Japan. J. Microbiol. Vol. 19(3), 167-172, 1975
Observations Obtained
on Nonconverting from
Clostridium
Phage,
a Nontoxigenic
c-n71,
Strain
botulinum
Type
of
C
Keiji OGUMA,Hiroo IIDA, and Katsuhiro INOUE Department of Bacteriology, Hokkaido University School of Medicine, Sapporo, andHokkaido Institute ofPublicHealth,Sapporo (Received for publication, July 9, 1974)
ABSTRACT
A nontoxigenic mutant (C-N71) obtained from a toxigenic strain of Clostridium botulinumtype C, Stockholm,with nitrosoguanidine treatment was found to be lysogenic by the lysis test. Although the filtrate of a passaged lysate of this nontoxigenic but lysogenic strain, C-N71, lysed cells of the nontoxigenic strain C-AO2 equally as well as the converting phage c-st obtained from the strain C-Stockholm,it did not convert C-AO2 to the toxigenic state. The lysis spectrum of this filtrate was the same as that of the c-st phage. The ability of the filtrate to lyse the indicator cells, C-AO2, was destroyed neither by trypsin nor DNase but was inactivated by heat treatment at 80 C for 10 min. This suggested that the agent which caused lysis was not boticin but probably a phage. An electron micrograph of the complete phage, c-n71, which was similar in morphology to that of the c-st phage was obtained from the filtrate of strain C-N71. Anti-c-n71 phage rabbit serum neutralized both the lytic and the converting activities of the c-st phage. These findings strongly suggest that the c-n71 phage is a mutant of the c-st phage which lacks the gene controlling production of botulinum type C toxin.
The authors were the first to suggest the phage conversion of toxigenicity in Clostridium botulinumtypes C and D [7-9]. Recently, Eklund et al verified and extended this by using purified phages from plaques [2-4]. In these experiments, all of the nontoxigenic strains which were converted to the toxigenic state by infection of specific phages were cured strains. Various methods were attempted to get nontoxigenic strains from the toxigenic strain C-Stockholm. Nontoxigenic mutants which retained their lysogenicities were isolated with nitrosoguanidine treatment. A complete phage, named c-n71, was found in the induced lysate of one of these strains. The results of comparative experiments on
c-n71
phage
this
and
c-st
MATERIALS Strains D
and
strains
type
are
described
6814,
151.
The
viously
[9].
strains
were
pH
type these
0.5%,
7.0) Isolations
in
transferred 1 %, yeast
cysteine
when
D,
203U-28, strains
strains
stored
medium and (lactalbumin
glucose
type
468U-31,
and of
reported
These (CM) medium
6816
added
167
to
at
a
to
incubate
C and
Toxigenic
Nontoxigenic
468U-16,
origin
types
follows:
Stockholm.
AO2,
6813,
as
139,
were
cooked into extract
hydrochloride
pre-
meat LYG 2%, 0.14%
required.
of
nontoxigenic
but
lysogenic
N-Methyl-N'-nitro-N-nitrosoguanidine
Requests for reprints should be addressed to Dr. Keiji Oguma, Department of Bacteriology, Hokkaido University School of Medicine, Nishi 7-chome, Kita 15, Kita-ku, Sapporo 060, Japan.
in
METHODS
C. botulinum
were
strain
strains
AND
media.
used
C,
C,
phage
report.
a young
concentration at
37
was
culture
of
of
500 ƒÊgirril
C for
mutants.
10
strain
min..
C-Stockholm and The
allowed culture
:
168
was
K.
centrifuged
the
pellet
at
was
and
incubated
The
culture
for
same the
C
procedure
petri light for
on
Isolated lated
CM
3
days.
for
fluid
was
neally
tested
of
toxigenic
strain,
mitomycin
C
treatment
for
trifugation
filter were
used of
ml
(0.D.
of
= 0.3)
2 ml
to
successive ceeded the
0.5
in plaque test
passaged c-st phage c-st
1000
phages. was about
and times
Lysis of
the
and
c-n71
a suitable Strain organism. was
added
suspension
0.2
we with used lytic
were
ml
had these
from at
each
not
suc-
phages,
to
determine
titers
of
these
titer of lytic titers
always
10
to
less. toxigenicity
phage
mixed
in
The converting 10-7 and the phages
a These
decreased
was
and
the
nm).
cell
finally
Since
method
and
which
formation
converting
thus
through
indicator
host
and
until
cen-
titers.
successively ml
passage.
tube
the
was
1 ml,
after hr
speed
times
filtrate
young
with
6
min)
phage the
the non-
lysates
450
four
each a
to
filtered size
as
of the
low
20
the
logarith-
treated
4
by
(pore
with
incubated
induced
passaged
was
10
test
and were
were
increase
volume
to
Lyso-
lysis
=0.2)
and
rpm,
37
intraperito-
the
(0.D.
