CHEMICAL COMPOSITION OF AUTOPLAST MEMBRANE OF CLOSTRIDIUM SACCHAROPERBUTYLACETONICUM

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1 J. Gen. App!. Microbiol., 28, (1982) CHEMICAL COMPOSITION OF AUTOPLAST MEMBRANE OF CLOSTRIDIUM SACCHAROPERBUTYLACETONICUM SEIYA OGATA, SADAZO YOSHINO, YUTAK_A OKUMA,* AND SHINSAKU HAYASHIDA Laboratory of Applied Microbiology, Department of Agricultural Chemistry, Kyushu University, Fukuoka 812, Japan *Lotte Co. Ltd., Tokyo, Japan (Received October 26, 1981) The chemical composition of bacterial protoplasmic membrane was studied using Clostridium saccharoperbutylacetonicum ATCC The membrane was prepared from the autoplasts. The purity of prepared membrane was certified by chemical analysis for the existence of diaminopimelic acid due to contaminated cell wall debris and of adenosinetriphosphatase (ATPase) activity in its preparation, and also by electron microscopic observation for the existence of contaminated cell wall debris. The membrane composition was made up of lipid (26 %), protein (65%), carbohydrate (2%) and nucleic acid (2%). The lipid consisted of neutral lipid (26 %), glycolipid (26%) and phospholipid (48 %). The main composition of phospholipid was phosphatidylethanolamine, phosphatidylglycerol and diphosphatidylglycerol. The membrane contained 22 kinds of proteins of different molecular weights. Bacterial protoplasts have frequently been used for the preparation of bacterial protoplasmic membrane. It is said that protoplasts produced autolytically are better for the preparation of protoplasmic membrane by eliminating the contamination of non-cellular supplements used for degradation of cell wall. From the advantages of autolytically produced protoplasts, JOSEPH and SHOCKMAN (1) called them "autoplasts," distinguishable from the protoplasts produced by using exogenous lytic enzymes or antibiotics. Clostridium saccharoperbutylacetonicum (ATCC 13564), previously used for acetone-butanol production, autolyses specifically in the presence of sucrose in hypertonic concentration ( M) (2, 3). The autolysis, i. e., sucrose-induced autolysis, is useful for the preparation of the protoplasts (autoplasts), the biological and physiological properties of which were investigated (3). We would like to determine composition of membrane prepared from the clostridial autoplasts, because that of clostridia, especially solvent-producing clostridia, is little known. 293

2 294 OGATA, YOSHINO, OKUMA, and HAYASHIDA VOL. 28 MATERIALS AND METHODS Organisms and cultural conditions. Clostridium saccharoperbutylacetonicum N 1-4 (ATCC 13564) (4) was used throughout the work. This organism was grown at 30 under reduced atmospheric conditions (5-10 mmhg) in TYA medium, as described in a previous paper (5). For the preparation of exponentially growing cells, the initial optical density (0D660) at 660 nm of the culture was adjusted to 0.1 in a fresh TYA medium and incubation was continued until the 0D660 became D660 of the culture was measured using a photoelectric colorimeter (model 7A, Tokyo Koden Ltd.). Preparation of autoplast. The cells of log phase were harvested by centrifugation (10,000 x g for 10 min at room temperature), washed with 1/30 M Na2HPO4 - KH2PO4 buffer (ph 6.0) containing 5 mm MgSO4 (PM-buffer), and suspended in the PM-buffer containing 0.35 M sucrose. The cell suspension was incubated at 30 for 2-4 hr for autolysis and formation of the autoplasts in the hypertonic solution, as described in previous papers (2, 3). Preparation of protoplasmic membrane. The autoplasts obtained were centrifuged at 3,000 x g for 20 min at room temperature, and the resultant pellet was suspended in 1/200 volume of the PM-buffer containing 0.35 M sucrose. The condensed autoplasts were poured into 50-fold chilled PM-buffer to burst the autoplasts by osmotic lysis, and deoxyribonuclease (5,ug/ml) and ribonuclease (5,ug/ ml) were then added to the suspension. The suspension was centrifuged at 1,000 x g for 30 min to remove whole cells. The supernatant containing the ghosts of autoplasts was washed with the PM-buffer for three times by centrifugation at 15,000>< g for 30 min. The washed ghosts were layered on 30 % (w/w) of buffered sucrose solution in a centrifugation tube, and centrifuged at 15,000 x g for 30 min to ensure the removal of whole cells. The membrane fraction was collected and washed with the PM-buffer three times. The membrane obtained was named "crude membrane." This crude membrane was layered over 10 ml of linear sucrose gradient (30-60 %), and the gradient was centrifuged at 32,000 x g for 2 hr at 4. The banded membrane fraction was collected and washed with the PMbuffer or 1/100 M Tris-HC1 buffer ph 7.0 three times by centrifugation at 15,000x g for 30 min. The Tris-HCl buffer was used in the case of measuring inorganic phosphate. The sucrose gradient centrifugation was repeated one more time. The membrane fraction obtained was named "purified membrane." This membrane suspension or lyophilized membrane was used throughout this work. Assay of amino acid composition of autoplast membrane. Five mg of lyophilized membrane was hydrolyzed in 3 ml of 4 N HCl for 24 hr at 105. The hydrolysate was evaporated in a reduced atmosphere to remove HC1, and then dissolved in 10 ml of 0.1 N succinate buffer of ph 2.2. The amino acid composition of the sample was analyzed by automatic amino acid analyzer (Type LC-R-2, Japan Electron Optics Lab. Ltd.).

