Sec Signal Peptides Research Articles

GFP fusions of Sec-routed extracellular proteins in Staphylococcus aureus reveal surface-associated coagulase in biofilms.

Staphylococcus aureus is a major human pathogen that utilises many surface-associated and secreted proteins to form biofilms and cause disease. However, our understanding of these processes is limited by challenges of using fluorescent protein reporters in their native environment, because they must be exported and fold correctly to become fluorescent. Here, we demonstrate the feasibility of using the monomeric superfolder GFP (msfGFP) exported from S. aureus. By fusing msfGFP to signal peptides for the Secretory (Sec) and Twin Arginine Translocation (Tat) pathways, the two major secretion pathways in S. aureus, we quantified msfGFP fluorescence in bacterial cultures and cell-free supernatant from the cultures. When fused to a Tat signal peptide, we detected msfGFP fluorescence inside but not outside bacterial cells, indicating a failure to export msfGFP. However, when fused to a Sec signal peptide, msfGFP fluorescence was present outside cells, indicating successful export of the msfGFP in the unfolded state, followed by extracellular folding and maturation to the photoactive state. We applied this strategy to study coagulase (Coa), a secreted protein and a major contributor to the formation of a fibrin network in S. aureus biofilms that protects bacteria from the host immune system and increases attachment to host surfaces. We confirmed that a genomically integrated C-terminal fusion of Coa to msfGFP does not impair the activity of Coa or its localisation within the biofilm matrix. Our findings demonstrate that msfGFP is a good candidate fluorescent reporter to consider when studying proteins secreted by the Sec pathway in S. aureus.

PvdM of fluorescent pseudomonads is required for the oxidation of ferribactin by PvdP in periplasmic pyoverdine maturation

Fluorescent pseudomonads such as Pseudomonas aeruginosa or Pseudomonas fluorescens produce pyoverdine siderophores that ensure iron-supply in iron-limited environments. After its synthesis in the cytoplasm, the nonfluorescent pyoverdine precursor ferribactin is exported into the periplasm, where the enzymes PvdQ, PvdP, PvdO, PvdN, and PtaA are responsible for fluorophore maturation and tailoring steps. While the roles of all these enzymes are clear, little is known about the role of PvdM, a human renal dipeptidase–related protein that is predicted to be periplasmic and that is essential for pyoverdine biogenesis. Here, we reveal the subcellular localization and functional role of PvdM. Using the model organism P. fluorescens, we show that PvdM is anchored to the periplasmic side of the cytoplasmic membrane, where it is indispensable for the activity of the tyrosinase PvdP. While PvdM does not share the metallopeptidase function of renal dipeptidase, it still has the corresponding peptide-binding site. The substrate of PvdP, deacylated ferribactin, is secreted by a ΔpvdM mutant strain, indicating that PvdM prevents loss of this periplasmic biosynthesis intermediate into the medium by ensuring the efficient transfer of ferribactin to PvdP in vivo. We propose that PvdM belongs to a new dipeptidase-related protein subfamily with inactivated Zn2+ coordination sites, members of which are usually genetically linked to TonB-dependent uptake systems and often associated with periplasmic FAD-dependent oxidoreductases related to d-amino acid oxidases. We suggest that these proteins are necessary for selective binding, exposure, or transfer of specific d- and l-amino acid–containing peptides and other periplasmic biomolecules in manifold pathways.

Biosensor-Based Optimization of Cutinase Secretion by Corynebacterium glutamicum.

