Download Translocation of proteins across the cell envelope of Gram

FEMS Microbiology Reviews 25 (2001) 437^454
www.fems-microbiology.org
Translocation of proteins across the cell envelope of
Gram-positive bacteria
Karel H.M. van Wely
a
a;1
, Jelto Swaving a , Roland Freudl b , Arnold J.M. Driessen
a;
*
Department of Microbiology, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Kerklaan 30,
9751 NN Haren, The Netherlands
b
Institut fu«r Biotechnologie 1, Forschungszentrum Ju«lich GmbH, D-52425 Ju«lich, Germany
Received 6 December 2000 ; received in revised form 8 May 2001; accepted 9 May 2001
First published online 5 June 2001
Abstract
In contrast to Gram-negative bacteria, secretory proteins of Gram-positive bacteria only need to traverse a single membrane to enter the
extracellular environment. For this reason, Gram-positive bacteria (e.g. various Bacillus species) are often used in industry for the
commercial production of extracellular proteins that can be produced in yields of several grams per liter culture medium. The central
components of the main protein translocation system (Sec system) of Gram-negative and Gram-positive bacteria show a high degree of
conservation, suggesting similar functions and working mechanisms. Despite this fact, several differences can be identified such as the
absence of a clear homolog of the secretion-specific chaperone SecB in Gram-positive bacteria. The now available detailed insight into the
organization of the Gram-positive protein secretion system and how it differs from the well-characterized system of Escherichia coli may in
the future facilitate the exploitation of these organisms in the high level production of heterologous proteins which, so far, is sometimes very
inefficient due to one or more bottlenecks in the secretion pathway. In this review, we summarize the current knowledge on the various steps
of the protein secretion pathway of Gram-positive bacteria with emphasis on Bacillus subtilis, which during the last decade, has arisen as a
model system for the study of protein secretion in this industrially important class of microorganisms. ß 2001 Federation of European
Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.
Keywords : Protein secretion ; Translocase ; Bacillus
Contents
1.
2.
3.
4.
5.
6.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . .
General features of protein translocation . . . . . .
Signal peptides . . . . . . . . . . . . . . . . . . . . . . . . .
Cytosolic factors . . . . . . . . . . . . . . . . . . . . . . . .
4.1. Molecular chaperones . . . . . . . . . . . . . . . . .
4.2. Signal recognition particle . . . . . . . . . . . . . .
4.3. SRP receptor . . . . . . . . . . . . . . . . . . . . . . .
The secretion machinery . . . . . . . . . . . . . . . . . .
5.1. SecA drives translocation . . . . . . . . . . . . . .
5.2. SecYEG forms a protein-conducting channel
5.3. SecDF are accessory membrane components
5.4. YidC and other factors . . . . . . . . . . . . . . . .
Signal peptidases . . . . . . . . . . . . . . . . . . . . . . . .
6.1. Type I signal peptidase . . . . . . . . . . . . . . . .
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* Corresponding author. Tel. : +31 (50) 3632164; Fax: +31 (50) 3632154. E-mail address: [email protected] (A.J.M. Driessen).
1
Present address: Department of Experimental Pathology, Josephine Nefkens Institute, Erasmus University Rotterdam, P.O. Box 1738, 3000 DR
Rotterdam, The Netherlands.
0168-6445 / 01 / $20.00 ß 2001 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.
PII: S 0 1 6 8 - 6 4 4 5 ( 0 1 ) 0 0 0 6 2 - 6
FEMSRE 722 20-8-01
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Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
448
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
448
7.
8.
6.2. Type II signal peptidase and other proteolytic
Extracellular factors . . . . . . . . . . . . . . . . . . . . . .
7.1. Competition between folding and degradation
7.2. Passage through the cell wall . . . . . . . . . . . . .
Concluding remarks . . . . . . . . . . . . . . . . . . . . . .
1. Introduction
All living cells need to target newly synthesized proteins
to their site of action. For extracellular proteins, this involves transport steps across one or more membranes, a
process called translocation. In bacteria, the ¢rst barrier
for protein translocation is the cytoplasmic membrane.
This membrane harbors an intricate machine that ensures
the proper delivery of a large number of proteins to the
external environment. During the last two decades bacterial protein secretion has been studied in great detail.
These studies were mainly focused on the Gram-negative
bacterium Escherichia coli, but more recently, Bacillus subtilis has become the model system for Gram-positive bacteria. Unlike in Gram-negative bacteria, which are surrounded by an outer membrane, proteins exported
across the cytoplasmic membrane of Gram-positive bacteria are released directly into the external environment.
Hence, Gram-positive bacteria are attractive hosts for
the industrial production of enzymes that occur naturally
in these organisms and that are secreted into the supernatant in large amounts. They may also function as excellent hosts for the secretory production of heterologous
proteins. Indeed, Bacilli can be used successfully for
this purpose, but sometimes the yields are disappointing.
Obviously, one or more bottlenecks in the secretion pathway reduce the amount of the desired product in the
supernatant. A detailed understanding of the export mechanism is crucial for a further optimization of Gram-positive bacteria with respect to heterologous protein secretion.
For some time, the mechanisms by which the two main
groups of bacteria secrete proteins was believed to be very
di¡erent because of the identi¢cation of the SecA protein
in E. coli [1] and the proposed existence of an S-complex
in B. subtilis [2]. At a later stage it became clear that the
S-complex is not related to protein secretion [3,4], and in
the mean time a large number of common components
have been identi¢ed [5^12]. The identi¢cation of the
components of the translocation machinery was accelerated by the availability of the complete genome sequence
of B. subtilis [13] and other Gram-positive and phylogenetically related bacteria, such as Mycoplasma genitalium
[14], Mycoplasma pneumoniae [15], Bacillus halodurans
[16], Lactococcus lactis [17], Mycobacterium tuberculosis
enzymes
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[18], Mycobacterium leprae [19], Ureaplasma urealyticum
[20], and Listeria monocytogenes (http://www.pasteur.
fr/recherche/unites/gmp/Gmp_europ_consor_lm.html). A
complete up to date list can be found at http://www.
tigr.org/tdb/mdb/mdbinprogress.html (or /mdbcomplete.
html).
It is now generally believed that Gram-positive and
Gram-negative bacteria secrete their proteins by similar
mechanisms as the genome sequences show that they share
the major components. Here, we discuss the recent progress in the study of the main pathway for protein export
in Gram-positive bacteria with the emphasis on some of
the remarkable di¡erences between Gram-negative and
Gram-positive bacteria. Since protein translocation is
best characterized in B. subtilis, we will mainly focus
on this organism. An overview of the components of the
general protein secretion machinery of B. subtilis is shown
in Fig. 1. We will not discuss the speci¢c protein export
pathways such as the Tat pathway, polypeptide translocating ABC transporters and the type IV prepilin-like pathway found in Gram-positive bacteria.
2. General features of protein translocation
The cell needs a means to distinguish cytosolic proteins
from secretory proteins. For this purpose, secretory proteins are synthesized as precursors with a cleavable aminoterminal signal peptide (for reviews see [21^23]). The signal
peptide ensures the proper targeting to the translocation
machinery at the cytosolic membrane and the initiation of
translocation. The general secretory pathway mediates the
translocation of proteins in an unfolded conformation.
