Structural and chemical characterization of the S layer of a Pseudomonas-like bacterium.

Sections and freeze-fractured preparations showed an S layer on the surface of Pseudomonas-like strain EU2. Polyacrylamide gel electrophoresis of cell envelopes extracted with 1% sodium dodecyl sulfate (SDS) at room temperature showed three proteins (45K, 55K, and 110K). The 55K protein was identified as the S-layer protein. Incubation in 1.5 M guanidine hydrochloride removed the S layer from cell envelopes and dissociated the structure into subunits. The soluble 55K protein reassembled into planar sheets upon removal of the guanidine hydrochloride by dialysis. Electron microscopy and image processing indicated that these sheets had p4 symmetry in projection with a lattice constant of 13.2 +/- 0.1 nm (corresponding to 9.3 nm between adjacent fourfold axes). In some instances these reassemblies appeared to form small three-dimensional crystals which gave particularly clear views of the structure in projection because of the superimposition of information from a number of layers. A model is proposed with molecules having rounded lobes connected by a narrower linker region and joining at the lobes to form the fourfold axes of the array. The pattern superficially resembles those of other bacterial S layers, such as those of Aeromonas salmonicida, Aeromonas hydrophila, and Azotobacter vinelandii. Extraction of cell envelopes with 1% SDS at 50 degrees C released the 110K protein from the envelopes and removed an amorphous backing layer from the S layer. The 45K protein displayed heat-modifiable migration in SDS-polyacrylamide gel electrophoresis and was insoluble in SDS at 50 degrees C or in high concentrations of guanidine hydrochloride, suggesting that it was associated with the peptidoglycan.     

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.     

Three-dimensional structure of the regular tetragonal surface layer of Azotobacter vinelandii.

Fragments of the Azotobacter vinelandii tetragonal surface (S) layer, free of outer membrane material, were obtained by treating whole cells with 100 microM EDTA. The three-dimensional structure of the S layer was reconstructed from tilted-view electron micrographs of the S-layer fragments, after computer-assisted image processing by correlation averaging. At a resolution of 1.7 nm, the S layer exhibited funnel-shaped subunits situated at one fourfold-symmetry axis and interconnected at the other fourfold-symmetry axis to form prominent cruciform linking structures. These data, in conjunction with a relief reconstruction of the surface of freeze-etched whole cells, indicated that the apex of the funnel-shaped subunit was associated with the outer membrane, while the funnel "opening" faced the environment; the cruciform linking structures were formed at the outermost surface of the S layer. Electron microscopy and image enhancement were used to compare the structure of the outer membrane-associated S layer with that of fragments of the S layer dislodged from the outer membrane. This analysis revealed an increase in the lattice constant of the S layer from 12.5 to 13.6 nm and an alteration in the position of the cruciform linking structures in the z direction. These conformational changes resulted in a reduction in the thickness of the S layer (minimum estimate, 5 nm) and an apparent increase in the size of the gaps between the subunits. In terms of the porosity of the S layer, this gave the appearance of a transition from a closed to a more open structure.     

Structure of the regular surface layer of Aquaspirillum serpens MW5.

The structure of the regular surface layer of Aquaspirillum serpens MW5 has been investigated by electon microscopy supplemented by computer image processing and least-squares analysis. The layer has a ribbed appearance, both on the bacterium and in isolated, negatively stained fragments. However, detailed analysis indicated that the layer was composed of two hexagonal sheets having p6mm symmetry and a = 16 nm. One sheet was staggered by one half repeat along a (1,0) line of the p6nm lattice relative to the second so that, in projection, the pattern of the composite layer was a translational moiré, characterized by a series of ribs spaced 16 nm apart. The ribbed layer had cmm symmetry with a = 32 nm and b = 18.5 nm. Analysis of this pattern indicated that the two p6nm hexagonal sheets were unevenly stained, and this was confirmed by using least-squares methods to simulate the observed pattern by combining two hexagonal patterns. The general structure of the layer was consistent with a role as a selective and protective barrier on the cell surface.     

