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