Isolation and in vitro assembly of the components of the outer S layer of Lampropedia hyalina

The outermost component of the S layer of Lampropedia hyalina, the punctate layer, is assembled onto an inner perforate layer. The punctate layer is composed of long, tapered cylindrical units centered on p6 symmetry axes and connected by six fine linking arms, joining at the axis of threefold symmetry to create a hexagonal layer with a lattice constant of 25.6 +/- 0.5 nm (J. A. Chapman, R. G. E. Murray, and M. R. J. Salton, Proc. R. Soc. London Ser. B 158:498-513, 1963; R. G. E. Murray, Can. J. Microbiol. 9:593-600, 1963). Extraction of cell envelopes with 100 mM Tris buffer (pH 8) containing 2% deoxycholate resulted in the release of several proteins, but left the S layers intact. The punctate layer was then extracted with 3 M guanidine hydrochloride or 6 M urea, leaving the perforate layer intact. This treatment led to the release of three polypeptides with molecular weights of 60,000, 66,000, and 240,000 (60K, 66K, and 240K polypeptides). These three polypeptides reassembled on the perforate layer as a template to form the S-layer complex or self-assembled to form the punctate layer alone after dialysis of the extract against 50 mM HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid) buffer (pH 7.5) containing 10 mM CaCl2. The self-assemblies were composed of a 240K polypeptide and a 60K polypeptide. The 240K and 60K polypeptides were separated by column chromatography and examined by electron microscopy. The 240K polypeptide appeared in negative stain as a long, flexible structure and assembled into loose arrays with sixfold symmetry with obvious Y-shaped linking elements, while fractions containing both the 60K and 240K polypeptides showed assemblies closely resembling the punctuate layer. Immunoelectron microscopy was used to confirm the presence of both the 60K and 240K polypeptides as components of the punctuate layer.

Lampropedia hyalina is an aerobic, chemoheterotrophic, gram-negative eubacterium characterized by its growth habit and a remarkable surface structure or S layer (25,28,29,33). The cells divide synchronously in one plane to form ordered square tablets, one cell thick, giving rise to ruffled square colonies on solid medium and a floating, hydrophobic pellicle on the surface of liquid medium. The S layer encloses whole tablets of cells, so that the daughter cells are separated by an "intercalated layer" but remain attached by fine fibers extending between the cells (13,24,26). As the generic and specific names suggest, the tablets appear flat and individual cells contain refractile inclusions of poly-p-hydroxybutyric acid, giving rise to "glistening tablets" (28). Each tablet of cells is enclosed by a complex S layer composed of an inner perforate layer and an outer punctate layer.
The S layer of L. hyalina is different from that of many cells in that it is disposed loosely around a group of cells rather than closely applied to the cell wall of individual cells and it is intricately structured (2,13,24,26). The outer component, the punctate layer, consists of spiny units connected by linking elements to make an array with hexagonal symmetry. The punctate layer is attached to the underlying perforate layer, so named because it has the appearance of a sheet perforated with regularly arranged holes, which is loosely connected to the outer membrane of the cells by random fibers in an intercalated layer (1,13,24,26). Such complex S layers have been observed on only a limited number of bacteria. We can now offer a more detailed analysis of the components of the outer punctate layer of L. hyalina. * Corresponding author.

MATERIALS AND METHODS
Organism and growth conditions. L. hyalina UWO 440 (originally obtained from R. E. Hungate, University of California, Davis, and of the same provenance as ATCC 43383) was used throughout and was chosen because it overproduced and shed its S layer into the growth medium. It was grown at 30°C on solid medium containing 0.3% each yeast extract and Bacto-peptone (Difco Laboratories, Detroit, Mich.), 0.05% sodium acetate (pH 7.3), and solidified with 1.5% Bacto-agar (Difco) (YPA agar). After 3 days the cells formed an uninterrupted lawn across the surface of the agar. Cells were also grown in YPA broth, with shaking at 150 rpm, at 30°C (Psychrotherm incubator-shaker; New Brunswick Scientific, New Brunswick, N.J.). Stock cultures were maintained on slants of YPA agar.
