Bacterial Twin-arginine Translocation Research Articles

Oligomerization state of the functional bacterial twin-arginine translocation (Tat) receptor complex

The twin-arginine translocation (Tat) system transports folded proteins across bacterial and plastid energy transducing membranes. Ion leaks are generally considered to be mitigated by the creation and destruction of the translocation conduit in a cargo-dependent manner, a mechanism that enables tight sealing around a wide range of cargo shapes and sizes. In contrast to the variable stoichiometry of the active translocon, the oligomerization state of the receptor complex is considered more consistently stable but has proved stubbornly difficult to establish. Here, using a single molecule photobleaching analysis of individual inverted membrane vesicles, we demonstrate that Tat receptor complexes are tetrameric in native membranes with respect to both TatB and TatC. This establishes a maximal diameter for a resting state closed pore. A large percentage of Tat-deficient vesicles explains the typically low transport efficiencies observed. This individual reaction chamber approach will facilitate examination of the effects of stochastically distributed molecules.

Oligomerization state of the functional bacterial twin arginine translocation (Tat) receptor complex

The twin-arginine translocation (Tat) system transports folded proteins important for growth and survival across bacterial and plastid energy transducing membranes. Ion leaks are generally considered to be mitigated by the creation and destruction of the translocation conduit in a cargo-dependent manner, a mechanism that enables tight sealing around a wide range of cargo shapes and sizes. Since the Tat system is conserved among bacteria and absent in mammalian cells, it is an attractive target for antibacterial therapies.

Cross-Species Functional Conservation and Possible Origin of the N-Terminal Specificity Domain of Mitochondrial Presequences.

Plants have two endosymbiotic organelles, chloroplast and mitochondrion. Although they have their own genomes, proteome assembly in these organelles depends on the import of proteins encoded by the nuclear genome. Previously, we elucidated the general design principles of chloroplast and mitochondrial targeting signals, transit peptide, and presequence, respectively, which are highly diverse in primary structure. Both targeting signals are composed of N-terminal specificity domain and C-terminal translocation domain. Especially, the N-terminal specificity domain of mitochondrial presequences contains multiple arginine residues and hydrophobic sequence motif. In this study we investigated whether the design principles of plant mitochondrial presequences can be applied to those in other eukaryotic species. We provide evidence that both presequences and import mechanisms are remarkably conserved throughout the species. In addition, we present evidence that the N-terminal specificity domain of presequence might have evolved from the bacterial TAT (twin-arginine translocation) signal sequence.

The early mature part of bacterial twin-arginine translocation (Tat) precursor proteins contributes to TatBC receptor binding

The twin-arginine translocation (Tat) pathway transports folded proteins across bacterial membranes. Tat precursor proteins possess a conserved twin-arginine (RR) motif in their signal peptides that is involved in the binding of the proteins to the membrane-associated TatBC receptor complex. In addition, the hydrophobic region in the Tat signal peptides also contributes to TatBC binding, but whether regions beyond the signal-peptide cleavage site are involved in this process is unknown. Here, we analyzed the contribution of the early mature protein part of the Escherichia coli trimethylamine N-oxide reductase (TorA) to productive TatBC receptor binding. We identified substitutions in the 30 amino acids immediately following the TorA signal peptide (30aa-region) that restored export of a transport-defective TorA[KQ]-30aa-MalE precursor, in which the RR residues had been replaced by a lysine-glutamine pair. Some of these substitutions increased the hydrophobicity of the N-terminal part of the 30aa-region and thereby likely enhanced hydrophobic substrate-receptor interactions within the hydrophobic TatBC substrate-binding cavity. Another class of substitutions increased the positive net charge of the region's C-terminal part, presumably leading to strengthened electrostatic interactions between the mature substrate part and the cytoplasmic TatBC regions. Furthermore, we identified substitutions in the C-terminal domains of TatB following the transmembrane segment that restored transport of various transport-defective TorA-MalE derivatives. Some of these substitutions most likely affected the orientation or conformation of the flexible, carboxy-proximal helices of TatB. Therefore, we propose that a tight accommodation of the folded mature region by TatB contributes to productive binding of Tat substrates to TatBC.