Translocation takes place through a con¢ned aqueous
channel composed of a set of integral membrane proteins
[24,25]. The general architecture of this channel is conserved among bacteria, archaea and eukaryotes [26]. In
addition, the channel proteins provide a binding site for
cytoplasmic components that drive targeting and translocation (Fig. 1) (for review see [27]).
Precursor proteins are synthesized at the ribosome as
extended polypeptides that have not yet folded into their
¢nal conformation. Cytosolic chaperones act to prevent
the tight folding or aggregation of the precursor protein
prior to its translocation across the membrane. Two pos-
FEMSRE 722 20-8-01
K.H.M. van Wely et al. / FEMS Microbiology Reviews 25 (2001) 437^454
439
warrants an optimal quality of the secreted proteins. Finally, the folded secreted proteins have to pass the cell
wall in order to be released into the external medium. A
Gram-positive organism like B. subtilis contains approximately 300 secretory proteins [23]. Almost all of these
proteins are translocated via the Sec pathway.
3. Signal peptides
Fig. 1. Schematic overview of the B. subtilis protein translocation machinery. The signal recognition particle (SRP) consists of scRNA, Ffh
and HBsu. See text for details. The dashed line arrow indicates an SRPindependent pathway, possibly mediated by SecA that shuttles between
the SecYEG-bound and free cytosolic state. Proteases that degrade the
secreted proteins are located near the membrane surface, in the cell wall
and free in the suspending medium.
sible routes can be followed at this stage. One possibility is
to bring the site of synthesis of the precursor in contact
with the translocation channel and couple translocation to
the synthesis of the secretory protein at the ribosome. This
mechanism, termed co-translational translocation, is used
for the transport of proteins into the endoplasmic reticulum (for review see [28]) and for a subset of bacterial
proteins [29^31]. The other possibility is to complete the
synthesis of the precursor protein prior to its translocation. This mechanism, termed post-translational translocation, requires that tight folding of the precursor in the
cytosol is prevented and thus involves molecular chaperones such as SecB, or the more general chaperones GroEL
or DnaK (for reviews see [32,33]). Some of the cytosolic
components are also involved in the speci¢c targeting of
the precursor to the translocation sites at the membrane
[27]. The relative importance of co- and post-transcriptional translocation may vary among organisms. In bacteria,
the driving force for both routes of translocation is provided by the hydrolysis of ATP by the SecA protein and
the proton motive force (pmf) across the membrane
[24,34]. Once the precursor has crossed the membrane,
maturation to its ¢nal folded conformation takes place.
The folding of the translocated polypeptide competes
with degradation by extracellular proteases. This process
To ensure the proper targeting and translocation across
the membrane, secretory proteins are equipped with a signal peptide that is proteolytically removed by signal peptidases during or shortly after translocation [35]. In the
general secretion pathway, two classes of signal peptides
can be identi¢ed, i.e., the general signal peptides (type I)
and the lipoprotein signal peptides (type II).
A signal peptide usually is 14^25 amino acids long and
consists of three identi¢able domains, i.e., the amino (N-),
hydrophobic (H-), and carboxy-terminal (C-) regions. The
N-region is rich in positively charged amino acids, and is
followed by a hydrophobic region that tends to organize
into an K-helical conformation when brought into contact
with the membrane lipid phase. The C-terminal region is
hydrophilic and contains the signal peptide cleavage site
that is recognized by the signal peptidase. This site conforms to the 33, 31 rule [36], and in many cases corresponds to an Ala-X-Ala cleavage site. On average, type I
signal peptides of Bacillus species are ¢ve to seven amino
acids longer than those of E. coli [36]. The extensions
occur in all three regions (N-, H- and C-regions). In addition, the N-region usually contains a higher number of
positively charged lysine and arginine residues. In Grampositive bacteria, cleavage by signal peptidase preferentially occurs seven to nine residues from the C-terminal
end of the H-region, whereas in E. coli, processing takes
place three to seven residues from the same position
[22]. Therefore, a longer C-region may re£ect di¡erences
in substrate speci¢city of the Gram-positive signal peptidases. Type II signal peptides largely resemble the type
I signal peptides, but contain within the C-region a socalled lipoprotein box with a Leu-Ala-Gly-Cys consensus
sequence. In the cytosolic membrane, the cysteine in
this sequence is covalently linked to a lipid [37,38].
After lipomodi¢cation, the type II signal peptide is recognized by signal peptidase II and cleaved [37]. The type I
and II signal peptidases [39] are further discussed in Section 6.
4. Cytosolic factors
4.1. Molecular chaperones
Post-translational translocation of a protein involves a
cytosolic intermediate of the precursor stabilized by a mo-
FEMSRE 722 20-8-01
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lecular chaperone to ensure a translocation-competent
state, i.e., an unfolded state that is compatible with translocation. B. subtilis, like all organisms, contains a number
of heat shock proteins or molecular chaperones such as
GroEL, DnaJ, and DnaK. Their genes have been characterized and their regulation has been studied in detail [40^
43]. However, the role of these chaperones in protein secretion has remained elusive. Inactivation of the hrcA
gene, the product of which is a negative regulator, results
in higher expression of the groE and dnaK operons [43].
Concurrently, the hrcA mutant secretes some heterologous
proteins to higher concentrations [44]. Formation of inclusion bodies is a problem often associated with high level
expression and secretion of heterologous secretory proteins. General molecular chaperones may prevent these
proteins from aggregation or may even act to dissolve
aggregates, thereby increasing the amount of protein available for translocation. A direct role of the general molecular chaperones in the secretion of endogenous proteins in
B. subtilis has, however, not been demonstrated.
Many Gram-negative organisms including E. coli contain a secretion-dedicated chaperone called SecB [27]. SecB
functions by rapidly binding to partially folded precursors
either free in the cytosol [45] or bound to the ribosome as
nascent chains [46]. Next, the binary SecB precursor protein is targeted to SecA. SecA binds SecB with high a¤nity, and the subsequent association of the signal peptide of
the precursor with SecA promotes the release of precursor
from SecB and its transfer to SecA [47,48]. SecB binds to
the carboxy-terminus of SecA. This region of SecA is
highly conserved among many Gram-negative and
Gram-positive bacteria, with the exception of Streptomyces, Mycoplasma and Mycobacterium species [47]. However, no homologs with sequence similarity to SecB are
present in any of the Gram-positive organisms of which
the genome sequence has been completed. In line with this
¢nding, the deletion of the carboxy-terminus of the B.
subtilis SecA yielded cells that in contrast to E. coli cells
[49] are normally viable, but exhibit a lowered expression
of some secreted proteins [50]. Although capable of interacting with E. coli SecB, the carboxy-terminus of B. subtilis SecA speci¢cally binds a single protein in cytosolic
extracts isolated from B. subtilis. This protein, MrgA,
has been reported to function in relation to oxidative
stress and is unrelated to secretion. Deletion of the mrgA
gene from the chromosome does not result in a secretion
defect. It therefore appears that the carboxy-terminus of
the B. subtilis SecA is involved in some regulatory mechanism of which the details are unknown.
When expressed in B. subtilis, the E. coli SecB has been
shown to enhance the heterologous secretion of the precursor of maltose binding protein (preMBP) of E. coli [51].
