Genetic toggling of alkaline phosphatase folding reveals signal peptides for all major modes of transport across the inner membrane of bacteria

Prediction of export pathway specificity in prokaryotes is a challenging endeavor due to the similar overall architecture of N-terminal signal peptides for the Sec-, SRP- (signal recognition particle), and Tat (twin arginine translocation)-dependent pathways. Thus, we sought to create a facile experimental strategy for unbiased discovery of pathway specificity conferred by N-terminal signals. Using a limited collection of Escherichia coli strains that allow protein oxidation in the cytoplasm or, conversely, disable protein oxidation in the periplasm, we were able to discriminate the specific mode of export for PhoA (alkaline phosphatase) fusions to signal peptides for all of the major modes of transport across the inner membrane (Sec, SRP, or Tat). Based on these findings, we developed a mini-Tn5 phoA approach to isolate pathway-specific export signals from libraries of random fusions between exported proteins and the phoA gene. Interestingly, we observed that reduced PhoA was exported in a Tat-independent manner when targeted for Tat export in the absence of the essential translocon component TatC. This suggests that initial docking to TatC serves as a key specificity determinant for Tat-specific routing of PhoA, and in its absence, substrates can be rerouted to the Sec pathway, provided they remain compatible with the Sec export mechanism. Finally, the utility of our approach was demonstrated by experimental verification that four secreted proteins from Mycobacterium tuberculosis carrying putative Tat signals are bona fide Tat substrates and thus represent potential Tat-dependent virulence factors in this important human pathogen.     

Export of Thermus thermophilus cytoplasmic beta-glycosidase via the E. coli Tat pathway

The Tat pathway is distinct from the Sec machinery given its unusual capacity to export folded proteins, which contain a twin-arginine (RR) signal peptide, across the plasma membrane. The functionality of the Tat pathway has been demonstrated for several Gram-negative and Gram-positive mesophilic bacteria. To assess the specificity of the Tat system, and to analyze the capacity of a mesophilic bacterial Tat system to translocate cytoplasmic proteins from hyperthermophilic bacteria, we fused the Thermus thermophilus beta-glycosidase (Glc) to the twin-arginine signal peptide of the E. coli TorA protein. When expressed in E. coli, the thermophilic RR-Glc chimera was successfully synthesized and efficiently translocated into the periplasm of the wild type strain. In contrast, the beta-glycosidase accumulated within the cytoplasm of all the tat mutants analyzed. The beta-glycosidase synthesized in these strains exhibited thermophilic properties. These results demonstrated, for the first time, the capacity of the E. coli Tat system to export cytoplasmic hyperthermophilic protein, implying an important potential of the Tat system for the production of thermostable enzymes used in bioprocessing applications.     

Export pathway selectivity of Escherichia coli twin arginine translocation signal peptides

The Escherichia coli genome encodes at least 29 putative signal peptides containing a twin arginine motif characteristic of proteins exported via the twin arginine translocation (Tat) pathway. Fusions of the putative Tat signal peptides plus six to eight amino acids of the mature proteins to three reporter proteins (short-lived green fluorescent protein, maltose-binding protein (MBP), and alkaline phosphatase) and also data from the cell localization of epitope-tagged full-length proteins were employed to determine the ability of the 29 signal peptides to direct export through the Tat pathway, through the general secretory pathway (Sec), or through both. 27/29 putative signal peptides could export one or more reporter proteins through Tat. Of these, 11 signal peptides displayed Tat specificity in that they could not direct the export of Sec-only reporter proteins. The rest (16/27) were promiscuous and were capable of directing export of the appropriate reporter either via Tat (green fluorescent protein, MBP) or via Sec (PhoA, MBP). Mutations that conferred a >or=+1 charge to the N terminus of the mature protein abolished or drastically reduced routing through the Sec pathway without affecting the ability to export via the Tat pathway. These experiments demonstrate that the charge of the mature protein N terminus affects export promiscuity, independent of the effect of the folding state of the mature protein.     