The industrial microbe Corynebacterium glutamicum is gaining substantial importance as a platform host for recombinant protein secretion. We recently developed a fluorescence-based (eYFP) C. glutamicum reporter strain for the quantification of Sec-dependent protein secretion by monitoring the secretion-related stress response and now demonstrate its applicability in optimizing the secretion of the heterologous enzyme cutinase from Fusarium solani pisi. To drive secretion, either the poor-performing PelSP or the potent NprESP Sec signal peptide from Bacillus subtilis was used. To enable easy detection and quantification of the secreted cutinase we implemented the split green fluorescent protein (GFP) assay, which relies on the GFP11-tag fused to the C-terminus of the cutinase, which can complement a truncated GFP thereby reconstituting its fluorescence. The reporter strain was transformed with different mutant libraries created by error-prone PCR, which covered the region of the signal peptide and the N-terminus of the cutinase. Fluorescence-activated cell sorting (FACS) was performed to isolate cells that show increased fluorescence in response to increased protein secretion stress. Five PelSP variants were identified that showed a 4- to 6-fold increase in the amount and activity of the secreted cutinase (up to 4,100 U/L), whereas two improved NprESP variants were identified that showed a ∼35% increase in secretion, achieving ∼5,500 U/L. Most of the isolated variants carried mutations in the h-region of the signal peptide that increased its overall hydrophobicity. Using site-directed mutagenesis it was shown that the combined mutations F11I and P16S within the hydrophobic core of the PelSP are sufficient to boost cutinase secretion in batch cultivations to the same level as achieved by the NprESP. Screening of a PelSP mutant library in addition resulted in the identification of a cutinase variant with an increased specific activity, which was attributed to the mutation A85V located within the substrate-binding region. Taken together the biosensor-based optimization approach resulted in a substantial improvement of cutinase secretion by C. glutamicum, and therefore represents a valuable tool that can be applied to any secretory protein of interest.

Toxoplasma gondii ROP38 protein: Bioinformatics analysis for vaccine design improvement against toxoplasmosis

Rhoptry proteins (ROPs) play a significant role in various stages of Toxoplasma gondii (T. gondii) life cycle, being critical for both invasion and intracellular survival. ROP38 is a key manipulator of host gene expression and has a function in tachyzoite to bradyzoite conversion. In this study, we've employed various bioinformatics online tools for immunogenicity prediction of ROP38 protein, comprising physico-chemical, antigenic and allergenic profiles, transmembrane domain, subcellular localization, post-translational modification (PTM) sites, secondary and 3D structure, B-cell, MHC-binding and cytotoxic T-lymphocyte (CTL) epitopes. The findings showed 54 PTM sites without a transmembrane domain. Also, ROP38 was proved a non-allergenic and antigenic protein. The protein had Sec signal peptide (Sec/SPI) with 0.8762 likelihood. The secondary structure included 52.68% random coil, 29.57% alpha helix and 17.74% extended strand. Based on Ramachandran plot output for refined model, 95.3%, 3.4%, and 1.4% of amino acid residues were incorporated in the favored, allowed, and outlier regions, respectively. B-cell epitopes TFPGDDIQTSS (67–72) and KAKNKWGRTRYTLQG (207–221) as well as T-cell epitope LSPVGFFTAL (6–15) possessed the highest antigenic index in the protein sequence. This paper is a premise for further researches, and provides insights for the development of a suitable vaccine against toxoplasmosis. More empirical studies are required using the ROP38 alone or in combination with other antigens/epitopes in the future.

Screening of a genome-reduced Corynebacterium glutamicum strain library for improved heterologous cutinase secretion.

The construction of microbial platform organisms by means of genome reduction is an ongoing topic in biotechnology. In this study, we investigated whether the deletion of single or multiple gene clusters has a positive effect on the secretion of cutinase from Fusarium solani pisi in the industrial workhorse Corynebacterium glutamicum. A total of 22 genome-reduced strain variants were compared applying two Sec signal peptides from Bacillus subtilis. High-throughput phenotyping using robotics-integrated microbioreactor technology with automated harvesting revealed distinct cutinase secretion performance for a specific combination of signal peptide and genomic deletions. The biomass-specific cutinase yield for strain GRS41_51_NprE was increased by~200%, although the growth rate was reduced by~60%. Importantly, the causative deletions of genomic clusters cg2801-cg2828 and rrnC-cg3298 could not have been inferred a priori. Strikingly, bioreactor fed-batch cultivations at controlled growth rates resulted in a complete reversal of the screening results, with the cutinase yield for strain GRS41_51_NprE dropping by~25% compared to the reference strain. Thus, the choice of bioprocess conditions may turn a 'high-performance' strain from batch screening into a 'low-performance' strain in fed-batch cultivation. In conclusion, future studies are needed in order to understand metabolic adaptations of C.glutamicum to both genomic deletions and different bioprocess conditions.