Superficial Macromolecular Arrays on the Cell Wall of Spirillum putridiconchylium

Electron microscopy of the cell envelope of Spirillum putridiconchylium, using negatively stained, thin-sectioned, and replicated freeze-etched preparations, showed two superficial wall layers forming a complex macromolecular pattern on the external surface. The outer structured layer was a linear array of particles overlying an inner tetragonal array of larger subunits. They were associated in a very regular fashion, and the complex was bonded to the outer, pitted surface of the lipopolysaccharide tripartite layer of the cell wall. The relationship of the components of the two structured layers was resolved with the aid of optical diffraction, combined with image filtering and reconstruction and linear and rotary integration techniques. The outer structural layer consisted of spherical 1.5-nm units set in double lines determined by the size and arrangement of 6- by 3-nm inner structural layer subunits, which bore one outer structural layer unit on each outer corner. The total effect of this arrangement was a double-ridged linear structure that was evident in surface replicas and negatively stained fragments of the whole wall. The packing of these units was not square but skewed by 2 degrees off the perpendicular so that the "unit array" described by optical diffraction and linear integration appeared to be a deformed tetragon. The verity of the model was checked by using a photographically reduced image to produce an optical diffraction pattern for comparison with that of the actual layers. The correspondence was nearly perfect.     

Structure of the Azotobacter vinelandii surface layer.

Electron microscopy of the Azotobacter vinelandii tetragonal surface array, negatively stained with ammonium molybdate in the presence of 1 mM calcium chloride, showed an apparent repeat frequency of 12 to 13 nm. Image processing showed dominant tetrad units alternating with low-contrast cruciform structures formed at the junction of slender linkers extending from corner macromolecules of four adjoining dominant units. The actual unit cell showed p4 symmetry, and a = b = 18.4 nm. Distilled water extraction of the surface array released a multimeric form of the single 60,000 molecular-weight protein (S protein) which constitutes the surface layer. The molecular weight of the multimer was estimated at 255,000 by gel filtration, indicating a tetrameric structure of four identical subunits and suggesting that this multimer was the morphological subunit of the S layer. Tetrameric S protein exhibited low intrinsic stability once released from the outer membrane, dissociating into monomers when incubated in a variety of buffers including those which served as the base for defined media used to cultivate A. vinelandii. The tetramer could not be stabilized in these buffers at any temperature between 4 and 30 degrees C, but the addition of 2 to 5 mM Ca2+ or Mg2+ completely prevented its dissociation into monomers. Circular dichroism measurements indicated that the secondary structure of the tetramer was dominated by aperiodic and beta-sheet conformations, and the addition of Ca2+ did not produce any gross changes in this structure. Only the tetrameric form of S protein was able to reassemble in vitro in the presence of divalent cations onto the surface of cells stripped of their native S layer.     

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.     

Surface protein composition of Aeromonas hydrophila strains virulent for fish: identification of a surface array protein.

The surface protein composition of members of a serogroup of Aeromonas hydrophila which exhibit high virulence for fish was examined. Treatment of whole cells of representative strain A. hydrophila TF7 with 0.2 M glycine buffer (pH 4.0) resulted in the release of sheets of a tetragonal surface protein array. Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis analysis showed that this sheet material was composed primarily of a protein of apparent molecular weight 52,000 (52K protein). A 52K protein was also the predominant protein in glycine extracts of other members of the high-virulence serogroup. Immunoblotting with antiserum raised against formalinized whole cells of A. hydrophila TF7 showed the 52K S-layer protein to be the major surface protein antigen, and impermeant Sulfo-NHS-Biotin cell surface labeling showed that the 52K S-layer protein was the only protein accessible to the Sulfo-NHS-Biotin label and effectively masked underlying outer membrane (OM) proteins. In its native surface conformation the 52K S-layer protein was only weakly reactive with a lactoperoxidase 125I surface iodination procedure. A UV-induced rough lipopolysaccharide (LPS) mutant of TF7 was found to produce an intact S layer, but a deep rough LPS mutant was unable to maintain an array on the cell surface and excreted the S-layer protein into the growth medium, indicating that a minimum LPS oligosaccharide size was required for A. hydrophila S-layer anchoring. The 52K S-layer protein exhibited hear-dependent SDS-solubilization behavior when associated with OM, but was fully solubilized at all temperatures after removal from the OM, indicating a strong interaction of the S layer with the underlying OM. The native S layer was permeable to 125I in the lactoperoxidase radiolabeling procedure, and two major OM proteins of molecular weights 30,000 and 48,000 were iodinated. The 48K species was a peptidoglycan-associated, transmembrane protein which exhibited heat-modifiable SDS solubilization behaviour characteristic of a porin protein. A 50K major peptidoglycan-associated OM protein which was not radiolabeled exhibited similar SDS heat modification characteristics and possibly represents a second porin protein.     