The Hairpin‐Hinge Model of Tat Protein Transport

The bacterial twin arginine translocation (Tat) pathway transports folded proteins across the cytoplasmic membrane. Biochemical evidence indicates that the TatBC receptor complex contains the signal peptide binding site and the interaction of the signal peptide with this complex triggers the assembly of a translocation channel. However, it is unclear how the signal peptide interacts with the receptor complex and how it enables the movement of cargo through the channel. Here we show using fluorescence quenching and disulfide crosslinking that TatBC complex inserts pre‐SufI as a hairpin that extends about half‐way across the membrane. Photo crosslinking studies indicated that the c‐terminal of the hairpin interacts with TatB. Furthermore, computational analysis of ~500 Tat signal peptides and docking simulations of TatBC and pre‐SufI revealed 3 common features of the hairpin structure: 1) a helical structure; 2) a helix destabilizing residue; and 3) a relatively disordered domain. A hinge motion at the hairpin bend would allow the signal peptide to be bound to TatC surface via double arginines (n‐terminal) and allow the cleavage site (c‐terminal) to migrate to the periplasmic side. Disulfide crosslinking of n‐ and c‐terminal hairpin ends inhibited the transport of the cargo suggesting a novel Hairpin‐Hinge Translocation for Tat pathwaySupport or Funding InformationNIH Grant GMO65534

An engineered genetic selection for ternary protein complexes inspired by a natural three-component hitchhiker mechanism.

The bacterial twin-arginine translocation (Tat) pathway is well known to translocate correctly folded monomeric and dimeric proteins across the tightly sealed cytoplasmic membrane. We identified a naturally occurring heterotrimer, the Escherichia coli aldehyde oxidoreductase PaoABC, that is co-translocated by the Tat translocase according to a ternary “hitchhiker” mechanism. Specifically, the PaoB and PaoC subunits, each devoid of export signals, are escorted to the periplasm in a piggyback fashion by the Tat signal peptide-containing subunit PaoA. Moreover, export of PaoA was blocked when either PaoB or PaoC was absent, revealing a surprising interdependence for export that is not seen for classical secretory proteins. Inspired by this observation, we created a bacterial three-hybrid selection system that links the formation of ternary protein complexes with antibiotic resistance. As proof-of-concept, a bispecific antibody was employed as an adaptor that physically crosslinked one antigen fused to a Tat export signal with a second antigen fused to TEM-1 β-lactamase (Bla). The resulting non-covalent heterotrimer was exported in a Tat-dependent manner, delivering Bla to the periplasm where it hydrolyzed β-lactam antibiotics. Collectively, these results highlight the remarkable flexibility of the Tat system and its potential for studying and engineering ternary protein interactions in living bacteria.

Optimizing recombinant antibodies for intracellular function using hitchhiker-mediated survival selection

The 'hitchhiker' mechanism of the bacterial twin-arginine translocation pathway has previously been adapted as a genetic selection for detecting pairwise protein interactions in the cytoplasm of living Escherichia coli cells. Here, we extended this method, called FLI-TRAP, for rapid isolation of intracellular antibodies (intrabodies) in the single-chain Fv format that possess superior traits simply by demanding bacterial growth on high concentrations of antibiotic. Following just a single round of survival-based enrichment using FLI-TRAP, variants of an intrabody against the dimerization domain of the yeast Gcn4p transcription factor were isolated having significantly greater intracellular stability that translated to yield enhancements of >10-fold. Likewise, an intrabody specific for the non-amyloid component region of α-synuclein was isolated that has ~8-fold improved antigen-binding affinity. Collectively, our results illustrate the potential of the FLI-TRAP method for intracellular stabilization and affinity maturation of intrabodies, all without the need for purification or immobilization of the antigen.

Functional characterization of Edwardsiella tarda twin-arginine translocation system and its potential use as biological containment in live attenuated vaccine of marine fish

Bacterial twin-arginine translocation (Tat) system contributes to translocate folded proteins to the periplasm and plays pleiotropic roles in physiological fitness. Here, we showed that the fish pathogen Edwardsiella tarda Tat pathway was functional and was essential for H2S production and hemolytic activity. E. tarda Tat mutant was more susceptible to diverse stresses such as high temperature, SDS, ethanol, and high-salt conditions. However, E. tarda Tat mutant displayed marginal in vivo virulence attenuation in fish models. Comparative proteomics analysis using two-dimensional gel electrophoresis (2-DGE) and matrix-assisted laser desorption/ionization time-of-flight/time-of-flight tandem mass spectrometry were performed to identify proteins undergoing changes in expression levels under high-salt conditons when the Tat pathway was mutilated. Of the 96 differently expressed proteins on the 2-DGE map, 15 proteins were successfully identified with a MASCOT score >45 (p < 0.05) and fold change higher than 2. These significantly differentially expressed proteins were functionally related to basal metabolism and the biosynthesis of proteins and macromolecules. The results of plate counting further confirmed that the Tat mutant was high-salt-sensitive, indicating that Tat mutant merits as a novel salt-sensitive biological containment system for live attenuated vaccine (LAV) in marine fish vaccinology. To test this, we deleted the type III secretion system genes and cured endogenous plasmid pEIB202 to construct a LAV candidate in the context of Tat abrogation in E. tarda. The results indicated that the LAV candidate was highly attenuated when injected intraperitoneally and elicited significant protection against challenge of wild-type E. tarda in turbot while being rapidly eliminated in seawater.