$15 ml of membrane fraction after sucrose gradient centrifugation was incubated with 0.15 ml of 5 mm adenosinetriphosphate and 0.15 ml of 5 m t MgC12 for 1 hr at 40.$

3 1982 Membrane Composition of C. saccharoperbutylacetonicuin 295 Assay of adenosinetriphosphatase (ATPase) activity of autoplast membrane. ATPase activity of membrane was measured as follows. A 0.15 ml of membrane fraction after sucrose gradient centrifugation was incubated with 0.15 ml of 5 mm adenosinetriphosphate and 0.15 ml of 5 m t MgC12 for 1 hr at 40. The reaction was stopped by addition of 0.25 ml of 8 % perchloric acid, and the resulting precipitate was removed by centrifugation at 3,000 x g for 15 min. Free inorganic phosphate of supernatant fluid was measured by the method of FISKE and SUB- BAROW (6). Estimation of protein contents. The amount of protein in the membrane was measured by the method of LoWRY et al. (7). Estimation of lipid contents. Lipid of the membrane was extracted by the method of BLIGH and DYER (8), and its weight was measured after vaporization of solvent under a stream of N2 gas. Estimation of carbohydrate contents. Carbohydrate in the membrane was measured by the anthron method (9) after hydrolysis with 2 N HCl for 5 hr at 100. Estimation of nucleic acid contents. Deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) were fractionated from the membrane by a modified STS method (10). RNA contents were estimated by the orcinol method, and DNA by the diphenylamine method (10). Estimation of fatty acid contents. Lipid extracted from the membrane by the method of BLIGH and DYER (8), as described above, was hydrolyzed by methanol - HCl (95:5) for 3 hr at 100. After the hydrolysis, 1 ml of water and 3 ml of petroleum ether were added to the hydrolysate, followed by vigorous agitation to extract the fatty acid methylesters into the ether layer. The ether layer was collected, and washed with distilled water three times. The washed ether layer was dehydrated using anhydrous sodium sulfate, and evaporated under a stream of N2 gas. The resulting fatty acid methylester was dissolved in n-hexane, and its contents were analyzed by gas-liquid chromatography (Diethylene glycol succinate, 15 % coated Chromosorb W, 170 ). Electron microscopy. The membrane was fixed with 1 % osmium tetraoxide containing veronal acetate buffer. The fixed membrane was embedded in a small block (1 mm2) of 2 % agar, and stained with 0.5 % uranyl acetate for 2 hr. The stained block was dehydrated in a graded series of 70 to 100% ethanol and propylene oxide. After dehydration, the block was embedded in Epon 812 and solidified at graded temperature from 40 to 60. The embedded sample was sectioned by ultramicrotome (MT-2, Sorvall) and stained with 2 % uranyl acetate and 1 % lead acetate. Electron microscopy was carried out using a JEM 100B instrument (Japan Electron Optics Lab. Ltd.). RESULT Purity of prepared autoplast membrane The amino acid composition of the crude and purified membrane prepara-

296 OGATA, YosHINO, OKUMA, and HAYASHIDA VOL. 28 Table 1. Amino acid composition of membrane preparation. Fig. 1. Electron micrograph of the purifi ed membrane. Scale in dicates 0.5 ~~m.