Two polar residues within C‐terminal domain of M1 are critical for the formation of influenza A Virions

The matrix protein 1 (M1) is the most abundant structural protein in influenza A virus particles. It oligomerizes to form the matrix layer under the lipid membrane, sustaining stabilization of the morphology of the virion. The present study indicates that M1 forms oligomers based on a fourfold symmetrical oligomerization pattern. Further analysis revealed that the oligomerization pattern of M1 was controlled by a highly conserved region within the C‐terminal domain. Two polar residues of this region, serine‐183 (S183) and threonine‐185 (T185), were identified to be critical for the oligomerization pattern of M1. M1 point mutants suggest that single S183A or T185A substitution could result in the production of morphologically filamentous particles, while double substitutions, M1‐S183A/T185A, totally disrupted the fourfold symmetry and resulted in the failure of virus production. These data indicate that the polar groups in these residues are essential to control the oligomerization pattern of M1. Thus, the present study will aid in determining the mechanisms of influenza A virus matrix layer formation during virus morphogenesis.     

Staphylococcus aureusα-Toxin: Characterization of Protein/Lipid Interactions, 2D Crystallization on Lipid Monolayers, and 3D Structure

Staphylococcus aureusα-toxin was characterized with respect to surface activity and its interaction with lipid monolayers. The protein alone had a detergent-like behavior at the air/water interface. Its affinity was higher for negatively charged than for neutral phospholipids. The interaction was pH dependent, showing a maximum increase at pH 7.0. Only a small part of the protein oligomer appeared to be inserted into the monolayers. Crystalline sheets of α-toxin were formed using negatively charged phospholipids. Electron microscopy of such areas, at different tilt angles, allowed reconstruction of a three-dimensional model following image processing. The sheets analyzed consisted of two protein layers arranged on a tetragonal lattice. Under the conditions used to grow the crystals the toxin formed 90-Å-wide cylinders with a height of 70 Å. One of the imposed fourfold axes running perpendicular to the plane of the crystalline layer is positioned at a protein-deficient region which forms a 25-Å-wide pore through the oligomer.     

Factor B Is the Second Lipopolysaccharide-binding Protease Zymogen in the Horseshoe Crab Coagulation Cascade

Factor B is a serine-protease zymogen in the horseshoe crab coagulation cascade, and it is the primary substrate for activated factor C, the LPS-responsive initiator of the cascade. Factor C is autocatalytically activated to α-factor C on LPS and is artificially converted to β-factor C, another activated form, by chymotrypsin. It is not known, however, whether LPS is required for the activation of factor B. Here we found that wild-type factor B expressed in HEK293S cells is activated by α-factor C, but not by β-factor C, in an LPS-dependent manner and that β-factor C loses the LPS binding activity of factor C through additional cleavage by chymotrypsin within the N-terminal LPS-binding region. Surface plasmon resonance and quartz crystal microbalance analyses revealed that wild-type factor B binds to LPS with high affinity comparable with that of factor C, demonstrating that factor B is the second LPS-binding zymogen in the cascade. An LPS-binding site of wild-type factor B was found in the N-terminal clip domain, and the activation rate of a clip domain deletion mutant was considerably slower than that of wild-type factor B. Moreover, in the presence of LPS, Triton X-100 inhibited the activation of wild-type factor B by α-factor C. We conclude that the clip domain of factor B has an important role in localizing factor B to the surface of Gram-negative bacteria or LPS released from bacteria to initiate effective proteolytic activation by α-factor C.     

