The Twin-Arginine Signal Peptide of Bacillus subtilis YwbN Can Direct either Tat- or Sec-Dependent Secretion of Different Cargo Proteins: Secretion of Active Subtilisin via the B. subtilis Tat Pathway

Proteins that are produced for commercial purposes in Bacillus subtilis are commonly secreted via the Sec pathway. Despite its high secretion capacity, the secretion of heterologous proteins via the Sec pathway is often unsuccessful. Alternative secretion routes, like the Tat pathway, are therefore of interest. Two parallel Tat pathways with distinct specificities have previously been discovered in B. subtilis. To explore the application potential of these Tat pathways, several commercially relevant or heterologous model proteins were fused to the signal peptides of the known B. subtilis Tat substrates YwbN and PhoD. Remarkably, the YwbN signal peptide directed secretion of active subtilisin, a typical Sec substrate, via the B. subtilis TatAyCy route. In contrast, the same signal peptide directed Tat-independent secretion of the Bacillus licheniformis α-amylase (AmyL). Moreover, the YwbN signal peptide directed secretion of SufI, an Escherichia coli Tat substrate, in a Tat-independent manner, most likely via Sec. Our results suggest that cytoplasmic protein folding prior to translocation is probably a major determinant of Tat-dependent protein secretion in B. subtilis, as is the case with E. coli. We conclude that future applications for the Tat system of B. subtilis will most likely involve commercially interesting proteins that are Sec incompatible.     

The Canonical Twin-Arginine Translocase Components Are Not Required for Secretion of Folded Green Fluorescent Protein from the Ancestral Strain of Bacillus subtilis

Cellular processes, such as the digestion of macromolecules, phosphate acquisition, and cell motility, require bacterial secretion systems. In Bacillus subtilis, the predominant protein export pathways are Sec (generalized secretory pathway) and Tat (twin-arginine translocase). Unlike Sec, which secretes unfolded proteins, the Tat machinery secretes fully folded proteins across the plasma membrane and into the medium. Proteins are directed for Tat-dependent export by N-terminal signal peptides that contain a conserved twin-arginine motif. Thus, utilizing the Tat secretion system by fusing a Tat signal peptide is an attractive strategy for the production and export of heterologous proteins. As a proof of concept, we expressed green fluorescent protein (GFP) fused to the PhoD Tat signal peptide in the laboratory and ancestral strains of B. subtilis. Secretion of the Tat-GFP construct, as well as secretion of proteins in general, was substantially increased in the ancestral strain. Furthermore, our results show that secreted, fluorescent GFP could be purified directly from the extracellular medium. Nonetheless, export was not dependent on the known Tat secretion components or the signal peptide twin-arginine motif. We propose that the ancestral strain contains additional Tat components and/or secretion regulators that were abrogated following domestication.     

The Escherichia coli TatABC system and a Bacillus subtilis TatAC-type system recognise three distinct targeting determinants in twin-arginine signal peptides

The Tat system transports folded proteins across bacterial and thylakoid membranes. In Gram-negative organisms, it is encoded by tatABC genes and the system recognizes substrates bearing signal peptides with a conserved twin-arginine motif. Most Gram-positive organisms lack a tatB gene, indicating major differences in organisation and/or mechanism. Here, we have characterized the essential targeting determinants that are recognized by a Bacillus subtilis TatAC-type system, TatAdCd. Substitution by lysine of either of the twin-arginine residues in the TorA signal peptide can be tolerated, but the presence of twin-lysine residues blocks export completely. We show that additional determinants can be as important as the twin-arginine motif. Replacement of the −1 serine by alanine, in either the TorA or DmsA signal peptide, almost blocks export by either the B. subtilis TatAdCd or Escherichia coli TatABC systems, firmly establishing the importance of this −1 residue in these signal peptides. Surprisingly, the +2 leucine in the DmsA signal peptide (sequence SRRGLV) appears to play an equally important role and substitution by alanine or phenylalanine blocks export by both the B. subtilis and E. coli systems. These data identify three distinct determinants, whose importance varies depending on the signal peptide in question. The data also show that the B. subtilis TatAdCd and E. coli TatABC systems recognize very similar determinants within their target peptides, and exhibit surprisingly similar responses to mutations within these determinants.     