clarified
fluids
membrane
The
at
mice.
additional
(4000
supernatant
C-A02
inocu-
supernatant
by
The
a
immedi-
up,
into
C-N71,
were
to
and
by
ml
(1 ƒÊg/m1),
occurred.
obtained
C. in
incubated
C-Stockholm,
an
37
plates.
toxicity
growth
strain,
host
LYG
obtained from C-Stockholm. of phage. Cells in the
toxigenic
filtrates
after
in at
picked
determined
phase
lysis
except
culture
resultant
for
Co.) under
Also
hr
cm
and
0.5
was
phage c-st Preparation
was
were
medium
injecting
genicity
50 agar
The
C
with ultraviolet GX-590G, 15 W)
of
blood
colonies
into
24
a young
a distance
37
above
for of
C.
agar
used
culture
irradiated Co., model
at
ml
37
Kogyo
2 hr.
treated
dish was (National sec
mic
was
incubated
streaked
C
described
2
45
at
at
was
as
and
blood
Hakko
5 ƒÊg/m1
addition,
ately
on
time the
it was
In
ml
(Kyowa
IIDA
medium
1 hr
streaked
H.
min
anaerobically
treatment
medium
20
LYG
additional
of
resuspending
of
an then
a concentration
that
for in
incubated
Mitomycin
the
rpm
resuspended
was
plates and for 2 days.
at
3000
OGUMA,
test.
(=filtrate with
2.5
ml
An
aliquot
of
passaged
of
actively
of
0.5
lysate) growing
AND K.
INOUE
cells (0.D. =0.2). This mixture was incubated at 37 C for 3 to 6 hr and the turbidity of the mixture was assayed using a Hitachi photometer at 520-nm wave length to detect the extent of lysis. At the same time, 0.2 ml of the mixture was transferred from the tube into 10 ml of CM medium and incubated at 37 C for 3 days. The culture fluid supernate was diluted ten times to remove the influence of the toxin which was transferred with the phage, and then 0.5 ml was intraperitoneally injected into mice to detect toxin production. When toxin was demonstrated in a CM tube culture, that culture was plated on blood agar plates and incubated anaerobically at 37 C for 2 days. Several colonies resulting from this plating were isolated and inoculated into fresh CM medium. Toxicity was tested by intraperitoneally injecting 0.5 ml of each supernatant into mice. The conversion rate was determined by the following method. After incubating with phage for 3 hr, the culture was centrifuged at 3000 rpm for 10 min. Immediately after centrifugation the sediment was streaked on blood agar plates and incubated at 37 C for 2 days in an anaerobic jar. Twelve colonies from each plate were isolated at random and incubated for 3 days in CM medium. The ratio of toxic colonies to total colonies tested was calculated and expressed as the conversion rate. Production of anti-c-n71 phage rabbit serum. Each 50 ml of growing cell culture of C-A02 was mixed with 1 ml of passaged lysate, c-n71, and incubated about 4 hr until complete lysis occurred. A total of 1500 ml of lysate thus obtained was centrifuged at 8000 rpm for 30 min. The supernatant was filtered through a membrane filter with a pore size of 450 nm, and then centrifuged at 29 000 rpm for 90 min. The pellet was resuspended in 30 ml of 0.01 M phosphate-buffered saline (PBS, pH 7.2). After dividing this phage suspension into six equal parts, each was used to immunize two rabbits. Three intravenous injections each giving 5 ml of the phage preparation were carried out at 2week intervals. The rabbits were bled on the third day after the last injection and the sera separated were pooled. Electron micrograph of the c-n71 phage. Ninety milliliters of the filtrate was ultra-
NONCONVERTING
Table
1.
Isolation
PHAGE
of nontoxigenic
FROM
strains
from
C. BO TULINUM
C-Stockholm
by
TYPE
various
C
169
treatments
were obtained through treatments with acridine orange [7], nitrosoguanidine, mitomycin C or UV irradiation (Table 1). Three out of six total nontoxigenic colonies obtained by exposure to nitrosoguanidine were found to be lysogenic, because the cells of these three strains were neither lysed nor converted to toxin production by the c-st phage. One of these strains was labeled C-N71 and used in the following experiments.
