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.     

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.     

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.     

Functional Analysis of the Twin-Arginine Translocation Pathway in Corynebacterium glutamicum ATCC 13869

Compared to those of other gram-positive bacteria, the genetic structure of the Corynebacterium glutamicum Tat system is unique in that it contains the tatE gene in addition to tatA, tatB, and tatC. The tatE homologue has been detected only in the genomes of gram-negative enterobacteria. To assess the function of the C. glutamicum Tat pathway, we cloned the tatA, tatB, tatC, and tatE genes from C. glutamicum ATCC 13869 and constructed mutants carrying deletions of each tat gene or of both the tatA and tatE genes. Using green fluorescent protein (GFP) fused with the twin-arginine signal peptide of the Escherichia coli TorA protein, we demonstrated that the minimal functional Tat system required TatA and TatC. TatA and TatE provide overlapping function. Unlike the TatB proteins from gram-negative bacteria, C. glutamicum TatB was dispensable for Tat function, although it was required for maximal efficiency of secretion. The signal peptide sequence of the isomaltodextranase (IMD) of Arthrobacter globiformis contains a twin-arginine motif. We showed that both IMD and GFP fused with the signal peptide of IMD were secreted via the C. glutamicum Tat pathway. These observations indicate that IMD is a bona fide Tat substrate and imply great potential of the C. glutamicum Tat system for industrial production of heterologous folded proteins.     

大肠杆菌Tat蛋白质转运体系

Tat是大肠杆菌中能够将折叠蛋白质跨膜转运的体系,其信号肽中含有一个高度保守的双精氨酸模体。Tat体系包括TatA、TatB、TatC和TatE4种蛋白质,它们的复合物在大肠杆菌质膜上形成转运通道。大肠杆菌Tat体系转运的底物蛋白质多为呼吸电子传递链组分,与大肠杆菌的许多生命活动有关。     

Bimolecular Fluorescence Complementation Analysis of Inducible Protein Interactions: Effects of Factors Affecting Protein Folding on Fluorescent Protein Fragment Association

Bimolecular fluorescence complementation (BiFC) analysis enables visualization of the subcellular locations of protein interactions in living cells. Using fragments of different fluorescent proteins, we investigated the temporal resolution and the quantitative accuracy of BiFC analysis. We determined the kinetics of BiFC complex formation in response to the rapamycin-inducible interaction between the FK506 binding protein (FKBP) and the FKBP-rapamycin binding domain (FRB). Fragments of yellow fluorescent protein fused to FKBP and FRB produced detectable BiFC complex fluorescence 10 min after the addition of rapamycin and a 10-fold increase in the mean fluorescence intensity in 8 h. The N-terminal fragment of the Venus fluorescent protein fused to FKBP produced constitutive BiFC complexes with several C-terminal fragments fused to FRB. A chimeric N-terminal fragment containing residues from Venus and yellow fluorescent protein produced either constitutive or inducible BiFC complexes depending on the temperature at which the cells were cultured. The concentrations of inducers required for half-maximal induction of BiFC complex formation by all fluorescent protein fragments tested were consistent with the affinities of the inducers for unmodified FKBP and FRB. Treatment with the FK506 inhibitor of FKBP-FRB interaction prevented the formation of BiFC complexes by FKBP and FRB fusions, but did not disrupt existing BiFC complexes. Proteins synthesized before the addition of rapamycin formed BiFC complexes with the same efficiency as did newly synthesized proteins. Inhibitors of protein synthesis attenuated BiFC complex formation independent of their effects on fusion protein synthesis. The kinetics at which they inhibited BiFC complex formation suggests that they prevented association of the fluorescent protein fragments, but not the slow maturation of BiFC complex fluorescence. Agents that induce the unfolded protein response also reduced formation of BiFC complexes. The effects of these agents were suppressed by cellular adaptation to protein folding stress. In summary, BiFC analysis enables detection of protein interactions within minutes after complex formation in living cells, but does not allow detection of complex dissociation. Conditional BiFC complex formation depends on the folding efficiencies of fluorescent protein fragments and can be affected by the cellular protein folding environment.     