Unlike in E. coli [52], considerable parts of proOmpA and
preMBP are secreted co-translationally by B. subtilis without the aid of SecB [51,53]. Co-expression of SecB partially shifts the secretion to a post-translational modus [51].
Replacing the E. coli signal peptides of proOmpA and
preMBP with that of Bacillus amyloliquefaciens alkaline
protease signi¢cantly increased the secretion rate and e¤ciency, probably by shifting secretion towards the cotranslational mode [53], concomitantly reducing the need
for SecB. This suggests that the requirement for a chaperone is not determined by the precursor alone but also by
the mode of secretion. With respect to the absence of a
SecB homolog in Gram-positive bacteria, it might is important to note that SecB is not essential for viability of E.
coli. It is needed only for the secretion of a speci¢c subset
of proteins [54], many of which are outer membrane proteins. Such proteins are absent in Gram-positive bacteria.
It has been suggested that SecB acts in particular on proteins that have a tendency to rapidly fold or aggregate
[55]. Gram-positive organisms lack an outer membrane,
and many of their secreted proteins seem to fold slowly.
For instance, in Bacillus some secreted proteins fold slowly
or incompletely in the absence of calcium [56,57], whereas
the presence of calcium at the external surface of the cell
allows them to rapidly adopt a native conformation [58^
60]. Thus, intracellular chaperones do not seem to be
needed to stabilize the unfolded conformation of these
proteins. This does not exclude a possible role in preventing aggregation or other non-speci¢c interactions.
Screening of a gene library of B. subtilis chromosomal
DNA for complementation of the E. coli secA51(ts) mutant has resulted in the cloning of the csaA gene [61].
CsaA is a cytoplasmic protein that is capable of complementing the growth defect of the E. coli secA51(ts),
SecB: :Tn5 null, dnaK756, dnaJ259 and grpE280 mutants
[61,62]. The presence of CsaA not only restores the growth
of the secA51(ts) mutant but also diminishes the cytosolic
accumulation of precursors at the non-permissive temperature, and restores the processing of proOmpA and pre-Llactamase [63]. CsaA may function as a molecular chaperone as it is able to prevent thermal denaturation of luciferase in vivo and in vitro [63]. Puri¢ed CsaA interacts
with unfolded precursor proteins and even binds to SecA
[62]. Depletion of CsaA has a moderate e¡ect on secretion
with the loss of only a limited number of secretory proteins from the culture supernatant. In this respect, CsaA
shows an activity reminiscent of the E. coli SecB, although
the two proteins share no sequence similarity. On the other hand, deletion of the csaA gene is lethal suggesting that
CsaA either is involved in the translocation of essential
proteins or plays a vital role in a process other than protein translocation. In this respect, the recently solved
three-dimensional structure of the Thermus thermophilus
CsaA protein has provided some new insights into the possible function of CsaA [64]. CsaA is a dimer, and on its
protein surface bears two identical, large hydrophobic cavities that may function as peptide binding sites. However,
CsaA shares structural and sequence similarity with tRNA
binding proteins, suggesting that it may possess tRNA
binding activity in addition to a chaperone function.
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Fig. 2. Domain organization of the B. subtilis Ffh protein. The three
parts of the GTP binding motif, the helical domains (H1, H2, H3), and
the sites of FtsY, precursor protein and scRNA interaction are indicated.
4.2. Signal recognition particle
The translocation of precursor proteins across the endoplasmic reticulum of eukaryotes depends on the signal
recognition particle (SRP). SRP consists of six proteins
that form a complex with an RNA molecule [65]. SRP
interacts with the signal peptide of a nascent polypeptide
when it emerges from the ribosome, and targets the complete nascent chain^ribosome complex to the SRP receptor
at the endoplasmic reticulum membrane [66]. Also in prokaryotes, homologs of some of the SRP components have
been identi¢ed but the SRP of prokaryotes seems of a
lesser complexity than its eukaryotic counterparts [67].
Central to the bacterial SRP is an RNA molecule ¢rst
identi¢ed as small cytoplasmic RNA or scRNA [68]. The
B. subtilis scRNA is 271 nucleotides long and consists of
three domains which correspond to domains I, II and IV
of the mammalian SRP 7S RNA. Studies with deletion
mutants revealed that only domain IV is required for vegetative growth [69], but domains I and II are necessary to
promote sporulation. The scRNA from Clostridium perfringens, which has a predicted secondary structure similar
to that of B. subtilis, allows for both growth and sporulation of a B. subtilis scRNA mutant [70]. The E. coli 4.5S
RNA is an equivalent of scRNA, but contains the conserved domain IV only. The E. coli 4.5S RNA can support
vegetative growth of B. subtilis but not sporulation, which
indicates additional functions for the other RNA domains
in B. subtilis (see also below). The central protein Ffh (fifty
four homolog), which is homologous to the main eukaryotic SRP subunit, SRP54, associates with scRNA. The B.
subtilis Ffh was found using an E. coli ¡h DNA fragment
as a probe [71]. It is a protein of 446 amino acids that
shows a well-de¢ned domain structure (Fig. 2). The amino-terminal part of Ffh, called the G-domain, contains
three GTP binding motifs and has been shown to interact
with FtsY, the SRP receptor homolog [72^74]. The carboxy-terminal M-domain is rich in methionine residues
and contains three amphipathic helices. The ¢rst of these
helices is situated close to the G-domain, and mediates the
speci¢c binding of Ffh to precursors of secreted proteins
[75]. The other two helices are in close vicinity to the
carboxy-terminus and bind the scRNA to form the SRP
[76].
441
Upon depletion of Ffh, proteome analysis indicates that
approximately 80% of extracellular proteins disappear
from the culture supernatant [39]. Based on this, Ffh
was suggested to be a central component of the general
secretory pathway of B. subtilis. However, care should be
taken with this hypothesis as Ffh depletion may indirectly
result in a protein secretion defect by a¡ecting membrane
insertion of one or more proteins that are essential for
protein translocation such as SecY or SecE. In B. subtilis,
Ffh is an essential protein for vegetative growth and spore
development. Only the vegetative growth of a conditionally lethal mutant of the B. subtilis ¡h gene can be restored
by the E. coli Ffh. Puri¢ed B. subtilis Ffh can be speci¢cally cross-linked to precursors of L-lactamase equipped
with di¡erent signal peptides in vitro, but does not interact
with mature L-lactamase [75,77]. The binding to the precursors of secreted proteins is an intrinsic property of the
Ffh protein and does not require scRNA [75]. A remarkable property of B. subtilis Ffh is that it binds to SecA
with high a¤nity. The binary complex of Ffh and precursor proteins is up to 30-fold more e¤ciently recognized by
SecA than the precursor protein alone [78], in a fashion
reminiscent of the SecA binding to the precursor^SecB
complex in E. coli [48]. It therefore appears that B. subtilis
Ffh functions as a general targeting factor for secretory
proteins, whereas the E. coli SRP pathway mainly seems
to function in the targeting of integral membrane proteins
[79,80]. In E. coli, the ability of precursors to interact with
Ffh depends on the signal peptide, with a strong preference for slightly longer and more hydrophobic signal peptides [81,82]. The relatively long signal peptides of Grampositive bacteria might therefore favor translocation via
the SRP-mediated pathway, possibly lowering or even
abolishing the need for chaperone-assisted translocation.
