Disruption of Bacterial Cells by a Synthetic Zeolite

Transcription

1 APPLIED MICROBIOLOGY, Sept. 1968, p American Society for Microbiology Vol. 16, No. 9 Printed in U.S.A. Disruption of Bacterial Cells by a Synthetic Zeolite GEORGE WISTREICH, MAX D. LECHTMAN, J. W. BARTHOLOMEW, AiND R. F. BILS Department of Biological Sciences, University of Southern California, Los Angeles, California Received for publication 17 May 1968 The use of a synthetic zeolite (type 4A, Union Carbide Corp., Linde Div., New York, N.Y.) in a procedure for the preparation of pure cell wall fractions proved successful for many gram-positive, gram-negative, and acid-fast bacteria, as well as for some fungi. The technique, however, was found to be limited in effectiveness for Rhodospirillum rubrum, Gaffkya tetragena, and Sarcina lutea, and not applicable to preparations of heat killed microorganisms. The possible mechanisms of zeolite action, together with the effect of the disruptive procedure on the chemical composition of cell wall fragments, were investigated also. The zeolite (type 4A) used in these experiments was obtained from the Union Carbide Corp., Linde Div., New York, N.Y. It is a synthetic crystalline zeolite, of a type not found in nature, that has a structure completely different from the gel type aluminosilicates usually referred to as zeolites. Its preparation and structure were first described by Breck et al. (1). Zeolite possesses a three-dimensional network of SiO4 and A104 tetrahedra, with each oxygen shared by another tetrahedron. Electrical neutrality is achieved by the inclusion of ions such as Na, K, Ca, or Ba. Its structure consists of large (0.11 pm) and small (0.06 pm) cavities connected by openings of 0.04 pm and 0.02 pm. In a dehydrated state, the structure is stable even at very high temperatures. Zeolite can be highly hydrated because the synthetic substance adsorbs up to 22.2% (by weight) of water. Moreover, the adsorption of water is highly exothermic and water is retained with great tenacity. Such a zeolite can be used also as a molecular sieve for small molecules because it will not admit larger molecules, such as benzene (1, 6). Many methods for the disruption of bacterial cells have proven successful (e.g., grinding with powdered glass or alumina, shaking with glass beads, extrusion of cells through a small orifice under high pressure, explosive release of pressure, alternate freezing and thawing, heat shock, and exposure to ultrasonic energy). Most of these methods have broad, but not universal, application to bacterial cells, and several require involved procedures or expensive apparatus. Recently, Person and Zipper (5, 13) suggested a new and simple method of cell rupture for yeast, posure of the majority of cells to the zeolite material. They used this procedure to release cytochrome oxidase from mitochondrial preparations from beef heart cells, and to disrupt whole cells of Candida utilis. Our interest in this new method arose because of a proposed study of the dye uptake properties of pure bacterial cell wall preparations, biochemical studies of cell walls of mycobacteria, and the suggestion of Zipper and Person (13) that the zeolite procedure might prove successful for some microorganisms that are difficult to disrupt by other methods. For example, Mycobacterium and Streptomyces cells tend to aggregate and clump both before and after disintegration in the Mickle apparatus, thus making difficult the isolation of suitable cell wall preparations (8). The use of zeolite seemed to be a simple and plausible way to obtain purified cell walls from a large variety of bacteria, providing that the method was applicable to many bacterial species. This paper reports the results of studies concerning the applicability of the zeolite method to 20 microbial species, some investigations of the possible ways in which zeolite exerts its disruptive effect, and the effect of this procedure on the