利用荧光蛋白对大肠杆菌蛋白质Tat转运系统的研究

探讨了荧光蛋白作为报告蛋白用于蛋白质转运系统研究的可行性 ,结果表明海葵红色荧光蛋白聚集在细胞质内 ,不能转运至周质空间。而水母绿色荧光蛋白在Tat信号肽和Tat转运酶的共同作用下 ,以折叠形式转运至周质空间。通过荧光定量分析表明信号肽保守序列中的双精氨酸是保证绿色荧光蛋白转运及转运效率所必需的 ,且第二个精氨酸比第一个精氨酸更为重要。同时 ,揭示了Tat信号肽需要一定的高级结构才能行使功能 ;Tat信号肽不仅引导蛋白质的转运 ,而且也参与蛋白质的折叠。因此 ,绿色荧光蛋白是非常理想的报告蛋白 ,可用于研究Tat系统 ,但是海葵红色荧光蛋白易于聚集而不适合于此目的。     

萤火素酶互补实验检测蛋白互作

蛋白质-蛋白质相互作用在真核生物的各项生命活动中发挥重要作用。与其它蛋白质互作研究技术相比,借助于烟草(Nicotiana benthamiana)瞬时表达系统的萤火素酶互补实验(LCA)具有简单、灵敏、可靠、高效和低背景等优点,并可轻松扩展为大规模蛋白质互作的筛选和验证研究。该文介绍了萤火素酶互补实验的具体操作过程,通过2种数据收集方法来定性并定量分析生物发光或发光强度,从而检测植物目标蛋白之间的相互作用。     

Cotranslational Translocation and Folding of a Periplasmic Protein Domain in Escherichia coli

In Gram-negative bacteria, periplasmic domains in inner membrane proteins are cotranslationally translocated across the inner membrane through the SecYEG translocon. To what degree such domains also start to fold cotranslationally is generally difficult to determine using currently available methods. Here, we apply Force Profile Analysis (FPA) – a method where a translational arrest peptide is used to detect folding-induced forces acting on the nascent polypeptide – to follow the cotranslational translocation and folding of the large periplasmic domain of the E. coli inner membrane protease LepB in vivo. Membrane insertion of LepB’s two N-terminal transmembrane helices is initiated when their respective N-terminal ends reach 45–50 residues away from the peptidyl transferase center (PTC) in the ribosome. The main folding transition in the periplasmic domain involves all but the ~15 most C-terminal residues of the protein and happens when the C-terminal end of the folded part is ~70 residues away from the PTC; a smaller putative folding intermediate is also detected. This implies that wildtype LepB folds post-translationally in vivo, and shows that FPA can be used to study both co- and post-translational protein folding in the periplasm.     

In Vivo Protein Interactions within the Escherichia coli DNA Polymerase III Core

The mechanisms that control the fidelity of DNA replication are being investigated by a number of approaches, including detailed kinetic and structural studies. Important tools in these studies are mutant versions of DNA polymerases that affect the fidelity of DNA replication. It has been suggested that proper interactions within the core of DNA polymerase III (Pol III) of Escherichia coli could be essential for maintaining the optimal fidelity of DNA replication (H. Maki and A. Kornberg, Proc. Natl. Acad. Sci. USA 84:4389–4392, 1987). We have been particularly interested in elucidating the physiological role of the interactions between the DnaE (α subunit [possessing DNA polymerase activity]) and DnaQ ( subunit [possessing 3′→5′ exonucleolytic proofreading activity]) proteins. In an attempt to achieve this goal, we have used the Saccharomyces cerevisiae two-hybrid system to analyze specific in vivo protein interactions. In this report, we demonstrate interactions between the DnaE and DnaQ proteins and between the DnaQ and HolE (θ subunit) proteins. We also tested the interactions of the wild-type DnaE and HolE proteins with three well-known mutant forms of DnaQ (MutD5, DnaQ926, and DnaQ49), each of which leads to a strong mutator phenotype. Our results show that the mutD5 and dnaQ926 mutations do not affect the subunit-α subunit and subunit-θ subunit interactions. However, the dnaQ49 mutation greatly reduces the strength of interaction of the subunit with both the α and the θ subunits. Thus, the mutator phenotype of dnaQ49 may be the result of an altered conformation of the protein, which leads to altered interactions within the Pol III core.     