The structure and associations of the double S layer on the cell wall of Aquaspirillum sinuosum

Aquaspirillum sinuosum cell walls bear two paracrystalline, proteinaceous surface layers (S layers). Each shows a different symmetry: the inner layer is closely apposed to the outer membrane and is a tetragonal array (90 degrees axes; 5-nm units; repeat frequency 8 nm); the outer layer is a hexagonal array on the external surface (14-nm units; repeat frequency 18 nm) and, although the units have a six-pointed stellate form, the linkage between units is not resolved. The outer layer consists of a major 130-kDa protein and a 180-kDa minor component; these co-extract, co-assemble, and are inseparable by hydroxylapatite chromatography or by recrystallization. The solubilizing effects of reagents suggest stabilization by hydrogen bonding and Ca2+. The two outer layer proteins are serologically related and show partial identity by peptide mapping. Periodic acid--Schiff staining of the 180-kDa band suggests that this may be a glycosylated form of the 130-kDa component. The inner layer components form a doublet of 75- and 80-kDa polypeptides with extreme resistance to extraction. Close apposition to the outer membrane, resistance to chaotropes, aqueous insolubility, and behaviour in charge-shift electrophoresis suggest hydrophobic interaction between subunits and an integral association with the outer membrane.     

Structure of the regular surface layer of Sporosarcina ureae.

Optical diffraction and computer image processing of electron micrographs were employed to analyze the structure of the regular surface layer of Sporosarcina ureae at high resolution. Negatively stained preparations of regular surface layer fragments showed two types of tetragonal pattern, each having p4 symmetry in projection with a = 12.8 nm. Although the two patterns differed greatly in overall appearance, both had a common pattern of areas of high stain density which we interpret as arising from gaps or holes in the structure. We speculate that these holes may be related to a protective role of the regular surface layer, whereby hostile environmental agents (such as muramidases) larger than about 2 nm would be screened from the underlying layers of the bacterial surface, while the free passage of nutrients and waste products into and out of the cell would still be allowed.     

Three-dimensional structure of the surface layer protein of Aquaspirillum serpens VHA determined by electron crystallography.

The three-dimensional structure of the protein which forms the S layer of Aquaspirillum serpens strain VHA has been determined by electron microscopy. Structures have been reconstructed to a resolution of about 1.6 nm for single-layered specimens and about 4 nm for two-layered specimens. The structure, which has hexagonal symmetry, consists of a core in the shape of a cup, with six projections arising from the rim of the cup to join adjacent subunits at the threefold symmetry axes. The model is consistent with edge views of the S layer which have been obtained in this and other work. It is now clear from this work and from three-dimensional reconstructions of other bacterial S layers that a wide diversity exists in the morphology of surface layers.     

Characterization of the lipopolysaccharide from a Rhizobium phaseoli mutant that is defective in infection thread development.

The lipopolysaccharide (LPS) from a Rhizobium phaseoli mutant, CE109, was isolated and compared with that of its wild-type parent, CE3. A previous report has shown that the mutant is defective in infection thread development, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis shows that it has an altered LPS (K. D. Noel, K. A. VandenBosch, and B. Kulpaca, J. Bacteriol. 168:1392-1462, 1986). Mild acid hydrolysis of the CE3 LPS released a polysaccharide and an oligosaccharide, PS1 and PS2, respectively. Mild acid hydrolysis of CE109 LPS released only an oligosaccharide. Chemical and immunochemical analyses showed that CE3-PS1 is the antigenic O chain of this strain and that CE109 LPS does not contain any of the major sugar components of CE3-PS1. CE109 oligosaccharide was identical in composition to CE3-PS2. The lipid A's from both strains were very similar in composition, with only minor quantitative variations. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of CE3 and CE109 LPSs showed that CE3 LPS separated into two bands, LPS I and LPS II, while CE109 had two bands which migrated to positions similar to that of LPS II. Immunoblotting with anti-CE3 antiserum showed that LPS I contains the antigenic O chain of CE3, PS1. Anti-CE109 antiserum interacted strongly with both CE109 LPS bands and CE3 LPS II and interacted weakly with CE3 LPS I. Mild-acid hydrolysis of CE3 LPS I, extracted from the polyacrylamide gel, showed that it contained both PS1 and PS2. The results in this report showed that CE109 LPS consists of only the lipid A core and is missing the antigenic O chain.     

