JOURNAL OF BACTERIOLOGY, June 1979, p. 999-1009 0021-9193/79/06-0999/11$02.00/0
Vol. 138, No. 3
Isolation, Characterization, and In Vitro Assembly of the Tetragonally Arrayed Layer of Bacillus sphaericus ANNETTE THOMAS HASTIEt* AND CHARLES C. BRINTON, JR. Department of Biological Sciences, University of Pittsburgh, Pittsburgh, Pennsylvania 15260 Received for publication 2 March 1979
The tetragonally arranged cell wall layer (T-layer) of Bacillus sphaericus NTCC 9602 was isolated and characterized. Parallel studies were made on a spontaneous variant of the wild-type strain which had a T-layer subunit of altered molecular weight. A purification method for the T-layers was devised which involved separation of the cell walls from the cytoplasmic contents, urea dissociation of the T-layer from the cell walls, removal of soluble contaminants by differential centrifugation, and finally selective adsorption of uncleaved subunits to sacculi. The purified subunits retained the capacity to form an assembly in vitro with the same lattice parameters as that observed on whole cells or cell walls and could readsorb to the cell walls from which they had been extracted. Both the wild-type and the variant subunits behaved as single, homogeneous polypeptide chains. Carbohydrate assay and isoelectric point determinations revealed that both subunit types were acidic glycoproteins. Values obtained for the buoyant density, isoelectric point, and extinction coefficient differed minimally; major differences were observed in the molecular weight and the characteristic width of cylinders formed by in vitro-assembled T-layer of the wild-type and variant. Assembled T-layer was subject to alkaline or acid dissociation and in acid titration dissociated at its isoelectric point.
Cell wall layers composed of regularly arrayed particles or subunits have been observed on a variety of microorganisms (2, 3, 7, 13, 18-21, 24, 27, 28, 31, 32). Usually the arrays are either hexagonally structured (3-5, 7, 30) or tetragonally structured (square array) (11, 21, 26, 31; C. C. Brinton, Jr., J. E. McNary, and J. Carnahan, Bacteriol. Proc., p. 48, 1969; J. Carnahan, J. E. McNary, and C. C. Brinton, Jr. Proc. 25th Meet. Electron Microsc. Soc. Am., p. 210-211, 1967). Brinton et al. (Bacteriol. Proc., p. 48, 1969) reported the isolation of a tetragonally arranged layer (T-layer) from Bacillus brevis P-1 (reclassified as Bacillus sphaericus P-1 [11]). The subunits composing the T-layer were capable of in vitro self-assembly without template or additional ions, and in the appropriate conditions, were observed in the form of cylinders. These results suggested the possible involvement of the T-layer in shape determination and perhaps shape maintenance in conjunction with the peptidoglycan layer (Brinton et al., Bacteriol. Proc., p. 48,1969; C. M. Henry, Ph.D. thesis, University of Pittsburgh, Pittsburgh, Pa., 1972; A. L. Thomas, Ph.D. thesis, University of Pittsburgh, Pittsburgh, Pa., 1975).
Generally the particles composing the regularly structured layers that have been purified are composed of single, homogeneous, acidic polypeptides with carbohydrate as a minor component and rarely lipid (2, 4, 19, 21, 24, 28, 32; C. M. Henry, Ph.D. thesis, University of Pittsburgh, Pittsburgh, Pa., 1972; A. L. Thomas, Ph.D. thesis, University of Pittsburgh, Pittsburgh, Pa., 1975). The molecular weights vary widely from 36,500 (24) to 140,000 (28). The bonds holding the subunits together and to the underlying layer are noncovalent, being disrupted by reagents such as urea, formamide, guanidine-hydrochloride, and sodium dodecyl sulfate (SDS) (3, 11, 21, 28, 32; McNary et al., Bacteriol. Proc., p. 65, 1968). Electrostatic forces are also involved in the attachment of these subunits to underlying cell wall layers (3, 4, 21, 32). For only a few of the regularly structured layers have the parameters of in vitro assembly been defined (2, 4, 28, 32; J. E. McNary, J. Carnahan, and C. C. Brinton, Jr., Bacteriol. Proc., p. 65, 1968). This paper reports the purification and characterization of the tetragonally arrayed layer (Tlayer) of B. sphaericus NTCC 9602. This bacterium was chosen since the regularly structured
t Present address: School of Hygiene and Public Health, layer composed more than 50% of the cell wall mass and comprehensive characterization of the Johns Hopkins University, Baltimore, MD 21205. 999
HASTIE AND BRINTON 1000 peptidoglycan, the other major component of the cell wall, had been accomplished (12). A variant strain with altered T-layer protein was isolated and compared with the wild-type. The purification and characterization of the B. sphaericus 9602 and 9602 lower-molecularweight (Lmw) T-layer proteins provide the foundation for further studies on its interaction with the peptidoglycan.
MATERIALS AND METHODS
Cultures, culture media, and methods. B. sphaericus NTCC 9602 wild type was obtained from D. J. Tipper. B. sphaericus 9602 Lmw occurred as a spontaneous variant in a frozen stock culture. The relatedness of the Lmw strain to the wild type was questioned due to the circumstances of its appearance. However, studies demonstrated that bacteriophage isolated on one strain would infect the other, antiserum prepared to each strain would agglutinate the other strain (A. L. Thomas, Ph.D. thesis, University of Pittsburgh, Pittsburgh, Pa., 1975) and isolated Tlayer of both strains would bind to the isolated peptidoglycan sacculus of the other (8). Furthermore, crossreactivity in these three categories was not found with the similar strain B. sphaericus P-1. These results were considered by us to be indicative of a close genetic relationship between the 9602 and 9602 Lmw strains. Storage and growth conditions were the same for the wild-type and variant strains. They were routinely grown from -70°C frozen stock cultures in 5.5% dimethyl sulfoxide in Z medium and inoculated into Z medium which consisted of 10 g of tryptone (Difco), 0.14 M NaCl, 5.5 mM glucose, 1 g of yeast extract (Difco), and 0.34 ml of 10 N NaOH per liter of water. To prepare radioactively labeled T-layer protein, the following procedure was used: 100 ml of synthetic medium was inoculated with 1 ml of frozen stock culture and incubated at 37°C with aeration for 8 h. Synthetic medium consists of 0.055 M glucose, 7.6 mM ammonium sulfate, 0.8 mM magnesium sulfate, 22 mM potassium phosphate monobasic, 40 mM potassium phosphate dibasic, 0.23 mM sodium citrate, 0.02 mM biotin, 0.03 mM thiamine hydrochloride, 0.13 mM Larginine, 0.76 mM L-asparagine, 0.38 mM L-aspartic acid, 0.13 mM L-cysteine-hydrochloride, 0.68 mM Lglutamic acid, 0.34 mM L-glutamine, 1.3 mM L-glycine, 0.11 mM L-histidine-hydrochloride, 0.15 mM L-isoleucine, 0.15 mM L-leucine, 0.44 mM L-lysine-hydrochloride, 0.067 mM L-methionine, 0.12 mM L-phenylalanine, 0.26 mM L-proline, 1.9 mM DL-serine, 0.67 mM DL-threonine, 0.098 mM L-tryptophan, 0.11 mM Ltyrosine, and 0.17 mM L-valine. The entire 100-ml starter culture was then inoculated into 2 liters of the synthetic medium containing 7.6 ,tM L-leucine and 5 ml of L-[3H]leucine (giving a final count of 1.85 x 106 cpm/ml or approximately 2 mCi total) in place of the 0.15 mM L-leucine in the starter culture broth. This culture was incubated at 37°C for 16 h with shaker aeration. The growth rate in synthetic medium was approximately one half that in Z or rich medium.
