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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
Harold Tjalsma,1,2
Haike Antelmann,3
Jan D.H. Jongbloed,1
Peter G. Braun,4
Elise Darmon,1
Ronald Dorenbos,4
Jean-Yves F. Dubois,4,5
Helga Westers,4
Geeske Zanen,4
Wim J. Quax,4
Oscar P. Kuipers,1
Sierd Bron,1*
Michael Hecker,3 and
Jan Maarten van Dijl4,5
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
Secretory proteins perform a variety of important
"remote-control" functions for bacterial survival in
the environment. The availability of complete genome sequences has
allowed us to make predictions about the composition of bacterial
machinery for protein secretion as well as the extracellular complement
of bacterial proteomes. Recently, the power of proteomics was
successfully employed to evaluate genome-based models of these
so-called secretomes. Progress in this field is well illustrated by the
proteomic analysis of protein secretion by the gram-positive bacterium
Bacillus subtilis, for which
90 extracellular
proteins were identified. Analysis of these proteins disclosed various
"secrets of the secretome," such as the residence of
cytoplasmic and predicted cell envelope proteins in the extracellular
proteome. This showed that genome-based predictions reflect only
50% of the actual composition of the extracellular
proteome of B. subtilis. Importantly, proteomics allowed the
first verification of the impact of individual secretion machinery
components on the total flow of proteins from the cytoplasm to the
extracellular environment. In conclusion, proteomics has yielded a
variety of novel leads for the analysis of protein traffic in B.
subtilis and other gram-positive bacteria. Ultimately, such leads
will serve to increase our understanding of virulence factor biogenesis
in gram-positive pathogens, which is likely to be of high medical
relevance.
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).
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SCOPE OF THIS REVIEW: THE PROTEOMICS OF PROTEIN SECRETION BY B. SUBTILIS
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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.

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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.
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PROTEIN SORTING IN B. SUBTILIS
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Although the soil bacterium B. subtilis
has a relatively simple cell structure, proteins can at least be
delivered to, or retained at, five (sub)cellular locations: the
cytoplasm, the cytoplasmic membrane, the membrane/cell wall interface,
the cell wall, and the growth medium
(129). The final
destination of a protein is governed by the presence or absence of
signal peptides and/or retention signals. Nearly all proteins of B.
subtilis lacking transport signals are retained in the cytoplasm
and fold, with or without the aid of chaperones, into their native
conformation. Other proteins contain membrane-spanning domains that are
required for their insertion into the cytoplasmic membrane. Most
proteins that are completely transported across the cytoplasmic
membrane are synthesized with an N-terminal signal peptide. Since
B. subtilis lacks an outer membrane, many of these proteins
are secreted directly into the growth medium. Other exported proteins
involved in processes, such as cell wall turnover, substrate binding,
and the folding and modification of translocated secretory proteins,
have to be retained at the membrane/cell wall interface to fulfill
their function. In the following sections, signal peptides, export
routes, and retention mechanisms that are known to be involved in
protein sorting in B. subtilis are discussed in the light of
recent findings from proteomic analyses.
Signal Peptides
Three distinct domains, N, H, and C, are generally
present in signal peptides
(148-151).
The N-domain contains at least one arginine or lysine residue, which
has been suggested to interact with the translocation machinery and the
negatively charged phospholipids in the lipid bilayer of the membrane
(1,
32). The H-region,
following the N-region, is formed by a stretch of hydrophobic residues
that can adopt an
-helical conformation in the membrane
(21). In the middle of
this hydrophobic core, helix-breaking glycine or proline residues are
often present to allow the formation of a hairpin-like structure that
can insert into the membrane. It was proposed that unlooping of this
hairpin results in insertion of the complete signal peptide into the
membrane (32).
Helix-breaking residues at the end of the H-domain are thought to
facilitate cleavage by a specific SPase
(88). The C-domain,
following the H-domain, contains the cleavage site for specific SPases
that remove signal peptides from the mature part of the exported
protein during or shortly after translocation. The signal peptide is
degraded by signal peptide peptidases and removed from the membrane.
