Sec System Research Articles

Single molecule studies of the E.coli protein export system (997.9)

The general secretory or Sec system is the major route of export for proteins from the cytosol of Escherichia coli. The pathway through the membrane is provided by the translocon SecYEG. At the membrane, ATPase SecA binds SecYEG and drives protein translocation. In this work we shed light on Sec system using atomic force microscopy (AFM), a single molecule technique that is well suited for studying membrane proteins in near native conditions. AFM images provided direct visualizations of the dynamic loops of SecYEG which orchestrate critical translocon functions as well as determination of the oligomeric state of SecYEG in lipid bilayers. Additionally, we studied structure‐function relationships of SecA‐SecYEG complexes. Our results show that SecA binds SecYEG in liposomes in two distinct modes. When SecA was added extraneously to liposomes containing SecYEG, the proteins showed a wide distribution of heights with no prominent peaks, indicating that SecA populates multiple binding states. In contrast to this broad distribution of heights, the sample of proteoliposomes in which SecY and SecA were co‐assembled had a predominant species centered at 40 Å in height above the lipid bilayer. Because these proteoliposomes contain approximately sixfold more active translocons than do the proteoliposomes to which SecA was added after assembly, we conclude that this structural state represents SecA bound to translocons that have been rendered active. Taken together this work provides new functional and structural insights into the Sec system. More generally, the assays developed here are adaptable to other membrane protein systems.Grant Funding Source: NSF CAREER Award , Burroughs Wellcome Fund Career Award, NIH

Energy efficient electromagnetic actuated CVT system

A continuously variable transmission (CVT) system transmits the engine/battery power to the car driving wheel smoothly and efficiently. Several types of CVT already been developed to improve the transmission losses while maintaining acceleration time. However, most of the CVT has some constraints in the actuation mechanism which led us to develop an innovative electromagnetic actuator for CVT. Simplified mathematical equations have been developed for the kinematics analysis of clamping forces of the CVT and electromagnetic forces of EMA. The EMA has been developed for ¼ scale car with two sets of solenoid. Each of the two sets has been equipped with primary and secondary pulleys for pushing and pulling the movable sheave. The solenoid is operated by controlling the supply current with a fuzzy logic controller. A simulation based fuzzy logic controller has been introduced here for identifying the desired current of the EMA actuation. The experimental results show that the EMA develops electromagnetic forces 301 N for the supply current of 3.37 amp, which makes the acceleration time of the car in the range of 2.5∼3.5 sec and electromagnetic actuated CVT system highly energy efficient.

TheXanthomonasAx21 protein is processed by the general secretory system and is secreted in association with outer membrane vesicles

Pattern recognition receptors (PRRs) play an important role in detecting invading pathogens and mounting a robust defense response to restrict infection. In rice, one of the best characterized PRRs is XA21, a leucine rich repeat receptor-like kinase that confers broad-spectrum resistance to multiple strains of the bacterial pathogen Xanthomonas oryzae pv. oryzae (Xoo). In 2009 we reported that an Xoo protein, called Ax21, is secreted by a type I-secretion system and that it serves to activate XA21-mediated immunity. This report has recently been retracted. Here we present data that corrects our previous model. We first show that Ax21 secretion does not depend on the predicted type I secretion system and that it is processed by the general secretion (Sec) system. We further show that Ax21 is an outer membrane protein, secreted in association with outer membrane vesicles. Finally, we provide data showing that ax21 knockout strains do not overcome XA21-mediated immunity.

Membrane Chaperone SecDF Plays a Role in the Secretion of Listeria monocytogenes Major Virulence Factors

