Centre for Ultrastructure Research and Ludwig Boltzmann-Institute for Molecular Nanotechnology, University of Agricultural Sciences, 1180 Vienna, Austria1
Institute of Applied Microbiology, University of Agricultural Sciences, 1190 Vienna, Austria2
Author for correspondence: Margit Sára. Tel: +43 1 47 654 2208. Fax: +43 1 47 89 112. e-mail: sara{at}edv1.boku.ac.at
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ABSTRACT |
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Keywords: heterologous expression, self assembly, peptidoglycan, secondary cell wall polymer, exoamylase
Abbreviations: GHCl, guanidine hydrochloride; GPC, gel-permeation chromatograpy; HMMA, high-molecular-mass exoamylase; SLH, S-layer homologous
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INTRODUCTION |
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The S-layer protein from Bacillus stearothermophilus ATCC 12980 assembles into an oblique lattice type (Egelseer et al., 1996 ). The gene encoding this S-layer protein (sbsC) was sequenced and cloned, showing that the full-length SbsC consists of 1099 aa, possesses a 30 aa signal peptide and has a calculated isoelectric point (pI) of 5·73 (Jarosch et al., 2000
). In the course of studies on the biological function, the oblique S-layer lattice was found to serve as an adhesion site for a cell-associated high-molecular-mass exoamylase (HMMA) which in the S-layer-deficient strain was bound to the peptidoglycan-containing rigid cell wall layer (Egelseer et al., 1996
). By using the whole S-layer protein, proteolytic cleavage fragments, native peptidoglycananionic polymer complex and peptidoglycan for affinity studies, it became apparent that the N-terminal part of SbsC (aa 31257) recognizes a distinct type of secondary cell wall polymer as the proper anchoring structure in the rigid cell wall layer (Egelseer et al., 1998
). This secondary cell wall polymer is composed of tetrasaccharide repeating units that contain glucose, N-acetylglucosamine and 2,3-dideoxy-2,3-diacetamido-D-mannuronic acid in the molar ratio of 1:1:2 and the polymer chains are covalently linked to the peptidoglycan backbone (Schäffer et al., 1999
). Due to the presence of uronic acids, this type of secondary cell wall polymer is strongly negatively charged and should be regarded as a member of the teichuronic acids (Archibald et al., 1993
). Typical of protein domains capable of interacting with carbohydrates (Transue et al., 1997
; Weis, 1997
), the N-terminal part of the mature SbsC has a high content of arginine, lysine and tyrosine. In contrast to the mature SbsC, the N-terminal part is positively charged, with a calculated pI of 9·13 (Jarosch et al., 2000
). Moreover, the N-terminal part of the mature SbsC shows 85% identity to the N-terminal region of the mature SbsA, which assembles into a hexagonally ordered lattice and represents the S-layer protein of B. stearothermophilus PV72/p6 (Sára et al., 1996
). According to a common functional principle, the N-terminal part of the mature SbsA recognizes an identical type of secondary cell wall polymer as binding site in the rigid cell wall layer (Egelseer et al., 1998
; Sára & Sleytr, 2000
).
In a previous study it was demonstrated that the S-layer protein isolated from B. stearothermophilus ATCC 12980 reassembles into small cylinders with a mean diameter of 100 nm, but independent of the applied experimental conditions, the self-assembly products did not exhibit a regular lattice structure in negatively stained preparations (Egelseer et al., 1996 , 1998
). The absence of the oblique lattice structure was found to be due to the fact that B. stearothermophilus ATCC 12980 coexpresses a C-terminally truncated S-layer protein showing an apparent molecular mass of 60000 on SDS-PAGE gels. This SbsC fragment, termed P60, was incorporated into the S-layer lattice on whole cells and led to the loss of its regular structure. The amount of P60 coexpressed with SbsC was strongly dependent on the growth conditions and on the growth stage of the cells. Moreover, the incorporation of P60 into the S-layer lattice on whole cells was linked to the release of HMMA into the culture fluid (Egelseer et al., 1996
), suggesting that appropriate exoenzyme-binding sites in the S-layer lattice became blocked. Since HMMA recognized S-layer-carrying whole cells, S-layer-carrying cell wall fragments as well as self-assembly products as an adhesion site, it was concluded that the enzyme is not incorporated into the crystal lattice and does not participate in the self-assembly process, but binds to protein domains that are exposed on the outer S-layer surface. This was in clear contrast to P60, which showed no affinity to the outer surface of the S-layer lattice (Egelseer et al., 1996
).
