THE ACTION OF LYSOZYME ON THE CELL WALL AND CAPSULE OFBACILLUS MEGATERIUM

H. J. WELSHIMERDepartment of Bacteriology and Parasitology, Medical College of Virginia, Richmond, Virginia

Received for publication February 20, 1953

Observations concerning the action of lysozymeon Bacillus megaterium have been suggestive,although not conclusive, in indicating that thebacterial cell wall is lysed (Welshimer andRobinow, 1949). Recently Tomesik and Guex-Holzer (1952) have supported this contention bya combination of immuno-chemical proceduresand phase contrast microscopy. In the experi-mental work here reported, a different approachhas been made toward demonstrating the actionof lysozyme on the external structure of thebacillus.

MATERIALS AND METHODS

The lysogenic Bacillus megaterium, strain 899,was used throughout these studies. Unless other-wise specified the organisms studied were ob-tained as follows: Sporulated cultures weresuspended in distilled water to the turbiditygiving a reading of 350 on the Klett-Summersonphotoelectric colorimeter with a no. 54 filter.Three-tenths of a milliliter of spore suspensionserved as the inoculum for each 2.5 by 20 cmtest tube containing 20 ml of medium. Themedium (designated as YWT broth) was com-posed of 2 per cent yeast extract and 0.2 per centtryptone adjusted to pH 7.2. Glucose at a concen-tration of 2 per cent was added to the YWTbroth in some of the experiments. The inoculatedYWT broth was placed at 34 C in a water bath,and air was bubbled through the suspensionuntil the desired turbidity was attained.

Previous observations (Welshimer and Ro-binow, 1949) have indicated the necessity forinhibiting the action of autolytic enzymes toavoid its being confused with lysozyme activity;consequently, the suspensions were treated withformalin to provide a final concentration of 15per cent formaldehyde and held at room tempera-ture for 1 hour, after which period the cells werewashed three times by centrifugation with physio-logical saline and ultimately suspended in M/15

phosphate buffer (pH 6.6). The cells were usedimmediately after this treatment.The lysozyme consisted of purified egg white

lysozyme (Armour) which was dissolved in M/15phosphate buffer (pH 6.6).

Capsules of the bacilli were demonstrated bythe method of White (1947) with slight modifica-tion consisting of the omission of serum andsubstitution of 1.6 per cent aqueous basic fuchsinfor methylene blue.

All preparations, other than those intended todemonstrate capsules, were air dried and fixed inBouin's fluid for at least 15 minutes. Afterfixationthe smears were stained with 0.05 per centVictoria blue 4R which is especially suitable as adifferential stain for the plasma membrane andcell wall (Robinow, 1948; Welshimer and Ro-binow, 1949; Robinow and Murray, 1953).

EXPERIMENTAL RESULTS

Webb (1951) observed that lysozyme promotedthe fission of chains of bacteria in which thechaining tendency was enhanced by cultivationin media containing low concentrations of mag-nesium.

Preparatory to the observation of the action oflysozyme on chains of B. megaterium, YWT brothwas inoculated with a spore suspension andaerated 18 hours at 34 C. Two-tenths of amilliliter of the suspension was introduced into 20ml of YWT broth which was aerated at 34 C for2.5 hours. The resulting suspension consisted offilaments of cells containing 15 or more bacilli perchain. After fixation with formalin at a concen-tration equivalent to 15 per cent formaldehyde,the bacilli were washed and suspended in M/15phosphate buffer (pH 6.6) to give a turbidityreading of 80 in the Klett-Summerson photo-electric colorimeter with the no. 54 filter. Thesuspension was divided into two portions. Oneportion was treated with lysozyme to give a finalconcentration of 4 ,ug per ml while the otherfraction was untreated. Both suspensions were

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LYSOZYME ON CELL WALL AND CAPSULE OF B. MEGATERIUM

maintained at 34 C, and samples were removed atintervals for microscopic examination. After 45minutes the bacilli in the untreated suspensionremained grouped in long chains (figure 1),whereas in the lysozyme solution the chains werefragmented to the extent that only single cellswere present (figure 2).

Other studies (Welshimer and Robinow, 1949)had suggested that the cell wall of the bacilluswas attacked early in the lytic process; therefore,the ability of lysozyme to cleave the bacillarychains might be interpreted as dissolution of thecell wall substance of adjoining organisms. Inorder to ascertain the validity of this hypothesis,

times; each time the centrifuge control was placedat maximum speed and the material was cen-trifuged for a period of 15 seconds from the timethe rotor began accelerating. The supernatantmaterial was removed after each centrifugationfor further treatment, and the sediment wasdiscarded. The supernatant material was cen-trifuged at maximum speed for 20 minutes. Thepellet from this centrifugation was saved andresuspended in 1 or 2 drops of phosphate buffer(pH 6.6). The resulting material (figures 3 and4) contained some intact bacilli together with cellwall fragments similar to those described byMurray and Robinow (1952).