HBsu is a histone-like protein of 10 kDa that was found
as another binding partner of scRNA [83,84], constituting
another component of the SRP. Like the other components of the SRP, HBsu is essential for growth of B. subtilis, but the exact role of this protein is unresolved so far.
Interestingly, unlike Gram-negative bacteria, the scRNA
of Gram-positive bacteria like B. subtilis contains the 5P
and 3P RNA domains. This region is termed the Alu domain, and in eukaryotes it binds the small SRP9 and
SRP14 subunits of SRP. The latter interaction is essential
for translational arrest that allows the ribosome^nascent
chain^SRP complex to dock at the translocation sites at
the membrane before chain elongation is continued. HBsu
can bind the RNA in a similar manner as SRP9 and
SRP14, which opens the exciting possibility that translocation arrest is a feature of protein translocation in these
bacteria.
4.3. SRP receptor
Although the B. subtilis SRP can form a ternary complex with a precursor protein and SecA directly and thus
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seems to function in targeting, a speci¢c SRP receptor is
also present in this organism [85]. FtsY is a homolog of
the SRK subunit of the mammalian SRP receptor [86,87]
but lacks the amino-terminal membrane anchor domain.
A mutant of B. subtilis in which an IPTG-dependent promoter controls the FtsY expression shows poor and ¢lamentous growth in the absence of inducer [30]. In this
strain, the kinetics of L-lactamase processing is lowered
upon FtsY depletion and the precursor accumulates inside
the cell. Depletion of FtsY during sporulation results in an
incomplete assembly of the outer coat [88]. The ftsY gene
is expressed during both the exponential phase and sporulation. In vegetative cells, ftsY is transcribed together
with two upstream genes, rncS and smc, which are under
the control of the major transcription factor cA . FtsY is
solely expressed during sporulation from a cK - and GerEcontrolled promoter that is located immediately upstream
of ftsY inside the smc gene. The ftsY genes from several
Mycoplasma species have also been cloned and functionally expressed in E. coli [73,89]. Binding of Ffh to FtsY
stimulates the endogenous GTPase activity of FtsY markedly [73]. This activity requires the amino-terminal part of
Ffh, which by itself also acts as a GTPase. The e¡ect of
the FtsY protein on the interaction between Ffh and SecA
in B. subtilis has not been investigated so far. These data
indicate that in B. subtilis only one targeting pathway
operates, in which SecA and SRP cooperate. In E. coli,
the SRP and SecB targeting pathways converge at the
translocase [90].
5. The secretion machinery
5.1. SecA drives translocation
SecA is a precursor protein-stimulated ATPase that performs a central role in bacterial protein secretion, as it is
the molecular motor that drives translocation. SecA is a
homodimeric protein with a subunit mass of about 100
kDa. During the last few years, genes encoding SecA homologs from many di¡erent Gram-positive organisms
have been cloned [91^95]. Strikingly, mycobacteria even
contain two secA genes [18,19,95], but it is not clear if
both genes are essential. The gene encoding the B. subtilis
SecA was initially identi¢ed through the div mutation,
which results in a temperature-dependent defect of division, sporulation, competence, and protein secretion [96^
98]. DivA encodes the B. subtilis SecA. By the use of the
divA mutant, it was shown that about 90% of all proteins
in the culture supernatant of B. subtilis depend on SecA
for secretion [39].
The conditional defect of the divA mutation can be restored by expression of the B. subtilis secA gene but not by
the E. coli secA gene [99]. On the other hand, the B. subtilis SecA is able to suppress the growth and secretion
defects of E. coli SecA conditionally lethal mutants when
expressed at a low level [100,101]. Truncated forms of the
B. subtilis SecA and chimeras of the B. subtilis and E. coli
SecA have been shown to complement the growth defect
of the E. coli secA52(ts) mutant that expresses a malfunctioning SecA protein [7,99,102,103]. De¢nite proof that
SecA is involved in protein translocation in Gram-positive
bacteria was obtained by the use of in vitro translocation
systems [104,105] that have only in recent years become
available for this group of organisms. From these studies
it also became evident that the B. subtilis and E. coli SecA
are only partially exchangeable, indicating some level of
species speci¢city. In vivo studies with secA genes from
other organisms point in the same direction, and suggest
that the barrier between Gram-positive organisms is smaller than that between Gram-positive and Gram-negative
bacteria [106,107]. In vitro translocation of B. subtilis alkaline phosphatase has demonstrated that, in some cases,
B. subtilis SecA is needed to e¤ciently drive precursor
protein translocation by the B. subtilis SecYEG complex
[108]. The combination of B. subtilis SecYEG and E. coli
SecA was only partially active for translocation of this
precursor, whereas the completely homologous E. coli system operated rather e¤ciently. The reason for the species
speci¢city of SecA is not entirely clear. Possibly, it relates
to a defective interaction with one or more of the other
components of the translocase, and/or poor recognition of
one or more essential secretory precursors in the heterologous hosts.
SecA seems to function as a molecular motor that couples the energy of ATP binding and hydrolysis to the
stepwise translocation of precursor proteins [34,101,109].
SecA binds precursor proteins through their signal peptide
and mature domain [105^112]. The SecA protein exhibits a
low endogenous ATPase activity, which can be stimulated
by the presence of membranes, the SecYEG complex, and
precursor proteins [100,109^111]. Whereas the E. coli SecA
shows high a¤nity for both the E. coli and B. subtilis
SecYEG complexes, the B. subtilis SecA bears a much
lower a¤nity for any of these [108,109] (J. Swaving, unpublished results). Nevertheless, this latter combination of
SecA and SecYEG supports the translocation of precursor
proteins. Tight binding of SecA to SecYEG may not be
needed for the proper function of the B. subtilis complex,
but the functional consequence of this di¡erence in interaction remains to be elucidated. Interestingly, although
protein crystallization attempts with the E. coli SecA
have been unsuccessful, the B. subtilis SecA has recently
î resolution in the presence of
been crystallized at 2.70 A
30% glycerol [113]. This could possibly relate to a di¡erence in solubility and/or conformational heterogeneity between the B. subtilis and E. coli SecA proteins. The lower
SecYEG binding a¤nity of the B. subtilis SecA points to
an important role of the cytosolic SecA fraction in the cell.
SecA contains two nucleotide binding folds [100,101,
109,114]. The high a¤nity nucleotide binding site
(NBS-I) (Kd; ADP W0.15 WM) is located in the amino-ter-
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minal region of the protein, and contains the typical
Walker A and B sequence signatures. This site is important for the activity of SecA. A second site, NBS-II, has
been proposed to bind nucleotides with low a¤nity
(Kd; ADP W340 WM) [114]. This region is also important
for ATP hydrolysis and protein translocation, but recent
data indicate that it is not an independent nucleotide binding site, but instead functions as an intramolecular regulator of ATP hydrolysis at NBS-I [115].
