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1 Institute of Genetics, University of Bayreuth, Bayreuth, Germany
2 Infection Pathogenesis Laboratory, Department of Viral Diseases and Immunology, National Public Health Institute, Helsinki, Finland
Correspondence
Thomas Wiegert
thomas.wiegert{at}uni-bayreuth.de
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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W regulon is induced by different stresses that most probably affect integrity of the cell envelope. The activity of the extracytoplasmic function (ECF) sigma factor W is modulated by the transmembrane anti-sigma factor RsiW, which undergoes stress-induced degradation in a process known as regulated intramembrane proteolysis, finally resulting in the release of W and the transcription of W-controlled genes. Mutations in the ecsA gene, which encodes an ATP binding cassette (ABC) of an ABC transporter of unknown function, block site-2 proteolysis of RsiW by the intramembrane cleaving protease RasP (YluC). In addition, degradation of the cell division protein FtsL, which represents a second RasP substrate, is blocked in an ecsA-negative strain. The defect in W induction of an ecsA-knockout strain could be partly suppressed by overproducing RasP. A B. subtilis rasP-knockout strain displayed the same pleiotropic phenotype as an ecsA knockout, namely defects in processing 
-amylase, in competence development, and in formation of multicellular structures known as biofilms.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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W constitute an antibiosis regulon (Helmann, 2002
, 2006) that responds to a variety of envelope stresses such as certain antibiotics (Cao et al., 2002) and antimicrobial peptides (Pietiäinen et al., 2005; Butcher & Helmann, 2006), and also alkaline shock and phage infection (Wiegert et al., 2001). Its activity is modulated by RsiW, a transmembrane anti-sigma factor that sequesters and inactivates W. Upon a stress signal, RsiW is degraded by the mechanism of regulated intramembrane proteolysis (RIP). In a concerted action, at least three proteases in three different compartments of the cell degrade the RsiW anti-sigma factor in a sequential manner, finally resulting in the release of W and the transcription of W-controlled genes. The first protease that has been identified to be involved in that process is RasP (regulating anti-sigma factor protease; formerly YluC). RasP belongs to the group of zinc-dependent intramembrane cleaving proteases (iClips) and cleaves RsiW in its transmembrane domain after the extracytoplasmic part of the anti-sigma factor has been removed by a site-1 protease (Schöbel et al., 2004). The site-2 degradation step catalysed by RasP uncovers a cryptic proteolytic tag that ensures further degradation of the RsiW remnant by cytoplasmic proteases, mainly ClpXP (Zellmeier et al., 2006). These two steps are related to RIP of the E anti-sigma factor RseA of Escherichia coli (Ades, 2004; Alba & Gross, 2004). RasP is an orthologue of E. coli RseP, and the concept of a cryptic proteolytic tag recognized by ClpXP was first described for that system (Akiyama et al., 2004; Flynn et al., 2004). However, there is a marked difference in the site-1 proteolytic step, because no dependency on B. subtilis Deg (Htr) proteases was found and the inducing stress is apparently different. The probable site-1 protease of RsiW was identified by two different approaches. First, B. subtilis clones suppressing the toxic effect of SdpC, a protein which is involved in a cannibalism process of killing sibling cells upon entry into the sporulation programme, were mapped to a gene now renamed as prsW (protease responsible for activating W, formerly ypdC) (Ellermeier & Losick, 2006). It has been shown that suppression is due to constitutive activation of the W regulon, which is known to confer resistance to the SdpC toxin in cells lacking the SdpI immunity protein (Butcher & Helmann, 2006). Second, a transposon screen with a reporter consisting of GFP fused to the amino terminus of RsiW was performed, and several transposon insertions in the prsW gene were identified that stabilize GFP–RsiW and prevent site-1 cleavage of the anti-sigma factor (Heinrich & Wiegert, 2006). In addition to prsW, transposon insertions in a second gene locus, ecsAB, show marked stabilization of the fluorescent reporter and prevent induction of W. Here, we show that mutations in the ecsA gene block site-2 proteolysis of RsiW by the intramembrane-cleaving protease RasP.