Preparation of cell envelopes. Isolation of the S-layer components was initiated from cell envelopes (i.e., a complex of plasma membrane, cell wall, and S layers). All steps were carried out at 4°C. Three-liter broth cultures in early stationary phase (24 h) were centrifuged (3,500 x g, 30 min). The pellet was washed once in 50 mM HEPES [N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid) buffer (Sigma Chemical Co., St. Louis, Mo.), pH 7.5, containing 10 mM CaCl2 and suspended in 25 ml of the same buffer. The cells were disrupted by two passages through a French pressure cell (Aminco) at 16,000 lbs/in2 (1 lb/in2 = 6.895 kPa). Cell envelopes were sedimented by centrifugation (48,000 x g, 30 min) and washed in 100 mM Tris buffer (pH 8) containing 150 mM NaCl and 2% sodium deoxycholate to remove traces of cytoplasmic membrane and loosely associated proteins, leaving the outer membrane and the S layers.
Isolation of the components of the punctate layer. Cell 3682 AUSTIN AND MURRAY envelopes were suspended in 3 M guanidine hydrochloride for 15 min at room temperature to dissolve the punctate layer. The suspension was centrifuged (105,000 x g for 1 h), and the supernatant, which contained the soluble components of the punctate layer, was dialyzed against 10 mM sodium phosphate buffer (pH 7.5). The soluble components of the punctate layer were separated by column chromatography on hydroxyapatite (Bio-Rad Econocolumn, 5 ml bed volume) by elution with an increasing gradient (10 to 200 mM) of sodium phosphate buffer (pH 7.5), with a total volume of 60 ml. Column fractions (1 ml) were collected and assayed for protein concentration with the Coomassie blue dye-binding assay of Bradford (8). Peaks containing protein were examined by electron microscopy and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Preparation of antisera and isolation of IgG. Fractions containing the 240,000-molecular-weight (240K) and 60K polypeptides were applied to SDS-PAGE gels, and the proteins were separated by electrophoresis. Bands containing the 240K and 60K polypeptides were excised, and antibodies to these proteins were raised in rabbits. Immunoglobulin G (IgG) was purified from sera containing anti-60K and anti-240K antibodies by affinity chromatography on protein A-Sepharose (Pharmacia) and was used for immunoelectron microscopy studies and Western immunoblots.
Electrophoresis. Discontinuous SDS-PAGE was done by the method of Laemmli (22). Samples were run in a miniature electrophoresis chamber (Hoefer Scientific, San Francisco, Calif.) with a constant current of 12.5 mA for 0.75mm-thick slabs and 25 mA for 1.5-mm-thick slabs. For protein staining, gels were fixed and stained in a solution of 7% acetic acid-25% methanol-0.1% Coomassie blue R-250 at room temperature. Gels were destained in several changes of 7% acetic acid-25% methanol.
Electron microscopy. Micrographs were routinely taken with a Philips EM300 or Philips EM400T electron microscope operated at an accelerating voltage of 60 kV.
For routine preparation for thin sectioning, cells were fixed by the method of Burdett and Murray (12). Fixed samples were washed with cacodylate buffer, enrobed in 2% Noble agar (Difco), and stained with 1.0% uranyl acetate (BDH Chemicals, Toronto, Ontario, Canada) in distilled water for 2 h. The samples were dehydrated through a 30 to 100% ethanol series and embedded in Spurr (Marivac Ltd., Halifax, Nova Scotia, Canada) or Vestopal W (Martin Jaeger Co., Geneva, Switzerland) embedding resin. Thin sections were cut on a Reichert OMU2 Ultra Microtome with glass knives and stained with lead citrate and uranyl acetate (31).
