BACILLUS MEGATERIUM. The lysozyme consisted of purified egg white

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1 THE ACTION OF LYSOZYME ON THE CELL WALL AND CAPSULE OF BACILLUS MEGATERIUM H. J. WELSHIMER Department of Bacteriology and Parasitology, Medical College of Virginia, Richmond, Virginia Received for publication February 20, 1953 Observations concerning the action of lysozyme on Bacillus megaterium have been suggestive, although not conclusive, in indicating that the bacterial cell wall is lysed (Welshimer and Robinow, 1949). Recently Tomesik and Guex- Holzer (1952) have supported this contention by a combination of immuno-chemical procedures and phase contrast microscopy. In the experimental work here reported, a different approach has been made toward demonstrating the action of lysozyme on the external structure of the bacillus. MATERIALS AND METHODS The lysogenic Bacillus megaterium, strain 899, was used throughout these studies. Unless otherwise specified the organisms studied were obtained as follows: Sporulated cultures were suspended in distilled water to the turbidity giving a reading of 350 on the Klett-Summerson photoelectric colorimeter with a no. 54 filter. Three-tenths of a milliliter of spore suspension served as the inoculum for each 2.5 by 20 cm test tube containing 20 ml of medium. The medium (designated as YWT broth) was composed of 2 per cent yeast extract and 0.2 per cent tryptone adjusted to ph 7.2. Glucose at a concentration of 2 per cent was added to the YWT broth in some of the experiments. The inoculated YWT broth was placed at 34 C in a water bath, and air was bubbled through the suspension until the desired turbidity was attained. Previous observations (Welshimer and Robinow, 1949) have indicated the necessity for inhibiting the action of autolytic enzymes to avoid its being confused with lysozyme activity; consequently, the suspensions were treated with formalin to provide a final concentration of 15 per cent formaldehyde and held at room temperature for 1 hour, after which period the cells were washed three times by centrifugation with physiological saline and ultimately suspended in M/ phosphate buffer (ph 6.6). The cells were used immediately after this treatment. The lysozyme consisted of purified egg white lysozyme (Armour) which was dissolved in M/15 phosphate buffer (ph 6.6). Capsules of the bacilli were demonstrated by the method of White (1947) with slight modification consisting of the omission of serum and substitution of 1.6 per cent aqueous basic fuchsin for methylene blue. All preparations, other than those intended to demonstrate capsules, were air dried and fixed in Bouin's fluid for at least 15 minutes. Afterfixation the smears were stained with 0.05 per cent Victoria blue 4R which is especially suitable as a differential stain for the plasma membrane and cell wall (Robinow, 1948; Welshimer and Robinow, 1949; Robinow and Murray, 1953). EXPERIMENTAL RESULTS Webb (1951) observed that lysozyme promoted the fission of chains of bacteria in which the chaining tendency was enhanced by cultivation in media containing low concentrations of magnesium. Preparatory to the observation of the action of lysozyme on chains of B. megaterium, YWT broth was inoculated with a spore suspension and aerated 18 hours at 34 C. Two-tenths of a milliliter of the suspension was introduced into 20 ml of YWT broth which was aerated at 34 C for 2.5 hours. The resulting suspension consisted of filaments of cells containing 15 or more bacilli per chain. After fixation with formalin at a concentration equivalent to 15 per cent formaldehyde, the bacilli were washed and suspended in M/15 phosphate buffer (ph 6.6) to give a turbidity reading of 80 in the Klett-Summerson photoelectric colorimeter with the no. 54 filter. The suspension was divided into two portions. One portion was treated with lysozyme to give a final concentration of 4,ug per ml while the other fraction was untreated. Both suspensions were