Twin-arginine translocase mutations that suppress folding quality control and permit export of misfolded substrate proteins

The bacterial twin-arginine translocation (Tat) pathway facilitates the transport of correctly folded proteins across the tightly sealed cytoplasmic membrane. Here, we report the isolation and characterization of suppressor mutations in the Tat translocase that allow export of misfolded proteins, which form structures that are not normally tolerated by the wild-type translocase. Selection of suppressors was enabled by a genetic assay that effectively linked in vivo folding and stability of a test protein with Tat export efficiency of a selectable marker protein, namely TEM-1 β-lactamase. By using a test protein named α(3)B-a designed three-helix-bundle protein that forms collapsed, stable molten globules but lacks a uniquely folded structure-translocase mutants that rescued export of this protein were readily identified. Each mutant translocase still efficiently exported folded substrate proteins, indicating that the substrate specificity of suppressors was relaxed but not strictly altered. A subset of the suppressors could also export other misfolded proteins, such as the aggregation-prone α(3)A protein and reduced alkaline phosphatase. Importantly, the isolation of genetic suppressors that inactivate the Tat quality-control mechanism provides direct evidence for the participation of the Tat translocase in structural proofreading of substrate proteins and reveals epitopes in the translocase that are important for this process.

Kinetics of Precursor Interactions with the Bacterial Tat Translocase Detected by Real-time FRET

The Escherichia coli twin-arginine translocation (Tat) system transports fully folded and assembled proteins across the inner membrane into the periplasmic space. Traditionally, in vitro protein translocation studies have been performed using gel-based transport assays. This technique suffers from low time resolution, and often, an inability to distinguish between different steps in a continuously occurring translocation process. To address these limitations, we have developed an in vitro FRET-based assay that reports on an early step in the Tat translocation process in real-time. The natural Tat substrate pre-SufI was labeled with Alexa532 (donor), and the fluorescent protein mCherry (acceptor) was fused to the C terminus of TatB or TatC. The colored Tat proteins were easily visible during purification, enabling identification of a highly active inverted membrane vesicle (IMV) fraction yielding transport rates with NADH almost an order of magnitude faster than previously reported. When pre-SufI was bound to the translocon, FRET was observed for both Tat proteins. FRET was diminished upon addition of nonfluorescent pre-SufI, indicating that the initial binding step is reversible. When the membranes were energized with NADH, the FRET signal was lost after a short delay. These data suggest a model in which a Tat cargo initially associates with the TatBC complex, and an electric field gradient is required for the cargo to proceed to the next stage of transport. This cargo migration away from the TatBC complex requires a significant fraction of the total transport time.

Engineering Antibody Fitness and Function Using Membrane-Anchored Display of Correctly Folded Proteins

A hallmark of the bacterial twin-arginine translocation (Tat) pathway is its ability to export folded proteins. Here, we discovered that overexpressed Tat substrate proteins form two distinct, long-lived translocation intermediates that are readily detected by immunolabeling methods. Formation of the early translocation intermediate Ti-1, which exposes the N- and C-termini to the cytoplasm, did not require an intact Tat translocase, a functional Tat signal peptide, or a correctly folded substrate. In contrast, formation of the later translocation intermediate, Ti-2, which exhibits a bitopic topology with the N-terminus in the cytoplasm and C-terminus in the periplasm, was much more particular, requiring an intact translocase, a functional signal peptide, and a correctly folded substrate protein. The ability to directly detect Ti-2 intermediates was subsequently exploited for a new protein engineering technology called MAD-TRAP (membrane-anchored display for Tat-based recognition of associating proteins). Through the use of just two rounds of mutagenesis and screening with MAD-TRAP, the intracellular folding and antigen-binding activity of a human single-chain antibody fragment were simultaneously improved. This approach has several advantages for library screening, including the unique involvement of the Tat folding quality control mechanism that ensures only native-like proteins are displayed, thus eliminating poorly folded sequences from the screening process.

