Institute of Biological Sciences, University of Tsukuba, Tsukuba-shi, Ibaraki 305, Japan1
Author for correspondence: Kunio Yamane. Tel: +81 298 53 6680. Fax: +81 298 53 6680. e-mail: kyamane{at}sakura.cc.tsukuba.ac.jp
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ABSTRACT |
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Keywords: Bacillus subtilis, extracellular proteins, proteome analysis, secA and ffh mutants, two-dimensional gel electrophoresis
Abbreviations: 2D, two dimensional; SRP, signal-recognition particle
The SWISS-PROT accession numbers for the N-terminal amino acid sequences reported in this paper are: P00691 for AmyE; P54507 for CotN; O07921 for Csn; P09124 for Gap; P26901 for KatA; P39116 for Pel; P39824 for PenP; P54375 for SodA; P29141 for Vpr; Q07833 for WapA; P54423 for WprA; P54327 for XkdG; Q45071 for XynD; P94421 for YclQ; O31803 for YcnM; O05512 for YdhT; O34952 for YflE; O06487 for YfnI; O31737 for YlqB; P96740 for YwtD; P42110 for YxaK; P94356 for YxkC.
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INTRODUCTION |
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Protein-secretion pathways have been extensively studied in Escherichia coli, yeast and mammals. A presecretory protein precursor in E. coli is thought to be recognized by a molecular chaperone such as SecB and is targeted to membrane-bound SecA, which is the peripheral ATPase subunit of translocase (Collier et al., 1988 , Hartle et al., 1990
, Valent et al., 1998
). The precursor is then translocated across the cytoplasmic membrane through a SecA ATPase-dependent translocase consisting of SecA, SecE, SecG, SecY and other membrane proteins (Douville et al., 1995
). SecA promotes protein translocation during cycles of SecA insertion into and removal from SecEYG on the membrane (Nishiyama et al., 1996
). Therefore, SecA plays a central role in the protein-secretion pathway in E. coli. A secA mutant of B. subtilis has been isolated as the cell-division mutation div341 (Miyakawa & Komano, 1980
). After cloning and characterizing the wild-type gene that can complement the div341 mutation, the wild-type gene was designated secA. The predicted amino acid sequence of the gene show 50% identity with that of E. coli SecA. The div341 mutation is a nucleotide replacement of C to T at position 1292 of secA (Sadaie et al., 1991
, Takamatsu et al., 1992
). SecA homologues have been found in all eubacteria and chroloplasts (Nohara et al., 1995
). Homologues of E. coli SecE, SecG, SecD, SecF and SecY have been also identified in B. subtilis, but not SecB (Bolhuis et al., 1998
; van Wely et al., 1999
).
In contrast, a SecA homologue has not been identified in yeast and mammalian cells (Rapoport et al., 1996 ). The SRP plays a central role in recognizing and targeting presecretory proteins to the endoplasmic reticulum membrane in mammalian cells (Walter & Blobel, 1982
). SRP is a ribonucleoprotein complex composed of one RNA (SRP 7S RNA) and six proteins (SRP9, SRP14, SRP19, SRP54, SRP68 and SRP72; Walter & Johnson, 1994
). SRP54 binds to the signal peptide of a nascent presecretory protein, probably through direct interaction of the hydrophobic region of its M-domain with the hydrophobic region of the signal peptide (Lutcke et al., 1992
; Lutcke, 1995
). Furthermore, homologues of SRP54, Ffh (fifty-four homologues) in bacteria, have also been identified in animals, plants, yeast, eubacteria, archaea and chloroplasts (Keenan et al., 1998
). B. subtilis Ffh forms an SRP-like particle complexed with small cytoplasmic RNA (scRNA, a homologue of SRP RNA) and HBsu, a histone-like protein (Nakamura et al., 1999
).