Listeria monocytogenes is a Gram-positive human intracellular pathogen that infects diverse mammalian cells. Upon invasion, L. monocytogenes secretes multiple virulence factors that target host cellular processes and promote infection. It has been presumed, but was not empirically established, that the Sec translocation system is the primary mediator of this secretion. Here, we validate an important role for SecDF, a component of the Sec system, in the secretion of several critical L. monocytogenes virulence factors. A ΔsecDF mutant is demonstrated to exhibit impaired membrane translocation of listeriolysin O (LLO), PlcA, PlcB, and ActA, factors that mediate L. monocytogenes phagosomal escape and spread from cell to cell. This impaired translocation was monitored by accumulation of the factors on the bacterial membrane and by reduced activity upon secretion. This defect in secretion is shown to be associated with a severe intracellular growth defect of the ΔsecDF mutant in macrophages and a less virulent phenotype in mice, despite normal growth in laboratory medium. We further show that SecDF is upregulated when the bacteria reside in macrophage phagosomes and that it is necessary for efficient phagosomal escape. Taken together, these data support the premise that SecDF plays a role as a chaperone that facilitates the translocation of L. monocytogenes virulence factors during infection.

Single-Molecule Studies of Bacterial Protein Translocation

In prokaryotes, a large share of the proteins are secreted from the cell through a process that requires their translocation across the cytoplasmic membrane. This process is mediated by the universally conserved Sec system with homologues in the endoplasmic reticulum and thylakoid membranes of eukaryotes. The Sec system also facilitates the membrane insertion of integral membrane proteins, an essential step along their folding pathway. In bacteria, the Sec system consists of the protein-conducting channel (SecYEG) that associates with soluble components, such as the motor protein SecA or translating ribosomes, and with integral membrane proteins, such as the heterotrimeric complex termed SecDFyajC and the YidC insertase. Over the past three decades, biochemical and structural studies have provided a comprehensive view of protein translocation, but the exact mechanistic details of this process remain to be resolved. For a number of other biomolecular systems, single-molecule biophysical analysis has efficiently complemented the conventional biochemical studies conducted in bulk, with high-sensitivity measurements probing the structure and dynamics of individual molecules in vitro and in vivo. Here, we review recent advances in studies of protein translocation employing single-molecule techniques with the aim of resolving molecular mechanisms, thereby providing a new and detailed view of the process.

Temperature-dependent size exclusion chromatography for the in situ investigation of dynamic bonding/debonding reactions

Polymers capable of dynamic bonding/debonding reactions are of great interest in modern day research. Potential applications can be found in the fields of self-healing materials or printable networks. Since temperature is often used as a stimulus for triggering reversible bonding reactions, an analysis operating at elevated temperatures is very useful for the in situ investigation of the reaction mechanism, as unwanted side effects can be minimized when performing the analyses at the same temperature at which the reactions occur. A temperature-dependent size exclusion chromatographic system (TD SEC) has been optimized for investigating the kinetics of retro Diels-Alder-based depolymerization of Diels-Alder polymers. The changing molecular weight distribution of the analyzed polymers during depolymerization gives valuable quantitative information on the kinetics of the reactions. Adequate data interpretation methods were developed for the correct evaluation of the chromatograms. The results are confirmed by high-temperature dynamic light scattering, thermogravimetric analysis, and time-resolved nuclear magnetic resonance spectroscopy at high temperatures. In addition, the SEC system and column material stability under application conditions were assessed using thermoanalysis methods, infrared spectroscopy, nitrogen physisorption, and scanning electron microscopy. The findings demonstrate that the system is stable and, thus, we can reliably characterize such dynamically bonding/debonding systems with TD SEC.

Leaving home ain't easy: protein export systems in Gram-positive bacteria

Transport of proteins into or across biological membranes is catalyzed by membrane-bound transport machineries. In Gram-positive bacteria, the vast majority of proteins are exported out of the cytosol by the conserved general secretion (Sec) system or, alternatively, by the twin-arginine translocation (Tat) system, that closely resemble their well-studied counterparts in Gram-negative bacteria. Besides these common major export routes, additional unique protein export systems (such as accessory Sec2 systems and/or type VII/WXG100 secretion systems) exist in some Gram-positive bacteria that are specifically involved in the secretion of limited subsets of proteins.