Samples for immunoelectron microscopy were fixed in 10 mM sodium phosphate buffer containing 2.5% glutaraldehyde (Polysciences, Warrington, Pa.) and 1% paraformaldehyde (BDH Chemicals) for 1.5 h. Cells to be used in immunolabeling experiments were usually not postfixed in OSO4. Fixed cells were enrobed in 2% Noble agar and washed several times with 10 mM sodium phosphate buffer (pH 7.5). The agar blocks were dehydrated through a graded series of ethanol to 95% ethanol and were infiltrated with L. R. White resin (Bio-Rad) overnight at room temperature. After several changes of the resin, blocks were polymerized at 60°C for 20 h.
Negative stains. To prepare the combined punctate and perforate layers for negative staining, a small amount of cells, grown on YPA agar, was vigorously suspended in a drop of water by aspirating the cells in a Pasteur pipette; this method appeared to release more of the combined layers. Carbon-and Formvar-coated grids were floated on the suspensions for 1 to 2 min and then floated on a drop of 1% ammonium molybdate-0.1% glycerol at pH 7.5 for ca. 2 min. Excess stain was removed by touching the edge of the grid to a torn edge of Whatman no. 1 filter paper. Uranyl acetate (pH 4.5) was not used because it disrupted the punctate layer.
Immunoelectron microscopy. For immunolabeling, sections of L. R. White embeddings were mounted on grids and incubated on a drop of 100 mM Tris buffer (pH 7.5) containing 150 mM NaCl, 0.1% bovine serum albumin, and 0.05% Tween 20 (Tris-NaCl-BSA-Tween-20) for 15 min to block nonspecific binding sites. The sections were then floated on a drop of a 1:10 dilution of stock IgG (OD280, 2.5) in Tris-NaCl-BSA-Tween-20 for 2 h and washed twice for 3 min each on drops of Tris-NaCl-BSA-Tween-20, followed by 2 h on a 1:20 dilution of protein A-colloidal gold in Tris-NaCl-BSA-Tween-20. The nonspecifically adhering protein A-gold was then removed with repeated washes with Tris-NaCl-BSA-Tween-20 and finally with three washes in distilled water. The sections were then stained for 10 min with uranyl acetate and 3 min with lead citrate (31).
Chemical treatments to cell envelopes. Cell envelopes, suspended in 100 mM HEPES buffer (pH 7.5) to an OD600 of 1.0, were mixed with an equal volume of the dissociating reagent in distilled water and incubated for various times at room temperature, as described in the Results section. The treated suspensions were centrifuged (15,000 x g, 15 min), and residual pellets were washed once in 50 mM HEPES buffer (pH 7.5); the state of the S layers was examined by electron microscopy after negative staining and by SDS-

PAGE.
Reassembly of the punctate layer. A solution of the punctate layer components was extracted from approximately 5 mg (total protein) of cell envelopes (preferably deoxycholate-extracted) by suspension in 10 ml of 3 M guanidine hydrochloride at room temperature. After 30 min of incubation, the suspension was centrifuged (150,000 x g) for 1 h, and the supernatant was used for experiments. The solution was dialyzed overnight at 4°C against 50 mM HEPES buffer (pH 7.5) containing 10 mM CaCl2 or SrCl2 to obtain assemblies and against 10 mM EDTA or 10 mM EGTA (ethylene glycol tetraacetic acid) to determine the importance of divalent cations.
Reassembly of the entire S-layer complex required the addition of a preparation of the isolated perforate layer (1) to the soluble punctate layer components and dialysis against reagents as above. Reassembly was assayed by electron microscopy of negatively stained preparations, while protein composition of the reassemblies was assayed by SDS-PAGE.