Visualizing multiple constrictions in spheroidal Escherichia coli cells

An Escherichia coli cell grows by elongation and divides in a perpendicular plane. Alternating planes of successive divisions in three dimensions can only be ascertained when multiple constrictions exist simultaneously in large, spheroidal cells (with extended constriction process), if the division signals are enhanced. Large, spheroidal cells are obtained by a brief mecillinam treatment, and more frequent divisions are achieved by manipulating the rate of chromosome replication without affecting cell mass growth rate. Such a procedure has recently been performed by thymine-limitation of E. coli K12 strain CR34 (Zaritsky et al., Microbiology 145 (1999), 1052-1022). Enhancing the replication rate in cells with multi-forked replicating chromosomes (by addition of deoxyguanosine) shortens the intervals between successive terminations and thus triggers divisions more frequently. Monoclonal antibodies against FtsZ were used to visualize the rings of secondary constrictions, but apparent shortage of FtsZ to complete rings over wide cells allowed assembly of arcs only. The arcs observed were not parallel nor perpendicular; the tilted constriction planes are consistent with our 3-D 'nucleoid segregation'model for division under conditions which relieve the cylindrical constraint for nucleoid segregation by the bacillari peptidoglycan sacculus (Woldringh et al. , J. Bacteriol. 176 (1994) 6030-6038). The shortage in FtsZ may explain the longer time required to complete the division process in wide cells with long circumferences, observed during thymine step-up. Overexpression of fusion protein FtsZ-GFP on a multi-copy plasmid should circumvent the shortage.     

Translocation of alpha-synuclein expressed in Escherichia coli

alpha-Synuclein is a major component of Lewy bodies in Parkinson's disease. Although no signal sequence is apparent, alpha-synuclein expressed in Escherichia coli is mostly located in the periplasm. The possibilities that alpha-synuclein translocated into the periplasm across the inner membrane by the SecA or the Tat targeting route identified in bacteria and that alpha-synuclein was released through MscL were excluded. The signal recognition particle-dependent pathway is involved in the translocation of alpha-synuclein. The C-terminal 99-to-140 portion of the alpha-synuclein molecule plays a signal-like role for its translocation into the periplasm, cooperating with the central 61-to-95 section. The N-terminal 1-to-60 region is not required for this translocation.     

Translocation of a Thioether-Bridged Azurin Peptide Fragment via the Sec Pathway in Lactococcus lactis

This study demonstrates for the first time that a thioether-containing peptide, an azurin fragment, can be translocated via the Sec pathway. This methyl-lanthionine was introduced by the nisin modification enzymes. The Sec pathway can therefore be a successful alternative for those cyclized peptides that are inefficiently transported via NisT.Azurin, a cupredoxin produced by Pseudomonas aeruginosa, can selectively enter human cancer cells and induce apoptosis (24) via binding to the tumor suppressor protein p53 (1). The azurin peptide fragment p28, containing amino acids 50 to 77 (LSTAADMQGVVTDGMASGLDKDYLKPDD), still enters human cancer cells and inhibits tumor proliferation (20). Importantly, novel cancer treatments can be based on azurin peptide fragments and derivatives thereof (T. Das Gupta and A. Chakrabarty, 20 March 2008, patent application WO2008033820). Although the pharmacokinetic property of therapeutic peptides is promising, lack of biostability is the major hurdle for their successful application. Consequently, it is very relevant to explore the possibilities for enhancing biostability of these peptides.In our group, we developed a technology to improve the stability of therapeutic peptides by exploiting the nisin synthetase enzymes NisB and NisC for the introduction of thioether bridges. We applied a two-plasmid expression system (7, 8, 12), in which the NisBTC-encoding plasmid is compatible with the substrate-peptide-encoding plasmid. Lactococcus lactis containing this expression system can secrete nonlantibiotic peptides which are dehydrated or stabilized by a thioether ring (8, 16). NisB dehydrates serines and threonines in substrate peptides, NisC couples dehydrated residues stereo- and regioselectively to cysteines, and NisT, the ABC transporter, translocates the modified peptides out of the cell (10, 11, 13, 15). The leader peptide is essential for targeting and modification of the propeptides (23).When transport via NisT is impaired or is less efficient, the Sec pathway of L. lactis is a successful alternative in translocation of dehydrated peptides. When the nisin leader is preceded by a Sec signal peptide or a Tat signal peptide 27 or 44 amino acids long, respectively, modification by NisB and NisC still occurs (12; G. N. Moll, A. Kuipers, R. Rink, A. J. M. Driessen, and O. P. Kuipers, 15 June 2006, patent application WO 2006062398). However, NisC-cyclized prenisin was not translocated via the Sec system (12). This is likely due to the dimensions of fully modified nisin (3), which is too large to fit in the SecY pore (12, 21). Here, we report for the first time that the Sec pathway of L. lactis can translocate a p28 azurin fragment analog with a thioether ring.We previously demonstrated that under culturing conditions, the highly reactive dehydroalanines can spontaneously couple to cysteines, either intra- or extracellularly, whereas the less reactive dehydrobutyrines do not (16). To exclude spontaneous thioether ring formation by dehydroalanines, we mutated serines in positions 51 and 66 to alanines, whereas a single dehydratable threonine was kept in position 52 of the azurin(50-77) peptide fragment. Position 56 was mutated to a cysteine to allow posttranslational introduction of a thioether bridge (Table ​(Table1;1; pNZ8048-derived plasmids).