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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. E. coli and B. subtilis strains were grown aerobically at 37 °C in Luria–Bertani (LB) medium. When necessary, LB medium was supplemented with ampicillin (100 µg ml–1), phleomycin (1 µg ml–1), neomycin (10 µg ml–1), spectinomycin (100 µg ml–1), chloramphenicol (10 µg ml–1), tetracycline (10 µg ml–1) or erythromycin (1 µg ml–1 or 100 µg ml–1). Xylose was added at a concentration of 2 % and vancomycin at a concentration of 2 µg ml–1 where indicated. Pellicle formation experiments were performed according to a published procedure (Branda et al., 2004). A 50 µl volume of mid-exponential-phase culture in LB medium was inoculated into 10 ml minimal MSgg medium [5 mM potassium phosphate (pH 7), 100 mM MOPS (pH 7), 2 mM MgCl2, 700 µM CaCl2, 50 µM MnCl2, 50 µM FeCl3, 1 µM ZnCl2, 2 µM thiamine, 0.5 % (v/v) glycerol, 0.5 % glutamate, 50 µg tryptophan ml–1, 50 µg phenylalanine ml–1 and 50 µg threonine ml–1] and incubated at 28 °C. For colony architecture analysis, 3 µl LB precultures were spotted onto minimal MSgg agar plates and incubated at 28 °C.
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-amylase assays, and Northern and Western blot analysis.-amylase activities of culture supernatants were measured as described previously (Wiegert et al., 2001; Pummi et al., 2002). Northern blot analysis was performed according to a published procedure (Homuth et al., 1997) with antisense RNAs against yuaG, pbpE and yhaU (Zellmeier et al., 2006). Western blots were performed as described previously (Homuth et al., 1996) using a semi-dry blotting procedure (Bio-Rad, Trans Blot SD). Blots were developed with polyclonal antibodies against RsiW at a dilution of 1 : 4000, B. subtilis FtsH and HtpG (1 : 10 000), and the FLAG epitope (1 : 5000).
NaOH-shock experiments and preparation of B. subtilis cell fractions.
NaOH-shock experiments were performed as described previously (Schöbel et al., 2004). Cells were harvested by centrifugation, washed, and suspended in 1 ml cold disruption buffer (50 mM Tris/HCl, 100 mM NaCl, pH 7.5) containing the Complete protease inhibitor cocktail (Roche). Samples were adjusted to the same OD578 by dilution with cold disruption buffer. Cell suspensions (1 ml) were sonicated (Cell Disrupter B15, Branson) on ice. A 100 µl aliquot was removed (whole-cell fraction, W) and the remaining 900 µl was centrifuged at 5000 g for 15 min at 4 °C to remove cell debris. Then, 800 µl of the supernatant was ultracentrifuged at 45 000 g for 1 h at 4 °C. The supernatant (soluble fraction, S) was removed and the resulting membrane pellet (membrane fraction, M) was washed with 500 µl disruption buffer, ultracentrifuged again (45 000 g, 30 min, 4 °C), dissolved in 100 µl Laemmli buffer (Sambrook & Russell, 2005) and heated for 5 min at 95 °C. The protein content of the W and S fractions was estimated by the Bradford method, and 10 µg total protein was loaded in each lane for SDS-PAGE and Western blotting. A volume equivalent to the S fraction was loaded for the M fraction, i.e. one-eighth of the volume of the S fraction, containing 10 µg soluble protein.
Pulse–chase experiments, and sporulation and competence tests.
Pulse–chase experiments were performed as described elsewhere (Leskela et al., 1999) using strain IH6531 and derivatives thereof obtained by transformation of chromosomal DNA of knockouts of ecsA (1012 ecsA : : spec), rasP (1012 rasP : : tet) and sigW (sigW : : erm of HB4246; Huang et al., 1998). Competence was analysed as the transformation efficiency (Kontinen & Sarvas, 1988), and the sporulation frequency according to a standard procedure (Nicholson & Setlow, 1990) using strain IH8209 and respective knockouts (see above).
Construction of B. subtilis ecsA- and rasP-negative strains, and SPP1 phage transduction.