Comparative analysis of twin-arginine (Tat)-dependent protein secretion of a heterologous model protein (GFP) in three different Gram-positive bacteria

In contrast to the general protein secretion (Sec) system, the twin-arginine translocation (Tat) export pathway allows the translocation of proteins across the bacterial plasma membrane in a fully folded conformation. Due to this feature, the Tat pathway provides an attractive alternative to the secretory production of heterologous proteins via the Sec system. In this study, the potential for Tat-dependent heterologous protein secretion was compared in the three Gram-positive bacteria Staphylococcus carnosus, Bacillus subtilis, and Corynebacterium glutamicum using green fluorescent protein (GFP) as a model protein. In all three microorganisms, fusion of a Tat signal peptide to GFP resulted in its Tat-dependent translocation across the corresponding cytoplasmic membranes. However, striking differences with respect to the final localization and folding status of the exported GFP were observed. In S. carnosus, GFP was trapped entirely in the cell wall and not released into the supernatant. In B. subtilis, GFP was secreted into the supernatant, however, in an inactive form. In contrast, C. glutamicum effectively secreted active GFP. Our results clearly demonstrate that a comparative evaluation of different Gram-positive host microorganisms is a crucial step on the way to an efficient Tat-mediated secretory production process for a desired heterologous target protein. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. This paper is dedicated to Hermann Sahm on the occasion of his 65th birthday.     

Presenting a foreign antigen on live attenuated Edwardsiella tarda using twin-arginine translocation signal peptide as a multivalent vaccine

The twin-arginine translocation (Tat) system is a major pathway for transmembrane translocation of fully folded proteins. In this study, a multivalent vaccine to present foreign antigens on live attenuated vaccine Edwardsiella tarda WED using screened Tat signal peptide was constructed. Because the Tat system increases the yields of folded antigens in periplasmic space or extracellular milieu, it is expected to contribute to the production of conformational epitope-derived specific antibodies. E. tarda Tat signal peptides fused with the green fluorescent protein (GFP) was constructed under the control of an in vivo inducible dps promoter. The resulting plasmids were electroporated into WED and the subcellular localizations of GFP were analyzed with Western blotting. Eight signal peptides with optimized GFP translocation efficiency were further fused to a protective antigen glyceraldehyde-3-phosphate dehydrogenase (GapA) from a fish pathogen Aeromonas hydrophila. Signal peptides of DmsA, NapA, and SufI displayed high efficiency for GapA translocation. The relative percent survival (RPS) of turbot was measured with a co-infection of E. tarda and A. hydrophila, and the strain with DmsA signal peptide showed the maximal protection. This study demonstrated a new platform to construct multivalent vaccines using optimized Tat signal peptide in E. tarda.     

Specificity of signal peptide recognition in tat-dependent bacterial protein translocation

