Department of Microbiology, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Kerklaan 30, 9751 NN Haren, The Netherlands1
Institut für Biotechnologie 1, Forschungszentrum Jülich GmbH, D-52425 Jülich, Germany2
Author for correspondence: Arnold J. M. Driessen. Tel: +31 50 3632164. Fax: +31 50 3632154. e-mail: a.j.m.driessen{at}biol.rug.nl
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ABSTRACT |
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Keywords: protein secretion, chaperones, SecA, SecB
Abbreviations: Cam, chloramphenicol; GST, Schistosoma japonicum glutathione S-transferase; GST-C, GST fusion bearing the C-terminal 22 amino acids of B. subtilis SecA; Phle, phleomycin; SRP, signal recognition particle
a Present address: Department of Experimental Pathology, Josephine Nefkens Institute, Erasmus University Rotterdam, PO Box 1738, 3000 DR Rotterdam, The Netherlands.
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INTRODUCTION |
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Studies in E. coli have demonstrated that prior to their interaction with SecA, precursor proteins may interact with the signal recognition particle (SRP) (Powers & Walter, 1997 ; Bunai et al., 1999
) or the export-dedicated molecular chaperone SecB (Kumamoto, 1989
, 1991
). Both pathways converge at the translocase (Valent et al., 1998
; Bunai et al., 1999
). The bacterial SRP consists of Ffh (Römisch et al., 1989
; Honda et al., 1993
) and 4·5S RNA (Struck et al., 1989
; Powers & Walter, 1997
). In E. coli, SRP interacts with hydrophobic signal sequences of nascent precursor proteins, and targets these nascent chainribosome complexes to the membrane through the SRP receptor, FtsY (Luirink et al., 1994
; Oguro et al., 1995
). The B. subtilis Ffh specifically binds to precursor proteins (Bunai et al., 1996
) and promotes their binding to SecA (Bunai et al., 1999
). Depletion of components of the SRP pathway results in a partial block in protein translocation that seems to vary depending on the precursor protein (Oguro et al., 1996
, Hirose et al., 2000
).
In E. coli, SecB supports the translocation of a subset of proteins, mainly precursors of outer-membrane proteins (Kumamoto & Francetiç, 1993 ). SecB binds to nascent precursor proteins (Randall et al., 1997
; Behrmann et al., 1998
), and holds them in a translocation-competent conformation (Lecker et al., 1989
). SecB subsequently targets these proteins to the SecYEG-bound SecA (Hartl et al., 1990
; Fekkes et al., 1997
). This targeting event is accomplished by the high-affinity binding of SecB to the carboxyl (C-) terminus of the SecYEG-bound SecA, whereas cytosolic SecA interacts with only poor affinity (Den Blaauwen et al., 1997
; Fekkes et al., 1997
). The interaction is stimulated by the presence of a precursor protein with a functional signal sequence (Fekkes et al., 1998
). During the ATP-dependent initiation of protein translocation, SecB is released from the SecYEGSecAprecursor complex and recycled to the cytosol (Fekkes et al., 1997
).
SecB seems to be present in Gram-negative bacteria only (Fekkes et al., 1998 ), in particular in Enterobacteriaceae. It is not required for viability of E. coli but its gene overlaps with gpsA, which encodes a biosynthetic sn-glycerol-3-phosphate dehydrogenase (Shimizu et al., 1997
). Disruption of the gpsA gene results in a severe growth defect when cells are grown on rich media (Shimizu et al., 1997
). On the other hand, the C-terminal SecB-interacting domain of E. coli SecA cannot be disrupted (Breukink et al., 1995
). SecA lacking this region still promotes protein translocation but no longer binds SecB nor supports SecB-dependent translocation (Fekkes et al., 1997
). Since SecB is obsolete, the observation that cells harbouring SecA with a deleted C-terminus are unable to grow is difficult to understand in terms of an impaired SecB function. SecB appears not to be present in Gram-positive bacteria. Strikingly, the C-terminal SecB binding domain is highly conserved among the bacterial SecA proteins (Fig. 1
). In the case of Mycoplasma tuberculosis SecA, the conserved region is found in the middle of the SecA protein sequence. The conserved cysteine and histidine residues present in this domain are involved in the coordination of a zinc ion that stabilizes a tertiary structure of SecA that is needed for the interaction with SecB (Fekkes et al., 1999
).
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METHODS |
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Chromosomal disruptions.