2 1953] LYSOZYME ON CELL WALL AND CAPSULE OF B. MEGATERIUM 113 maintained at 34 C, and samples were removed at intervals for microscopic examination. After 45 minutes the bacilli in the untreated suspension remained grouped in long chains (figure 1), whereas in the lysozyme solution the chains were fragmented to the extent that only single cells were present (figure 2). Other studies (Welshimer and Robinow, 1949) had suggested that the cell wall of the bacillus was attacked early in the lytic process; therefore, the ability of lysozyme to cleave the bacillary chains might be interpreted as dissolution of the cell wall substance of adjoining organisms. In order to ascertain the validity of this hypothesis, A~~~~~~~~~~~ '1~~~~~~~~~~~~~~~~~~~~4 times; each time the centrifuge control was placed at maximum speed and the material was centrifuged for a period of 15 seconds from the time the rotor began accelerating. The supernatant material was removed after each centrifugation for further treatment, and the sediment was discarded. The supernatant material was centrifuged at maximum speed for 20 minutes. The pellet from this centrifugation was saved and resuspended in 1 or 2 drops of phosphate buffer (ph 6.6). The resulting material (figures 3 and 4) contained some intact bacilli together with cell wall fragments similar to those described by Murray and Robinow (1952). Figure 1. (left) Bacillus megaterium chains; nonlysozyme treated. Victoria blue 4R. X 160. Figure 2. (right) Bacillus megaterium chains after exposure to lysozyme for 45 minutes. Victoria blue 4R. X 160. cell walls were removed from the bacteria and subjected to lysozyme as described below. Formalinized bacilli were centrifuged, and the sedimented organisms were placed between two optical flat glass disks (64 mm diameter; 3 mm thick) maintained in a horizontal position. The bottom disk was held stationary on a rubber mat, and the upper disk (to which a no. 6 rubber stopper had been cemented) was rotated 10 to 15 times by hand while exerting firm downward pressure. The cells were washed from the surfaces of the disks in 2 ml of buffer. The suspension of crushed bacilli was subjected to centrifugation in an ordinary angle head, clinical centrifuge four A drop of the cell wall preparation was mixed with a drop of lysozyme solution (conc 16,ug per ml) and incubated at 34 C in a 9 by 75 mm test tube capped with parafilm. Samples were removed from the tube after 1 hour by means of an inoculating loop, placed on clean coverslips to dry, fixed in Bouin's fluid, and stained in Victoria blue 4R. In contrast to control preparations (figure 4) which displayed numerous cell wall elements, only unfragmented cells remained in the suspension treated with lysozyme (figure 5). The cells remaining in the treated suspension were stained intensely and nondifferentially with the Victoria blue, in contrast to the control

3 ...: v _. t 114 H. J. WELSHIMER [VOL. 66 preparation wherein the nonfragmented cells were lysozyme may be due to direct action on the cell stained heavily in the cell wall and plasma wall there remains another possibility. An encompassing slime or capsular substance may be membrane layers and lightly within the cytoplasm. As noted before (Welshimer and Robinow, holding the bacilli together in chains; indeed, AE. Ut *:-' _~~~~~~ Figure S. Bacillus megaterium after having been crushed between glass disks. Cell wall elements and intact bacilli are present. The cytoplasm of the intact cells stains more intensely than is observed with normal (uncrushed) cells. Victoria blue 4R. X( 1,200. i.... s., _ d a ^:2=S,.'.. ~ ~ :....v *b:.s: '. :. ^:.).:.>ti!:.d, W*.0 *>~*9' *.r 6 z {*E * F :.:\ 4.,,,0t t :. 8 * * 0, I.: ^.,. ti': i *it; s 05VE *S :* #:.s 2 r ev, W.~4N, $,..%:. J X., tik -1...m Figure 4. Bacillus megaterium after having been crushed between glass disks; nonlysozyme treated. Victoria blue 4R. X ) with bacilli pretreated with 15 per cent formaldehyde, the cells exposed to lysozyme had undergone a remarkable decrease in size, especially after incubation for 3 hours. Although the disruption of bacillary chains by capsules have been demonstrated for B. megaterium by Aubert and Millet (1950) employing dyes and Tomesik and Guex-Holzer (1951) who studied the effect of specific antiserum on B. megaterium by phase contrast microscopy.

4 1953] LYSOZYME ON CELL WALL AND CAPSULE OF B. MEGATERIUM 115 Accordingly, spores of B. megaterium were introduced into YWT broth containing 2 per cent glucose and aerated for 3 hours at 34 C. When stained with the modified White technique the bacilli exhibited remarkable capsules. Following prefixation with 15 per cent formaldehyde and repeated washing with saline, lysozyme was added at 34 C is that of bacilli with the cytoplasm stained lightly and the transverse septa stained intensely with basic fuchsin. The capsular material is unstained and delineated by the blue background of the acidified Congo red. Treatment with lysozyme for 15 minutes causes changes in the appearance of the encapsulated N *9 I k I % / I f- I\' v \\ Nk Figure 5. Bacillus megaterium after having been crushed between glass disks and suspended in lysozyme for 60 minutes. Cell wall fragments are no longer visible. Victoria blue 4R. X 600. Figure 6. Bacillus megaterium suspended in phosphate buffer for 2 hours. Modified B. White Stain, X 2,000. to buffered (ph 6.6) suspensions of the encapsulated bacilli (adjusted to read 80 with the no. 54 filter in Klett-Summerson photoelectric colorimeter) to give a final concentration of 4,ug per ml. Capsule stains of the bacilli were made at intervals during incubation at 34 C. The appearance of untreated control preparations (figure 6) at the end of 2 hours' incubation /4*" \ \k I -Y "IN N I -p0 :--. bacilli (figure 7). The chains are disrupted and bacilli are found to occur singly or doubly within capsules that have been reduced somewhat. The bacilli proper stain evenly and intensely with the basic fuchsin, indicating some change of permeability. With continued incubation, the treated cells progressively show an increased affinity for the acidified Congo red. After 2 hours of exposure I

