S-layer protein gene of Acetogenium kivui: cloning and expression in Escherichia coli and determination of the nucleotide sequence.

Acetogenium kivui is anaerobically growing thermophilic bacterium with a gram-positive type of cell wall structure. The outer surface is covered with a hexagonally packed surface (S) layer. The gene coding for the S-layer polypeptide was cloned in Escherichia coli on two overlapping fragments by using the plasmid pUC18 as the vector. It was expressed under control of a cloned Acetogenium promoter or the lacZ gene. We determined the complete sequence of the structural gene. The mature polypeptide comprises 736 amino acids and is preceded by a typical procaryotic signal sequence of 26 amino acids. It i weakly acidic, weakly hydrophilic, and contains a relatively high proportion of hydroxyamino acids, including two clusters of serine and threonine residues. An N-terminal region of about 200 residues is homologous to the N-terminal part of the middle wall protein, one of the two S-layer proteins of Bacillus brevis, and there is also an internal homology within the N-terminal region of the A. kivui polypeptide.     

The tetragonal surface layer of Clostridium aceticum: three-dimensional structure and comparison with the hexagonal layer of Clostridium thermohydrosulfuricum

The regular surface layer (S-layer) of Clostridium aceticum has been isolated and the three-dimensional structure determined to a resolution of 2.0 nm from tilt series of negatively stained preparations. It has tetragonal symmetry with a lattice constant of 12 nm and a thickness of 6 nm; there are probably 4 protein monomers per unit cell. A large proportion of the protein is concentrated in massive "cores" at the major four-fold axes which are situated towards the inner surface of the layer. From these cores, delicate arms extend towards the minor four-fold axes, where secondary connectivity is established near the exterior surface. When viewed from the outside, each of the cores appears to have a large central depression, rather than a true "pore". Since this general pattern of mass distribution is shared by the hexagonal S-layer of Clostridium thermohydrosulfuricum, some consideration has been given to the possible evolutionary steps leading to changes in symmetry. From modelling experiments, it is evident that the change from four-fold to six-fold symmetry in this instance could be accomplished simply by the loss of a structural "domain" from the protomer.     

Three-dimensional structure of the surface protein layer (MW layer) of Bacillus brevis 47

The three-dimensional (3D) structure of one surface protein layer from Bacillus brevis 47, the middle wall (MW) layer, has been reconstructed from tilted-view electron micrographs after correlation averaging to a resolution of 2 nm. The MW layer has p6 symmetry with a center-to-center spacing of 18.3 nm and a minimum thickness of 5.5 nm. The reconstruction reveals a distinct domain structure: the heavier domain of six monomers jointly forms a massive core centered at the sixfold symmetry axis, and lighter domains interconnect adjacent unit cells. In addition, the larger domains collectively form a pore by making contact with each other towards the inner surface, while the smaller domains establish a second connectivity towards the outer surface of the S layer. The MW layer of B. brevis resembles the S layer of Acetogenium kivui in various aspects: they have very similar lattice parameters and highly reminiscent 3D structures; the pores penetrate through the whole core and appear to determine the porosity of the S layers.     

Isolation,gene structure,and comparative analysis of the S-layer gene <Emphasis Type="Italic">sslA</Emphasis> of <Emphasis Type="Italic">Sporosarcina ureae</Emphasis> ATCC 13881

The surface (S)-layer of Sporosarcina ureae strain ATCC 13881, a periodic ordered structure with p4 square type symmetry, was recently reported to be an excellent biotemplate for the formation of highly ordered metal clusters. The S-layer is formed by self-assembly of a single subunit, the 116 kDa SslA protein. Here we report on the isolation and sequence analysis of the sslA gene. The protein sequence reveals a high degree of similarity to the sequences of other S-layer proteins that form self-assembly lattices with the p4 square type symmetry, especially to those of Bacillus sphaericus. Two conserved surface layer homology (SLH) domains in the extreme aminoterminal portion are likely to mediate attachment of the protein to secondary cell wall polymers. A central HisXXXHis motif and a cysteine residue in the carboxyl-terminal part of the protein, both extremely rare in S-layer proteins, may contribute to the high affinity for metal ions. The strong bias in the codon usage may explain that heterologous expression of SslA in E. coli is not very intense. With respect to the regulatory region we notice several features that are also present in other S-layer genes. The distance between the −35/−10 region and the ATG initiation codon is unusually long, and a 41 bp palindromic sequence is present in the immediate vicinity of the −35/−10 region.     

