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.     

Asp2 and Asp3 interact directly with GspB, the export substrate of the Streptococcus gordonii accessory Sec System

GspB is a serine-rich glycoprotein adhesin of Streptococcus gordonii that is exported to the bacterial surface by the accessory Sec system. This dedicated export pathway is comprised of seven components (SecA2, SecY2, and five accessory Sec proteins [Asp1 to Asp5]). The latter proteins have no known homologs beyond the Asps of other species. Asp1 to Asp3 are absolutely required for export of the substrate GspB, but their roles in this process are unknown. Using copurification analysis and far-Western blotting, we found that Asp2 and Asp3 could individually bind the serine-rich repeat (SRR) domains of GspB. Deletion of both SRR regions of GspB led to a decrease in its export, suggesting that binding of the Asps to the SRR regions is important for GspB transport by the accessory Sec system. The Asps also bound a heterologous substrate for the accessory Sec system containing a slow-folding MalE variant, but they did not bind wild-type MalE. The combined results indicate that the Asps may recognize the export substrate through preferential interactions with its unstructured or unfolded regions. Glycosylation of the SRR domains on GspB prevented Asp binding, suggesting that binding of the Asps to the preprotein occurs prior to its full glycosylation. Together, these findings suggest that Asp2 and Asp3 are likely to function in part as chaperones in the early phase of GspB transport.     

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.     

Glycine residues in the hydrophobic core of the GspB signal sequence route export toward the accessory Sec pathway

The Streptococcus gordonii cell surface glycoprotein GspB mediates high-affinity binding to distinct sialylated carbohydrate structures on human platelets and salivary proteins. GspB is glycosylated in the cytoplasm of S. gordonii and is then transported to the cell surface via a dedicated transport system that includes the accessory Sec components SecA2 and SecY2. The means by which the GspB preprotein is selectively recognized by the accessory Sec system have not been characterized fully. GspB has a 90-residue amino-terminal signal sequence that displays a traditional tripartite structure, with an atypically long amino-terminal (N) region followed by hydrophobic (H) and cleavage regions. In this report, we investigate the relative importance of the N and H regions of the GspB signal peptide for trafficking of the preprotein. The results show that the extended N region does not prevent export by the canonical Sec system. Instead, three glycine residues in the H region not only are necessary for export via the accessory Sec pathway but also interfere with export via the canonical Sec route. Replacement of the H-region glycine residues with helix-promoting residues led to a decrease in the efficiency of SecA2-dependent transport of the preprotein and a simultaneous increase in SecA2-independent translocation. Thus, the hydrophobic core of the GspB signal sequence is responsible primarily for routing towards the accessory Sec system.     

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.     

Two additional components of the accessory sec system mediating export of the Streptococcus gordonii platelet-binding protein GspB

The gspB-secY2A2 locus of Streptococcus gordonii strain M99 encodes the platelet-binding glycoprotein GspB, along with proteins that mediate its glycosylation and export. We have identified two additional components of the accessory Sec system (Asp4 and Asp5) encoded just downstream of gtfB in the gspB-secY2A2 locus. These proteins are required for GspB export and for normal levels of platelet binding by M99. Asp4 and Asp5 may be functional homologues of SecE and SecG, respectively.     

Genes in the accessory sec locus of Streptococcus gordonii have three functionally distinct effects on the expression of the platelet-binding protein GspB

Platelet binding by Streptococcus gordonii strain M99 is strongly correlated with the expression of the large surface glycoprotein GspB. A 14 kb chromosomal region downstream of gspB was previously shown to be required for the expression of this protein. The region encodes SecA2 and SecY2, which are components of an accessory secretion system dedicated specifically to the export of GspB. The region also includes three genes (gly, nss and gtf) that encode proteins likely to function in carbohydrate metabolism, and four genes (orf1-4) that encode proteins of unknown function. In this report, we have investigated the role of these genes in GspB expression. We found that disruption of orf1, orf2 or orf3 resulted in a loss of GspB export and the intracellular accumulation of GspB. As they are apparently essential components of the accessory secretion system, these genes were renamed asp1-3 (for accessory secretory protein). In gtf and orf4 mutants, gspB was transcribed, but no GspB was detected. These results suggest that Gtf and Orf4 are required for the translation or for the stability of GspB. In contrast, gly and nss mutants were able to express and export GspB. However, disruption of these genes appeared to affect the carbohydrate composition of this glycoprotein. As asp1-3, gtf and orf4, but not gly and nss, are conserved in the accessory sec loci of several staphylococcal and streptococcal species, these genes may also have crucial roles in the expression and export of GspB homologues in the other Gram-positive bacteria.     

