Japan. J. Microbiol.

Vol. 17(5), 353-360, 1973

A Lysin(s) in Lysates of Clostridium botulinum A190

Induced by Ultraviolet Ray or Mitomycin C

Noriko MITSUI, Kazuyoshi KIRITANI, and Shoki NISHIDA

Departmentof Bacteriology,Schoolof Medicine,Kanazawa University, Kanazawa

(Received for publication, January 10, 1973)

ABSTRACT

When cells of the Clostridium botulinum A190 strain were subjected to treatment with mitomvcin C or to
irradiation with ultraviolet ray and then grown at 37C, an induced lytic agent(s) was produced in the
resultant lysate. Production of the lytic agent in the induced bacterial culture was inhibited in the
presence of chloramphenicol. A crude preparation of the phage-free lytic agent readily lysed freeze-
thawed or acetone-treated cells of C. botulinum B-NIH19, and heated or chloroform-treated cells to a
lesser extent at the optimal pH of 6.8. The lytic agent was stable at or below 37C, but unstable
above 37C. A crude preparation of the lytic agent did not exhibit a detectable effect on the viability of
vegetative cells.

It is well known that lysins, which lyse
bacterial cells, are produced in bacterial
cells of various genera when multiplication
of virulent or temperate phages is provoked
[1-3, 5, 7, 12, 14-16]. These lysins are
usually released in the culture media follow-
ing cellular lysis. In general, phage-asso-
ciated lysins are enzymes capable of dissolv-
ing cell walls of the producer strains and their
related strains. Many lytic agents are pro-
duced in cultures of proteolytic Clostridium
botulinum types A, B and F, and Clostridium
sporogenesthrough cellular lysis by treatment
with mitomycin C, although presence or
absence of phage multiplication is not yet
known [11]. We conducted a detailed
investigation to examine the utility of the
lytic agent for purposes of taxonomic studies
and described some properties of the lytic
agent induced in a C. botulinum A190 culture.
MATERIALS AND METHODS
Bacterial strains. Unless otherwise indicated
Please address requests of reprints to Dr. Kazuyoshi
Kiritani, Department of Bacteriology, School of
Medicine, Kanazawa University, 13-1 Takara-machi,
Kanazawa 920, Japan.
C. botulinum A190 strain was used for pro-
duction of the lytic agent and C. botulinum
B-NIH19, a consistently toxigenic substrain
isolated from C. botulinum B-NIH strain [8],
as the indicator strain to the lytic agent.
Medium. TYG broth consisted of trypti-
case (BBL), 3%; yeast extract (Difco), 2%;
glucose, 1%; and sodium thioglycollate, 0.1%
(pH7.0). One and a half per cent agar was
added to the broth for the agar medium.
Bacteria grown in liver broth overnight were
provided as the inoculum for further cul-
tivation in TYG medium.
Preparation of indicator cellsfor the lytic agent.
For preparation of freeze-thawed cells (FT),
exponentially growing bacteria in TYG
broth at 37C were harvested by centri-
fugation and immediately stored at -20C.
Acetone-dried cells were prepared from TYG
cultures in the exponential phase incubated
at 37C. The acetone-dried cells were ground
to a fine powder with a mortar and pestle,
and stored at room temperature in a vacuum
desiccator. Bacteria for heat treatment were
collected by centrifugation from TYG cul-
tures in the exponential phase incubated at
37C. After being washed three times with

353
 354 N. MITSUI, K. KIRITANI AND S. NISHIDA

0.05M NaCl, the cells were resuspended in Bausch & Lomb spectronic 20 colorimeter .
0.05M phosphate buffer (pH6.8) and the Indicator cells were suspended in 0.05M

optical density (O. D.) of the suspension at phosphate buffer (pH6.8) in a 1•~10cm
535nm was adjusted to 0.6 using a 1•~10cm tube to give an optical density at 535 urn of

tube. Five-milliliter samples, distributed in 0.4 to 0.5. The assay was carried out at 37C

1.5•~16cm tubes, were incubated in a water by adding 0.5ml of an appropriately diluted

bath at 52, 60 and 100C for 10min, re- lysate with TYG broth to 3.0ml of indicator

spectively. These suspensions were cooled cell suspension. The cell suspension without

in an ice bath. lysate was also incubated for the control.

