This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Tjalsma, H. Articles by van Dijl, J. M. Search for Related Content PubMed PubMed Citation Articles by Tjalsma, H. Articles by van Dijl, J. M.

 Previous Article  |  Next Article 

Microbiology and Molecular Biology Reviews, June 2004, p. 207-233, Vol. 68, No. 2
1092-2172/04/$08.00+0     DOI: 10.1128/MMBR.68.2.207-233.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Proteomics of Protein Secretion by Bacillus subtilis: Separating the "Secrets" of the Secretome

Department of Genetics, Groningen Biomolecular Sciences and Biotechnology Institute, 9751 NN Haren,¹ Department of Clinical Chemistry/564, University Medical Centre Nijmegen, 6500 HB Nijmegen,² Department of Pharmaceutical Biology, University of Groningen, 9713 AV Groningen,⁴ Department of Molecular Bacteriology, University of Groningen, 9700 RB Groningen, The Netherlands,⁵ Institut für Mikrobiologie und Molekularbiologie, Ernst-Moritz-Arndt-Universiät Greifswald, D-17487 Greifswald, Germany³

SUMMARY

GENERAL INTRODUCTION

SCOPE OF THIS REVIEW: THE PROTEOMICS OF PROTEIN SECRETION BY B. SUBTILIS

PROTEIN SORTING IN B. SUBTILIS

Signal Peptides

Signal Peptide Prediction and Classification

Twin-arginine (RR/KR-type) signal peptides.

Secretory (Sec-type) signal peptides.

Lipoprotein signal peptides.

Pseudopilin-like signal peptides.

Signal peptides of pheromones and bacteriocins.

Retention Signals

Transmembrane domains.

Lipid modification.

Pseudopilin assembly.

Cell wall-binding repeats.

Covalent attachment to the cell wall.

PROTEOMICS OF PROTEIN SECRETION BY B. SUBTILIS

Extracellular Proteome of B. subtilis 168

Toward an Extracellular Zymoproteome of B. subtilis 168

Cell Wall Proteome of B. subtilis 168

Verification of Secretome Predictions

CONTRIBUTION OF THE Sec MACHINERY TO THE EXTRACELLULAR PROTEOME

Cytoplasmic Targeting Factors

Ffh depletion.

Sec Translocase

SecA limitation.

SecA inhibition by sodium azide.

SecDF deletion and SpoIIIJ and YqjG depletion.

Type I Signal Peptidases

SPase I deletions.

Lipoprotein Modification and Processing

SPase II deletion.

Lgt deletion.

Folding Catalysts

PrsA depletion.

Bdb mutations.

Quality Control Factors

Modulation of HtrA and HtrB levels.

WprA deletion.

Extracellular Proteases

CONTRIBUTION OF Sec-INDEPENDENT PROTEIN EXPORT TO THE EXTRACELLULAR PROTEOME

Twin-Arginine Translocation Machinery

TatC and total-Tat deletions.

MECHANISMS FOR EXTRACELLULAR ACCUMULATION OF PROTEINS

Protein Secretion via the Sec and Tat Pathways

Release of Membrane Proteins by Proteolysis

Release of Lipoproteins by Proteolytic Shaving and/or Shedding

Release of Cell Wall Proteins by Proteolytic Shaving and Cell Wall Turnover

Release of Extracellular Proteins without Typical Export Signals

EXTRACELLULAR PROTEOMES OF OTHER GRAM-POSITIVE BACTERIA

Bacillus cereus

Clostridium difficile

Staphylococcus aureus

Group A Streptococcus

Mycobacterium tuberculosis

PERSPECTIVES

ACKNOWLEDGMENTS

REFERENCES

Top Next References

GENERAL INTRODUCTION

Top Previous Next References

 
Protein export from the cytoplasm to destinations outside the cell is a phenomenon that takes place in all domains of life. Most bacterial proteins destined to leave the cytoplasm are exported via the highly conserved SecA-YEG (Sec) pathway. In addition, more specialized bacterial export pathways are used for the export of specific subsets of extracellular proteins. Most exported proteins are synthesized as precursors with an N-terminal signal peptide (151, 152). These preproteins are first recognized by soluble targeting factors for their transport to the translocation machinery in the cell membrane. Next, the polypeptide chain is transported through a proteinacious channel in the membrane, a process driven by a translocation motor that binds and hydrolyzes nucleotide triphosphates. Finally, the signal peptide is removed, resulting in the release of the mature protein from the membrane. The mature protein folds into its native conformation shortly after the release from the translocase, unless it is translocated in a folded state. These basic principles of protein transport across membranes apply to most eukaryotic and prokaryotic organisms (35, 93, 102, 111, 129).