Differential translocation of green fluorescent protein fused to signal sequences of Ruminococcus albus cellulases by the Tat and Sec pathways of Escherichia coli

Ruminococcus albus is a Gram-positive bacterium that degrades plant cell walls in the rumen of herbivores. It was described to synthesize two major glycoside-hydrolases (Cel9B and Cel48A), which are exported and anchored at the cell surface. In bacteria, proteins destined to cross the cytoplasmic membrane are synthesized as precursors and possess a signal sequence (SS) directing them to the 'Sec' (general secretory) or 'Tat' (twin arginine translocation) pathway. SS composition of Cel9B and Cel48A suggests that these two enzymes translocate using different secretory pathways. In order to confirm this hypothesis, the SSs of Cel9B and Cel48A were fused to the green fluorescent protein (GFP) and expressed in wild-type Escherichia coli and in its Tat and Sec isogenic mutants. The SS cleavage and the formation of the mature protein were then followed by Western blot and fluorescence microscopy. This study shows that the SS of Cel9B directs the preprotein to the 'Tat' translocation pathway while the GFP fused to the SS of Cel48A is exported through the 'Sec' machinery. These observations suggest that R. albus possess a Tat pathway, in addition to the general secretory pathway.     

Efficient export of prefolded,disulfide‐bonded recombinant proteins to the periplasm by the Tat pathway in Escherichia coli CyDisCo strains

Numerous high‐value therapeutic proteins are produced in Escherichia coli and exported to the periplasm, as this approach simplifies downstream processing and enables disulfide bond formation. Most recombinant proteins are exported by the Sec pathway, which transports substrates across the plasma membrane in an unfolded state. The Tat system also exports proteins to the periplasm, but transports them in a folded state. This system has attracted interest because of its tendency to transport correctly folded proteins, but this trait renders it unable to export proteins containing disulfide bonds since these are normally acquired only in the periplasm; reduced substrates tend to be recognized as incorrectly folded and rejected. In this study we have used a series of novel strains (termed CyDisCo) which oxidise disulfide bonds in the cytoplasm, and we show that these cells efficiently export a range of disulfide‐containing proteins when a Tat signal peptide is attached. These test proteins include alkaline phosphatase (PhoA), a phytase containing four disulfide bonds (AppA), an antiinterleukin 1β scFv and human growth hormone. No export of PhoA or AppA is observed in wild‐type cells lacking the CyDisCo factors. The PhoA, AppA and scFv proteins were exported in an active form by Tat in the CyDisCo strain, and mass spectrometry showed that the vast majority of the scFv protein was disulfide‐bonded and correctly processed. The evidence indicates that this combination of Tat + CyDisCo offers a novel means of exporting active, correctly folded disulfide bonded proteins to the periplasm. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 30:281–290, 2014     

Exploration of twin‐arginine translocation for expression and purification of correctly folded proteins in Escherichia coli

Historically, the general secretory (Sec) pathway of Gram‐negative bacteria has served as the primary route by which heterologous proteins are delivered to the periplasm in numerous expression and engineering applications. Here we have systematically examined the twin‐arginine translocation (Tat) pathway as an alternative, and possibly advantageous, secretion pathway for heterologous proteins. Overall, we found that: (i) export efficiency and periplasmic yield of a model substrate were affected by the composition of the Tat signal peptide, (ii) Tat substrates were correctly processed at their N‐termini upon reaching the periplasm and (iii) proteins fused to maltose‐binding protein (MBP) were reliably exported by the Tat system, but only when correctly folded; aberrantly folded MBP fusions were excluded by the Tat pathway's folding quality control feature. We also observed that Tat export yield was comparable to Sec for relatively small, well‐folded proteins, higher relative to Sec for proteins that required cytoplasmic folding, and lower relative to Sec for larger, soluble fusion proteins. Interestingly, the specific activity of material purified from the periplasm was higher for certain Tat substrates relative to their Sec counterparts, suggesting that Tat expression can give rise to relatively pure and highly active proteins in one step.     