In B. subtilis, SecA and Ffh are essential for normal cell growth and protein translocation. SecA or Ffh depletion inhibits the secretion of extracellular -amylase, ß-lactamase and ß-lactamase fusion proteins carrying the signal peptide of B. subtilis alkaline protease, penicillin-binding protein 5* or cyclodextrin glucanotransferase (Honda et al., 1993
, Bunai et al., 1996
, Takamatsu et al., 1997
). Furthermore, SecA and Ffh can intrinsically bind to precursors of ß-lactamase and its fusion proteins in vitro but not to their mature forms. Since Ffh interacts with SecA and Ffh enhances the binding of SecA to precursors, we have proposed that the co-operation of B. subtilis SecA and Ffh constitutes an efficient protein-secretion pathway (Bunai et al., 1999
).
In this study, we predict that 150 to 180 B. subtilis proteins are extracellular. By comparing 2D extracellular protein profiles from B. subtilis 168, and SecA and Ffh conditional mutants, 17 out of 23 proteins where the N-terminal amino acid sequence was determined were extracellular, and two were membrane proteins that appeared to be librated into the medium after processing. The appearance of these 19 proteins depended on both SecA and Ffh. In contrast, Hag and Gap appeared even under SecA- and Ffh-deficient conditions and SodA appeared as a large spot under Ffh deficient conditions.
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METHODS |
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Extracellular sample preparation for the proteome analysis.
A final concentration of 3 mM PMSF was added to culture medium (100 ml each) at the late-exponential phase of growth to prevent proteolytic digestion, then cells were removed by centrifugation followed by filtration through a 0·2 nm nitrocellulose filter (Millipore). Then 4 ml 50% TCA was added to the medium, mixed well and placed on ice for 30 min. The aggregated proteins were precipitated by centrifugation, washed three times in cold 70% ethanol (-20 °C), dried and dissolved in sample solution consisting of 8 M urea, 1% Triton X-100, 15 mM DTT and 5 mM PMSF.
Analytical 2D gels.
The first dimension in the 2D gel electrophoresis was isoelectrically foucused on an Electro Immobiline Dry Strip pH 310L (11cm) (Amersham Pharmacia). Extracellular preparations containing 20 µg protein mixture dissolved in the sample solution containing 1% (v/v) Formalyta 310·5 was applied to the first dimension. Gels were focused for 15 h at 400V followed by 1 h at 600V using a Multiphor II (Amersham Pharmacia). After placing in equilibration buffer A [50 mM Tris/HCl, pH 6·8, containing 6 M urea, 30 % (v/v) glycerol, 2·5% SDS and 0·25% DTT] for 15 min and buffer B [50 mM Tris/HCl, pH 6·8, 6 M urea, 30% (v/v) glycerol, 2·5% SDS, 0·25% DTT and 4·5% iodoacetic acid] for 5 min, the isoelectric focusing gels were embedded in 0·25 M Tris/HCl, pH 6·8, 0·25% SDS, 1% agarose onto 12% SDS polyacrylamide gels (14x14x0·1 cm), and the proteins were resolved in the second dimension by a constant current of 20 mA until the bromphenol blue marker entered the stacking gel, followed by 40 mA for 3 h (Rabilloud et al., 1997 ). The gels were fixed in an 80% ethanol/20% acetic acid mixture and silver stained as described by Morrissey (1981)
.
Preparative 2D gel electrophoresis and N-terminal amino acid sequencing.
For preparative 2D gel electrophoresis, 200250 µg proteins from the extracellular preparations were separated by 2D gel electrophoresis as described above. The protein spots in the gels were transferred onto PVDF membranes (Millipore) by the method of Matsudaira (1987) , then stained with 0·1% Coomassie Brilliant Blue R250. Spots were excised from the membrane and sequenced using an Applied Biosystems 492 protein sequencer.
Detection of SecA and Ffh.
