Protein secretion and membrane insertion systems in gram-negative bacteria

In contrast to other organisms, gram-negative bacteria have evolved numerous systems for protein export. Eight types are known that mediate export across or insertion into the cytoplasmic membrane, while eight specifically mediate export across or insertion into the outer membrane. Three of the former secretory pathway (SP) systems, type I SP (ISP, ABC), IIISP (Fla/Path) and IVSP (Conj/Vir), can export proteins across both membranes in a single energy-coupled step. A fourth generalized mechanism for exporting proteins across the two-membrane envelope in two distinct steps (which we here refer to as type II secretory pathways [IISP]) utilizes either the general secretory pathway (GSP or Sec) or the twin-arginine targeting translocase for translocation across the inner membrane, and either the main terminal branch or one of several protein-specific export systems for translocation across the outer membrane. We here survey the various well-characterized protein translocation systems found in living organisms and then focus on the systems present in gram-negative bacteria. Comparisons between these systems suggest specific biogenic, mechanistic and evolutionary similarities as well as major differences.     

细菌脂肪酶分泌的研究进展

细菌脂肪酶是一类重要的工业用酶,其分泌系统有着严谨的机制。革兰阳性细菌利用Sec-转运系统使脂肪酶跨过质膜完成分泌;革兰氏阴性细菌的外泌蛋白通过Sec-转运系统、Tat-转运系统或其他机制跨越内膜后,还必须利用Ⅰ型、Ⅱ型、Ⅲ型、Ⅳ型与Ⅴ型分泌系统来完成跨外膜分泌。详细介绍细菌脂肪酶分泌主要依赖的Sec-或Tat-跨内膜的转运系统及革兰氏阴性细菌的Ⅰ型、Ⅱ型与Ⅴ型自分泌系统的3种不同分泌方式。细菌脂肪酶分泌的研究对人们认识其分泌机制,并利用基因工程的手段提高其外泌产量等具有重要的指导意义。     

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

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

Requirement for YaeT in the outer membrane assembly of autotransporter proteins

Autotransporters constitute the largest group of secreted proteins in gram-negative bacteria. Autotransporter secretion involves the insertion of a carboxy-terminal beta barrel into and the translocation of an amino-terminal domain across the outer membrane. Here, we demonstrate that secretion of autotransporters from several organisms requires the outer membrane assembly factor YaeT.     

Two-partner secretion of gram-negative bacteria: a single β-barrel protein enables transport across the outer membrane

The mechanisms of protein secretion by pathogenic bacteria remain poorly understood. In gram-negative bacteria, the two-partner secretion pathway exports large, mostly virulence-related "TpsA" proteins across the outer membrane via their dedicated "TpsB" transporters. TpsB transporters belong to the ubiquitous Omp85 superfamily, whose members are involved in protein translocation across, or integration into, cellular membranes. The filamentous hemagglutinin/FhaC pair of Bordetella pertussis is a model two-partner secretion system. We have reconstituted the TpsB transporter FhaC into proteoliposomes and demonstrate that FhaC is the sole outer membrane protein required for translocation of its cognate TpsA protein. This is the first in vitro system for analyzing protein secretion across the outer membrane of gram-negative bacteria. Our data also provide clear evidence for the protein translocation function of Omp85 transporters.     

Export of beta-lactamase is independent of the signal recognition particle

In Escherichia coli, three different types of proteins engage the SecY translocon of the inner bacterial membrane for translocation or insertion: 1) polytopic membrane proteins that prior to their insertion into the membrane are targeted to the translocon using the bacterial signal recognition particle (SRP) and its receptor; 2) secretory proteins that are targeted to and translocated across the SecY translocon in a SecA- and SecB-dependent reaction; and 3) membrane proteins with large periplasmic domains, requiring SRP for targeting and SecA for the translocation of the periplasmic moiety. In addition to its role as a targeting device for membrane proteins, a function of the bacterial SRP in the export of SecB-independent secretory proteins has also been postulated. In particular, beta-lactamase, a hydrolytic enzyme responsible for cleavage of the beta-lactam ring containing antibiotics, is considered to be recognized and targeted by SRP. To examine the role of the SRP pathway in beta-lactamase targeting and export, we performed a detailed in vitro analysis. Chemical cross-linking and membrane binding assays did not reveal any significant interaction between SRP and beta-lactamase nascent chains. More importantly, membrane vesicles prepared from mutants lacking a functional SRP pathway did block the integration of SRP-dependent membrane proteins but supported the export of beta-lactamase in the same way as that of the SRP-independent protein OmpA. These data demonstrate that in contrast to previous results, the bacterial SRP is not involved in the export of beta-lactamase and further suggest that secretory proteins of Gram-negative bacteria in general are not substrates of SRP.     