Breaking on through to the other side: protein export through the bacterial Sec system

More than one-third of cellular proteomes traffic into and across membranes. Bacteria have invented several sophisticated secretion systems that guide various proteins to extracytoplasmic locations and in some cases inject them directly into hosts. Of these, the Sec system is ubiquitous, essential and by far the best understood. Secretory polypeptides are sorted from cytoplasmic ones initially due to characteristic signal peptides. Then they are targeted to the plasma membrane by chaperones/pilots. The translocase, a dynamic nanomachine, lies at the centre of this process and acts as a protein-conducting channel with a unique property; allowing both forward transfer of secretory proteins but also lateral release into the lipid bilayer with high fidelity and efficiency. This process, tightly orchestrated at the expense of energy, ensures fundamental cell processes such as membrane biogenesis, cell division, motility, nutrient uptake and environmental sensing. In the present review, we examine this fascinating process, summarizing current knowledge on the structure, function and mechanics of the Sec pathway.

Differential Localization of the Streptococcal Accessory Sec Components and Implications for Substrate Export

The accessory Sec system of Streptococcus gordonii is comprised of SecY2, SecA2, and five proteins (Asp1 through -5) that are required for the export of a serine-rich glycoprotein, GspB. We have previously shown that a number of the Asps interact with GspB, SecA2, or each other. To further define the roles of these Asps in export, we examined their subcellular localization in S. gordonii and in Escherichia coli expressing the streptococcal accessory Sec system. In particular, we assessed how the locations of these accessory Sec proteins were altered by the presence of other components. Using fluorescence microscopy, we found in E. coli that SecA2 localized within multiple foci at the cell membrane, regardless of whether other accessory Sec proteins were expressed. Asp2 alone localized to the cell poles but formed a similar punctate pattern at the membrane when SecA2 was present. Asp1 and Asp3 localized diffusely in the cytosol when expressed alone or with SecA2. However, these proteins redistributed to the membrane in a punctate arrangement when all of the accessory Sec components were present. Cell fractionation studies with S. gordonii further corroborated these microscopy results. Collectively, these findings indicate that Asp1 to -3 are not integral membrane proteins that form structural parts of the translocation channel. Instead, SecA2 serves as a docking site for Asp2, which in turn attracts a complex of Asp1 and Asp3 to the membrane. These protein interactions may be important for the trafficking of GspB to the cell membrane and its subsequent translocation.

Biogenesis of β-barrel integral proteins of bacterial outer membrane

Gram-negative bacteria are enveloped by two membranes, the inner (cytoplasmic) (CM) and the outer (OM). The majority of integral outer membrane proteins are arranged in β-barrels of cylindrical shape composed of amphipathic antiparallel β-strands. In bacteria, β-barrel proteins function as water-filled pores, active transporters, enzymes, receptors, and structural proteins. Proteins of bacterial OM are synthesized in the cytoplasm as unfolded polypeptides with an N-terminal sequence that marks them for transport across the CM. Precursors of membrane proteins move through the aqueous medium of the cytosol and periplasm under the protection of chaperones (SecB, Skp, SurA, and DegP), then cross the CM via the Sec system composed of a polypeptide-conducting channel (SecYEG) and ATPase (SecA), the latter providing the energy for the translocation of the pre-protein. Pre-protein folding and incorporation in the OM require the participation of the Bam-complex, probably without the use of energy. This review summarizes current data on the biogenesis of the β-barrel proteins of bacterial OM. Data on the structure of the proteins included in the multicomponent system for delivery of the OM proteins to their destination in the cell and on their complexes with partners, including pre-proteins, are presented. Molecular models constructed on the basis of structural, genetic, and biochemical studies that describe the mechanisms of β-barrel protein assembly by this molecular transport machinery are also considered.

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 bc(1) and cytochrome b(6)f 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.

The Accessory Sec Protein Asp2 Modulates GlcNAc Deposition onto the Serine-Rich Repeat Glycoprotein GspB