The ecsA and rasP genes were inactivated by a deletion, and by insertion of a spectinomycin-resistance gene, respectively. To delete ecsA, the gene and 5'/3'-flanking regions were PCR-amplified using primers (1) 5'-GGCCATGTCGACCACCTCATTTGACAATTTGCTTCA-3' and (2) 5'-GGCCATAAGCTTCCTGCTTCAAGTAAGGCTCCAT-3' and chromosomal DNA of B. subtilis 1012 as a template. The PCR product was restricted with SalI/HindIII and ligated to the pBR322 vector fragment cut with the same enzymes, yielding plasmid pBRecsA. An internal 450 bp ecsA fragment was replaced by restricting pBRecsA with BamHI/SacI and inserting the spectinomycin-resistance gene cut with the same enzymes that had been PCR-amplified with primers (3) 5'-GGCCATAAGCTTGGATCCATCGATTTGACATTTTTCTTGTGGA-3' and (4) 5'-GGCCATAAGCTTGAGCTCGTAAGCACCTGTTATTGCAATAAAA-3' and plasmid pK2-spec (Härtl et al., 2001) as a template, resulting in plasmid pJH07. The ecsA : : spec construct was PCR-amplified with the above primers (1) and (2), and the product was used to transform B. subtilis 1012. Chromosomal DNA of transformants (1012 ecsA : : spec) resistant to spectinomycin was checked by PCR and by Southern blotting using a DIG-labelled DNA probe for ecsA according to standard procedures (Sambrook & Russell, 2005). The ecsA knockout was combined with a transcriptional fusion of the W-controlled yuaF promoter to lacZ by transforming chromosomal DNA of 1012 ecsA : : spec into B. subtilis JAH12, which is a derivative of BFS233 containing the empty xylose control system of plasmid pX (Kim et al., 1996) integrated in amyQ. As it was not possible to transduce the rasP : : tet construct into B. subtilis strain NCIB3610, a rasP : : spec construct was cloned by replacing the ecsA-5' and ecsA-3' regions of pJH07 with the respective rasP up- and downstream regions that were PCR-amplified using primers (5) 5'-GGCCATGTCGACTGTGATCGCACAGCTCGGAAC-3', (6) 5'-GGCCATGGATCCTATAACTGTATTCACGAACATACCA-3', (7) 5'-GGCCATGAGCTCTTGTCACATGGAACGATATCCAG-3' and (8) 5'-GGCCATGAATTCCCTCATCGCGAACAAGCGAAG-3', resulting in plasmid pJH08. B. subtilis 1012 rasP : : spec was constructed as described above.
B. subtilis NCIB3610 does not become competent; therefore, respective knockouts were introduced via SPP1 phage transduction as described elsewhere (Kearns & Losick, 2003).
Complementation and site-directed mutagenesis of ecsA.
To complement the B. subtilis ecsA : : spec deletion strain, the ecsA gene was ectopically expressed under IPTG control. The ecsA gene was PCR-amplified with primers (9) 5'-GGCCATAGATCTATGTCTCTGCTATCGGTAAAAGAC-3' and (10) 5'-GGCCATGCATGCTTATTCATGGCCAGCGTCTTCC-3', restricted with BglII and SphI, and ligated to the vector fragment of plasmid pAL-FLAGrsiW (Schöbel et al., 2004) cut with BamHI and SphI. The resulting plasmid pJH67 encodes a translational fusion of the 3xFLAG epitope tag to the amino terminus of EcsA, which was integrated at the lacA locus using B. subtilis strain 1012 amyE : : PyuaF-lacZ lacA : : spec (Schöbel et al., 2004). For a double crossover event, transformants were screened for erythromycin resistance and spectinomycin sensitivity, resulting in strain 1012 amyE : : PyuaF-lacZ lacA : : pAL-FLAGecsA. Finally, the ecsA gene was deleted by transformation of chromosomal DNA of the 1012 ecsA : : spec strain. The resulting strain was named JAH67. An allele of ecsA with a mutation of the catalytic glutamate residue of the Walker B ATPase motif to glutamine (E160Q) was constructed by a two-step PCR mega-primer method, essentially as described previously (Schöbel et al., 2004), using primer (11) 5'-CCTGCGCTCTACATTATTGATCAGCCTTTTCTAGGGCTTGATC-3' and primers (9) and (10), yielding plasmid pJH68, which was then transformed into B. subtilis as described above for pJH67. The resulting strain (1012 ecsA : : spec amyE : : PyuaF-lacZ lacA : : pAL-FLAGecsAE160Q) was named JAH68.
