Chemical Composition and Ultrastructure of Native and Reaggregated Membranes from Protoplasts of Bacillus cereus

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1 JOURNAL OF BACTERIOLOGY, Mar. 1974, p Copyright American Society for Microbiology Vol. 117, No. 3 Printed in U.S.A. Chemical Composition and Ultrastructure of Native and Reaggregated Membranes from Protoplasts of Bacillus cereus TEOFILA CABRERA BEAMAN, H. STUART PANKRATZ, AND PHILIPP GERHARDT Department of Microbiology and Public Health, Michigan State University, East Lansing, Michigan Received for publication 19 October 1973 Conditions were defined for producing protoplasts with lysozyme and isolating the protoplast membranes from cells of Bacillus cereus T harvested late in the exponential growth phase just before sporogenesis. The membranes contained approximately 60% protein, 30% lipid, 6% carbohydrate, and 1% ribonucleic acid. Seventeen proteins were distinguished by molecular size in the membrane solubilized with sodium dodecyl sulfate, and 12 in that with phenol and acetic acid. The lipid fraction consisted of neutral lipids (28%) and phospholipids (72%). Four phospholipids were identified: diphosphatidyl glycerol, phosphatidyl ethanolamine, phosphatidyl glycerol, and lysophosphatidyl ethanolamine. Eighteen fatty acids were identified, with a predominance of branched C,5 and C17 and of normal C,, acids. The carbohydrate fraction consisted of neutral hexoses. A clear supernatant solution from the solubilized preparation became reaggregated into membrane by dialysis in the presence of MgCl2. The reaggregated membrane had the same main components as the native membrane, but the amount and ratio of protein and lipid depended on the buffer and the MgCl2 concentration. By electron microscopy, the reaggregated membranes appeared as vesicles or sheets, depending on the MgCl2 concentration. Hexagonal lattices were occasionally detected in the negatively stained ultrastructure of both native and reaggregated membrane fragments. The cytoplasmic membrane in vegetative cells of Bacillus cereus is of particular interest because of its role in sporulation (7, 17) and its possible chemical similarity to the exosporium membrane of this widely studied species (14). The detection of paracrystallinity (9) and the solubilization and reassembly of exosporium (2) suggested that the application of similar procedures might provide useful information about the ultrastructure of the vegetative cell membrane. It first became necessary to determine conditions in which protoplasts of this species could be produced, free of cell wall residues. Then the protoplast membranes were released by plasmoptysis, separated by differential centrifugation, examined electronmicroscopically, and analyzed chemically. The membranes were then solubilized and reaggregated, and the resulting preparation also was characterized physically and chemically. MATERIALS AND METHODS The terminalas (T) strain of B. cereus was grown in modified G-medium in the same way as for spore production (10, 14). However, vegetative cells were harvested at stage T. (17), i.e., at the end of the exponential growth phase just before the onset of sporogenesis. The cells were harvested by centrifugation at 27,000 x g for 5 min. Protoplasts were obtained by resuspension of the cells in tris(hydroxymethyl)aminomethane (Tris)- hydrochloride buffer (0.1 M, ph 7.5), containing sucrose (0.5 M) and lysozyme (1 mg/ml), and were incubated for 90 to 120 min at 37 C. After the conversion was complete, deoxyribonuclease (5,ug/ ml) was added, and incubation was continued for another 15 min. The protoplast suspension then was sedimented for 5 min at 20,000 x g, and the pellet was suspended in Tris buffer (0.05 M) without sucrose. The membranes thus released by plasmoptysis were washed in the buffer four successive times by centrifugation for 20 min at 20,000 x g. Separation of the membranes from most of the cytoplasmic granules of poly-,b-hydroxybutyrate (PHB) was accomplished by differential centrifugation at 1,200 x g for 30 min in a swinging-bucket rotor. Samples for chemical analysis were further washed four times in distilled water and lyophilized. Samples for solubilization and reaggregation were suspended and washed in Tris buffer (0.05 M, ph 7.5) or in a 1: 20 (vol/vol) dilution of, buffer (0.15 M NaCl, 0.05 M Tris, 0.01 M,8-mercaptoethanol; ph 7.4). The membranes in suspension [1 mg (dry weight)/ ml] were disintegrated by incubation in the presence of 0.01 M sodium dodecyl sulfate (SDS) at 37 C for