TABLE 1.

Bacterial strains and plasmids used in this study

The Glove-like Structure of the Conserved Membrane Protein TatC Provides Insight into Signal Sequence Recognition in Twin-Arginine Translocation

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Highlights? Crystal structures of TatC from the thermophile Aquifex aeolicus are presented ? The architecture of TatC generates a glove-shaped pocket buried in the bilayer ? Molecular dynamics reveal destabilization of the membrane around the pocket ? Correlation to biochemical results suggest the signal sequence binds in this pocket     

抗汉坦病毒核蛋白单链抗体的构建与表达

汉坦病毒核蛋白在病毒各个基因组片段核糖核蛋白(RNP)复合物的形成,以及RNP复合物装配入病毒颗粒中发挥重要作用。体外研究表明,核蛋白与病毒RNA的特异性结合结构域位于第175—217个氨基酸残基,羧基端其它部分为非特异性结合结构域。为了在细胞内病毒复制的正常过程中研究核蛋白的功能,应用噬菌体表面呈现技术,分别从鼠杂交瘤细胞和人外周血淋巴细胞天然抗体库中,筛选出两株不同抗原结合位点的抗汉坦病毒核蛋白抗体L13 F3和H34Fab抗体,并在大肠杆菌中进行表达。L13 F3 Fab抗体的抗原结合位点位于核蛋白氨基端25—65个氨基酸之间,H34的抗原结合位点位于核蛋白的羧基端的一半。分别使重链可变区羧基端与轻链可变区氨基端通过9个氨基酸寡肽连接起来,构建成单链抗体,并在大肠杆菌内进行表达。通过酶联免疫吸附试验(EL-SIA)、间接免疫荧光试验(IFA)和Western blot检测,确定所构建单链抗体与母抗体的抗原结合特性无差别。单链抗体分子量小,易于操作并保留了全部的抗原结合活性,是在细胞内探索汉坦病毒核蛋白功能的良好工具。     

Genetic Analysis of Transfer by the Escherichia coli Sex Factor F, Using P1 Transductional Complementation

P1 transduction has been used to perform a complementation analysis of a series of transfer-deficient mutants of Flac. The results define ten cistrons and are consistent with the results of a conjugational analysis presented in an accompanying report. Both sets of results are summarized here. Between them, they define eleven cistrons, traA through traK, necessary for conjugational deoxyribonucleic acid (DNA) transfer. Mutants in traI and traD and some in traG still make F-pili, although traD mutants are resistant to f2 phage; their products may be involved in conjugational DNA metabolism. Other mutants in traG and all mutants in the remaining eight cistrons do not make F-pili. One of these, traJ, may be a control cistron, and the others may specify a biosynthetic pathway responsible for synthesis and modification of the F-pilin subunit protein and its assembly into the F-pilus.