Construction of a B. subtilis GFP–FtsL reporter fusion.
To test for stability of FtsL, a fusion of GFP to the amino terminus of FtsL was constructed, essentially as described previously (Zellmeier et al., 2006). The ftsL coding region was PCR-amplified using primers (12) 5'-GGCCATAGATCTATGAGCAATTTAGCTTACCAACCAGAG-3' and (13) 5'-GGCCATAGATCTAGCGCTTCATTCCTGTATGTTTTTCACTTT-3' and chromosomal DNA of B. subtilis 1012 as a template, and the product was restricted with BglII and ligated into plasmid pTW700 cut with the same enzyme. Correct orientation of the insert was checked by restriction analysis and DNA sequencing, and the resulting plasmid was named pJH66.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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W-controlled genes). Different transposon insertions in ecsA or ecsB causing stabilization of the GFP–RsiW reporter and abolishing induction of a transcriptional fusion of lacZ to the strong W-controlled yuaF promoter were obtained. These genes encode the ABC and the transmembrane part of an ABC transporter that has been characterized previously (Leskela et al., 1996), orthologues of which can be found in a great variety of bacterial species. However, the function of EcsAB is still unknown. To analyse the obvious influence of the EcsAB ABC transporter on degradation of RsiW, a B. subtilis ecsA-knockout mutant was constructed by replacing the major part of ecsA by a spectinomycin-resistance gene (ecsA : : spec). When compared with the wild-type, the ecsA-knockout strain did not induce the yuaF–lacZ reporter following alkaline shock, a stress condition that strongly activates the W regulon. (Fig. 1a, lanes 1 and 2 compared with 7 and 8). Northern blot analysis with riboprobes against the W-controlled genes yuaG and pbpE revealed that the ecsA : : spec strain is completely unable to induce the W regulon, similar to rasP- or sigW-knockout strains (Fig. 1b). In contrast, alkali induction of the W-independent yhaU gene remained unaffected.
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W in the ecsA : : spec strain is due to a polar effect on downstream genes, ecsA was ectopically expressed as an amino-terminally 3xFLAG epitope-tagged protein under IPTG control. Alkali induction of the yuaF reporter fusion could be measured in the absence of IPTG (Fig. 2a, lanes 1 and 2), whereas full induction was restored in the presence of IPTG (Fig. 2a, lanes 3 and 4). To examine whether the effect of EcsA on W induction was due to the absence of its catalytic activity or the absence of EcsA protein, an ecsA allele mutated in the catalytic Walker B ATPase domain (E160Q) was expressed. Whereas both 3xFLAG–EcsA and 3xFLAG–EcsA-E160Q were detectable in Western blots after IPTG addition (Fig. 2b), the E160Q mutant was unable to induce the yuaF reporter (Fig. 2a, lanes 5–8). Spot-on-lawn assays according to a published procedure (Butcher & Helmann, 2006) showed that a sublancin-producing strain spotted onto a lawn of a sublancin-negative ecsA : : spec strain generates a larger halo than on a lawn of the corresponding ecsA wild-type strain, indicating that W-mediated sublancin immunity is absent in the ecsA-negative strain. Similarly, treatment with vancomycin, an antibiotic that targets cell wall biosynthesis and induces the B. subtilis W regulon (Cao et al., 2002), did not induce the W-controlled pbpE and yuaG genes in an ecsA-negative strain, in contrast to the ecsA wild-type (data not shown). These experiments clearly demonstrate that EcsAB ABC-transporter activity is crucial for induction of W, and that the failure of induction is not limited to specific W-inducing stress signals.