SCOPE OF THIS REVIEW: THE PROTEOMICS OF PROTEIN SECRETION BY B. SUBTILIS

Top Previous Next References

 
Bacterial secretory proteins are known to perform several very important "remote-control" functions, such as the provision of nutrients, cell-to-cell communication, detoxification of the environment, and killing of potential competitors. More specifically, extracellular proteins of pathogenic bacteria seem to play critical roles in virulence (53, 59, 105). The fact that exported Bacillus subtilis proteins are not retained by an outer membrane, as encountered in gram-negative bacteria, makes this gram-positive bacterium a preferred organism for the proteomic analysis of protein secretion. In addition, the availability of the complete genome sequence (58) and about 3,000 "y"-mutants constructed within the Bacillus subtilis Functional Analysis program (54, 115) make B. subtilis an ideal model organism for research on gram-positive bacteria. Furthermore, previous studies have predicted the composition of the so-called secretome of B. subtilis, which, by our definition, includes both the secreted proteins and the protein secretion machinery (129). These predictions showed that at least four distinct pathways for protein export from the cytoplasm and approximately 300 proteins with the potential to be exported could be distinguished. By far the largest number of exported proteins was predicted to follow the major Sec pathway for protein secretion. In contrast, the recently identified twin-arginine translocation Tat pathway (51, 52), a pseudopilin export pathway for competence development, and pathways using ATP-binding cassette (ABC) transporters, can be regarded as special-purpose pathways through which only few proteins appear to be transported (Fig. 1) (129). In this review, we discuss the latest views of protein secretion by B. subtilis as obtained from recent proteomic studies that were aimed at defining the extracellular complement of the B. subtilis secretome. Using different growth conditions and mutant strains, about 200 extracellular proteins could be visualized by two-dimensional (2D) gel electrophoresis, of which almost 50% could be identified by mass spectrometry (3-6, 46, 51, 52). In summary, these studies showed that in addition to the known mechanisms for protein export, B. subtilis also makes use of alternative mechanisms to release proteins into the external environment. Furthermore, the proteomic data could be used to verify genome-wide predictions concerning the secretome. Even though the process of protein secretion by B. subtilis had been documented fairly well by more classical genetic and biochemical approaches (129, 145), various secretome secrets were unveiled by proteomic approaches. These include the apparent export of cytoplasmic proteins, processing of native membrane proteins by type I signal peptidases (SPases), and the release of normally cell-associated lipoproteins and cell wall proteins into the growth medium.

View larger version (37K): [in this window] [in a new window]   FIG. 1. Protein export pathways in B. subtilis. Ribosomally synthesized proteins can be sorted to various destinations depending on the presence (+SP) or absence (–SP) of an N-terminal signal peptide and specific retention signals. Proteins devoid of a signal peptide remain in the cytoplasm. Proteins that have to be retained at the extracytoplasmic side of the membrane can contain either a transmembrane segment (TM) or a lipid modification (+lipobox). They are exported via the Sec or Tat pathway. Pseudopilins are exported by the Com system. Proteins that need to be retained in the cell wall can be exported via either the Sec or Tat pathway. To be retained in the cell wall, the mature parts of these proteins contain cell wall-binding repeats (+CWB). Proteins can be secreted into the medium via the Sec or Tat pathway or by ABC transporters.

Top Previous Next References

Twin-arginine (RR/KR-type) signal peptides. Signal peptide predictions resulted in the identification of 180 potential substrates for type I SPases. A twin-arginine motif, containing at least three residues of the consensus sequence R/K-R-X-#-# (where # is a hydrophobic residue) was found in 44 of these signals (12 RR and 32 KR signal peptides; [51]). The presence of such twin-arginine motifs was initially interpreted as an indication that the corresponding preproteins could be directed into the Tat pathway for protein export, possibly in a Sec-independent manner. The predicted twin-arginine signal peptides with a consensus R-R-X-#-# motif have an average length of 36 amino acid residues. Thus, they are significantly longer than typical Sec-type signal peptides. This is mainly because the N-domains of these R-R-X-#-# containing signal peptides have an average length of 14 amino acid residues, almost twice as long as the N-domain of the regular (Sec-type) signals (Fig. 2). Furthermore, these N-domains contain, on average, more positively charged residues than do those of Sec-type signal peptides (129). In contrast, the average features of predicted twin-arginine signal peptides with a K-R-X-#-# motif are similar to those of Sec-type signal peptides (51, 52, 129).

View larger version (22K): [in this window] [in a new window]   FIG. 2. Classification of cleavable N-terminal signal peptides. On the basis of SPase cleavage sites and the export pathways via which the preproteins are exported, predicted signal peptides (129) were divided into five distinct classes: twin-arginine (RR/KR) signal peptides, secretory (Sec-type) signal peptides, lipoprotein signal peptides, pseudopilin-like signal peptides, and bacteriocin and pheromone signal peptides. The export pathways via which the preproteins are exported and the SPases responsible for their cleavage are indicated. Most signal peptides have a tripartite structure: a positively charged N-domain (N), containing lysine and/or arginine residues (indicated by +), a hydrophobic H-domain (H, indicated by a gray box), and a C-domain (C) that specifies the cleavage site for their specific SPase. The length of the signal peptides and their subdomains is drawn to the same scale. Furthermore, helix-breaking residues, mostly glycine or proline (G/P), in the H-domain of Sec-type signal peptides are indicated. These residues are, respectively, thought to facilitate loopwise membrane insertion and cleavage by SPase I (129). Finally, where appropriate, the most frequently occurring first amino acid of the mature protein (+1) is indicated.