Fig. 1. Lysis curves with passaged lysates, c-st and c-n71. Two milliliters of a passaged lysate, either c-st or c-n71, or the LYG medium (as control) was added to three separate tubes containing 80 ml of C-A02 culture (0 hr), and incubated at 37 C. Three milliliters was sampled from each culture at 0, 0.5, 1, 1.5, 2, 3, 4 and 5 hr, and the turbidities were determined.
centrifuged (25 000 rpm, 90 min) and the pellet was resuspended in 1 ml of a 0.1 M ammonium acetate solution. This suspension was then stained with 2% phosphotungstic acid with the pH adjusted to 6.8 with KOH. RESULTS
Isolationof Nontoxigenicbut LysogenicStrains Nontoxigenic strains cured of their phages
Lysis Curvesand Lysis Spectra of c-st and c-n71 Phages Lysis curves of c-st and c-n71 phages were obtained by the following method. A total of 30 ml of actively growing C-A02 cells was distributed into three tubes each containing 70 ml of the LYG medium. The first and the second tubes were then mixed with 2 ml of passaged lysates, c-st and c-n71, respectively. The third was mixed with 2 ml of the LYG medium for the control. All tubes were then incubated at 37 C. An aliquot of 3 ml was sampled from each culture after 0.5, 1, 1.5, 2, 3, 4 and 5 hr, and then turbidities were determined. Lysis spectra were obtained by changing the indicator strains in the lysis test described above. The results clearly showed that the lysis curves and the lysis spectra of these phages were identical (Figs. 1, 2). However the nontoxigenic strains which were lysed by the c-st and c-n71 phages, were converted to the toxigenic state only by the c-st phage. Effect of Trypsin in
Trypsin, (2 •~
sterile
0.01
DNase cryst., M
and Heat Treatments Sigma) was dissolved
PBS
concentration
of 200 ƒÊg/ml.
free,
was
Sigma)
also
(pH
8.2)
at
DNase dissolved
a
final
(RNasein
0.01
170
PBS
K.
(pH
7.2)
Phages of
at
were
these
and
twice
the
effects
verting
phages
The
C
same
for
2
phages buffered
these the
the
37
same
of
and
c-n71
at
were
IIDA
hr.
abilities
Heatdiluted
saline
(pH
7.2).
on
the
con-
of
c-st
and
observed.
relationships
between
heat
AND K.
INOUE
and lysis and conversion were not clear in the beginning. For example, with the experiment on heat treatment at 60 C for 30 min, the O.D. value was not different from at 80 C for 10 min (Table 2, expt. I). These cultures were filtered and repeated for the conversion and lysis tests. As shown in Table 2 (expt. II), conversion and lysis were observed after heating the phage c-st at 60 C
volumes
were
treatments lytic
H.
of 40 ƒÊgiml.
with
control
with
The
a concentration
treated
solutions
treated
OGUMA,
treatment
Fig. 2. Lysis test with filtrates of passaged lysates, c-st and c-n71. Five tenths milliliters of either filtrate, c-st or c-n71, or the LYG medium (as control) was mixed separately with 2.5 ml of actively growing cells shown in the figure. The mixture were incubated at 37 C for 3 to 6 hr and the turbidities were determined. Table
2.
Filtrate,
either
200 ƒÊg/ml, then 10
pH
min.
c-st
ment
at
Five at
I was
indicator
or
37
37
II).
C
d,
mixed
(RNase-free, for
2 hr.
separately Sigma,
these
treated
3 hr,
and
the
turbidities
the O.D.
death;
conversion values
and of the
s, survival.
with
filtrates phages
were
were lysis
initial
and
tests
lysis by phages
equal
40 ƒÊg/ml,
Twice-diluted
of
and The
was
on conversion
of each
C for
filtered
treatments
c-n71,
DNase
tenths
strain. (Expt.
of various
8.2),
incubated
incubated
0.20
Effect
volumes
pH were
7.2)
60
C for
with
2.5
ml
of an
turbidities
(Expt. repeated of indicator
I).
Each
(Expt.
II). strain
c-n71
(2 •~
medium
at
mixed
and
of trypsin LYG
heated
determined were
or
c-st
30
culture
min
or
and
80
C
strain fluid
was 0.17
Sigma,
control),
indicator
C-A02 were
cryst.,
(as
for and
of experiused
(Expt.
as I)
the and
NONCONVERTING
PHAGE
tbr 30 min but not observed after heating at 80 C for 10 min. Summing-up, the lytic and converting abilities of these filtrates were not destroyed by trypsin and DNase but partially and completely inactivated by the heat treatments at 60 C for 30 min and 80 C for 10 min, respectively. These findings strongly suggested that the agent which caused lysis was not boticin but a phage [5]. Although we tried to obtain an inactivation curve for the phage by changing the times of treatment at 60 C, a linear line was not obtained. However, it was observed that the degree of lysis and the conversion rate were both decreased by the beat treatment (Table 3).
FROM
C. BOTIILINITM
TYPE
C
171
Electron Microscopy of the c-n71 Phage An electron micrograph of a complete phage which was similar in its morphology to that of the c-st phage was obtained from the induced lysate of strain C-N71. This phage Table
Effect
3.
on
of the heat conversion
treatment and
of phages
lysis
Filtrates, c-st and c-n71, were treated at 60 C for various periods, and then they were mixed with the indicator strain. After incubation at 37 C for 3 hr, the 0.D. values of the mixtures and the conversion rates of the c-st filtrates were determined. The O.D. value of the indicator strain, C-A02, was 0.15 at the beginning of the experiment. Table rabbit
4.