Salt Sensitivity of Minimal Twin Arginine Translocases

Bacterial twin arginine translocation (Tat) pathways have evolved to facilitate transport of folded proteins across membranes. Gram-negative bacteria contain a TatABC translocase composed of three subunits named TatA, TatB, and TatC. In contrast, the Tat translocases of most Gram-positive bacteria consist of only TatA and TatC subunits. In these minimal TatAC translocases, a bifunctional TatA subunit fulfils the roles of both TatA and TatB. Here we have probed the importance of conserved residues in the bifunctional TatAy subunit of Bacillus subtilis by site-specific mutagenesis. A set of engineered TatAy proteins with mutations in the cytoplasmic hinge and amphipathic helix regions were found to be inactive in protein translocation under standard growth conditions for B. subtilis or when heterologously expressed in Escherichia coli. Nevertheless, these mutated TatAy proteins did assemble into TatAy and TatAyCy complexes, and they facilitated membrane association of twin arginine precursor proteins in E. coli. Interestingly, most of the mutated TatAyCy translocases were salt-sensitive in B. subtilis. Similarly, the TatAC translocases of Bacillus cereus and Staphylococcus aureus were salt-sensitive when expressed in B. subtilis. Taken together, our present observations imply that salt-sensitive electrostatic interactions have critical roles in the preprotein translocation activity of certain TatAC type translocases from Gram-positive bacteria.

Folding and Self-Assembly of the Pore-Forming Unit Tat-A of the Bacterial Twin-Arginine Translocase

The bacterial Twin-arginine translocation pathway is able to transport fully folded proteins across membranes. In B. subtilis it consists only of two components: TatCd, which serves as a receptor for the signal peptide, and the pore forming unit TatAd, which occurs in high stoichiometric excess. According to circular dichroism TatA contains a transmembrane segment, an amphiphilic helix, and an unstructured C-terminus [1]. Its detailed moelcular structure was resolved by solid-state NMR spectroscopy in oriented bilayers [2]. A striking pattern on the monomeric protein surface allowed us to assemble several units into protomers and into an open oligomeric pore. The stability of these complexes was supported by all-atom MD simulations and using structure-based modeling [3]. The observed interactions suggest that a novel motif for folding and self-assembly motif is present in this membrane-bound transport system, which allows reversible pore formation. Our comprehensive three-dimensional model thus reconciles for the first time TatA transport with a pore size of variable diameter, which can open and close by an energetically feasible mechanism.[1] Muller, S.D., A.A. De Angelis, T.H. Walther, S.L. Grage, C. Lange, S.J. Opella & A.S. Ulrich (2007) Biochim. Biophys. Acta 1768: 3071-3079[2] Walther, T.H., S.L. Grage, N. Roth & A.S. Ulrich (2010) J. Am. Chem. Soc., in press[3] Grage, S.L., T.H. Walther, M. Wolf, A. Vargiu, M.J. Klein, P. Ruggerone, W. Wenzel, A.S. Ulrich (2010) submitted∼

Functional Characterization of Vibrio alginolyticus Twin-Arginine Translocation System: Its Roles in Biofilm Formation, Extracellular Protease Activity, and Virulence Towards Fish

The bacterial twin-arginine translocation (Tat) system contributes to translocate folded proteins and plays pleiotropic roles in growth, motility, and the secretion of some virulent factors. In this study, the authors identified the Tat gene cluster in fish pathogen Vibrio alginolyticus and explored its roles in pathogenesis toward fish. Vibrio alginolyticus Tat mutants showed growth deficiency in TMAO medium, while the complement strain restored the ability to grow in the medium, demonstrating the conservative function of the Tat system in translocation of redox enzymes or cofactors in this bacterium. In V. alginolyticus, deletion of the tatABC genes led to a drastic decrease in biofilm biogenesis. Interestingly, the secretion of extracellular protease Asp, an established exotoxin of the bacterium, was significantly decreased in the TatC mutant, suggesting that TatC might play a part in the production of virulence factors in the bacterium. Furthermore, the Tat mutants displayed attenuated virulence toward the fish model and EPC cells. These findings suggest that the Tat secretion related to the extracellular protease activity as well as virulence in V. alginolyticus provided new insights into the pathogenesis of vibriosis in fish.