Lipoprotein signal peptides. Lipoprotein signal peptide predictions resulted in the identification of 114 potential substrates for the lipoprotein-specific (type II) SPase (Lsp) (133). Signal peptides from lipoproteins have an average length of 19 residues. These are therefore considerably shorter than RR/KR- and Sec-type signal peptides. This is because both the N-domain (average of 4 residues) and the H-domain (average of 12 residues) are shorter than the corresponding domains in RR/KR- and Sec-type signal peptides. Furthermore, helix-breaking residues are not conserved in the H-region of lipoprotein signal peptides. The C-domain contains a so-called lipobox with the consensus sequence L-(A/S)-(A/G)-C. The invariable cysteine residue of the lipobox is the target for lipid modification and the first residue of the mature lipoprotein after cleavage by SPase II (Fig. 2) (129). In fact, this lipid modification is indispensable for signal peptide cleavage by SPase II. Finally, although some lipoprotein signal peptides contain an RR/KR motif (51), so far export of lipoproteins via the Tat pathway has not been reported.

Pseudopilin-like signal peptides. Only four proteins (ComGC, ComGD, ComGE, and ComGG) with pseudopilin signal peptides have been identified in B. subtilis (129). These pseudopilin signal peptides have an average length of 33 residues. Strikingly, the C-domain of pseudopilin signal peptides, with the consensus sequence K-G-F at positions –2 to +1 relative to the SPase cleavage site, is located between the N- and H-domains (Fig. 2). This is in line with the observation that the pseudopilin signal peptidase (ComC) acts at the cytoplasmic side of the membrane (64). In addition to processing, ComC is responsible for aminomethylation of the phenylalanine at position +1 relative to the cleavage site. Although pseudopilin signal peptides show structural similarity to the previously described signal peptides, pseudopilin precursors bypass the Tat and Sec pathways and are transported via the specific Com pathway (26, 27, 129).

Signal peptides of pheromones and bacteriocins. Pheromones and antimicrobial peptides form a distinct group of exported proteins with cleavable N-terminal signal peptides, often called leader peptides. These leader peptides consist of only N- and C-domains and completely lack a hydrophobic H-domain (Fig. 2). It has been described that parts of the mature protein are also required for export by a dedicated ABC transporter. Moreover, the leader peptide has an important function in the prevention of premature antimicrobial activity and is required for the posttranslational modification of lantibiotics (141, 144). The two known leader peptides of this type in B. subtilis 168 direct the secretion of sublancin 168 (89) and ComX (67). Like leader peptides of other lantibiotics (23, 84), the sublancin 168 leader peptide contains a double-glycine motif (GS) N-terminally of the SPase cleavage site. Interestingly, the ABC transporter SunT is likely to play a dual role in the secretion of sublancin 168 since it belongs to a class of ABC transporters that are responsible for both the removal of the leader peptide and the translocation of the mature lantibiotic across the cytoplasmic membrane (33). Although not documented, it seems likely that an ABC transporter is also responsible for the processing and secretion of the ComX pheromone. This pheromone is involved in the density-controlled onset of competence development, and, similar to sublancin 168, it is ribosomally synthesized as a precursor and modified before secretion (119).

Transmembrane domains. Membrane proteins with large extracytoplasmic domains are translocated across the membrane by the Sec or Tat machinery. Due to the presence of one or more transmembrane domains and the absence of an SPase cleavage site, such proteins remain anchored to the membrane. The N-terminal transmembrane domain with an N_in-C_out topology is regarded as an uncleaved signal peptide, and the absence of a proper SPase I cleavage site is regarded as a determinant for retention in the membrane. Furthermore, certain proteins containing cleavable N-terminal signal peptides contain additional transmembrane domains in their C terminus that can function as membrane anchors (5, 51, 129). It should be noted that proteins with predicted putative transmembrane domains were regarded as nonsecretory proteins in previous secretome predictions (129, 143).

Lipid modification. In Gram-positive bacteria, lipid modification of exported proteins can serve to retain these proteins at the extracytoplasmic membrane surface. This may explain why 32 lipoproteins of B. subtilis are homologues of periplasmic high-affinity substrate-binding proteins from gram-negative bacteria (136). Lipid-modified proteins are synthesized as prelipoproteins and have to be modified by the diacylglyceryl transferase (Lgt) (62) before the lipoprotein precursor can be processed by SPase II. The diacylglyceryl group, attached to the cysteine residue at position +1 of the mature lipoprotein, inserts into the lipid bilayer of the cytoplasmic membrane, preventing release of the protein into the environment. It is noteworthy that some lipoproteins, such as CtaC (12) and QoxA (5), contain transmembrane segments in addition to a lipoprotein signal peptide. In these cases, lipid modification seems to be required for optimal functionality rather than for cell retention.

Pseudopilin assembly. A specific class of exported B. subtilis proteins that remain attached to the cytoplasmic membrane consists of the above-mentioned pseudopilins ComGC, ComGD, ComGE, and ComGG. These proteins are required for the binding and uptake of exogenous DNA during genetic competence (34). These resemble type IV pilins of various gram-negative bacteria that are synthesized as precursors with cleavable signal peptides. After cleavage and modification, the hydrophobic H-domains represent the N termini of mature pseudopilins, which are thought to form pilin-like structures that are attached to the cytoplasmic membrane (98).

