Department of Genetics, Groningen Biomolecular Sciences and Biotechnology Institute, PO Box 14, 9750 AA Haren, The Netherlands1
Author for correspondence: Jan Maarten van Dijl. Tel: +31 50 363 3079. Fax: +31 50 363 2348. e-mail: J.M.VAN.DIJL{at}FARM.RUG.NL
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ABSTRACT |
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Keywords: Bacillus subtilis, protein secretion, SipS, SipC
Abbreviations: Bam, Bacillus amyloliquefaciens; Bsu, Bacillus subtilis; CBB, Coomassie brilliant blue R; pre-A13i-Bla, pre(A13i)-ß-lactamase; pre-A2-AmyL, pre(A2)--amylase; SPase, signal peptidase
a Present address: Department of Pharmaceutical Biology, University of Groningen, Antonius Deusinglaan 1, 9713 AV Groningen, The Netherlands.
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INTRODUCTION |
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Recent studies have provided compelling evidence that the type I SPase of Escherichia coli, also known as leader peptidase, makes use of a serinelysine catalytic dyad (Tschantz et al., 1993 ; Paetzel et al., 1998
). This is also probably true for SipS, SipT, SipU and SipV of B. subtilis and for the SPases from other eubacteria and organelles that are related to the SPase I of E. coli (van Dijl et al., 1995
; Dalbey et al., 1997
). Nevertheless, apart from the residues involved in catalysis (Paetzel et al., 1998
; Bolhuis et al., 1999c
), very little is known of those factors that determine the activity of SPases from eubacteria other than E. coli. Considering the fact that the type I SPases of B. subtilis display different substrate specificities in vivo, these enzymes appeared to be attractive models for the identification of important determinants of SPase activity. Furthermore, these enzymes are structurally very different from the SPase of E. coli as they are much smaller (e.g. SipS of B. subtilis comprises 184 residues but the E. coli SPase has 323 residues; Fig. 1
) and contain only one membrane anchor instead of two (for review, see Dalbey et al., 1997
). Notably, like the SPases from B. subtilis, the majority of known eubacterial SPases contain only one N-terminal membrane anchor (Dalbey et al., 1997
).
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METHODS |
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The sipC gene of B. caldolyticus was cloned as previously described for the cloning of the sipS gene of B. subtilis (van Dijl et al., 1992 ). First, chromosomal DNA of B. caldolyticus was cleaved with Sau3A and ligated into the unique BclI site of plasmid pGDL46. Subsequently, E. coli MC1061 was transformed with the ligation mixture and kanamycin- and ampicillin-resistant transformants were selected. Finally, transformants that were able to process the hybrid precursor pre-A13i-Bla, and release mature A13i-Bla into the surrounding growth medium, were selected using a plate (halo) assay for ß-lactamase activity (van Dijl et al., 1992
). Notably, the E. coli SPase I is unable to process pre-A13i-Bla in vivo. Consequently, this hybrid precursor remains attached to the membrane and no mature A13i-Bla is released into the surrounding medium, unless a heterologous SPase is expressed that is able to process pre-A13i-Bla. As shown by DNA sequencing, the plasmid pGDL46.36 thus selected contained the sipC gene of B. caldolyticus (SWISS-PROT # P41027).