Finally, the mature part of the protein is released from the membrane
and can fold into its native conformation. Despite the similar
structure of signal peptides, apparently small variations can result in
transport to different destinations and/or export via different
pathways, as described below.
Signal Peptide Prediction and Classification
Predictions showed that
300 proteins with the potential to be exported could be distinguished
in B. subtilis
(129). On the basis of
SPase cleavage sites and the export pathways by which these preproteins
are (predicted to be) exported, signal peptides can be divided into
five distinct classes: (i) twin-arginine (RR/KR) signal peptides, (ii)
secretory (Sec-type) signal peptides, (iii) lipoprotein signal
peptides, (iv) pseudopilin-like signal peptides, and (v) bacteriocin
and pheromone signal peptides. The first group of signal peptides
contains a so-called twin-arginine (RR/KR) motif, which serves to
direct proteins into the Tat pathway
(51). The second, and
most abundant, class is composed of typical secretory signal peptides
(lacking an RR/KR-motif) that direct proteins into the Sec pathway.
Both the twin-arginine and secretory signal peptides appear to be
cleaved by one of the various type I SPases of B. subtilis
(130). The third class
of signal peptides is present at the N terminus of prelipoproteins that
are exported via the Sec pathway, lipid modified, and cleaved by the
type II SPase (Lsp)
(136). The fourth class
is formed by signal peptides of pseudopilins which, in B.
subtilis, are cleaved by the SPase ComC
(64). Finally, the fifth
class of signal peptides is found on ribosomally synthesized pheromones
and lantibiotics that are exported and cleaved by ABC transporters
(80). It should be noted
that this specific class of signal peptides is often referred to as
"leader peptides."
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).

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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.
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Secretory (Sec-type) signal peptides.
The 135
predicted signal peptides lacking RR/KR motifs have an average length
of 28 residues and contain two or three positively charged lysine (L)
or arginine (R) residues in their N-domain. The hydrophobic core
(H-domain) has an average length of 19 residues, and about 60%
of the predicted Sec-type signal peptides contains a helix-breaking
residue (mostly glycine) in the middle of this domain. The C-domain of
the predicted signal peptides carries a type I SPase cleavage site,
with the consensus sequence A-S-A at positions 3 to 1
relative to the cleavage site. About 50% of these signal
peptides contain a helix-breaking residue (proline or glycine) at
positions 7 to 4 relative to the predicted processing
site for SPase I
(129).
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).
Retention Signals
In gram-negative bacteria, the outer membrane confines
numerous proteins to the periplasm. The membrane/cell wall interface of
B. subtilis defines a cellular area that is analogous to the
gram-negative periplasm and contains many proteins that fulfill
important functions (72,
94). Proteins retained at
the membrane/cell wall interface include substrate-binding proteins,
chaperones for protein secretion, RNases, DNases, enzymes involved in
the synthesis of peptidoglycan (penicillin-binding proteins), and cell
wall hydrolases, which are involved in cell wall turnover during cell
growth, cell division, sporulation, and germination
(10,
14,
39,
77,
95,
129). To prevent the
loss of these proteins, various retention mechanisms are employed by
the cell.
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
Nin-Cout 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.
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PROTEOMICS OF PROTEIN SECRETION BY B. SUBTILIS
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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.

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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.
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Extracellular Proteome of B. subtilis 168
From the approximately 200 visible
extracellular protein spots, 75 different proteins could be identified
as marked in the 2D master gel for the extracellular proteome (Fig.