In order to investigate the self-assembly properties of SbsC in the absence of P60 and to elucidate the structurefunction relationship of distinct segments of this S-layer protein, the PCR products encoding the mature SbsC and three N- and seven C-terminal truncations were cloned and expressed in Escherichia coli HMS174(DE3). After isolation and purification of the various SbsC truncations, their ability to self-assemble, to bind to the native peptidoglycananionic polymer complex as well as to peptidoglycan and to function as adhesion site for HMMA was investigated.
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METHODS |
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Other strains, plasmid, culture conditions and DNA manipulations.
E. coli TG1 was used for transformations with the plasmid pET3a. For expression, E. coli HMS174(DE3) was chosen as a host strain for derivatives of pET3a as described by Studier et al. (1990) . E. coli was grown on LuriaBertani medium (Gibco-BRL Life Technologies) or on modified M9ZB medium (Studier et al., 1990
) at 37 °C. For selection of transformants harbouring pET3a, ampicillin was added to the medium to a final concentration of 50 µg ml-1. Chromosomal DNA of B. stearothermophilus ATCC 12980 was prepared by using Genomic Tips 100 (QIAGEN) according to the manufacturers instructions. Digestion of PCR fragments with restriction endonucleases, separation of DNA fragments by agarose gel electrophoresis, ligation of DNA fragments and transformation procedures were performed as described by Sambrook et al. (1989)
.
PCR amplification, plasmid construction, cloning and expression.
Derived from the repeated amino acid sequences within SbsC and from sequence comparison with other S-layer proteins (Jarosch et al., 2000 ), various N- or C-terminal SbsC truncations (rSbsC2581099, rSbsC3431099, rSbsC4471099, rSbsC31713, rSbsC31844, rSbsC31860, rSbsC31880, rSbsC31900, rSbsC31920 and rSbsC31930) were constructed by PCR amplification of the corresponding DNA fragments using the oligonucleotides listed in Table 1
. For cloning, NdeI and BamHI restriction sites were introduced during PCR at the 5' and 3' end, respectively. PCR amplification was performed under conditions given in a previous study (Jarosch et al., 2000
). The PCR products encoding the mature SbsC (rSbsC311099) or the various truncated SbsC forms were inserted into the corresponding restriction sites of pET3a and transformed into E. coli TG1 and E. coli HMS174(DE3). The plasmid stability test and expression were performed as described by Jarosch et al. (2000)
. Samples of cultures from E. coli HMS174(DE3) were taken 28 h after induction of expression. Preparation of biomass samples and SDS-PAGE were carried out as described by Laemmli (1970)
. Immunoblotting with polyclonal rabbit antiserum raised against the S-layer protein of B. stearothermophilus ATCC 12980 and N-terminal sequencing was performed as described by Egelseer et al. (1996)
. Whole cells of E. coli HMS174(DE3) induced to express the various pET3a constructs were fixed, embedded in Spurr resin and subjected to ultrathin sectioning according to procedures given by Messner et al. (1984)
. Preparations were examined with a Philips CM 100 transmission electron microscope, operated at 80 kV and equipped with a 30 µm objective aperture.
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Investigation of the self-assembly properties of rSbsC311099 and of the various N- or C-terminal SbsC truncations.
To evaluate the self-assembly properties, 1 mg of the purified and lyophilized S-layer proteins was dissolved per ml 2 M GHCl in 50 mM Tris/HCl buffer (pH 7·2). The solutions were dialysed against distilled water, 10 mM CaCl2 or 10 mM KCl for at least 24 h at 20 °C. The suspensions were negatively stained and prepared for ultrathin sectioning according to procedures described by Messner et al. (1984) . Labelling of self-assembly products formed by rSbsC311099 with polycationized ferritin as a positively charged topographical marker was carried out as described in a previous study (Messner et al., 1986
).
Isolation of HMMA from the S-layer deficient strain, and affinity studies.