An accessory sec locus of Streptococcus gordonii is required for export of the surface protein GspB and for normal levels of binding to human platelets

The translocation of proteins across the bacterial cell membrane is carried out by highly conserved components of the Sec system. Most bacterial species have a single copy of the genes encoding SecA and SecY, which are essential for viability. However, Streptococcus gordonii strain M99 encodes SecA and SecY homologues that are not required for viability or for the translocation of most exported proteins. The genes (secA2 and secY2) reside in a region of the chromosome required for the export of GspB, a 286 kDa cell wall-anchored protein. Loss of GspB surface expression is associated with a significant reduction in the binding of M99 to human platelets, suggesting that it may be an adhesin. Genetic analyses indicate that M99 has a second, canonical SecA homologue that is essential for viability. At least two other Gram-positive species, Streptococcus pneumoniae and Staphylococcus aureus, encode two sets of SecA and SecY homologues. One set is more similar to SecA and SecY of Escherichia coli, whereas the other set is more similar to SecA2 and SecY2 of strain M99. The conserved organization of genes in the secY2-secA2 loci suggests that, in each of these Gram-positive species, SecA2 and SecY2 may constitute a specialized system for the transport of a very large serine-rich repeat protein.     

Direct identification of the site of binding on the chaperone SecB for the amino terminus of the translocon motor SecA

Protein export mediated by the general secretory Sec system in Escherichia coli proceeds by a dynamic transfer of a precursor polypeptide from the chaperone SecB to the SecA ATPase motor of the translocon and subsequently into and through the channel of the membrane‐embedded SecYEG heterotrimer. The complex between SecA and SecB is stabilized by several separate sites of contact. Here we have demonstrated directly an interaction between the N‐terminal residues 2 through 11 of SecA and the C‐terminal 13 residues of SecB by isothermal titration calorimetry and analytical sedimentation velocity centrifugation. We discuss the unusual binding properties of SecA and SecB in context of a model for transfer of the precursor along the pathway of export.     

Determinants of the streptococcal surface glycoprotein GspB that facilitate export by the accessory Sec system

GspB is a large cell-surface glycoprotein expressed by Streptococcus gordonii M99 that mediates binding of this organism to human platelets. This adhesin is glycosylated in the cytoplasm, and is then transported to the cell surface via an accessory Sec system. To assess the structural features of GspB that are needed for export, we examined the effects of altering the carbohydrate moieties or the polypeptide backbone of GspB. Truncated, glycosylated variants of GspB were exported exclusively via the accessory Sec pathway. When glycosylation was abolished, the GspB variants were still exported by this pathway, but minor amounts could also be transported by the canonical Sec system. GspB variants with in-frame insertions or deletions in the N-terminus were not secreted, indicating that this domain is necessary for export. However, the N-terminus is not sufficient for the transport of heterologous proteins, because C-terminal fusion of passenger proteins to this domain hindered export. In contrast, fusion of GspB to a canonical signal peptide resulted in the efficient export of non-glycosylated forms of the fusion protein via the canonical Sec pathway, whereas glycosylated forms could not be exported. Thus, the carbohydrate moieties and the atypical signal sequence of GspB interfere with export via the canonical pathway, and direct GspB towards the accessory Sec system.     

The Streptococcus gordonii platelet binding protein GspB undergoes glycosylation independently of export