Neutralizing
serum
on
Twofold-diluted was mixed with
Fig. from culture.
3.
Electron a
filtrate
micrograph of
a
Magnification •~255
lysed
of C.
the
phage,
botulinum
type
000.
c-n71, C,
N71
titer
conversion c-st phage
of the and
anti-c-n71 equal volumes
anti-c-n71
lysis by
the
phage scrum of either c-st
or c-n71 phage. After the mixtures were incubated at 37 C for 1 hr, both conversion and lysis tests were carried out. Arrows indicate the final dilutions of the antiserum capable of neutralizing the lytic and the converting activities
of the
phages.
K. OGUMA,
172
exhibited diameter, contained 15 nm in 30 nm in
H. IIDA
a hexagonal head, 100 nm in with a long flexible tail which a tail tube, 350 nm in length and diameter, surrounded by a sheath, diameter (Fig. 3).
Antigenicityof the c-n71 Phage The anti-c-n71 phage rabbit serum was diluted in twofold dilutions with the LYG medium, and mixed with equal volumes of either the c-st or c-n71 phage. Conversion and lysis tests were carried out after the mixtures were incubated at 37 C for 1 hr. As shown in Table 4, anti-c-n71 phage serum neutralized both lytic and converting activities of the c-st phage. DISCUSSION
A nontoxigenic mutant of C. botulinum type C, N71, produced phage c-n71 which had the same lytic activities as the c-st phage but no converting activity. The lytic spectrum of the c-n71 phage seemed to be identical to that of the c-st phage from experiments with various indicator strains. The morphology of the c-n7l phage was similar to that of the c-st phage and anti-c-n71 phage serum neutralized both the lytic and the converting activities of the c-st phage. These findings strongly suggested that the phage c-n71 should be the same as c-st except that it lacked the genetic information required for toxin production. Most of the nontoxigenic strains obtained. from the toxigenic strain by various treatment methods were lysed by the phage from the parent toxigenic strain. However, some nontoxigenic strains such as C-N71 were not lysed by the same phage. From the data described above, we presumed that strain C-N71 was immune to the c-st phage because it had a phage, c-n71, which was closely associated with the c-st phage. Since strain C-N71 is lysogenized by this nonconverting phage, it exhibits immunity to the converting phage c-st. Eklund et al reported that some strains of type C and D cured of their prophages
AND K. INOUE
ceased to produce their dominant toxins but continued to produce low levels of another toxin designated as C2 [1]. We are now studying the production of the C2 toxin using these nontoxigenic strains. In Corynebacteriumdiphtheriae, it was reported that the substance which competed the active site with toxin was produced by the cells infected with the mutant phages [10, 11]. We have not yet obtained a substance in a culture of C-N71 which produces antibody to neutralize the toxin or which competes the active site with the toxin. REFERENCES
[ 1]
Eklund, M.W., and Poysky, F.T. 1972. Activation of a toxic component of C. botulinum types C and D by trypsin. Appl. Microbiol. 24: 108-113. [ 2 ] Eklund, M.W., and Poysky, F.T. 1974. Interconversion of types C and D strains of C. botulinum by specific bacteriophages. Appl. Microbiol. 27: 251-258. [ 3 ] Eklund, M.W., Poysky, F.T., and Reed, S.M. 1972. Bacteriophage and the toxigenicity of C. botulinum type D. Nature (New Biol.) 235: 16-17. [ 4 ] Eklund, M.W., Poysky, F.T., and Smith, C.A. 1971. Bacteriophage and the toxigenicity of C. botulinumtype C. Science 172: 480-482. [ 5 ] Ellison, J.S., and Kautter, T.A. 1970. Purification and some properties of two boticins. J. Bacteriol. 104: 19-26. [ 6 ] Inoue, K., and Iida, H. 1968. Bacteriophages of C. botulinum.J. Virol. 2: 537-540. [ 7 ] Inoue, K., and Iida, H. 1970. Conversion of toxigenicity in C. botulinumtype C. Japan. J. Microbiol. 14: 87-89. [ 8 ] Inoue, K., and Iida, H. 1971. Phage-conversion of toxigenicity in C. botulinum types C and D. Japan. J. Med. Sci. Biol. 24: 53-56. [ 9 ] Oguma, K., Iida, H., and Inoue, K. 1973. Bacteriophage and toxigenicity in C. botulinum: An additional evidence for phage conversion. Japan. J. Microbiol. 17: 425-426. [10] Uchida, T., Gill, D.M., and Pappenheimer, A.M., Jr. 1971. Mutation in the structural gene for diphtheria toxin carried by temperate phage. Nature (New Biol.) 233: 8-11. [11] Uchida, T., Pappenheimer, A.M., Jr., and Harper, A.A. 1972. Reconstitution of diphtheria toxinfrom nontoxic cross-reacting mutant proteins. Science 175: 901-903.