3; Tables
1 and
2). Therefore, B. subtilis 168 cells were grown in Luria-Bertani
broth and extracellular proteins were harvested from the medium
2 h after entry into the postexponential growth phase
(5). In the medium of
phosphate-starved cells, eight additional extracellular proteins were
identified (3,
5,
52). When B.
subtilis cells were grown in minimal media, much lower levels of
extracellular proteins were detected
(46). Nevertheless, these
studies resulted in the identification of three additional
extracellular proteins. In total, 90 extracellular proteins were
identified, including 53 proteins to which a function has been assigned
previously and 37 "Y-proteins" of unknown function
(Tables 1 and
2). A possible function
could be attributed to 20 Y-proteins based on their amino acid sequence
similarity to proteins with a known function. In summary, the
identified extracellular proteins of B. subtilis 168 include
enzymes related to the metabolism of carbohydrates, proteases, or
peptidases, enzymes involved in the metabolism of amino acids, enzymes
involved in the decay of DNA or RNA, lipases, alkaline phosphatases,
phosphodiesterases, enzymes involved in cell wall biogenesis,
lipoproteins (many of which are substrate-binding components of various
transport systems), proteins involved in detoxification,
flagellum-related proteins, putative transcriptional regulators,
proteins involved in protein synthesis and folding, prophage-related
proteins, sporulation-specific proteins, and proteins of unknown
function. In addition, Chu et al.
(25) identified five
extracellular proteins of B. subtilis strain K-1, which were
specifically induced by growth in xylan-containing medium. These are a
xylose isomerase homologous to XylA of B. subtilis 168, two
endoxylanases homologous to XynA and XynD of B. subtilis 168,
a dehydroquinate dehydratase homologous to AroC of B. subtilis
168, and a regulatory protein homologous to GltC of B.
subtilis 168. The latter proteins are not included in Tables
1 and
2, which list only the
extracellular proteins of the B. subtilis 168
strain.
Toward an Extracellular Zymoproteome of B. subtilis 168
In addition to the
identification of extracellular proteins, proteomics can be used to
attribute functions to extracellular proteins. An early exploration in
this area was performed by Park et al.
(92), who used a
proteomic approach to detect fibrinolytic enzymes in the medium of
B. subtilis 168. For this purpose, images of 2D PAGE gels were
superimposed to detect extracellular protein spots that coincided with
clearing zones on a 2D zymogram gel containing bovine fibrinogen. In
this way, four protein spots with fibrinolytic activity were
identified. These spots were shown to correspond to differently
processed forms of the serine proteases WprA and Vpr. The processed
form of WprA with proteolytic activity is usually referred to as
CWBP52.
Cell Wall Proteome of B. subtilis 168
The unexpected identification of relatively
large quantities of several proteins with cell wall-binding domains
(e.g., LytD, WapA, YocH, YvcE, and YwtD [Tables
1 and
2]) in the
extracellular proteome may relate to the well-known fact that B.
subtilis undergoes cell wall turnover
(7). This finding prompted
Antelmann et al. (6) to
define the protein composition of the cell wall, which revealed that
seven LiCl-extractable proteins are present in this compartment of the
B. subtilis cell (Table
3). These proteins include the known cell wall-bound proteins LytB and LytC
(both involved in cell wall biogenesis), the CWBP23- and
CWBP52-processing products of WprA (cell wall-located protease), and
processed forms of WapA (structural cell wall-binding protein).
Furthermore, the flagellum-related protein Hag and two proteins with
unknown functions, YwsB and YqgA, are present in the cell wall
proteome. It should be noted that although processed forms of WprA are
known as cell wall proteins, they lack the typical cell
wall-binding repeats that are present in LytB, LytC, and
WapA (6,
68,
129). Surprisingly, two
additional cell wall-located proteins, YwsB and YqgA, also lack known
cell wall retention motifs. Finally, it was remarkable that
extracellular proteins with cell wall-binding motifs, such as LytD,
YocH, YvcE, and YwtD, were apparently absent from the cell wall
proteome. It has to be emphasized that LytD, YvcE, and YwtD are
abundantly present in the extracellular proteome under the growth
conditions used to identify cell wall-associated proteins
(5), showing that the
absence of these proteins from the wall proteome is not due to a lack
of expression. Probably, the same is true for YocH, but this is less
clearly evident from the published data
(6).