RESULTS
General appearance of the cells and the S layer. Individual cells of L. hyalina were bounded by a normal cell wall of gram-negative profile. The envelope was covered by a complex bipartite S layer separated from the underlying cell wall by a fibrous intercalated layer (Fig. 1A). The strain L.
hyalina UWO 440 produced the composite punctate layer and perforate layers in excess, and large pieces, often larger than 1 ,um across, were shed during growth of the organism on solid medium, allowing visualization of the layers as isolated fragments by negative stains (Fig. 1B). Despite this natural separation, we found that isolation in quantity required extraction from an envelope fraction derived from disrupted cells. Effect of detergents on the cell envelope. Envelopes incubated at room temperature for up to 16 h with various nonionic detergents or bile salts still possessed a wellordered punctate and perforate layer (Table 1), although the membrane components disappeared after detergent extraction of the envelopes. Detergent treatment proved useful as a pretreatment before extraction of the S-layer proteins. While nonionic detergents and bile salts had no effect on the S layer, most anionic and cationic detergents disrupted the punctate layer, leaving the perforate layer intact. The dipolar-ionic derivative of cholic acid, CHAPS (3-[(3-cholamidopropyl) dimethylammonio] -1propanesulfonate), did not affect the structure of the punctate layer. The punctate layer was disrupted by lithium 3,5-diiodosalicylate (LIS). Several cationic detergents, including cetyltrimethylammonium bromide (CTAB), dodecyltrimethylammonium bromide (DTAB), and tetradecyltrimethylammonium bromide (TDTAB), selectively disrupted the punctate layer, causing it to form long fibers about 3 nm in thickness ( Fig. 2A). These fibers were seen only in extracts with cationic detergents which removed the 60K polypeptide from envelopes (Fig. 3, lane 7). Incubation of the S layer in 100 mM Tris buffer (pH 8.0) containing 10 mM EDTA or 10 mM EGTA disorganized but did not appreciably dissolve the punctate layer (Fig. 2B). Detergent extraction of cell envelopes was assayed by SDS-PAGE of extracted cell envelopes (Fig. 3). Very few of the envelope proteins were dissolved by nonionic detergents. The bile salts (sodium deoxycholate and cholic acid) and the cationic detergents removed more proteins than did the nonionic detergents, but bile salts did not release the 60K polypeptide. Extraction of cell envelopes with 2% deoxycholate offered the best method to remove contaminating proteins while keeping the S layer intact.
Effect of urea and guanidine hydrochloride on cell envelopes. Treatment with urea or guanidine hydrochloride released the punctate layer from cell envelopes. The predominant polypeptides dissolved by urea and guanidine hydrochloride were 32K, 60K, 66K, and 240K polypeptides (as shown in a preliminary study [2]). Electron microscopy indicated that extraction of cell envelopes with 3 M guanidine hydrochloride or 6 M urea caused disappearance of the punctate layer and partial solubilization of the perforate layer, the latter effect leading to the appearance of the 32K polypeptide (1). The insoluble residue of the envelope remaining after extraction with 3 M guanidine hydrochloride consisted of perforate layer and outer membrane pieces with strands, perhaps from the intercalated layer, radiating from them (Fig. 4A). Extraction with 3 M urea led to only partial dissolution of the punctate layer (Fig. 4B), which was easily recognized because of its distinctive structure (1, 13, 24). Coomassie blue increased after it had been exposed to either urea or guanidine hydrochloride (data not shown).
Fractionation of the components. The components of the punctate layer were obtained in solution by treating sodium deoxycholate-extracted cell envelopes in 3 M guanidine hydrochloride for 15 min at room temperature. The suspension was centrifuged, and the supernatant containing the soluble punctate layer was dialyzed overnight versus 10 mM sodium phosphate buffer (pH 7.5) and fractionated on a hydroxyapatite column. The bound proteins were eluted with a linear gradient of sodium phosphate. Four peaks were obtained (Fig. 5A). Peak 1 contained small amounts of protein which did not adsorb to the hydroxyapatite. The remaining peaks contained the components of the punctate layer. SDS-PAGE (Fig. 5B) indicated that peak 2 (fraction 25) contained the isolated 240K polypeptide, peak 3 contained predominantly the 66K polypeptide, and peak 4 contained a mixture of the 240K and 60K polypeptides.