Cells were harvested by centrifugation and cell lysates were prepared as described by Takamatsu et al. (1994 , 1997
). Total lysate proteins (10 mg each) were resolved by SDS-PAGE and blotted onto PVDF membranes. Bands for SecA and Ffh were detected using anti-B. subtilis SecA antibody and anti-B. subtilis Ffh antibody, respectively, by immunoblotting as described by Takamatsu et al. (1994
, 1997
)
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RESULTS |
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2D analysis of the extracellular proteome
Bacillus subtilis 168 produces high levels of extracellular proteins at the early stationary phase of growth. We made extracellular preparations at the late-exponential phase of growth in minimal medium containing 0·4% glucose to avoid possible contamination with cytoplasmic proteins due to partial cell lysis. The protein composition of the samples was then examined by 2D gel electrophoresis (Fig. 1). B. subtilis 168 generated 100110 detectable spots on the gel. Among these, thirty eight obvious spots were selected and assigned names as shown in Fig. 1
. Their N-terminal amino acid sequences were analysed; the results of 20 of these are summarized in Table 1
. After searching the database, spots C1 and C2 were defined as the gene products of pel and spots J and C4 were those of wprA. Therefore, a total of eighteen gene products were identified. The N-terminal amino acids of spots J and C4 corresponded to Ala-414 and Ala-32 of the predicted amino acid sequence of wprA, respectively. Therefore, spot J was CWBP52 serine protease and spot C4 was CWBP23 (Margot & Karamata, 1996
; Stephenson & Harwood, 1998
). Spot B8 was the predicted gene product of wapA. The predicted amino acid sequence of wapA indicates that a signal peptide (Met-1 to Ala-28) is located in its precursor. However, the N-terminal amino acid of spot B8 corresponded to Tyr-98 of the sequence. Therefore, WapA is processed after secretion (Foster, 1993
). The N-terminal amino acid of spot O corresponded to Gly-31 of the predicted sequence of yclQ. Met-1 to Ala-20 of the precursor of YclQ was the predicted signal peptide. Therefore Cys-21 to Gly-30 constitutes the propeptide. These results indicate that 13 of the 18 gene products are secretion proteins. However the 18 gene products contained two membrane proteins (YflE and YfnI), flagellin monomer (Hag), the intracellular protein Gap (glyceraldehyde-3-phosphate dehydrogenase) and XkdG, an extracellular protein encoded in the PBSX genome.
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A comparison of the 2D profiles of strains 168, TB301 at 30 °C and 42 °C and DF46 in the presence or absence of IPTG, classified the 38 spots (Fig. 1) into three groups as follows. Thirty-one spots completely disappeared under both SecA- and Ffh-deficient conditions and the 5 spots marked with asterisks (spots M2, E3, E, E4 and M3) disappeared under SecA-deficient conditions and were faint in the absence of Ffh. These 36 proteins seem to be extracellular, although two (YflE, spot U, and YfnI, spot E) were predicted membrane proteins. These results indicate that both SecA and Ffh function in the secretion of most extracellular proteins.
On the other hand, Hag and Gap were not inhibited by the absence of SecA or Ffh. In E. coli, the production of Hag in the culture medium depends on a specific pathway (Hueck, 1998 , Namba et al., 1989
). Therefore, the extracellular production of Hag in B. subtilis may also be independent of the SRP/Sec protein-secretion system. Gap is a predicted cytosolic protein (Graumann et al., 1996)
.
Effect of carbon source on the protein composition of the extracellular preparations
We did not identify any spots for typical extracellular enzymes such as -amylase and proteases. We surmised that this was caused by using 0·4% glucose as the carbon source in the medium, which would cause catabolite repression.