Verification of Secretome Predictions
The availability of accumulating
proteomic data concerning the extracellular complement of the secretome
of B. subtilis 168 has allowed proteomic verification of the
genome-based predictions of the composition of the secretome as
previously performed
(129). Intriguingly,
only 48 (53%) of the 90 identified extracellular proteins are
expected to be released into the medium, as judged by the presence of
predicted signal peptides and a lack of retention signals (Tables
1 and
2). A potential RR/KR
motif is present in the N-domains of 14 signal peptides of the latter
group of proteins, suggesting their potential transport via the Tat
pathway. The remaining 34 proteins contain a Sec-type signal peptide
and are most probably exported by the Sec pathway of B.
subtilis. Strikingly, 47% of the extracellular proteome
currently cannot be predicted to end up at this location
(129). This unpredicted
fraction consists of proteins which have an N-terminal lipoprotein
signal peptide (cleaved by SPase II) or potential transmembrane
segments according to the TMHMM algorithm
(28). Both groups of
proteins are supposed to be retained in or at the cytoplasmic membrane.
In addition, some predicted preproteins with a type I SPase
cleavage site contain typical cell wall-binding repeats and therefore
have a predicted cell wall localization. As listed in Table
2, 24 proteins found in
the medium of B. subtilis are in fact proteins that lack a
typical export signal. The latter include flagellum-related
proteins, prophage-related proteins, and proteins with known or
predicted enzymatic activities in the cytoplasm. The possible
mechanisms by which these proteins are released from the cell are
discussed in "Mechanisms for extracellular accumulation of
proteins" (below).
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
|
|---|
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.

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|
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.
|
|
Cytoplasmic Targeting Factors
Since
B. subtilis lacks a secretion-specific targeting factor
similar to the SecB protein of Escherichia coli
(99), an important role
in this process has been attributed to the highly conserved signal
recognition particle (SRP) pathway
(129). An important
component of this pathway is the Ffh protein (for "fifty-four
homologue"), a GTPase that is homologous to the
54-kDa subunit of the eukaryotic signal recognition
particle (SRP54) (47).
This protein forms a complex (denoted SRP) with the small cytoplasmic
RNA that is functionally related to the eukaryotic 7S RNA
(78) and HBsu, a
histone-like protein of B. subtilis
(79). This SRP complex of
B. subtilis binds to the signal peptides of nascent chains
emerging from the ribosome and is targeted to the membrane with the aid
of the FtsY protein, a homologue of the eukaryotic SRP receptor
-subunit (SR
)
(87). Both Ffh and FtsY
are essential for SRP-dependent protein secretion and cell viability
(54).
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.
Sec Translocase
The preprotein translocation machinery of the B. subtilis
Sec pathway consists of at least four proteins: SecA, which is the
translocation motor, and the integral membrane proteins SecE, SecG, and
SecY. In the current model for preprotein translocation in B.
subtilis, which has many similarities to that of E. coli,
several successive steps in the translocation of proteins occur
(36,
38,
73,
129,
145). First, SecA binds
to the SecYEG translocase in the cytoplasmic membrane. Next,
preproteins are transferred from a targeting factor (i.e., SRP or CsaA)
to SecA dimers that are bound to the SecYEG complex. The binding of ATP
by SecA leads to insertion of the C terminus of SecA through the pore
of a SecYEG complex in the membrane, causing the translocation of a
short stretch of the preprotein. Next, ATP is hydrolyzed by SecA,
leading to the release of the preprotein and deinsertion of
SecA. The latter step can be specifically inhibited by low
concentrations of the ATPase inhibitor sodium azide. Further
translocation is driven by both repeated cycling of SecA through ATP
binding and hydrolysis and the proton motive force. Two proteomic
approaches were performed to determine the effects of SecA limitation
on the composition of the extracellular complement of the secretome.