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Incorporation of radioactivity was 45% of total added counts for wild-type whole cells and 65% for variant whole cells. The culture at this point was treated the same as Z-grown cultures for isolation of T-layer. Cell wall preparation. Cell wall preparations from B. sphaericus or other bacteria were obtained by the following method: 4 liters of Z broth overnight cultures (12 h) were centrifuged at 10,000 x g for 30 min. All centrifugation was performed at 4°C in a Sorvall RC2B centrifuge unless stated otherwise. Cell pellets were resuspended in 100 to 150 ml of chilled distilled water and sonicated at an output of 80 to 95 on a Branson Sonifier model W140D with large horn for 6 to 7.5 min in a 180-ml Rosett cooling tube in an ice-water bath. The sonicated suspension was centrifuged at 17,000 x g for 20 min. The soft cell wall pellet was resuspended in 40 ml of chilled, sterile, distilled water, leaving behind hard-packed unbroken cells, and placed in fresh centrifuge tubes for washing three or four more times. The washed cell walls were then used for the isolation of T-layer protein. Isolation and purification of T-layer. Cell wall pellets from 4 liters of culture were resuspended in 30 to 40 ml of unbuffered 6 M urea and agitated for 2 to 4 h at 37°C. The cell walls were pelleted at 48,000 x g for 30 min. (The pellet was either discarded or resuspended in water for sacculi preparation.) The supernatant was centrifuged again at 153,100 x g in a Beckman L3-50 centrifuge to remove remaining cell wall fragments. The supernatant was dialyzed against several changes of 0.02 M Tris-hydrochloride buffer (pH 8.0) at 4°C. All dialysis steps were performed in this way. The T-layer was concentrated and further purified by ammonium sulfate precipitation at 0.2 to 0.25 saturation. The precipitate was pelleted at 27,000 x g for 15 min at 30 min after the final addition of ammonium sulfate. The pellet was resuspended in 4 to 6 ml of 0.02 M Tris-hydrochloride buffer (pH 8.0) and dialyzed. The material at this point is referred to as a crude preparation of T-layer. Assembled material in the dialyzed suspension was pelleted at 27,000 x g for 20 min. The pelleted T-layer material was then recycled through 6 M urea dissociation, dialysis, and ammonium sulfate precipitation at 0.55 saturation. The precipitate was pelleted at 27,000 x g for 15 min; the pellet was resuspended in one half the volume of the crude preparation and dialyzed. A volume of T-layer suspension at a concentration of 20 to 30 mg/ml was added to an equal volume of cell walls in 12 M urea (0.2 g [wet weight] of cell walls per ml), and then dialyzed. Sacculi with adsorbed Tlayer protein were pelleted at 48,000 x g for 15 min. The pellets were washed with buffer, and T-layer was re-extracted in an equal volume of 6 M urea at 37°C for 1 to 2 h. Differential centrifugation at 48,000 x g for 30 min and dialysis of the T-layer-urea supernatant were followed by ammonium sulfate precipitation of the T-layer at a saturation of 0.5. The precipitate was resuspended in a small volume of buffer after centrifugation at 48,000 x g for 10 min and finally dialyzed. This suspension of T-layer was considered "pure". Preparation of purified cell walls (sacculi). A modification of the method of Schwarz et al. (25) was used to isolate the mucopeptide layer of the cell walls.
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Cell walls in water at a concentration of 0.1 g/ml (wet weight) were added dropwise over a 5-min period to a volume of boiling 4% SDS five times the volume of the cell wall suspension. After final addition of the cell wall suspension, boiling was continued for 10 min more. The suspension was then centrifuged at 48,000 x g for 15 to 20 min. The pellet was resuspended in 10 to 20 ml of sterile distilled water and repelleted as before. The sacculi were washed four or five more times and stored in water at 4°C for future use. SDSpolyacrylamide gel electrophoresis of concentrated sacculi revealed no protein bands throughout the entire gel. SDS-polyacrylamide gel electrophoresis. The methods and reagents of Maizel (17) for high-pHdissociating (SDS) system in 5% disc gels were used. Samples were treated with 0.1% SDS, 0.005 M Trishydrochloride (Sigma; pH 8.0), and 0.01 dithiothreoitol (Calbiochem) and heated in boiling water for 1.5 min, or 8 M urea was added to the samples to give a final concentration of 4 to 6 M urea to effect dissociation without boiling. The number and position of the protein bands remained the same with either treatment. Bromophenol blue (0.0015%, Fisher) and glycerol (20%, Difco) were always added to the samples. The current was usually set at 5 to 7 mA/gel tube. Gels were removed from the tubes and cut in the middle of the tracking dye band. Staining was carried out in Coomassie brilliant blue dye for 4 to 12 h. Electrophoretic destaining proceeded for 2 h at 200 V. Destained gels were stored in destaining buffer. Polyacrylamide slab gels were run in an apparatus modeled after Reid and Bieleski (22) and the SDSbuffer system of Laemmli and Maizel (14, 17) with a 7.5% separating and 5% stacking polyacrylamide gel. Maizel's procedure for electrophoresis, staining, destaining, and drying of gels was followed (17). Molecular weight marker proteins in both systems were ,B-galactosidase (Sigma), bovine serum albumin, ovalbumin, human immunoglobulin, trypsin, pepsin (Schwarz/Mann molecular weight marker kit), and Escherichia coli RNA polymerase, a gift from Dai Nakada (later purchased from Sigma). Isoelectric focusing. The following method was adapted for the T-layer system by the methods of Righetti and Drysdale (23) and Vesterberg (33). Pyrex tubes (15 cm long by 7-mm inner diameter) were filled with 3 ml of the following solution: 2 ml of 20% acrylamide:bis-acrylamide (Bio-Rad), 0.6 ml of a 40% solution of pH 3 to 10 or pH 3 to 6 ampholytes (LKB), 5 ml of 8 M urea, 10 ,tl of TEMED (N,N,N',N'-tetramethylethylenediamine; Eastman), and 0.8 ml of 0.5% ammonium persulfate. After the addition of approximately one half of the solution to the tube, the sample in 4 M urea was added (a volume of 2 to 3 pl, usually, but no larger than 25 ,Il), and then the rest of the ampholyte-gel solution was poured. After the gels had solidified, they were placed in a disc gel electrophoresis apparatus with 8% NaOH in the top cathode tank and 5% phosphoric acid in the bottom anode tank. The entire apparatus was placed at 4°C for cooling purposes. Current was set at 400 V for 20 h for focusing (25 mA initially, dropping to 1 or 2 mA after 15 to 30 min). The gels were removed from the tubes, frozen at
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-70°C or on dry ice, and then sliced into 1-mm slices (approximately 120 slices per gel). Every two slices were combined as one fraction and eluted in 1 ml of water overnight. pH's of the fractions were read. Then 10 ml of scintillation fluid (6 g of Packard Permablend III, 333 ml of Triton X-100, 667 ml of toluene) was added to the vials, and the vials were counted in a Packard scintillation counter. Electron microscopy. Samples for electron microscopic observation were prepared by placing a drop of an aqueous preparation on a collodion-carbon-filmcoated copper mesh grid. The excess fluid was then drawn off with a piece of filter paper. The sample was stained with a drop of 0.3 to 2% potassium phosphotungstic acid, pH 7.2, and the excess stain was drawn off with filter paper. The grid was allowed to air dry and then was observed in a Phillips EM 300 microscope at an accelerating voltage of 60 kV. Micrographs were made on Kodak Electron Image film EM 4489 and developed by manufacturer's suggested methods. Other stains were tried, including uranyl formate, ammonium molybdate, and silico-tungstate, but none was as useful in observing the T-layer pattern on or off the cell walls as the potassium phosphotungstic acid, although the T-layer pattern was discernible. Magnification was determined by known spacing in crystalline catalase (34). Cesium chloride gradients. Optically pure CsCl (Schwarz/Mann) was weighed out just before use and dissolved in an amount of buffer or sterile distilled water dependent upon the volume of the sample to be added. The sample was added, and the volume of the CsCl-protein solution was brought to 12 to 12.5 ml with buffer or water. An equilibrium run was usually centrifuged at 272,500 x g (Beckman L2-65B or L3-50 centrifuge) from 40 to 48 h in duration. The samples were fractionated by puncturing the bottom of the tube with a needle and collecting drops in test tubes (approximately 0.25 ml/tube). Refractive indexes were read with an Abbe refractometer on every other or every third fraction, and the densities were read from a table. Optical densities of dilutions of various fractions were read at 275 nm (Cary 14 spectrophotometer). Protein determination. Protein concentration was estimated by the procedure of Lowry et al. (16) with bovine serum albumin as a standard. Later, the method modified by Low et al. (15) for use in the presence of urea was employed. Carbohydrate assay. Staining of SDS-polyacrylamide slab gels for carbohydrate followed the procedure of Zacharius et al. (35). Carbohydrate content was quantitatively measured by the phenol-sulfuric acid reaction. This reaction is not affected by the presence of large amounts of protein (1). Glucose was used as a standard for comparison, and bovine serum albumin was used as a negative control. The samples were scanned from 520 to 400 nm in a Cary 14 spectrophotometer. Dissociation by low or high pH. The purified Tlayer protein suspension was diluted 1/20 into portions of 0.05 M pyrophosphate or 0.05 M potassium phosphate monobasic-0.05 M potassium tetraborate buffers at various pH's. The optical density at 660 nm
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was read and then the pH was read. Samples were taken for electron microscopic observation.
