ON THE MECHANISM OF SONIC LYSIS OF BACTERIrl BORIS ROTMAN Institute for Enzyme Research and the Department of Genetics, University of Wisconsin, Madison, Wisconsin Reprinted from .JOURNAL OF BACTERIOLOGY Vol. 72, No. 6, pp. 627-830, December, 1956 Printed in U.S.A. 827 823 ROT1I.IS j \I ,I_. 7" 19561 MECHANISM OF SONIC LYSIS OF BACTERL4 829 Viability of sonic-treated cells Optical density before lysis = 2.10 Optical density after lysis = 0.286 TABLE 1 TABLE 3 Lysis inhibition Assay by addition of 0.01 ml of ~/2 K-PO,, pH 72 to 0.99 ml of sonic-treated cells Viable Cells/ml _______ Before sonic vibration. 7.0 1.0 x 1010 Before sonic vibration. 5.6 7.8 X loo After sonic vibration. 7.0 4.3 x 10' After sonic vibration. 5.6 2.7 x lo6 TABLE 2 pH eject: 0.01 ml of N/B Na-PO4 buffer was added to 2.99 ml of sonic-treated cells of oplical density (O.D.) O.D. = 0.656 -- pH of Buffer 3.D. after 1 min ( -- No buffer added 0.635 4.75 0.640 5.5 0.585 6.0 0.480 6.5 0.308 7.25 0.275* 7.7 0.277* 8.2 0.271* N/2 NaCl 0.540 20% glucose 0.635 - - * Optical density. - Final pH 6.25 5.8 6.2 6.6 7.1 7.65 7.95 8.15 - - * No intact cells were found in the lysates by microscopic examination. have been used with similar effects. Only ghosts (as depicted in figure 3) were found by micro- scopic examination after lysis of the suspension. No intact cells were seen. The viability of sonic treated cells, if plated without addition of buffer, i. e., prior to lysis, was about 5-fold greater on nutrient agar at pH 5.6 than at pH 7.0, as shown in table 1. The results obtained with deionized cells with or without addition of citric acid were similar. The only detectable difference was that extensive lysis occurred during vibration in the absence of citric acid and therefore the yield of "intact" cells was greatly reduced. Table 2 shows that lysis is inhibited at pH values below 6. Nevertheless, pH alone does not determine lysis but a certain ionic concen- tration seems to be required. This is clear from comparison of lines 1 and 3 of table 2. In con- trast to cells without buffer, cells wit'h buffer b&T A B C - -- - _ - Treatment 0. D: after 1 Min Initial O.D. = 0.549 No treatment 4 min at 70 C 8 min at 70 C 12 min at 70 C 0.270 0.460 0.477 0.520 -- Initial O.D. = 0.850 No treatment 7 min at 30 C in 7 X 10-a M TEPP 0.430 0.370 (PH 5.5) 240 min at 30 C in 7 X 10-J M 0.875 TEPP (pH 5.5) 240 min at 30 C 240 min at 30 C in ~/50 HCl 30 min in ~/loo0 NaN, 5 min in 0.1% TC.4 at 25 C 0.475 0.360 0.430 0.425 When buffer was added t,o a mixture 1:l of cells without heat treatment and cells held 8 min at 70 C, the O.D. after lysis was 0.375. The calculated value assuming no lysis of the latter ones is 0.365. pH 5.5 show a degree of lysis in spite of the simi- lar pH attained by both suspensions at the end of the experiment. From line 9 of table 2 it can be inferred that neutral salts alone do not cause lysis. Lysis inhibition by heat treat,ment and other agents is described in table 3. Even storage in the refrigerator for several days caused some inhibition. Among the chemicals only tetra- ethyl-pyrophosphate (TEPP) was found t,o be inhibitory at relatively high concentrations compared with those used for proteolytic en- zymes (Jansen et al., 1951). Metabolic inhibitors like azide, cyanide, arsenic or dinitrophenol and protein denaturants such as trichloroacetic acid, mercuric chloride or hydrochloric acid did not inhibit lysis. Glucose, sucrose and lactose did not inhibit nor cause lysis even at high con- centrations (10 per cent). Experiments were conducted to determine whether heat shock inhibits an enzymatic lytic reaction. Sonic-treated cells mere mixed wit)h an equal volume of the same suspension which had cytoplasm even at pH values of 6.6 or in the been submitted to heat shock after vibration. As presence of m1/300 NaCl. The possibility that the indicated in table 3, no lysis of the heat-treated cytoplasm is gelated under these conditions can- cells was observed