The exact molecular mechanism by which SecA acts as a
motor protein is only poorly understood. Microcalorimetric studies on the B. subtilis SecA protein have shown that
the protein consists of two thermodynamically distinct domains that correspond to amino- and carboxy-terminal
regions of the proteins [116,117]. Binding of ADP to the
amino-terminal NBS-I was shown to stabilize the carboxyterminal domain, and promotes the formation of a more
compact conformation of the B. subtilis SecA protein
[117]. The size of these two domains of B. subtilis SecA
di¡ers from stable proteolytic fragments that can be derived from the E. coli protein, i.e., an amino- and carboxyterminal region of 65 and 30 kDa, respectively [118]. Studies with the E. coli SecA suggest that its carboxy-terminal
region penetrates the membrane when ATP is bound to
the protein [119,120]. This `membrane-inserted' state of
SecA is sensed by an intramolecular regulator located in
the carboxy-terminal 30-kDa fragment, allowing hydrolysis of ATP [121]. In its ADP-bound state, SecA adopts a
surface-bound, `membrane-deinserted' conformation.
Based on these ¢ndings, a model has been proposed in
which binding and hydrolysis of ATP alternates SecA
between extended and compact conformations allowing
the stepwise translocation of polypeptide segments [34,
116,117,121,122] (Fig. 3).
Site-directed mutagenesis of the conserved lysine of the
Walker A site of NBS-I of the B. subtilis SecA has demonstrated that this residue is critical for ATP hydrolysis
and translocation. This mutant (K106N) still binds but no
longer releases the precursor protein [109], consistent with
the notion that this step requires the hydrolysis of ATP.
Moreover, the K106N mutant interferes with the SecAmediated translocation of precursor proteins in vivo and
in vitro [100,109]. A similar defect in protein translocation
can be created by the addition of non-hydrolyzable analogs of ATP such as AMP-PNP, which competes with
ATP for binding to SecA, or azide, an inhibitor of the
ATPase activity of SecA [105,109]. Inhibition by azide
can be overcome by point mutations [106,123] that render
the ATPase activity of SecA less sensitive to this compound. In vivo, both the pmf and the hydrolysis of ATP
by SecA are needed for e¤cient protein secretion
[124,125]. In vitro, the pmf only contributes to the e¤ciency of translocation and appears not to be essential
[105,122].
In E. coli, the cytosolic level of SecA is regulated
through an autoregulatory mechanism involving the bind-
443
Fig. 3. Model for SecA-driven protein translocation across the membrane. ADP-bound SecA and precursor are associated on the membrane
at the SecYEG complex (1a). ADP is exchanged for ATP, changing the
conformation of SecA and driving membrane insertion (2). Upon hydrolysis of ATP, SecA deinserts from the membrane and releases the bound
precursor (3a). This step is blocked by azide, non-hydrolyzable analogs
of ATP, and in the K106N mutant that is unable to hydrolyze ATP.
Membrane-bound SecA can exchange with the free cytosolic pool of
SecA, but this event requires the hydrolysis of another ATP molecule
(3b). Finally, SecA can re-bind the precursor and return to the initial
position (1b).
ing of SecA to its own mRNA [126]. The E. coli SecA
possess an RNA helicase activity, and it contains the
DEAD motif typical of the helicase superfamily II [127].
The autoregulatory mechanism responds to a high secretory demand. When a secretory defect occurs, an increasing amount of the cellular SecA is associated with precursor proteins, and the repression of the SecA expression is
relieved. Various mutations in Sec proteins that cause a
defect in protein translocation have been reported to result
in an elevated SecA expression. The SecA level in B. subtilis and other Gram-positive organisms seems not to be
controlled by an autoregulatory mechanism. For instance,
in the B. subtilis degU32Hy mutant, which produces high
levels of exoenzyme throughout the exponential and stationary phases of growth, SecA levels are identical to those
observed in the wild-type [128]. The SecA level gradually
increases during the exponential phase of growth, reaching
its maximum at the transition stage to the stationary phase
of growth. As a result, the expression of SecA is coordinately regulated with the production of exoenzymes that
also reaches a maximum at the end of the exponential
phase of growth. Transcription of the bicistronic operon
containing the secA gene occurs from a promoter that
depends on cA , the major vegetative sigma factor. This
promoter is mainly active during the exponential phase
of growth, after which its activity drops to a basal level
[129]. However, due to the stability of SecA, high levels
can be detected far into the stationary phase. Regulation
of the Sec genes has also been studied in Streptomyces
lividans [130] and Streptomyces griseus [131] as a function
of growth conditions. The mRNA levels of secA, but also
of the other Sec genes (secD, secE, secF, and secY) are
almost constant during the early and exponential phases
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of growth, but show a marked decrease at the beginning of
the stationary phase. A full recovery of the mRNA levels
takes place in the late stationary phase concomitantly with
the di¡erentiation of Streptomyces.
SecA seems to in£uence the transcription of other genes
in B. subtilis. The temperature-sensitive secA341 mutation
not only a¡ects the secretion of proteins and related
events, but also blocks transcriptional expression of a
number of cH -dependent genes including kinA, spoOA,
spoVG, phrC and citG [132]. Similar results have been
reported with the secA12 mutant that is not temperature-sensitive [98]. The carboxy-terminus of SecA is highly
conserved among bacterial SecA proteins, and in E. coli it
is involved in the binding of SecB and lipid interactions
(for review see [27]). Since B. subtilis and other Grampositive bacteria lack a SecB homolog, the function of
this cysteine-rich thionine-like domain is unclear (see
also Section 4 on cytosolic factors). In a B. subtilis strain
that expresses a SecA truncate that lacks the carboxy-terminal 22 amino acids, the mrgA, ahpC, ahpF and katA
genes become induced, leading to a situation reminiscent
of a response to oxidative stress [50]. At the same time, a
number of cytosolic and secretory proteins were repressed.
The mechanism by which the regulation of the respective
genes is altered by the SecA truncate is unknown.
5.2. SecYEG forms a protein-conducting channel
Proteins are not secreted directly through the hydrophobic environment of the cytosolic membrane, but through
an aqueous channel formed by the SecY, SecE and SecG
polypeptides [25,133,134].
SecY is the largest subunit of the membrane domain of
the protein translocase (Fig. 4). The secY gene can be
readily identi¢ed in bacteria through the conserved localization in the spc operon, which contains the genes encoding ribosomal proteins [6,135^145]. Involvement of the B.
subtilis SecY in protein secretion could be shown by its
regulated depletion, which strongly a¡ected the growth
and secretion of a number of secretory proteins [141].
Some temperature-sensitive sporulation mutants map in
Fig. 4. Secondary structure prediction of the B. subtilis SecY, SecE and
SecG proteins.
SecY [142]. SecY of B. subtilis is a 423-amino acid polypeptide that, like all other known SecY proteins, is predicted to span the cytoplasmic membrane 10 times (for
review see [143]). The B. subtilis SecY protein can support
the export of some proteins from an E. coli secY temperature-sensitive mutant at the non-permissive temperature,
but the two SecY proteins do not appear to be completely
exchangeable in vivo [135,144] or in vitro [108]. High level
expression of B. subtilis SecY even inhibits growth of E.
coli at the permissive temperature, probably due to titration of its obligatory partner protein SecE (see below).
The sporulation de¢ciency of the B. subtilis SecA12 mutant can be suppressed by mutations in SecY [138]. Strikingly, these mutations localize at sites that correspond to
the prlA suppressor phenotype of E. coli SecY. PrlA suppressors restore the translocation of precursors with a defective signal peptide, by promoting a more stable interaction between SecY and SecA [145]. The interaction
between the S. griseus SecA and SecY has been demonstrated by a ligand a¤nity blot assay [146].