Degree of cell lysis occurring for the first

Preparation of lysates. To induce production

of the lytic agent, exponentially growing cells 5min was measured and defined as the lytic

activity of the lysate. One unit of lytic activity

at 37C in TYG broth were treated with
was expressed by a decrease in optical density
mitomycin C (Kyowa Hakko Kogyo Co.)
of 1.0per hr per ml of lysate.
(A) or irradiated with UV (B). (A) Mito-
Minimum lethal dose (MLD) for mice. A
mycin C (final concentration; 1ƒÊg/ml) was

added to the culture when the optical density phage-free lysate was prepared according to

of bacterial turbidity at 560nm reached the method described above. For the control,

0.1-0.15 (about 1•~108cells/ml). (B) Bac- a supernatant fraction was prepared from a

cell-free extract of an uninduced culture by

teria in the exponential phase harvested by
ultracentrifugation. Two tenths milliliters
centrifugation were suspended in 0.05M phos-
of an appropriately diluted sample with
phate buffer (pH6.8), and the optical density
0.05M phosphate buffer (pH6.8) was injected
of the suspension at 560nm was adjusted to
intraperitoneally into a mouse. Two mice
0.45-0.5. After UV (germicidal lamp of
were used with each dilution. Deaths of mice
Toshiba Co., 15 watt) irradiation was carried

out over the bacterial suspension in a petri were recorded for 2 days. The extent of

dish at a distance of 100cm for 25 sec, 1ml maximum dilution to kill two mice was

determined and defined as the MLD of the

of the suspension was transferred into 10ml
lysate or the extract.
of TYG broth. Each culture was further

incubated for 3.5 to 4hr until bacterial lysis Test for the bactericidal ability of the lysate.

was complete. The lysed culture was used Supernatant and pellet fractions were pre-

as the lysate. Untreated bacteria were also pared from a cell-free lysate as described
above. Five tenths milliliters of the super-
grown for the identical period as controls.
The culture was then sonicated for 10min natant fraction, or the pellet fraction, was

added to 3ml of exponentially growing cells

using a Tomy ultrasonic vibrator (model
in TYG broth (O. D. at 560nm=0.3), and
UR-200P, 20kc) to obtain an extract. Each
then incubated at 37C for 20min. A cell
lysate and extract were cleared of bacterial
culture with 0.5ml of TYG broth added was
debris by centrifugation (3000 •~g, 15min),
also incubated for the control. Each culture
and the supernatants were used as cell-free
was then diluted with saline, and 0.1ml of
lysate and cell-free extract, respectively. The
each dilution was plated on a TYG agar
cell-free lysate was fractionated into a super-
medium. Duplicate plates were incubated
natant fraction (phage-free lysate) and a
for 24hr at 37C under anaerobic conditions
pellet by ultracentrifugation (100000•~g,
and colonies formed were counted.
60min). The pellet was resuspended in
Chemicals. Trypsin (Difco, Bact), pronase
0.5ml of TYG broth, and the suspension was

used as the pellet fraction. These cell-free (Kaken Co.), chloramphenicol (Sankyo Co.),
tetracycline (Lederle Ltd.), streptomycin
and phage-free lysates could be stored at 4C

for at least two weeks without measurable (Meiji Seika Co.), penicillin G (Takeda Ltd.),
and D-cycloserine (Meiji Seika Co.) were
loss of their lyticc activities.
purchased from the respective companies.
Assay of the lytic activity. The method des-