Deletion of the 22 C-terminal amino acids of SecA (Fig. 1) was accomplished as follows. A gene fragment corresponding to amino acids 670 to 819, followed by a stop codon, was amplified from chromosomal DNA from strain DB104 as a SacIXbaI PCR fragment, and cloned into pBluescript SK+. Subsequently, a XbaIPstI fragment containing a phleomycin (Phle) resistance marker and the 1·1 kb PstINcoI chromosomal downstream region were cloned downstream of the partial secA gene, resulting in plasmid pDELC. This vector contains the DB104 chromosomal region with the C-terminal 22 amino acids of the secA gene replaced by a Phle resistance marker. Vector pDELC was linearized with PvuII to yield a 2·5 kb secA
C::phleo fragment and subsequently transformed into B. subtilis DB104 by natural competence (Young, 1967
). Phle-resistant colonies resulting from a double crossover were selected. The correct position of the chromosomal replacement was confirmed by PCR. In the resulting strain, DB104
C, the secA gene encodes only the first 819 amino acids of the protein.
The mrgA gene of B. subtilis DB104 was disrupted as follows. Regions immediately upstream and downstream of mrgA were amplified from the chromosome of strain DB104 as BamHIXbaI and KpnIHincII cassettes, respectively, and cloned into pBluescript SK+. Subsequently, a BglII-PvuII-digested chloramphenicol (Cam) resistance marker was placed between the BamHI and HincII sites, yielding pDELM. This vector contains the DB104 chromosomal region with the mrgA gene replaced by the Cam resistance marker. Vector pDELM was linearized with PvuII to yield a 2·8 kb fragment containing the mrgA::cam region and subsequently transformed into B. subtilis DB104 by natural competence. Cam-resistant colonies resulting from a double crossover were selected. The correct position of the chromosomal replacement was confirmed by PCR. In the resulting strain, DB104M, the Cam resistance gene replaced the mrgA gene while leaving the flanking regions intact. Strain DB104
CM, carrying both mutations, was generated by transforming DB104
M with chromosomal DNA from strain DB104
C and selecting for Cam and Phle resistance. Since the mutations cause a complete deletion, no selective pressure is needed after the initial selection.
A vector expressing a fusion product of Schistosoma japonicum glutathione S-transferase (GST) and the C-terminal 22 amino acids of SecA was generated by ligation of a 102 bp EcoRVNarI fragment from pMKL4 (Klose et al., 1993 ) into SmaI-AccI-digested pGEX4T2 (Pharmacia), yielding pET446.
Binding of cytosolic proteins to the C-terminus of SecA.
GST and GST-C were purified from cells bearing plasmids pGEX4T2 and pET446, respectively, according to the manufacturers procedures. After purification, glutathione was removed by dialysis against 50 mM potassium phosphate, pH 7·0, 100 mM NaCl (buffer A). For the preparation of a cytosolic lysate, B. subtilis cells were broken by French pressure treatment [three times at 8000 p.s.i. (55 MPa)] and debris was removed by centrifugation at 60000 g for 45 min. For the binding experiments, GST or GST-C (5 mg) was mixed with the supernatant fraction of the lysate (100 mg total protein) in buffer A, and incubated for 30 min on ice. Subsequently, GST or GST-C was reisolated on glutathione Sepharose, washed with 20 column volumes of buffer A, and eluted with one column volume of 25 mM reduced glutathione or alternatively with 50 mM EDTA. The eluted fractions were precipitated with 10% TCA (final concentration), washed twice with cold acetone and analysed by SDS-PAGE.
Analysis of cellular and secreted proteins.
B. subtilis DB104 and its derivatives were grown at 37 °C in liquid medium. Overnight cultures were diluted 1:50 into fresh medium and grown to the late exponential phase. Cultures were cooled on ice and fractionated into a cellular and a medium fraction by centrifugation. The medium fraction was precipitated with 10% TCA, washed twice with cold acetone and analysed by SDS-PAGE (van Wely et al., 1999 ). Cellular pellets were resuspended in sample buffer, sonicated and analysed by SDS-PAGE.
Semi-quantitative RT-PCR.
Total RNA was isolated from exponentially growing cultures using a total RNA isolation kit (QIAGEN) according to the manufacturers indications. First-strand synthesis was accomplished in a single reaction using the access RT-PCR system (Promega) and with primer pairs that specify wapA (forward, 5'-TGTTAAGTCATGGAACTCCGG-3'; backward, 5'-ATCTAATGCCAATTCAGCTCC-3'), yweA (forward, 5'-TCTTACTTCCTTCGGGCCAAGC-3'; backward, 5'-CCCAAACCGTTTACTACATCGCC-3') or yolA (forward, 5'-TTGCTCTTCTAGCAGTTGTTGC-3'; backward, 5'-TTTATCCCAGCCTATTACCACC-3'). Subsequently, the relative levels of cDNA were determined in a multiplex PCR in the presence of [32P]dATP with the gap (glyceraldehyde-3-phosphate dehydrogenase) primer pair (forward, 5'-TGCACAACAAACTGCCTTGCGC-3'; backward, 5'-TTTACCATGCTGCCTTCCATAAC-3') alone or in combination with one of the other primer pairs. The PCR products formed after 20 cycles of amplification were separated by electrophoresis on a 6% denaturing acrylamide gel and quantitated by phosphor-imaging and ImageQuant software (Molecular Dynamics). Values were corrected for gel loading.