Plasmid pT7dC, specifying a soluble form of SipC, was constructed by ligating an EcoRI- and BamHI-cleaved PCR-amplified fragment of sipC into the corresponding sites of pT713. The sipC-specific fragment was amplified with the primers SipC002 (5'-GGAATTCGTCGACGGAGGACAATTATGCGGTTGTTCGTGTTCAGCAATTACG-3') and SipC004 (5'-GGAATTCGGATCCATAGAAGCGGAAGACTCC-3'), using pGDL46.36 DNA as a template. Plasmid pT7dCH, specifying a hexa-histidine-tagged soluble form of SipC, was constructed by ligating an EcoRI- and SalI-cleaved, PCR-amplified fragment of sipC into the corresponding sites of pT712. The sipC-specific fragment was amplified with the primers SipC002 and SipCHis004 (5'-GCTCTAGAATTCTTAGTGATGGTGATGGTGATGAAACTGAAAGGCGAACTGTTTAAACGG-3'), using plasmid pGDL46.36 DNA as a template. Plasmid pT7dS, specifying a soluble form of SipS from B. subtilis (Bsu), was constructed by ligating a SalI-and BamHI-cleaved, PCR-amplified fragment of sipS (Bsu) into the corresponding sites of pT712. The sipS (Bsu)-specific fragment was amplified by PCR with the primers SipS001 (5'-GACTAGTCGACGGAGGACAATTATGCGCAACTTTATTTTTGC-3') and Lbs91 (5'-CGGGATCCCGGGACTAATTTGTTTTGCGC-3') using B. subtilis 168 chromosomal DNA as a template. Plasmid pT7dAH, specifying a hexa-histidine-tagged soluble form of SipS from B. amyloliquefaciens (Bam), was constructed by ligating an EcoRI- and SalI-cleaved PCR-amplified fragment of sipS (Bam) into the corresponding sites of plasmid pT712. The sipS (Bam)-specific fragment was amplified by PCR with the primers SipA001 (5'-GGAATTCGTCGACGGAGGACAATTATGCGCAACTTTTTATTTGCTCC-3') and SipAHis02 (5'-GGAATTCTTAGTGATGGTGATGGTGATGGATCGATTTCGTCTTGCGAA-3') using pGDL46.21 as a template. To prevent the selection of non-overexpressing variants of pT7dC, pT7dCH, pT7dS and pT7dAH, these plasmids were first constructed using E. coli MC1061, which does not contain the gene for the T7 RNA polymerase. Subsequently, E. coli BL21(de3) was used for the production of soluble SPases specified by pT7dC, pT7dCH, pT7dS and pT7dAH.
Protein overproduction and purification.
For overproduction and subsequent purification of the hexa-histidine-tagged soluble form of SipS (Bam), denoted sf-SipS-His (Bam), E. coli BL21(de3) was transformed with plasmid pT7dAH. Transformants were grown overnight in TY medium at 37 °C. Next, 1 litre of fresh TY medium was inoculated with 10 ml of this overnight culture and incubated at 37 °C. When the culture reached an OD600 of 0·60·9, the production of sf-SipS-His (Bam) was induced by adding IPTG to a final concentration of 0·5 mM. Cells were collected by centrifugation about 3 h after induction. The cell pellet was resuspended in 10 ml lysis buffer (50 mM Tris/HCl, 300 mM NaCl, 1 mM phosphoramidon, 1 mM PMSF, pH 8·0) and disrupted by three passages through a chilled French pressure cell at 10000 p.s.i. (69 MPa). Cells and debris were removed from the extract by centrifugation at 5000 g (15 min, 4 °C). To separate the soluble sf-SipS-His (Bam) from the membrane-bound E. coli SPase I, membranes were removed from the supernatant by two subsequent ultracentrifugation steps (100000 g, 30 min, 4 °C). Finally, sf-SipS-His (Bam) was isolated by metal-affinity chromatography, using a column containing 5 ml TALON resin (CLONTECH Laboratories) that was pre-equilibrated with lysis buffer. The column was washed with 25 ml lysis buffer and sf-SipS-His (Bam) was eluted with elution buffer (i.e. lysis buffer with 300 mM imidazole). To verify the level of purification, samples from 0·5 ml fractions were analysed by SDS-PAGE and subsequent staining with Coomassie brilliant blue R (CBB). Fractions containing the purified sf-SipS-His (Bam) were pooled and transferred to a buffer containing 50 mM HEPES, 50 mM NaCl, 0·1 mM EDTA, 1 mM DTT (pH 8·0) by gel filtration with a PD-10 Sephadex G-25 M column (Amersham Pharmacia Biotech). Pure sf-SipS-His (Bam) was stored either at 4 °C or at -80 °C in the presence of 20% (v/v) glycerol.