Negative stains of samples from these fractions showed distinctive components of the punctate layer; the fractions containing the isolated 240K polypeptide contained long, slender, and slightly curved structures (Fig. 6A). Fractions containing both the 60K and the 240K proteins contained assemblies which closely resembled the native punctate layer. The center of each unit cell had a well-defined stain-excluding region in the shape of a ring, and these rings were connected by Y-shaped linking elements. In contrast, the assemblies formed by the isolated 240K protein had centers which appeared to contain the junction of six linking elements without the central ring.
Antibody made to the 240K polypeptide reacted only with that protein and showed no reaction, in Western blots, with either the 66K or 60K polypeptide (data not shown; J. W. Austin, Ph.D. thesis, University of Western Ontario, London, Ontario, Canada, 1989). There was, therefore, no indication that the larger protein was a multimeric form of Immunoelectron microscopy. When sections of L. hyalina were labeled with anti-240K or anti-60K IgG, the antibody bound specifically to the punctate layer ( Fig. 7A and B). All dilutions of IgG could be used without nonspecific binding to the resin or other cell constituents. Some label was found in the cytoplasm, and clusters of gold particles were often observed in the area of the cytoplasmic membrane or periplasm. Since fixation in aldehydes without postfixation in OS04 gave poor contrast for the cell envelope layers, cells were processed for section immunolabeling with postfixation in 1% OS04 in 10 mM sodium phosphate buffer (pH 7.5). The cell envelope layers were then stainable with lead citrate and uranyl acetate. Cells fixed in 1% OS04 labeled intensely with anti-240K IgG but did not label with anti-60K IgG, suggesting that the epitopes on the 60K polypeptide were destroyed by osmium tetroxide.
Reassembly of the punctate layer. When the soluble punctate layer proteins were mixed with intact perforate layer and dialyzed versus 50 mM HEPES buffer (pH 7.5) containing either 10 mM CaCl2 or 10 mM SrCI2, the punctate layer reassembled onto the perforate layer (Fig. 8A). Dialysis against 50 mM HEPES buffer (pH 7.5) without divalent cations resulted in very limited reassembly, in the form of small patches (Fig. 8B). Dialysis against 50 mM tetrasodium EDTA in 50 mM HEPES buffer (pH 7.5) did not allow any assembly of the punctate layer.
The punctate layer self-assembled without the perforate layer present as a template when the soluble proteins were dialyzed against 50 mM HEPES buffer (pH 7.5) containing 10 mM CaCl2 (Fig. 8C). Addition of 150 mM NaCl to the HEPES buffer-Ca2' did not affect the reassembly. The assemblies were collected in a centrifuged pellet and proved to consist almost entirely of the 240K and 60K polypeptides. Reassembled punctate layer could be negatively stained with 1% ammonium molybdate or 1% phosphotungstate; uranyl acetate could not be used because the low pH disrupted the reassemblies.
Very limited reassembly could be obtained by dialysis of the guanidine hydrochloride-soluble extract against 50 mM HEPES buffer (pH 7.5) without added CaCl2, probably the result of small amounts of calcium remaining in the extract. This calcium may have remained bound to the protein or dissolved at trace levels. Addition of EDTA or EGTA to 50 mM HEPES buffer prevented all reassembly.

DISCUSSION
The identification of the components of a visibly complex S layer was done by differential extraction and monitored by electron microscopy, associated with SDS-PAGE analysis of the polypeptides in each fraction. The insolubility of the S layer in several detergents, including sodium deoxycholate, allowed extraction of membrane proteins and loosely associated proteins but left the S layer intact. Further extraction with guanidine hydrochloride or urea dissolved the structural proteins of the S layer with little contamination from other envelope proteins. It is not unusual for nonionic detergents and bile salts to remove specifically some envelope proteins without affecting the S layer, because these detergents do not usually affect protein-protein interactions and do not usually denature proteins (23). The S layers of Aeromonas salmonicida A400 and A461 (18) and Aquaspirillum serpens VHA (20) also are not soluble in sodium deoxycholate.