Cell wall-binding repeats. Several B. subtilis enzymes involved in cell wall turnover contain a variable number of repeated domains (129) in their noncatalytic C termini, which have affinity for components of the cell wall (41, 69, 100). These repeats are thought to direct enzymes for cell wall assembly and turnover to specific sites, where cell wall synthesis and/or hydrolysis take place, as was shown for Staphylococcus aureus (8, 9). The targeting to a specific location is most probably promoted by certain components of the cell wall, such as choline, which is a receptor for several cell wall proteins of Streptococcus pneumoniae (106, 109, 110).

Covalent attachment to the cell wall. A specific group of surface proteins from gram-positive organisms is covalently anchored to the cell wall via the C terminus (112, 113). Cell wall anchoring of a variety of surface proteins in S. aureus requires, in addition to an N-terminal signal peptide, a C-terminal cell wall sorting signal consisting of the so-called LPxTG motif, a C-terminal hydrophobic domain, and a positively charged tail (82, 83, 114). A specific transpeptidase, the sortase A (SrtA), is responsible for both cleavage of the cell wall sorting signal (between the Thr and Gly residues of the LPxTG motif) and covalent attachment of the carboxyl group of the Thr residue to the cell wall (137, 138). A second, and structurally related, C-terminal cell wall sorting signal in S. aureus, Bacillus halodurans, and Bacillus anthracis contains the NPQTN motif. This sorting signal is most probably cleaved between the Thr and Asn residues by sortase B (SrtB), a paralogue of SrtA (70). Two sortase homologues, YhcS and YwpE, were identified in B. subtilis, suggesting that sortase-like enzymes for the cleavage and cell wall linkage of surface proteins are present in B. subtilis. However, no exported B. subtilis proteins with LPxTG or NPQTN motifs were identified (129). This indicates either that B. subtilis does not use this cell wall retention mechanism or that YhcS and YwpE recognize a cell wall sorting signal with a different amino acid sequence.

PROTEOMICS OF PROTEIN SECRETION BY B. SUBTILIS

Top Previous Next References

 
The first proteomic approaches to define the extracellular complement of the secretome of B. subtilis 168 were made by Hirose et al. (46). In their study, cells were grown in minimal media with glucose, maltose, cellobiose, or starch. Extracellular proteins were separated by 2D polyacrylamide gel electrophoresis (2D PAGE) and identified by N-terminal sequencing. In subsequent studies by Antelmann et al. (3) and Jongbloed et al. (52), B. subtilis was grown under conditions of phosphate starvation, or in Luria-Bertani (LB) broth (4, 5, 6). Extracellular proteins separated by 2D PAGE were identified by matrix-assisted laser desorption ionization/time-of-flight (MALDI-TOF) mass spectrometry. The highest levels of protein secretion are usually observed when cells of B. subtilis are grown in rich media, in particular during the postexponential growth phase (see Fig. 3). Moreover, the relative amounts of most identified extracellular proteins were significantly increased during the postexponential growth phase (5). The importance of protein secretion during postexponential growth was highlighted by the fact that the extracellular levels of a subset of 13 degradative enzymes are strongly increased in the extracellular proteome of a B. subtilis degU32(hy) mutant (5, 61). Recent transcript profiling experiments (66) have confirmed that the genes encoding these degradative enzymes are indeed under the positive control of DegU-phosphate, causing their increased expression after the end of the exponential growth phase.

View larger version (134K): [in this window] [in a new window]   FIG. 3. Master gel for the extracellular proteome of B. subtilis 168. Cells of B. subtilis 168 were grown in LB broth, and extracellular proteins were harvested 1 h after entry into the stationary growth phase. After precipitation with trichloroacetic acid, the extracellular proteins were separated by 2D PAGE and stained with Sypro Ruby as described by Jongbloed et al. (51). The proteins identified by mass spectrometry and/or N-terminal amino acid sequencing are indicated on the gel and listed in Table 1. The extracellular proteins found specifically during growth in minimal media (i.e., YdhT, YflE, and GapA) (46) and those specifically found during growth in phosphate starvation medium (i.e., GlpQ, PhoA, PhoB, PhoD, PstS, YdhF, YcdH, and YrpE) (5, 6, 51, 52) cannot be seen on this gel.

Similarly, only about half of the identified cell wall proteins are predicted to be retained at this subcellular location, since Hag, the WprA-processing products CWBP23 and CWBP52, YqgA, and YwsB lack known cell wall-binding motifs. The last two proteins are, in fact, found exclusively in the cell wall, like the known cell wall-bound proteins LytB and LytC. This suggests that an as yet undefined cell wall retention signal is present in YqgA and YwsB. Conversely, the remarkable observation that four proteins with typical cell wall-binding domains (i.e., LytD, YocH, YvcE, and YwtD) are found extracellularly, but not cell wall bound, might indicate that the presence of a cell wall-binding repeat is not a guarantee for retention at this location. In this respect, it may be relevant that YwtD exclusively cleaves extracellular -polyglutamic acid whereas it cannot use cell wall peptidoglycan as a substrate (127). However, the possibilities that LytD, YocH, YvcE, and YwtD are not properly extracted from the wall with LiCl, or that these proteins are degraded during the extraction procedure, cannot be excluded.