The bacterial twin arginine translocation (Tat) pathway translocates across the cytoplasmic membrane folded proteins which, in most cases, contain a tightly bound cofactor. Specific amino-terminal signal peptides that exhibit a conserved amino acid consensus motif, S/T-R-R-X-F-L-K, direct these proteins to the Tat translocon. The glucose-fructose oxidoreductase (GFOR) of Zymomonas mobilis is a periplasmic enzyme with tightly bound NADP as a cofactor. It is synthesized as a cytoplasmic precursor with an amino-terminal signal peptide that shows all of the characteristics of a typical twin arginine signal peptide. However, GFOR is not exported to the periplasm when expressed in the heterologous host Escherichia coli, and enzymatically active pre-GFOR is found in the cytoplasm. A precise replacement of the pre-GFOR signal peptide by an authentic E. coli Tat signal peptide, which is derived from pre-trimethylamine N-oxide (TMAO) reductase (TorA), allowed export of GFOR, together with its bound cofactor, to the E. coli periplasm. This export was inhibited by carbonyl cyanide m-chlorophenylhydrazone, but not by sodium azide, and was blocked in E. coli tatC and tatAE mutant strains, showing that membrane translocation of the TorA-GFOR fusion protein occurred via the Tat pathway and not via the Sec pathway. Furthermore, tight cofactor binding (and therefore correct folding) was found to be a prerequisite for proper translocation of the fusion protein. These results strongly suggest that Tat signal peptides are not universally recognized by different Tat translocases, implying that the signal peptides of Tat-dependent precursor proteins are optimally adapted only to their cognate export apparatus. Such a situation is in marked contrast to the situation that is known to exist for Sec-dependent protein translocation.     

Selective contribution of the twin-arginine translocation pathway to protein secretion in Bacillus subtilis

The availability of the complete genome sequence of Bacillus subtilis has allowed the prediction of all exported proteins of this Gram-positive eubacterium. Recently, approximately 180 secretory and 114 lipoprotein signal peptides were predicted to direct protein export from the cytoplasm. Whereas most exported proteins appear to use the Sec pathway, 69 of these proteins could potentially use the Tat pathway, as their signal peptides contain RR- or KR-motifs. In the present studies, proteomic techniques were applied to verify how many extracellular B. subtilis proteins follow the Tat pathway. Strikingly, the extracellular accumulation of 13 proteins with potential RR/KR-signal peptides was Tat-independent, showing that their RR/KR-motifs are not recognized by the Tat machinery. In fact, only the phosphodiesterase PhoD was shown to be secreted in a strictly Tat-dependent manner. Sodium azide-inhibition of SecA strongly affected the extracellular appearance of de novo synthesized proteins, including the lipase LipA and two other proteins with predicted RR/KR-signal peptides. The SecA-dependent export of pre-LipA is particularly remarkable, because its RR-signal peptide conforms well to stringent criteria for the prediction of Tat-dependent export in Escherichia coli. Taken together, our observations show that the Tat pathway makes a highly selective contribution to the extracellular proteome of B. subtilis.     

A TatABC-Type Tat Translocase Is Required for Unimpaired Aerobic Growth of Corynebacterium glutamicum ATCC13032

The twin-arginine translocation (Tat) system transports folded proteins across the cytoplasmic membrane of bacteria and the thylakoid membrane of plant chloroplasts. Escherichia coli and other Gram-negative bacteria possess a TatABC-type Tat translocase in which each of the three inner membrane proteins TatA, TatB, and TatC performs a mechanistically distinct function. In contrast, low-GC Gram-positive bacteria, such as Bacillus subtilis, use a TatAC-type minimal Tat translocase in which the TatB function is carried out by a bifunctional TatA. In high-GC Gram-positive Actinobacteria, such as Mycobacterium tuberculosis and Corynebacterium glutamicum, tatA, tatB, and tatC genes can be identified, suggesting that these organisms, just like E. coli, might use TatABC-type Tat translocases as well. However, since contrary to this view a previous study has suggested that C. glutamicum might in fact use a TatAC translocase with TatB only playing a minor role, we reexamined the requirement of TatB for Tat-dependent protein translocation in this microorganism. Under aerobic conditions, the misassembly of the Rieske iron-sulfur protein QcrA was identified as a major reason for the severe growth defect of Tat-defective C. glutamicum mutant strains. Furthermore, our results clearly show that TatB, besides TatA and TatC, is strictly required for unimpaired aerobic growth. In addition, TatB was also found to be essential for the secretion of a heterologous Tat-dependent model protein into the C. glutamicum culture supernatant. Together with our finding that expression of the C. glutamicum TatB in an E. coli ΔtatB mutant strain resulted in the formation of an active Tat translocase, our results clearly indicate that a TatABC translocase is used as the physiologically relevant functional unit for Tat-dependent protein translocation in C. glutamicum and, most likely, also in other TatB-containing Actinobacteria.     

