Legionella pneumophila Philadelphia-1 tatB and tatC affect intracellular replication and biofilm formation

Legionella pneumophila is a facultative intracellular human pathogen and an important cause of Legionnaires' disease, a severe form of pneumonia. Recently, we showed the presence of a putative twin-arginine translocation (Tat) pathway in L. pneumophila Philadelphia-1. This secretion pathway is used to transport completely folded proteins across the cytoplasmic membrane. The importance of the Tat pathway in L. pneumophila was investigated by constructing a tatB and a tatC mutant. Functionality of the Tat pathway was shown using a proven heterologous Tat substrate. It was shown that tatB and tatC are involved in intracellular replication in Acanthamoeba castellanii and differentiated U937 cells, and in biofilm forming ability. A putative Legionella Tat substrate was identified via 2D gel electrophoresis.     

The twin-arginine translocation pathway is necessary for correct membrane insertion of the Rieske Fe/S protein in Legionella pneumophila

The twin-arginine translocation (Tat) pathway translocates folded proteins across the cytoplasmic membrane. Proteins transported through this secretion system typically carry two arginine residues in their signal peptide that is cleaved off during translocation. Recently, we demonstrated the presence of the Tat pathway in Legionella pneumophila Philadelphia-1 and the Rieske Fe/S protein PetA was one of the predicted Tat substrates. Because we observed that the signal peptide of PetA is not processed and that this protein is still membrane associated in the tat mutants, correct membrane insertion was assayed using a trypsin sensitivity assay. We conclude that the Tat pathway is necessary for correct membrane insertion of L. pneumophila PetA.     

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.     

Development and Application of a Novel Signal Peptide Probe Vector with PGA as Reporter in Bacillus subtilis WB700: Twenty-Four Tat Pathway Signal Peptides from Bacillus subtilis were Monitored

In this study, we have developed a novel, versatile signal peptide probe vector driven by promoter P43 in Bacillus subtilis WB700, using Penicillin G Acylase (PGA) as reporter. Twenty-four signal peptides considered belonging to twin-arginine translocation (Tat) pathway were cloned into the probe vector to direct the secretion expression of PGA, respectively. Through 6-nitro-3-phenylacetamidobenzoic acid (NIPAB) filter paper assay, four signal peptides (AmyX, AlbB, LipA, and YmzC) were chosen for further investigation. The extracellular production of PGA demonstrated that these recombinants mediated efficient secretion expression in B. subtilis WB700, in which the maximum activity reached 0.11, 0.21, 0.08, and 0.26 U/mL, respectively. Thus, we provided an efficient tool for easy detection of the signal peptides in B. subtilis, and demonstrated the efficiency of Tat pathway signal peptides via PGA secretion in B. subtilis WB700.     

Targeting of proteins to the twin-arginine translocation pathway

The twin-arginine protein transport (Tat pathway) is found in prokaryotes and plant organelles and transports folded proteins across membranes. Targeting of substrates to the Tat system is mediated by the presence of an N-terminal signal sequence containing a highly conserved twin-arginine motif. The Tat machinery comprises membrane proteins from the TatA and TatC families. Assembly of the Tat translocon is dynamic and is triggered by the interaction of a Tat substrate with the Tat receptor complex. This review will summarise recent advances in our understanding of Tat transport, focusing in particular on the roles played by Tat signal peptides in protein targeting and translocation.     

Translocation of aprotinin, a therapeutic protease inhibitor, into the thylakoid lumen of genetically engineered tobacco chloroplasts

Aprotinin, a bovine protease inhibitor of important therapeutic value, was expressed in tobacco plastid transformants. This disulphide bond-containing protein was targeted to the lumen of thylakoids using signal peptides derived from nuclear genes which encode lumenal proteins. Translocation was attempted via either the general secretion (Sec) or the twin-arginine translocation (Tat) pathway. In both cases, this strategy allowed the production of genuine aprotinin with its N-terminal arginine residue. The recombinant protease inhibitor was efficiently secreted within the lumen of thylakoids, accumulated in older leaves and was bound to trypsin, suggesting that the three disulphide bonds of aprotinin are correctly folded and paired in this chloroplast compartment. Mass spectrometric analysis indicated that translocation via the Sec pathway, unlike the Tat pathway, led predominantly to an oxidized protein. Translocation via the Tat pathway was linked to a slightly decreased growth rate, a pale-green leaf phenotype and supplementary expression products associated with the thylakoids.     