Miscellaneous methods.
Pulsechase experiments with B. subtilis DB104 and DB104C expressing E. coli proOmpA or Bacillus licheniformis preAmyL were carried out as described before (Meens et al., 1993
). Protein concentrations were determined by the method of Lowry, using BSA as standard. For N-terminal sequencing of polypeptides, samples were separated by SDS-PAGE and blotted onto PVDF. Sequencing was done by the NAPS facility at the University of British Columbia (Vancouver, Canada).
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RESULTS |
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The C-terminus of B. subtilis SecA binds E. coli SecB and B. subtilis MrgA
When fused to GST, the C-terminal 22 amino acids of the E. coli SecA have been shown to constitute a genuine SecB-binding site (Fekkes et al., 1997 ). To identify a possible binding partner in B. subtilis, the C-terminal 22 amino acids of B. subtilis SecA were fused to GST to yield GST-C. Both GST and GST-C were expressed in E. coli and purified by glutathione-affinity chromatography. E. coli SecB specifically co-purified with the GST-C fusion protein as detected by Coomassie-stained SDS-PAGE (data not shown) and Western blotting (Fig. 5
). The same binding was detected before with the GST fusion protein harbouring the C-terminal 22 amino acids of the E. coli SecA (Fekkes et al., 1997
). Binding studies with purified E. coli SecB confirmed the interaction with the GST-C fusion protein harbouring the B. subtilis sequence (data not shown). It is, therefore, concluded that the C-terminus of the B. subtilis SecA provides a valid binding site for SecB.
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AhpF functions together with AphC in the reduction of alkyl hydroperoxides (Chen et al., 1995 ), whereas KatA is involved in the reduction of hydrogen peroxide (Bsat et al., 1996
). MrgA is synthesized as a 17·3 kDa polypeptide, but has been reported to exist in a high-molecular-mass complex that does not completely dissociate even after boiling in SDS (Chen & Helmann, 1995
). The high-molecular-mass form of MrgA confers resistance to oxidative challenge, whereas the form bound by GST-C seems to exclusively represent the monomeric form (Fig. 6
). These data indicate that the deletion of the C-terminus of SecA elicits a strong oxidative stress response.
MrgA is not involved in protein secretion
The proposed function of MrgA is to bind to DNA under conditions of oxidative stress and to protect the DNA against damage (Chen & Helmann, 1995 ). To gain insight into the possible functional relation between SecA and MrgA, the mrgA gene was deleted from the chromosome in both DB104 and DB104
C, yielding strains DB104
M and DB104
CM, respectively. These strains had normal viability under the conditions tested. Inactivation of the mrgA gene in the DB104
C strain indeed resulted in the loss of the 120 kDa polypeptide, confirming that this protein band corresponds to a high-molecular-mass complex of MrgA (Fig. 3b
, lane 4). The DB104
CM strain, however, still overproduced the AhpC and AhpF proteins to the level observed in strain DB104
C (Fig. 3b
). Comparison of the culture supernatant of the parental strains and mrgA deletion mutants did not reveal any specific differences in the polypeptide pattern except that the overall level of secreted proteins was somewhat reduced (Fig. 3a
, lane 4). This demonstrates that MrgA is not directly involved in protein secretion, nor needed for the oxidative stress response induced by the loss of the C-terminus of SecA.
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DISCUSSION |
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The screen with the C-terminus of B. subtilis SecA as bait points to the MrgA protein as a cytosolic binding partner. The interaction between the E. coli SecA and SecB is stabilized by a zinc ion (Fekkes et al., 1999 ). By analogy, chelators could disrupt the observed binding of MrgA to the C-terminus of the B. subtilis SecA. MrgA is not an analogue of SecB, since the deletion of the mrgA gene from the chromosome did not result in any noticeable secretion defect. The chromosomal replacement of the B. subtilis SecA by a truncate that lacks the putative SecB-binding domain resulted in the loss of specific proteins from the culture supernatant. In combination with the disruption of the mrgA gene, only a slight overall reduction of secretion was observed, but no additional loss of protein bands from the culture supernatant fraction. Since the specific loss of protein bands could be accounted for by a reduction of expression levels, it appears that the interaction of the C-terminus of SecA with MrgA is not directly related to protein secretion.