densitometer. Figure 3 demonstrates the progressive removal of lower-molecular-weight material thus far in the T-layer isolation. The yield RESULTS at this point was 6 to 8 ml of approximately 20 Intial observations. B. sphaericus 9602 is to 40 mg of protein per ml from 4 liters of culture. a gram-positive, flagellated, spore-forming, rod- Electron microscopic observation showed mashaped bacterium. The outermost layer of the terial assembled into sheets or pieces of the same cell wall is a tetragonally pattemed layer of tetragonal pattern as seen on cells and cell wals. seemingly globular units as seen in Fig. 1. The Also observed were open-ended cylindrical wild-type cell has the ability to form endospores structures, the majority of which appeared to in a swollen club-shaped sporangium seen in have unifonn diameters (Fig. 4A and B).
both phase-contrast and electron microscopy. The flagella are peritrichous, and the organism is motile. The variant, B. sphaericus 9602 Lmw, is nonmotile, with peritrichous, straight flagella. The variant is asporogenous and is never observed by phase-contrast microscopy to forn spores under the conditions that nornally induce the wild type to sporulate. The orientation of the T-layer pattern is parallel to the long and short axis of the cell for both the wild type and the variant. The pattern is difficult to discern on whole cells, but is easily seen on lysed cells, cell walls, and cell wall fragments, as evidenced by Fig. 2. The pattern generally remains oriented on the cell wall fragments as it is on whole cells. Isolation and puriflcation of T-layer. Tlayer protein extracted to the stage referred to as crude preparation was 60 to 80% of the total protein as determined by scan of stained polyacrylamide slab gels on a Joyce-Loebel micro-
Extraction of the wild-type T-layer by 2 M guanidine-hydrochloride or 50% formamide was possible but produced lower yields than urea. Tlayer of the variant was as easily removed by formamide as by urea. However, T-layer of both strains extracted in formamide became degraded much more quickly than in urea. Final purification was achieved by taking advantage of two useful properties of T-layer. The first of these properties was that the extracted subunits in crude preparations spontaneously self-assembled. Once the assemblies were large enough, they were separable from soluble contaminants by differential centrifugation. The major remaining contaminant after this step was a protein banding in SDS-polyacrylamide gels just below that of the T-layer band. The only method found for separation of T-layer subunits and this lower-molecular-weight material capitalizes on the ability of native T-layer
FIG. 1. Negatively stained cell of wild-type B. sphaericus 9602. T-layer pattern was easily seen on this partially lysed cell. The cell has a flat appearance with disorganized and fragmentary internal contents. Bar represents I gim.
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FIG. 2. Negatively stained preparation of B. sphaericus 9602 wild-type cell walls. The orientation of the pattern was parallel with respect to the longitudinal axis of the cell wall and the circumference. The orientation of the T-layer array varies on the hemispherical ends. Bar represents 1 ,um.
subunits to readsorb to peptidoglycan sacculi (cell walls lacking noncovalently bound material), subsequent re-extraction with urea, and concentration (8). The yield of T-layer protein at this point was approximately 3 to 4 ml of 20 mg/ml. Densitometer scans of stained SDSpolyacrylamide slab gels of the final suspension of T-layer protein gave a value of 87% purity for the wild type and 98% purity for the variant (Fig. 5). Electron microscopy showed that the protein was assembled into pieces of the same tetragonal array as viewed on whole cells, cell walls, and in the crude preparation (Fig. 6). This was considered pure T-layer protein at this stage. Chemical and physical properties. Molecular weight from gel electrophoresis. The
molecular weights of the wild type and the variant were determined by SDS-polyacrylamide gel electrophoresis with E. coli RNA polymerase (whose f,B,' subunits are 165,000 and 155,000, respectively), 8B-galactosidase (130,000), bovine serum albumin (68,000), and human immunoglobulin (heavy chain, 50,000; light chain, 23,500) as molecular weight standards. An average value of 142,000 ± 4,000 daltons was obtained for the wild-type T-layer protein. The molecular weight of the variant T-layer subunit was 120,000 ± 3,000. The large molecular weight was also confirmed by results from gel filtration studies. In 6 M urea the T-layer protein was not retained by a Sephadex G-200 column, but eluted in the void volume (A. L. Thomas, Ph.D. thesis, University of Pittsburgh, Pittsburgh, Pa., 1975).
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A B C
FIG. 3. SDS-polyacrylamide disc gels of various solutions or suspensions of T-layer in 6 M urea extraction method: (A) cell walls ofwild-type B. sphaericus, (B) supernatant of urea wash of cell walls, and (C) resuspension of precipitate of 0.2 saturation of ammonium sulfate of urea wash supernatant.
Ultraviolet spectra. The ultraviolet spectra for purified T-layer samples of both wild-type and variant strains are shown in Fig. 7. Since at neutral pH the subunits self-assembled, creating interfering scatter, the spectra were made at pH 2.0 and 12.0. The maximum absorption at pH 2 occurs at 275 nm with a minimum at 250 nm. The molar extinction coefficient was calculated at = 5.25 x 104 liters/mol. cm for the wild-type T-layer protein and e = 4.09 x 104 liters/mol. cm for the variant T-layer protein. At alkaline pH the peak of the maximum absorption shifted to 283 nm for the wild-type protein. Although there is slightly more absorption at the longer wavelengths, the peak of maximum absorption shifted only slightly to 277 nm for the variant Tlayer protein. This may indicate a difference in the number of tyrosine-tryptophan residues or perhaps a difference in the folding of the polypeptide chain between these two proteins. Carbohydrate content. SDS-polyacrylamide slab gels stained for carbohydrate revealed that T-layer is a glycoprotein. No extraneous carbohydrate bands were observed. A phenol-sulfuric acid assay for carbohydrate gave a value of 0.4% carbohydrate content for both wild-type and variant T-layer subunits. This represents approximately three hexose equivalents per molecule. The spectra scans for the Tlayer samples resembled that ofimmunoglobulin with two maximums occurring at 485 and 405 E
nm.
Buoyant density. In initial equilibrium cesium chloride gradients at neutral pH's (6.5 to 8.0), multiple peaks were seen for the T-layer proteins. These same samples rebanded in 0.05 M pyrophosphate (pH 2.0) buffer in cesium chloride gradients each produced a single sharp peak.