Characterization of a dynamic S layer on Bacillus thuringiensis.

The surfaces of three Bacillus thuringiensis strains possess an S layer composed of linear arrays of small particles arranged with p2 symmetry and with a = 8.5 nm, b = 7.2 nm, and gamma = 73 degrees. Platinum shadows of whole cells and S-layer fragments revealed the outer surface of the array to be smooth and the inner surface to be corrugated. Treatment with 2 M guanidine hydrochloride at pH 2.5 to 4 best removed the S layer for chemical characterization; it was a relatively hydrophilic 91.4-kilodalton protein with a pI of 5, no detectable carbohydrate, cysteine, methionine or tryptophan, and 21.2% nonpolar residues. No N-terminal homology with other S-layer proteins was evident. Antibody labeling experiments confirmed that the amount of S layer was proportional to the growth phase in broth cultures. Late-exponential- and stationary-growth-phase cells typically sloughed off fragments of S layer, and this may be the result of wall turnover. Indigenous autolytic activity in isolated walls rapidly digested the wall fabric, liberating soluble S-layer protein. At the same time, proteases frequently reduced the molecular weight of the 91.4-kilodalton protein, but these polypeptides could still be identified as S-layer components by immunoblotting. As cultures were serially subcultured, the frequency of appearance of the S layer diminished, and it was eventually lost. The dynamic nature of this S layer makes it atypical of most previously identified S layers and made it unusually difficult to characterize.     

An S-layer heavy chain camel antibody fusion protein for generation of a nanopatterned sensing layer to detect the prostate-specific antigen by surface plasmon resonance technology

The bacterial cell surface layer (S-layer) protein of Bacillus sphaericus CCM 2177 assembles into a square lattice structure and recognizes a distinct type of secondary cell wall polymer (SCWP) as the proper anchoring structure in the rigid cell wall layer. For generating a nanopatterned sensing layer with high density and well defined distance of the ligand on the outermost surface, an S-layer fusion protein incorporating the sequence of a variable domain of a heavy chain camel antibody directed against prostate-specific antigen (PSA) was constructed, produced, and recrystallized on gold chips precoated with thiolated SCWP. The S-layer protein moiety consisted of the N-terminal part which specifically recognized the SCWP as binding site and the self-assembly domain. The PSA-specific variable domain of the camel heavy chain antibody was selected by several rounds of panning from a phage display library of an immunized dromedary, and was produced by heterologous expression in Escherichia coli. For construction of the S-layer fusion protein, the 3'-end of the sequence encoding the C-terminally truncated form rSbpA(31)(-)(1068) was fused via a short linker to the 5'-end of the sequence encoding cAb-PSA-N7. The S-layer fusion protein had retained the ability to self-assemble into the square lattice structure. According to the selected fusion site in the SbpA sequence, the cAb-PSA-N7 moiety remained located on the outer surface of the protein lattice. After recrystallization of the S-layer fusion protein on gold chips precoated with thiolated SCWP, the monomolecular protein lattice was exploited as sensing layer in surface plasmon resonance biochips to detect PSA.     

Pullulanase of Thermoanaerobacterium thermosulfurigenes EM1 (Clostridium thermosulfurogenes): molecular analysis of the gene, composite structure of the enzyme, and a common model for its attachment to the cell surface.