Extraction of HMMA from cell wall fragments of the S-layer-deficient strain of B. stearothermophilus ATCC 12980 with 4 M GHCl in 50 mM Tris/HCl buffer (pH 7·2) was performed as described by Egelseer et al. (1996) . After centrifugation of the suspension at 36000 g for 10 min at 4 °C, the clear supernatant was diluted with 50 mM Tris/HCl buffer to a final GHCl concentration of 2 M and the solution was subjected to GPC using a Sephacryl S-200 column for separation. Fractions containing HMMA were pooled, dialysed against 50 mM Tris/HCl buffer (pH 7·2) for 24 h at 4 °C and the protein content of the dialysed solution was adjusted to 100 µg per ml buffer. The purity of HMMA was checked by SDS-PAGE and the amylolytic activity was visualized in situ on SDS-gels by the method of Lacks & Springhorn (1980)
. To perform the binding assay, 1 mg lyophilized self-assembly products consisting of rSbsC311099, rSbsC31880 or rSbsC2581099 were dissolved in 1 ml 2 M GHCl in 50 mM Tris/HCl buffer, and the solutions were dialysed for 18 h at 20 °C against 50 mM Tris/HCl buffer. After the addition of 100 µl HMMA solution, the suspensions were incubated for 20 min at 20 °C, centrifuged at 36000 g for 10 min at 4 °C and the pellets were washed twice with buffer. The pellets and the supernatants were collected and analysed by SDS-PAGE. For comparison, self-assembly products formed by the S-layer protein from Bacillus sphaericus CCM 2177 were used (Ilk et al., 1999
). The affinity of the HMMA to the native peptidoglycananionic polymer complex and to peptidoglycan devoid of the secondary cell wall polymer from B. stearothermophilus ATCC 12980, B. stearothermophilus PV72/p2 (Sára et al., 1996
) and B. sphaericus CCM 2177 (Ilk et al., 1999
) was also investigated.
Investigation of the affinity of rSbsC311099 and of the various N- or C-terminal SbsC truncations to the native peptidoglycananionic polymer complex as well as to peptidoglycan.
For affinity studies, 1 mg lyophilized rSbsC311099 and of the self-asssembling SbsC truncations was dissolved in 1 ml 2 M GHCl in 50 mM Tris/HCl buffer (pH 7·2) and either 1 mg lyophilized native peptidoglycananionic polymer complex or 1 mg peptidoglycan (HF-extracted peptidoglycananionic polymer complex) was added. The suspensions were then stirred for 15 min at 20 °C. After dialysis against distilled water for at least 24 h at 4 °C, the suspensions were negatively stained and prepared for ultrathin sectioning. To investigate the binding properties of the soluble truncated S-layer proteins, 1 mg of the lyophilized material was dissolved in 1 ml 50 mM Tris/HCl buffer (pH 7·2) and 1 mg native peptidoglycananionic polymer complex or peptidoglycan was added. The suspensions were incubated for 30 min at 20 °C and centrifuged at 36000 g for 20 min at 4 °C. Subsequently, the pellets and the supernatants were analysed by SDS-PAGE.
DNA sequencing.
PCR products encoding the mature SbsC or the various truncated SbsC forms were sequenced as described elsewhere (Jarosch et al., 2000 ).
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RESULTS |
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As observed with the C-terminal truncations rSbsC31880, rSbsC31900 and rSbsC31920, the length in this part of the sequence had a significant influence on the formation of the oblique lattice structure. The C-terminal truncation rSbsC31880 reassembled into flat sheets or open-ended cylinders and the self-assembly process was as quantitative as that of rSbsC311099, since no S-layer protein could be detected in the supernatant after the removal of self-assembled material by centrifugation. In contrast to rSbsC311099, self-assembly products formed by rSbsC31880 did not exhibit a regular lattice structure (Fig. 3d). Self-assembly products consisting of rSbsC31900 (Fig. 3e
), rSbsC31920 (Fig. 3f
) or rSbsC31930 (not shown) had a comparable shape, but differences were observed in the quality of the oblique lattice structure. For example, self-assembly products formed by rSbsC31900 exhibited only a diffuse lattice structure in negatively stained preparations (Fig. 3e
), while self-assembly products consisting of rSbsC31920 (Fig. 3f
) or rSbsC31930 clearly showed the oblique lattice structure. Considering the water solublity of rSbsC31860 and the formation of first self-assembly products by rSbsC31880, an elongation by 20 aa was sufficient to establish the contact sites necessary for subunit-to-subunit interactions. Based on the results obtained with rSbsC31880 and rSbsC31920, only 40 additional aa in the C-terminal part were required to completely restore the oblique lattice structure (Fig. 3e
, f
).