Observations Obtained
on Nonconverting from
Clostridium
Phage,
a Nontoxigenic
c-n71,
Strain
botulinum
Type
of
C
Keiji OGUMA,Hiroo IIDA, and Katsuhiro INOUE Department of Bacteriology, Hokkaido University School of Medicine, Sapporo, andHokkaido Institute ofPublicHealth,Sapporo (Received for publication, July 9, 1974)
ABSTRACT
A nontoxigenic mutant (C-N71) obtained from a toxigenic strain of Clostridium botulinumtype C, Stockholm,with nitrosoguanidine treatment was found to be lysogenic by the lysis test. Although the filtrate of a passaged lysate of this nontoxigenic but lysogenic strain, C-N71, lysed cells of the nontoxigenic strain C-AO2 equally as well as the converting phage c-st obtained from the strain C-Stockholm,it did not convert C-AO2 to the toxigenic state. The lysis spectrum of this filtrate was the same as that of the c-st phage. The ability of the filtrate to lyse the indicator cells, C-AO2, was destroyed neither by trypsin nor DNase but was inactivated by heat treatment at 80 C for 10 min. This suggested that the agent which caused lysis was not boticin but probably a phage. An electron micrograph of the complete phage, c-n71, which was similar in morphology to that of the c-st phage was obtained from the filtrate of strain C-N71. Anti-c-n71 phage rabbit serum neutralized both the lytic and the converting activities of the c-st phage. These findings strongly suggest that the c-n71 phage is a mutant of the c-st phage which lacks the gene controlling production of botulinum type C toxin.
The authors were the first to suggest the phage conversion of toxigenicity in Clostridium botulinumtypes C and D [7-9]. Recently, Eklund et al verified and extended this by using purified phages from plaques [2-4]. In these experiments, all of the nontoxigenic strains which were converted to the toxigenic state by infection of specific phages were cured strains. Various methods were attempted to get nontoxigenic strains from the toxigenic strain C-Stockholm. Nontoxigenic mutants which retained their lysogenicities were isolated with nitrosoguanidine treatment. A complete phage, named c-n71, was found in the induced lysate of one of these strains. The results of comparative experiments on
c-n71
phage
this
and
c-st
MATERIALS Strains D
and
strains
type
are
described
6814,
151.
The
viously
[9].
strains
were
pH
type these
0.5%,
7.0) Isolations
in
transferred 1 %, yeast
cysteine
when
D,
203U-28, strains
strains
stored
medium and (lactalbumin
glucose
type
468U-31,
and of
reported
These (CM) medium
6816
added
167
to
at
a
to
incubate
C and
Toxigenic
Nontoxigenic
468U-16,
origin
types
follows:
Stockholm.
AO2,
6813,
as
139,
were
cooked into extract
hydrochloride
pre-
meat LYG 2%, 0.14%
required.
of
nontoxigenic
but
lysogenic
N-Methyl-N'-nitro-N-nitrosoguanidine
Requests for reprints should be addressed to Dr. Keiji Oguma, Department of Bacteriology, Hokkaido University School of Medicine, Nishi 7-chome, Kita 15, Kita-ku, Sapporo 060, Japan.
in
METHODS
C. botulinum
were
strain
strains
AND
media.
used
C,
C,
phage
report.
a young
concentration at
37
was
culture
of
of
500 ƒÊgirril
C for
mutants.
10
strain
min..
C-Stockholm and The
allowed culture
:
168
was
K.
centrifuged
the
pellet
at
was
and
incubated
The
culture
for
same the
C
procedure
petri light for
on
Isolated lated
CM
3
days.
for
fluid
was
neally
tested
of
toxigenic
strain,
mitomycin
C
treatment
for
trifugation
filter were
used of
ml
(0.D.
of
= 0.3)
2 ml
to
successive ceeded the
0.5
in plaque test
passaged c-st phage c-st
1000
phages. was about
and times
Lysis of
the
and
c-n71
a suitable Strain organism. was
added
suspension
0.2
we with used lytic
were
ml
had these
from at
each
not
suc-
phages,
to
determine
titers
of
these
titer of lytic titers
always
10
to
less. toxigenicity
phage
mixed
in
The converting 10-7 and the phages
a These
decreased
was
and
the
nm).
cell
finally
Since
method
and
which
formation
converting
thus
through
indicator
host
and
until
cen-
titers.
successively ml
passage.
tube
the
was
1 ml,
after hr
speed
times
filtrate
young
with
6
min)
phage the
the non-
lysates
450
four
each a
to
filtered size
as
of the
low
20
the
logarith-
treated
4
by
(pore
with
incubated
induced
passaged
was
10
test
and were
were
increase
volume
to
Lyso-
lysis
=0.2)
and
rpm,
37
intraperito-
the
(0.D.
clarified
fluids
membrane
The
at
mice.