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W, and the strong fluorescence of the GFP–RsiW reporter in transposon-insertion strains, suggested that stress-induced intramembrane proteolysis of RsiW is impaired in the absence of EcsAB activity. Therefore, the ecsA : : spec mutation was introduced into strains expressing GFP–RsiW and a GFP–RsiW
1 reporter, respectively. GFP–RsiW1 is a constitutive substrate for RasP and not dependent on site-1 proteolysis for degradation (Zellmeier et al., 2006). Colonies of both strains were highly fluorescent, whereas in the ecsA wild-type background only weak fluorescence was visible (data not shown). In Western blots, the site-1-clipped truncated form of GFP–RsiW was the main form detectable in membrane fractions (Fig. 3a, compare lanes 3 and 9). GFP–RsiW1, which is absent in the wild-type due to proteolysis by RasP and further cytoplasmic proteases (Zellmeier et al., 2006), was stabilized in the ecsA : : spec background (Fig. 3a, lanes 6 and 12).
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, lanes 3–6). For the wild-type control, the full-length RsiW was detectable only in non-shocked cells (Fig. 3b, lanes 1, 2). In conclusion, RasP-catalysed intramembrane site-2 proteolysis of RsiW does not take place in the absence of EcsAB activity, concomitantly with deregulated site-1 proteolysis that takes place without an external stress.
The intramembrane cleaving protease RasP is inactive in a B. subtilis ecsA-negative strain
To investigate whether the inability of RasP to attack site-1-clipped RsiW or RsiW1 in the absence of EcsAB is substrate-specific for RsiW, degradation of a second RasP substrate was analysed. Recently, it has been demonstrated that FtsL, a transmembrane protein of the B. subtilis cell division machinery, is attacked by RasP (Bramkamp et al., 2006). Therefore, in analogy to the GFP–RsiW reporter, a GFP–FtsL fusion was constructed and expressed in different genetic backgrounds. Stability of the GFP–FtsL reporter protein was monitored by using the fluorescence of whole cells grown on LB agar plates. Fluorescence in the wild-type and prsW-knockout background was low, indicating that GFP–FtsL is unstable and degraded in a PrsW-independent manner (Fig. 3c). The rasP-negative and ecsA-negative strains were highly fluorescent, indicating that GFP–FtsL is proteolysed in a RasP-dependent manner, and that this degradation is dependent on EcsAB activity. Interestingly, GFP–FtsL was also stabilized in a clpP-negative background, meaning that the alanine residues in its transmembrane domain constitute a cryptic proteolytic tag, as has been described previously for RsiW (Zellmeier et al., 2006). Taken together, the failure to degrade both RsiW and FtsL suggests that, in the absence of EcsAB activity, RasP is not functional.
There might be two different explanations for this finding. First, that RasP activity itself is directly or indirectly dependent on EcsAB. For example, EcsAB is known to influence correct localization of secretory proteins (Leskela et al., 1999), and therefore could be involved in correct membrane insertion of RasP. Second, that an EcsAB substrate mislocated in the absence of the ABC transporter activity inhibits RasP. To address this question, we overexpressed rasP under the control of a strong xylose-inducible promoter in the ecsA : : spec background and analysed alkali induction of the W-controlled yuaF–lacZ fusion. In the ecsA wild-type background, alkali induction of the reporter fusion was already detectable in the absence of xylose (Fig. 1a, columns 11–14), which is due to leakiness of the xylA promoter (Schöbel et al., 2004). As expected, the isogenic ecsA-knockout strain did not induce lacZ. However, after overproduction of RasP in the presence of xylose, there was a significant increase in β-galactosidase activity after alkaline shock (Fig. 1a, columns 15–18). One possible explanation for these findings is that RasP is competitively inhibited by an EcsAB substrate, and that this is alleviated by increasing the concentration of RasP.