A~~~~~~~~~~~'1~~~~~~~~~~~~~~~~~~~~4

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 andsubjected to lysozyme as described below.

Formalinized bacilli were centrifuged, and thesedimented organisms were placed between twooptical flat glass disks (64 mm diameter; 3 mmthick) maintained in a horizontal position. Thebottom disk was held stationary on a rubber mat,and the upper disk (to which a no. 6 rubberstopper had been cemented) was rotated 10 to 15times by hand while exerting firm downwardpressure. The cells were washed from the surfacesof the disks in 2 ml of buffer. The suspension ofcrushed bacilli was subjected to centrifugation inan ordinary angle head, clinical centrifuge four

A drop of the cell wall preparation was mixedwith a drop of lysozyme solution (conc 16 ,ugper ml) and incubated at 34 C in a 9 by 75 mmtest tube capped with parafilm. Samples wereremoved from the tube after 1 hour by means ofan inoculating loop, placed on clean coverslipsto dry, fixed in Bouin's fluid, and stained inVictoria blue 4R. In contrast to control prepara-tions (figure 4) which displayed numerous cellwall elements, only unfragmented cells remainedin the suspension treated with lysozyme (figure 5).The cells remaining in the treated suspension

were stained intensely and nondifferentially withthe Victoria blue, in contrast to the control

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H. J. WELSHIMER

preparation wherein the nonfragmented cells werestained heavily in the cell wall and plasmamembrane layers and lightly within the cyto-plasm. As noted before (Welshimer and Robinow,

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lysozyme may be due to direct action on the cellwall there remains another possibility. An en-compassing slime or capsular substance may beholding the bacilli together in chains; indeed,

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Figure S. Bacillus megaterium after having been crushed between glass disks. Cell wall elements andintact bacilli are present. The cytoplasm of the intact cells stains more intensely than is observed withnormal (uncrushed) cells. Victoria blue 4R. X( 1,200.. t

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Figure 4. Bacillus megaterium after having been crushed between glass disks; nonlysozyme treated.Victoria blue 4R. X 600.

1949) with bacilli pretreated with 15 per centformaldehyde, the cells exposed to lysozyme hadundergone a remarkable decrease in size, es-pecially after incubation for 3 hours.Although the disruption of bacillary chains by

capsules have been demonstrated for B. mega-terium by Aubert and Millet (1950) employingdyes and Tomesik and Guex-Holzer (1951) whostudied the effect of specific antiserum on B.megaterium by phase contrast microscopy.

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LYSOZYME ON CELL WALL AND CAPSULE OF B. MEGATERIUM

Accordingly, spores of B. megaterium wereintroduced into YWT broth containing 2 per centglucose and aerated for 3 hours at 34 C. Whenstained with the modified White technique thebacilli exhibited remarkable capsules. Followingprefixation with 15 per cent formaldehyde andrepeated washing with saline, lysozyme was added

at 34 C is that of bacilli with the cytoplasmstained lightly and the transverse septa stainedintensely with basic fuchsin. The capsular ma-terial is unstained and delineated by the bluebackground of the acidified Congo red.Treatment with lysozyme for 15 minutes causes

changes in the appearance of the encapsulated

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Figure 5. Bacillus megaterium after having been crushed between glass disks and suspended in lyso-zyme 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 en-

capsulated bacilli (adjusted to read 80 with theno. 54 filter in Klett-Summerson photoelectriccolorimeter) to give a final concentration of 4 ,ugper ml. Capsule stains of the bacilli were made atintervals during incubation at 34 C.The appearance of untreated control prepara-

tions (figure 6) at the end of 2 hours' incubation

bacilli (figure 7). The chains are disrupted andbacilli are found to occur singly or doubly withincapsules that have been reduced somewhat. Thebacilli proper stain evenly and intensely with thebasic fuchsin, indicating some change of per-meability. With continued incubation, the treatedcells progressively show an increased affinity forthe acidified Congo red. After 2 hours of exposure

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H. J. WELSHIMER

the capsular material is removed completely fromthe bacilli, and they are no longer stained withbasic fuchsin. On the contrary, they appear blue-black from the Congo red, thus differing littlefrom the background which is colored also by theacid reaction of the Congo red (figure 8).

Figure 7.X 2,000.