additional
(4000
supernatant
C-A02
inocu-
supernatant
by
The
a
immedi-
up,
into
C-N71,
were
to
and
by
ml
(1 ƒÊg/m1),
occurred.
obtained
C. in
incubated
C-Stockholm,
an
37
plates.
toxicity
growth
strain,
host
LYG
obtained from C-Stockholm. of phage. Cells in the
toxigenic
filtrates
after
in at
picked
determined
phase
lysis
except
culture
resultant
for
Co.) under
Also
hr
cm
and
0.5
was
phage c-st Preparation
was
were
medium
injecting
genicity
50 agar
The
C
with ultraviolet GX-590G, 15 W)
of
blood
colonies
into
24
a young
a distance
37
above
for of
C.
agar
used
culture
irradiated Co., model
at
ml
37
Kogyo
2 hr.
treated
dish was (National sec
mic
was
incubated
streaked
C
described
2
45
at
at
was
as
and
blood
Hakko
5 ƒÊg/m1
addition,
ately
on
time the
it was
In
ml
(Kyowa
IIDA
medium
1 hr
streaked
H.
min
anaerobically
treatment
medium
20
LYG
additional
of
resuspending
of
an then
a concentration
that
for in
incubated
Mitomycin
the
rpm
resuspended
was
plates and for 2 days.
at
3000
OGUMA,
test.
(=filtrate with
2.5
ml
An
aliquot
of
passaged
of
actively
of
0.5
lysate) growing
AND K.
INOUE
cells (0.D. =0.2). This mixture was incubated at 37 C for 3 to 6 hr and the turbidity of the mixture was assayed using a Hitachi photometer at 520-nm wave length to detect the extent of lysis. At the same time, 0.2 ml of the mixture was transferred from the tube into 10 ml of CM medium and incubated at 37 C for 3 days. The culture fluid supernate was diluted ten times to remove the influence of the toxin which was transferred with the phage, and then 0.5 ml was intraperitoneally injected into mice to detect toxin production. When toxin was demonstrated in a CM tube culture, that culture was plated on blood agar plates and incubated anaerobically at 37 C for 2 days. Several colonies resulting from this plating were isolated and inoculated into fresh CM medium. Toxicity was tested by intraperitoneally injecting 0.5 ml of each supernatant into mice. The conversion rate was determined by the following method. After incubating with phage for 3 hr, the culture was centrifuged at 3000 rpm for 10 min. Immediately after centrifugation the sediment was streaked on blood agar plates and incubated at 37 C for 2 days in an anaerobic jar. Twelve colonies from each plate were isolated at random and incubated for 3 days in CM medium. The ratio of toxic colonies to total colonies tested was calculated and expressed as the conversion rate. Production of anti-c-n71 phage rabbit serum. Each 50 ml of growing cell culture of C-A02 was mixed with 1 ml of passaged lysate, c-n71, and incubated about 4 hr until complete lysis occurred. A total of 1500 ml of lysate thus obtained was centrifuged at 8000 rpm for 30 min. The supernatant was filtered through a membrane filter with a pore size of 450 nm, and then centrifuged at 29 000 rpm for 90 min. The pellet was resuspended in 30 ml of 0.01 M phosphate-buffered saline (PBS, pH 7.2). After dividing this phage suspension into six equal parts, each was used to immunize two rabbits. Three intravenous injections each giving 5 ml of the phage preparation were carried out at 2week intervals. The rabbits were bled on the third day after the last injection and the sera separated were pooled. Electron micrograph of the c-n71 phage. Ninety milliliters of the filtrate was ultra-
NONCONVERTING
Table
1.
Isolation
PHAGE
of nontoxigenic
FROM
strains
from
C. BO TULINUM
C-Stockholm
by
TYPE
various
C
169
treatments
were obtained through treatments with acridine orange [7], nitrosoguanidine, mitomycin C or UV irradiation (Table 1). Three out of six total nontoxigenic colonies obtained by exposure to nitrosoguanidine were found to be lysogenic, because the cells of these three strains were neither lysed nor converted to toxin production by the c-st phage. One of these strains was labeled C-N71 and used in the following experiments.
Fig. 1. Lysis curves with passaged lysates, c-st and c-n71. Two milliliters of a passaged lysate, either c-st or c-n71, or the LYG medium (as control) was added to three separate tubes containing 80 ml of C-A02 culture (0 hr), and incubated at 37 C. Three milliliters was sampled from each culture at 0, 0.5, 1, 1.5, 2, 3, 4 and 5 hr, and the turbidities were determined.