chemical composition of the cell wall fragments that were obtained. MATERIALS AND METHODS Preparation oforganisms. Of the 20 organisms that were used, 18 were obtained from the stock culture collection of the Department of Bacteriology, University of Southern California. These were Bacillus cereus, Bacillus megaterium, Bacillus polymyxa, Bacillus subtilis, Clostridium sporogenes, Corynebacterium which involved mixing a synthetically produced pseudodiphtheriticum, Escherichia coli, Gaffkya tetragena, Micrococcus roseus, Mycobacterium phlei, and dehydrated zeolite preparation with cell paste, and grinding just enough to insure ex- Rhodospirillum rubrum, Pseudomonasfluorescens, Pro- 1269

2 1270 WISTREICH ET AL. APPL. MICROBIOL. teus vulgaris, Saccharomyces cerevisiae, Sarcina lutea, Serratia marcescens, Staphylococcus aureus, and Streptomyces lavendulae. Mycobacterium smegmatis was supplied by S. Froman of the Olive View Hospital, Olive View, Calif., and preparations of killed whole cells of Mycobacteriwn tuberculosis (H37RV) were supplied by Parke Davis & Co., Detroit, Mich. With the exception of M. smegmatis, S. cerevisiae, and S. lavendulae, all microorganisms were grown in Trypticase Soy Broth (BBL) at 30 C for 24 to 48 hr. The yeast culture was grown in McClary's medium (3), or on Sabouraud Dextrose Agar (Difco) slants, and incubated at room temperature for a period of 7 to 12 days. M. smegmatis was cultured in a modified Sauton's medium (11) at 37 C for 2 weeks. Disruption ofcells with zeolite. The method used by Zipper and Person (13) was slightly modified as follows. Microbial pellets were obtained from cells that were harvested by centrifugation at 5,000 X g, and washed three times with sterile distilled water. Washed pellets of the same organism were pooled, weighed while wet, and placed in a mortar that was kept cold by an ice-water bath. Zeolite 4A was added, in small quantities, to the microbial material to achieve a final proportion of approximately 2.0 g of zeolite to 1.0 g of cells. Hand pestle grinding was performed to incorporate the zeolite into the cell mass. After the final addition of zeolite, the mixture was ground for an additional 3 min. Approximately 100 ml of cold, sterile, distilled water was then added slowly to the resulting powdery preparation and centrifuged at approximately 2,000 X g for 10 min. This pellet was discarded, and the supernatant fluid was centrifuged at 10,000 X g for 30 min. The latter pellet was washed twice, and a final suspension was made for the preparation of grids for electron microscopy. The procedure was modified slightly for the yeast, mycobacteria, and streptomycete cultures; for these, the first centrifugation was at the slower speed of 500 X g for 10 min. Chemical analysis of cell wall material. One of the most impressive of the results obtained with the zeolite procedure occurred in the case of mycobacteria, which were known to have presented difficulties when the production of pure cell wall fragments was attempted. The mixing of zeolite and cells produces heat, and Salton (7) demonstrated that cell wall preparations produced by exposure to 100 C were not chemically identical to those produced by a procedure using the Mickle apparatus. A question arose, therefore, concerning the effect of the zeolite disruption procedure on the chemical composition of the cell wall fragments obtained. Because of the exceptional success achieved with cell walls of mycobacteria prepared by zeolite treatment, these preparations were chosen for chemical analysis. Organic phosphorus was determined by the method of Chen et al. (2). The amino acid composition was determined by use of an Amino Acid Analyzer, model 120B (Beckman Instruments, Inc., Fullerton, Calif.). The cell wall hydrolysates were prepared by a procedure based on that of Takeya et al. (12). The hydrolysate residues, obtained in 0.5 ml of distilled water, were lyophilized four times by the