SecE is a small integral membrane protein that is essential for translocation and viability. The E. coli SecE consists of 127 amino acid residues that form three transmembrane segments (TMS). SecE genes of Gram-positive
bacteria have been cloned through their conserved localization in the chromosomal region that contains the nusG
gene that encodes a transcription antitermination factor
[9,147^149]. However, SecE proteins from Gram-positive
bacteria are considerably smaller than the E. coli SecE,
i.e., about 60 amino acid residues long (Fig. 4). Their sequence is homologous to the carboxy-terminal part of the
E. coli SecE, which corresponds to the highly conserved
cytosolic loop region and the third TMS. With respect to
this, the ¢rst two TMS of the E. coli SecE are found to be
non-essential for its function [150^152]. The B. subtilis and
Staphylococcus carnosus SecE can complement cold-sensitive E. coli secE mutants [9,147]. Furthermore, SecE can
accept a number of mutations in the conserved region
before it loses its functionality [152,153].
SecE associates with SecY [154] and prevents SecY from
being degraded by FtsH, an ATP-dependent proteinase
present in the cytoplasmic membrane [155,156]. In E.
coli, uncomplexed SecY is rapidly degraded by FtsH
[154]. Since FtsH is widely distributed in bacteria
[157,158] it likely ful¢lls a general role in the degradation
of uncomplexed SecY. The B. subtilis SecY is readily degraded by FtsH when expressed in E. coli in the absence of
SecE [108]. In vitro translocation experiments using hybrid
SecYEG complexes consisting of E. coli and B. subtilis
subunits have been conducted [108]. This study demonstrated that heterologous SecE^SecY complexes could be
formed, but that these complexes are either non-functional
or less active. This suggests that stabilization of SecY is
not the only function of SecE, but that SecE also contributes to the speci¢city and catalytic activity of the translocase.
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SecG is the third component of the integral membrane
domain of the translocase. In E. coli, a protein termed p12
was found to stimulate SecYE-mediated protein translocation in vitro [10], and appeared identical to band 1, a
protein that co-puri¢es with the SecYE complex [133]. Inactivation of the gene encoding p12 results in a pleiotropic
export defect, and therefore the gene was named secG. By
means of sequence analysis and database searches, putative Gram-positive SecG homologs, which show a weak
but signi¢cant sequence similarity with the E. coli SecG,
have been identi¢ed. One of these genes, the B. subtilis
yvaL, has been functionally characterized and shown to
encode a SecG homolog [12]. The chromosomal localization of the secG gene is not conserved. The E. coli secG
gene is located downstream of ftsH, while the B. subtilis
and M. leprae secG genes are located downstream of the
genes encoding the 10S ribosomal RNA and phosphoenolpyruvate carboxylase, respectively. SecG proteins from
Gram-positive bacteria tend to be shorter than their
Gram-negative counterparts, as the SecG proteins of
Gram-negative bacteria contain a carboxy-terminal extension of about 80 amino acids. SecG consists of two TMS
that are interconnected through a glycine-rich loop (Fig.
4). Unlike SecY and SecE, SecG is not essential for viability and protein translocation [10,12]. In some genetic
backgrounds, a deletion of the secG genes results in a
cold-sensitive growth defect [10]. For the B. subtilis secG
null strain, cold sensitivity is only observed when secretory
stress is imposed, for instance by overproduction of the
precursor of K-amylase, preAmyL [12]. This observation is
consistent with the notion that SecG is not required for
translocation of proteins, but that it contributes to the
e¤ciency of the reaction. In vivo [12] and in vitro
[108,159] studies have demonstrated that the SecG proteins of E. coli and B. subtilis are not readily functionally
exchangeable, similar to the situation of SecY. The B.
subtilis SecG cannot complement the cold-sensitive phenotype of an E. coli secG null strain, while the E. coli SecG
can partially restored growth at low temperature of a B.
subtilis secG null strain under conditions where K-amylase
is overexpressed [12].
Since the B. subtilis SecG cannot complement E. coli
secG null mutants, attempts to clone the gene from B.
subtilis in this way have not been successful. However,
these attempts resulted in the cloning of the B. subtilis
gene for phosphatidylglycerol synthetase, pgsA [160]. The
same strategy identi¢ed the B. subtilis scgR, a gene that
regulates the cardiolipin^phosphatidylglycerol ratio [161].
Anionic phospholipids are essential for protein translocation and SecA ATPase activity of the B. subtilis translocase [162]. An increase in the amount of anionic phospholipids in the membrane improves the translocation
e¤ciency, and possibly explains the compensatory e¡ect
of phospholipid biosynthetic genes on the secretion defect
of the secG null strain [163]. In addition to anionic phospholipids, non-bilayer lipids appear to be essential for
445
Fig. 5. Secondary structure prediction of the B. subtilis SecDF protein.
In B. subtilis, the secDF gene encodes a single continuous open reading
frame of 571 amino acids with homology to the E. coli SecD and SecF
proteins. In most bacteria, SecD and SecF are expressed as two individual proteins.
protein translocation in B. subtilis [162], while in E. coli
they only contribute to the e¤ciency of translocation
[164]. The reconstituted B. subtilis translocase functions
optimally in a lipid environment that mimics the polar
headgroup composition of the native Bacillus membrane,
i.e., about 30% phosphatidylethanolamine and 70% phosphatidylglycerol [162].
The puri¢ed B. subtilis SecYE complex forms ring-like
structures in detergent solution and after reconstitution in
membranes [165]. This structure most likely corresponds
to an E. coli SecYEG dimer, which represents an idle,
non-assembled state of the translocase [25]. In E. coli,
pore formation requires the activation of SecA by a precursor protein and ATP. Under those conditions, the SecA
dimer recruits four SecYEG units to form an oligomer
with a large central opening that may function as the
protein-conducting pore [25]. The B. subtilis SecYEG complex likely functions in a similar manner.
5.3. SecDF are accessory membrane components
SecD and SecF are two large integral membrane proteins with an accessory function in protein translocation
[8]. In E. coli, these two proteins have been implicated in
the modulation of the catalytic cycle of the SecA protein
[119,120,166] while another study suggests that SecD and
SecF are needed for the maintenance of the pmf [167].
SecD and SecF form a complex with YajC, a membrane
protein without a known function in protein translocation.
This heterotrimeric SecDFYajC complex loosely associates
with SecYEG to form a supramolecular translocase complex [166]. In B. subtilis and some other organisms, SecD
and SecF are fused into a single membrane protein of 571
amino acids that is predicted to span the membrane 12
times (Fig. 5) [11]. However, in B. halodurans, SecD and
SecF are two separate membrane proteins [16]. It has been
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suggested that the genome of Lactococcus lactis lacks a
clear SecDF homolog [17], which so far is the only exception of a bacterium that may lack SecD and SecF. Unlike
the other Sec genes, which are widely dispersed throughout the chromosome, secD and secF of E. coli are organized in an operon together with the yajC gene and located
downstream of two genes involved in guanosine synthesis
[8]. The YajC homolog of B. subtilis is encoded by the
yrbF gene and separated from secDF by two pairs of divergently transcribed genes [11].