5 116 the capsular material is removed completely from the bacilli, and they are no longer stained with basic fuchsin. On the contrary, they appear blueblack from the Congo red, thus differing little from the background which is colored also by the acid reaction of the Congo red (figure 8). H. J. WELSHIMER [VOL. 66 a mucoid in the capsule and cell of B. megaterium. Aubert (1949, 1951) studied the nature of the polysaccharide components in the capsule of B. megaterium. Employing immuno-chemical procedures, Tomcsik (1951) and Tomcsik and Guex- Holzer (1951, 1952) disclosed the presence of Figure 7. X 2,000. Figure 8. 2,000. (left) Bacillus megaterium exposed to lysozyme for 15 minutes. Modified B. White stain. (right) Bacillus megaterium exposed to lysozyme for 2 hours. Modified B. White stain. DISCUSSION The lytic action of lysozyme on the capsular substance and the segmentation of the chains of B. megaterium occur independently. The disruption of chains occurs very early in the course of lysis, while the capsular material remains intact though possibly diminished in volume. In view of the demonstration of the action of lysozyme directly on the cell wall elements, disruption of the bacillary chain must result from the lysis of the cell wall about contiguous bacilli. Lysozyme has been demonstrated to depolymerize and hydrolyze a mucopolysaccharide consisting of an acetyl aminopolysaccharide (Meyer et al., 1936). The lytic action of lysozyme on the capsular and cell wall substances of the B. megaterium indicates that these structures consist, at least in part, of an acetyl aminopolysaccharide. The studies of others support the existence of polypeptide as well as polysaccharide substances in the capsular material. The latter authors found immuno-chemical evidence that the cell wall contained polysaccharide substances. The findings in the present study have corroborated the observations of Tomcsik and Guex- Holzer (1952), who followed the action of lysozyme on B. megaterium with phase contrast microscopy and reported lysis of capsule and cell wall. ACKNOWLEDGMENTS It is a pleasure to acknowledge the helpful suggestions of Dr. C. F. Robinow and the assistance of Mr. M. C. Shaffer in taking the photomicrographs. SUMMARY The action of lysozyme on cells of Bacillus megaterium, strain 899, killed with 15 per cent formaldehyde was observed and described. X

6 1953] LYSOZYME ON CELL WALL AND CAPSULE OF B. MEGATERIUM 117 The capsule and cell wall substances are attacked by lysozyme indicating the presence of an acetyl aminopolysaccharide component of these structures. Chains of the bacilli are disrupted by the lysozyme as a result of the lysis of the walls of adjoining cells. REFERENCES AuBERT, J. P De l'existence d'un polyoside chez Bacillus megatherium. Compt. rend. soc. biol., 229, AUBERT, J. P Atude biochimique du rendement materiel de croissance d'une bacterie aerobie: Bacillus megatherium. Ann. inst. Pasteur, 80, AUBERT, J. P., AND MILLET, J Existence d'une capsule glucidique chez Bacillus megatherium. Ann. inst. Pasteur, 79, MEYER, K., PALMER, J. W., THOMPSON, R., AND KHORAZO, D On the mechanism of lysozyme action. J. Biol. Chem., 113, MURRAY, R. G. E., AND RoBINow, C. F A demonstration of the disposition of the cell wall of Bacillus cereus. J. Bact., 63, ROBINOW, C. F Proc. Soc. Am. Bact., 1, 13. ROBINOW, C. F., AND MURRAY, R. G. E The differentiation of cell wall, cytoplasmic membrane and cytoplasm of gram positive bacteria by selective staining. Exptl. Cell Research, 4, in press. ToMcSIK, J Complex structures of the bacterial capsule in the genus Bacillus. Experientia, 7, ToMcsIK, J., AND GUEX-HOLZER, S Anthrax-Polypeptid und andere spezies-spezifische Substanzen der Kapsel in der Bazillus- Gruppe. Schweiz. Z. Allgem. Path. u Bakt., 14, ToMcSIK, J., AND GUEX-HOLZER, S Anderung der Struktur der Bakterienzelle im Verlauf der Lysozym-Einwirkung. Schweiz. Z. Ailgem. Path. u Bakt., 15, WEBB, M The influence of magnesium on cell division. 6. The action of certain hydrolytic enzymes on the filamentous and chain forms of gram-positive rod-shaped organisms. J. Gen. Microbiol., 5, WELSHIMER, H. J., AND ROBINOW, C. F The lysis of Bacillus megatherium by lysozyme. J. Bact., 57, WHITE, P. B A method for combined positive and negative staining of bacteria. J. Path. Bact., 59, Downloaded from on April 10, 2018 by guest