enzymes required for anaerobic respiration and enzymes involved in peptidoglycan remodeling during cell division. Multiple studies have demonstrated the susceptibility of tat mutants to antimicrobial compounds such as SDS and bile; however, in this work, we use growth curves and viable plate counts to demonstrate that cell wall targeting antibiotics (penicillins, carbapenems, cephalosporins and fosfomycin) have increased killing against a Δtat strain. Further, we demonstrate that this increased killing is primarily due to defects in translocation of critical Tat substrates: MepK, AmiA, AmiC and SufI. Finally, we show that a ΔhyaAB ΔhybABC ΔhydBC strain has an altered ΔΨ that impacts proper secretion of critical Tat substrates in aerobic growth conditions.

Insights into substrate-mediated assembly of the chloroplast TAT receptor complex

The Twin Arginine Transport (TAT) system translocates fully folded proteins across the thylakoid membrane in the chloroplast (cp) and the cytoplasmic membrane of bacteria. In chloroplasts, cpTAT transport is achieved by three components: Tha4, Hcf106, and cpTatC. Hcf106 and cpTatC function as the substrate recognition/binding complex while Tha4 is thought to play a significant role in forming the translocation pore. Recent studies challenged this idea by suggesting that cpTatC-Hcf106-Tha4 function together in the active translocase. Here, we have mapped the inter-subunit contacts of cpTatC-Hcf106 during the resting state and built a cpTatC-Hcf106 structural model based on our crosslinking data. In addition, we have identified a substrate-mediated reorganization of cpTatC-Hcf106 contact sites during active substrate translocation. The proximity of Tha4 to the cpTatC-Hcf106 complex was also identified. Our data suggest a model for cpTAT function in which the transmembrane helices of Hcf106 and Tha4 may each contact the fifth transmembrane helix of cpTatC while the insertion of the substrate signal peptide may rearrange the cpTatC-Hcf106-Tha4 complex and initiate the translocation event.One sentence summaryProtein subunits of the thylakoidal twin arginine transport complex function together during substrate recognition and translocase assembly.

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.

Novel Tat-Dependent Protein Secretion

ABSTRACT The transcription initiation signal elicited by the binding of ferric citrate to the outer membrane FecA protein is transmitted by the FecR protein across the cytoplasmic membrane to the FecI extracytoplasmic function (ECF) sigma factor. In this issue of Journal of Bacteriology, I. J. Passmore, J. M. Dow, F. Coll, J. Cuccui, et al. (J Bacteriol 202:e00541-19, 2020, https://doi.org/10.1128/JB.00541-19) report that the FecR sequence contains both the twin-arginine signal motif and the secretory (Sec) avoidance motif typical of proteins secreted by the twin-arginine translocation (TAT) system. The same study shows that FecR is indeed secreted by Tat and represents a new class of bitopic Tat-dependent membrane proteins.

Twin-Arginine Translocation System Is Involved in Citrobacter rodentium Fitness in the Intestinal Tract

ABSTRACT The twin-arginine translocation (Tat) system is involved in not only a wide array of cellular processes but also pathogenesis in many bacterial pathogens; thus, this system is expected to become a novel therapeutic target to treat infections. To the best of our knowledge, involvement of the Tat system has not been reported in the gut infection caused by Citrobacter rodentium. Here, we studied the role of Tat in C. rodentium gut infection, which resembles human infection with enterohemorrhagic Escherichia coli (EHEC) and enteropathogenic E. coli (EPEC). A C. rodentium Tat loss-of-function mutant displayed prolonged gut colonization, which was explained by reduced inflammatory responses and, particularly, neutrophil infiltration. Further, the Tat mutant had colonization defects upon coinfection with the wild-type strain of C. rodentium. The Tat mutant also became hypersensitive to bile acids, and an increase in fecal bile acids fostered C. rodentium clearance from the gut lumen. Finally, we show that the chain form of C. rodentium cells, induced by a Tat-dependent cell division defect, exhibits impaired resistance to bile acids. Our findings indicate that the Tat system is involved in gut colonization by C. rodentium, which is associated with neutrophil infiltration and resistance to bile acids. Interventions that target the Tat system, as well as luminal bile acids, might thus be promising therapeutic strategies to treat human EHEC and EPEC infections.