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The buoyant density was determined to be 1.26 for the wild type and 1.27 for the variant. We postulate that the multiple peaks seen in neutral pH gradients represent various aggregates of the protein which disassembled at low pH (see below). Isoelectric point determination. Radioactively labeled T-layer subunits purified to the crude preparation stage were used for establishing the isoelectric point of the wild type and variant. To insure dissociation of the subunits, urea was added to the gel-ampholyte solution to a final concentration of 4 M. The isoelectric point of the wild type was found to be 5.35; the isoelectric point of the variant was 5.55. The isoelectric points of the wild type and variant Tlayer extracted by formamide were essentially the same: 5.35 and 5.50, respectively. Dissociation at low and high pH. The state of assembly and aggregation of the two T-layer proteins was studied as a function of pH. The first significant decrease in turbidity of a suspension of assembled T-layer occurred when the pH dropped below the isoelectric point for that particular T-layer protein (5.35 for the wild type and 5.55 for the variant). The midpoints did not differ. Electron microscopic observations for both revealed that as the turbidity decreased the assemblies of T-layer subunits disappeared. High-pH dissociation demonstrated the same features as low-pH dissociation. Parameters of assembly. The most striking feature of T-layer protein was its ability to selfassemble into structures that may contain many hundreds of subunits. Observed in the electron microscope were two basic configurations, either a planar sheet with uneven edges or an openended cylinder with parallel sides and unevenend edges (Fig. 2). The sizes of the planar sheet structures found in a given sample varied widely. The cylindrical structures had random distributions of lengths, growing longer with time. However, the width (actually one-half the circumference) of these cylindrical structures appeared constant for either wild-type T-layer or variant T-layer protein (see Fig. 4). Note also that the axis of the assembly was at an approximate 450 angle with respect to the axis of the cylinder. Variant T-layer cylinder widths have a mean of 1.062 ± 0.154 ,m as compared with 0.460 ± 0.065 ,tm for the wild-type cylinders. This difference in average cylinder width between wild-type and variant T-layer assemblies was not attributable to differences in cell width. Log-phase cells had a mean width of 1.02 ,um with a standard deviation of ±0.039 ,um for the wild type and a mean width of 0.997 + 0.057 ,um for the variant. Cell walls (Fig. 2) which are more uniformly flattened during processing for
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FIG. 4. (A) Crude preparation of wild-type B. sphaericus 9602 T-layer (resuspended precipitate of 0.20 saturation of ammonium sulfate of the urea wash supernatant). Note the cylinders with oriented arrays at 450 to the circumference and length of the cylinder. The larger cylinder was representative of the upper range in width that was observed; the smaller cylinder represented the usual width. Also, sheets of assembled T-layer subunits were observable as well as some amorphous material. Bar represents 1 jim. (B) Crude preparation of variant B. sphaericus 9602 Lmw T-layer. Cylinders and sheets were seen as in the wild-type preparation; however, the width of the cylinders was approximately twice that observed for the wild-type T-layer in A. Bar represents 1 ,um.
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A
B
C
D
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E
F
FIG. 5. SDS-polyacrylamide slab gel of crude preparation and further purification steps of wild type and variant T-layer. (A) Crude preparation of wild-type T-layer (cell walls extracted with 6 M urea and then the supernatant precipitated with ammonium sulfate); 60% purity determined from scan on Joyce-Loebel microdensitometer. (B) Crude preparation of variant T-layer extracted and treated as in A; 80% purity determined from scan. (C) Assembled material in A after pelleting; urea dissociated and reassociated by dialysis, and ammonium sulfate precipitated; 65.5% purity. (D) Assembled material in B treated as described for C; 84% purity. (E) C preparation of wild-type T-layer readsorbed onto sacculi; urea re-extracted and ammonium sulfate precipitated; 87% purity. (F) D preparation of variant Tlayer treated as described for E; 98% purity.
electron microscopy were also measured. There slight difference in the cell wall widths of wild type and variant (1.24 ± 0.038 ,um for the wild-type and 1.15 ± 0.059 ,um for the variant cell walls). Measurements of center-to-center spacing of subunits were made on planar sheets, cylinders, whole cells, and cell walls in one direction and in a direction 900 to the first. A distance of 25 to 100 tetramers in an array was measured and then divided by the total number of tetramers in that distance. No significant differences could be found. The average center-to-center spacing based on an internal standard of catalase crystals was found to be 13.5 ± 0.9 nm for the wild-type T-layer protein and 13.5 ± 0.6 nm for the variant T-layer protein. was a
DISCUSSION A method for the purification of the T-layer of B. sphaericus was developed capitalizing on four characteristics of the regularly structured layer. The T-layer constitutes more than 50% of the cell wall mass (12) such that separation of cell walls and cytoplasmic contents yielded con-
siderable purification. The subunits were noncovalently bound to the cell wall and can be removed by urea, formamide, or guanidine-hydrochloride. Subunits thus extracted self-assembled into macromolecular structures in vitro upon removal of the disrupting agent and were separable from other soluble proteins by differential centrifugation. Finally, native uncleaved subunits readsorbed to the cell walls from which they have been isolated, thereby providing a mechanism of separation from degraded subunits (8). The subunits were not substantially altered by the urea extraction, since they retained the ability to form in vitro assemblies with the same lattice parameters as the structure exhibits on whole cells and cell walls, and could readsorb to the cell walls from which extracted. It was also found that the subunits have essentially the same isoelectric point whether extracted by urea or formamide, further indicating that the properties of the subunits were not changed by extraction. However, prolonged exposure (three times the usual) of the subunits to urea appeared to alter the ability of the subunits to reassemble (A. L. Thomas, Ph.D. thesis, University of Pittsburgh, Pittsburgh, Pa., 1975), probably by the reaction of cyanate (produced in equilibrium from urea at neutral pH) with lysine residues in the protein (29). The purified T-layer subunit of both wild type and variant was a single homogeneous, large, acidic glycoprotein, as determined by SDS-polyacrylamide gel electrophoresis, isoelectric focusing, cesium chloride equilibrium gradients, and carbohydrate assay. Thus, B. sphaericus 9602 and 9602 Lmw T-layer proteins exhibited similarities to biochemical and physical characteristics described for other regularly structured layers (4, 11, 24, 28, 32; C. M. Henry, Ph.D. thesis, University of Pittsburgh, Pittsburgh, Pa., 1972). In vitro-assembled subunits were reversibly dissociated below their isoelectric point by acid titration to pH 2. Alkaline dissociation to pH 12 appeared to denature the protein irreversibly. However, it was not possible to release the subunits from cell walls by acid titration (A. L. Thomas, Ph.D. thesis, University of Pittsburgh, Pittsburgh, Pa., 1975). This is in agreement with the results of Sleytr with Clostridium thermosaccharolyticum and Clostridium thermohydrosulfuricum (26). These observations suggest that the bonds holding the subunits together differed somewhat from those binding the subunits to the underlying layer of the cell wall. Therefore, although the cell wall was not necessary for assembly of the subunits, it appeared to stabilize bound assemblies. Two forms of extracted T-layer assembly, sheets and cylinders, do not necessarily imply
VOL. 138, 1979
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FIG. 6. (A) Negatively stained sample of purified wild-type B. sphaericus T-layer. Large aggregates of assembled T-layer of varying sizes were present. Bar represents 1 ,um. (B) Negatively stained sample of variant T-layer. Varying sizes of sheets of assembled T-layer were observed as well as small aggregates. Bar represents I tun. two different populations of subunits. In fact,
different subunit types. For example, native unactually possess cleaved subunits may assemble into sheets, curvature not detectable unless complete closure whereas proteolytically cleaved subunits cominto a cylinder has occurred. However, it is pos- pose cylinders; as Henry had observed for B. sible that these two states of assembly represent sphaericus P-1 (C. M. Henry, Ph.D. thesis, Uni-
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versity of Pittsburgh, Pittsburgh, Pa., 1972). Slight variations in physical and biochemical \properties between the wild-type and variant Tlayer protein were observed despite a 15% larger wild-type subunit molecular weight. A major difference was found in characteristic cylinder width. The twofold-larger average cylinder \ ^. width for variant subunits could not be attributed to a difference with the wild-type in intersubunit distance. Nor did it represent a change in the underlying cell wall layer since the Tlayer needed no template for assembly. Therefore, a critical change in subunit structure probably occurred which was reflected in subunitsubunit interaction at a inacromolecular level. Some flexibility in subunit-subunit bonds could account for cylinders of different widths, as ob:served in Fig. 4A. However, this flexibility is \ Xlimited since the bell-shaped distributions of wild-type and variant cylinder widths hardly
02
overlapped.
0.1
The most important observation is that the T-layer proteins of B. sphaericus 9602 and 9602 Lmw self-assembled in vitro and, in doing so, were capable of forming seamless curved structures of uniform width. Brinton et al. (Bacteriol. Proc., p. 48, 1969) has proposed on the basis of B findings a morphopoietic role for regularly Q.9 ON .,these .