The binding of bacteria and platelets may play a central role in the pathogenesis of infective endocarditis. Platelet binding by Streptococcus gordonii strain M99 is predominantly mediated by the 286-kDa cell wall-anchored protein GspB. This unusually large protein lacks a typical amino-terminal signal peptide and is translocated from the cytoplasm via a dedicated transport system. A 14-kb segment just downstream of gspB encodes SecA2 and SecY2, two components of the GspB-specific transport system. The downstream segment also encodes several putative glycosyl transferases that may be responsible for the posttranslational modification of GspB. In this study, we compared the abilities of M99 and two GspB(-) mutant strains to bind various lectins. GspB was found to have affinity for lectins that bind N-acetylglucosamine. We also examined variant forms of GspB that lack a carboxy-terminal cell wall-anchoring domain and thus are free of covalent linkage to cell wall peptidoglycan. Like native GspB, these truncated proteins appear to be heavily glycosylated, as evidenced by migration during sodium dodecyl sulfate-polyacrylamide gel electrophoresis with an apparent molecular mass >100 kDa in excess of the predicted mass, negligible staining with conventional protein stains, and reactivity with hydrazide following periodate oxidation. Furthermore, analysis of the carbohydrate associated with the GspB variants by high-pH anion-exchange chromatography revealed the presence of approximately 70 to 100 monosaccharide residues per GspB polypeptide (primarily N-acetylglucosamine and glucose). Analysis of GspB in protoplasts of secA2 or secY2 mutant strains, which do not export GspB, indicates that GspB is glycosylated in the cytoplasm of these strains. The combined data suggest that the native GspB is a glycoprotein and that it may be glycosylated prior to export.     

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.     

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.     

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.     

Characterization of the accessory Sec system of Staphylococcus aureus

The SraP adhesin of Staphylococcus aureus is a member of a highly conserved family of serine-rich surface glycoproteins of gram-positive bacteria. For streptococci, export of the SraP homologs requires a specialized transport pathway (the accessory Sec system). Compared to streptococci, however, SraP is predicted to differ in its signal peptide and glycosylation, which may affect its dependence on a specialized system for transport. In addition, two genes (asp4 and asp5) essential for export in Streptococcus gordonii are missing in S. aureus. Thus, the selectivity of the accessory Sec system in S. aureus may also differ compared to streptococci. To address these issues, the five genes encoding the putative accessory Sec system (secY2, secA2, and asp1-3) were disrupted individually in S. aureus ISP479C, and the resultant mutants were examined for SraP export. Disruption of secA2 resulted in the near complete loss of SraP surface expression. Similar results were seen with disruption of secY2 and asp1, asp2, or asp3. To assess whether the accessory Sec system transported other substrates, we compared secreted proteomes of ISP479C and a secA2 isogenic mutant, by two-dimensional fluorescence difference gel electrophoresis. Although two consistent differences in proteome content were noted between the strains, neither protein appeared to be a likely substrate for accessory Sec export. Thus, the accessory Sec system of S. aureus is required for the export of SraP, and it appears to be dedicated to the transport of this substrate exclusively.     

Selective transport by SecA2: An expanding family of customized motor proteins

The SecA2 proteins are a special class of transport-associated ATPases that are related to the SecA component of the general Sec system, and are found in an increasingly large number of Gram-positive bacterial species. The SecA2 substrates are typically linked to the cell wall, but may be lipid-linked, peptidoglycan-linked, or non-covalently associated S-layer proteins. These substrates can have a significant impact on virulence of pathogenic organisms, but may also aid colonization by commensals. The SecA2 orthologues range from being highly similar to their SecA paralogues, to being distinctly different in apparent structure and function. Two broad classes of SecA2 are evident. One transports multiple substrates, and may interact with the general Sec system, or with an as yet unidentified transmembrane channel. The second type transports a single substrate, and is a component of the accessory Sec system, which includes the SecY paralogue SecY2 along with the accessory Sec proteins Asp1-3. Recent studies indicate that the latter three proteins may have a unique role in coordinating post-translational modification of the substrate with transport by SecA2. Comparative functional and phylogenetic analyses suggest that each SecA2 may be uniquely adapted for a specific type of substrate. This article is part of a Special Issue entitled: Protein trafficking and secretion in bacteria. Guest Editors: Anastassios Economou and Ross Dalbey.     

<Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis> possesses two <Emphasis Type="Italic">secA</Emphasis> homologous genes that are essential for viability

SecA is a central component of the bacterial Sec preprotein translocase. Besides the housekeeping SecA (SecA1), some mostly pathogenic Gram-positive bacteria possess an accessory SecA (SecA2) that is involved in the export of a few substrates only. Here we show that neither of the two secA homologous genes present in the genome of the non-pathogenic bacterium Corynebacterium glutamicum can be deleted, unless a copy of the respective gene is provided in trans on a plasmid. This finding is in marked contrast to all other cases examined so far making C. glutamicum the first reported bacterium possessing two essential SecA proteins.     