Expression and secretion of a lytic polysaccharide monooxygenase by a fast-growing cyanobacterium

BackgroundCyanobacteria have the potential to become next-generation cell factories due to their ability to use CO2, light and inorganic nutrients to produce a range of biomolecules of commercial interest. Synechococcus elongatus UTEX 2973, in particular, is a fast-growing, genetically tractable, cyanobacterium that has garnered attention as a potential biotechnological chassis. To establish this unique strain as a host for heterologous protein production, we aimed to demonstrate expression and secretion of the industrially relevant TfAA10A, a lytic polysaccharide monooxygenase from the Gram-positive bacterium Thermobifida fusca.ResultsTwo variations of TfAA10A were successfully expressed in S. elongatus UTEX 2973: One containing the native N-terminal, Sec-targeted, signal peptide and a second with a Tat-targeted signal peptide from the Escherichia coli trimethylamine-N-oxide reductase (TorA). Although the TorA signal peptide correctly targeted the protein to the plasma membrane, the majority of the TorA-TfAA10A was found unprocessed in the plasma membrane with a small fraction of the mature protein ultimately translocated to the periplasm. The native Sec signal peptide allowed for efficient secretion of TfAA10A into the medium with virtually no protein being found in the cytosol, plasma membrane or periplasm. TfAA10A was demonstrated to be correctly cleaved and active on the model substrate phosphoric acid swollen cellulose. Additionally, expression and secretion only had a minor impact on cell growth. The secretion yield was estimated at 779 ± 40 µg L−1 based on densitometric analysis. To our knowledge, this is the highest secretion yield ever registered in cyanobacteria.ConclusionsWe have shown for the first time high-titer expression and secretion of an industrially relevant and catalytically active enzyme in S. elongatus UTEX 2973. This proof-of-concept study will be valuable for the development of novel and sustainable applications in the fields of bioremediation and biocatalysis.

Export of a Vibrio parahaemolyticus toxin by the Sec and type III secretion machineries in tandem.

Many Gram-negative pathogens utilize dedicated secretion systems to export virulence factors such as exotoxins and effectors1-4. Several exotoxins are synthesized as precursors containing amino-terminal Sec signal peptides and are exported through the inner-membrane-bound Sec machinery to the periplasm, followed by secretion across the outer membrane to the exterior using a type II secretion system (T2SS)3,5. Here, we report that thermostable direct haemolysin (TDH), an exotoxin of the food-borne pathogen Vibrio parahaemolyticus, can be exported through the type III secretion system (T3SS), which engages in one-step secretion of effectors4, despite possessing a Sec signal peptide and being mainly secreted via the T2SS. Although the precursor of TDH is targeted to the Sec pathway, a fraction of mature TDH was observed to re-enter the bacterial cytoplasm. The N terminus of mature TDH comprises a T3SS signal sequence, allowing it to be loaded into the T3SS. We also show that T3SS-delivered TDH as an effector contributes to intestinal fluid accumulation in a rabbit diarrhoeal model of V. parahaemolyticus infection. Thus, our results show that an unconventional export mechanism for a bacterial toxin via the T3SS in tandem with the Sec machinery facilitates the virulence trait of V. parahaemolyticus.

Combinatorial impact of Sec signal peptides from Bacillus subtilis and bioprocess conditions on heterologous cutinase secretion by Corynebacterium glutamicum.

The impact of Sec signal peptides (SPs) from Bacillus subtilis in combination with isopropyl-β- d-1-thiogalactopyranoside concentration and feeding profile was investigated for heterologous protein secretion performance by Corynebacterium glutamicum using cutinase as model enzyme. Based on a comprehensive data set of about 150 bench-scale bioreactor cultivations in fed-batch mode and choosing the cutinase yield as objective, it was shown that relative secretion performance for bioprocesses remains very similar, irrespective of the applied SP enabling Sec-mediated cutinase secretion. However, to achieve the maximal absolute cutinase yield, careful adjustment of bioprocess conditions was found to be necessary. A model-based, two-step multiple regression approach resembled the collected data in a comprehensive way. The corresponding results suggest that the choice of the heterologous Sec SP and its interaction with the adjusted exponential feeding profile is highly relevant to maximize absolute cutinase yield in this study. For example, the impact of Sec SP is high at low growth rates and low at high growth rates. However, promising Sec SPs could be inferred from less complex batch cultivations. The extensive data were also evaluated in terms of cutinase productivity, highlighting the well-known trade-off between yield and productivity in bioprocess development in detail. Conclusively, only the right combination of target protein, Sec SP, and bioprocess conditions is the key to success.

Use of a Sec signal peptide library from Bacillus subtilis for the optimization of cutinase secretion in Corynebacterium glutamicum.