Identification of Streptomyces lividans proteins secreted by the twin-arginine translocation pathway following growth with different carbon sources

Genome-based signal peptide predictions classified Streptomyces coelicolor as the microorganism that secretes the most proteins through the twin-arginine translocation (Tat)-dependent secretion pathway. Availability of a DeltatatC mutant of the closely related strain Streptomyces lividans impaired Tat-dependent protein secretion and enabled identification of many extracellular proteins that are secreted via the Tat pathway. Proteomic techniques were applied to analyze proteins from the supernatants of log-phase cultures. Since the bacterial secretome depends mainly on the carbon sources available during growth, xylose, glucose, chitin, and soil extracts were used. A total of 63 proteins were identified, among which 7 were predicted by the TATscan program, and 20 were not predicted but contained a potential Tat signal motif. Thirteen proteins having no signal sequence could be co-transported by Tat-dependent proteins because the genes that encode these proteins are in close proximity in the genome. Finally, the presence of 23 proteins lacking signal peptides was difficult to explain. More secreted proteins could be identified as Tat substrates in varying carbon sources.     

TatC is a specificity determinant for protein secretion via the twin-arginine translocation pathway

The recent discovery of a ubiquitous translocation pathway, specifically required for proteins with a twin-arginine motif in their signal peptide, has focused interest on its membrane-bound components, one of which is known as TatC. Unlike most organisms of which the genome has been sequenced completely, the Gram-positive eubacterium Bacillus subtilis contains two tatC-like genes denoted tatCd and tatCy. The corresponding TatCd and TatCy proteins have the potential to be involved in the translocation of 27 proteins with putative twin-arginine signal peptides of which approximately 6-14 are likely to be secreted into the growth medium. Using a proteomic approach, we show that PhoD of B. subtilis, a phosphodiesterase belonging to a novel protein family of which all known members are synthesized with typical twin-arginine signal peptides, is secreted via the twin-arginine translocation pathway. Strikingly, TatCd is of major importance for the secretion of PhoD, whereas TatCy is not required for this process. Thus, TatC appears to be a specificity determinant for protein secretion via the Tat pathway. Based on our observations, we hypothesize that the TatC-determined pathway specificity is based on specific interactions between TatC-like proteins and other pathway components, such as TatA, of which three paralogues are present in B. subtilis.     

Adaptation of protein secretion to extremely high-salt conditions by extensive use of the twin-arginine translocation pathway

Halophilic archaea thrive in environments with salt concentrations approaching saturation. However, little is known about the way in which these organisms stabilize their secreted proteins in such 'hostile' conditions. Here, we present data suggesting that the utilization of protein translocation pathways for protein secretion by the Halobacteriaceae differs significantly from that of non-haloarchaea, and most probably represents an adaptation to the high-salt environment. Although most proteins are secreted via the general secretion (Sec) machinery, the twin-arginine translocation (Tat) pathway is mainly used for the secretion of redox proteins and is distinct from the Sec pathway, in that it allows cytoplasmic folding of secreted proteins. tatfind (developed in this study) was used for systematic whole-genome analysis of Halobacterium sp. NRC-1 and several other prokaryotes to identify putative Tat substrates. Our analyses revealed that the vast majority of haloarchaeal secreted proteins were predicted substrates of the Tat pathway. Strikingly, most of these putative Tat substrates were non-redox proteins, the homologues of which in non-haloarchaea were identified as putative Sec substrates. We confirmed experimentally that the secretion of one such putative Tat substrate depended on the twin-arginine motif in its signal sequence. This extensive utilization of the Tat pathway in haloarchaea suggests an evolutionary adaptation to high-salt conditions by allowing cytoplasmic folding of secreted proteins before their secretion.     

Oligomeric properties and signal peptide binding by Escherichia coli Tat protein transport complexes

The Escherichia coli Tat apparatus is a protein translocation system that serves to export folded proteins across the inner membrane. The integral membrane proteins TatA, TatB and TatC are essential components of this pathway. Substrate proteins are directed to the Tat apparatus by specialized N-terminal signal peptides bearing a consensus twin-arginine sequence motif. Here we have systematically examined the Tat complexes that can be purified from overproducing strains. Our data suggest that the TatA, TatB and TatC proteins are found in at least two major types of high molecular mass complex in detergent solution, one consisting predominantly of TatA but with a small quantity of TatB, and the other based on a TatBC unit but also containing some TatA protein. The latter complex is shown to be capable of binding a Tat signal peptide. Using an alternative purification strategy we show that it is possible to isolate a TatABC complex containing a high molar excess of the TatA component.     