cribed by Meinke and Jones [13] was slightly

modified. The lytic activity in lysates was

measured turbidimetrically using a Shimadzu-

 LYSIN IN LYSATES OF C. BOTULINUM 355

tained in an uninduced cell-free extract was

RESULTS very low (0.72 units per ml per hr). In a
Lytic Activity in the Induced Lysate and in the control experiment, FT cells autolysed grad-
UninducedCell-Free Extract ually during the incubation period. The
As shown in Fig. 1, a cell-free lysate pre- amount of decrease in optical density at
pared by treatment with mitomycin C con- 535nm was usually 0.02 to 0.03 after 5min
tained a lytic agent against FT cells (9.8 of incubation at 37C. In general, when
units per ml per hr). When the lysate was bacterial cells were induced with mitomycin
fractionated by ultracentrifugation, full activ- C, lytic activity found in the lysates ranged
ity was found in the supernatant fraction from 7 to 13 units per ml per hr, and with UV
(phage-free lysate) and no or, if any, little irradiation, 15 to 25 units per ml per hr.
activity in the pellet fraction. The lytic agent Lytic activities in uninduced cell-free extracts
in these cell-free and phage-free lysates lysed were so low that the values estimated might
FT cells linearly for an initial reaction time or might not be significant (generally 0.24-
of 5min. When a lysate having a lytic ac- 0.72 units per ml per hr).
tivity of more than 10 units per ml per hr was
used, it was diluted 2- to 5-fold with TYG Inductionof the Lytic Agent
broth for measurement of the activity. It Irradiation with UV of C. botulinumA190
should be noted that the lytic activity in cells resulted in bacterial lysis of the culture
a diluted sample was inversely proportional accompanied by the release of a lytic agent
to the degree of dilution. Lytic activity ob- (Fig. 2). The irradiated bacteria grew at a
slower rate (generation time=120min) than
that of the unirradiated cells (generation
time=80min) for an initial 2 to 3hr and then

Fig. 1. Changes in the optical density of suspensions

of FT cells after the addition of an induced lysate,

or uninduced extract, of a C. botulinurn A190 cul-

ture. The cell-free lysate was prepared from a bac-

terial culture treated with mitomycin C (1ƒÊg/ml).

Supernatant and pellet fractions were prepared Fig. 2. Effect of UV-irradiation on bacterial growth
by ultracentrifugation of the cell-free lysate. The and the production of a lytic agent. After UV-
cell-free extract was obtained from an uninduced irradiation at 0 time, bacteria were grown in 20ml
bacterial culture (O. D. at 560nm=0.6) by sonic of TYG broth. At each interval 2ml of the bacterial
oscillation and subsequent centrifugation. Changes cultures was sampled, sonicated, and then the lytic ac
in ontical densities caused by the addition of cell-
tivities in the cell-free culture media were mea-
free lysate (〇), supernatant fraction (□), Pellet sured. Growth of the bacteria; UV-irradiate (〇),

fraction (△), cell-free extract (●), or TYG broth unirradiated (△). Lytic activity; UV-irradiated

(control) (▲) were recorded at intervals. (●), unirradiated (▲).

 356 N. MITSUI, K. KIRITANI AND S. NISHIDA

Table 1. Effect of pH of the culture medium

on the production of the lytic agent

Bacteria were induced with 1ƒÊg of mitomy-

cin C per ml. At the end of the incubation,

remaining bacteria in these cultures were soni-

cated and the lytic activities released in the

cell-free culture media were assayed.

lysed within the nest hour. The  lytic activity-

Fig. 3. The effect of chloramphenicol addition on
in the irradiated culture started to increase
the induction of the lytic agent. Irradiation with
after 1.5-to 2-hr incubation and reached a UV was carried out at 0 time. Time of chloram-
maximum level after 3 to 3.5hr, whereas the phenicol addition after UV irradiation; 30min
activity was very low in the unirradiated (△), 60min (◇), 90min(●), 120min (▲),
150min (◆), control without chloramphenicol
culture. Similar results could be obtained
(〇). Lytic activities (units/ml/hr) liberated in
when bacteria were grown in the presence of
cell-free culture media by sonic oscillation are
1 to 5μg mitomycia C per ml. A maximum indicated in parenthesis.

yield ofthe lytic agent in the culture medium

was obtained at pH 6.5 and 7.0 (Table 1). To test whether the production of botu-
To determine the existence of free phages linum toxin was increased in an irradiated
in the cell-free lysate, 10ml of the induced culture, toxicities of the induced lysates and
lysate was centrifuged at 100000×g for 1hr. of the uninduced extracts were compared.
The resulting pellet was resuspendcd in 0.1ml The toxicities of these preparations were
of 0.1ml ammonium acetate (pH6.8) and found to be in a similar range of 100 to 1000
examined in an etectron microscope using the MLD.
negative-staining technique with 2% phos- Thus a phage-associated lytic agent(s) was
photungstic acid, adjusted to pH 6.8 with induced in cells of C. botulinum A190 by the
KOH. The pellet fraction contained phage action of UV or mitomycin C, while the
particles with hexagonal heads and tails botulinum toxin was not.
covered with sheaths. The induction process is sensitive to cldor-