CONTRIBUTION OF THE Sec MACHINERY TO THE EXTRACELLULAR PROTEOME

Top Previous Next References

 
Protein secretion via the Sec pathway in B. subtilis can be divided into three functional stages: targeting, translocation, and folding and release. The following components have known or predicted functions in these stages. Cytoplasmic chaperones, such as SRP/FtsY (47) and CsaA (75, 76), keep the precursors in a translocation competent state and facilitate their targeting to the translocase in the membrane. The translocation machinery consists of SecA (motor), SecYEG (pore), and SecDF. Possibly, YrbF and SpoIIIJ/YqjG are also part of this machinery (17, 129, 135, 145). During or shortly after translocation, the preprotein is cleaved by one of the five type I signal peptidases (SipS to SipW)(130) or lipid-modified by the diacylglyceryl transferase (Lgt) (62) and cleaved by the lipoprotein-specific signal peptidase (Lsp; 133, 136). SppA and TepA may be involved in the degradation of cleaved signal peptides (16). The folding of several secreted proteins depends on the activities of PrsA (55), BdbBCD (18, 71), and/or SpoIIIJ/YqjG (135). HtrA and HtrB (85), as well as WprA (68), are involved in the quality control of secretory proteins. Importantly, HtrA and HtrB have the potential to assist in the folding or, if folding is impossible, degradation of malfolded secretory proteins. A model for the function of these main components of the Sec machinery of B. subtilis is depicted in Fig. 4. Using proteomic approaches, the extracellular proteomes of B. subtilis mutants that are affected in different stages in protein secretion have been analyzed. In the following sections, we review the currently available proteome data concerning B. subtilis strains lacking, or depleted of, various components involved in Sec-dependent protein export. These data are summarized in Table 4.

View larger version (49K): [in this window] [in a new window]   FIG. 4. Components involved in Sec-dependent protein export in B. subtilis. Secretory proteins are ribosomally synthesized as precursor proteins with an N-terminal signal peptide (SP). Cytoplasmic chaperones, such as SRP/FtsY (47) and CsaA (75, 76), keep the precursors in a translocation-competent state and facilitate their targeting to the translocase in the membrane, consisting of SecA, SecY, SecE, SecG, and SecDF (17, 129). During or shortly after translocation, the preprotein is cleaved by one of the type I signal peptidases (SipS-W) (130) or lipid modified by the diacylglyceryl-transferase (Lgt) (62) and cleaved by the lipoprotein-specific signal peptidase (Lsp) (136). SppA and TepA may be involved in the degradation of cleaved signal peptides (16), whereas the folding of several secreted proteins depends on the activities of PrsA (55), BdbBC (18), and/or SpoIIIJ/YqjG (135). HtrA, HtrB (85), and WprA (68, 124) are involved in the quality control of secretory proteins. It should be noted that for reasons of simplicity, HtrAB are depicted in the cell wall, although HtrA is detected in both the membrane and the medium (5). On passage through the cell wall, the mature protein is released into the environment.

Ffh depletion. The effect of Ffh depletion on the composition of the extracellular proteome was studied by Hirose et al. (46), using a strain in which cellular Ffh levels were controlled by the isopropyl-ß-D-thiogalactopyranoside (IPTG)-inducible Pspac promoter. When this strain was grown in minimal medium without IPTG, 31 protein spots were missing and 5 spots were significantly reduced in intensity in the extracellular proteome compared to the case for the same strain grown with IPTG. Only the level of the cytoplasmic protein SodA was increased under these conditions. Of the proteins that were unaffected or only mildly affected by Ffh depletion, three were identified as Hag and GapA (both unaffected) and XkdG (mildly affected). The fact that the extracellular accumulation of the flagellin Hag is not affected by Ffh depletion is understandable since this protein is exported by a specific flagellin assembly pathway (48). In addition, GapA is a cytoplasmic protein (44) that is released into the medium by an unknown mechanism. Furthermore, XkdG is a prophage-related protein that is probably exported by a specific prophage PBSX-encoded holin system. These data suggest that flagellin assembly, release of certain cytoplasmic proteins, and export of certain phage-related proteins are not (strictly) SRP dependent. In contrast, all identified extracellular proteins with cleavable Sec-type signal peptides, Csn, Pel, PenP, TasA, WapA, WprA, XynD, YclQ, YlqB, YncM, YwtD, YxaK, and YxkC, were completely absent from the medium of Ffh-depleted cells. These data strongly suggest that signal peptide-dependent protein secretion via the Sec pathway is, directly or indirectly, SRP dependent. In this respect, it is important to consider the possibility that membrane proteins critical for protein translocation, such as certain components of the Sec translocase, could be inserted SRP dependently into the membrane. If so, SRP depletion would indirectly have a negative impact on the secretion of proteins with Sec-type signal peptides. Finally, YfnI and YflE are two paralogous transmembrane proteins whose C-terminal domain is released into the medium (5, 46). The release of YfnI was mildly affected on Ffh depletion, whereas the release of YflE was completely inhibited under these conditions. This might indicate that compared to protein secretion, lower levels of SRP are required for the insertion of certain transmembrane proteins into the cytoplasmic membrane.