Hirose et al. (46) used a
strain that contains a temperature-sensitive SecA (SecAts)
protein, while Jongbloed et al.
(51) used sodium azide to
inhibit SecA activity. It should be emphasized that SecA limitation by
inactivation of SecAts at elevated temperatures and SecA
inhibition by azide represent two distinct approaches, both of which
have their limitations. When SecA activity is inhibited by sodium
azide, the initial targeting and translocation steps can possibly still
take place, whereas these initial stages in protein transport as well
as later SecA-dependent steps are affected significantly on SecA
limitation. If so, initial stages in the translocation of
"azide-resistant" secretory proteins may be driven by
SecA while later stages in the translocation of these proteins could be
more strongly dependent on the proton motive force than on SecA
activity.
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).
Type I Signal Peptidases
SPases remove signal peptides from secretory
preproteins when the C-domain of the signal peptide emerges at the
extracytoplasmic side of the membrane. This enzymatic reaction is a
prerequisite for the release of the mature secretory protein from the
membrane (29,
30,
129). One of the most
remarkable features of the B. subtilis protein secretion
machinery is the presence of multiple, paralogous, type I SPases. This
is in contrast to many other bacteria and archaea and the endoplasmic
reticulum (ER) of yeast, in which just one type I SPase seems to be
sufficient for the processing of secretory preproteins
(129,
130). In B.
subtilis five sip genes for type I SPases are located on
the chromosome (denoted sipS, sipT, sipU,
sipV, and sipW
[130,
134]).
Interestingly, SipW is homologous to SPases found in sporulating
gram-positive bacteria, archaea, and the ER membrane of eukaryotes,
which, together, form the subfamily of ER-type SPases. The uniqueness
of SipW was further underscored by the observation that this SPase is
solely required for the processing of the spore-associated
protein TasA (126,
131). In contrast, all
other B. subtilis SPases are of the prokaryotic type (P-type).
Such P-type SPases are typically present in eubacteria, mitochondria,
and chloroplasts (130).
Although all chromosomally encoded SPases in B. subtilis can
process secretory preproteins, only SipS and SipT are of major
importance for preprotein processing and cell viability. In contrast,
SipU, SipV, and SipW play a minor role in protein secretion and have
substrate specificities that differ at least in part from those of SipS
and SipT (130,
134).
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).
Lipoprotein Modification and Processing
Although lipoproteins are
transported via the general Sec pathway, specific enzymes for their
modification (Lgt) and processing (SPase II) are required. In contrast
to the type I SPases, B. subtilis contains only one gene for a
type II SPase (lspA)
(97,
136), which is
specifically required for the processing of lipid-modified preproteins.
Interestingly, B. subtilis cells lacking SPase II are viable
under standard laboratory conditions. This indicates that processing of
lipoproteins by SPase II is not strictly required for lipoprotein
function, since at least one lipoprotein, PrsA, is essential for
viability (55). The fact
that processing of lipoproteins by SPase II is not strictly required
for lipoprotein function is probably due to activity of uncleaved
lipoproteins, as was recently shown to be the case for lipoprotein
precursors in Lactococcus lactis
(146). In B.
subtilis cells lacking SPase II, lipoprotein precursors are
subject to alternative N-terminal processing by as yet unidentified
proteases (132,
136). The cumulative
activity of unprocessed and alternatively processed (mature-like)
lipoproteins is in many cases strongly reduced compared to that of
their corresponding mature form
(12,
136). In B.
subtilis cells lacking SPase II, the secretion of the
nonlipoprotein AmyQ was strongly impaired, which could be attributed to
malfunctioning of the precursor and mature forms of the lipoprotein
PrsA, an extracytoplasmic folding catalyst for many secretory proteins
(136).
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.
Folding Catalysts
After translocation in an unfolded state, Sec-dependent secretory
proteins have to fold into their native conformation. Even though
proteins can fold spontaneously in vitro, their folding in