when buffer was added to the not be excluded. mixture. Analogous experiments with cells A certain proportion of the sonic treated treated with benzene, lysozyme, or heat gave cells can give rise to clones if plated under condi- identical results. tions w-hich inhibit lysis, i. e., nutrient agar at Experiments with A. v&elan&i did not differ pH 5.6. This could be interpreted as recovery from those with E. coli. from sonic injury, although the type of injury could be entirely different from that suffered by DISCUSSION the cells shown in figure 2. The primary action of sonic vibrations ap- pears to be damage of the cell structure most ACKNOWLEDGMENTS clearly demonstrated by the appearance of the The author gratefully acknowledges the in- cell wall under the electron microscope. terest and suggestions given by Dr. R. M. The quest'ion left open by the experiments Pengra, the generous hospitality of Professor P. described here concerns the nature of the lysis W. Wilson's laboratory, and thanks Professors following sonic treatment. Two alternat,ives H. A. Lardy and J. Lederberg for reviewing the were considered but none could be excluded: manuscript. (1) The lysis is enzymatic in nature. The sonic vibrations would put the digestive enzymes in SUMMARY contact with their substrates by breakage of Bacterial suspensions (Escherichia coli, Azoto- physical barriers or would permit the normally batter vinelandii) do not lyse under sonic vibra- inactive enzymes to be reached by activators or tions when suspended in medium of low ionic to release bound inhibitors. strength or at low pH. After sonic treatment, (2) The lysis occurs by dissolut'ion of the lysis can bc brought about by adding neutral cytoplasm. The sonic vibrations would desbroy buffer. Sonic vibrations are shown to cause the physical barriers between the cytoplasm structural damage to the cell but no leakage of and the medium. The cytoplasm would undergo intracellular material occurs under the condi- a gel-sol transition after contact with salts at tions described. The nature of the lytic process neutral pH. following vibration is discussed in terms of The enzymatic hypothesis is supported by the enzymatic lysis or of solation of the cytoplasm. following: (a) Lysis does not occur at pH values below 6.0; (b) Heat treatment inhibits lpsis; REFERENCES (c) Incubation with 7 X 1OW M TEPP inhibits Davrs, B. D. 1919 Isolation of biochemically lysis; (d) It is known that ribonuclease and deficient mutants of bacteria by means of autolytic enzymes become active upon drstruc- penicillin. Proc. Natl. Acnd. Sci. U. S., 36, tion of the cell (Manson, 1953); (e) The lysis 1-3. described here resembles the Nakamura effect GRUL.~, E. ii. AND HARTSELL, s. F,. l%i LySO- with lysozyme as modified by Gruls and Hart- zyme action and its relation t,o the Nakamura sell (1954). In their experiments, cells previously effect. J. Becteriol., 68, 302-306. treated with lysozyme were lysed when the pH JANSEW, E. F., CURL, A. L., AND BALLS, A. K. 1951 Reaction of a-chymotrypsin with was raised to about 10 with NaOH. analoguea of di-iso-propyl-fluorophosphate. On the other hand, the fact that under no J. Biol. Chem., 190, 557-561. conditions could b&eriolytic activity of the MAXSON, L. A. 1953 The metabolism of ribo- lysates be demonstrated does not sustain the nucleic acid in normal and bacteriophage enzymatic hypothesis unless one postulates that infected Escherichia toll. J. Bacterial., 66, the bacteriolytic enzyme(s) has to act from 703-711. within or t,hat' it becomes extremely labile under ROTMAX, B. 1955 4 simplified device for con- extracellular condit,ions. tinuous growth of microorganisms. J. Bac- To accept t,hat dissolution of the coagulated teriol., 70, k%-486. cytoplasm is the cause of lysis requires prior WILSON, P. W. AND KNIGHT, S. G. 1952 Ex- periments in Bacterial Physiology. Burgess explanation of how broken cells retain their Publishing Co., Minneapolis, Minn. 830 ROTMAN [VOL. 72