The complete pullulanase gene (amyB) from Thermoanaerobacterium thermosulfurigenes EM1 was cloned in Escherichia coli, and the nucleotide sequence was determined. The reading frame of amyB consisted of 5,586 bp encoding an exceptionally large enzyme of 205,991 Da. Sequence analysis revealed a composite structure of the pullulanase consisting of catalytic and noncatalytic domains. The N-terminal half of the protein contained a leader peptide of 35 amino acid residues and the catalytic domain, which included the four consensus regions of amylases. Comparison of the consensus regions of several pullulanases suggested that enzymes like pullulanase type II from T. thermosulfurigenes EM1 which hydrolyze alpha-1,4- and alpha-1,6-glycosidic linkages have specific amino acid sequences in the consensus regions. These are different from those of pullulanases type I which only cleave alpha-1,6 linkages. The C-terminal half, which is not necessary for enzymatic function, consisted of at least two different segments. One segment of about 70 kDa contained two copies of a fibronectin type III-like domain and was followed by a linker region rich in glycine, serine, and threonine residues. At the C terminus, we found three repeats of about 50 amino acids which are also present at the N-termini of surface layer (S-layer) proteins of, e.g., Thermus thermophilus and Acetogenium kivui. Since the pullulanase of T. thermosulfurigenes EM1 is known to be cell bound, our results suggest that this segment serves as an S-layer anchor to keep the pullulanase attached to the cell surface. Thus, a general model for the attachment of extracellular enzymes to the cell surface is proposed which assigns the S-layer a new function and might be widespread among bacteria with S-layers. The triplicated S-layer-like segment is present in several enzymes of different bacteria. Upstream of amyB, another open reading frame, coding for a hypothetical protein of 35.6 kDa, was identified. No significant similarity to other sequences available in DNA and protein data bases was found.     

The cell envelope of Thermoproteus tenax: three-dimensional structure of the surface layer and its role in shape maintenance

The sulphur-dependent archaebacterium Thermoproteus tenax has a cylindrical cell shape variable in length, but constant in diameter. Its whole surface is covered by a regular protein layer (S-layer). The lattice has p6 symmetry and a lattice constant of 32.8 nm. The three-dimensional reconstruction from a tilt series of isolated and negatively stained S-layer shows a complex mass distribution of the protein: a prominent, pillar-shaped protrusion is located at the 6-fold crystallographic axis with radiating arms connecting neighbouring hexamers in the vicinity of the 3-fold axis. The base vectors of the S-layer lattice have a preferred orientation with respect to the longitudinal axis of the cell. The layer can be seen as a helical structure consisting of a right-handed, two-stranded helix, with the individual chains running parallel. Supposing that new S-layer protein is inserted at lattice faults (wedge disclinations) near the poles, growing of the layer would then proceed by moving a disclination at the end of the helix. The constant shape of the cell, as well as the particular structure of the layer, strongly suggest that this S-layer has a shape-maintaining function.     

Three-dimensional structure of the tetragonal surface layer of Sporosarcina ureae.

The three-dimensional structure of the regular surface layer of Sporosarcina ureae has been determined to a resolution of 1.7 nm by electron microscopy and image reconstruction. The S-layer has p4 symmetry, a lattice constant of 12.9 nm, and a minimum thickness of 6.6 nm. The reconstruction reveals a distinct domain structure: a massive core, arms connecting adjacent unit cells, and spurs which make contact at the subsidiary fourfold symmetry axes. In the z-direction the domains appear to be arranged in three planes, creating two entirely different surface reliefs. The S-layer has a complex pattern of pores and gaps that are 2 to 3 nm wide. In addition, the secondary-structure composition has been determined by infrared spectroscopy: about 35% of the polypeptide appears to have a beta-structure conformation.     

The S-Layer Proteins of Two Bacillus stearothermophilus Wild-Type Strains Are Bound via Their N-Terminal Region to a Secondary Cell Wall Polymer of Identical Chemical Composition

Two Bacillus stearothermophilus wild-type strains were investigated regarding a common recognition and binding mechanism between the S-layer protein and the underlying cell envelope layer. The S-layer protein from B. stearothermophilus PV72/p6 has a molecular weight of 130,000 and assembles into a hexagonally ordered lattice. The S-layer from B. stearothermophilus ATCC 12980 shows oblique lattice symmetry and is composed of subunits with a molecular weight of 122,000. Immunoblotting, peptide mapping, N-terminal sequencing of the whole S-layer protein from B. stearothermophilus ATCC 12980 and of proteolytic cleavage fragments, and comparison with the S-layer protein from B. stearothermophilus PV72/p6 revealed that the two S-layer proteins have identical N-terminal regions but no other extended structurally homologous domains. In contrast to the heterogeneity observed for the S-layer proteins, the secondary cell wall polymer isolated from peptidoglycan-containing sacculi of the different strains showed identical chemical compositions and comparable molecular weights. The S-layer proteins could bind and recrystallize into the appropriate lattice type on native peptidoglycan-containing sacculi from both organisms but not on those extracted with hydrofluoric acid, leading to peptidoglycan of the A1γ chemotype. Affinity studies showed that only proteolytic cleavage fragments possessing the complete N terminus of the mature S-layer proteins recognized native peptidoglycan-containing sacculi as binding sites or could associate with the isolated secondary cell wall polymer, while proteolytic cleavage fragments missing the N-terminal region remained unbound. From the results obtained in this study, it can be concluded that S-layer proteins from B. stearothermophilus wild-type strains possess an identical N-terminal region which is responsible for anchoring the S-layer subunits to a secondary cell wall polymer of identical chemical composition.     