Affinity studies with HMMA
In addition to self-assembly products formed by rSbsC311099, those consisting of rSbsC2581099 or rSbsC31880, which represented either the shortest N- or the shortest C-terminal truncation capable of self-assembly, were used for affinity studies with HMMA. As shown by SDS-PAGE, the exoenzyme (Fig. 4, lanes 1 and 2) could bind to self-assembly products formed by rSbsC311099 (Fig. 4
, lanes 3 and 6) or rSbsC31880 (Fig. 4
, lanes 4 and 7), but not to those consisting of rSbsC2581099 (Fig. 4
, lanes 5 and 8), which indicated that the N-terminal part of SbsC must comprise the binding region for HMMA. Actually, two sequences of 6 and 7 aa (AKAALD and KAAYEAA), which are located between aa 223228 and 254260 in the N-terminal part of SbsC, were previously identified in the amylase-binding protein AbpA of Streptococcus gordonii (Rogers et al., 1998
). Further affinity studies revealed that HMMA recognized the native peptidoglycananionic polymer complex from B. stearothermophilus ATCC 12980, as well as pure peptidoglycan of the A1
-chemotype from which the secondary cell wall polymer had been extracted, as binding site. Moreover, HMMA could bind to the native peptidoglycananionic polymer complex of B. stearothermophilus PV72/p2, which possesses the same peptidoglycan chemotype (A1
), but reveals a different type of secondary cell wall polymer (Sára & Sleytr, 2000
). In contrast, the exoenzyme did not bind to native peptidoglycananionic polymer complex or to peptidoglycan from B. sphaericus CCM 2177, which has a different peptidoglycan chemotype (A4
) (Ilk et al., 1999
) as well as a different type of secondary cell wall polymer than B. stearothermophilus ATCC 12980 (Egelseer et al., 1998
). Furthermore, the exoenzyme did not bind to self-assembly products formed by the S-layer protein from B. sphaericus CCM 2177 (not shown).
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DISCUSSION |
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As demonstrated in previous studies, a proteolytic cleavage fragment of the S-layer protein SbsC from B. stearothermophilus ATCC 12980 lacking the N-terminal 227 aa had lost the ability to bind to the native peptidoglycananionic polymer complex, but was still able to self-assemble (Egelseer et al., 1998 ; Jarosch et al., 2000
). From affinity studies it became apparent that the N-terminal part of SbsC recognizes the secondary cell wall polymer as the proper anchoring structure in the rigid cell wall layer. Studies on the biological function revealed that the oblique S-layer lattice from B. stearothermophilus ATCC 12980 serves as an adhesion site for HMMA, an exoamylase that was produced by the S-layer-carrying as well as by the S-layer-deficient strain (Egelseer et al., 1996
). Several studies indicated that HMMA does not participate in the self-assembly process but recognizes protein domains that are exposed on the outer S-layer surface (Egelseer et al., 1996
, 1997
).
To obtain more insight into the structurefunction relationship of distinct segments of the S-layer protein SbsC, the PCR products encoding three N- or seven C-terminal truncations were cloned and expressed in E. coli HMS174(DE3). In agreement with the results obtained from previous studies (Egelseer et al., 1998 ; Jarosch et al., 2000
) with the proteolytic cleavage fragment of the S-layer protein SbsC lacking the N-terminal 227 aa, all N-terminal truncations had lost the ability to bind to the native peptidoglycananionic polymer complex, while all C-terminal truncations recognized them as a binding site. Thus, it is obvious that the N-terminal region comprising aa 31257 is responsible for cell wall binding.