Crystalline layers and three-dimensional structure of Staphylococcus aureus alpha-toxin

Interaction of the pore-forming protein alpha-toxin from Staphylococcus aureus with lipid components from platelet membranes induces crystal formation of the toxin oligomers. Structure analysis of crystalline areas in either sodium phosphotungstic acid or a sodium phosphotungstic acid/glucose mixture has been performed with electron microscopy and image processing. Ordered domains extending up to a few micrometers were observed, particularly after application of alpha-toxin to pre-formed lipid layers. The crystals, showing tetragonal symmetry, formed either separate two-dimensional sheets or three-dimensional piles of layers. The corresponding unit cell parameter of the single layer was a = b = 109.4 A (standard deviation 2.1 A, n = 21). Incubation of the toxin with intact membranes or extracted lipids as well as application of the lipid layer technique resulted in congruous crystalline properties. The projected averaged alpha-toxin oligomer shows cyclic symmetry with a stain-filled space in the centre. The bulk of the three-dimensional model consists of four asymmetric protein units forming a ring. In addition, a small domain covers the central cavity at the face of the protein opposite to the underlying lipid. The conditions under which the tetragonal arrays are formed on the lipid layers suggest that the alpha-toxin molecule is in a conformation binding to a hydrophobic surface rather than fully inserted into a lipid bilayer.     

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.     

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.     

Physical and functional S-layer reconstitution in Aeromonas salmonicida.

The various functions attributed to the S-layer of Aeromonas salmonicida have been previously identified by their conspicuous absence in S-layer-defective mutants. As a different approach to establish the multifunctional nature of this S-layer, we established methods for reconstitution of the S-layer of A. salmonicida. Then we investigated the functional competence of the reconstituted S-layer. S-layers were reconstituted in different systems: on inert membranes or immobilized lipopolysaccharide (LPS) from purified S-layer protein (A-protein) or on viable cells from either A-protein or preassembled S-layer sheets. In the absence of divalent cations and LPS, purified A-protein in solution spontaneously assembled into tetrameric oligomers and, upon concentration by ultrafiltration, into macroscopic, semicrystalline sheets formed by oligomers loosely organized in a tetragonal arrangement. In the presence of Ca2+, purified A-protein assembled into normal tetragonal arrays of interlocked subunits. A-protein bound with high affinity (Kd, 1.55 x 10(-7) M) and specificity to high-molecular-weight LPS from A. salmonicida but not to the LPSs of several other bacterial species. In vivo, A-protein could be reconstituted only on A. salmonicida cells which contained LPS, and Ca2+ affected both a regular tetragonal organization of the reattached A-protein and an enhanced reattachment of the A-protein to the cell surface. The reconstitution of preformed S-layer sheets (produced by an S-layer-secreting mutant) to an S-layer-negative mutant occurred consistently and efficiently when the two mutant strains were cocultured on calcium-replete solid media. Reattached A-protein (exposed on the surface of S-layer-negative mutants) was able to bind porphyrins and an S-layer-specific phage but largely lacked regular organization, as judged by its inability to bind immunoglobulins. Reattached S-layer sheets were regularly organized and imparted the properties of porphyrin binding, hydrophobicity, autoaggregation, adherence to and invasion of fish macrophages and epithelial cells, and resistance to macrophage cytotoxicity. However, cells with reconstituted S-layers were still sensitive to complement and insensitive to the antibiotics streptonigrin and chloramphenicol, indicating incomplete functional reconstitution.     

An investigation of Chromatium vinosum high-potential iron-sulfur protein by EPR and Mossbauer spectroscopy; evidence for a freezing-induced dimerization in NaCl solutions.

The high-potential iron-sulfur protein (HiPIP) from Chromatium vinosum contains a cubane prosthetic group that shuttles between the [4Fe-4S]3+,2+ states. We find that the EPR spectra from this protein can be explained as a sum of two components, a major one with g = 2.02; 2.04; 2.12, and a minor one with g = 2.04; 2.07; approximately 2.13. In the presence of 0.1-2.0 M NaCl, freezing induces polymerization of the protein (presumably dimers), which is detected as intercluster spin-spin interaction in the EPR. The observed spin-spin interactions are interpreted as being due to two very similar dimeric structures in an approx. 1:2 ratio. Computer simulation of the X- and Q-band EPR spectra shows that the z-components of the g-tensors in each dimer pair must be co-linear, with center-to-center distances between the clusters of approximately 13 A and approximately 16 A. Inspection of possible dimeric structures of C. vinosum HiPIP by standard molecular graphics procedures revealed that the Fe/S cluster is exposed toward a flattened surface and is accessible to solvent. Moreover, the Fe/S clusters in two HiPIP molecules can easily achieve a center-to-center distance of approximately 14 A when approaching along a common 3-fold axis that extends through the S4 sulfur atom of the cubane; the z-component of the EPR g-tensor is co-linear with this symmetry axis.