The B. subtilis chromosome (Kunst et al., 1997 ) contains another protein, YccF, that bears at its N-terminus an almost identical sequence as the C-terminus of SecA. The function of YccF is unknown, but its deletion from the chromosome has no notable effect on the growth of B. subtilis (J. M. van Dijl, personal communication). The conserved sequence present in YccF may also represent a zinc-binding site that is involved in the binding of another protein or even MrgA. Since YccF has not been implicated in protein translocation, we have not analysed this possibility further.
A striking observation is the highly elevated expression of the cytosolic protein AhpC (and tentatively the AhpF and KatA proteins) in the SecAC strain. AhpC and AhpF mediate the enzymic reduction of various alkyl hydroperoxides, whereas KatA mainly reduces hydrogen peroxide (Bsat et al., 1996
). MrgA confers resistance to oxidative stress by the protection of DNA when present in a high-molecular-mass complex that is stable in SDS-PAGE (Chen & Helmann, 1995
). Together, ahpCF, katA and mrgA form the peroxide regulon. The peroxide regulon is normally induced during oxidative stress or metal limitation (Völker et al., 1994
; Chen et al., 1995
; Bsat et al., 1996
), although AhpC is induced under various other conditions (Völker et al., 1994
; Antelmann et al., 1996
). Even though oxidative stress is the strongest known inducer of the peroxide regulon, the expression of ahpCF, mrgA and katA is under control of a complex regulatory regime and is influenced by other factors. Expression of these proteins is growth-phase dependent, i.e. induced upon entry into stationary phase (Bol & Yasbin, 1994
; Chen et al., 1995
). Mutation of AhpC leads to an increased expression of the other proteins in the regulon (Antelmann et al., 1996
; Bsat et al., 1996
). Finally, the growth-phase-dependent but not the peroxide-induced expression of KatA is abolished by mutation of spoOA (Bol & Yasbin, 1994
). The exact mechanism by which deletion of the C-terminus of SecA induces expression of the peroxide regulon is unknown. The same mutation however results in the loss of a number of distinct polypeptides in the culture supernatant. One of these proteins is WapA, which is secreted as a very large (2334 amino acids) precursor that is subsequently processed into a number of smaller polypeptides with various functions (Foster, 1993
). The loss of these proteins from the supernatant of strain DB104
C probably results from a regulatory process. Synthesis of preWapA is controlled by the DegSDegU system (Dartois et al., 1998
) and affected by salt stress like that of many other secretory proteins (Kunst & Rapoport, 1995
). WapA, as well as YweA and YolA, may also be repressed by the stress response that is evoked in strain DB104
C. Mutations in the secA gene of B. subtilis have been shown to affect transcription from spoOA-dependent promoters (Asai et al., 1997
, 1998
), and thus probably affect transcription of the peroxide regulon (Bol & Yasbin, 1994
). In the case of SecA
C, a similar common mechanism could cause the stress response and the repression of a set of secretory proteins.
The question arises whether there is a need for molecular chaperones such as SecB in protein secretion in B. subtilis. Although this question cannot be answered at this time, there are a number of notable differences between E. coli and B. subtilis. Most of the secretory proteins that interact with SecB in E. coli are outer-membrane proteins (Kumamoto & Francetiç, 1993 ). Due to their hydrophobic nature, these proteins tend to aggregate when not stabilized by chaperones in solution. B. subtilis completely lacks such proteins, and mainly produces soluble exoenzymes that fold rapidly only outside the cell when in contact with calcium (Leloup et al., 1997
). A recent report indicates that most exported proteins in B. subtilis require Ffh for secretion (Hirose et al., 2000
). The B. subtilis Ffh has been shown to bind specifically to precursor proteins and stimulates their association with the soluble form of SecA (Bunai et al., 1996
, 1999
). This has led to the suggestion that Ffh fulfils a general chaperone role in the export of secretory protein in B. subtilis, unlike the E. coli Ffh that is primarily needed for the targeting of nascent membrane proteins to the translocase (Valent et al., 1997
, 1998
).
In conclusion, our data suggest that the conserved C-terminal domain of B. subtilis SecA is not essential for protein secretion and viability, and indicate a regulatory function rather than a role in chaperone binding.
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ACKNOWLEDGEMENTS |
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Received 31 March 2000;
revised 23 June 2000;
accepted 6 July 2000.