In vivo assessment of the Tat signal peptide specificity in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Tat- and Sec-targeting signal peptides are specific for the cognate Tat or Sec pathways. Using two reporter proteins, the specificity and convertibility of a Tat signal peptide were assessed in vivo. The specific substitutions by RK, KR and KK for the RR motif of the TorA signal peptide had no effect on the exclusive Tat-dependent export of colicin V (ColV). By introducing multiple substitutions in a typical Tat signal peptide, altered signal peptides lacking the twin-arginine motif were obtained. Interestingly, some of these signal peptides preserved Tat-pathway targeting capacity, but resulted in a loss of exclusivity. In addition, further increasing the hydrophobicity of the n-region without modifying the h-region converted the Tat signal peptides to Sec signal peptides in the ColV transport. Replacement of positively charged residues in the c-region also abolished the Tat-exclusive targeting of ColV or green fluorescent protein (GFP), but the folded GFP could be transported only through the Tat pathway. These results strongly suggest that the overall hydrophobicity of the n-region is one of the determinants of Tat-targeting exclusivity.     

Degradation of the Twin-Arginine Translocation Substrate YwbN by Extracytoplasmic Proteases of Bacillus subtilis

Bacterial twin-arginine translocases can export fully folded proteins from the cytoplasm. Such proteins are usually resistant to proteolysis. Here we show that multiple extracellular proteases degrade the B. subtilis Tat substrate YwbN. This suggests either that secreted YwbN is not fully folded or that folded YwbN exposes protease cleavage sites.     

Phage Display of an Intracellular Carboxylesterase of Bacillus subtilis: Comparison of Sec and Tat Pathway Export Capabilities

Using the phage display technology, a protein can be displayed at the surface of bacteriophages as a fusion to one of the phage coat proteins. Here we describe development of this method for fusion of an intracellular carboxylesterase of Bacillus subtilis to the phage minor coat protein g3p. The carboxylesterase gene was cloned in the g3p-based phagemid pCANTAB 5E upstream of the sequence encoding phage g3p and downstream of a signal peptide-encoding sequence. The phage-bound carboxylesterase was correctly folded and fully enzymatically active, as determined from hydrolysis of the naproxen methyl ester with K_m values of 0.15 mM and 0.22 mM for the soluble and phage-displayed carboxylesterases, respectively. The signal peptide directs the encoded fusion protein to the cell membrane of Escherichia coli, where phage particles are assembled. In this study, we assessed the effects of several signal peptides, both Sec dependent and Tat dependent, on the translocation of the carboxylesterase in order to optimize the phage display of this enzyme normally restricted to the cytoplasm. Functional display of Bacillus carboxylesterase NA could be achieved when Sec-dependent signal peptides were used. Although a Tat-dependent signal peptide could direct carboxylesterase translocation across the inner membrane of E. coli, proper assembly into phage particles did not seem to occur.     

A facile reporter system for the experimental identification of twin-arginine translocation (Tat) signal peptides from all kingdoms of life

We have developed a reporter protein system for the experimental verification of twin-arginine signal peptides. This reporter system is based on the Streptomyces coelicolor agarase protein, which is secreted into the growth medium by the twin-arginine translocation (Tat) pathway and whose extracellular activity can be assayed colorimetrically in a semiquantitative manner. Replacement of the native agarase signal peptide with previously characterized twin-arginine signal peptides from other Gram-positive and Gram-negative bacteria resulted in efficient Tat-dependent export of agarase. Candidate twin-arginine signal peptides from archaeal proteins as well as plant thylakoid-targeting sequences were also demonstrated to mediate agarase translocation. A naturally occurring variant signal peptide with an arginine-glutamine motif instead of the consensus di-arginine was additionally recognized as a Tat-targeting sequence by Streptomyces. Application of the agarase assay to previously uncharacterized candidate Tat signal peptides from Bacillus subtilis identified two further probable Tat substrates in this organism. This is the first versatile reporter system for Tat signal peptide identification.     