B. subtilis ecsA- and rasP-negative strains display similar pleiotropic phenotypes
A point mutation causing defective ATPase activity of EcsA (ecsA26) had originally been isolated in a screen for B. subtilis mutants unable to secrete overproduced -amylase (AmyQ) (Kontinen & Sarvas, 1988). Further characterization of this mutation revealed a pleiotropic phenotype. The mutant is impaired in processing of pre-AmyQ and three other secretory proteins, and its ability to sporulate and to become competent is decreased (Leskela et al., 1999; Pummi et al., 2002). In addition, a mutant for ecsB has been described that is unable to produce a biofilm (Branda et al., 2004). As RasP seems to be inactive in the ecsA-negative strain, we wondered whether at least some of the defects listed above are correlated with rasP. The rasP : : tet deletion, and sigW : : erm (Huang et al., 1998) and ecsA : : spec as controls, were introduced into strain IH6531, which harbours plasmid pKTH10 for amyQ overexpression (Kontinen & Sarvas, 1988). The AmyQ activity of culture supernatants was determined. For the wild-type strain and the isogenic sigW-negative strain, high AmyQ activity was detectable, whereas the rasP-negative and ecsA-negative strains showed only about 10 % of the wild-type level (Fig. 4a). In pulse–chase experiments it became obvious that almost no processing of preAmyQ to its mature form took place when rasP was deleted (Fig. 4b), as has been described for the ecsA26 mutant strain (Leskela et al., 1999). Next, the transformation efficiency of the respective strains was determined. For both rasP : : tet and ecsA : : spec, transformation efficiency was only about 0.2 % of that of the wild-type; for the sigW-knockout strain, an efficiency 10 % of that of the wild-type was measured. Sporulation tests were performed by plating serial dilutions of samples of cells grown in sporulation medium with and without heat treatment. To our surprise, the sporulation rate of the ecsA : : spec strain, and also of the rasP : : tet strain, was similar to that of the wild-type in three individual experiments. An additional control of an ftsH : : cat strain, which is known to be defective for sporulation (Deuerling et al., 1997), displayed a very low sporulation rate, confirming the validity of the test. Therefore, earlier observations of a sporulation defect in an ecsA-negative strain (Kontinen & Sarvas, 1988) have to be revised. However, we observed rapid lysis of the ecsA : : spec and the rasP : : tet strains upon prolonged incubation in LB medium in stationary phase (data not shown), which might have been the reason for misinterpretation of sporulation rates. The last phenotype that we checked was the ability of respective strains to form structured multicellular communities, known as biofilms. To that purpose, the knockout mutations were introduced into B. subtilis strains 168 and into the undomesticated NCIB3610 isolate, which has been described as forming a characteristic pellicle on liquid medium and having a complex colony morphology on solid medium (Branda et al., 2004). Both the rasP and the ecsA knockouts were clearly handicapped in forming structured biofilms and colonies (Fig. 4c). In summary, a rasP deletion strain displays the same pleiotropic phenotype as an ecsA deletion strain, making it reasonable that at least some of the defects in the ecsA mutant are related to the inactivity of RasP.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). In bacteria, ABC transporters function either as import systems, usually assisted by a soluble or membrane-anchored solute-binding protein, or as exporters of surface components (capsular polysaccharides, lipopolysaccharides, teichoic acid), proteins involved in bacterial pathogenesis (haemolysin, haem-binding protein, alkaline protease), peptide antibiotics, haem, drugs and siderophores. For B. subtilis, at least 78 ABC transporters can be discerned, with 38 importers and 40 exporters. EcsAB has been grouped in a subfamily of extruders related to antibiotic-resistance systems, with similarities to possible efflux pumps for peptide antibiotics (Quentin et al., 1999). The actual substrate for EcsAB and its function is unknown. The most interesting phenotype of an EcsA-negative mutant is its inability to properly secrete AmyQ. The preAmyQ precursor protein remains cell-associated but is accessible to tryptic digestion in protoplasts, pointing to a defect in its processing by signal peptidases. Moreover, overexpression of the sipT signal peptidase gene enhances secretion of AmyQ in the ecsA26 strain (Pummi et al., 2002).