Vol. 138, No. 3
Isolation, Characterization, and In Vitro Assembly of the Tetragonally Arrayed Layer of Bacillus sphaericus ANNETTE THOMAS HASTIEt* AND CHARLES C. BRINTON, JR. Department of Biological Sciences, University of Pittsburgh, Pittsburgh, Pennsylvania 15260 Received for publication 2 March 1979
The tetragonally arranged cell wall layer (T-layer) of Bacillus sphaericus NTCC 9602 was isolated and characterized. Parallel studies were made on a spontaneous variant of the wild-type strain which had a T-layer subunit of altered molecular weight. A purification method for the T-layers was devised which involved separation of the cell walls from the cytoplasmic contents, urea dissociation of the T-layer from the cell walls, removal of soluble contaminants by differential centrifugation, and finally selective adsorption of uncleaved subunits to sacculi. The purified subunits retained the capacity to form an assembly in vitro with the same lattice parameters as that observed on whole cells or cell walls and could readsorb to the cell walls from which they had been extracted. Both the wild-type and the variant subunits behaved as single, homogeneous polypeptide chains. Carbohydrate assay and isoelectric point determinations revealed that both subunit types were acidic glycoproteins. Values obtained for the buoyant density, isoelectric point, and extinction coefficient differed minimally; major differences were observed in the molecular weight and the characteristic width of cylinders formed by in vitro-assembled T-layer of the wild-type and variant. Assembled T-layer was subject to alkaline or acid dissociation and in acid titration dissociated at its isoelectric point.
Cell wall layers composed of regularly arrayed particles or subunits have been observed on a variety of microorganisms (2, 3, 7, 13, 18-21, 24, 27, 28, 31, 32). Usually the arrays are either hexagonally structured (3-5, 7, 30) or tetragonally structured (square array) (11, 21, 26, 31; C. C. Brinton, Jr., J. E. McNary, and J. Carnahan, Bacteriol. Proc., p. 48, 1969; J. Carnahan, J. E. McNary, and C. C. Brinton, Jr. Proc. 25th Meet. Electron Microsc. Soc. Am., p. 210-211, 1967). Brinton et al. (Bacteriol. Proc., p. 48, 1969) reported the isolation of a tetragonally arranged layer (T-layer) from Bacillus brevis P-1 (reclassified as Bacillus sphaericus P-1 [11]). The subunits composing the T-layer were capable of in vitro self-assembly without template or additional ions, and in the appropriate conditions, were observed in the form of cylinders. These results suggested the possible involvement of the T-layer in shape determination and perhaps shape maintenance in conjunction with the peptidoglycan layer (Brinton et al., Bacteriol. Proc., p. 48,1969; C. M. Henry, Ph.D. thesis, University of Pittsburgh, Pittsburgh, Pa., 1972; A. L. Thomas, Ph.D. thesis, University of Pittsburgh, Pittsburgh, Pa., 1975).
Generally the particles composing the regularly structured layers that have been purified are composed of single, homogeneous, acidic polypeptides with carbohydrate as a minor component and rarely lipid (2, 4, 19, 21, 24, 28, 32; C. M. Henry, Ph.D. thesis, University of Pittsburgh, Pittsburgh, Pa., 1972; A. L. Thomas, Ph.D. thesis, University of Pittsburgh, Pittsburgh, Pa., 1975). The molecular weights vary widely from 36,500 (24) to 140,000 (28). The bonds holding the subunits together and to the underlying layer are noncovalent, being disrupted by reagents such as urea, formamide, guanidine-hydrochloride, and sodium dodecyl sulfate (SDS) (3, 11, 21, 28, 32; McNary et al., Bacteriol. Proc., p. 65, 1968). Electrostatic forces are also involved in the attachment of these subunits to underlying cell wall layers (3, 4, 21, 32). For only a few of the regularly structured layers have the parameters of in vitro assembly been defined (2, 4, 28, 32; J. E. McNary, J. Carnahan, and C. C. Brinton, Jr., Bacteriol. Proc., p. 65, 1968). This paper reports the purification and characterization of the tetragonally arrayed layer (Tlayer) of B. sphaericus NTCC 9602. This bacterium was chosen since the regularly structured
t Present address: School of Hygiene and Public Health, layer composed more than 50% of the cell wall mass and comprehensive characterization of the Johns Hopkins University, Baltimore, MD 21205. 999
HASTIE AND BRINTON 1000 peptidoglycan, the other major component of the cell wall, had been accomplished (12). A variant strain with altered T-layer protein was isolated and compared with the wild-type. The purification and characterization of the B. sphaericus 9602 and 9602 lower-molecularweight (Lmw) T-layer proteins provide the foundation for further studies on its interaction with the peptidoglycan.
MATERIALS AND METHODS
Cultures, culture media, and methods. B. sphaericus NTCC 9602 wild type was obtained from D. J. Tipper. B. sphaericus 9602 Lmw occurred as a spontaneous variant in a frozen stock culture. The relatedness of the Lmw strain to the wild type was questioned due to the circumstances of its appearance. However, studies demonstrated that bacteriophage isolated on one strain would infect the other, antiserum prepared to each strain would agglutinate the other strain (A. L. Thomas, Ph.D. thesis, University of Pittsburgh, Pittsburgh, Pa., 1975) and isolated Tlayer of both strains would bind to the isolated peptidoglycan sacculus of the other (8). Furthermore, crossreactivity in these three categories was not found with the similar strain B. sphaericus P-1. These results were considered by us to be indicative of a close genetic relationship between the 9602 and 9602 Lmw strains. Storage and growth conditions were the same for the wild-type and variant strains. They were routinely grown from -70°C frozen stock cultures in 5.5% dimethyl sulfoxide in Z medium and inoculated into Z medium which consisted of 10 g of tryptone (Difco), 0.14 M NaCl, 5.5 mM glucose, 1 g of yeast extract (Difco), and 0.34 ml of 10 N NaOH per liter of water. To prepare radioactively labeled T-layer protein, the following procedure was used: 100 ml of synthetic medium was inoculated with 1 ml of frozen stock culture and incubated at 37°C with aeration for 8 h. Synthetic medium consists of 0.055 M glucose, 7.6 mM ammonium sulfate, 0.8 mM magnesium sulfate, 22 mM potassium phosphate monobasic, 40 mM potassium phosphate dibasic, 0.23 mM sodium citrate, 0.02 mM biotin, 0.03 mM thiamine hydrochloride, 0.13 mM Larginine, 0.76 mM L-asparagine, 0.38 mM L-aspartic acid, 0.13 mM L-cysteine-hydrochloride, 0.68 mM Lglutamic acid, 0.34 mM L-glutamine, 1.3 mM L-glycine, 0.11 mM L-histidine-hydrochloride, 0.15 mM L-isoleucine, 0.15 mM L-leucine, 0.44 mM L-lysine-hydrochloride, 0.067 mM L-methionine, 0.12 mM L-phenylalanine, 0.26 mM L-proline, 1.9 mM DL-serine, 0.67 mM DL-threonine, 0.098 mM L-tryptophan, 0.11 mM Ltyrosine, and 0.17 mM L-valine. The entire 100-ml starter culture was then inoculated into 2 liters of the synthetic medium containing 7.6 ,tM L-leucine and 5 ml of L-[3H]leucine (giving a final count of 1.85 x 106 cpm/ml or approximately 2 mCi total) in place of the 0.15 mM L-leucine in the starter culture broth. This culture was incubated at 37°C for 16 h with shaker aeration. The growth rate in synthetic medium was approximately one half that in Z or rich medium.