In contrast to rSbsC311099 and to the self-assembling C-terminal truncations which formed in vitro monolayers as well as double-layer self-assembly products, the N-terminal truncation rSbsC2581099 reassembled exclusively into monolayer cylinders exhibiting an oblique lattice structure (Fig. 3c). This observation indicated that the N-terminal region of SbsC is responsible for double-layer formation, which was independent of the presence of bivalent cations. In a previous study, conditions leading to the formation of monolayer or double-layer self-assembly products, or both, were investigated in detail for the wild-type S-layer glycoprotein from B. stearothermophilus NRS 2004/3a (Messner et al., 1986
). Interestingly, in double-layer self-assembly products the individual monolayers had bound to each other with the inner S-layer surface. Double-layer formation occurred only in the presence of bivalent cations. Because polycationized ferritin was densely bound by the monolayer self-assembly products, but not at all by the double layers, it was concluded that the inner S-layer surface has a net negative charge. These findings are in clear contrast to the results obtained in the present study for the self-assembly products consisting of rSbsC311099, since on the one hand, the monolayers did not bind polycationized ferritin, while on the other hand, the formation of double-layer self-assembly products was independent of the presence of bivalent cations. Moreover, sequence analysis revealed that the N-terminal part of SbsC which must be located on the inner S-layer surface and is conserved among S-layer proteins from B. stearothermophilus wild-type strains (Sára & Sleytr, 2000
) reveals a positive net charge with a pI>9. These data strongly indicate that the net negative charge determined on the inner S-layer surface of self-assembly products formed by the wild-type S-layer proteins might result from adherent secondary cell wall polymer (Egelseer et al., 1998
; Sára et al., 1998
; Sára & Sleytr, 2000
).
Affinity studies with the HMMA and self-assembly products formed by rSbsC311099, rSbsC2581099 or rSbsC31880 demonstrated that the exoenzyme requires the N-terminal part for binding to this S-layer protein. Comparison of the N-terminal part of SbsC with the amylase-binding protein AbpA from Streptococcus gordonii (Rogers et al., 1998 ) revealed the existence of common sequences comprising 6 aa (AKAALD) and 7 aa (KAAYEAA). In SbsC, the 7 aa sequence is located in the N-terminal part between aa 254 and 260, and carries a glutamic acid residue which is attacked by endoproteinase Glu-C and can therefore be expected to be exposed on the surface of the S-layer subunits (Egelseer et al., 1998
). Derived from secondary-structure prediction, the N-terminal part is mainly organized as
-helices connected by loops and seems to fold independently of the remainder of the protein sequence. Although the N-terminal part is involved in cell wall binding and a major portion must therefore be located on the inner S-layer surface, it cannot be excluded that certain sequences are exposed on the outer S-layer surface, thereby functioning as adhesion site for HMMA. It was interesting to observe that the 6 and 7 aa sequences which were identical to those on AbpA of S. gordonii (Rogers et al., 1998
) were located at the end of the N-terminal part, which connects this structurally defined domain to that part of the sequence responsible for the formation of the oblique lattice structure. In contrast to the S-layer protein, HMMA could bind to peptidoglycan of the A1
-chemotype, but it is not currently known whether the exoenzyme also shows affinity to the secondary cell wall polymer. The pullulanase of Thermoanaerobacterium thermosulfurigenes EM1 was demonstrated to bind via its SLH motifs to the native peptidoglycananionic polymer complex. However, a reduced amount of this exoenzyme was able to attach to the HF-extracted peptidoglycananionic polymer complex, which most probably represented pure peptidoglycan (Brechtel & Bahl, 1999
). Surface plasmon resonance, a method to determine molecular interactions, further revealed that HMMA immobilized on the sensor chips could bind the native peptidoglycananionic polymer complex as well as the peptidoglycan, but the exoenzyme was not able to interact with non-assembled monomeric rSbsC311099 (unpublished results). These findings strongly indicated that in addition to appropriate HMMA-binding sites on the S-layer subunits, the oblique lattice structure or at least the dimers are required for exoenzyme binding. This is in agreement with observations on whole cells of B. stearothermophlius ATCC 12980 (Egelseer et al., 1996
).
As described for the S-layer proteins of A. hydrophila and C. glutamicum (Thomas et al., 1992 ; Chami et al., 1997
), the C-terminal part of SbsC is of major importance for the self-assembly process and for the formation of the oblique lattice structure. With the various C-terminal SbsC truncations it could be demonstrated that the C-terminal 219 aa do not determine the shape of the self-assembly products and can be deleted without interfering with the self-assembly process, while the C-terminal 179 aa are not required for the formation of the oblique lattice structure (Fig. 7
).
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ACKNOWLEDGEMENTS |
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Received 29 September 2000;
revised 8 January 2001;
accepted 30 January 2001.