Environmental salinity determines the specificity and need for Tat-dependent secretion of the YwbN protein in Bacillus subtilis

Twin-arginine protein translocation (Tat) pathways are required for transport of folded proteins across bacterial, archaeal and chloroplast membranes. Recent studies indicate that Tat has evolved into a mainstream pathway for protein secretion in certain halophilic archaea, which thrive in highly saline environments. Here, we investigated the effects of environmental salinity on Tat-dependent protein secretion by the Gram-positive soil bacterium Bacillus subtilis, which encounters widely differing salt concentrations in its natural habitats. The results show that environmental salinity determines the specificity and need for Tat-dependent secretion of the Dyp-type peroxidase YwbN in B. subtilis. Under high salinity growth conditions, at least three Tat translocase subunits, namely TatAd, TatAy and TatCy, are involved in the secretion of YwbN. Yet, a significant level of Tat-independent YwbN secretion is also observed under these conditions. When B. subtilis is grown in medium with 1% NaCl or without NaCl, the secretion of YwbN depends strictly on the previously described "minimal Tat translocase" consisting of the TatAy and TatCy subunits. Notably, in medium without NaCl, both tatAyCy and ywbN mutants display significantly reduced exponential growth rates and severe cell lysis. This is due to a critical role of secreted YwbN in the acquisition of iron under these conditions. Taken together, our findings show that environmental conditions, such as salinity, can determine the specificity and need for the secretion of a bacterial Tat substrate.     