J. BACTERIOL.
Incorporation of radioactivity was 45% of total added counts for wild-type whole cells and 65% for variant whole cells. The culture at this point was treated the same as Z-grown cultures for isolation of T-layer. Cell wall preparation. Cell wall preparations from B. sphaericus or other bacteria were obtained by the following method: 4 liters of Z broth overnight cultures (12 h) were centrifuged at 10,000 x g for 30 min. All centrifugation was performed at 4°C in a Sorvall RC2B centrifuge unless stated otherwise. Cell pellets were resuspended in 100 to 150 ml of chilled distilled water and sonicated at an output of 80 to 95 on a Branson Sonifier model W140D with large horn for 6 to 7.5 min in a 180-ml Rosett cooling tube in an ice-water bath. The sonicated suspension was centrifuged at 17,000 x g for 20 min. The soft cell wall pellet was resuspended in 40 ml of chilled, sterile, distilled water, leaving behind hard-packed unbroken cells, and placed in fresh centrifuge tubes for washing three or four more times. The washed cell walls were then used for the isolation of T-layer protein. Isolation and purification of T-layer. Cell wall pellets from 4 liters of culture were resuspended in 30 to 40 ml of unbuffered 6 M urea and agitated for 2 to 4 h at 37°C. The cell walls were pelleted at 48,000 x g for 30 min. (The pellet was either discarded or resuspended in water for sacculi preparation.) The supernatant was centrifuged again at 153,100 x g in a Beckman L3-50 centrifuge to remove remaining cell wall fragments. The supernatant was dialyzed against several changes of 0.02 M Tris-hydrochloride buffer (pH 8.0) at 4°C. All dialysis steps were performed in this way. The T-layer was concentrated and further purified by ammonium sulfate precipitation at 0.2 to 0.25 saturation. The precipitate was pelleted at 27,000 x g for 15 min at 30 min after the final addition of ammonium sulfate. The pellet was resuspended in 4 to 6 ml of 0.02 M Tris-hydrochloride buffer (pH 8.0) and dialyzed. The material at this point is referred to as a crude preparation of T-layer. Assembled material in the dialyzed suspension was pelleted at 27,000 x g for 20 min. The pelleted T-layer material was then recycled through 6 M urea dissociation, dialysis, and ammonium sulfate precipitation at 0.55 saturation. The precipitate was pelleted at 27,000 x g for 15 min; the pellet was resuspended in one half the volume of the crude preparation and dialyzed. A volume of T-layer suspension at a concentration of 20 to 30 mg/ml was added to an equal volume of cell walls in 12 M urea (0.2 g [wet weight] of cell walls per ml), and then dialyzed. Sacculi with adsorbed Tlayer protein were pelleted at 48,000 x g for 15 min. The pellets were washed with buffer, and T-layer was re-extracted in an equal volume of 6 M urea at 37°C for 1 to 2 h. Differential centrifugation at 48,000 x g for 30 min and dialysis of the T-layer-urea supernatant were followed by ammonium sulfate precipitation of the T-layer at a saturation of 0.5. The precipitate was resuspended in a small volume of buffer after centrifugation at 48,000 x g for 10 min and finally dialyzed. This suspension of T-layer was considered "pure". Preparation of purified cell walls (sacculi). A modification of the method of Schwarz et al. (25) was used to isolate the mucopeptide layer of the cell walls.
VOL. 138, 1979
TETRAGONAL LAYER OF B. SPHAERICUS
Cell walls in water at a concentration of 0.1 g/ml (wet weight) were added dropwise over a 5-min period to a volume of boiling 4% SDS five times the volume of the cell wall suspension. After final addition of the cell wall suspension, boiling was continued for 10 min more. The suspension was then centrifuged at 48,000 x g for 15 to 20 min. The pellet was resuspended in 10 to 20 ml of sterile distilled water and repelleted as before. The sacculi were washed four or five more times and stored in water at 4°C for future use. SDSpolyacrylamide gel electrophoresis of concentrated sacculi revealed no protein bands throughout the entire gel. SDS-polyacrylamide gel electrophoresis. The methods and reagents of Maizel (17) for high-pHdissociating (SDS) system in 5% disc gels were used. Samples were treated with 0.1% SDS, 0.005 M Trishydrochloride (Sigma; pH 8.0), and 0.01 dithiothreoitol (Calbiochem) and heated in boiling water for 1.5 min, or 8 M urea was added to the samples to give a final concentration of 4 to 6 M urea to effect dissociation without boiling. The number and position of the protein bands remained the same with either treatment. Bromophenol blue (0.0015%, Fisher) and glycerol (20%, Difco) were always added to the samples. The current was usually set at 5 to 7 mA/gel tube. Gels were removed from the tubes and cut in the middle of the tracking dye band. Staining was carried out in Coomassie brilliant blue dye for 4 to 12 h. Electrophoretic destaining proceeded for 2 h at 200 V. Destained gels were stored in destaining buffer. Polyacrylamide slab gels were run in an apparatus modeled after Reid and Bieleski (22) and the SDSbuffer system of Laemmli and Maizel (14, 17) with a 7.5% separating and 5% stacking polyacrylamide gel. Maizel's procedure for electrophoresis, staining, destaining, and drying of gels was followed (17). Molecular weight marker proteins in both systems were ,B-galactosidase (Sigma), bovine serum albumin, ovalbumin, human immunoglobulin, trypsin, pepsin (Schwarz/Mann molecular weight marker kit), and Escherichia coli RNA polymerase, a gift from Dai Nakada (later purchased from Sigma). Isoelectric focusing. The following method was adapted for the T-layer system by the methods of Righetti and Drysdale (23) and Vesterberg (33). Pyrex tubes (15 cm long by 7-mm inner diameter) were filled with 3 ml of the following solution: 2 ml of 20% acrylamide:bis-acrylamide (Bio-Rad), 0.6 ml of a 40% solution of pH 3 to 10 or pH 3 to 6 ampholytes (LKB), 5 ml of 8 M urea, 10 ,tl of TEMED (N,N,N',N'-tetramethylethylenediamine; Eastman), and 0.8 ml of 0.5% ammonium persulfate. After the addition of approximately one half of the solution to the tube, the sample in 4 M urea was added (a volume of 2 to 3 pl, usually, but no larger than 25 ,Il), and then the rest of the ampholyte-gel solution was poured. After the gels had solidified, they were placed in a disc gel electrophoresis apparatus with 8% NaOH in the top cathode tank and 5% phosphoric acid in the bottom anode tank. The entire apparatus was placed at 4°C for cooling purposes. Current was set at 400 V for 20 h for focusing (25 mA initially, dropping to 1 or 2 mA after 15 to 30 min). The gels were removed from the tubes, frozen at
1001
-70°C or on dry ice, and then sliced into 1-mm slices (approximately 120 slices per gel). Every two slices were combined as one fraction and eluted in 1 ml of water overnight. pH's of the fractions were read. Then 10 ml of scintillation fluid (6 g of Packard Permablend III, 333 ml of Triton X-100, 667 ml of toluene) was added to the vials, and the vials were counted in a Packard scintillation counter. Electron microscopy. Samples for electron microscopic observation were prepared by placing a drop of an aqueous preparation on a collodion-carbon-filmcoated copper mesh grid. The excess fluid was then drawn off with a piece of filter paper. The sample was stained with a drop of 0.3 to 2% potassium phosphotungstic acid, pH 7.2, and the excess stain was drawn off with filter paper. The grid was allowed to air dry and then was observed in a Phillips EM 300 microscope at an accelerating voltage of 60 kV. Micrographs were made on Kodak Electron Image film EM 4489 and developed by manufacturer's suggested methods. Other stains were tried, including uranyl formate, ammonium molybdate, and silico-tungstate, but none was as useful in observing the T-layer pattern on or off the cell walls as the potassium phosphotungstic acid, although the T-layer pattern was discernible. Magnification was determined by known spacing in crystalline catalase (34). Cesium chloride gradients. Optically pure CsCl (Schwarz/Mann) was weighed out just before use and dissolved in an amount of buffer or sterile distilled water dependent upon the volume of the sample to be added. The sample was added, and the volume of the CsCl-protein solution was brought to 12 to 12.5 ml with buffer or water. An equilibrium run was usually centrifuged at 272,500 x g (Beckman L2-65B or L3-50 centrifuge) from 40 to 48 h in duration. The samples were fractionated by puncturing the bottom of the tube with a needle and collecting drops in test tubes (approximately 0.25 ml/tube). Refractive indexes were read with an Abbe refractometer on every other or every third fraction, and the densities were read from a table. Optical densities of dilutions of various fractions were read at 275 nm (Cary 14 spectrophotometer). Protein determination. Protein concentration was estimated by the procedure of Lowry et al. (16) with bovine serum albumin as a standard. Later, the method modified by Low et al. (15) for use in the presence of urea was employed. Carbohydrate assay. Staining of SDS-polyacrylamide slab gels for carbohydrate followed the procedure of Zacharius et al. (35). Carbohydrate content was quantitatively measured by the phenol-sulfuric acid reaction. This reaction is not affected by the presence of large amounts of protein (1). Glucose was used as a standard for comparison, and bovine serum albumin was used as a negative control. The samples were scanned from 520 to 400 nm in a Cary 14 spectrophotometer. Dissociation by low or high pH. The purified Tlayer protein suspension was diluted 1/20 into portions of 0.05 M pyrophosphate or 0.05 M potassium phosphate monobasic-0.05 M potassium tetraborate buffers at various pH's. The optical density at 660 nm
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was read and then the pH was read. Samples were taken for electron microscopic observation.