Intrinsic GTPase activity of a bacterial twin‐arginine translocation proofreading chaperone induced by domain swapping

The bacterial twin-arginine translocation (Tat) system is a protein targeting pathway dedicated to the transport of folded proteins across the cytoplasmic membrane. Proteins transported on the Tat pathway are synthesised as precursors with N-terminal signal peptides containing a conserved amino acid motif. In Escherichia coli, many Tat substrates contain prosthetic groups and undergo cytoplasmic assembly processes prior to the translocation event. A pre-export 'Tat proofreading' process, mediated by signal peptide-binding chaperones, is considered to prevent premature export of some Tat-targeted proteins until all other assembly processes are complete. TorD is a paradigm Tat proofreading chaperone and co-ordinates the maturation and export of the periplasmic respiratory enzyme trimethylamine N-oxide reductase (TorA). Although it is well established that TorD binds directly to the TorA signal peptide, the mechanism of regulation or control of binding is not understood. Previous structural analyses of TorD homologues showed that these proteins can exist as monomeric and domain-swapped dimeric forms. In the present study, we demonstrate that isolated recombinant TorD exhibits a magnesium-dependent GTP hydrolytic activity, despite the absence of classical nucleotide-binding motifs in the protein. TorD GTPase activity is shown to be present only in the domain-swapped homodimeric form of the protein, thus defining a biochemical role for the oligomerisation. Site-directed mutagenesis identified one TorD side-chain (D68) that was important in substrate selectivity. A D68W variant TorD protein was found to exhibit an ATPase activity not observed for native TorD, and an in vivo assay established that this variant was defective in the Tat proofreading process.

Mining mammalian genomes for folding competent proteins using Tat‐dependent genetic selection in Escherichia coli

Recombinant expression of eukaryotic proteins in Escherichia coli is often limited by poor folding and solubility. To address this problem, we employed a recently developed genetic selection for protein folding and solubility based on the bacterial twin-arginine translocation (Tat) pathway to rapidly identify properly folded recombinant proteins or soluble protein domains of mammalian origin. The coding sequences for 29 different mammalian polypeptides were cloned as sandwich fusions between an N-terminal Tat export signal and a C-terminal selectable marker, namely beta-lactamase. Hence, expression of the selectable marker and survival on selective media was linked to Tat export of the target mammalian protein. Since the folding quality control feature of the Tat pathway prevents export of misfolded proteins, only correctly folded fusion proteins reached the periplasm and conferred cell survival. In general, the ability to confer growth was found to relate closely to the solubility profile and molecular weight of the protein, although other features such as number of contiguous hydrophobic amino acids and cysteine content may also be important. These results highlight the capacity of Tat selection to reveal the folding potential of mammalian proteins and protein domains without the need for structural or functional information about the target protein.

An Essential Role for the DnaK Molecular Chaperone in Stabilizing Over-expressed Substrate Proteins of the Bacterial Twin-arginine Translocation Pathway

All secreted proteins in Escherichia coli must be maintained in an export-competent state before translocation across the inner membrane. In the case of the Sec pathway, this function is carried out by the dedicated SecB chaperone and the general chaperones DnaK-DnaJ-GrpE and GroEL-GroES, whose job collectively is to render substrate proteins partially or entirely unfolded before engagement of the translocon. To determine whether these or other general molecular chaperones are similarly involved in the translocation of folded proteins through the twin-arginine translocation (Tat) system, we screened a collection of E. coli mutant strains for their ability to transport a green fluorescent protein (GFP) reporter through the Tat pathway. We found that the molecular chaperone DnaK was essential for cytoplasmic stability of GFP bearing an N-terminal Tat signal peptide, as well as for numerous other recombinantly expressed endogenous and heterologous Tat substrates. Interestingly, the stability conferred by DnaK did not require a fully functional Tat signal as substrates bearing translocation defective twin lysine substitutions in the consensus Tat motif were equally unstable in the absence of DnaK. These findings were corroborated by crosslinking experiments that revealed an in vivo association between DnaK and a truncated version of the Tat substrate trimethylamine N-oxide reductase (TorA502) bearing an RR or a KK signal peptide. Since TorA502 lacks nine molybdo-cofactor ligands essential for cofactor attachment, the involvement of DnaK is apparently independent of cofactor acquisition. Finally, we show that the stabilizing effects of DnaK can be exploited to increase the expression and translocation of Tat substrates under conditions where the substrate production level exceeds the capacity of the Tat translocase. This latter observation is expected to have important consequences for the use of the Tat system in biotechnology applications where high levels of periplasmic expression are desirable.