centrifuged (25 000 rpm, 90 min) and the pellet was resuspended in 1 ml of a 0.1 M ammonium acetate solution. This suspension was then stained with 2% phosphotungstic acid with the pH adjusted to 6.8 with KOH. RESULTS
Isolationof Nontoxigenicbut LysogenicStrains Nontoxigenic strains cured of their phages
Lysis Curvesand Lysis Spectra of c-st and c-n71 Phages Lysis curves of c-st and c-n71 phages were obtained by the following method. A total of 30 ml of actively growing C-A02 cells was distributed into three tubes each containing 70 ml of the LYG medium. The first and the second tubes were then mixed with 2 ml of passaged lysates, c-st and c-n71, respectively. The third was mixed with 2 ml of the LYG medium for the control. All tubes were then incubated at 37 C. An aliquot of 3 ml was sampled from each culture after 0.5, 1, 1.5, 2, 3, 4 and 5 hr, and then turbidities were determined. Lysis spectra were obtained by changing the indicator strains in the lysis test described above. The results clearly showed that the lysis curves and the lysis spectra of these phages were identical (Figs. 1, 2). However the nontoxigenic strains which were lysed by the c-st and c-n71 phages, were converted to the toxigenic state only by the c-st phage. Effect of Trypsin in
Trypsin, (2 •~
sterile
0.01
DNase cryst., M
and Heat Treatments Sigma) was dissolved
PBS
concentration
of 200 ƒÊg/ml.
free,
was
Sigma)
also
(pH
8.2)
at
DNase dissolved
a
final
(RNasein
0.01
170
PBS
K.
(pH
7.2)
Phages of
at
were
these
and
twice
the
effects
verting
phages
The
C
same
for
2
phages buffered
these the
the
37
same
of
and
c-n71
at
were
IIDA
hr.
abilities
Heatdiluted
saline
(pH
7.2).
on
the
con-
of
c-st
and
observed.
relationships
between
heat
AND K.
INOUE
and lysis and conversion were not clear in the beginning. For example, with the experiment on heat treatment at 60 C for 30 min, the O.D. value was not different from at 80 C for 10 min (Table 2, expt. I). These cultures were filtered and repeated for the conversion and lysis tests. As shown in Table 2 (expt. II), conversion and lysis were observed after heating the phage c-st at 60 C
volumes
were
treatments lytic
H.
of 40 ƒÊgiml.
with
control
with
The
a concentration
treated
solutions
treated
OGUMA,
treatment
Fig. 2. Lysis test with filtrates of passaged lysates, c-st and c-n71. Five tenths milliliters of either filtrate, c-st or c-n71, or the LYG medium (as control) was mixed separately with 2.5 ml of actively growing cells shown in the figure. The mixture were incubated at 37 C for 3 to 6 hr and the turbidities were determined. Table
2.
Filtrate,
either
200 ƒÊg/ml, then 10
pH
min.
c-st
ment
at
Five at
I was
indicator
or
37
37
II).
C
d,
mixed
(RNase-free, for
2 hr.
separately Sigma,
these
treated
3 hr,
and
the
turbidities
the O.D.
death;
conversion values
and of the
s, survival.
with
filtrates phages
were
were lysis
initial
and
tests
lysis by phages
equal
40 ƒÊg/ml,
Twice-diluted
of
and The
was
on conversion
of each
C for
filtered
treatments
c-n71,
DNase
tenths
strain. (Expt.
of various
8.2),
incubated
incubated
0.20
Effect
volumes
pH were
7.2)
60
C for
with
2.5
ml
of an
turbidities
(Expt. repeated of indicator
I).
Each
(Expt.
II). strain
c-n71
(2 •~
medium
at
mixed
and
of trypsin LYG
heated
determined were
or
c-st
30
culture
min
or
and
80
C
strain fluid
was 0.17
Sigma,
control),
indicator
C-A02 were
cryst.,
(as
for and
of experiused
(Expt.
as I)
the and
NONCONVERTING
PHAGE
tbr 30 min but not observed after heating at 80 C for 10 min. Summing-up, the lytic and converting abilities of these filtrates were not destroyed by trypsin and DNase but partially and completely inactivated by the heat treatments at 60 C for 30 min and 80 C for 10 min, respectively. These findings strongly suggested that the agent which caused lysis was not boticin but a phage [5]. Although we tried to obtain an inactivation curve for the phage by changing the times of treatment at 60 C, a linear line was not obtained. However, it was observed that the degree of lysis and the conversion rate were both decreased by the beat treatment (Table 3).
FROM
C. BOTIILINITM
TYPE
C
171
Electron Microscopy of the c-n71 Phage An electron micrograph of a complete phage which was similar in its morphology to that of the c-st phage was obtained from the induced lysate of strain C-N71. This phage Table
Effect
3.
on
of the heat conversion
treatment and
of phages
lysis
Filtrates, c-st and c-n71, were treated at 60 C for various periods, and then they were mixed with the indicator strain. After incubation at 37 C for 3 hr, the 0.D. values of the mixtures and the conversion rates of the c-st filtrates were determined. The O.D. value of the indicator strain, C-A02, was 0.15 at the beginning of the experiment. Table rabbit
4.