The accessory Sec system is a specialized transport system that exports serine-rich repeat (SRR) glycoproteins of Gram-positive bacteria. This system contains two homologues of the general secretory (Sec) pathway (SecA2 and SecY2) and several other essential proteins (Asp1 to Asp5) that share no homology to proteins of known function. In Streptococcus gordonii, Asp2 is required for the transport of the SRR adhesin GspB, but its role in export is unknown. Tertiary structure predictions suggest that the carboxyl terminus of Asp2 resembles the catalytic region of numerous enzymes that function through a Ser-Asp-His catalytic triad. Sequence alignment of all Asp2 homologues identified a highly conserved pentapeptide motif (Gly-X-Ser(362)-X-Gly) typical of most Ser-Asp-His catalytic triads, where Ser forms the reactive residue. Site-directed mutagenesis of residues comprising the predicted catalytic triad of Asp2 of S. gordonii had no effect upon GspB transport but did result in a marked change in the electrophoretic mobility of the protein. Lectin-binding studies and monosaccharide content analysis of this altered glycoform revealed an increase in glucosamine deposition. Random mutagenesis of the Asp2 region containing this catalytic domain also disrupted GspB transport. Collectively, our findings suggest that Asp2 is a bifunctional protein that is essential for both GspB transport and correct glycosylation. The catalytic domain may be responsible for controlling the glycosylation of GspB, while other surrounding regions are functionally required for glycoprotein transport.

The Tat System for Membrane Translocation of Folded Proteins Recruits the Membrane-stabilizing Psp Machinery in Escherichia coli

Tat systems transport folded proteins across energized membranes of bacteria, archaea, and plant plastids. In Escherichia coli, TatBC complexes recognize the transported proteins, and TatA complexes are recruited to facilitate transport. We achieved an abstraction of TatA from membranes without use of detergents and observed a co-purification of PspA, a membrane-stress response protein. The N-terminal transmembrane domain of TatA was required for the interaction. Electron microscopy displayed TatA complexes in direct contact with PspA. PspB and PspC were important for the TatA-PspA contact. The activator protein PspF was not involved in the PspA-TatA interaction, demonstrating that basal levels of PspA already interact with TatA. Elevated TatA levels caused membrane stress that induced a strictly PspBC- and PspF-dependent up-regulation of PspA. TatA complexes were found to destabilize membranes under these conditions. At native TatA levels, PspA deficiency clearly affected anaerobic TMAO respiratory growth, suggesting that energetic costs for transport of large Tat substrates such as TMAO reductase can become growth limiting in the absence of PspA. The physiological role of PspA recruitment to TatA may therefore be the control of membrane stress at active translocons.

A Specific Interaction between SecA2 and a Region of the Preprotein Adjacent to the Signal Peptide Occurs during Transport via the Accessory Sec System

The accessory Sec systems of streptococci and staphylococci mediate the transport of a family of large, serine-rich glycoproteins to the bacterial cell surface. These systems are comprised of SecA2, SecY2, and three core accessory Sec proteins (Asp1-3). In Streptococcus gordonii, transport of the serine-rich glycoprotein GspB requires both a unique 90-residue N-terminal signal peptide and an adjacent 24-residue segment (the AST domain). We used in vivo site-specific photo-cross-linking to identify proteins that interact with the AST domain during transport. To facilitate this analysis, the entire accessory Sec system of S. gordonii was expressed in Escherichia coli. The determinants of GspB trafficking to the accessory Sec system in E. coli matched those in S. gordonii, establishing the validity of this approach. When the photo-cross-linker was placed within the AST domain, the preprotein was found to cross-link to SecA2. Importantly, no cross-linking to SecA was detected. Cross-linking of the N-terminal end of the AST domain to SecA2 occurred regardless of whether Asp1-3 were present. However, cross-linking to the C-terminal end was dependent on the Asps. The combined results indicate that full engagement of the AST domain by SecA2 is modulated by one or more of the Asps, and suggest that this process is important for initiating transport.

Broadening the Spectrum of β-Lactam Antibiotics through Inhibition of Signal Peptidase Type I