BackgroundTechnical bulk enzymes represent a huge market, and the extracellular production of such enzymes is favorable due to lowered cost for product recovery. Protein secretion can be achieved via general secretion (Sec) pathway. Specific sequences, signal peptides (SPs), are necessary to direct the target protein into the translocation machinery. For example, >150 Sec-specific SPs have been identified for Bacillus subtilis alone. As the best SP for a target protein of choice cannot be predicted a priori, screening of homologous SPs has been shown to be a powerful tool for different expression organisms. While SP libraries between closely related species were successfully applied to optimize recombinant protein secretion, this was not investigated for distantly related species. Therefore, in this study a Sec SP library from low-GC firmicutes B. subtilis is investigated to optimize protein secretion in high-GC actinobacterium Corynebacterium glutamicum using cutinase from Fusarium solani pisi as model protein.ResultsA homologous SP library (~150 SP) for recombinant cutinase secretion in B. subtilis was successfully transferred to C. glutamicum as alternative secretion host. Cutinase secretion in C. glutamicum was quantified using an automated micro scale cultivation system for online growth monitoring, cell separation and cutinase activity determination. Secretion phenotyping results were correlated to those from a previous study, in which the same SP library was used to optimize secretion of the same cutinase but using B. subtilis as host. Strikingly, behavior of specific SP-cutinase combinations was changed dramatically between B. subtilis and C. glutamicum. Some SPs showed comparable cutinase secretion performances in both hosts, whereas other SPs caused diametrical extracellular cutinase activities.ConclusionThe optimal production strain for a specific target protein of choice still cannot be designed in silico. Not only the best SP for a target protein has to be evaluated each time from scratch, the expression host also affects which SP is best. Thus, (heterologous) SP library screening using high-throughput methods is considered to be crucial to construct an optimal production strain for a target protein.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-016-0604-6) contains supplementary material, which is available to authorized users.

Corynebacterium glutamicum possesses β-N-acetylglucosaminidase

BackgroundIn Gram-positive Corynebacterium glutamicum and other members of the suborder Corynebacterianeae, which includes mycobacteria, cell elongation and peptidoglycan biosynthesis is mainly due to polar growth. C. glutamicum lacks an uptake system for the peptidoglycan constituent N-acetylglucosamine (GlcNAc), but is able to catabolize GlcNAc-6-phosphate. Due to its importance in white biotechnology and in order to ensure more sustainable processes based on non-food renewables and to reduce feedstock costs, C. glutamicum strains have previously been engineered to produce amino acids from GlcNAc. GlcNAc also is a constituent of chitin, but it is unknown if C. glutamicum possesses chitinolytic enzymes.ResultsChitin was shown here not to be growth substrate for C. glutamicum. However, its genome encodes a putative N-acetylglucosaminidase. The nagA2 gene product was active as β-N-acetylglucosaminidase with 0.27 mM 4-nitrophenyl N,N’-diacetyl-β-D-chitobioside as substrate supporting half-maximal activity. NagA2 was secreted into the culture medium when overproduced with TAT and Sec dependent signal peptides, while it remained cytoplasmic when overproduced without signal peptide. Heterologous expression of exochitinase gene chiB from Serratia marcescens resulted in chitinolytic activity and ChiB secretion was enhanced when a signal peptide from C. glutamicum was used. Colloidal chitin did not support growth of a strain secreting exochitinase ChiB and β-N-acetylglucosaminidase NagA2.ConclusionsC. glutamicum possesses β-N-acetylglucosaminidase. In the wild type, β-N-acetylglucosaminidase activity was too low to be detected. However, overproduction of the enzyme fused to TAT or Sec signal peptides led to secretion of active β-N-acetylglucosaminidase. The finding that concomitant secretion of endogenous NagA2 and exochitinase ChiB from S. marcescens did not entail growth with colloidal chitin as sole or combined carbon source, may indicate the requirement for higher or additional enzyme activities such as processive chitinase or endochitinase activities.Electronic supplementary materialThe online version of this article (doi:10.1186/s12866-016-0795-3) contains supplementary material, which is available to authorized users.

A Lysine Cluster in Domain II of Bacillus subtilis PBP4a Plays a Role in the Membrane Attachment of This C1-PBP.