Genetic selection for protein solubility enabled by the folding quality control feature of the twin‐arginine translocation pathway

One of the most vexing problems facing structural genomics efforts and the biotechnology enterprise in general is the inability to efficiently produce functional proteins due to poor folding and insolubility. Additionally, protein misfolding and aggregation has been linked to a number of human diseases, such as Alzheimer's. Thus, a robust cellular assay that allows for direct monitoring, manipulation, and improvement of protein folding could have a profound impact. We report the development and characterization of a genetic selection for protein folding and solubility in living bacterial cells. The basis for this assay is the observation that protein transport through the bacterial twin-arginine translocation (Tat) pathway depends on correct folding of the protein prior to transport. In this system, a test protein is expressed as a tripartite fusion between an N-terminal Tat signal peptide and a C-terminal TEM1 beta-lactamase reporter protein. We demonstrate that survival of Escherichia coli cells on selective medium expressing a Tat-targeted test protein/beta-lactamase fusion correlates with the solubility of the test protein. Using this assay, we isolated solubility-enhanced variants of the Alzheimer's Abeta42 peptide from a large combinatorial library of Abeta42 sequences, thereby confirming that our assay is a highly effective selection tool for soluble proteins. By allowing the bacterial Tat pathway to exert folding quality control on expressed target protein sequences, we have generated a powerful tool for monitoring protein folding and solubility in living cells, for molecular engineering of solubility-enhanced proteins or for the isolation of factors and/or cellular conditions that stabilize aggregation-prone proteins.

Twin-Arginine Translocation of Active Human Tissue Plasminogen Activator in Escherichia coli

When eukaryotic proteins with multiple disulfide bonds are expressed at high levels in Escherichia coli, the efficiency of thiol oxidation and isomerization is typically not sufficient to yield soluble products with native structures. Even when such proteins are secreted into the oxidizing periplasm or expressed in the cytoplasm of cells carrying mutations in the major intracellular disulfide bond reduction systems (e.g., trxB gor mutants), correct folding can be problematic unless a folding modulator is simultaneously coexpressed. In the present study we explored whether the bacterial twin-arginine translocation (Tat) pathway could serve as an alternative expression system for obtaining appreciable levels of recombinant proteins which exhibit complex patterns of disulfide bond formation, such as full-length human tissue plasminogen activator (tPA) (17 disulfides) and a truncated but enzymatically active version of tPA containing nine disulfides (vtPA). Remarkably, targeting of both tPA and vtPA to the Tat pathway resulted in active protein in the periplasmic space. We show here that export by the Tat translocator is dependent upon oxidative protein folding in the cytoplasm of trxB gor cells prior to transport. Whereas previous efforts to produce high levels of active tPA or vtPA in E. coli required coexpression of the disulfide bond isomerase DsbC, we observed that Tat-targeted vtPA and tPA reach a native conformation without thiol-disulfide oxidoreductase coexpression. These results demonstrate that the Tat system may have inherent and unexpected benefits compared with existing expression strategies, making it a viable alternative for biotechnology applications that hinge on protein expression and secretion.

Secretion of active xylanase C from Streptomyces lividans is exclusively mediated by the Tat protein export system

The bacterial twin-arginine translocation (Tat) pathway transports folded proteins across the cytoplasmic membrane. The precursors targeted to the Tat pathway have signal peptides bearing the consensus motif (S/T-R-R-X-F-L-K). The xylanase C (XlnC) of Streptomyces lividans is a 20-kDa secreted enzyme. The XlnC signal peptide is 49 amino acids long and contains the S-R-R-G-F-L-G sequence, which is similar to the twin-arginine consensus motif. In S. lividans, XlnC secretion was impaired in a tatC insertion mutant, which is unable to secrete proteins that are dependent on the Tat system. When the signal peptide of XlnC was replaced by the Sec-dependent signal peptide of xylanase A, XlnC was secreted as an inactive form and demonstrated rapid proteolytic degradation in the culture supernatant, thus indicating that XlnC was specifically secreted through the Tat system. Deletions of the n-region of the XlnC signal sequence showed that a minimum of six amino acids residues preceding the twin-arginine motif was required to secrete XlnC. Replacement of one or both arginines by lysine residues in the twin arginine motif decreased four- and sevenfold, respectively, the enzyme production but did not abolish it. However, pulse chase experiments showed that the half-life of the precursor was from 2 to 3 h instead of 11 min for the wild- type precursor. Since XlnC is not associated with cofactors to exhibit activity, it is therefore a newly identified prokaryotic non-redox Tat substrate.