Neutralizing
serum
on
Twofold-diluted was mixed with
Fig. from culture.
3.
Electron a
filtrate
micrograph of
a
Magnification •~255
lysed
of C.
the
phage,
botulinum
type
000.
c-n71, C,
N71
titer
conversion c-st phage
of the and
anti-c-n71 equal volumes
anti-c-n71
lysis by
the
phage scrum of either c-st
or c-n71 phage. After the mixtures were incubated at 37 C for 1 hr, both conversion and lysis tests were carried out. Arrows indicate the final dilutions of the antiserum capable of neutralizing the lytic and the converting activities
of the
phages.
K. OGUMA,
172
exhibited diameter, contained 15 nm in 30 nm in
H. IIDA
a hexagonal head, 100 nm in with a long flexible tail which a tail tube, 350 nm in length and diameter, surrounded by a sheath, diameter (Fig. 3).
Antigenicityof the c-n71 Phage The anti-c-n71 phage rabbit serum was diluted in twofold dilutions with the LYG medium, and mixed with equal volumes of either the c-st or c-n71 phage. Conversion and lysis tests were carried out after the mixtures were incubated at 37 C for 1 hr. As shown in Table 4, anti-c-n71 phage serum neutralized both lytic and converting activities of the c-st phage. DISCUSSION
A nontoxigenic mutant of C. botulinum type C, N71, produced phage c-n71 which had the same lytic activities as the c-st phage but no converting activity. The lytic spectrum of the c-n71 phage seemed to be identical to that of the c-st phage from experiments with various indicator strains. The morphology of the c-n7l phage was similar to that of the c-st phage and anti-c-n71 phage serum neutralized both the lytic and the converting activities of the c-st phage. These findings strongly suggested that the phage c-n71 should be the same as c-st except that it lacked the genetic information required for toxin production. Most of the nontoxigenic strains obtained. from the toxigenic strain by various treatment methods were lysed by the phage from the parent toxigenic strain. However, some nontoxigenic strains such as C-N71 were not lysed by the same phage. From the data described above, we presumed that strain C-N71 was immune to the c-st phage because it had a phage, c-n71, which was closely associated with the c-st phage. Since strain C-N71 is lysogenized by this nonconverting phage, it exhibits immunity to the converting phage c-st. Eklund et al reported that some strains of type C and D cured of their prophages
AND K. INOUE
ceased to produce their dominant toxins but continued to produce low levels of another toxin designated as C2 [1]. We are now studying the production of the C2 toxin using these nontoxigenic strains. In Corynebacteriumdiphtheriae, it was reported that the substance which competed the active site with toxin was produced by the cells infected with the mutant phages [10, 11]. We have not yet obtained a substance in a culture of C-N71 which produces antibody to neutralize the toxin or which competes the active site with the toxin. REFERENCES
[ 1]
Eklund, M.W., and Poysky, F.T. 1972. Activation of a toxic component of C. botulinum types C and D by trypsin. Appl. Microbiol. 24: 108-113. [ 2 ] Eklund, M.W., and Poysky, F.T. 1974. Interconversion of types C and D strains of C. botulinum by specific bacteriophages. Appl. Microbiol. 27: 251-258. [ 3 ] Eklund, M.W., Poysky, F.T., and Reed, S.M. 1972. Bacteriophage and the toxigenicity of C. botulinum type D. Nature (New Biol.) 235: 16-17. [ 4 ] Eklund, M.W., Poysky, F.T., and Smith, C.A. 1971. Bacteriophage and the toxigenicity of C. botulinumtype C. Science 172: 480-482. [ 5 ] Ellison, J.S., and Kautter, T.A. 1970. Purification and some properties of two boticins. J. Bacteriol. 104: 19-26. [ 6 ] Inoue, K., and Iida, H. 1968. Bacteriophages of C. botulinum.J. Virol. 2: 537-540. [ 7 ] Inoue, K., and Iida, H. 1970. Conversion of toxigenicity in C. botulinumtype C. Japan. J. Microbiol. 14: 87-89. [ 8 ] Inoue, K., and Iida, H. 1971. Phage-conversion of toxigenicity in C. botulinum types C and D. Japan. J. Med. Sci. Biol. 24: 53-56. [ 9 ] Oguma, K., Iida, H., and Inoue, K. 1973. Bacteriophage and toxigenicity in C. botulinum: An additional evidence for phage conversion. Japan. J. Microbiol. 17: 425-426. [10] Uchida, T., Gill, D.M., and Pappenheimer, A.M., Jr. 1971. Mutation in the structural gene for diphtheria toxin carried by temperate phage. Nature (New Biol.) 233: 8-11. [11] Uchida, T., Pappenheimer, A.M., Jr., and Harper, A.A. 1972. Reconstitution of diphtheria toxinfrom nontoxic cross-reacting mutant proteins. Science 175: 901-903.