4 296 OGATA, YosHINO, OKUMA, and HAYASHIDA VOL. 28 Table 1. Amino acid composition of membrane preparation. Fig. 1. Electron micrograph of the purifi ed membrane. Scale in dicates 0.5 ~~m. tions was examined to ensure their purities. As shown in Table 1, the crude membrane contained a slight amount of diaminopimelic acid that might have originated from cell wall debris. The purified membrane preparation, which was obtained after the sucrose gradient centrifugation, contained no diaminopimelic acid. The other amino acids of the purified membrane did not differ from those of the crude membrane. Electron microscopic observation of the purified membrane showed various sizes of membrane vesicles, but no cell wall debris, as shown in Fig. 1.

5 1982 Membrane Composition of C. sacc haroperbutylacetonicum 297 Fig. 2. Sucrose gradient analysis of the purified membrane. The purified membrane was layered on the sucrose gradient described in MATERIALS AND METHODS., adsorption at activity. and centrifuged as 0D280; 0, ATPase The distribution of membrane on the second sucrose gradient centrifugation was detected at an optical density of 280 nm (0D280) and from the ATPase activity of each fraction; ATPase activity was known to be of membrane bound enzyme (11). As shown in Fig. 2, ATPase activity of each fraction coincided with the pattern of the adsorption at 0D280, and no other peaks of ATPase activity were found. From these results, we confirmed that the membrane fraction obtained was pure. Chemical composition of autoplast membrane The chemical composition of the purified membrane was determined. As shown in Table 2, protein and lipid were the main components, and small amounts of carbohydrate and nucleic acid were also present. The chemical composition was very similar to that of Bacillus megaterium (12). The percentages of lipid contents of the membrane was also very similar to those for many aerobes reported (13). Fractionation of lipid Lipid extracted by the method of BLEIGH and DYER (8) was applied on Unisil column and eluted with chloroform, acetone and then methanol, as described by RoUSER et al. (14). These eluents obtained corresponded to neutral lipid, glycolipid and phospholipid, respectively. As shown in Table 3, the lipid consisted of neutral

6 298 OGATA, YOSHINO, OKUMA, and HAYASHIDA VOL. 28 Table 2. Chemical composition of membrane preparation. Table 3. Composition of membrane lipid. lipid (26 %), glycolipid (26 %) and phospholipid (48 %) in dry weight after evaporation of solvents. Phospholipid composition Phospholipid composition was determined by thin-layer chromatography, using Kieselgel F625 (Merck) with the solvent system of chloroform-methanol-acetic acid (65:25:10). As shown in Fig. 3, Rf values of spots 1, 2 and 3 coincided with those of cardiolipin, phosphatidylglycerol and phosphatidylethanolamine of Escherichia coil, respectively. All spots stained blue with DITTMER-LESTER reagent (15) and spot 3 stained purple with ninhydrin reagent (16). Spot 4 was unknown. Though it stained yellow with Dragendroff reagent (17), its Rf value did not coincide with that of phosphatidylcholine derived from egg yolk. As shown in Table 3, the phosphatidylethanolamine content, was predominant on the phosphorus content of each spot being determined (18). Fatty acid composition of membrane lipid Fatty acid composition was determined by gas-liquid chromatography. As shown in Table 4, fatty acids of C16 were predominant, and accounted for over 65 % of total fatty acids. The contents of C-odd fatty acids were very low. Protein composition of autoplast membrane Molecular weights of membrane proteins were determined by SDS-polyacrylamide gel electrophoresis (19). The gel after electrophoresis was stained with

7 1982 Membrane Composition of C, saccharoperbutylacetonicum 299 Fig. 3. Thin-layer chromatogram of phospholipids fractionated by Unisil column chromatography. The thin-layer chromatography was carried out as described in MATERIALS AND METHODS. Spots revealed by charring after spraying with 50% sulfuric acid and some other reagents are described in the text. Table 4. Fatty acid composition of membrane lipid % coomassie brilliant blue - 50% methanol -10 % acetic acid overnight at room temperature. Twenty two bands of protein were observed, and their molecular weights varied from 9,400 to 180,000, as shown in Fig. 4. Proteins of molecular weights of 56,000 and 170,000 were determined as glycoprotein by method of ZACHARIUs et al. (20).