In PBP4a, a Bacillus subtilis class-C1 penicillin-binding protein (PBP), four clustered lysine (K) residues, K86, K114, K119, and K265, protrude from domain II. Replacement of these amino acids with glutamine (Q) residues by site-directed mutagenesis yielded Mut4KQ PBP4a. When produced in Escherichia coli without its predicted Sec-signal peptide, wild-type (WT) PBP4a was found mainly associated with the host cytoplasmic membrane, whereas Mut4KQ PBP4a remained largely unbound. After purification, the capacities of the two proteins to bind to B. subtilis membranes were compared. The results were similar to those obtained in E. coli: in vitro, a much higher percentage of WT PBP4a than of Mut4KQ PBP4a was found to interact with B. subtilis membranes. Immunodetection of PBP4a in B. subtilis membrane extracts revealed that a processed form of this PBP (as indicated by its size) associates with the B. subtilis cytoplasmic membrane. In the absence of any amphiphilic peptide in PBP4a, the crown of positive charges on the surface of domain II is likely responsible for the cellular localization of this PBP and its attachment to the cytoplasmic membrane.

Secretion of Tat-dependent halolysin SptA capable of autocatalytic activation and its relation to haloarchaeal growth.

Halolysins are Tat-dependent extracellular subtilases of haloarchaea. Whether halolysins can be activated before transport across the cytoplasmic membrane in a folded state and how haloarchaea minimize the risk of intracellular activation of halolysins and proteolysis of cellular proteins are unknown. Here, we report that both the precursor and proform of halolysin SptA from Natrinema sp. J7-2 mature autocatalytically, and the SptA maturation proceeds less efficiently in the presence of KCl than NaCl. When produced in Haloferax volcanii, most SptA molecules are secreted into the culture medium, but a small number of molecules can be activated intracellularly, affecting the cell's growth. Furthermore, retardation of SptA secretion in Hfx. volcanii via mutation of the Tat signal peptide leads to intracellular accumulation of the active enzyme and subsequent cell death. Although the Sec signal peptide can mediate SptA secretion in Hfx. volcanii, the secreted protein undergoes proteolysis. In Natrinema sp. J7-2, SptA is secreted primarily during stationary phase, and the intracellular accumulation of mature enzyme occurs during the stationary and death phases. The growth phase-dependent synthesis of SptA, highly efficient secretion system, and high intracellular KCl concentration, contribute to the suppression of premature activation of this enzyme in Natrinema sp. J7-2.

Proteomic Analysis of the Secretome of Haloarchaeon Natrinema sp. J7–2

Although in silico predictions have revealed that haloarchaea can be distinguished from other organisms in that the Tat pathway is used more extensively than the Sec pathway for haloarchaeal protein secretion, only a few haloarchaeal-secreted proteins have been experimentally confirmed. Here, the culture supernatant and membrane fraction of the haloarchaeon Natrinema sp. J7-2 grown at 23% salt concentration were subjected to RPLC-ESI-MS/MS analysis. In total, 46 predicted Tat substrates, 14 predicted Sec substrates, and 3 class III signal peptide-bearing proteins were detected. Approximately 65% of the detected Tat substrates contain lipoboxes, emphasizing the role of the Tat pathway in haloarchaeal lipoprotein secretion. Most of the detected Tat substrates are extracellular substrate (solute)-binding proteins and redox proteins. Despite the small number of Sec substrates, two of them, a cell surface glycoprotein and a putative lipoprotein carrier protein, were identified to be high-abundance secreted proteins. While limited proteins were detected in the culture supernatant, most of the secreted proteins were found in the membrane fraction. The anchoring of secreted proteins to the cell surface via a lipobox or a PGF-CTERM seems to be an adaptation strategy of haloarchaea to handle the harsh extracellular environment. Additionally, ∼15% of the integral membrane proteins (IMPs) detected in the membrane fraction possess putative Sec signal peptides or signal anchors, implying that the Sec pathway is important for membrane insertion of IMPs. This is the first report to describe the experimental secretome of haloarchaea and provide new information for better understanding of haloarchaeal protein secretion patterns.