SecA limitation. The effect of SecA limitation was studied by growing a strain with a temperature-sensitive SecA protein at 30°C (permissive temperature) or 42°C (nonpermissive temperature) in minimal medium (46). Of the 39 detected proteins in the medium of this strain grown at 30°C, 36 were completely absent from the medium of cells grown at 42°C (SecA limitation). Only three proteins were not affected by SecA limitation: the cytoplasmic proteins GapA and SodA, and the flagellin Hag. The latter finding suggests that flagellum assembly is both SRP (see the previous section) and SecA independent. Proteins that were completely absent from the medium of SecA-depleted cells included the signal peptide-containing proteins Csn, Pel, PenP, TasA, XynD, YclQ, YlqB, YncM, YwtD, YxaK, and YxkC, as well as the processing products of WapA and WprA. These proteins were also absent from the medium of Ffh-depleted cells (see the previous section). Furthermore, SecA limitation completely inhibited not only the release of the C-terminal domain of the transmembrane protein YflE but, unlike Ffh depletion, also that of the C-terminal domain of its paralogue YfnI. As expected, these data confirm that SecA is indispensable for protein secretion via the Sec pathway. Notably, the secretion of all identified secretory proteins with cleavable signal peptides depends on both SRP (see the previous section) and SecA, confirming the general view that SRP and the Sec machinery of B. subtilis cooperate in this process. Furthermore, these data indicate that the insertion and/or proteolytic processing of at least two transmembrane proteins does require the Sec pathway. Likewise, the provoked export inhibition by SecA limitation of the prophage-encoded protein XkdG, which lacks a typical signal peptide, may be caused by an impaired membrane insertion of certain components of the XkdG export pathway (e.g., holins [see Mechanisms for extracellular accumulation of proteins below). Thus, the fact that the export of YfnI and XkdG is unaffected or only mildly affected by Ffh depletion might indicate that at least some transmembrane proteins that are Sec-dependently inserted into the membrane can bypass the SRP pathway.

For E. coli, it was proposed that the SRP route facilitates primarily the cotranslational targeting of inner membrane proteins, which contain longer and more hydrophobic (uncleaved) signal peptides than do secretory proteins (11, 118, 140). Subsequent initial transmembrane domain insertion steps seem to be independent of SecA in E. coli (118). The fact that signal peptides of B. subtilis are, on average, longer and more hydrophobic than those of E. coli (129) might explain why the majority of secretory B. subtilis proteins are secreted in an SRP-dependent manner. Remarkably, the studies by Hirose et al. (46) suggest that the Sec-dependent insertion of transmembrane segments of some integral membrane proteins of B. subtilis could be rather SRP independent. Possibly, nascent chain-ribosome complexes can, in certain cases, dock directly onto the Sec translocase of B. subtilis without the aid of SRP. Thus, the process of membrane protein biogenesis in B. subtilis may be organized somewhat differently from the equivalent process in E. coli.

SecA inhibition by sodium azide. The proteomic studies by Hirose et al. (46) suggested that the secretion of the majority of extracellular proteins by B. subtilis is strongly SecA dependent. However, the use of a temperature-sensitive secA mutant strain, which stops growing and dies after a temperature upshift, might influence the results. Furthermore, only a limited number of extracellular proteins were detected, since this strain was grown in minimal medium. Therefore, Jongbloed et al. (51) used a different approach, which was based on the inhibition of SecA activity by sodium azide in cells grown in a rich medium. For this purpose, it was essential to study the secretion of de novo-synthesized proteins because, otherwise, the kinetic effects of sodium azide on protein secretion would be overshadowed by the large amounts of extracellular proteins that accumulate in the growth medium of the azide-treated strain. Thus, postexponentially growing B. subtilis cells were separated from the growth medium, and grown for 20 min in fresh medium with or without sodium azide. This procedure resulted in the visualization of extracellular proteins that normally accumulate in the growth medium at relatively high levels (51). Of the 26 identified de novo-synthesized proteins in the medium of untreated cells, protein spots belonging to LipA, WapA, YolA, YvcE, YweA, and YxaL were almost completely absent from the medium of cells grown in the presence of sodium azide. Furthermore, Csn and XynA were secreted at significantly reduced levels. Notably, all eight extracellular proteins that were affected by SecA inhibition contained N-terminal signal peptides (Table 1). In contrast, no significant effect of SecA inhibition was observed on the extracellular appearance of 18 other de novo-synthesized proteins. This group of proteins consisted of the flagellum-related proteins FliD and Hag; the cytoplasmic proteins RocF, YwjH, and KatA; the membrane proteins YflE and YfnI; the lipoproteins MntA, OppA, and YclQ; and the AbnA, AprE, YbdN, YlqB, YncM, YrpD, YwtD, and YxkC proteins, which are synthesized with typical Sec-type signal peptides. These data, obtained by proteomics, support the view that the secretion of different secretory proteins depends to different extents on the activity of SecA. Accordingly, previous research has shown a difference in the SecA requirements of the -amylase AmyE (requiring low levels of SecA activity) and the levansucrase SacB (requiring high levels of SecA activity) for secretion into the medium of B. subtilis (60). Notably, the signal peptides of SacB and AmyE are quite different (129, 143). The H-domain of the signal peptide of AmyE is longer than that of SacB (23 and 17 residues, respectively), and its overall hydrophobicity is higher (1.8 and 1.1 residues, respectively). It was therefore proposed that these differences in the H-domains could be responsible for the difference in SecA requirement of pre-AmyE and pre-SacB (60). Although AmyE and SacB were not detected in the proteomic analysis of SecA-dependent (i.e., azide-sensitive) protein secretion (51), these studies provided a good opportunity to evaluate the above-mentioned hypothesis. Primary amino acid sequence analysis showed, however, that the H-domains of the signal peptides of both azide-sensitive and azide-resistant secretory proteins have an average length of 22 residues and an average hydrophobicity between 1.5 and 1.6. Thus, it seems unlikely that the H-domain is the main determinant for the SecA requirement of a preprotein. In contrast, the N-domains of the signal peptides of the eight highly azide-sensitive secretory proteins are on average shorter (7 versus 11 residues) and more hydrophilic (–1.4 versus –1.1) than those of the eight azide-resistant secretory proteins. Nevertheless, the number of positively charged residues in the N-regions of signal peptides of azide-sensitive and azide-resistant secretory proteins did not significantly differ (3.6 on average). Although this finding might indicate that the N-domain of signal peptides is a determinant for the SecA requirement of a preprotein, a larger data set and site-directed mutagenesis approaches are needed to pinpoint the relevant features of signal peptides in relation to the extent of SecA requirement of the corresponding preprotein. It is conceivable, however, that specific properties of the mature parts of particular secretory preproteins are more important in determining their SecA requirement than are the properties of their signal peptides.