The resistance of methicillin-resistant Staphylococcus aureus (MRSA) to all β-lactam classes limits treatment options for serious infections involving this organism. Our goal is to discover new agents that restore the activity of β-lactams against MRSA, an approach that has led to the discovery of two classes of natural product antibiotics, a cyclic depsipeptide (krisynomycin) and a lipoglycopeptide (actinocarbasin), which potentiate the activity of imipenem against MRSA strain COL. We report here that these imipenem synergists are inhibitors of the bacterial type I signal peptidase SpsB, a serine protease that is required for the secretion of proteins that are exported through the Sec and Tat systems. A synthetic derivative of actinocarbasin, M131, synergized with imipenem both in vitro and in vivo with potent efficacy. The in vitro activity of M131 extends to clinical isolates of MRSA but not to a methicillin-sensitive strain. Synergy is restricted to β-lactam antibiotics and is not observed with other antibiotic classes. We propose that the SpsB inhibitors synergize with β-lactams by preventing the signal peptidase-mediated secretion of proteins required for β-lactam resistance. Combinations of SpsB inhibitors and β-lactams may expand the utility of these widely prescribed antibiotics to treat MRSA infections, analogous to β-lactamase inhibitors which restored the utility of this antibiotic class for the treatment of resistant Gram-negative infections.

Canonical SecA Associates with an Accessory Secretory Protein Complex Involved in Biogenesis of a Streptococcal Serine-Rich Repeat Glycoprotein

Fap1, a serine-rich repeat glycoprotein (SRRP), is required for bacterial biofilm formation of Streptococcus parasanguinis. Fap1-like SRRPs are found in many gram-positive bacteria and have been implicated in bacterial fitness and virulence. A conserved five-gene cluster, secY2-gap1-gap2-gap3-secA2, located immediately downstream of fap1, is required for Fap1 biogenesis. secA2, gap1, and gap3 encode three putative accessory Sec proteins. SecA2 mediates export of mature Fap1, and Gap1 and Gap3 are required for Fap1 biogenesis. Interestingly, gap1 and gap3 mutants exhibited the same phenotype as a secA2 mutant, implying that Gap1 and Gap3 may interact with SecA2 to mediate Fap1 biogenesis. Glutathione S-transferase pulldown experiments revealed a direct interaction between SecA2, Gap1, and Gap3 in vitro. Coimmunoprecipitation analysis demonstrated the formation of a SecA2-Gap1-Gap3 complex. Homologues of SecA2, Gap1, and Gap3 are conserved in many streptococci and staphylococci. The corresponding homologues from Streptococcus agalactiae also interacted with each other and formed a protein complex. Furthermore, the Gap1 homologues from S. agalactiae and Streptococcus sanguinis rescued the Fap1 defect in the Gap1 mutant, indicating the functional conservation of the accessory Sec complex. Importantly, canonical SecA interacted with the accessory Sec protein complex, suggesting that the biogenesis of SRRPs mediated by the accessory Sec system is linked to the canonical Sec system.

Analysis of branched polymers by high resolution multidetector size exclusion chromatography: Separation of the effects of branching and molecular weight distribution

AbstractThis article presents the SEC analysis of branched polyisobutylene PIB and polystyrene PS with high molecular weight and broad multimodal molecular weight distribution. Both polymers were synthesized using an inimer technique, which results in long‐chain branched polymers with statistical branching and broad multimodal distributions. Using high resolution multidetector Size Exclusion Chromatography SEC the polymers were analyzed based on three branching factors: g = (Rz,br/Rz,lin)Mw; h = (〈Rh〉z,br/〈Rh〉z,lin)Mw ; and ρ = (R 1/2/〈Rh〉z). It is generally accepted that for monodisperse branched polymers g and h < 1. In the case of our polydisperse PIB and PS, it was seen that g and h > 1, and ρ increases with molar mass and the number of chain ends as predicted earlier. The multidetector SEC system allowed for the separation of branching and polydispersity, reported here for the first time experimentally. The g parameter as a function of DPi was compared to the theory developed by Zimm and Stockmayer. The plots followed a similar trend, but were shifted by a factor related to the average chain length between branching points. The ρ parameter decreased with increasing DPi, as predicted theoretically by Kajiwara. © 2011 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem, 2012

Genetic organization and molecular characterization of secA2 locus in Listeria species

The translocation of proteins across the bacterial cell wall is carried out by the general secretory (Sec) system. Most bacteria have a single copy of the secA gene, with the exception of a few Gram-positive bacteria, which have an additional copy of secA, designated secA2. secA2 is present in Listeria monocytogenes and is responsible for secretion and translocation of several proteins including virulence factors; however, little is known about the secA2 gene and its genetic organization in nonpathogenic members of the genus Listeria. The goal of this study was to determine the presence of secA2 locus and analyze the genetic relatedness among pathogenic and nonpathogenic Listeria species. Cloning experiments revealed that secA2 is present in all analyzed pathogenic (L. monocytogenes and L. ivanovii) and nonpathogenic (L. welshimeri, L. innocua, L. seeligeri, L. grayi and L. marthii) Listeria species except L. rocourtiae. Likewise, SecA2 transcripts were also detected in all species. Sequence analysis further revealed that 2331 nucleotides (776 amino acids) are conserved in L. monocytogenes, L. welshimeri, L. innocua and L. marthii. Three nucleotides are deleted in L. ivanovii and L. seeligeri and six in L. grayi, resulting in amino acid counts of 775, 775 and 774, respectively. secA2 is flanked upstream by iap (encoding p60) and downstream by a putative membrane protein (lmo0583, lmo f2365_0613) in all analyzed Listeria species, demonstrating conserved genetic organization of the secA2 locus in pathogenic and nonpathogenic species. Deletion of secA2 in L. innocua impaired accumulation of SecA2 substrate, N-acetyl muramidase (NamA) in the cell wall, providing evidence for the presence of functional SecA2 in nonpathogenic Listeria.

SSPred: A prediction server based on SVM for the identification and classification of proteins involved in bacterial secretion systems

Protein secretion systems used by almost all bacteria are highly significant for the normal existence and interaction of bacteria with their host. The accumulation of genome sequence data in past few years has provided great insights into the distribution and function of these secretion systems. In this study, a support vector machine (SVM)- based method, SSPred was developed for the automated functional annotation of proteins involved in secretion systems further classifying them into five major sub-types (Type-I, Type-II, Type-III, Type-IV and Sec systems). The dataset used in this study for training and testing was obtained from KEGG and SwissProt database and was curated in order to avoid redundancy. To overcome the problem of imbalance in positive and negative dataset, an ensemble of SVM modules, each trained on a balanced subset of the training data were used. Firstly, protein sequence features like amino-acid composition (AAC), dipeptide composition (DPC) and physico-chemical composition (PCC) were used to develop the SVM-based modules that achieved an average accuracy of 84%, 85.17% and 82.59%, respectively. Secondly, a hybrid module (hybrid-I) integrating all the previously used features was developed that achieved an average accuracy of 86.12%. Another hybrid module (hybrid-II) developed using evolutionary information of a protein sequence extracted from position-specific scoring matrix and amino-acid composition achieved a maximum average accuracy of 89.73%. On unbiased evaluation using an independent data set, SSPred showed good prediction performance in identification and classification of secretion systems. SSPred is a freely available World Wide Web server at http//www.bioinformatics.org/sspred.

Clostridium difficile Has Two Parallel and Essential Sec Secretion Systems

Protein translocation across the cytoplasmic membrane is an essential process in all bacteria. The Sec system, comprising at its core an ATPase, SecA, and a membrane channel, SecYEG, is responsible for the majority of this protein transport. Recently, a second parallel Sec system has been described in a number of gram-positive species. This accessory Sec system is characterized by the presence of a second copy of the energizing ATPase, SecA2; where it has been studied, SecA2 is responsible for the translocation of a subset of Sec substrates. In common with many pathogenic gram-positive species, Clostridium difficile possesses two copies of SecA. Here, we describe the first characterization of the C. difficile accessory Sec system and the identification of its major substrates. Using inducible antisense RNA expression and dominant-negative alleles of secA1 and secA2, we demonstrate that export of the S-layer proteins (SLPs) and an additional cell wall protein (CwpV) is dependent on SecA2. Accumulation of the cytoplasmic precursor of the SLPs SlpA and other cell wall proteins was observed in cells expressing dominant-negative secA1 or secA2 alleles, concomitant with a decrease in the levels of mature SLPs in the cell wall. Furthermore, expression of either dominant-negative allele or antisense RNA knockdown of SecA1 or SecA2 dramatically impaired growth, indicating that both Sec systems are essential in C. difficile.