Cell biology of Legionella pneumophila

Legionella pneumophila is the causative agent of a potentially fatal form of pneumonia named Legionnaires' disease. L. pneumophila survives and replicates inside macrophages by preventing phagosome-lysosome fusion. A large number of L. pneumophila genes, called dot or icm, have been identified that are required for intracellular growth. It has recently been shown that the dot/icm genes code for a putative large membrane complex that forms a type IV secretion system used to alter the endocytic pathway.     

Haloferax volcanii twin-arginine translocation substates include secreted soluble, C-terminally anchored and lipoproteins

Recent in silico and in vivo studies have suggested that the majority of proteins destined for secretion in the haloarchaea are trafficked through the twin-arginine translocation (Tat) pathway. The presence of lipobox motifs in most haloarchaeal Tat signal sequences is intriguing as: (i) bioinformatic searches of archaeal genomes have not identified lipoprotein biogenesis enzymes and (ii) there are no known Tat substrates containing both a twin-arginine and a bona fide lipobox. We have examined six computationally designated Tat substrates in the haloarchaeon Haloferax volcanii to verify previous computational predictions and to initiate studies of lipoprotein biogenesis via the Tat pathway. Our results confirmed that the six candidate proteins were not only Tat substrates, but also belonged to diverse classes of secretory proteins. Analysis of predicted lipoprotein Tat substrates revealed that they are anchored to the archaeal membrane in a cysteine-dependent manner. Interestingly, despite the absence of an archaeal lipoprotein signal peptidase II (SPase II) homologue, the SPase II inhibitor globomycin impeded cell growth and specifically prevented maturation of lipoproteins. Together, this work not only represents the first experimental demonstration of a lipoprotein Tat substrate, but also indicates the presence of an unidentified lipoprotein biogenesis pathway in archaea.     

Twin-Arginine Translocation Pathway in Streptomyces lividans

The recently discovered bacterial twin-arginine translocation (Tat) pathway was investigated in Streptomyces lividans, a gram-positive organism with a high secretion capacity. The presence of one tatC and two hcf106 homologs in the S. lividans genome together with the several precursor proteins with a twin-arginine motif in their signal peptide suggested the presence of the twin-arginine translocation pathway in the S. lividans secretome. To demonstrate its functionality, a tatC deletion mutant was constructed. This mutation impaired the translocation of the Streptomyces antibioticus tyrosinase, a protein that forms a complex with its transactivator protein before export. Also the chimeric construct pre-TorA-23K, known to be exclusively secreted via the Tat pathway in Escherichia coli, could be translocated in wild-type S. lividans but not in the tatC mutant. In contrast, the secretion of the Sec-dependent S. lividans subtilisin inhibitor was not affected. This study therefore demonstrates that also in general in Streptomyces spp. the Tat pathway is functional. Moreover, this Tat pathway can translocate folded proteins, and the E. coli TorA signal peptide can direct Tat-dependent transport in S. lividans.     

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.     

Pathfinders and trailblazers: a prokaryotic targeting system for transport of folded proteins

The twin-arginine (Tat) protein translocase is a highly unusual protein transport machine that is dedicated to the movement of folded proteins across the bacterial cytoplasmic membrane. Proteins are targeted to the Tat pathway by means of N-terminal signal peptides harbouring a distinctive twin-arginine motif. In this minireview, we describe our current knowledge of the Tat system, paying particular attention to the function of the TatA protein and to the often overlooked step of signal peptide cleavage.     