The three-dimensional structures of S-layers of two novel Eubacterium species isolated from inflammatory human processes

The three-dimensional structures of the crystalline surface layers of two species of Eubacteria have been determined by electron microscopy and computerized image processing. The S-layer of Eubacterium sp. ES4C has tetragonal symmetry, with a unit cell spacing of 10.6 nm and a thickness of 9.5 nm, while that of Eubacterium sp. AHN 990 has hexagonal symmetry a = b = 15.7 nm and a thickness of 13 nm. The resolutions in the reconstructions were 2.5 nm and 1.8 nm, respectively. The reconstruction of the S-layer of strain ES4C reveals a distinct domain structure: a major tetramer, arms connecting adjacent unit cells, and a minor tetramer. The S-layer of strain AHN 990, on the other hand, has a rather complex arrangement, centred around the six-fold axis.     

The S-layer of Caulobacter crescentus: three-dimensional image reconstruction and structure analysis by electron microscopy.

The regular surface protein structure (S-layer) of Caulobacter crescentus was analyzed by electron microscopy and three-dimensional image reconstruction to a resolution of 2 nm. Projections showed that the S-layer is an array of ring structures, each composed of six subunits that are arranged on a lattice with p6 symmetry. Three-dimensional reconstructions showed that the ring subunits were approximately rod-shaped structures and were perpendicular to the plane of the array, with a linker arm emanating from approximately the middle of the rod, accounting for the connections between the rings. The calculated subunit mass was ca. 100 kDa, very close to the size of RsaA (the protein known to be at least the predominant species in the S-layer) predicted from the DNA sequence of the rsaA gene. The core region of the rings creates an open pore 2.5 to 3.5 nm in diameter. The size of the gaps between the neighboring unit cells is in the same range, suggesting a uniform porosity predicted to exclude molecules larger than ca. 17 kDa. Attempts to remove membrane material from S-layer preparations with detergents revealed that the structure spontaneously rearranged into a mirror-image double layer. Negative-stain and thin-section electron microscopy examination of colonies of C. crescentus strains with a mutation in a surface molecule involved in the attachment of the S-layer showed that shed RsaA protein organized into large sheets. The sheets in turn organized into stacks that tended to accumulate near the upper surface of the colony. Image reconstruction indicated that these sheets were also precise mirror-image double layers, and thickness measurements obtained from thin sections were consistent with this finding. The sheets were absent when these mutant strains were grown without calcium, supporting other data that calcium is involved in attachment of the S-layer to a surface molecule and perhaps in subunit-subunit interactions. We propose that when the membrane is removed from S-layer fragments by detergents or the attachment-related surface molecule is absent, the attachment sites of the S-layer align precisely to form a double layer via a calcium interaction.     

Cell wall of Thermoanaerobacterium thermosulfurigenes EM1: isolation of its components and attachment of the xylanase XynA

Thermoanaerobacterium thermosulfurigenes EM1 has a gram-positive type cell wall completely covered by a surface layer (S-layer) with hexagonal lattice symmetry. The components of the cell envelope were isolated, and the S-layer protein was purified and characterized. S-layer monomers assembled in vitro into sheets with the same hexagonal symmetry as in vivo. Monosaccharide analysis revealed that the S-layer is associated with fucose, rhamnose, mannosamine, glucosamine, galactose, and glucose. The N-terminal 31 amino acid residues of the S-layer protein showed significant similarity to SLH (S-layer homology) domains found in S-layer proteins of different bacteria and in the exocellular enzymes pullulanase, polygalacturonate hydrolase, and xylanase of T. thermosulfurigenes EM1. The xylanase from T. thermosulfurigenes EM1 was copurified with the S-layer protein during isolation of cell wall components. Since SLH domains of some structural proteins have been shown to anchor these proteins noncovalently to the cell envelope, we propose a common anchoring mechanism for the S-layer protein and exocellular enzymes via their SLH domains in the peptidoglycan-containing layer of T. thermosulfurigenes EM1. Received: 23 October 1998 / Accepted: 21 December 1998     