Table 2. Effect of various antibiotics and D-cycloserine on the production

of the lyric agent by C. bolulinum A190 cells

The antibiotic or D-cycloserine was added to an exponentially growing bacterial culture (O. D. at 560
nm=0.1-0.15), and removed after 30min of contact by harvesting the cells and resuspending them
in fresh TYG broth. The bacteria were allowed to continue to grow for an additional 3.5hr, then
sonicated and assayed for lytic activity released in the cell-free culture medium .
a) Cellular lysis was observed in the bacterial culture . b) Bacterial growth was inhibited.
 LYSIN IN LYSATES OF C . BOTULINUM 357

amphenicol (Fig. 3). To UV-irradiated To see if a substance like that activated by

bacterial cultures, chloramphenicol in a cellular protease was present in the bacterial

concentration of 50ƒÊg/ml was added at cells, a bacterial culture (O . D. at 560nm=
different intervals. Chloramphenicol was 0.7) in the exponential phase was sonicated
,
fully effective in preventing bacterial growth then the disrupted fluid was divided into
and cellular lysis if added during the first 90 several tubes and treated with various
min, although the growth inhibition became amounts of trypsin or pronase (0 to 100ƒÊg
apparent with about a 30min lag after the
per ml) for 1hr at 37C. The lytic activities
addition of the antibiotic. Production of the in these digested fluids were not different
lytic agent in bacteria was also inhibited by from that of the undigested sonicate used as
chloramphenicol. the control (0.48 to 0.72 units per ml per hr) .
Inducibility of the lytic agent by several It should be noted that the lytic activities

antibiotics and D-cycloserine was also ex- in induced lysates were resistant to the treat-

amined. When bacteria were cultivated in ment of trypsin or pronase when the concen-

a medium in the presence of an antibiotic or tration of the enzyme was 30ƒÊg per ml or
D-cvcloserine throughout the incubation less. However, the lytic agent was inactivated

with a dose of more than 30μg/m1.

period of 4hr, bacterial growth was inhibited
and lytic activity in the resultant culture was

not increased. To minimize the effect of the Properties of the Lytic Agent
antibiotic on the protein-synthesizing activity, Although FT cells prepared from a C.
contact time of the bacteria with the anti- botulinum A190 culture were susceptible to
biotic was limited to 30min. As shown in the action of the induced lytic agent pro-
Table 2, none of these antibiotics enhanced duced by the identical strain, the susceptibility
the yield of the lytic agent, although bacterial was 1/5 to 1/8 that of the FT cells obtained
lysis like that induced by UV or mitomycin from C. botulinum B-NIH 19 culture. Because
C occurred in many cultures Within several of the high susceptibility and negligible auto-
hours after treatment with the antibiotics.

When treated with 50ƒÊg streptomycin per

ml, partial inhibition of bacterial growth was

observed in the culture, and with 100ƒÊg,

bacterial lysis. Treatment with 50ƒÊg chlor-

amphenicol per ml inhibited bacterial

growth completely, and 100ƒÊg induced

cellular lysis. Partial bacterial lysis was

provoked by treatment with penicillin or

tetracycline at each concentration used.

Bacterial growth was not influenced by treat-

ment With D-cvcloserine.