2 1336 BEAMAN, PANKRATZ, AND GERHARDT J. BACTERIOL. min, with an 85 to 90% decrease in turbidity. A minimally effective concentration of SDS was used to prevent possible precipitation during the subsequent dialysis. From this preparation, a clear supernatant fraction containing the solubilized membrane was obtained by centrifugation at 27,000 x g for 30 min with the pellet containing the PHB granules. Reaggregation of the clear supernatant fraction into membranes was accomplished by dialysis at 4 C for several days with Tris or diluted f, buffer containing 0.01 M or 0.02 M MgCl2. In one procedure, disc gel electrophoresis was accomplished by the use of 7.5% (wt/vol) acrylamide gel after the membranes (2 mg/ml) were solubilized in 0.01 M sodium phosphate buffer (ph 7.0) containing M SDS and 1% (vol/vol), mercaptoethanol (2, 27). The proteins distributed in the gel were stained with Coomassie brilliant blue R250 by using the method of Weber and Osborn (26) or that of Dunker and Rueckert (6). In an alternative procedure (22), electrophoresis was accomplished in the acrylamide gel plus 35% (wt/vol) acetic acid and 5 M of urea after the membranes were solubilized with a phenol-acetic acid-water mixture (2:1:0.5 vol/vol). The proteins in the gel were stained with 1% (wt/vol) acetic acid, and the excess stain was removed electrophoretically. The peaks in densitometric tracings of the stained gels were considered to represent protein species only if distinct and reproducible, even though small. The molecular weight of each protein species was determined from a plot of mobilities with the following reference proteins: bovine serum albumin, 68,000; ovalalbumin, 43,000; pepsin, 35,000; trypsin, 23,000; and lysozyme, 14,300. The analytical procedures were essentially the same as those used previously for characterization of exosporium (14). Protein was analyzed by the method of Lowry et al. (13); carbohydrate, by the differential anthrone procedure of Toennies and Kolb (26) after hydrolysis in 2 N H2SO4; ribonucleic acid (RNA), by the orcinol method (15); total amino sugars, by the method of Rondle and Morgan (23) after hydrolysis in 6 N HCl for 6 h, with correction for loss in glucosamine; diaminopimelic acid, by paper chromatography (21) after hydrolysis in 6 N HCl for 6 h and removal of HCl by flash evaporation; and PHB by the method of Law and Slepecky (12). Lipid was extracted with a solution of chloroform in methanol (2:1 vol/vol), washed with 1 M KCl and then with distilled water, dried, and weighed (8). Phospholipid was determined from the amount of phosphorus in the chloroform-methanol extract, as measured by the procedure of Bartlett (1). Individual phospholipids were identified by thin-layer chromatography (14) on plates coated with Silica Gel G (Merck & Co., Rahway, N.J.), with commercial reference standards (Supelco, Inc., Bellafonte, Pa.). Fatty acids in the lipid extract were separated by gas-liquid chromatography (14) after methanolysis with 15% boron trifluoride-methanol reagent (16). Samples for electron microscopy were negatively stained with 1 to 2% (wt/vol) potassium phosphotungstate or with 2% (wt/vol) ammonium molybdate, both at ph 7.0, and examined with a Philips 300 instrument. RESULTS AND DISCUSSION Protoplasts of B. cereus T were successfully prepared by using cells harvested late in the exponential growth phase. The protoplast preparation by electron microscopy appeared to be homogeneous and free of residual cell wall material and, by chemical analysis, contained no diaminopimelic acid and only a trace of hexosamines. Consequently, they were considered true protoplasts rather than spheroplasts. The gross chemical composition of the membranes isolated from the protoplasts is shown in Table 1, in comparison with that of the exosporium from spores of the same species and with that of the cell membrane from other species of Bacillus. The predominant components of the B. cereus membrane, protein (60%) and lipid (30%), occurred in amounts similar to those in TABLE 1. Chemical composition of membranes isolated from protoplasts of B. cereus Tcompared with that of membranes from other Bacillus species and with that of the exosporium from spores of B. cereus T Percentage of dry weight Membrane from Membrane Membrane Membrane Exosporium Component Membrane B. megaterium KM from from from B. from from B. megaterium M B. subtilis 168 licheniformis B. cereus T B. cereus T Ref. 30 Ref. 29 Ref. 28 Ref. 4 Ref. 25 Ref. 14 Protein Lipid Carbohydrate Ash 3.8 RNA PHB Trace Other 2.4 Total

