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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 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 438 438 439 439 439 441 441 442 442 444 445 446 446 446 * 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 20-8-01 438 K.H.M. van Wely et al. / FEMS Microbiology Reviews 25 (2001) 437^454 . . . . . 446 447 447 447 448 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 ....... ....... ....... ....... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . [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 20-8-01 440 K.H.M. van Wely et al. / FEMS Microbiology Reviews 25 (2001) 437^454 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. FEMSRE 722 20-8-01 K.H.M. van Wely et al. / FEMS Microbiology Reviews 25 (2001) 437^454 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 FEMSRE 722 20-8-01 442 K.H.M. van Wely et al. / FEMS Microbiology Reviews 25 (2001) 437^454 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- FEMSRE 722 20-8-01 K.H.M. van Wely et al. / FEMS Microbiology Reviews 25 (2001) 437^454 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 FEMSRE 722 20-8-01 444 K.H.M. van Wely et al. / FEMS Microbiology Reviews 25 (2001) 437^454 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. FEMSRE 722 20-8-01 K.H.M. van Wely et al. / FEMS Microbiology Reviews 25 (2001) 437^454 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 FEMSRE 722 20-8-01 446 K.H.M. van Wely et al. / FEMS Microbiology Reviews 25 (2001) 437^454 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 FEMSRE 722 20-8-01 K.H.M. van Wely et al. / FEMS Microbiology Reviews 25 (2001) 437^454 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 20-8-01 448 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. [1] Oliver, D.B. and Beckwith, J. 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