densitometer. Figure 3 demonstrates the progressive removal of lower-molecular-weight material thus far in the T-layer isolation. The yield RESULTS at this point was 6 to 8 ml of approximately 20 Intial observations. B. sphaericus 9602 is to 40 mg of protein per ml from 4 liters of culture. a gram-positive, flagellated, spore-forming, rod- Electron microscopic observation showed mashaped bacterium. The outermost layer of the terial assembled into sheets or pieces of the same cell wall is a tetragonally pattemed layer of tetragonal pattern as seen on cells and cell wals. seemingly globular units as seen in Fig. 1. The Also observed were open-ended cylindrical wild-type cell has the ability to form endospores structures, the majority of which appeared to in a swollen club-shaped sporangium seen in have unifonn diameters (Fig. 4A and B).
both phase-contrast and electron microscopy. The flagella are peritrichous, and the organism is motile. The variant, B. sphaericus 9602 Lmw, is nonmotile, with peritrichous, straight flagella. The variant is asporogenous and is never observed by phase-contrast microscopy to forn spores under the conditions that nornally induce the wild type to sporulate. The orientation of the T-layer pattern is parallel to the long and short axis of the cell for both the wild type and the variant. The pattern is difficult to discern on whole cells, but is easily seen on lysed cells, cell walls, and cell wall fragments, as evidenced by Fig. 2. The pattern generally remains oriented on the cell wall fragments as it is on whole cells. Isolation and puriflcation of T-layer. Tlayer protein extracted to the stage referred to as crude preparation was 60 to 80% of the total protein as determined by scan of stained polyacrylamide slab gels on a Joyce-Loebel micro-
Extraction of the wild-type T-layer by 2 M guanidine-hydrochloride or 50% formamide was possible but produced lower yields than urea. Tlayer of the variant was as easily removed by formamide as by urea. However, T-layer of both strains extracted in formamide became degraded much more quickly than in urea. Final purification was achieved by taking advantage of two useful properties of T-layer. The first of these properties was that the extracted subunits in crude preparations spontaneously self-assembled. Once the assemblies were large enough, they were separable from soluble contaminants by differential centrifugation. The major remaining contaminant after this step was a protein banding in SDS-polyacrylamide gels just below that of the T-layer band. The only method found for separation of T-layer subunits and this lower-molecular-weight material capitalizes on the ability of native T-layer
FIG. 1. Negatively stained cell of wild-type B. sphaericus 9602. T-layer pattern was easily seen on this partially lysed cell. The cell has a flat appearance with disorganized and fragmentary internal contents. Bar represents I gim.
VOL. 138, 1979
TETRAGONAL LAYER OF B. SPHAERICUS
1003
Me
.1 t
-4. I
,
1
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i
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FIG. 2. Negatively stained preparation of B. sphaericus 9602 wild-type cell walls. The orientation of the pattern was parallel with respect to the longitudinal axis of the cell wall and the circumference. The orientation of the T-layer array varies on the hemispherical ends. Bar represents 1 ,um.
subunits to readsorb to peptidoglycan sacculi (cell walls lacking noncovalently bound material), subsequent re-extraction with urea, and concentration (8). The yield of T-layer protein at this point was approximately 3 to 4 ml of 20 mg/ml. Densitometer scans of stained SDSpolyacrylamide slab gels of the final suspension of T-layer protein gave a value of 87% purity for the wild type and 98% purity for the variant (Fig. 5). Electron microscopy showed that the protein was assembled into pieces of the same tetragonal array as viewed on whole cells, cell walls, and in the crude preparation (Fig. 6). This was considered pure T-layer protein at this stage. Chemical and physical properties. Molecular weight from gel electrophoresis. The
molecular weights of the wild type and the variant were determined by SDS-polyacrylamide gel electrophoresis with E. coli RNA polymerase (whose f,B,' subunits are 165,000 and 155,000, respectively), 8B-galactosidase (130,000), bovine serum albumin (68,000), and human immunoglobulin (heavy chain, 50,000; light chain, 23,500) as molecular weight standards. An average value of 142,000 ± 4,000 daltons was obtained for the wild-type T-layer protein. The molecular weight of the variant T-layer subunit was 120,000 ± 3,000. The large molecular weight was also confirmed by results from gel filtration studies. In 6 M urea the T-layer protein was not retained by a Sephadex G-200 column, but eluted in the void volume (A. L. Thomas, Ph.D. thesis, University of Pittsburgh, Pittsburgh, Pa., 1975).
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HASTIE AND BRINTON
A B C
FIG. 3. SDS-polyacrylamide disc gels of various solutions or suspensions of T-layer in 6 M urea extraction method: (A) cell walls ofwild-type B. sphaericus, (B) supernatant of urea wash of cell walls, and (C) resuspension of precipitate of 0.2 saturation of ammonium sulfate of urea wash supernatant.
Ultraviolet spectra. The ultraviolet spectra for purified T-layer samples of both wild-type and variant strains are shown in Fig. 7. Since at neutral pH the subunits self-assembled, creating interfering scatter, the spectra were made at pH 2.0 and 12.0. The maximum absorption at pH 2 occurs at 275 nm with a minimum at 250 nm. The molar extinction coefficient was calculated at = 5.25 x 104 liters/mol. cm for the wild-type T-layer protein and e = 4.09 x 104 liters/mol. cm for the variant T-layer protein. At alkaline pH the peak of the maximum absorption shifted to 283 nm for the wild-type protein. Although there is slightly more absorption at the longer wavelengths, the peak of maximum absorption shifted only slightly to 277 nm for the variant Tlayer protein. This may indicate a difference in the number of tyrosine-tryptophan residues or perhaps a difference in the folding of the polypeptide chain between these two proteins. Carbohydrate content. SDS-polyacrylamide slab gels stained for carbohydrate revealed that T-layer is a glycoprotein. No extraneous carbohydrate bands were observed. A phenol-sulfuric acid assay for carbohydrate gave a value of 0.4% carbohydrate content for both wild-type and variant T-layer subunits. This represents approximately three hexose equivalents per molecule. The spectra scans for the Tlayer samples resembled that ofimmunoglobulin with two maximums occurring at 485 and 405 E
nm.
Buoyant density. In initial equilibrium cesium chloride gradients at neutral pH's (6.5 to 8.0), multiple peaks were seen for the T-layer proteins. These same samples rebanded in 0.05 M pyrophosphate (pH 2.0) buffer in cesium chloride gradients each produced a single sharp peak.