The punctate layer was selectively extracted from cell envelopes by urea or guanidine hydrochloride; the latter was preferable because it extracted predominantly the punctate layer, leaving the perforate layer intact. The guanidine hydrochloride extract contained three polypeptides (60K, 66K, and 240K polypeptides). Reassemblies of the punctate layer showed that two of these three polypeptides, the 60K and 240K polypeptides, were essential to that structure. The precise role of the 66K polypeptide in the punctate layer has not been determined, but preliminary evidence indicates that the reassemblies of the punctate layer, formed by using the perforate layer as a template, contained the 66K polypeptide in addition to the 60K and 240K polypeptides. Sequential assembly-dissolution-assembly experiments were persuasive that these were the necessary components of the S-layer complex. Immunocytochemical studies with antibodies to both the 60K and 240K polypeptides showed that the punctate layer contains both of these polypeptides.
Chromatography on hydroxyapatite allowed separation of the 240K, 66K, and 60K polypeptides in dilute mixtures of unassembled punctate layer components. The eluted protein was concentrated, and this probably facilitated the observed reassembly of the isolated components. The fractions con-taining the isolated 240K polypeptide showed long, slightly curved molecules in negative stain. The limited and loose assemblies of these were convincing evidence that this molecule comprises both the delicate Y-shaped linking ele- ments and the spines of the punctate layer but not the central structure of the natural unit. When the 60K protein was present, the reassembly was enhanced and closely resembled the native punctate layer. The 66K polypeptide was only incorporated in self-assemblies of the punctate layer when the perforate layer was present. This suggests that the 66K polypeptide attaches and forms a bridge between the punctate and perforate layers. While addition of the perforate layer as a template improved the reassembly of the punctate layer, the two layers exhibited a degree of independence. This is in contrast to the behavior of the outer layers of both Aquaspirillum serpens MW5 (19) and Bacillus brevis (36,39), which fail to reassemble into even small patches if the inner layer is not present as a template.
Addition of exogenous CaCl2 assisted but was not necessary for assembly of the punctate layer; however, chelation of Ca2+ with EDTA or EGTA inhibited reassembly and denatured the punctate layer. It is likely that enough Ca2r emained bound to the soluble proteins to allow proper reassembly, and when this Ca2' was removed by chelation, the proteins were unable to assemble into the punctate layer.
Complex morphological units in S layers have been described, notably those of the S layer of Flexibacter polymorphus (32), which was shown to consist of four major polypeptides, 80K, 74K, 29K, and 13K. Others have been observed, but not characterized biochemically, on the surfaces of Amoebobacter bacillosus (14), Methylomonas albus (30), and Chromatium buderi (14,30). The freshwater photosynthetic bacteria Chromatium weissei and C. okenii possess hexagonal S layers made up of hollow cone-shaped units 25 nm long and 13 nm in diameter, with a centerto-center spacing of 19 nm (16).
L. hyalina has an unusual and complex S layer that is amenable to isolation and analysis. The function of this complex structure remains unknown. Variants of L. hyalina which lack the S layer grow as individual cells rather than in tablets (24), suggesting that the S layer somehow holds the cells together within a tablet. These naked variants are susceptible to predation by Bdellovibrio bacteriovorus, while the covered strains are resistant to B. bacteriovorus (S. Lanys, master's thesis, University of Western Ontario, London, Ontario, Canada, 1972). While the S layer is not required for growth in culture, the layer must have an important function in nature, especially when one considers the large investment in energy required to synthesize such a complex S layer.