Identification of new secreted proteins and secretion of heterologous amylase by <Emphasis Type="Italic">C. glutamicum</Emphasis>

In this study, secreted Corynebacterium glutamicum proteins were investigated by two-dimensional gel electrophoresis. Around 100 spots observed in the pH range 4.5–5.5 had molecular masses that varied from 10 to 50 kDa. Upon N-terminal amino acid sequence analysis by Edman degradation, two of them were hits to two hypothetical proteins encoded by cgR_1176 and cgR_2070 on C. glutamicum R genome, respectively. Active-form α-amylase derived from Geobacillus stearothermophilus was successfully secreted by using the predicted cgR_1176 and cgR_2070 signal sequences, indicating that these hypothetical proteins were secreted proteins. Analysis using a disruption mutant of the twin-arginine translocation (Tat) export pathway machinery of C. glutamicum suggested that one is Tat pathway dependent secretion while the other is independent of the pathway. Our results demonstrate that C. glutamicum can secrete exoproteins by using its own signal sequences, indicating its potential as a host for protein productions.     

Functional analysis of TatA and TatB in Streptomyces lividans

Recently, genes encoding TatA, TatB, and TatC homologues were identified in Streptomyces lividans and the functionality of the twin-arginine translocation (Tat) pathway was demonstrated. Previously, we have shown that TatC is indispensable for Tat-dependent secretion in S. lividans. In the present work, we demonstrate that as TatB, S. lividans TatA is important but not essential for efficient secretion of xylanase C and tyrosinase. The results presented here indicate that in the presence of TatC, still partially functional translocation systems composed of TatAC or TatBC can be formed, suggesting that TatA and TatB have at least partially overlapping activities. However, the dissimilar effect caused by a tatA deletion or a tatB deletion on Tat-dependent secretion together with the fact that TatA cannot fully functionally substitute TatB and vice versa indicates that in S. lividans TatA and TatB are not functionally equivalent. Interestingly, soluble GST-tagged TatA and TatB were able to specifically bind Tat-dependent preproteins. The ability to bind Tat-dependent preproteins together with their cytoplasmic localization in S. lividans strongly suggests that both TatA and TatB, independently or associated, serve to recruit Tat-dependent preproteins to the translocase.     

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.     

Role of the Escherichia coli Tat pathway in outer membrane integrity

The Escherichia coli Tat system serves to export folded proteins harbouring an N-terminal twin-arginine signal peptide across the cytoplasmic membrane. Previous work has demonstrated that strains mutated in genes encoding essential Tat pathway components are highly defective in the integrity of their cell envelope. Here, we report the isolation, by transposon mutagenesis, of tat mutant strains that have their outer membrane integrity restored. This outer membrane repair of the tat mutant arises as a result of upregulation of the amiB gene, which encodes a cell wall amidase. Overexpression of the genes encoding the two additional amidases, amiA and amiC, does not compensate for the outer membrane defect of the tatC strain. Analysis of the amiA and amiC coding sequences indicates that the proteins may be synthesized with plausible twin-arginine signal sequences, and we demonstrate that they are translocated to the periplasm by the Tat pathway. A Tat+ strain that has mislocalized AmiA and AmiC proteins because of deletion of their signal peptides displays an identical defective cell envelope phenotype. The presence of genes encoding amidases with twin-arginine signal sequences in the genomes of other Gram-negative bacteria suggests that a similar cell envelope defect may be a common feature of tat mutant strains.     

The Tat system of Gram-positive bacteria

The twin-arginine protein translocation (Tat) system has a unique ability to translocate folded and co-factor-containing proteins across lipid bilayers. The Tat pathway is present in bacteria, archaea and in the thylakoid membranes of chloroplasts and, depending on the organism and environmental conditions, it can be deemed important for cell survival, virulence or bioproduction. This review provides an overview of the current understanding of the Tat system with specific focus on Gram-positive bacteria. The ‘universal minimal Tat system’ is composed of a TatA and a TatC protein. However, this pathway is more commonly composed of two TatA-like proteins and one TatC protein. Often the TatA-like proteins have diverged to have two different functions and, in this case, the second TatA-like protein is usually referred to as TatB. The correct folding and/or incorporation of co-factors are requirements for translocation, and the known quality control mechanisms are examined in this review. A number of examples of crosstalk between the Tat system and other protein transport systems, such as the Sec–YidC translocon and signal peptidases or sheddases are also discussed. Further, an overview of specific Gram-positive bacterial Tat systems found in monoderm and diderm species is detailed. Altogether, this review highlights the unique features of Gram-positive bacterial Tat systems and pinpoints key questions that remain to be addressed in future research. This article is part of a Special Issue entitled: Protein trafficking and secretion in bacteria. Guest Editors: Anastassios Economou and Ross Dalbey.