addition of distilled water each time the residues became dry. The final residues were stored in a lyophilized state at 5 C until the analyses could be performed. Procedures used to study the mechanism of zeolite action. Person and Zipper (5) observed that mitochondria from beef heart cells and whole Candida cells were not disrupted if wet (instead of dry) zeolite was used, if lyophilized mitochondrial or whole cell preparations were used. These authors postulated that the action of zeolite involved the removal of water from lipoprotein complexes, which resulted in a structural instability. Therefore, rupture of mitochondria or cells was due to the loss of lipoprotein membrane structures. The applicability of this concept to bacterial cells in general was tested in B. megaterium, E. coli, M. phlei, and S. marcescens. These microorganisms were exposed to wet zeolite, with dry zeolite as the control. Wet zeolite was prepared by adding an excess of distilled water and subsequently cooling the zeolite, which settled out as a thick paste. The role of grinding was determined by comparing preparations in which the grinding step was omitted to those exposed to the usual grinding procedure. Preparations for electron microscopy. Suspensions of the various zeolite-treated and control bacterial preparations were placed on collodion-coated copper grids (200 mesh). The grids were allowed to air-dry, and then they were shadowed with either platinum or platinum-palladium alloy and examined with an RCA (EMU 3-f) electron microscope operated at 50 kv. RESULTS Disruption of cells with zeolite 4A. When dry zeolite 4A was mixed with cell paste (prepared as described in Materials and Methods) by grinding for 3 min, good cell wall preparations were obtained from 17 of the 20 cultures studied. The only exceptions were G. tetragena, R. rubrum S. lutea, and all heat-killed preparations. Therefore, this comparatively simple, easy to use zeolite method can be said to have a broad application for bacterial cells. Electron microscope photographs of representative preparations are shown in Fig. 1, 2, 5D, and 6D. No attempt was made to determine whether the use of younger cultures or the introduction of procedural modifications would produce good preparations from the three organisms for which the method was not wholly successful. In addition to the electron microscope examinations, preparations of M. phlei, M. smegmatis, and E. coli were washed with 1.0 M sodium chloride and distilled water, and then studied spectrophotometrically with a Bausch and Lomb 505 recording spectrophotometer. The results with mycobacteria yielded a smooth curve corresponding to that of the ultraviolet absorption spectra of purified cell walls described by Salton and Horne (10). Also described by Salton and Home (10), the cell wall preparations from E. coli showed a shoulder from 260 to 280 nm. This shoulder

3 VOL. 16, 1968 ZEOLITE DISRUPTION OF BACIERIAL CELL WALLS 1271.~~~~~~~~~~~~~~~~~~~... '~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~...~~~~~~~~~~~~~~~~~~~~~~~~~~~~... FIG Microbial cell wall preparations obtained by the zeolite procedure. X 32,000. FIG. 1. Corynebacterium pseudodiphtheriticum. FIG. 2. Streptomyces lavendulae. FiG. 3. Zeolite-treated Rhodospirillum rubrum. FiG. 4. Ruptured Gaffkya tetragena cell walls adhering to whole cells.

4 ... - ;....;f:'...:_ s_ w0 X '.fi : 1272 WISTREICH ET AL. APPL. MICROBIOL..'! :; :).: w.: j i < si :.' X 1_.:, *.:';i. :. i2s. { : :_.: tt *.: t. 'R._ S_ g *E.>.... l,. t... *: l:.:.m&... o..... s, i :i g.. e w _Sfi _..,> >. *.: s.' it: i_ * : Z..... ; FIG. 5. Mechanism of zeolite action investigated with Mycobacterium phlei. X 32,000. (A) The effect of wet zeolite when added by mixing only. (B) The effect of wet zeolite when added by the usual grinding procedure. (C) The effect ofdry zeolite when added by mixing only. (D) The effect ofdry zeolite when added by the usual grinding procedure.