3 VOL. 117, 1974 other membranes, but the carbohydrate and RNA amounts varied among the several species. The exosporium membrane clearly was different in chemical composition from the others. The protein fraction of the B. cereus membrane after solubilization with SDS was comprised of 10 main species and at least 7 minor ones (Fig. 1A). After solubilization of the membrane with phenol and acetic acid, a total of 12 protein species was detected (cf. T. C. Beaman and P. Gerhardt, Bacteriol. Proc., p. 51, 1970). A direct comparison was not possible with other bacilli because of the different methods applied but, for example, 14 proteins occur in B. subtilis membrane solubilized by sodium deoxycholate (18). In exosporium, only two protein species occur after SDS solubilization, only eight occur after phenol and acetic acid solubilization, and the main protein species are different than in the vegetative cell membrane (14). The lipid fraction of the B. cereus membrane was comprised of phospholipid (72%) and neutral lipid (28%). Four phospholipids were identified: phosphatidyl ethanolamine, phosphatidyl glycerol, diphosphatidyl glycerol, and lysophosphatidyl ethanolamine. The neutral lipids were not characterized. Eighteen fatty acids were identified, with branched C,5 and C,7 (anteiso) and normal C1, (saturated and unsaturated) fatty acids representing approximately 60% of the total (Table 2). Branched C,, fatty acids and several of the same phospholipids also predominate in the membranes of B. subtilis (4) and B. megaterium (30). However, normal C1, fatty acids and diphosphatidyl glycerol predominate in the exosporium of B. cereus spores (14). The carbohydrate fraction (6.3%) of the B. cereus membrane was comprised of neutral hexoses, as with other bacillus membranes. In contrast, the B. cereus exosporium contains a significant amount of glucosamine and rhamnose and a substantially greater total amount of carbohydrate (14). The RNA content of the B. cereus membrane was small (1.2%) but apparently represented an actual component, because RNA also occurred in the same amount in the membrane forms reaggregated after solubilization (see below). With other Bacillus membranes, the RNA content sometimes has been reported to occur in a relatively large amount which may reflect the presence of ribosomes (Table 1). The most difficult problem of contamination occurred with the granules of PHB (1.7%), because they remained trapped within the collapsed membranes after other cytoplasmic constituents were washed away. The membrane preparations appeared homogeneous by electron microscopy, except for the PROTOPLAST MEMBRANES OF B. CEREUS E C 0 0In I~z w0 -J > w E c 0 0in cn - z 0 -J 4 0 w 4 -J w l iui' l * I? - RELATIVE MIGRATION -- + L I RELATIVE MIGRATION.-m & FIG. 1. Comparative electrophoretic distribution of size-identified protein species in native (A) and reaggregated (in, buffer and 0.02 M MgCI12) membranes (B). Before electrophoresis, both types of membrane were solubilized by treatment with SDS. The numbered protein species correspond to molecular weights as follows: 1, 103,000; 2, 98,000; 3, 92,000; 4, 87,000; 5, 82,000; 6, 74,000; 7, 65,000; 8, 60,000; 9, 56,000; 10, 48,000; 11, 43,000; 12, 39,000; 13, 34,000; 14, 28,000; 15, 24,000; 16, 18,000; 17, 13,000. A B 1337

4 1338 BEAMAN, PANKRATZ, AND GERHARDT J. BACTERIOL. TABLE 2. Fatty acid constituents in the lipids from native and reaggregated membranes Percentage of total Peak Fatty acid fatty acids no. constituent Native Reaggregated membrane membranea 1 n-c1o Trace 2 n-cll Trace 3 i-ci n-c n-c a-c i-c n-c i-c a-c n-c i-ci n-c n-c16s a-c n-c17 Trace n-c n-c181= Trace Trace Total a Reaggregated in, buffer and 0.02 M MgCl,. PHB granules. A ruptured, but whole, protoplast membrane looked like a collapsed sphere, whereas fragments were irregular in shape. Although usually without evidence of ultrastructure, occasionally a fragment at high magnification contained an ordered lattice with hexagonal periodicity of 7.6 nm between the dark centers (Fig. 2). The periodicity was detected in several preparations of membranes. The isolated B. cereus cell membrane was disintegrated by treatment with SDS, and the clear supernatant fraction was reaggregated into membrane by dialysis in the presence of MgCl2. The chemical composition of the reaggregated membrane included the same principal components as in the native membrane, but the amount and ratio of protein and lipid varied with the buffer and the concentration of MgCl2 used in dialysis (Table 3). The highest ratio occurred when the solubilized membrane fraction was reaggregated in the presence of the higher magnesium concentration and in f# rather than Tris buffer. However, the reaggregated membrane still had less protein, more lipid, and a lower ratio than in the native membrane. Similar effects have been obtained in the reaggregation of B. cereus exosporium (2), Mycoplasma membrane (24), and Micrococcus lysodeikticus membrane (5). The ten main protein species, and all but three of the seven minor ones in the native B. cereus membrane, were incorporated into the reaggregated membrane (Fig. 1B). The four phospholipids in the native membrane also were incorporated into the reaggregated membrane in about the same relative amounts. Similarly, nearly all of the original ti FIG. 2. Electron micrographs of a negatively stained fragment of a protoplast membrane. A, The entire fragment; B, enlargement of the area with hexagonal periodicity indicated by the arrow in A. Bars, 100 nm.