Role of the Twin-Arginine Translocation Pathway in Staphylococcus

In Staphylococcus, the twin-arginine translocation (Tat) pathway is present only in some species and is composed of TatA and TatC. The tatAC operon is associated with the fepABC operon, which encodes homologs to an iron-binding lipoprotein, an iron-dependent peroxidase (FepB), and a high-affinity iron permease. The FepB protein has a typical twin-arginine (RR) signal peptide. The tat and fep operons constitute an entity that is not present in all staphylococcal species. Our analysis was focused on Staphylococcus aureus and S. carnosus strains. Tat deletion mutants (ΔtatAC) were unable to export active FepB, indicating that this enzyme is a Tat substrate. When the RR signal sequence from FepB was fused to prolipase and protein A, their export became Tat dependent. Since no other protein with a Tat signal could be detected, the fepABC-tatAC genes comprise not only a genetic but also a functional unit. We demonstrated that FepABC drives iron import, and in a mouse kidney abscess model, the bacterial loads of ΔtatAC and Δtat-fep mutants were decreased. For the first time, we show that the Tat pathway in S. aureus is functional and serves to translocate the iron-dependent peroxidase FepB.The Sec pathway is the major secretion system that exports the majority of extracytosolic proteins in pro- and eukaryotes. Proteins are translocated through this pathway in a more or less unfolded state. A second protein export pathway was identified first in the chloroplast thylakoid membrane (8) and later in several bacteria (4, 14, 35). This pathway has been designated the twin-arginine translocation system (Tat), as the preproteins targeted to this pathway carry a characteristic amino acid motif, including two consecutive arginine residues, which are essential for the recognition by the Tat translocon. The Tat pathway operates independently of the Sec pathway and exports exoproteins across the bacterial cytoplasmic membrane, apparently in a fully folded conformation (3). Many of these proteins are complexed with cofactors.Studies of several bacterial species, including Escherichia coli (37), Bacillus subtilis (22, 23), Pseudomonas aeruginosa (31), Legionella pneumophila (10, 11), and Mycobacterium smegmatis (29), have demonstrated that they possess a functional Tat export pathway. In E. coli, the TatA, TatB, and TatC proteins have been demonstrated to be essential for Tat-dependent protein translocation (4). However, several bacterial and archaeal species lack a TatB-like protein. For example, the B. subtilis genome encodes three TatA- and two TatC-like proteins. Thus, at least one copy of the TatA homologue and one copy of the TatC homologue are required for a functional Tat pathway. In Bacillus subtilis, several proteins were predicted that could potentially use the Tat pathway, as their signal peptides (SPs) contain RR or KR motifs. However, proteomic analysis revealed that 13 proteins with potential RR/KR SPs were Tat independent, showing that the Tat machinery does not recognize their RR/KR motifs. In fact, only the phosphodiesterase PhoD and the newly identified YwbN protein were shown to be secreted in a strictly Tat-dependent manner (22). YwbN is part of the YwbLMN operon product and is involved in the uptake of free ferric iron (32).For staphylococci, very little information regarding the function of the Tat system exists. A tatC-deficient mutant showed little difference from the wild type (WT) in its extracellular protein pattern (48). In another study, green fluorescent protein (GFP) was fused with the SP of the E. coli TorA protein and the subcellular localization of the hybrid protein was compared in Staphylococcus carnosus and its tatC mutant (30). The results showed that GFP was secreted in neither the WT nor the tatC mutant but was stuck in the cell wall fraction of the WT. However, no Tat-transported protein has been identified in staphylococci so far.Here we show that the tat operon encoding TatA and TatC is present in Staphylococcus aureus, S. carnosus, and Staphylococcus haemolyticus but is missing in a number of other staphylococcal species. By comparative analysis of WT and tatAC mutant strains, the functionality of the Tat pathway in S. carnosus and S. aureus was demonstrated, and it was found that the iron-dependent peroxidase (FepB) is translocated by the Tat system. The Tat system can efficiently translocate heterologous proteins such as lipase or protein A when it is fused with the SP of FepB. ⁵⁵Fe transport experiments demonstrated that FepABC is involved in iron uptake.     

Escherichia coli twin arginine (Tat) mutant translocases possessing relaxed signal peptide recognition specificities

The twin arginine (Tat) secretion pathway allows the translocation of folded proteins across the cytoplasmic membrane of bacteria. Tat-specific signal peptides contain a characteristic amino acid motif ((S/T)RRXFLK) including two highly conserved consecutive arginine residues that are thought to be involved in the recognition of the signal peptides by the Tat translocase. Here, we have analyzed the specificity of Tat signal peptide recognition by using a genetic approach. Replacement of the two arginine residues in a Tat-specific precursor protein by lysine-glutamine resulted in an export-defective mutant precursor that was no longer accepted by the wild-type translocase. Selection for restored export allowed for the isolation of Tat translocases possessing single mutations in either the amino-terminal domain of TatB or the first cytosolic domain of TatC. The mutant Tat translocases still efficiently accepted the unaltered precursor protein, indicating that the substrate specificity of the translocases was not strictly changed; rather, the translocases showed an increased tolerance toward variations of the amino acids occupying the positions of the twin arginine residues in the consensus motif of a Tat signal peptide.     

Subcellular Localization of TatAd of Bacillus subtilis Depends on the Presence of TatCd or TatCy