Lytic lctivities in the Autolysaie of C. botulinum

A190 Cells and in the Sonicated Culture

Digested with Trypsin or Pronuse

To study the lytic activity in the autolysate,

exponentially growing cells harvested by

centrifugation from a TYG culture (O. D.

at 560nm=0.7) were suspended in 0.05M Fig. 4. The susceptibility of indicator cells prepared
phosphate buffer (pH6.8) in the same by different methods to the action of the lytic agent.
volume as that of the original culture, then Treatment of cells; freeze-thawed (FT) (〇),

acetone-powdered (△), chloroform-treated (□),

incubated at 37C. After several hours, the
10min at 52C (●), 10min at 60C (▲), 10min
suspension was clarified by autolysis of 90%
at 100
C (■), control (vegetative cells) (×). The
of the cells. Lytic activity measured in the lytic activity of phage-free lysate against FT cells

lysate was less than 0.24 units per ml per hr. was 14.4units/ml/hr.
 358 N. MITSUI, K. KIRITANI AND S. NISHIDA

Fig. 6. Stability of the lyric agent as a function of

time at temperatures ranging from 37 to 50C.

Phage-free lysate was heated in a tube (1.5•~

16cm) at each temperature. Samples (1ml) re-

moved at intervals were immediately chilled in

Fig. 5. Effect of pH on the activity of the lytic agent. ice, and assayed for their lytic activities. The lytic

FT cells were suspended in 3ml of 0.05M phos- activity of unheated lysate against FT cells was

phate buffer adjusted at various pHs as indicated. 15.6units/ml/hr.

lysis of FT cells, cells of C. botulinum B-NIH 19
were chosen as the indicator of the induced
lytic agent. Figure 4 shows that FT cells and
acetone-powdered cells are markedly suscepti-
ble to the action of the lytic agent, while ve-
getative (untreated) cells are resistant. Heat-
ing cells resulted in the loss of susceptibility.
The susceptibility of chloroform-treated cells
was low. When phage-free lysate was added to
suspensions of treated cells, absorbancy-time
plots yielded curves. It should be noted that
the numbers of viable cells in the FT prepara-
tion were usually less than 0.1%. When FT
cells were suspended in 0.05M phosphate
buffer (pH6.8) containing 0.5M sucrose,
spheroplasts were formed within 5min at
37C with the addition of the induced lysate.
Maximum activity of the lytic agent was
obtained at a pH between 6.5 and 6.8 (Fig. 5).
As shown in Fig. 6, the lytic agent was stable
at or below 37C. Whereas, heating the
phage-free lysates at a temperature above
37C resulted in a rapid loss of the lytic
activity for a certain initial period followed
by a subsequent slow loss. As presented in
Table 3, neither the supernatant fraction
(phage-free lysate) nor the pellet fraction
of the cell-free lysate exhibited a killing
action on vegetative cells of C. botulinum
B-NIH 19.

Table 3. Effect of the induced lytic agent on viability of exponentially growing

cells of C. botulinum B-NIH19

a) Lytic activity of the indicated fractions against FT cells is listed .