Fig. 5 (a
, b
and c
) shows the 2D profiles of the extracellular proteins prepared from strain 168 cultured in minimal medium containing 0·4% cellobiose, 0·4% maltose or 0·4% soluble starch, respectively. Growth rates in cellobiose were similar to those in glucose, but were slightly delayed in maltose and soluble starch. Spots for Hag and Gap were detected in all three 2D profiles, and the other spots were essentially identical to the profile of strain 168 (Fig. 1
). Nineteen spots (arrowed) that were absent from strain 168 cultured in glucose, were novel. Spots for NG1 to NG4, CS1, ML1 and SS1 to SS4 were extracted and their N-terminal amino acids were sequenced (Table 2
). This analysis revealed AmyE, Vpr, KatA and YhdT (mannan endo-1,4-ß-mannosidase). AmyE and Vpr are extracellular proteins and YdhT was also predicted to be extracellular. KatA is a cytoplasmic protein that locates in the culture medium during the stationary phase of growth (Bol & Yasbin, 1991
). Spots SS1 to SS4 were degradation products of Hag. Spot ML1 could not be transferred onto PVDF membranes. These spots were not found in the 2D profiles preparations obtained from TB301 cultured in 0·4% maltose and soluble starch at 42 °C and from DF46 cultured in 0·4% maltose in the absence of IPTG.
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DISCUSSION |
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Weiner et al. (1998) and Sargent et al. (1998)
reported a fourth secretion pathway in E. coli, called the Tat system, for twin-arginine leader peptides of a group of secretory proteins. B. subtilis counterparts of E. coli tatA, C, D and E were identified as ydiI/ycbB, ydiJ/ycbI, yabD and ynzA, respectively, by a computer search of the databases. Therefore a similar protein pathway to the E. coli Tat system may also be present in B. subtilis. However, we found two signal peptide amino acid sequences of WprA and WapA precursors, which were similar to the twin-arginine leader motif, (S/T)RRXFLK (Berks, 1996
). Their production into the culture medium was dependent on SecA and Ffh. Therefore, a B. subtilis Tat system may be involved in the secretion and membrane location of some specific protein groups.
We ascertained that the AmyE precursor accumulates in the absence of SecA or Ffh (Takamatsu et al., 1992 ). Since a homologue of E. coli SecB has not been identified in B. subtilis either by individual investigation and/or the genome project, SRP in B. subtilis must recognize presecretory proteins as a chaperone and target them to Sec protein translocase for transport across the membrane. Therefore the co-operation of SRP and Sec translocase should be the major protein-secretion pathway in B. subtilis, although we have not yet confirmed the accumulation of precursors of extracellular proteins other than AmyE. Furthermore, we have to ascertain whether or not the disappearance of these extracellular proteins is due to a primary effect of the depletion of SecA or Ffh.
Ulbrandt et al. (1997) and Valent et al. (1998)
reported that E. coli SRP is mainly required for the localization of inner-membrane proteins. The requirement of SRP for the localization of membrane proteins has not been analysed in B. subtilis. However it is quite possible that the location of membrane protein is due to the function of SRP and Sec protein translocase, as well as the secretion of extracellular proteins as shown in Table 1
, where the production and liberation of membrane proteins YfnI and YflE also depend on SecA and Ffh.
On the other hand, Hag migrated as the largest spot in 2D gels of extracellular protein preparations. Hag is another protein, the secretion of which is not due to SRP and Sec translocase in E. coli (Hueck, 1998 ; Namba et al., 1989
). Therefore the appearance of Hag in the absence of both SecA and Ffh does not conflict with the disappearance of many extracellular proteins under these conditions. Hag in B. subtilis must be secreted via a specific pathway like that in E. coli. The appearance of Gap, KatA and SodA in the extracellular preparations was probably due to leakage of cytoplasmic proteins.
A computer search of the databases and the similarity of the N-terminal amino acid sequences to typical signal peptides of extracellular proteins led to the selection of 138 secretory-protein candidates. We identified only 17 proteins that migrated as obvious spots on 2D gel electrophoresis as secretory proteins. However, YwtD and XynD in Table 1 were not included in the candidates. Approximately 120 to 140 spots were detected in our 2D gel system overall and many more proteins will be identifiable by studies using mutants with other features such as high protein-secretion ability and by using various culture conditions. These results indicate that B. subtilis 168 will produce 150 to 180 proteins into culture medium. This number is fully consistent with the earlier estimate of 180 secreted proteins by Tjalsma et al. (1999)
.
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ACKNOWLEDGEMENTS |
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Received 23 June 1999;
revised 16 September 1999;
accepted 20 September 1999.