Figure 8.2,000.

a mucoid in the capsule and cell of B. megaterium.Aubert (1949, 1951) studied the nature of thepolysaccharide components in the capsule ofB. megaterium. Employing immuno-chemical pro-cedures, Tomcsik (1951) and Tomcsik and Guex-Holzer (1951, 1952) disclosed the presence of

(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. X

DISCUSSION

The lytic action of lysozyme on the capsularsubstance and the segmentation of the chains ofB. megaterium occur independently. The dis-ruption of chains occurs very early in the course

of lysis, while the capsular material remainsintact though possibly diminished in volume. Inview of the demonstration of the action oflysozyme directly on the cell wall elements,disruption of the bacillary chain must result fromthe lysis of the cell wall about contiguous bacilli.Lysozyme has been demonstrated to de-

polymerize and hydrolyze a mucopolysaccharideconsisting of an acetyl aminopolysaccharide(Meyer et al., 1936). The lytic action of lysozymeon the capsular and cell wall substances of theB. megaterium indicates that these structuresconsist, at least in part, of an acetyl amino-polysaccharide.The studies of others support the existence of

polypeptide as well as polysaccharide substancesin the capsular material. The latter authors foundimmuno-chemical evidence that the cell wallcontained polysaccharide substances.The findings in the present study have cor-

roborated the observations of Tomcsik and Guex-Holzer (1952), who followed the action oflysozyme on B. megaterium with phase contrastmicroscopy and reported lysis of capsule and cellwall.

ACKNOWLEDGMENTS

It is a pleasure to acknowledge the helpfulsuggestions of Dr. C. F. Robinow and the as-sistance of Mr. M. C. Shaffer in taking thephotomicrographs.

SUMMARY

The action of lysozyme on cells of Bacillusmegaterium, strain 899, killed with 15 per centformaldehyde was observed and described.

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LYSOZYME ON CELL WALL AND CAPSULE OF B. MEGATERIUM

The capsule and cell wall substances are

attacked by lysozyme indicating the presence ofan acetyl aminopolysaccharide component ofthese structures.

Chains of the bacilli are disrupted by thelysozyme as a result of the lysis of the walls ofadjoining cells.

REFERENCES

AuBERT, J. P. 1949 De l'existence d'un polyo-side chez Bacillus megatherium. Compt.rend. soc. biol., 229, 477-478.

AUBERT, J. P. 1951 Atude biochimique durendement materiel de croissance d'unebacterie aerobie: Bacillus megatherium. Ann.inst. Pasteur, 80, 644-658.

AUBERT, J. P., AND MILLET, J. 1950 Existenced'une capsule glucidique chez Bacillus mega-

therium. Ann. inst. Pasteur, 79, 468-469.MEYER, K., PALMER, J. W., THOMPSON, R., AND

KHORAZO, D. 1936 On the mechanism oflysozyme action. J. Biol. Chem., 113, 479-486.

MURRAY, R. G. E., AND RoBINow, C. F. 1952A demonstration of the disposition of thecell wall of Bacillus cereus. J. Bact., 63,

298-300.

ROBINOW, C. F. 1948 Proc. Soc. Am. Bact.,1, 13.

ROBINOW, C. F., AND MURRAY, R. G. E. 1953The differentiation of cell wall, cytoplasmicmembrane and cytoplasm of gram positivebacteria by selective staining. Exptl. CellResearch, 4, in press.

ToMcSIK, J. 1951 Complex structures of thebacterial capsule in the genus Bacillus.Experientia, 7, 459-460.

ToMcsIK, J., AND GUEX-HOLZER, S. 1951 An-thrax-Polypeptid und andere spezies-spezi-fische Substanzen der Kapsel in der Bazillus-Gruppe. Schweiz. Z. Allgem. Path. u Bakt.,14, 515-522.

ToMcSIK, J., AND GUEX-HOLZER, S. 1952 Ander-ung der Struktur der Bakterienzelle im Ver-lauf der Lysozym-Einwirkung. Schweiz. Z.Ailgem. Path. u Bakt., 15, 517-525.

WEBB, M. 1951 The influence of magnesium oncell division. 6. The action of certainhydrolytic enzymes on the filamentous andchain forms of gram-positive rod-shaped or-ganisms. J. Gen. Microbiol., 5, 496-501.

WELSHIMER, H. J., AND ROBINOW, C. F. 1949The lysis of Bacillus megatherium bylysozyme. J. Bact., 57, 489-499.

WHITE, P. B. 1947 A method for combinedpositive and negative staining of bacteria.J. Path. Bact., 59, 334-335.

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