Signal peptidase activity.
The in vitro assay for Bacillus SPase activity was performed essentially as described previously (Vehmaanperä et al., 1993 ). An S-135 extract for the in vitro synthesis of [35S]methionine-labelled pre-A2-AmyL (Smith et al., 1987
, 1988
) was prepared from B. subtilis WB600, a sixfold protease-negative strain (Wu et al., 1991
). The energy-regenerating system for in vitro transcriptiontranslation was from the Promega E. coli S-30 Extract System for Linear Templates. To assay SPase activity, 2 µl of the in vitro transcriptiontranslation mix, with labelled pre-A2-AmyL, was diluted in 16 µl reaction buffer (50 mM HEPES, 5 mM MgSO4, pH 7·5, 37 °C). Next, 2 µl purified sf-SipS-His (Bam) (7·5 µM) or reaction buffer (negative control) was added. If required, 0·5% (v/v) Triton X-100 was added to the reaction buffer. Reactions were terminated by the addition of sample buffer for SDS-PAGE (20 µl) and subsequent boiling for 5 min. Finally, the samples were analysed by SDS-PAGE, fluorography and scanning of films as described by Vehmaanperä et al. (1993)
.
Extracts of E. coli that were used to verify the absence of SPase-like activities in the cytoplasm were prepared as described by van Dijl et al. (1991a) . The in vitro assay for SPase activity, based on the pre-A2-AmyL synthesized in extracts of E. coli, was performed as described by van Dijl et al. (1991b)
.
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RESULTS |
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To characterize the enzymic activity of sf-SipS-His (Bam), a hybrid precursor, denoted pre-A2-AmyL (Smith et al., 1987 , 1988
) was used. This precursor contains the first 60 residues of the YvcE protein of B. subtilis, including a typical signal peptide (Bolhuis et al., 1999b
). The 35S-labelled pre-A2-AmyL was generated by in vitro transcriptiontranslation in a B. subtilis S-135 extract and incubated for 16 h with purified sf-SipS-His (Bam), in the absence of added phospholipids and in the absence or presence of 0·5% Triton X-100. As shown in Fig. 4(a)
, a significant fraction of the labelled pre-A2-AmyL was processed to the mature form, irrespective of the presence of Triton X-100. Notably, no mature A2-AmyL was produced upon synthesis and incubation of the corresponding precursor (which was competent for processing by the SPase I of E. coli) in extracts of E. coli (Fig. 4b
). This was true even when pre-A2-AmyL was incubated for 16 h in an E. coli extract (data not shown), showing that SPase-like activities are absent from the cytoplasm of E. coli. Similarly, the wild-type ß-lactamase (Bla) precursor and the hybrid precursor pre-A2-Bla (Smith et al., 1987
, 1988
) remained unprocessed in extracts of E. coli (van Dijl et al., 1991b
), confirming the absence of cytoplasmic SPase-like activities in E. coli. Taken together, these observations demonstrate that the purified sf-SipS-His (Bam) was active, even in the absence of added phospholipids or detergents.