The B. subtilis SecDF protein is unable to complement
cold-sensitive E. coli secD and secF mutants [11]. Like the
secG null strain, depletion of SecDF in B. subtilis results in
a weak cold sensitivity of growth and a reduced e¤ciency
of the secretion of neutral protease. Only under conditions
of high level overproduction of a secretory protein, i.e.,
pre-K-amylase, a strong cold sensitivity of growth and a
secretion defect became apparent. Therefore, it appears
that SecDF only contributes to the e¤ciency of protein
secretion. The B. subtilis SecDF is maximally expressed
at the onset of the post-exponential phase of growth
when cells are cultured on rich medium [11]. On minimal
medium, however, expression appears rather constant
throughout the growth.
5.4. YidC and other factors
Recently, an integral membrane protein termed YidC
was found in E. coli, which is involved in the insertion
of hydrophobic sequences into the membrane [168].
YidC associates with the SecYEGDF^YajC complex
[168], but can also act independently of SecYEG for the
insertion of a special subset of membrane proteins [169].
YidC is an essential protein in E. coli [169] and is homologous to OXA1p of mitochondria [170] and ALBINO3 in
chloroplasts [171]. In B. subtilis two homologs of YidC are
found: SpoIIIJ and YqjG. Both proteins have a lipoprotein signature and are predicted to span the membrane ¢ve
times. Mutations in the SpoIIIJ protein were found to
block the sporulation at an intermediate stage, probably
by interfering with the cG function [172]. A yqjG disruption mutant constructed during the B. subtilis functional
analyses program (Japan Functional Analysis Network for
Bacillus subtilis) (http://bacillus.genome.ad.jp/BSORF-DB.
html) showed that this gene is not essential. Therefore,
SpoIIIJ and YqjG probably are proteins with overlapping
functions necessary for the insertion of speci¢c subsets of
membrane proteins or other so far unknown functions.
lacking signal peptidase activity are normally not viable
[173,174]. The ¢rst type I signal peptidase from a Grampositive organism was found by using L-lactamase
equipped with a non-natural signal peptide, which is
slowly processed in B. subtilis but not at all in E. coli
[175]. By using a halo assay that allows for screening of
colonies that produce high amounts of processed enzyme
the sipS gene was identi¢ed. SipS is a protein of 184 amino
acids that spans the membrane once and exposes its catalytic site to the outside of the cell. Surprisingly, deletion of
the sipS gene had no e¡ect on the growth of B. subtilis,
implying the presence of additional signal peptidases. By
various methods, four additional type I signal peptidases
(sipT, sipV, sipU, sipW) have been identi¢ed in B. subtilis
[174]. Furthermore, a sixth signal peptidase (sipP), located
on a plasmid, was found in some Bacillus strains [176].
The reason for this redundancy is not entirely clear but
some of the signal peptidases di¡er in speci¢city [177,178].
B. subtilis strains that lack up to four signal peptidases are
still viable, but the combination of a sipS and sipT deletion appears lethal [174]. The latter two genes are regulated in a temporal fashion, whereas sipV and sipU are
transcribed at a low constitutive level under all conditions
[178]. The plasmid-borne SipP can rescue the otherwise
lethal combination of sipS and sipT deletions when expressed at a high level [179]. Four of the ¢ve type I signal
B. subtilis peptidases, SipS, T, U, and V, closely resemble
each other and bear the same membrane topology. These
sip genes are part of the P-type class of signal peptidases
usually found in bacteria, whereas SipW belongs to the
family of ER-type signal peptidases mostly found in archaea and the endoplasmic reticulum of eukaryotes [180].
The main di¡erences between the two subfamilies reside in
the spacing of boxes of conserved amino acid residues and
a single change of a conserved lysine for a histidine [181].
SipW is involved in the processing of a limited number of
sporulation-speci¢c precursors, such as the TasA protein,
which is secreted and subsequently incorporated into
spores [182^185]. Since SipW carries out such a specialized
task, it is dispensable for vegetative growth [186]. In
Gram-positive bacteria, the occurrence of multiple type I
signal peptidases within a single species is very common.
Two type I signal peptidases have been cloned and sequenced from B. amyloliquefaciens [187], and four distinct
type I signal peptidase have been cloned from S. lividans
[188^190]. The exact reason for this multiplicity is unclear,
but this phenomenon could relate to a certain degree of
speci¢city and/or cell cycle-related processes.
6.2. Type II signal peptidase and other proteolytic enzymes
6. Signal peptidases
6.1. Type I signal peptidase
Processing is a prerequisite for the release of the precursor protein from the membrane, and therefore cells
A Gram-positive type II signal peptidase gene was ¢rst
cloned from Staphylococcus aureus by complementation of
a conditionally lethal E. coli mutant [191]. It is an 18.3kDa membrane protein with four TMS. In contrast to the
large number of type I signal peptidases, the B. subtilis
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chromosome contains only a single signal peptidase II
gene, lsp [192]. Deletion of this gene yielded a strain that
is viable at intermediate, but not at low or high growth
temperatures. Lsp is, however, essential for E. coli and
needed for the processing and sorting of lipoproteins of
the outer membrane [37]. The B. subtilis lsp null strain
accumulates lipomodi¢ed proteins with molecular masses
corresponding to precursor but also to mature forms of
PrsA, a lipoprotein involved in maturation of some secretory proteins [193]. Disruption of the gene encoding prelipoprotein diacylglycerol transferase, lgt, results in secreted PrsA and L-lactamase proteins that are not
lipomodi¢ed. These proteins were, however, normally processed and released into the medium [194]. The B. subtilis
strain lacking the lgt gene was therefore normally viable.
Upon processing, the mature region of the precursor
protein is released and the remaining signal peptide is
degraded by signal peptide peptidases. The fate of the
signal peptide after processing is not exactly known, but
they are likely degraded by proteases. B. subtilis contains
two cytosolic proteins, SppA and TepA, with similarity to
the signal peptide peptidase of E. coli. Deletion of the
sppA gene from the B. subtilis chromosome resulted in a
mild secretion defect only under conditions of hypersecretion, whereas the deletion of tepA caused a secretion defect
also under normal conditions [195]. TepA has been named
`translocation enhancing protein' as in its absence a translocation defect occurs which may be caused by the accumulation of released signal peptides that could potentially
interfere with protein secretion. Alternatively, TepA is involved in clearing of the translocation machinery of
wrongly folded precursor proteins. Proteolytic activity of
SppA and TepA against signal peptides or proteins remains to be demonstrated.
7. Extracellular factors
Once the secretory protein starts to emerge at the extracellular side of the translocase, it needs to fold into its
active conformation. Since B. subtilis contains a multitude
of extracellular proteinases, newly secreted proteins rapidly need to acquire their active, native conformation, as
the unfolded state is highly prone to degradation.