Another interesting observation from the azide inhibition experiments is that all three lipoproteins that are detectable in the extracellular proteome of untreated cells are present in equal or even larger amounts in the medium of cells treated with sodium azide. This suggests that the transport of lipoproteins via the Sec pathway requires less SecA activity than does the transport of secretory proteins. Furthermore, the insertion and release of the C-terminal domains of the transmembrane proteins YflE and YfnI do not seem to be affected by SecA inhibition with sodium azide. This is in marked contrast to the results obtained by Hirose et al. (46), who showed that the release of YflE, YfnI, and the lipoprotein YclQ into the medium of a temperature-sensitive secA mutant strain was completely blocked by SecA limitation. Similarly, the extracellular appearance of secretory proteins whose export was not (completely) inhibited by sodium azide (e.g., Csn, YwtD, YxkC, YncM and YlqB [51]) was completely blocked by SecA depletion (46). This shows that the secA mutation employed by Hirose et al. (46) is more effective in reducing the SecA translocation motor activity than is sodium azide.

SecDF deletion and SpoIIIJ and YqjG depletion. In addition to the heterotrimeric SecYEG subcomplex, the E. coli Sec machinery contains a second heterotrimeric subcomplex that is composed of the SecD, SecF, and YajC proteins. This second subcomplex is likely to form a part of the B. subtilis Sec machinery as well, although this has not been demonstrated experimentally. In B. subtilis, this complex would be composed of the SecDF protein (a natural fusion protein of SecD and SecF) and YrbF (a homologue of E. coli YajC). The precise role of SecDF-YajC in protein export is presently not clear, but a variety of possible functions have been proposed. These include (i) removal of cleaved signal peptides or transmembrane segments from the SecYEG translocation channel; (ii) release of translocated proteins from the translocation channel; (iii) regulation of SecA cycling; and (iv) prevention of preprotein backsliding (86). Unlike SecD and SecF of E. coli, SecDF of B. subtilis 168 was shown to have little impact on cell viability and protein export, at least under standard laboratory conditions (17). A secretion defect in a secDF mutant strain was observed only under conditions of high-level expression of secretory proteins, such as AmyQ of Bacillus amyloliquefaciens. Accordingly, the disruption of secDF had no detectable influence on the composition of the extracellular proteome (H. Antelmann, unpublished observations).

A final component that can associate with the Sec translocase of E. coli is the YidC protein, which is involved in the membrane insertion of newly synthesized membrane proteins (65, 108, 116). Interestingly, YidC seems to be linked to the SecYEG subcomplex of the translocase through the SecDF-YajC subcomplex (86). B. subtilis contains two homologues of YidC, known as SpollIJ and YqjG. Remarkably, the biogenesis of a variety of integral membrane proteins in B. subtilis is only mildly affected in cells depleted of both SpoIIIJ and YqjG (135). In contrast, the simultaneous removal of SpoIIIJ and YqjG has a severe impact on (as yet undefined) posttranslocational stages in the secretion of proteins, such as AmyQ, LipA, and E. coli PhoA (135). Unfortunately, proteomic studies with SpoIIIJ/YqjG-depleted cells turned out to be difficult, since the combined activities of these proteins are essential for cell viability. The extracellular proteomes of spoIIIJ and yqjG single mutants, which display no growth defects, revealed no significant changes compared to that of the parental strain (H. Antelmann and H. Tjalsma, unpublished observations).