High-yield export of a native heterologous protein to the periplasm by the tat translocation pathway in Escherichia coli

Numerous high‐value recombinant proteins that are produced in bacteria are exported to the periplasm as this approach offers relatively easy downstream processing and purification. Most recombinant proteins are exported by the Sec pathway, which transports them across the plasma membrane in an unfolded state. The twin‐arginine translocation (Tat) system operates in parallel with the Sec pathway but transports substrate proteins in a folded state; it therefore has potential to export proteins that are difficult to produce using the Sec pathway. In this study, we have produced a heterologous protein (green fluorescent protein; GFP) in Escherichia coli and have used batch and fed‐batch fermentation systems to test the ability of the newly engineered Tat system to export this protein into the periplasm under industrial‐type production conditions. GFP cannot be exported by the Sec pathway in an active form. We first tested the ability of five different Tat signal peptides to export GFP, and showed that the TorA signal peptide directed most efficient export. Under batch fermentation conditions, it was found that TorA‐GFP was exported efficiently in wild type cells, but a twofold increase in periplasmic GFP was obtained when the TatABC components were co‐expressed. In both cases, periplasmic GFP peaked at about the 12 h point during fermentation but decreased thereafter, suggesting that proteolysis was occurring. Typical yields were 60 mg periplasmic GFP per liter culture. The cells over‐expressed the tat operon throughout the fermentation process and the Tat system was shown to be highly active over a 48 h induction period. Fed‐batch fermentation generated much greater yields: using glycerol feed rates of 0.4, 0.8, and 1.2 mL h^?1, the cultures reached OD₆₀₀ values of 180 and periplasmic GFP levels of 0.4, 0.85, and 1.1 g L^?1 culture, respectively. Most or all of the periplasmic GFP was shown to be active. These export values are in line with those obtained in industrial production processes using Sec‐dependent export approaches. Biotechnol. Bioeng. 2012; 109: 2533–2542. © 2012 Wiley Periodicals, Inc.     

SecA specificity for different signal peptides

SecA performs a critical function in the recognition, targeting, and transport of secretory proteins across the cytoplasmic membrane of Escherichia coli. In this study we investigate the substrate specificity of SecA, including the influence of the early mature region of the preprotein on SecA interactions, and the extent to which SecA recognizes targeting signals from different transport pathways. A series of fusion proteins were generated which involved the tandem expression of GST, signal peptide, and the first 30 residues from alkaline phosphatase. These were purified and evaluated for their ability to promote SecA ATPase activity. No significant difference in the stimulation of SecA-lipid ATPase activity between the synthetic wild-type alkaline phosphatase signal peptide and a fusion that also contains the first 30 residues of alkaline phosphatase was observed. The incorporation of sequence motifs in the mature region, which confer SecB dependence in vivo, had no impact on SecA activation in vitro. These results suggest that the early mature region of alkaline phosphatase does not affect the interactions between SecA and the signal peptide. Sec, Tat, and YidC signal peptide fusions were also assayed for their ability to stimulate SecA ATPase activity in vitro and further analyzed in vivo for the Sec dependence of the transport of the corresponding signal peptide mutants of alkaline phosphatase. Our results demonstrate that E. coli Sec signals give the highest level of SecA activation; however, SecA-signal peptide interactions in vitro are not the only arbiter of whether the preprotein utilizes the Sec pathway in vivo.     

Involvement of the twin-arginine translocation system in protein secretion via the type II pathway.

The general secretory pathway (GSP) is a two-step process for the secretion of proteins by Gram-negative bacteria. The translocation across the outer membrane is carried out by the type II system, which involves machinery called the secreton. This step is considered to be an extension of the general export pathway, i.e. the export of proteins across the inner membrane by the Sec machinery. Here, we demonstrate that two substrates for the Pseudomonas aeruginosa secreton, both phospholipases, use the twin-arginine translocation (Tat) system, instead of the Sec system, for the first step of translocation across the inner membrane. These results challenge the previous vision of the GSP and suggest for the first time a mosaic model in which both the Sec and the Tat systems feed substrates into the secreton. Moreover, since P.aeruginosa phospholipases are secreted virulence factors, the Tat system appears to be a novel determinant of bacterial virulence.     