b) Cell-free lysate induced with mitomycin C or UV was fractionated into supernatant (phage-free lysate)
and pellet fractions by ultracentrifugation. Details are described in the Materials and Methods.
 LYSIN IN LYSATES
DISCUSSION
Data presented in this report indicate that
a phage-associated lytic agent(s), which is
able to lyse freeze-thawed (FT) cells or
acetone-treated cells of the indicator strain,
is induced in cells of C. botulinum A190 by
treatment with mitomycin C or UV irradia-
tion. The lytic agent(s) is released in the
culture medium following cellular lysis.
Inoue and Iida [6], and Dolman and Chang
[4] showed photographs of phage particles
liberated in the induced lysates ofC. botulinum
A19 cultures. The presence of phage parti-
cles in the lysate was also verified by us using
an electron microscope. However, a search
for sensitive bacteria to the phage has so far
been unsuccessful. Antibiotics, which inhibit
biosynthesis of protein or cell wall, do not
induce the production of a lytic agent in
bacterial cells, although they frequently in-
duce bacterial lysis. As discussed by Rogers
and Forsberg [17], cellular lysis provoked
by antibiotics might be due to the breakdown
of the cell wall by the action of pre-existing
autolytic enzymes. We presume that the lytic
agent found in the induced lysate may be
a kind of lytic enzyme(s) on the basis of the
following facts; activity of the lytic agent
is heat-sensitive, depends upon pH, and
is inactivated by trypsin or pronase. It
should be noted that the lytic agent was
a nondialvzable material. Because the
induced lytic agent is associated with phage
propagation, it may be a phage induced
endolysin [1-3, 5, 7, 12, 14-16].
According to studies made by Kawata
et al [9, 10, 18], actively growing cells of C.
botulinum A190 can autolyse in 0.05M phos-
phate buffer, pH 7.0, and liberate autolysins,
N-acetylmuramyl-L-alanine amidase andhex-
osaminidase in the resultant lysate. The
partially purified enzymes are active on
heated or SDS-treated cell-walls prepared
from proteolytic C. botulinum types A and B
at around pH 6.8, but not from nonproteo-
lytic strains of types B and E. The induced
lytic agent of this report seems to resemble the
autolysin with respect to the optimal pH
value and to the activity spectrum; bacterial
lysis provoked with the lytic agent is express-
ed in cells of confined groups belonging to
OF C. BOTULLNUM 35O
proteolytic C. botulinum and C. sporogoics [11].
Kawata and Takumi [10] reported that
trypsin and nagase could stimulate activity
of the partially purified autolysin(s) of the
C. botulinum A190 to a certain extent. We
suspected, therefore, that a pre-existing pre-
cursor of the autolysin(s), or the autolysin(s)
per se, might be activated by a cellular pro-
tease during the induction process. However,
the lytic activity liberated in phosphate buffer
by autolysis of vegetative cells was very low,
and the lytic activity in the crude extract of
uninduced cells was not increased either by
trypsin or pronase. Furthermore, the induc-
tion process of the lytic agent was sensitive
to chloramphenicol, although the antibiotic
did not stop bacterial growth immediately.
Our findings suggested that the lytic agent
alight presumably be synthesized de novo in
induced bacterial cells.
Presence of an autolysin in indicator cells
also gave us difficulty in analyzing the results.
As one of the possibilities, it was supposed that
the action of the inducedlytic agent, cooperat-
ing with that of the cell-bound autolysin,
might cause FT cells to lyse. A decrease in the
susceptibility of the indicator cells by heating
or treatment with chloroform might be
reflecting a decrease of the autolytic activity.
Attempts to inactivate the autolysin existing
in indicator cells without lowering the sus-
ceptibility have so far been unsuccessful.
Ueda and Takagi [19] reported that the
proteolytic C. botulinum B strains 39 and T
produced bacteriocin-like substances upon
induction with UV irradiation. These
substances were sedimentable by ultracentri-
fugation and active against vegetative cells
and germination of spores of the proteolytic
C. botulinum strains especially. Since the
induced lytic agent obtained by us is not
sedimentable, it appears that the agent is
different from the bacteriocin-like substances.
More detailed studies regarding these
matters mentioned above are necessary after
purification of the lytic agent(s) has been
accomplished. As will be mentioned in a
subsequent paper, however, a crude prepara-
tion of the induced lytic agent(s) obtained
from C. botulinum A190 culture showed a
strong specificity on cells of C. sporogenesand
proteolytic C. botulinum strains and proved
 360 N. MITSUI, K. KIRITANI AND S. NISHIDA

to be utilizable for taxonomic purposes to [9] Kawata, T., and Takumi, K. 1971. Autolytic
enzyme system of Clostridium botulinum. I. Partial
demonstrate the close relationship between
purification and characterization of an autolvsin
the two species [11]. ofClostridium botulinum type A. Japan. J. Microbiol,
15: 1-10.
ACKNOWLEDGEMENT
[10] Kawata, T., Takumi, K., Sato, S., and Yama-
shita, H. 1968. Autolytic formation of spheroplasts
We thank Dr. Genji Sakaguchi, Department of
and autolysis of cell walls in Clostridium botulinum
Public Health, College of Agriculture, the University
type A. Japan. J. Microbiol. 12: 445-455.
of Osaka Prefecture, for C. botulimmn B NIH strain

and Dr. Hiron Iida, Department of Bacteriology,

[11] Kiritani, K., Mitsui, N., Nakamura, S., and
Nishida, S. 1973. Numerical taxonomy of Clostri-
School of Medicine, Hokkaido University, for C.
dium botulinum and Clostridium sporogenes strains,
botulinum A190 strain.
and their susceptibilities to induced lysins and to
mitomycin C. Japan. J. Microbiol. 17: 361-372.
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