8 300 OGATA, YosHINO, OKUMA, and HAYASHIDA VOL. 28 Fig. 4. Molecular weights of membrane proteins. The SDS-polyacrylamide gel electrophoresis was carried out as described in the text. Dots indicate separated proteins. Arrowed bands corresponded to the PAS staining. DISCUSSION Studies of clostridial membrane have been little seen. Therefore, we compared our results with those for aerobic bacteria. The chemical composition of the membrane of C. saccharoperbutylacetonicum was very similar to that of B. megaterium, and also not so different from that of other aerobes. However, the content of glycolipid in this clostridial membrane was larger than that for aerobic bacteria (13). We found phosphatidylcholine-like lipid in phospholipids, but its Rf did not coincide with that of authentic sample. The overall fatty acid composition of cells has been investigated in many proteolytic clostridia (21). We expected some difference in the fatty acid composition of C. saccharoperbutylacetonicum from that of those clostridia, since C. saccharoperbutylacetonicum is a strain which produces a high concentration of butanol and acetone, and has some resistance to those solvents. However, its composition was very similar to that of proteolytic clostridia. The resistance to solvents might be due to other factors. We thank Prof. M. Hongo, The Kumamoto Institute of Technology, Kumamoto, Japan and Dr. K. H. Choi, Hyou-Sung Women's Univ., Taegu, Korea, for their helpful discussions.

9 1982 Membrane Composition of C, saccharoperbutylacetonicum 30.1 REFERENCE 1) R. JOSEPH and G. D. SHOCKMAN, J. Bacteriol.,118, 735 (1974). 2) S. OGATA, K. H. CHUI and M. HoNGO, Microbiol. Immunol., 24, 393 (1980). 3) S. OGATA, K. H. CHOI and S. HAYASHIDA, Nippon Nogei Kagaku Kaishi, 54, 753 (1980). 4) M. HoNGO and A. MURATA, Agric. Biol. Chem., 29, 1135 (1965). 5) S. OGATA and M. HoNGO, J. Gen. Microbiol., 81, 315 (1974). 6) C. H. FISKE and Y. SUBBAROW, J. Biol. Chem., 66, 158 (1930). 7) 0. H. LoWRY, N. J. ROSEBROUGH, A. L. FARR, and J. Randall, J. Biol. Chem., 193, 265 (1951). 8) E. G. BLIGH and W. J. DYER, Can. J. Biochem. Physiol., 37, 911 (1959). 9) S. FUKUI, Kangento no Teiryo Ho (Quantitative Analysis of Reducing Sugar), University of Tokyo Press, Tokyo, p. 49 (1969). 10) S. MIzuNo, Kakusan no Ippan Teki Bunri-Teiryo Ho (General Methods for Isolation and Determination of Nucleic Acids), University of Tokyo Press, Tokyo, p.19 (1969). 11) M. R. J. SALTON, Ann. Rev. Microbiol., 21, 417 (1967). 12) T. YAMAGUCHI, G. TAMURA, and K. ARIMA, J. Bacteriol., 93, 483 (1967). 13) N. S. GEL'MAN, M. A. LUKOYNOVA, and D. N. OSTROvSKII, In Biomembranes, Vol. 6, ed. by L. A. MANSON, Plenum Press, New York and London (1975), p ) G. RoUSER, G. KRITCHEVSKY, and A. YAMAMOTO, In Lipid Chromatographic Analysis, ed. by G. V. MARINETTI, Marcel Dekker, New York (1967), p ) J. D. DITTMER and R. L. LESTER, J. Lipid Res., 5, 129 (1964). 16) V. S. SKIPSKI, R. F. PETERSON, and H. BARCLAY, J. Lipid Res., 3, 497 (1962). 17) H. K. MANGOLD, J. Am. Oil. Chem. Soc., 38, 708 (1961). 18) P. S. CHEN, JR., T. Y. TORIBARA and H. WARNER, Anal. Chem., 28, 1756 (1956). 19) K. WEBER and M. OSBORN, J. Biol. Chem., 244, 4406 (1969). 20) R. M. ZACHARIUS, T. E. ZELL, T. H. MoRRISON, and J. J. WOODLOCK, Anal. Biochem., 30, 148 (1969). 21) S. R. ELSDEN, M. G. HILTON, K. R. PARSLEY, and R. SELF, J. Gen. Microbiol., 118, 115 (1980).

Work-flow: protein sample preparation Precipitation methods Removal of interfering substances Specific examples:

Dr. Sanjeeva Srivastava IIT Bombay Work-flow: protein sample preparation Precipitation methods Removal of interfering substances Specific examples: Sample preparation for serum proteome analysis Sample