The regular surface layer of Acetogenium kivui: some structural, developmental and evolutionary aspects

Abstract The structure of the regular surface layer of Acetogenium kivui has been investigated by electron microscopy in conjunction with image processing techniques. From the averaged unit cell structure a tentative model of the subunit organization is derived. The occurrence of mono- and polycrystalline S-layer sheets is interpreted in terms of transitions related to the growth cycle of cell. The question is raised as to whether the striking structural similarity of S-layers, even from evolutionary rather distant species, is due to divergent or convergent evolution.     

Structural research on surface layers: a focus on stability, surface layer homology domains, and surface layer-cell wall interactions

Surface layers (S-layers) from Bacteria and Archaea are built from protein molecules arrayed in a two-dimensional lattice, forming the outermost cell wall layer in many prokaryotes. In almost half a century of S-layer research a wealth of structural, biochemical, and genetic data have accumulated, but it has not been possible to correlate sequence data with the tertiary structure of S-layer proteins to date. In this paper, some highlights of structural aspects of archaeal and bacterial S-layers that allow us to draw some conclusions on molecular properties are reviewed. We focus on the structural requirements for the extraordinary stability of many S-layer proteins, the structural and functional aspects of the S-layer homology domain found in S-layers, extracellular enzymes and related functional proteins, and outer membrane proteins, and the molecular interactions of S-layer proteins with other cell wall components. Finally, the perspectives and requirements for structural research on S-layers, which indicate that the investigation of isolated protein domains will be a prerequisite for solving S-layer structures at atomic resolution, are discussed.     

Comparative studies of S-layer proteins from Bacillus stearothermophilus strains expressed during growth in continuous culture under oxygen-limited and non-oxygen-limited conditions.

The specific properties of S-layer proteins from three different Bacillus stearothermophilus strains revealing oblique, square, or hexagonal lattice symmetry were preserved during growth in continuous culture on complex medium only under oxygen-limited conditions in which glucose was used as the sole carbon source. When oxygen limitation was relieved, amino acids became metabolized, cell density increased, and different S-layer proteins from wild-type strains became rapidly replaced by a new common type of S-layer protein with an apparent subunit molecular weight of 97,000 which assembled into an identical oblique (p2) lattice type. During switching from wild-type strains to variants, patches of the S-layer lattices characteristics for wild-type strains, granular regions, and areas with oblique lattice symmetry could be observed on the surface of individual cells from all organisms. The granular regions apparently consisted of mixtures of the S-layer proteins from the wild-type strains and the newly synthesized p2 S-layer proteins from the variants. S-layer proteins from wild-type strains possessed identical N-terminal regions but led to quite different cleavage products upon peptide mapping, indicating that they are encoded by different genes. Chemical analysis including N-terminal sequencing and peptide mapping showed that the oblique S-layer lattices synthesized under increased oxygen supply were composed of identical protein species.     

Generation of a functional monomolecular protein lattice consisting of an s-layer fusion protein comprising the variable domain of a camel heavy chain antibody

Crystalline bacterial cell surface layer (S-layer) proteins are composed of a single protein or glycoprotein species. Isolated S-layer subunits frequently recrystallize into monomolecular protein lattices on various types of solid supports. For generating a functional protein lattice, a chimeric protein was constructed, which comprised the secondary cell wall polymer-binding region and the self-assembly domain of the S-layer protein SbpA from Bacillus sphaericus CCM 2177, and a single variable region of a heavy chain camel antibody (cAb-Lys3) recognizing lysozyme as antigen. For construction of the S-layer fusion protein, the 3'-end of the sequence encoding the C-terminally truncated form rSbpA(31)(-)(1068) was fused via a short linker to the 5'-end of the sequence encoding cAb-Lys3. The functionality of the fused cAb-Lys3 in the S-layer fusion protein was proved by surface plasmon resonance measurements. Dot blot assays revealed that the accessibility of the fused functional sequence for the antigen was independent of the use of soluble or assembled S-layer fusion protein. Recrystallization of the S-layer fusion protein into the square lattice structure was observed on peptidoglycan-containing sacculi of B. sphaericus CCM 2177, on polystyrene or on gold chips precoated with thiolated secondary cell wall polymer, which is the natural anchoring molecule for the S-layer protein in the bacterial cell wall. Thereby, the fused cAb-Lys3 remained located on the outer S-layer surface and accessible for lysozyme binding. Together with solid supports precoated with secondary cell wall polymers, S-layer fusion proteins comprising rSbpA(31)(-)(1068) and cAbs directed against various antigens shall be exploited for building up monomolecular functional protein lattices as required for applications in nanobiotechnology.     