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DISCUSSION |
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The soluble derivative of SipS (Bam) was shown to have optimal activity at alkaline pH and a similar result was obtained with the wild-type form of this enzyme (our unpublished observations). These observations are consistent with the proposed catalytic mechanism of type I SPases, involving a serinelysine catalytic dyad (Dalbey et al., 1997 ; Paetzel & Dalbey, 1997
; Paetzel et al., 1998
), as an alkaline pH would allow the active-site lysine residue to act as a general base in catalysis. Compared to the E. coli SPase, which has optimal activity between pH 8·5 and 9 (Zwizinski et al., 1981
), sf-SipS-His (Bam) displayed a slightly higher pH optimum (pH 10). Similarly, the lipoprotein-specific (type II) SPase of E. coli, which appears to belong to a novel family of aspartic proteases (Tjalsma et al., 1999b
), was shown to have optimal activity at alkaline pH (Tokunaga et al., 1985
). Even though the high pH optima of these SPases are consistent with their proposed catalytic mechanisms, they are in apparent conflict with the fact that the catalytic sites of type I and type II SPases are probably located at the outer surface of the cytoplasmic membrane (Dalbey et al., 1997
; Tjalsma et al., 1999b
), which has an acidic pH. In fact, the pH at the outer surface of the membrane is likely to be lower than 6, due to the transmembrane H+ gradient that is present in living cells. At such low pH values, the type I SPases at least are barely active, suggesting that their activity could be regulated by the pH. Thus, the potentially deleterious proteolysis of membrane proteins by SPases would be minimized in the acidic environment at the outer membrane surface, until such enzymes are activated to perform their specific task by interactions with pre-proteins or other, as yet unidentified, components of the protein export machinery. Alternatively, the pKa of the active-site lysine residue of type I SPases could be lowered by the hydrophobic interactions with membrane components, such as phospholipids. Consistent with the latter hypothesis, the crystal structure of the E. coli SPase indicates that hydrophobic interactions between the side-chain of the active-site lysine residue and various other residues, such as phenylalanine 133 and tyrosine 143, are important for catalysis (Paetzel et al., 1998
). This view is supported by the recent observation that the equivalents of the latter two residues in the B. subtilis SPase SipS (i.e. leucine 74 and tyrosine 81) are required for the catalytic activity of SipS (Bolhuis et al., 1999a
). Whether the hydrophobic environment of the membrane contributes to the lowering of the pKa of the active-site lysine residue of type I SPases is presently unknown.
Zwizinski et al. (1981) reported that Mg2+ can have an inhibitory effect on the activity of the E. coli SPase I. Interestingly, 5 mM Mg2+ does not seem to inhibit sf-SipS (Bam). Thus, the presence of MgSO4 cannot be the reason for the observed incomplete processing of pre-A2-AmyL shown in Fig. 4(a)
. Similar levels of processing were detected in the same buffer (HEPES, pH 7·0 or pH 8·0) without MgSO4, as shown in Fig. 5(a)
. The most likely explanation of why pre-A2-AmyL preparations are not completely processed is that some molecules of this precursor become incompetent for processing. In vitro this can be due to (micro)aggregation of the precursor, which leads to translocation and processing incompetence (van Wely et al., 1998
). In fact, this would explain why the use of different precursor preparations resulted in different maximum levels of processing. Moreover, even in the living cell, pre-proteins can become incompetent for processing due to folding of the mature protein before processing (van Dijl et al., 1991b
; Bolhuis et al., 1999c
). Notably, the pH-, temperature- and time-dependence of processing was not influenced when different pre-protein preparations were used (data not shown), indicating that the incomplete processing of pre-A2-AmyL, as observed in the present studies, has no bearing on our conclusions.
Finally, our present results show that sf-SipS-His (Bam) is prone to degradation and that this seems to be even worse for sf-SipC, sf-SipC-His and sf-SipS (Bsu). This suggests that the latter three proteins are more sensitive to degradation than sf-SipS-His (Bam). However, we cannot exclude the possibility that the different production levels reflect different expression levels. Most likely, the degradation of the soluble SPase mutant proteins used in these studies is due both to self-cleavage and to cleavage by cytosolic proteases of E. coli (van Roosmalen et al., 2000 ). In fact, as exemplified by sf-SipS-His (Bam), proteolysis is a major bottleneck for the overproduction, purification and structural analysis of soluble derivatives of Bacillus SPases. At present, we are trying to develop novel strategies to overcome this problem.
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ACKNOWLEDGEMENTS |
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Received 17 July 2000;
revised 13 November 2000;
accepted 7 December 2000.