SPase I deletions. The availability of proteomic techniques created a new opportunity to further investigate possible differences in the substrate specificities of the type I SPases of B. subtilis. Therefore, Antelmann et al. (5) analyzed the extracellular proteomes of single, double, triple, and quadruple SPase I mutants lacking sipS, sipT, sipU, sipV, or sipW or combinations thereof. Surprisingly, apart from the expected absence of TasA in the medium of a sipW mutant strain, no major differences in the extracellular protein patterns of these mutants were observed. This observation confirms the view that the presence of either SipS or SipT is sufficient for efficient precursor processing and that the type I SPases of B. subtilis have largely overlapping specificities (130). The only notable exception was the SipTV-dependent cleavage of the membrane protein YfnI. This observation was remarkable not only because YfnI is a polytopic membrane protein but also because the cleavage site is located 44 residues C-terminally of the fifth transmembrane segment of this protein. This suggests that despite its distant position relative to the transmembrane segment, the SPase I cleavage site of YfnI, and possibly that of the paralogous proteins YflE, YqgS, and YvgJ (46), is accessible to the catalytic sites of SipT and SipV at the extracytoplasmic membrane surface (5).

SPase II deletion. To explore the full impact of the absence of SPase II on the extracellular proteome, Antelmann et al. (5) analyzed the extracellular proteome of an lspA mutant strain. These studies showed that two abundant extracellular proteins of the parental strain, AmyE and YolA, were completely absent from the extracellular proteome of the lspA mutant (5). Furthermore, the relative amounts of a variety of other extracellular proteins were strongly reduced, as exemplified by the secretory proteins Csn, Epr, LipA, GlpQ, LytD, PenP, YncM, YrpD, YwoF, YxaK, and YxkC (Table 1) and the phage-related proteins XepA, XkdK, XkdM, and XlyA (Table 2). Unexpectedly, significantly increased levels of the two processed forms (CWBP23 and CWBP52) of the cell wall protease WprA were found in the medium of the lspA mutant strain. Similarly, the extracellular levels of two typical lipoproteins, MntA and YxeB, were strongly increased, showing that these proteins were not effectively retained in the membrane of the lspA mutant. In contrast, the extracellular levels of the lipoproteins OppA and YclQ were not affected by the lspA mutation. Taken together, these findings show that the absence of SPase II has rather pleiotropic effects on the composition of the extracellular proteome.

Lgt deletion. Analysis of the extracellular proteome of B. subtilis 168 showed that at least nine different potential lipoproteins are released into the medium (Table 1); six of these can be observed when cells are grown in LB medium (MntA, OppA, YclQ, YfmC, YqiX, and YrpE), and five are present when cells are grown in phosphate starvation medium (PstS, YcdH, YdhF, YqiX, and YrpE). Moreover, elevated levels of MntA and YxeB are found in the extracellular proteome of the lspA mutant strain (see the previous section). To further investigate the factors required for lipoprotein processing and retention in the cell, the composition of the extracellular proteome of an Igt mutant, defective in the lipid modification of prelipoproteins (62), was analyzed by Antelmann et al. (5). Unexpectedly, the extracellular protein pattern of the Igt mutant grown in LB medium was completely different not only from that of the parental strain but also from the extracellular proteome of the lspA mutant. In fact, the extracellular proteome of the Igt mutant exhibited about 35 additional spots that were absent or only very weakly present in the medium of the parental strain. Furthermore, the extracellular levels of the predicted lipoproteins OppA and MntA, along with several proteins related to autolytic activities, such as the (predicted) enzymes LytD, YvcE, XepA, and XlyA and the autolysin regulator YwtF (Tables 1 and 2), were significantly increased by the Igt mutation. Of the additional extracellular proteins appearing in the medium of the Igt mutant, nine were identified as (putative) lipoproteins. These were FeuA, FhuD, MsmE, PbpC, RbsB, YfiY, YodJ, YusA, and YxeB (Table 5). By growing the Igt mutant strain in phosphate starvation medium, it was shown that the extracellular levels of the phosphate starvation-induced lipoproteins, OpuAC, PstS, YcdH, YdhF, YqiX, and YrpE, were also significantly increased in the absence of Lgt. Of the latter lipoproteins, OpuAC is the only one not detected in the extracellular proteome of the parental strain, 168 (Table 5). Finally, as shown for LB medium, the extracellular levels of the putative lipoproteins OppA, PbpC, YfiY, YusA, and YxeB were also significantly increased when the Igt mutant was grown in phosphate starvation medium (5). Taken together, these studies showed that cells lacking Lgt shed lipoproteins into their growth medium. Since these lipoproteins are, by and large, retained at the cytoplasmic membrane of the parental strain 168, these observations demonstrate that lipid modification by Lgt is the key determinant for lipoprotein retention in B. subtilis. In contrast, the cleavage by SPase II seems to be required mainly to fully activate the lipid-modified proteins of this organism. In this respect, it should be kept in mind that in the absence of Lgt, unmodified lipoprotein precursors cannot be cleaved by SPase II. Therefore, lipoprotein shedding by B. subtilis Igt can be envisaged to take place in at least two ways. First, unmodified translocated prelipoproteins, as demonstrated for OpuAC and PrsA (5), could either leak from the membrane or be actively released into the growth medium by a (hypothetical) release factor. The released prelipoproteins could form micelle-like structures or could be subject to amino-terminal proteolysis. The latter would result in the presence of mature forms in the growth medium, as observed for MntA, OppA, YclQ, YfiY, YfmC, and YxeB (5). Alternatively, these unmodified prelipoproteins could first be retained in the membrane by their uncleaved signal peptide and subsequently released from the membrane by amino-terminal proteolysis.