Surface-layer (S-layer) proteins sap and EA1 govern the binding of the S-layer-associated protein BslO at the cell septa of Bacillus anthracis

The Gram-positive pathogen Bacillus anthracis contains 24 genes whose products harbor the structurally conserved surface-layer (S-layer) homology (SLH) domain. Proteins endowed with the SLH domain associate with the secondary cell wall polysaccharide (SCWP) following secretion. Two such proteins, Sap and EA1, have the unique ability to self-assemble into a paracrystalline layer on the surface of bacilli and form S layers. Other SLH domain proteins can also be found within the S layer and have been designated Bacillus S-layer-associated protein (BSLs). While both S-layer proteins and BSLs bind the same SCWP, their deposition on the cell surface is not random. For example, BslO is targeted to septal peptidoglycan zones, where it catalyzes the separation of daughter cells. Here we show that an insertional lesion in the sap structural gene results in elongated chains of bacilli, as observed with a bslO mutant. The chain length of the sap mutant can be reduced by the addition of purified BslO in the culture medium. This complementation in trans can be explained by an increased deposition of BslO onto the surface of sap mutant bacilli that extends beyond chain septa. Using fluorescence microscopy, we observed that the Sap S layer does not overlap the EA1 S layer and slowly yields to the EA1 S layer in a growth-phase-dependent manner. Although present all over bacilli, Sap S-layer patches are not observed at septa. Thus, we propose that the dynamic Sap/EA1 S-layer coverage of the envelope restricts the deposition of BslO to the SCWP at septal rings.     

Comparative study on the cell wall structure of protein-producing Bacillus brevis

Abstract Among eight strains of protein-producing Bacillus brevis , three morphological groups have been identified according to the structure of the cell walls.

• (I)

  Cell wall consisting of a peptidoglycanlayer
• (II)

  Two-layered cell wall consisting of a peptidoglycan-layer and an S-layer
• (III)

  Three-layered cell wall consisting of a peptidoglycan-layer and two S-layers

Group I and group II cell walls have not been described yet for protein-producing bacteria. The S-layers observed in this study all had hexagonal symmetry and lattice constants of approximately 18 nm. The immunological relation between the S-layer proteins of the newly isolated B. brevis strains and those of B. brevis 47 has been examined using antisera against both S-layer-proteins of B. brevis 47. S-layers from protein-producing B. brevis strains, which were adjacent to the peptidoglycan-layer, were similar to each other, whether they were the outermost cell wall layer (group II) or not (group III). However, no similarity was found between these layers and the outermost S-layer of B. brevis 47 (group III).     

Distinct affinity of binding sites for S-layer homologous domains in Clostridium thermocellum and Bacillus anthracis cell envelopes

Binding parameters were determined for the SLH (S-layer homologous) domains from the Clostridium thermocellum outer layer protein OlpB, from the C. thermocellum S-layer protein SlpA, and from the Bacillus anthracis S-layer proteins EA1 and Sap, using cell walls from C. thermocellum and B. anthracis. Each SLH domain bound to C. thermocellum and B. anthracis cell walls with a different KD, ranging between 7.1 x 10(-7) and 1.8 x 10(-8) M. Cell wall binding sites for SLH domains displayed different binding specificities in C. thermocellum and B. anthracis. SLH-binding sites were not detected in cell walls of Bacillus subtilis. Cell walls of C. thermocellum lost their affinity for SLH domains after treatment with 48% hydrofluoric acid but not after treatment with formamide or dilute acid. A soluble component, extracted from C. thermocellum cells by sodium dodecyl sulfate treatment, bound the SLH domains from C. thermocellum but not those from B. anthracis proteins. A corresponding component was not found in B. anthracis.