Escherichia coli “TatExpress” strains super‐secrete human growth hormone into the bacterial periplasm by the Tat pathway

Numerous high‐value proteins are secreted into the Escherichia coli periplasm by the General Secretory (Sec) pathway, but Sec‐based production chassis cannot handle many potential target proteins. The Tat pathway offers a promising alternative because it transports fully folded proteins; however, yields have been too low for commercial use. To facilitate Tat export, we have engineered the TatExpress series of super‐secreting strains by introducing the strong inducible bacterial promoter, ptac , upstream of the chromosomal tatABCD operon, to drive its expression in E. coli strains commonly used by industry (e.g., W3110 and BL21). This modification significantly improves the Tat‐dependent secretion of human growth hormone (hGH) into the bacterial periplasm, to the extent that secreted hGH is the dominant periplasmic protein after only 1 hr induction. TatExpress strains accumulate in excess of 30 mg L^?1 periplasmic recombinant hGH, even in shake flask cultures. A second target protein, an scFv, is also shown to be exported at much higher rates in TatExpress strains.

     

The superoxide dismutase SodA is targeted to the periplasm in a SecA-dependent manner by a novel mechanism

The manganese/iron-type superoxide dismutase (SodA) of Rhizobium leguminosarum bv. viciae 3841 is exported to the periplasm of R. l. bv. viciae and Escherichia coli. However, it does not possess a hydrophobic cleaved N-terminal signal peptide typically present in soluble proteins exported by the Sec-dependent (Sec) pathway or the twin-arginine translocation (TAT) pathway. A tatC mutant of R. l. bv. viciae exported SodA to the periplasm, ruling out export of SodA as a complex with a TAT substrate as a chaperone. The export of SodA was unaffected in a secB mutant of E. coli, but its export from R. l. bv. viciae was inhibited by azide, an inhibitor of SecA ATPase activity. A temperature-sensitive secA mutant of E. coli was strongly reduced for SodA export. The 10 N-terminal amino acid residues of SodA were sufficient to target the reporter protein alkaline phosphatase to the periplasm. Our results demonstrate the export of a protein lacking a classical signal peptide to the periplasm by a SecA-dependent, but SecB-independent targeting mechanism. Export of the R. l. bv. viciae SodA to the periplasm was not limited to the genus Rhizobium, but was also observed in other proteobacteria.     

Export of active green fluorescent protein to the periplasm by the twin-arginine translocase (Tat) pathway in Escherichia coli

The twin-arginine translocation (Tat) system targets cofactor-containing proteins across the Escherichia coli cytoplasmic membrane via distinct signal peptides bearing a twin-arginine motif. In this study, we have analysed the mechanism and capabilities of the E. coli Tat system using green fluorescent protein (GFP) fused to the twin-arginine signal peptide of TMAO reductase (TorA). Fractionation studies and fluorescence measurements demonstrate that GFP is exported to the periplasm where it is fully active. Export is almost totally blocked in tat deletion mutants, indicating that the observed export in wild-type cells occurs predominantly, if not exclusively, by the Tat pathway. Imaging studies reveal a halo of fluorescence in wild-type cells corresponding to the exported periplasmic form; the GFP is distributed uniformly throughout the cytoplasm in a tat mutant. Because previous work has shown GFP to be incapable of folding in the periplasm, we propose that GFP is exported in a fully folded, active state. These data also show for the first time that heterologous proteins can be exported in an active form by the Tat pathway.     

