LYSOZYME ACTION AND ITS RELATION TO THE NAKAMURA EFFECT'

E. A. GRULA AND S. E. HARTSELL

Laboratories of Bacteriology, Department of Biological Sciences, Purdue University, Lafayette, Indiana

Received for publication March 2, 1954

Nakamura (1923) found that when alkali wasadded to a lysozyme lysed, but not completelycleared, suspension of bacteria immediate clear-ing resulted. This phenomenon of alkali clearing,called the Nakamura effect, has been employedby several investigators to evaluate lysozymeaction (Epstein and Chain, 1940; Meyerholtz andHartsell, 1952; Warren and Durso, 1952; Stone,1952).

It has generally been agreed, as shown byN\akamura, that lysozyme first must be presentand act on the cells before any degree of clearingcan be obtained upon the addition of alkali. Themechanism of clearing of lysozyme lysed cells andnonclearing of cells which have not been in con-tact with lysozyme, however, has never beenexplained.

This report is an evaluation of this phenom-enon along with some observations regarding thepossible mechanism of gram staining as relatedto the lysozyme substrate in Micrococcus lysodeik-ticus.

MATERIALS AND METHODS

The materials and techniques employed in thisinvestigation were the same as reported in a pre-vious communication (Grula and Hartsell, 1954),except for a few additions. Hydrochloric acid andsodium hydroxide were used for all pH adjust-ments except where otherwise stated. The Huckermodification of the gram staining procedure wasemployed to determine the gram reaction.

RESULTS

In a previous communication (Grula andHartsell, 1954), it was indicated that the clearingof a suspension of M. lysodeikticus was not solelydue to the dissolving of the cytoplasm per se bylysozyme. After lysozyme had removed the cellwall, the factors of chemical solubility and auto-lytic digestion were also of considerable im-

' Grateful acknowledgement is expressed to TheEli Lilly Company, Indianapolis, Indiana, for thefellowship which made much of this work possible.

portance. The effect of pH on this sequence ofchanges is indicated in table 1.

If the results were read with respect to theper cent T after one hour of incubation, lysiswA-ould appear to be inhibited at all pH levelsexcept that in tube 9. When alkali was added toall tubes, after incubation, immediate and com-plete clearing occurred only in the tubes whichcontained lysozyme. Such clearing shows that theaction of lysozyme permits subsequent solubiliza-tion of the cells.The electron microscopy of all cells exposed to

lysozyme showed that the enzyme had acted eventhough the suspension had not cleared in theconventional manner. The removal or partialdigestion of the cell wall and some solubilizationof the cytoplasm occurred at all pH levels. Thecells were either less dense in appearance or par-tially disrupted; however, the lower the pH, theless drastic was the total effect on the cell (fig-ures 1 to 4).Lysozyme acting directly on cell wall prepara-

tions at pH levels of 5 to 7 soon caused them todisappear. At a lower pH level, e.g. 3.4, cell walls

TABLE 1Lysis of Micrococcus lysodeikticus at different

pH levels*

TUBE pH ZERO %T %T AFTER %T AFTER FINAL pH1O.tHOUR ALKALI

1 3.4 30 35 100 10.52 3.4 30 30 40 10.53 4.0 30 36 100 10.54 4.0 30 29 39 10.55 4.8 30 36 100 10.56 4.8 30 29 39 10.57 5.2 30 35 100 10.58 5.2 30 31 41 10.59 6.8 30 80 100 10.5

10 6.8 30 32 36 10.5

* Read at 540 m,u.t All tubes contained 3 ml of washed cells.

Tubes 1, 3, 5, 7, and 9 also contained 3 ml oflysozyme (1-10,000), whereas tubes 2, 4, 6, 8, and10 contained 3 ml of water instead of lysozyme.

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LYSOZYME ACTION AND RELATION TO NAKAMURA EFFECT

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Figure 1. Normal cells of Micrococcus lysodeikticus after one hour at pH 4.0.Figure 2. Action of lysozyme at pH 4.0. Note the decrease in density and unmasking of the granules.*Figure S. Action of lysozyme at pH 4.8Figure 4 Action of Iysozyme at pH 68?Density is greatly decreased the cells are more flattened and

disrupted.Figure 5. Action of lysozyme directly on cell walls at pH 3.4. Compare to figure 10.Figure 6. "Ghost" fraction remaining after lysozyme action and solubilization with alkali.* The magnification in figures 2 to 6 is given by the micron markers. Distance between the markers is

1 micron. The other figures have a magnification of approximately 8,500.

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IIFigure 7. Cytoplasmic residue remaining after lysozyme has digested the cell walls at pH 6.8. Compare

to figure 8.Figure 8. Residue and cell walls after mechanical disruption and washing. Note the presence of the

cytoplasmic granules in the debris.Figure 9. Appearance of the cell walls and cytoplasmic residue after alkaline solubilization. Not

washed. The residue has been solubilized. The "Jack Frost" pattern is due mostly to NaOH.Figure 10. Cell walls prepared by the technique of mechanical disruption and alkaline solubilization.Figure 11. Appearance of cell walls after saturation with Alcian Blue.Figure 12. Appearance of Alcian Blue saturated cell walls after incubation with lysozyme for one hour

at room temperature. Compare to figure 11.

were partially, though not completely, digested(figure 5, compare to figure 10).