There is a clear defect in the intramembrane cleaving protease RasP activity in the ecsA-knockout background, but the molecular basis for this defect remains enigmatic and at this stage we are only able to speculate. As there are no antibodies to RasP available and we were not able to detect epitope-tagged RasP in membrane fractions of B. subtilis at all, it is not clear whether this defect is due to improper membrane insertion of RasP in the ecsA-negative strain. However, the fact that rasP, controlled by a xylose-inducible promoter, does not restore full RasP activity reveals that the absence of RasP activity is not due to a regulatory effect that ecsA might have at the transcriptional level (Pummi et al., 2002). In addition, the fact that RasP activity is restored when the protein is overexpressed favours a model of inhibition of existing RasP in the absence of the EcsAB ABC transporter. Which substance(s) EcsAB transports is an intriguing question, and it is conceivable that it transports peptides, possibly peptides that insert into the cytoplasmic membrane and which therefore could interfere with RasP. Note that it has been suggested that ABC transporters are able to remove peptides like lantibiotics from the cytoplasmic membrane (Otto & Götz, 2001), and a ‘hydrophobic vacuum cleaner’ model has been proposed (Otto & Götz, 2001; Stein et al., 2005).
Another observation is that a rasP-negative strain, like the ecsA knockout, does not process preAmyQ, and that this is not caused by their common inability to induce the W regulon. It is not clear what effect RasP might have on the processing of overexpressed AmyQ, and whether the processing defect in the ecsA-negative strain is due to the inactivity of RasP. The only direct role that RasP could play in secretion is a possible function as a signal peptide peptidase, as has been proposed for the RasP orthologue RseP of E. coli because of its ability to cleave the β-lactamase signal peptide (Akiyama et al., 2004). For prokaryotes in general, little is known about the removal of signal peptides from the membrane. E. coli SppA (protease IV) has been shown to degrade the processed signal sequence of the major lipoprotein, but it is not the only protease that is responsible for signal peptide digestion in the cell envelope (Suzuki et al., 1987). For B. subtilis, three proteins similar to SppA have been described (TepA, SppA and YqeZ; Bolhuis et al., 1999; Helmann, 2002). Both, SppA and YqeZ are W-controlled (Huang et al., 1999; Wiegert et al., 2001), and for YqeZ a function for immunity against sublancin rather than degradation of signal peptides has been shown (Butcher & Helmann, 2006). For SppA (YteI) and TepA (YmfB), a function in degradation of proteins or (signal) peptides that are inhibitory to protein translocation has been proposed (Bolhuis et al., 1999). Signal peptide peptidases described for eukaryotes belong to the group of intramembrane cleaving proteases, and attack certain signal peptides after they have been clipped from newly synthesized secretory or membrane proteins (Weihofen & Martoglio, 2003). One function of RasP might be to degrade certain signal peptides in the membrane, and in the absence of RasP activity the accumulation of these peptides might inhibit signal sequence processing by leader peptidases, as has been shown in vitro for synthetic signal peptides (Wickner et al., 1987). We have tried to discern (signal) peptides in isolated membranes of B. subtilis rasP-negative and other strains several times using different methods, but so far the methods have proved to be problematic.
The inability of the rasP-negative strains to produce a biofilm is another interesting feature. Biofilm formation in B. subtilis is a complex programme that requires a variety of regulatory proteins and differential regulation of D- and H-dependent autolysins expressed at specific stages during pellicle formation (Kobayashi, 2007). RasP might play an important role in that process, and this points to a central role of the iClip. It is noteworthy that a triple mutant of the ECF sigma factors W, X and M is also unable to produce structured communities (Mascher et al., 2007), and it will be interesting to see whether the induction of X and M is dependent on regulated intramembrane proteolysis by RasP as well.
Taken together, we are at an early stage and a lot of questions remain to be answered. However, it is an intriguing question to unravel the roles of and connections between EcsA and RasP, as both of these proteins can be found in a great variety of prokaryotes. Both might represent good targets for new antimicrobial agents, as, for example, orthologues of RasP are involved in pathogenic processes (Makinoshima & Glickman, 2005, 2006).
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ACKNOWLEDGEMENTS |
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Edited by: M. Hecker
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Received 18 March 2008;
accepted 14 April 2008.
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