5 VOL. 117,1974 TABLE 3. PROTOPLAST MEMBRANES OF B. CEREUS Chemical composition of reaggregated membranes as affected by the buffer and the concentration of magnesium ions used in dialysis. Dialysis solution Percentage of dry weight Membrane Molarity of Protein Lipid Carbohydrate RNA Buffer MgCl2 Poen Lii abhyrt N 1339 Reaggregated Tris Tris Native lipids were incorporated into the reaggregated membrane from L-phase cells of Streptobacillus moniliformis (19). The same main fatty acids, except six of the shorter-chained ones in the native membrane, were incorporated into the reaggregated membrane (Table 2). The physical appearance of the reaggregated membranes was affected by the conditions of dialysis during reaggregation. When the reaggregated membranes contained a relatively high protein-to-lipid ratio, e.g., with 0.02 M of MgCl2 and d buffer (Table 3), they appeared rounded and vesicular. When the reaggregated membranes contained a relatively low protein-to-lipid ratio, e.g., with 0.01 M of MgCl2 and Tris buffer, they appeared irregular and sheetlike. Among the latter forms, periodic ultrastructure occasionally was detected to be the same as that in the native membrane (Fig. 3). In both instances, the infrequency in detection of periodicity made it uncertain whether the phenomenon was an uncontrolled artifact of the preparation procedures or a real, but masked, characteristic of the membrane. The occurrence of periodicity in the original preparation might be attributed to contamination with another type of membrane, but the recurrence in the reaggregated membrane preparation seemed to preclude that possibility. A hexagonal lattice with approximately the same periodicity occurs in the B. cereus exosporium (9), and a lattice also occurs in certain membranes from mammalian cells (3, 11). However, to our knowledge, an ordered ultrastructure has not been reported heretofore in a bacterial cell membrane, and consequently the phenomenon seemed worth describing. The presence of periodic ultrastructure implied a process of self-assembly in the formation of the native, as well as the reaggregated, membrane. ACKNOWLEDGMENTS This investigation was supported by Public Health Service grant no. AI from the National Institute of Allergy and Infectious Diseases and by contract no. Nonr-2587 from the Office of Naval Research. This paper was assigned journal article no from the Michigan Agricultural Experiment Station. LITERATURE CITED 1. Bartlett, G. R Phosphorus assay in column chromatography. J. Biol. Chem. 234: Beaman, T. C., H. S. Pankratz, and P. Gerhatdt Paracrystalline sheets reaggregated from solubilized exosporium of Bacillus cereus. J. Bacteriol. 107: Benedetti, E. L., and P. Emmelot Hexagonal array of subunits in tight junctions separated from isolated rat liver plasma membranes. J. Cell Biol. 38: Bishop, D. G., L. Rutberg, and B. Samuelson The chemical composition of the cytoplasmic membrane of Bacillus subtilis. Eur. J. Biochem. 2: Butler, T. F., G. L. Smith, and E. A. Grula Bacterial cell membranes. I. Reaggregation of membrane subunits from Micrococcus lysodeikticus. Can. J. Microbiol. 13: Dunker, A. K., and R. R. Rueckert Observations on molecular weight determinations on polyacrylamide gel. J. Biol. Chem. 244: Felix, J. A., and D. G. Lundgren Electron transport system associated with membranes of Bacillus cereus during vegetative growth and sporulation. J. Bacteriol. 115: Folch, J., M. Lees, and G. H. Sloane-Stanley A simple method for the isolation and purification of total lipides from animal tissues. J. Biol. Chem. 226: Gerhardt, P., and E. Ribi Ultrastructure of the exosporium enveloping spores of Bacillus cereus. J. Bacteriol. 88: Hashimoto, T., S. H. Black, and P. Gerhardt Development of fine structure, thermostability, and dipicolinate during sporogenesis in a Bacillus. Can. J. Microbiol. 6: Hicks, R. M., and B. Ketterer Isolation of the plasma membrane of the luminal surface of rat bladder epithelium and the occurrence of hexagonal lattice of subunits both in negatively stained whole mounts and in sectioned membranes. J. Cell Biol. 45: Law, J. H., and R. A. Slepecky Assay of poly-fhydroxybutyric acid. J. Bacteriol. 82: Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193: Matz, L. L., T. C. Beaman, and P. Gerhardt Chemical composition of exosporium from spores of Bacillus cereus. J. Bacteriol. 101: Mejbaum, W tyber die Bestimmung kleiner Pentosemengen insbesondere in Derivaten der Adenylsa-

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