The gram-positive bacterium Bacillus subtilis contains two minimal Tat translocases, TatAdCd and TatAyCy, which are each involved in the secretion of one or more specific protein substrates. We have investigated the subcellular localization of the TatA components by employing C-terminal green fluorescent protein (GFP) fusions and fluorescence microscopy. When expressed from a xylose-inducible promoter, the TatA-GFP fusion proteins displayed a dual localization pattern, being localized peripherally and showing bright foci which are predominantly located at the division sites and/or poles of the cells. Importantly, the localization of TatAd-GFP was similar when the protein was expressed from its own promoter under phosphate starvation conditions, indicating that these foci are not the result of artificial overexpression. Moreover, the TatAd-GFP fusion protein was shown to be functional in the translocation of its substrate PhoD, provided that TatCd is also present. Furthermore, we demonstrate that the localization of TatAd-GFP in foci depends on the presence of the TatCd component. Remarkably, however, the TatAd-GFP foci can also be observed in the presence of TatCy, indicating that TatAd can interact not only with TatCd but also with TatCy. These results suggest that the formation of TatAd complexes in B. subtilis is controlled by TatC.The bacterial twin-arginine translocation (Tat) machinery is able to transport folded proteins across the cytoplasmic membrane (26). Preproteins translocated by the Tat pathway are characterized by a twin-arginine (RR) motif in their signal sequences.In Escherichia coli, the Tat system consists of three components, the TatA, TatB, and TatC proteins. In the currently favored model for its mode of action, a TatB-TatC complex is involved in initial RR signal peptide recognition and binding of precursor proteins. Multiple TatA subunits then associate with this complex to form a protein-conducting channel (1). TatA, which is homologous to TatB, can be found complexed to TatBC but also forms a wide range of large, homooligomeric complexes (7, 23). In a few cases, the TatB protein can be functionally replaced by the TatA protein, indicating that TatA and TatC are able to form an active, minimal translocase (6, 10).Most gram-positive bacteria contain only two types of Tat subunit, a TatC protein and a TatA protein which has characteristics and the ability to perform the function of both TatA and TatB of E. coli (2, 13). Bacillus subtilis contains two substrate-specific Tat systems: a TatAyCy translocase that is required for translocation of the iron-dependent DyP peroxidase YwbN and a TatAdCd translocase which translocates the phosphodiesterase PhoD (12). In addition, B. subtilis contains a third TatA component, designated TatAc. This protein is dispensable for Tat-dependent translocation of YwbN or PhoD, and its function is currently unknown.TatAd is the most-studied TatA component of B. subtilis, and like TatA of E. coli, it is able to form both homooligomeric complexes and complexes with TatCd (2, 31). Despite the fact that it contains an N-terminal transmembrane segment (17), TatAd was also found in the cytosol, where it appears to interact with its substrate, pre-PhoD, via the signal sequence (24). TatCd was proposed to act as a receptor for the anchoring at and subsequent incorporation into the membrane of this TatAd-PhoD complex (28).The subcellular localization of Tat components in E. coli has been extensively investigated by fluorescence microscopy. Green fluorescent protein (GFP) fusions of TatA were localized at the periphery of the cells, but punctate regions of fluorescence were also reported (4, 25). In these studies, TatB was localized all over the membrane, with some accumulation at the cell poles. TatC was mainly distributed evenly throughout the periphery of the cells, with some small punctate regions. Recently, the oligomeric state of TatA-yellow fluorescent protein (YFP) in living E. coli cells was determined by single-molecule imaging (18). TatA complexes with a broad range of stoichiometries were observed as fluorescent foci, and TatA was also present in a dispersed state in the membrane.For B. subtilis, the subcellular localization of only one Tat component has been reported so far. Both N- and C-terminal fusions of GFP to TatCy were shown to be localized throughout the membrane, with frequent foci at the cell poles and division septa, and this localization pattern was classified as “polar” (20).In this study, we have investigated the subcellular localization of the three TatA proteins of B. subtilis by using GFP fusions, functionality assessments, and fluorescence microscopy. TatAc and TatAd showed a dual localization pattern, with fluorescence in the membrane as well as in foci which were enriched at the cell poles. Notably, the localization of TatAd-GFP in foci was shown to depend on the presence of a TatC component, suggesting that TatC drives complex formation by TatAd.     