Influence of tat mutations on the ribose-binding protein translocation in Escherichia coli

Proteins are exported across the bacterial cytoplasmic membrane either as unfolded precursors via the Sec machinery or in folded conformation via the Tat system. The ribose-binding protein (RBP) of Escherichia coli is a Sec-pathway substrate. Intriguingly, it exhibits fast folding kinetics and its export is independent of SecB, a general chaperone protein dedicated for protein secretion. In this study, we found that the quantity of RBP was significantly reduced in the periplasm of tat mutants, which was restored by in trans expression of the tatABC genes. Pulse-chase experiments showed that significant amount of wild-type RBP was processed in a secY mutant in the presence of azide (SecA inhibitor), whereas the processing of a slow folding RBP derivative was almost completely blocked under the same conditions. These results would suggest that under the Sec-defective conditions the export of a portion of folded RBP could be rescued by the Tat system.     

Signal peptide-dependent protein transport in Bacillus subtilis: a genome-based survey of the secretome.

One of the most salient features of Bacillus subtilis and related bacilli is their natural capacity to secrete a variety of proteins into their environment, frequently to high concentrations. This has led to the commercial exploitation of bacilli as major "cell factories" for secreted enzymes. The recent sequencing of the genome of B. subtilis has provided major new impulse for analysis of the molecular mechanisms underlying protein secretion by this organism. Most importantly, the genome sequence has allowed predictions about the composition of the secretome, which includes both the pathways for protein transport and the secreted proteins. The present survey of the secretome describes four distinct pathways for protein export from the cytoplasm and approximately 300 proteins with the potential to be exported. By far the largest number of exported proteins are predicted to follow the major "Sec" pathway for protein secretion. In contrast, the twin-arginine translocation "Tat" pathway, a type IV prepilin-like export pathway for competence development, and ATP-binding cassette transporters can be regarded as "special-purpose" pathways, through which only a few proteins are transported. The properties of distinct classes of amino-terminal signal peptides, directing proteins into the various protein transport pathways, as well as the major components of each pathway are discussed. The predictions and comparisons in this review pinpoint important differences as well as similarities between protein transport systems in B. subtilis and other well-studied organisms, such as Escherichia coli and the yeast Saccharomyces cerevisiae. Thus, they may serve as a lead for future research and applications.     

A bacterial two-hybrid system based on the twin-arginine transporter pathway of E. coli

We have developed a bacterial two-hybrid system for the detection of interacting proteins that capitalizes on the folding quality control mechanism of the Twin Arginine Transporter (Tat) pathway. The Tat export pathway is responsible for the membrane translocation of folded proteins, including proteins consisting of more than one polypeptide, only one of which contains a signal peptide ("hitchhiker export"). Here, one protein (bait) is expressed as a fusion to a Tat signal peptide, whereas the second protein (prey) is fused to a protein reporter that can confer a phenotype only after export into the bacterial periplasmic space. Since the prey-reporter fusion lacks a signal peptide, it can only be exported as a complex with the bait-signal peptide fusion that is capable of targeting the Tat translocon. Using maltose-binding protein as a reporter, clones expressing interacting proteins can be grown on maltose minimal media or on MacConkey plates. In addition, we introduce the use of the cysteine disulfide oxidase DsbA as a reporter. Export of a signal peptide-prey:bait-DsbA complex into the periplasm allows complementation of dsbA(-) mutants and restores the formation of active alkaline phosphatase, which in turn can be detected by a chromogenic assay.     

NOVEL MARKERS FOR CONSTITUTIVE SECRETION USED TO SHOW THAT TISSUE PLASMINOGEN ACTIVATOR IS SORTED TO THE REGULATED PATHWAY IN TRANSFECTED PC12 CELLS

The rat pheochromocytoma cell line PC12 contains two distinct pathways of protein secretion. Proteins secreted via the regulated pathway are stored in secretory vesicles and exocytosed only in response to a specific signal, whereas proteins secreted via the constitutive pathway are exported continuously. Analysis of regulated secretion of a heterologous protein in this system often relies on comparison of secretion rates with those of endogenous proteins known to be secreted via the constitutive route. In order to improve these controls, we have evaluated a number of secreted enzymes, selected for the sensitivity and convenience of their assays, as transgenic markers for the constitutive pathway. We show that both human-secreted placental alkaline phosphatase (SEAP) and bacterial β-lactamase operate in this way in transfected PC12 cells. In contrast, transfected human tissue plasminogen activator (tPA) is shown to be sorted to the regulated pathway.     