Penetration into membrane of amino‐terminal region of SecA when associated with SecYEG in active complexes

The general secretory (Sec) system of Escherichia coli translocates both periplasmic and outer membrane proteins through the cytoplasmic membrane. The pathway through the membrane is provided by a highly conserved translocon, which in E. coli comprises two heterotrimeric integral membrane complexes, SecY, SecE, and SecG (SecYEG), and SecD, SecF, and YajC (SecDF/YajC). SecA is an associated ATPase that is essential to the function of the Sec system. SecA plays two roles, it targets precursors to the translocon with the help of SecB and it provides energy via hydrolysis of ATP. SecA exists both free in the cytoplasm and integrally membrane associated. Here we describe details of association of the amino‐terminal region of SecA with membrane. We use site‐directed spin labelling and electron paramagnetic resonance spectroscopy to show that when SecA is co‐assembled into lipids with SecYEG to yield highly active translocons, the N‐terminal region of SecA penetrates the membrane and lies at the interface between the polar and the hydrophobic regions, parallel to the plane of the membrane at a depth of approximately 5 Å. When SecA is bound to SecYEG, preassembled into proteoliposomes, or nonspecifically bound to lipids in the absence of SecYEG, the N‐terminal region penetrates more deeply (8 Å). Implications of partitioning of the SecA N‐terminal region into lipids on the complex between SecB carrying a precursor and SecA are discussed.     

Two Nonredundant SecA Homologues Function in Mycobacteria

The proper extracytoplasmic localization of proteins is an important aspect of mycobacterial physiology and the pathogenesis of Mycobacterium tuberculosis. The protein export systems of mycobacteria have remained unexplored. The Sec-dependent protein export pathway has been well characterized in Escherichia coli and is responsible for transport across the cytoplasmic membrane of proteins containing signal sequences at their amino termini. SecA is a central component of this pathway, and it is highly conserved throughout bacteria. Here we report on an unusual property of mycobacterial protein export--the presence of two homologues of SecA (SecA1 and SecA2). Using an allelic-exchange strategy in Mycobacterium smegmatis, we demonstrate that secA1 is an essential gene. In contrast, secA2 can be deleted and is the first example of a nonessential secA homologue. The essential nature of secA1, which is consistent with the conserved Sec pathway, leads us to believe that secA1 represents the equivalent of E. coli secA. The results of a phenotypic analysis of a Delta secA2 mutant of M. smegmatis are presented here and also indicate a role for SecA2 in protein export. Based on our study, it appears that SecA2 can assist SecA1 in the export of some proteins via the Sec pathway. However, SecA2 is not the functional equivalent of SecA1. This finding, in combination with the fact that SecA2 is highly conserved throughout mycobacteria, suggests a second role for SecA2. The possibility exists that another role for SecA2 is to export a specific subset of proteins.     

Suppressor Analysis Reveals a Role for SecY in the SecA2-Dependent Protein Export Pathway of Mycobacteria

All bacteria use the conserved Sec pathway to transport proteins across the cytoplasmic membrane, with the SecA ATPase playing a central role in the process. Mycobacteria are part of a small group of bacteria that have two SecA proteins: the canonical SecA (SecA1) and a second, specialized SecA (SecA2). The SecA2-dependent pathway exports a small subset of proteins and is required for Mycobacterium tuberculosis virulence. The mechanism by which SecA2 drives export of proteins across the cytoplasmic membrane remains poorly understood. Here we performed suppressor analysis on a dominant negative secA2 mutant (secA2 K129R) of the model mycobacterium Mycobacterium smegmatis to better understand the pathway used by SecA2 to export proteins. Two extragenic suppressor mutations were identified as mapping to the promoter region of secY, which encodes the central component of the canonical Sec export channel. These suppressor mutations increased secY expression, and this effect was sufficient to alleviate the secA2 K129R phenotype. We also discovered that the level of SecY protein was greatly diminished in the secA2 K129R mutant, but at least partially restored in the suppressors. Furthermore, the level of SecY in a suppressor strongly correlated with the degree of suppression. Our findings reveal a detrimental effect of SecA2 K129R on SecY, arguing for an integrated system in which SecA2 works with SecY and the canonical Sec translocase to export proteins.