J. BACTERIOL.
The buoyant density was determined to be 1.26 for the wild type and 1.27 for the variant. We postulate that the multiple peaks seen in neutral pH gradients represent various aggregates of the protein which disassembled at low pH (see below). Isoelectric point determination. Radioactively labeled T-layer subunits purified to the crude preparation stage were used for establishing the isoelectric point of the wild type and variant. To insure dissociation of the subunits, urea was added to the gel-ampholyte solution to a final concentration of 4 M. The isoelectric point of the wild type was found to be 5.35; the isoelectric point of the variant was 5.55. The isoelectric points of the wild type and variant Tlayer extracted by formamide were essentially the same: 5.35 and 5.50, respectively. Dissociation at low and high pH. The state of assembly and aggregation of the two T-layer proteins was studied as a function of pH. The first significant decrease in turbidity of a suspension of assembled T-layer occurred when the pH dropped below the isoelectric point for that particular T-layer protein (5.35 for the wild type and 5.55 for the variant). The midpoints did not differ. Electron microscopic observations for both revealed that as the turbidity decreased the assemblies of T-layer subunits disappeared. High-pH dissociation demonstrated the same features as low-pH dissociation. Parameters of assembly. The most striking feature of T-layer protein was its ability to selfassemble into structures that may contain many hundreds of subunits. Observed in the electron microscope were two basic configurations, either a planar sheet with uneven edges or an openended cylinder with parallel sides and unevenend edges (Fig. 2). The sizes of the planar sheet structures found in a given sample varied widely. The cylindrical structures had random distributions of lengths, growing longer with time. However, the width (actually one-half the circumference) of these cylindrical structures appeared constant for either wild-type T-layer or variant T-layer protein (see Fig. 4). Note also that the axis of the assembly was at an approximate 450 angle with respect to the axis of the cylinder. Variant T-layer cylinder widths have a mean of 1.062 ± 0.154 ,m as compared with 0.460 ± 0.065 ,tm for the wild-type cylinders. This difference in average cylinder width between wild-type and variant T-layer assemblies was not attributable to differences in cell width. Log-phase cells had a mean width of 1.02 ,um with a standard deviation of ±0.039 ,um for the wild type and a mean width of 0.997 + 0.057 ,um for the variant. Cell walls (Fig. 2) which are more uniformly flattened during processing for
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FIG. 4. (A) Crude preparation of wild-type B. sphaericus 9602 T-layer (resuspended precipitate of 0.20 saturation of ammonium sulfate of the urea wash supernatant). Note the cylinders with oriented arrays at 450 to the circumference and length of the cylinder. The larger cylinder was representative of the upper range in width that was observed; the smaller cylinder represented the usual width. Also, sheets of assembled T-layer subunits were observable as well as some amorphous material. Bar represents 1 jim. (B) Crude preparation of variant B. sphaericus 9602 Lmw T-layer. Cylinders and sheets were seen as in the wild-type preparation; however, the width of the cylinders was approximately twice that observed for the wild-type T-layer in A. Bar represents 1 ,um.
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HASTIE AND BRINTON
A
B
C
D
J. BACTERIOL.
E
F
FIG. 5. SDS-polyacrylamide slab gel of crude preparation and further purification steps of wild type and variant T-layer. (A) Crude preparation of wild-type T-layer (cell walls extracted with 6 M urea and then the supernatant precipitated with ammonium sulfate); 60% purity determined from scan on Joyce-Loebel microdensitometer. (B) Crude preparation of variant T-layer extracted and treated as in A; 80% purity determined from scan. (C) Assembled material in A after pelleting; urea dissociated and reassociated by dialysis, and ammonium sulfate precipitated; 65.5% purity. (D) Assembled material in B treated as described for C; 84% purity. (E) C preparation of wild-type T-layer readsorbed onto sacculi; urea re-extracted and ammonium sulfate precipitated; 87% purity. (F) D preparation of variant Tlayer treated as described for E; 98% purity.
electron microscopy were also measured. There slight difference in the cell wall widths of wild type and variant (1.24 ± 0.038 ,um for the wild-type and 1.15 ± 0.059 ,um for the variant cell walls). Measurements of center-to-center spacing of subunits were made on planar sheets, cylinders, whole cells, and cell walls in one direction and in a direction 900 to the first. A distance of 25 to 100 tetramers in an array was measured and then divided by the total number of tetramers in that distance. No significant differences could be found. The average center-to-center spacing based on an internal standard of catalase crystals was found to be 13.5 ± 0.9 nm for the wild-type T-layer protein and 13.5 ± 0.6 nm for the variant T-layer protein. was a
DISCUSSION A method for the purification of the T-layer of B. sphaericus was developed capitalizing on four characteristics of the regularly structured layer. The T-layer constitutes more than 50% of the cell wall mass (12) such that separation of cell walls and cytoplasmic contents yielded con-
siderable purification. The subunits were noncovalently bound to the cell wall and can be removed by urea, formamide, or guanidine-hydrochloride. Subunits thus extracted self-assembled into macromolecular structures in vitro upon removal of the disrupting agent and were separable from other soluble proteins by differential centrifugation. Finally, native uncleaved subunits readsorbed to the cell walls from which they have been isolated, thereby providing a mechanism of separation from degraded subunits (8). The subunits were not substantially altered by the urea extraction, since they retained the ability to form in vitro assemblies with the same lattice parameters as the structure exhibits on whole cells and cell walls, and could readsorb to the cell walls from which extracted. It was also found that the subunits have essentially the same isoelectric point whether extracted by urea or formamide, further indicating that the properties of the subunits were not changed by extraction. However, prolonged exposure (three times the usual) of the subunits to urea appeared to alter the ability of the subunits to reassemble (A. L. Thomas, Ph.D. thesis, University of Pittsburgh, Pittsburgh, Pa., 1975), probably by the reaction of cyanate (produced in equilibrium from urea at neutral pH) with lysine residues in the protein (29). The purified T-layer subunit of both wild type and variant was a single homogeneous, large, acidic glycoprotein, as determined by SDS-polyacrylamide gel electrophoresis, isoelectric focusing, cesium chloride equilibrium gradients, and carbohydrate assay. Thus, B. sphaericus 9602 and 9602 Lmw T-layer proteins exhibited similarities to biochemical and physical characteristics described for other regularly structured layers (4, 11, 24, 28, 32; C. M. Henry, Ph.D. thesis, University of Pittsburgh, Pittsburgh, Pa., 1972). In vitro-assembled subunits were reversibly dissociated below their isoelectric point by acid titration to pH 2. Alkaline dissociation to pH 12 appeared to denature the protein irreversibly. However, it was not possible to release the subunits from cell walls by acid titration (A. L. Thomas, Ph.D. thesis, University of Pittsburgh, Pittsburgh, Pa., 1975). This is in agreement with the results of Sleytr with Clostridium thermosaccharolyticum and Clostridium thermohydrosulfuricum (26). These observations suggest that the bonds holding the subunits together differed somewhat from those binding the subunits to the underlying layer of the cell wall. Therefore, although the cell wall was not necessary for assembly of the subunits, it appeared to stabilize bound assemblies. Two forms of extracted T-layer assembly, sheets and cylinders, do not necessarily imply
VOL. 138, 1979
TETRAGONAL LAYER OF B. SPHAERICUS
ts
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1007
A
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." 4
FIG. 6. (A) Negatively stained sample of purified wild-type B. sphaericus T-layer. Large aggregates of assembled T-layer of varying sizes were present. Bar represents 1 ,um. (B) Negatively stained sample of variant T-layer. Varying sizes of sheets of assembled T-layer were observed as well as small aggregates. Bar represents I tun. two different populations of subunits. In fact,
different subunit types. For example, native unactually possess cleaved subunits may assemble into sheets, curvature not detectable unless complete closure whereas proteolytically cleaved subunits cominto a cylinder has occurred. However, it is pos- pose cylinders; as Henry had observed for B. sible that these two states of assembly represent sphaericus P-1 (C. M. Henry, Ph.D. thesis, Uni-
planar-appearing sheets
may
1008
z/ as90.9 0.8
0.7
0.6 z 0.5 0Q4
0 0.3
0.2
J. BACTERIOL.
HASTIE AND BRINTON
versity of Pittsburgh, Pittsburgh, Pa., 1972). Slight variations in physical and biochemical \properties between the wild-type and variant Tlayer protein were observed despite a 15% larger wild-type subunit molecular weight. A major difference was found in characteristic cylinder width. The twofold-larger average cylinder \ ^. width for variant subunits could not be attributed to a difference with the wild-type in intersubunit distance. Nor did it represent a change in the underlying cell wall layer since the Tlayer needed no template for assembly. Therefore, a critical change in subunit structure probably occurred which was reflected in subunitsubunit interaction at a inacromolecular level. Some flexibility in subunit-subunit bonds could account for cylinders of different widths, as ob:served in Fig. 4A. However, this flexibility is \ Xlimited since the bell-shaped distributions of wild-type and variant cylinder widths hardly
02
overlapped.
0.1
The most important observation is that the T-layer proteins of B. sphaericus 9602 and 9602 Lmw self-assembled in vitro and, in doing so, were capable of forming seamless curved structures of uniform width. Brinton et al. (Bacteriol. Proc., p. 48, 1969) has proposed on the basis of B findings a morphopoietic role for regularly Q.9 ON .,these .