7.1. Competition between folding and degradation
A comprehensive genetic screen of mutants that show a
reduced e¤ciency of protein secretion has led to the identi¢cation of the prsA gene [196]. PrsA is a lipoprotein of
292 amino acid residues that resides on the extracellular
side of the membrane. The protein shares 30% identity
with PrtM of L. lactis, a protein that is essential for the
maturation of the secreted proteinase PrtP [197,198]. PrsA
mutants exhibit pleiotropic secretion defects that concern
many exoproteins, including the protease subtilisin and K-
447
amylase [199,200]. Mutants defective in lsp and lgt, genes
encoding proteins that are involved in the lipomodi¢cation
of PrsA, also a¡ect secretion [179,194]. Overproduction of
PrsA results in increased yields of homologous as well as
heterologous secretory proteins [200]. Secreted proteins in
a prsA mutant are subjected to massive degradation [199]
suggesting that PrsA acts as an extracellular chaperone
that assists secreted proteins in acquiring their native conformation. PrsA and PrtM bear some sequence similarity
to a small peptidyl-prolyl cis/trans isomerase from E. coli,
indicating that they may belong to this family of chaperones [201]. So far, a peptidyl-prolyl cis/trans isomerase
activity of PrsA has not been biochemically demonstrated.
Alternatively, PrsA may protect secreted proteins against
degradation by acting as a proteinase inhibitor or by
shielding the newly secreted proteins from the proteinases.
For an e¤cient acquisition and maintenance of a native
conformation, many proteins need the formation of disul¢de bonds. In vivo, this process is catalyzed by thiol-disul¢de oxido-reductases. In B. subtilis, there is only one
secreted protein known that contains a disul¢de bond.
This is ComC, a protein involved in natural competence
[202]. For the heterologous expression and secretion of the
E. coli alkaline phosphatase in B. subtilis, BdbB and BdbC
are needed for stability and thus presumably for the formation of the two disul¢de bonds [203]. E. coli TEM-Llactamase with one disul¢de bond only requires BdbC. On
the basis of homology with the E. coli Dsb proteins, BdbB
and BdbC have been putatively designated membrane-localized, thiol-disul¢de oxido-reductases. The third putative
enzyme belonging to this group, BdbA, is a secreted protein with a molecular mass of 15 kDa. It is homologous to
Bacillus brevis Bdb, an extracellular protein for which a
thiol-disul¢de oxido-reductase activity has been biochemically demonstrated [204]. BdbA seems not to be needed for
the disul¢de bond formation in alkaline phosphatase or
L-lactamase. Mutation of a single bdb gene in B. subtilis
does not result in an increased sensitivity to reducing
agents as observed for E. coli [203]. Although this may
relate to some redundancy in the functioning of the Bdb
proteins, it likely re£ects the general lack of disul¢de
bonds in secreted proteins of Bacillus.
7.2. Passage through the cell wall
The cell wall of Gram-positive bacteria is a large structure that surrounds the cell, which besides acting as a
molecular sieve signi¢cantly in£uences the folding and
stability of newly translocated secretory proteins. A major
factor important for the post-translocational protein folding is the presence of metal ions in the cell wall area. Two
secreted proteins of Bacillus licheniformis and B. subtilis,
K-amylase and levansucrase, exhibit a weak a¤nity for
calcium, and the presence of this divalent cation facilitates
their folding [56,57,59]. Mutant proteins that exhibit a
greater dependence on calcium for their folding have
FEMSRE 722 20-8-01
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K.H.M. van Wely et al. / FEMS Microbiology Reviews 25 (2001) 437^454
been characterized [60]. These proteins are in general produced with a lower yield and are more susceptible to proteolysis. Ideally, translocated proteins and calcium should
be in close vicinity to ensure rapid folding. To guarantee
the presence of metal ions close to the cellular membrane,
the cell wall seems to function as a reservoir for calcium in
some organisms [56]. Removal of the calcium, either by
chelators or by breakage of the cell wall, dramatically
decreases the release of folded protein [59]. Other organisms, e.g. Mycoplasma and Mycobacterium species, manage to secrete proteins without a cell wall. In this case, the
metal ions may be provided directly by the exterior environment.
Following their secretion across the cytoplasmic membrane, processed secretory proteins of B. subtilis must fold
into their native conformation prior to translocation
through the cell wall and release into the culture medium.
Proteins that fail to fold in time are prone to digestion by
cell wall-associated and/or secreted proteases in the supernatant extracellular proteases [205,206], an observation
that is made frequently in the case of translocated (heterologous) proteins [199,207,208]. A possible cause for the
rapid degradation is the serine protease WprA that is
bound to the cell wall. A reduced expression of WprA
results in an increased yield of secreted K-amylase [209].
Also the presence of a prosequence can increase the yield
of secreted proteins [206]. The prosequence may a¡ect
secretion either by acceleration of extracellular folding or
by faster passage through the cell wall. In this respect, the
negative charge of the cell wall was found to slow down
the secretion of positively charged proteins [210]. One of
the suppressors of a PrsA de¢ciency localizes in the dlt
operon whose gene products are involved in the D-alanylation of teichoic acids [211]. It has been shown that inactivation of the dlt genes stabilizes an otherwise unstable
PrsA mutant protein and rescues various heterologous
proteins from degradation. These data indicate that the
main in£uence of the increased net negative charge of
the wall caused by the absence of D-alanine is to increase
the rate of post-translocational folding of exported proteins. Taken together, these data demonstrate that interactions with the cell wall are critical for the folding and
stability of newly translocated proteins and most likely
are a major bottleneck in the secretion of heterologous
proteins, which normally are not adapted to such interactions.
functions and working mechanisms. Complementation
studies, however, indicate species-speci¢c functioning of
the translocase subunits. These may relate to optimized
interactions between the various subunits of the translocase and/or the precursor proteins of the respective organisms. The mechanisms by which precursor proteins are
targeted to the translocase appear di¡erent for the two
groups of bacteria. For instance, the chaperone-mediated
pathway seems to be underdeveloped if not absent in the
Gram-positive microorganisms. On the other hand, an
elaborate system has evolved around the signal recognition
particle. On the external cell surface, the release and folding of the secreted proteins has to compete with a degradation pathway that guarantees an optimal quality of the
secreted proteins. In this respect, other Gram-positive bacteria such as S. carnosus and L. lactis are less proteolytic.
In addition, protein translocation in B. subtilis seems to be
regulated by di¡erent mechanisms than in E. coli. B. subtilis develops a high secretion capacity near the end of the
exponential phase of growth. Furthermore, at the onset of
sporulation other requirements may exist, as some of the
mutations described are located in Sec proteins. Little is
known about the mechanisms underlying this speci¢c subtype of the secretion pathway.
B. subtilis is well amenable to proteome analysis by twodimensional protein gel electrophoresis [35,212^214].
Extracellular proteins (secretome analysis) have been identi¢ed by matrix-assisted laser desorption ionization^time
of £ight mass spectrometry and amino acid sequence analysis. This now provides a powerful tool for the further
analysis of the role of the Sec system in protein secretion
and as yet unidenti¢ed factors that may arise from functional analysis studies of the genes with unknown functions. Future work may yield new insights into the unique
aspects of the Gram-positive protein secretion pathway
and facilitate a better exploitation of these organisms for
the high level production of heterologous proteins by engineering of the secretion pathway.
8. Concluding remarks
References
Comparison between the protein translocation pathways in Gram-positive and Gram-negative bacteria has
de¢ned common principles underlying protein translocation, but also revealed some striking di¡erences. The central components of the protein translocation machinery
show a high degree of conservation, suggesting similar
Acknowledgements
We would like to thank all collaborators in the EC
project on the optimization of Bacillus protein secretion.
This work was supported by CEC Biotech Grants BIO2
CT 930254, BIO4 CT 960097 and QLK3-CT-1999-00413.
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