Characterization and identification of weissellicin Y and weissellicin M, novel bacteriocins produced by Weissella hellenica QU 13

To identify and characterize novel bacteriocins from Weissella hellenica QU 13. Weissella hellenica QU 13, isolated from a barrel used to make Japanese pickles, produced two novel bacteriocins termed weissellicin Y and weissellicin M. The primary structures of weissellicins Y and M were determined, and their molecular masses were determined to be 4925·12 and 4968·40 Da, respectively. Analysis of the DNA sequence encoding the bacteriocins revealed that they were synthesized and secreted without N-terminal extensions such as leader sequences or sec signal peptides. Weissellicin M showed significantly high and characteristic homology with enterocins L50A and L50B, produced by Enterococcus faecium L50, while weissellicin Y showed no homology with any other known bacteriocins. Both bacteriocins showed broad antimicrobial spectra, with especially high antimicrobial activity against species, which contaminate pickles, such as Bacillus coagulans, and weissellicin M showed relatively higher activity than weissellicin Y. Furthermore, the stability of weissellicin M against pH and heat was distinctively higher than that of weissellicin Y. Weissella hellenica QU 13 produced two novel leaderless bacteriocins, weissellicin Y and weissellicin M, and weissellicin M exhibited remarkable potency that could be employed by pickle-producing industry. This study is the first report, which represents a complete identification and characterization of novel leaderless bacteriocins from Weissella genus.

Combined prediction of Tat and Sec signal peptides with hidden Markov models

Computational prediction of signal peptides is of great importance in computational biology. In addition to the general secretory pathway (Sec), Bacteria, Archaea and chloroplasts possess another major pathway that utilizes the Twin-Arginine translocase (Tat), which recognizes longer and less hydrophobic signal peptides carrying a distinctive pattern of two consecutive Arginines (RR) in the n-region. A major functional differentiation between the Sec and Tat export pathways lies in the fact that the former translocates secreted proteins unfolded through a protein-conducting channel, whereas the latter translocates completely folded proteins using an unknown mechanism. The purpose of this work is to develop a novel method for predicting and discriminating Sec from Tat signal peptides at better accuracy. We report the development of a novel method, PRED-TAT, which is capable of discriminating Sec from Tat signal peptides and predicting their cleavage sites. The method is based on Hidden Markov Models and possesses a modular architecture suitable for both Sec and Tat signal peptides. On an independent test set of experimentally verified Tat signal peptides, PRED-TAT clearly outperforms the previously proposed methods TatP and TATFIND, whereas, when evaluated as a Sec signal peptide predictor compares favorably to top-scoring predictors such as SignalP and Phobius. The method is freely available for academic users at http://www.compgen.org/tools/PRED-TAT/.

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.

The Thylakoid Proton Gradient Promotes an Advanced Stage of Signal Peptide Binding Deep within the Tat Pathway Receptor Complex

Tat (twin arginine translocation) systems transport folded proteins across the thylakoid membrane of chloroplasts and the plasma membrane of most bacteria. Tat precursors are targeted by hydrophobic cleavable signal peptides with twin arginine (RR) motifs. Bacterial precursors possess an extended consensus, (S/T)RRXFLK, of which the two arginines and the phenylalanine are essential for efficient transport. Thylakoid Tat precursors possess twin arginines but lack the consensus phenylalanine. Here, we have characterized two stages of precursor binding to the thylakoid Tat signal peptide receptor, the 700-kDa cpTatC-Hcf106 complex. The OE17 precursor tOE17 binds to the receptor by RR-dependant electrostatic interactions and partially dissociates during blue native gel electrophoresis. In addition, the signal peptide of thylakoid-bound tOE17 is highly exposed to the membrane surface, as judged by accessibility to factor Xa of cleavage sites engineered into signal peptide flanking regions. By contrast, tOE17 containing a consensus phenylalanine in place of Val(-20) (V - 20F) binds the receptor more strongly and is completely stable during blue native gel electrophoresis. Thylakoid bound V - 20F is also completely protected from factor Xa at the identical sites. This suggests that the signal peptide is buried deeply in the cpTatC-Hcf106 binding site. We further provide evidence that the proton gradient, which is required for translocation, induces a tighter interaction between tOE17 and the cpTat machinery, similar to that exhibited by V - 20F. This implies that translocation involves a very intimate association of the signal peptide with the receptor complex binding site.