5 VOL. 16, 1968 ZEOLITE DISRUPTION OF BACTERIAL CELL WALLS probably is related to the polypeptide composition of the E. coli cell wall, rather than being indicative of contamination with cytoplasmic material. From the criteria of both electron microscopy and spectrophotometry, the cell wall preparations obtained with the zeolite method appeared to be of high purity. Attempts to disrupt R. rubrum were relatively unsuccessful. The cell wall itself was somewhat disrupted, but the internal cytoplasmic material remained intact within the cell (Fig. 3). Suitable variations of the technique might produce success with this organism; however, no such variations were attempted. Electron microscopic examinations of G. tetragena and S. lutea preparations suggested why the method was limited in its effectiveness for these organisms. Ruptured cell walls had adhered to whole cells (Fig. 4); therefore, they had been deposited with the whole cells on the first centrifugation. A similar phenomenon was reported by Salton and Home (9) from their attempts to produce good cell wall preparations by exposure of intact cells to mild heat shock. Attempts were made to apply the zeolite method to heat-killed (100 C for 10 min) M. phlei and M. tuberculosis preparations. These attempts yielded cells that looked much like R. rubrum (Fig. 3) and that exhibited thickened or coagulated internal regions, which did not leave the cell after rupture of the cell envelopes. No attempt was made to separate the internal contents from the cell wall material of such preparations. For their mild heat-shock preparations, Salton and Home (10) used successfully a method that involved mild shaking with 0.3-cm glass beads. Mechanism of zeolite action. The two factors that seemed to be important in the ability of zeolite to disrupt cells were (i) the adsorption of water and (ii) the grinding process by which the zeolite particles were brought into close proximity with the bacterial cell. Figures 5A and 6A show the results when zeolite that had been saturated with water was used in the procedure, but without grinding. No cell rupture occurred. When the water-saturated zeolite was used with the usual grinding procedure, a small amount of cell rupture was produced (Fig. 5B and 6B). These results indicated that cell rupture was not caused primarily by a mechanical puncturing of the cell envelope by the zeolite particles. Thus, neither wet zeolite nor grinding was an effective force by itself in producing cell rupture. When dry zeolite was mixed with cells, with no grinding (Fig. 5C and 6C), only a small amount of cell rupture was produced. Good rupture was produced only when dry zeolite was 1273 used with a grinding step (Fig. 5D and 6D). These results could indicate that both a close contact of the zeolite with the cell and the ability to avidly adsorb water are necessary for good breaking of the cell envelope. When dry zeolite adsorbs water, the process is highly exothermic. The procedure used and the photographs obtained do not necessarily give any information about the possible role of heat in the rupturing process. During these experiments, regardless of the cooling procedures used, a temperature rise to 45 or 50 C often occurred. This fact indicated that intense local heating of the cell might have taken place, which might have played a significant role in the rupturing effect by burning a hole through the cell envelope before the heat could be dispersed. If the role of dry zeolite is to adsorb water from lipoprotein structures, thus rendering them unstable, as proposed by Person and Zipper (5), then structures, such as the stroma of human red blood cells that are almost completely lipoprotein, should be molecularly disrupted by the action of zeolite. The results of such a study will be reported later. The effect of the zeolite procedure on cell wall components. Salton and Home (9, 10) reported a method of cell rupture, applicable to gramnegative bacteria, that involves heat shock at 100 C for 10 min. After comparing this method with that of the Mickle apparatus, which uses glass beads, Salton (7) reported that for Salmonella pullorum the heat method affected the chemical composition of the cell wall material obtained. Pure cell walls prepared by the glass bead method were higher in total nitrogen and contained diaminopimelic acid, which was absent in cell walls produced by the heat shock method. For this reason, Salton recommended that the heat shock method not be used for any procedure that involved the chemical analysis of cell wall material. Because dry zeolite produces heat when mixed with cell paste, a question arose concerning the effect of such heating on cell wall composition. Analyses were made of the organic phosphorous and amino acid content of zeolite-prepared cell wall fractions for M. smegmatis and M. phlei. These results were compared with those previously reported (4, 11) in which glass beads were used as the disruptive agent. The molar ratios of alanine and diaminopimelic acid to glutamic acid were comparable for both methods, and no major differences in the kinds or amounts of amino acids were noted. The organic phosphorous content was higher for the zeolite-prepared cell walls than that previously reported for glass bead preparations. These analyses did not reveal a