The complete general secretory pathway in gram-negative bacteria.

The unifying feature of all proteins that are transported out of the cytoplasm of gram-negative bacteria by the general secretory pathway (GSP) is the presence of a long stretch of predominantly hydrophobic amino acids, the signal sequence. The interaction between signal sequence-bearing proteins and the cytoplasmic membrane may be a spontaneous event driven by the electrochemical energy potential across the cytoplasmic membrane, leading to membrane integration. The translocation of large, hydrophilic polypeptide segments to the periplasmic side of this membrane almost always requires at least six different proteins encoded by the sec genes and is dependent on both ATP hydrolysis and the electrochemical energy potential. Signal peptidases process precursors with a single, amino-terminal signal sequence, allowing them to be released into the periplasm, where they may remain or whence they may be inserted into the outer membrane. Selected proteins may also be transported across this membrane for assembly into cell surface appendages or for release into the extracellular medium. Many bacteria secrete a variety of structurally different proteins by a common pathway, referred to here as the main terminal branch of the GSP. This recently discovered branch pathway comprises at least 14 gene products. Other, simpler terminal branches of the GSP are also used by gram-negative bacteria to secrete a more limited range of extracellular proteins.     

The net charge of the first 18 residues of the mature sequence affects protein translocation across the cytoplasmic membrane of gram-negative bacteria

This statistical study shows that in proteins of gram-negative bacteria exported by the Sec-dependent pathway, the first 14 to 18 residues of the mature sequences have the highest deviation between the observed and expected net charge distributions. Moreover, almost all sequences have either neutral or negative net charge in this region. This rule is restricted to gram-negative bacteria, since neither eukaryotic nor gram-positive bacterial exported proteins have this charge bias. Subsequent experiments performed with a series of Escherichia coli alkaline phosphatase mutants confirmed that this charge bias is associated with protein translocation across the cytoplasmic membrane. Two consecutive basic residues inhibit translocation effectively when placed within the first 14 residues of the mature protein but not when placed in positions 19 and 20. The sensitivity to arginine partially reappeared again 30 residues away from the signal sequence. These data provide new insight into the mechanism of protein export in gram-negative bacteria and lead to practical recommendations for successful secretion of hybrid proteins.     

Channel-tunnels: outer membrane components of type I secretion systems and multidrug efflux pumps of Gram-negative bacteria

For translocation across the cell envelope of Gram-negative bacteria, substances have to overcome two permeability barriers, the inner and outer membrane. Channel-tunnels are outer membrane proteins, which are central to two distinct export systems: the type I secretion system exporting proteins such as toxins or proteases, and efflux pumps discharging antibiotics, dyes, or heavy metals and thus mediating drug resistance. Protein secretion is driven by an inner membrane ATP-binding cassette (ABC) transporter while drug efflux occurs via an inner membrane proton antiporter. Both inner membrane transporters are associated with a periplasmic accessory protein that recruits an outer membrane channel-tunnel to form a functional export complex. Prototypes of these export systems are the hemolysin secretion system and the AcrAB/TolC drug efflux pump of Escherichia coli, which both employ TolC as an outer membrane component. Its remarkable conduit-like structure, protruding 100 ? into the periplasmic space, reveals how both systems are capable of transporting substrates across both membranes directly from the cytosol into the external environment. Proteins of the channel-tunnel family are widespread within Gram-negative bacteria. Their involvement in drug resistance and in secretion of pathogenic factors makes them an interesting system for further studies. Understanding the mechanism of the different export apparatus could help to develop new drugs, which block the efflux pumps or the secretion system. Electronic Publication     

Transport and proofreading of proteins by the twin-arginine translocation (Tat) system in bacteria

The twin-arginine translocation (Tat) system operates in plant thylakoid membranes and the plasma membranes of most free-living bacteria. In bacteria, it is responsible for the export of a number of proteins to the periplasm, outer membrane or growth medium, selecting substrates by virtue of cleavable N-terminal signal peptides that contain a key twin-arginine motif together with other determinants. Its most notable attribute is its ability to transport large folded proteins (even oligomeric proteins) across the tightly sealed plasma membrane. In Gram-negative bacteria, TatABC subunits appear to carry out all of the essential translocation functions in the form of two distinct complexes at steady state: a TatABC substrate-binding complex and separate TatA complex. Several studies favour a model in which these complexes transiently coalesce to generate the full translocase. Most Gram-positive organisms possess an even simpler "minimalist" Tat system which lacks a TatB component and contains, instead, a bifunctional TatA component. These Tat systems may involve the operation of a TatAC complex together with a separate TatA complex, although a radically different model for TatAC-type systems has also been proposed. While bacterial Tat systems appear to require the presence of only a few proteins for the actual translocation event, there is increasing evidence for the operation of ancillary components that carry out sophisticated "proofreading" activities. These activities ensure that redox proteins are only exported after full assembly of the cofactor, thereby avoiding the futile export of apo-forms. This article is part of a Special Issue entitled Protein translocation across or insertion into membranes.     

Protein secretion in Bacillus species.

Bacilli secrete numerous proteins into the environment. Many of the secretory proteins, their export signals, and their processing steps during secretion have been characterized in detail. In contrast, the molecular mechanisms of protein secretion have been relatively poorly characterized. However, several components of the protein secretion machinery have been identified and cloned recently, which is likely to lead to rapid expansion of the knowledge of the protein secretion mechanism in Bacillus species. Comparison of the presently known export components of Bacillus species with those of Escherichia coli suggests that the mechanism of protein translocation across the cytoplasmic membrane is conserved among gram-negative and gram-positive bacteria differences are found in steps preceding and following the translocation process. Many of the secretory proteins of bacilli are produced industrially, but several problems have been encountered in the production of Bacillus heterologous secretory proteins. In the final section we discuss these problems and point out some possibilities to overcome them.     

Secretion by Pseudomonas aeruginosa: fate of a cloned gram-positive lipoprotein deletion mutant.

In gram-positive organisms, glyceride-cysteine thioether lipoproteins are frequently associated with secretion. They constitute membrane-bound forms retained by the cell but releasable late in growth phase. Most gram-negative organisms secrete very few proteins to the culture fluid; thioether lipoproteins in such organisms, typified by the enteric bacterium Escherichia coli, are integral outer membrane components for the most part. Unusual among gram-negative organisms, however, are Pseudomonas strains, known for extracellular export of a number of proteins. To examine whether a fundamental difference exists between the processing of lipoproteins in Pseudomonas strains and in nonsecretory gram-negative organisms, we examined the fate in Pseudomonas aeruginosa and E. coli of a cloned gram-positive secretory lipoprotein, Bacillus licheniformis penicillinase. A nonlipoprotein deletion mutant of the same gene was also examined in P. aeruginosa, and its processing was compared with that in E. coli. No important differences were found between P. aeruginosa and E. coli for either the lipoprotein or its deletion mutant. Thus, the contrast in secretory abilities of the two organisms does not appear to result from a difference in their general secretory systems.     

A universal system for the transport of redox proteins: early roots and latest developments

The transport of proteins binding redox cofactors across a biological membrane is complicated by the fact that insertion of the redox cofactor is often a cytoplasmic process. These cytoplasmically assembled redox proteins must thus be transported in partially or completely folded form. The need for a special transport system for redox proteins was first recognized for periplasmic hydrogenases in gram-negative bacteria. These enzymes, which catalyze the reaction H2 <--> 2H+ + 2e, are composed of a large and a small subunit. Only the small subunit has an unusually long signal sequence of 30-50 amino acid residues, characterized by a conserved motif (S/T)-R-R-x-F-L-K at the N-terminus. This sequence directs export of the large and small subunit complex to the periplasm. Sequencing of microbial genes and genomes has shown that signal sequences with this conserved motif, now referred to as twin-arginine leaders, occur ubiquitously and export different classes of redox proteins, containing iron sulfur clusters, molybdopterin cofactors, polynuclear copper sites or flavin adenine dinucleotide. Mutations in an Escherichia coli operon referred to as mtt (membrane targeting and translocation) or tat (twin arginine translocation) are pleiotropic, i.e. these prevent the expression of a variety of periplasmic oxido-reductases in functional form. The Mtt or Tat pathway is distinct from the well-known Sec pathway and occurs ubiquitously in prokaryotes. The fact that its component proteins share sequence homology with proteins of the delta pH pathway for protein transport associated with chloroplast thylakoid assembly, illustrates the universal nature of this novel protein translocation system.     

Protein secretion in the absence of ATP: the autotransporter, two-partner secretion and chaperone/usher pathways of gram-negative bacteria (review)

Bacteria secrete a wide variety of proteins, many of which play important roles in virulence. In gram-negative bacteria, these proteins must cross the cytoplasmic or inner membrane, periplasm, and outer membrane to reach the cell surface. Gram-negative bacteria have evolved multiple pathways to allow protein secretion across their complex envelope. ATP is not available in the periplasm and many of these secretion pathways encode components that harness energy available at the inner membrane to drive secretion across the outer membrane. In contrast, the autotransporter, two-partner secretion and chaperone/usher pathways are comparatively simple systems that allow secretion across the outer membrane without the need for input of energy from the inner membrane. This review will present overviews of these 'self-sufficient' pathways, focusing on recent advances and secretion mechanisms. Similarities among the pathways and with other protein translocation mechanisms will be highlighted.     

Two regions of EpsL involved in species-specific protein-protein interactions with EpsE and EpsM of the general secretion pathway in Vibrio cholerae

Extracellular secretion of proteins via the type II or general secretion pathway in gram-negative bacteria requires the assistance of at least 12 gene products that are thought to form a complex apparatus through which secreted proteins are translocated. Although this apparatus is specifically required only for the outer membrane translocation step during transport across the bacterial cell envelope, it is believed to span both membranes. The EpsE, EpsL, and EpsM proteins of the type II apparatus in Vibrio cholerae are thought to form a trimolecular complex that is required to either control the opening and closing of the secretion pore or to transduce energy to the site of outer membrane translocation. EpsL is likely to play an important role in this relay by interacting with both the cytoplasmic EpsE protein and the cytoplasmic membrane protein EpsM, which is predominantly exposed on the periplasmic side of the membrane. We have now extended this model and mapped the separate regions within EpsL that contain the EpsE and EpsM binding domains. By taking advantage of the species specificity of the type II pathway, we have used chimeric proteins composed of EpsL and its homologue, ExeL, from Aeromonas hydrophila together with either EpsE or its Aeromonas homologue, ExeE, to complement the secretion defect in both epsL and exeL mutant strains. These studies have mapped the species-specific EpsE binding site to the N-terminal cytoplasmic region between residues 57 and 216 of EpsL. In addition, the species-specific EpsM binding site was mapped to the C-terminal half of EpsL by coimmunoprecipitation of EpsM with different EpsL-ExeL chimeras. This site is present in the region between amino acids 216 and 296, which contains the predicted membrane-spanning segment of EpsL.     

Two novel proteins in the mitochondrial outer membrane mediate beta-barrel protein assembly

Mitochondrial outer and inner membranes contain translocators that achieve protein translocation across and/or insertion into the membranes. Recent evidence has shown that mitochondrial beta-barrel protein assembly in the outer membrane requires specific translocator proteins in addition to the components of the general translocator complex in the outer membrane, the TOM40 complex. Here we report two novel mitochondrial outer membrane proteins in yeast, Tom13 and Tom38/Sam35, that mediate assembly of mitochondrial beta-barrel proteins, Tom40, and/or porin in the outer membrane. Depletion of Tom13 or Tom38/Sam35 affects assembly pathways of the beta-barrel proteins differently, suggesting that they mediate different steps of the complex assembly processes of beta-barrel proteins in the outer membrane.     

A new twist on an old pathway--accessory Sec [corrected] systems

The export of proteins from their site of synthesis in the cytoplasm across the inner membrane is an important aspect of bacterial physiology. Because the location of extracytoplasmic proteins is ideal for host-pathogen interactions, protein export is also important to bacterial virulence. In bacteria, there are conserved protein export systems that are responsible for the majority of protein export: the general secretion (Sec) pathway and the twin-arginine translocation pathway. In some bacteria, there are also specialized export systems dedicated to exporting specific subsets of proteins. In this review, we discuss a specialized export system that exists in some Gram-positive bacteria and mycobacteria - the accessory Sec system. The common element to the accessory Sec system is an accessory SecA protein called SecA2. Here we present our current understanding of accessory Sec systems in Streptococcus gordonii, Streptococcus parasanguinis, Mycobacterium smegmatis, Mycobacterium tuberculosis and Listeria monocytogenes, making an effort to highlight apparent similarities and differences between the systems. We also review the data showing that accessory Sec systems can contribute to bacterial virulence.     

Type V secretion: From biogenesis to biotechnology

The two membranes of Gram-negative bacteria contain protein machines that have a general function in their assembly. To interact with the extra-cellular milieu, Gram-negatives target proteins to their cell surface and beyond. Many specialized secretion systems have evolved with dedicated translocation machines that either span the entire cell envelope or localize to the outer membrane. The latter act in concert with inner-membrane transport systems (i.e. Sec or Tat). Secretion via the Type V secretion system follows a two-step mechanism that appears relatively simple. Proteins secreted via this pathway are important for the Gram-negative life-style, either as virulence factors for pathogens or by contributing to the survival of non-invasive environmental species. Furthermore, this system appears well suited for the secretion of biotechnologically relevant proteins. In this review we focus on the biogenesis and application of two Type V subtypes, the autotransporters and two-partner secretion (TPS) systems. For translocation across the outer membrane the autotransporters require the assistance of the Bam complex that also plays a generic role in the assembly of outer membrane proteins. The TPS systems do use a dedicated translocator, but this protein shows resemblance to BamA, the major component of the Bam complex. Interestingly, both the mechanistic and more applied studies on these systems have provided a better understanding of the secretion mechanism and the biogenesis of outer membrane proteins. This article is part of a Special Issue entitled: Protein trafficking and secretion in bacteria. Guest Editors: Anastassios Economou and Ross Dalbey.     

Channel properties of TpsB transporter FhaC point to two functional domains with a C-terminal protein-conducting pore

Integral outer membrane transporters of the Omp85/TpsB superfamily mediate the translocation of proteins across, or their integration into, the outer membranes of Gram-negative bacteria, chloroplasts, and mitochondria. The Bordetella pertussis FhaC/FHA couple serves as a model for the two-partner secretion pathway in Gram-negative bacteria, with the TpsB protein, FhaC, being the specific transporter of its TpsA partner, FHA, across the outer membrane. In this work, we have investigated the structure/function relationship of FhaC by analyzing the ion channel properties of the wild type protein and a collection of mutants with varied FHA secretion activities. We demonstrated that the channel is formed by the C-terminal two-thirds of FhaC most likely folding into a beta-barrel domain predicted to be conserved throughout the family. A C-proximal motif that represents the family signature appears essential for pore function. The N-terminal 200 residues of FhaC constitute a functionally distinct domain that modulates the pore properties and may participate in FHA recognition.     

Type I secretion and multidrug efflux: transport through the TolC channel-tunnel

The crystal structure of TolC from Escherichia coli was recently determined to 2.1-A resolution and shows a unique type of channel architecture: a 12-stranded beta-barrel spans the outer membrane and is attached to a long alpha-helical channel that penetrates far into the periplasm. The structure suggests a mechanism for its role in secretion of proteins and in efflux of toxic small molecules. The TolC export pathway is compared with several import pathways of gram-negative bacteria where the outer membrane protein structures are also known.