Bacillus anthracis secretome time course under host-simulated conditions and identification of immunogenic proteins

BackgroundThe secretion time course of Bacillus anthracis strain RA3R (pXO1+/pXO2-) during early, mid, and late log phase were investigated under conditions that simulate those encountered in the host. All of the identified proteins were analyzed by different software algorithms to characterize their predicted mode of secretion and cellular localization. In addition, immunogenic proteins were identified using sera from humans with cutaneous anthrax.ResultsA total of 275 extracellular proteins were identified by a combination of LC MS/MS and MALDI-TOF MS. All of the identified proteins were analyzed by SignalP, SecretomeP, PSORT, LipoP, TMHMM, and PROSITE to characterize their predicted mode of secretion, cellular localization, and protein domains. Fifty-three proteins were predicted by SignalP to harbor the cleavable N-terminal signal peptides and were therefore secreted via the classical Sec pathway. Twenty-three proteins were predicted by SecretomeP for secretion by the alternative Sec pathway characterized by the lack of typical export signal. In contrast to SignalP and SecretomeP predictions, PSORT predicted 171 extracellular proteins, 7 cell wall-associated proteins, and 6 cytoplasmic proteins. Moreover, 51 proteins were predicted by LipoP to contain putative Sec signal peptides (38 have SpI sites), lipoprotein signal peptides (13 have SpII sites), and N-terminal membrane helices (9 have transmembrane helices). The TMHMM algorithm predicted 25 membrane-associated proteins with one to ten transmembrane helices.Immunogenic proteins were also identified using sera from patients who have recovered from anthrax. The charge variants (83 and 63 kDa) of protective antigen (PA) were the most immunodominant secreted antigens, followed by charge variants of enolase and transketolase.ConclusionThis is the first description of the time course of protein secretion for the pathogen Bacillus anthracis. Time course studies of protein secretion and accumulation may be relevant in elucidation of the progression of pathogenicity, identification of therapeutics and diagnostic markers, and vaccine development. This study also adds to the continuously growing list of identified Bacillus anthracis secretome proteins.

Sec-mediated transport of posttranslationally dehydrated peptides in Lactococcus lactis.

Nisin is a lanthionine-containing antimicrobial peptide produced by Lactococcus lactis. Its (methyl)lanthionines are introduced by two posttranslational enzymatic steps involving the dehydratase NisB, which dehydrates serine and threonine residues, and the cyclase NisC, which couples these dehydrated residues to cysteines, yielding thioether-bridged amino acids called lanthionines. The prenisin is subsequently exported by the ABC transporter NisT and extracellularly processed by the peptidase NisP. L. lactis expressing the nisBTC genes can modify and secrete a wide range of nonlantibiotic peptides. Here we demonstrate that in the absence of NisT and NisC, the Sec pathway of L. lactis can be exploited for the secretion of dehydrated variants of therapeutic peptides. Furthermore, posttranslational modifications by NisB and NisC still occur even when the nisin leader is preceded by a Sec signal peptide or a Tat signal peptide 27 or 44 amino acids long, respectively. However, transport of fully modified prenisin via the Sec pathway is impaired. The extent of NisB-mediated dehydration could be improved by raising the intracellular concentration NisB or by modulating the export efficiency through altering the signal sequence. These data demonstrate that besides the traditional lantibiotic transporter NisT, the Sec pathway with an established broad substrate range can be utilized for the improved export of lantibiotic enzyme-modified (poly)peptides.

Prediction of twin-arginine signal peptides

BackgroundProteins carrying twin-arginine (Tat) signal peptides are exported into the periplasmic compartment or extracellular environment independently of the classical Sec-dependent translocation pathway. To complement other methods for classical signal peptide prediction we here present a publicly available method, TatP, for prediction of bacterial Tat signal peptides.ResultsWe have retrieved sequence data for Tat substrates in order to train a computational method for discrimination of Sec and Tat signal peptides. The TatP method is able to positively classify 91% of 35 known Tat signal peptides and 84% of the annotated cleavage sites of these Tat signal peptides were correctly predicted. This method generates far less false positive predictions on various datasets than using simple pattern matching. Moreover, on the same datasets TatP generates less false positive predictions than a complementary rule based prediction method.ConclusionThe method developed here is able to discriminate Tat signal peptides from cytoplasmic proteins carrying a similar motif, as well as from Sec signal peptides, with high accuracy. The method allows filtering of input sequences based on Perl syntax regular expressions, whereas hydrophobicity discrimination of Tat- and Sec-signal peptides is carried out by an artificial neural network. A potential cleavage site of the predicted Tat signal peptide is also reported. The TatP prediction server is available as a public web server at .