Co-operation between different targeting pathways during integration of a membrane protein

Membrane protein assembly is a fundamental process in all cells. The membrane-bound Rieske iron-sulfur protein is an essential component of the cytochrome bc₁ and cytochrome b₆f complexes, and it is exported across the energy-coupling membranes of bacteria and plants in a folded conformation by the twin arginine protein transport pathway (Tat) transport pathway. Although the Rieske protein in most organisms is a monotopic membrane protein, in actinobacteria, it is a polytopic protein with three transmembrane domains. In this work, we show that the Rieske protein of Streptomyces coelicolor requires both the Sec and the Tat pathways for its assembly. Genetic and biochemical approaches revealed that the initial two transmembrane domains were integrated into the membrane in a Sec-dependent manner, whereas integration of the third transmembrane domain, and thus the correct orientation of the iron-sulfur domain, required the activity of the Tat translocase. This work reveals an unprecedented co-operation between the mechanistically distinct Sec and Tat systems in the assembly of a single integral membrane protein.     

Bacterial twin-arginine signal peptide-dependent protein translocation pathway: evolution and mechanism

The recently identified bacterial Tat pathway is capable of exporting proteins with a peculiar twin-arginine signal peptide in folded conformation independently of the Sec machinery. It is structurally and mechanistically similar to the delta pH-dependent pathway used for importing chloroplast proteins into the thylakoid. The tat genes are not ubiquitously present and are absent from half of the completely sequenced bacterial genomes. The presence of the tat genes seems to correlate with genome size and with the presence of important enzymes with a twin-arginine signal peptide. A minimal Tat system requires a copy of tatA and a copy of tatC. The composition and gene order of a tat locus are generally conserved within the same taxonomy group but vary considerably to other groups, which would exclude an acquisition of the Tat system by recent horizontal gene transfer. The tat genes are also found in the genomes of chloroplasts and plant mitochondria but are absent from animal mitochondrial genomes. The topology of evolution trees suggests a bacterial origin of the Tat system. In general, the twin-arginine signal peptide is capable of targeting any passenger protein to the Tat pathway. However, a structural signal carried by the mature part of a passenger protein can override targeting information in a signal peptide under certain circumstances. Tat systems show a substrate-Tat component specificity and a species specificity. The pore size of the Tat channel is estimated as being between 5 and 9 nm. Operational models of the Tat system are proposed.     

Behaviour of topological marker proteins targeted to the Tat protein transport pathway

The Escherichia coli Tat system mediates Sec-independent export of protein precursors bearing twin arginine signal peptides. Formate dehydrogenase-N is a three-subunit membrane-bound enzyme, in which localization of the FdnG subunit to the membrane is Tat dependent. FdnG was found in the periplasmic fraction of a mutant lacking the membrane anchor subunit FdnI, confirming that FdnG is located at the periplasmic face of the cytoplasmic membrane. However, the phenotypes of gene fusions between fdnG and the subcellular reporter genes phoA (encoding alkaline phosphatase) or lacZ (encoding beta-galactosidase) were the opposite of those expected for analogous fusions targeted to the Sec translocase. PhoA fusion experiments have previously been used to argue that the peripheral membrane DmsAB subunits of the Tat-dependent enzyme dimethyl sulphoxide reductase are located at the cytoplasmic face of the inner membrane. Biochemical data are presented that instead show DmsAB to be at the periplasmic side of the membrane. The behaviour of reporter proteins targeted to the Tat system was analysed in more detail. These data suggest that the Tat and Sec pathways differ in their ability to transport heterologous passenger proteins. They also suggest that caution should be observed when using subcellular reporter fusions to determine the topological organization of Tat-dependent membrane protein complexes.     

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.