An alternative model of the twin arginine translocation system

The twin arginine translocation (Tat) system is a machinery which can translocate folded proteins across energy transducing membranes. Currently it is supposed that Tat substrates bind directly to Tat translocon components before a ApH-driven translocation occurs. In this review, an alternative model is presented which proposes that membrane integration could precede Tat-dependent translocation. This idea is mainly supported by the recent observations of Tat-independent membrane insertion of Tat substrates in vivo and in vitro. Membrane insertion may allow i) a quality control of the folded state by membrane bound proteases like FtsH, ii) the recognition of the membrane spanning signal peptide by Tat system components, and iii) a pulling mechanism of translocation. In some cases of folded Tat substrates, the membrane targeting process may require ATP-dependent N-terminal unfolding-steps.     

Evaluation of TatABC overproduction on Tat- and Sec-dependent protein secretion in Streptomyces lividans

The majority of bacterial proteins are exported across the cytoplasmic membrane via the Sec pathway, but also the more recently discovered twin-arginine translocation (Tat) route seems to play an important role for protein secretion in Streptomyces lividans in whose genome tatA, tatB and tatC have been identified. In the present work we showed that simultaneous overproduction of TatABC improved the Tat-dependent secretion capacity as could be concluded from the increased amount of secreted xylanase C, an exclusive Tat-dependent substrate. This result demonstrates that next to the availability of energy to drive secretion, also the number of translocases can be rate-limiting for Tat-dependent secretion. On the other hand, tatABC overexpression was found to diminish secretion of the Sec-dependent proteins xylanase B and subtilisin inhibitor in S. lividans. These results reveal cross-talk between both pathways in S. lividans.     

Novel synthetic signal peptides for the periplasmic secretion of green fluorescent protein in Escherichia coli

New secretion vectors containing synthetic signal peptides were constructed to study the periplasmic translocation of green fluorescent protein (GFP) in Escherichia coli. These constructs encode synthetic signal peptides spA and spD fused to the amino terminal end of GFP, and expressed from T7/lac promoter in the BL21DE3 strain by induction with IPTG. The recombinant protein was detected in both the cytoplasmic and periplasmic fractions. Fluorescence analysis revealed that recombinant proteins with signal peptides were not fluorescent, indicating translocation to periplasmic space. In contrast, recombinant proteins without signal peptide were fluorescent. These results indicate that the expressed recombinant proteins were translocated into the periplasm. Therefore, the synthetic signal peptides derived from signal peptides of Bacillus sp. could efficiently secrete the heterologous proteins to the periplasmic space of E. coli.     

DnaK plays a pivotal role in Tat targeting of CueO and functions beside SlyD as a general Tat signal binding chaperone

The Tat (twin-arginine translocation) system from Escherichia coli transports folded proteins with N-terminal twin-arginine signal peptides across the cytoplasmic membrane. The influence of general chaperones on Tat substrate targeting has not been clarified so far. Here we show that the chaperones SlyD and DnaK bind to a broad range of different Tat signal sequences in vitro and in vivo. Initially, SlyD and GroEL were purified from DnaK-deficient extracts by their affinity to various Tat signal sequences. Of these, only SlyD bound Tat signal sequences also in the presence of DnaK. SlyD and DnaK also co-purified with Tat substrate precursors, demonstrating the binding to Tat signal sequences in vivo. Deletion of dnaK completely abolished Tat-dependent translocation of CueO, but not of DmsA, YcdB, or HiPIP, indicating that DnaK has an essential role specifically for CueO. DnaK was not required for stability of the CueO precursor and thus served in some essential step after folding. A CueO signal sequence fusion to HiPIP was Tat-dependently transported without the need of DnaK, indicating that the mature domain of CueO is responsible for the DnaK dependence. The overall results suggest that SlyD and DnaK are in the set of chaperones that can serve as general Tat signal-binding proteins. DnaK has additional functions that are indispensable for the targeting of CueO.