Therefore, it is evident that alkali clears a bac-terial suspension only after lysozyme has re-moved or damaged the cell wall to the pointwhere contact between cytoplasm and alkali is

possible. The alkaline solubilization leaves afaintly defined "ghost" (figure 6). That the alkalineed not be specific can be shown by substitutingKOH, NH40H, or Li2CO3 for NaOH.When cells were disrupted mechanically with

glass beads, two insoluble fractions were spun

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LYSOZYME ACTION AND RELATION TO NAKAMURA EFFECT

out (Grula and Hartsell, 1954). One was yellow(cytoplasmic debris), and the other was white(cell walls) (figures 7 to 9). When these fractionsare washed and resuspended in double distilledwater at pH 10.5 and then immediately centri-fuged, only the cell walLs (figure 10) are sedi-mented since the yellow fraction has been solu-bilized by the alkali. They can be stained withAlcian Blue, or they can be digested by lysozyme.However, if the enzyme is added to the resus-pended fraction instead of alkali, the cell wallsare digested and only the yellow fraction can besedimented (figure 7).Alian Blue observatiowns. Grula and Hartsell

(1954) reported that saturation of whole cells ofM. ly8odeikticus with Alcian Blue resulted in thecomplete inhibition of lysis by lysozyme. Simi-larly saturated cells were prepared. Addition ofalkali neither cleared the suspension nor causedany morphological changes observable with theelectron microscope.

Cell wall preparations, when saturated withAlcian Blue and incubated with lysozyme for onehour at room temperature, showed that this dyehad inhibited the action of lysozyme. Intact cellwalls were still evident (figures 11 and 12).Gram staining observatios. Lysozyme action on

many gram positive cells renders them gramnegative. This has led to the interpretation thatthe lysozyme substrate (specific carbohydrate) ispart of the gram positive complex which acts asa chemical acceptor of the basic dye (Webb, 1948).In these experiments, all preparations of cell wallsor cytoplasmic debris were gram negative. Theseresults agree with the generally accepted opinionthat damaged or crushed cells stain gram nega-tive. However, if the lysozyme substrate is anintegral part of the gram positive complex, thenthe separated cell walls of this organism shouldstain gram positive since they still contain thelysozyme substrate. Because this does not happen,it is evident that the lysozyme substrate is in-volved in the gram reaction only in the sense that,when it is removed, the porosity of the cell isaffected.

These results agree with the theory of the gramstain proposed by Bartholomew and Mittwer(1952). However, it appears neeessary to im-plicate the cell wall rather than the cell mem-brane alone in regard to porosity and mainte-nance of an intact cell structure.

DISCUSSION

The Nakamura effect and its relation to lyso-zyme action involve changes in the morphologyof M. lysodeikticus. Contact between the cyto-plasm and alkali normally is prevented by theresistant cell wall of this organism. However,once the cell wall has been digested or sufficientlydegraded by lysozyme, alkali enters causing im-mediate solubilization of the cytoplasm. Thus,the action of the enzyme and the base comple-ment each other to permit maximum clearing.Unless alkali is added to lysozyme treatedbacterial suspensions or unless electron micro-scopic examination of whole cells or cell wall prep-arations is made, erroneous interpretations couldbe made regarding the extent of lysozyme action.

It is possible that some components may beleached from the cell wall under conditions ofmechanical disruption and alkali solubilization;however, such leaching neither destroys thelysozyme substrate nor alters the gram reactionin cells which are left intact throughout thepreparational procedures.

SUMMARY

Clearing of a suspension does not appear to bea valid measurement of lysozyme action unlessenvironmental conditions relating to pH are ad-justed to permit maximum solubility. WithMicrococcU8 lysodeikticus, little or no clearingwas observed at low pH ranges, yet the cellsshowed the Nakamura effect. Electron micro-scopic observation of cells exposed to lysozymeat low pH ranges showed that considerableenzymatic action had occurred. The solubiliza-tion of the cytoplasm by alkali (Nakamura effect)was shown to have been possible only after thecell wall had been removed or sufficiently de-graded by lysozyme, to permit contact betweenalkali and the cytoplasm.

Saturation of cell wall preparations or intactcells with Alcian Blue results in the completeinhibition of lysozyme action.The gram positive state is not dependent

(chemically) on the presence of the lysozymesubstrate in the cell wall of M. lysodeikticus;these gram positive cells become gram negativeafter removal of the lysozyme substrate becauseof changes induced in cell wall permeability.

REFERENCESBARTHOLOMEW, J. W., AND MITTWER, T. 1952

The gram stain. Bacteriol. Revs., 16, 1-31.

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EPSTEIN, L. A., AND CHAIN, E. 1940 Some ob-servations on the preparation and propertiesof the substrate of lysozyme. Brit. J. Exptl.Pathol., 21, 339-55.

GEuIA, E. A., AND HARTBELL, S. E. 1954 Ly-sozyme and morphological alterations inducedin Micrococcus lysodeikticu. J. Bacteriol.,68, 171-177.

MEYERHOLTZ, L., AND HARTSELL, S. E. 1952Lysozyme and gram negative bacteria. Bac-teriol. Proc., 34-35.

NAKAMUR, 0. 1923 Ueber lysozymwirkungen.Z. ImmunitAtsforsch., 38, 425-449.

STONE, J. L. 1952 The effect of lysozyme on theproduction of tetanus toxin. I. Studies withflocculation. J. Bacteriol., 64, 299-303.

WARREN, G. H., AND DuRso, J. G. 1952 Theeffect of lysozyme on the cell structure ofAchromobacterftcheri. J. Bacteriol., 64, 483-

487.WIEBB, M. 1948 The action of lysozyme on heat-

killed gram-positive microorganisms. J. Gen.Microbiol., 2, 260-274.

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