6 1274 WISTREICH ET AL. APPL. MICROBIOL. FIG. 6. Mechanism ofzeolite action investigated with Escherichia coli. X 32,000. (A) The effect of wet when added zeolite by mixing only. (B) The effect of wet zeolite when added by the usual grinding procedure. effect of (C) The dry zeolite when added by mixing only. (D) The effect ofdry zeolite when added by the usual procedure. grinding

7 VOL. 16,51968 ZEOLITE DISRUPTION OF BACTERIAL CELL WALLS 1275 loss of any important amino acid cell wall component when the zeolite method was used. DIscussIoN The zeolite method for the disruption of bacterial cells applies to many bacterial species, and it is superior to other methods for mycobacteria. The method is simple; it does not require expensive equipment; and it produces cell wall preparations in which the material seems to represent almost the entire cell envelope, rather than the fragments obtained by sonic treatment. The procedure is highly recommended for the preparation of pure cell wall material. The mechanism by which zeolite ruptures cell envelopes is unknown. The rupture is not due entirely to mechanical puncture because only a small amount of grinding is involved, and wet zeolite does not produce disruption even with grinding. The ineffectiveness of wet zeolite indicates that the high water-adsorbing characteristic of zeolite is clearly involved, but the reason that this characteristic produces the cellular rupture is not clear. It is not possible to determine from the electron micrographs the manner of zeolite action; however, limited conclusions can be drawn. The damage usually appears as large or small ruptures in the cell wall (Fig. 1, 3, 5D, and 6D). A possible explanation is that the zeolite produces pits and grooves in the cell wall during grinding, which then allow a closer association of the zeolite particle with the cytoplasmic membrane and the water inside the cell. The force by which the water is extracted and the local heat produced possibly cause major ruptures of the cell wall and of the cytoplasmic membrane, thus releasing the cellular contents into the supernatant fluid. The results of this study of the method of zeolite action clearly indicate that zeolite must be brought into close contact with the cell and that the zeolite must be able to adsorb water. ACKNOWLEDGEMENT We thank L. A. Bavetta, H. Slavkin, and Charles Lyons of the Department of Biochemistry, School of Dentistry, University of Southern California, for the amino acid analyses. LITERATURE CiTm 1. Breck, D. W., E. G. Eversole, R. M. Milton, T. B. Reed, and T. W. Thomas Crystalline zeolites. I. The properties of a new synthetic zeolite, type A. J. Am. Chem. Soc. 78: Chen, P. S., T. Y. Toribara, and H. Warner Microdetermination of phosphorus. Anal. Chem. 28: McClary, D. O., W. D. Bowers, Jr., and G. R. Miller Ultraviolet microscopy of budding Saccharomyces. J. Bacteriol. 83: Misaki, A., S. Yukawa, K. Tsuchiya, and T. Yamasaki Studies on cell walls of mycobacteria. I. Chemical and biological properties of the cell walls and the mucopeptide of B. C. G. J. Biochem. Tokyo 59: Person, P., and H. Zipper Disruption of mitochondria and solubilization of cytochrome oxidase by a synthetic zeolite. Biochem. Biophys. Res. Commun. 17g Reed, T. B., and D. W. Breck Crystalline zeolites. II. Crystal structure of synthetic zeolite, type A. J. Am. Chem. Soc. 78: Salton, M. R. J Studies of the bacterial cell wall. IV. The composition of the cell walls of some gram-positive and gram-negative bacteria. Biochim. Biophys. Acta 10: Salton, M. R. J The bacterial cell wall. Elsevier Publishing Co., Amsterdam. 9. Salton, M. R. J., and R. W. Home Studies of the bacterial cell wall. I. Electron microscopical observations on heated bacteria. Biochim. Biophys. Acta 7: Salton, M. R. J., and R. W. Home Studies of the bacterial cell wall. II. Methods of preparation and some properties of cell walls. Biochim. Biophys. Acta 7: Takeya, K., and K. Hisatsune Mycobacterial cell walls. I. Methods of preparation and treatment with various chemicals. J. Bacteriol. 85: Takeya, K., K. Hisatsune, and Y. Inoue Mycobacterial cell walls. II. Chemical composition of the "basal layer". J. Bacteriol. 85: Zipper, H., and P. Person Rapid disruption of intact yeasts by synthetic zeolite. J. Bacteriol. 92: