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Factors inducing mesophilic bacteria to grow at 55°C
Vol. 6, No. 3, 1961
BIOCHEMICAL
FACTORS
Department
INDUCING
MESOPHILIC
Received Organisms range Davis,
been
1952;
mesophilic
to grow shown
to require
was
Brooklyn,
and Sobotka,
culture
of several
able
AT
55”C.*
New
York
New Jersey
to grow
Further
as well
studies
when
factor(s)
of mesophilic
yeast
revealed
(Maas that
from
supported
of
(Albimi
medium several
and
a strain
autolysate
that
fluid
which
biothermal
shown
to the culture
as the supernatant
contained
strains
added
their
supplements
we have
at 55°C.
was
than
growth
Recently,
York)
1961).
also
other
City,
higher
additional
1957).
New
preparations
bacterial
at temperatures
et al.,
bacteria
mercial
H. Baker of Medicine, Jersey
College
Hutner
Laboratory,
TO GROW
8, 1961
able
have
Baker
Hall
September
BACTERIA
E. H. C. Sie and H. Sobotka The Mount Sinai Hospital, New York,
of Chemistry,
Seton
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
(Sie, other
com-
a thermophilic grwoth
at 55°C.
bacteria.
Methods To obtain
the supernatant,
B-194,
was
grown
defined
medium
hydrolysate.
aeration et al.,
used
as inoculum.
at 50,000
to one liter
r. p. m.
To test
for 5 ml.
work
The
of test
ml.
portions
Fernbach
flasks;
suspension
was
is supported
G-17492, 205
1 percent
of a 16 hour
casein
culture
by a refrigerated
autoclaved factor(s)
consisting
by grant
with
in a chemically
of the thermophikmedium
clarified was
stearothermophilus
7-9 hours
one ml.
of temperature
medium,
for
supplemented
and the supernatant
the activity
Bacillus
at 55°C.
1953),
Three-hundred-fifty
added
*his
without (Baker
were
natant,
the thermophile,
the same
National
centrifuge day.
in the thermophile
of supernatant
and base, Science
was
superpH 6. 5,was
Foundation.
Vol. 6, No. 3‘1961 added for
BIOCHEMICAL
to 10 ml.
30 minutes
water
Fernbach
flasks,
at 16 psi.
They
suspension
were
carried
medium
et. were
were
1953).
Strains
maintained
Bacillus
strains
alvei,
bacteria
agar
7068;
B.
megatherium,
B.
subtilis,
B.
sphaericus,
coli
9637.
58;
medium
autolysate, philes 0.5 plus
7-hour
at 55°C. percent
proteose
a combination
equalled
or surpassed
B.
is directly
addition
of thermophile
M.
B.
(Baker study
in four
In yeast
extract
at 55°C.
with
when (Table
I).
thermo-
Unlike
yeast
the growth beef
good overnight
Growth
the growth of yeast
206
I).
of meso-
extract
or thermophile peptone,
RA-91,
regular
percent
to the concentration
concentrations
sphaericus,
(Table
incubated
70544;
- B. megatherium
but not with
0.5
842;
and Rscherichia
and B.
autolysate
increases
sphaericus,
did not support
and proteose
media,
B.
strains
7067;
B. macerans,
B. polymyxa,
Allen);
peptone
supplemented
proportional
at low
less;
supernatant
supernatant
medium
as
study:
7064;
(lysogenic);
subtilis
thermophile
that
probe
in this
B. cereus’,
observed
and proteose
In the same
at 55”C.,
Dr.
extract
of beef
6 days.
pronounced
was
less;
being
units
the present
included
subtilis-B2
from
peptone.
required
most
(obtained
without
for
basal
density
a red-sensitive
were 604;
B.
or thermophile
or beef
in optical
megatherium
7067;
subtilis-Ba
autolysate
B.
growth
Assays
and Discussion
B. brevis,
subtilis
RA-91
B.
yeast
phile
B.
at 55°C. and inoculated
selected
bacteria
7056;
At 55°C.)
(lysogenic); with
662;
of a distilled
slants.
of mesophilic
(ATCC)
recorded with
and autoclaved
one drop
uninoculated
equipped
Results Thirteen
was
of mesophilic
on nutrient
with
caps,
and incubated
with
Growth
Densichron,
aluminum
inoculated
Flasks
as controls.
with
bacteria,
out in triplicate.
by a Welch
al.,
covered
of the mesophilic
served
measured
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
supernatant growth
at 55°C.
at 37°C.)
of these
four
of yeast
autolyeate
response.
The
autolysate
and
growth
mesophiles,
effect
(i. e. 1 percent);
alone; is growth
Vol. 6, No. 3,196l
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
TABLEI
r
GIiOWTIiOFMFZ3OPHILICBACTERIAAT 37' C. Am 55%. IN THEHMOPHILE SIJP%,NATAEJT AhD IN YEASTAUTOLYSATE f
Growth1 of
7 hr. supenatant (v/v) (in percent)
J+ eubtllisa
lh beef extract l protease peptone 0
;iliS zt
B. subt TAe
37’c. 15 hrs.
0.08
0.42
0.10
0.26
1.x)
0.28
0.95
0.17
1.18
0.66
1.28
I 0.59
1.07
0.68
0.95
0.85
1.43
0.44
1.27
0.80
1.22
0.47
1.10
0.18
1.37
o.l.4
0.94
0.12
1.28
Ob33
1.44
0.43
1.66
0.15
1.60
0.28
1.63
0.73
1.56
0.86
1.60
0.52
2.35
1.25
1.78
1.lJ.l
1.55
0.00
0.003
0.00
0.003
0.00
1.75
0.00
0.003
0.9
0.68
0.60
2.10
0.44
2.80
0.55
1.91
0.66
1.20
0.82
2.29
0.62
3.30
0.75
2.13
1.21
2.30
1.21
2.71
1.76
3.40
1.28
2.&S
0.10
0.22
0.08
0.26
0.08
0.42
0.10
0.26
0.38
1.60
0.24
1.22
0.61
2.00
0.47
0.52
lLJ.4
1.70
1.94
1.33
0.10
0.26
10
0.25
25
50
b
.08
0.26
Yeast autolysati
In thermophil medium 0
Inbeefextract + Pro*oaepeptone
' Growth was recorded in optical density units. 2 A vitamin B, requiring strain. 3 Good growth (O.D. above 1,s) wz.s observed after
207
48 hours of incubation.
Vol. 6, No. 3,196l is also
increased
growth
previously
tone.
No growth
occurs
or thermophilic At 55°C. grow
in liver
factor(s)
autoclaving.
showed
much
Enzymatic and its trypein,
plus
beef
at 37X.
chymotrypsin isolation
The
authors
extract
(10e4
its effect
to
on the
of
autolysate
or by
and proteoee
containing
no yeast
and proteose and B.
hydrolysate,
autolysate
peptone.
subtilis,
suggesting
pep-
7067,
that
can also
the temperature
substances.
in the yeast
autolysate
by 2 volumes (1 N HCl, (Table
is dialyzable of EtOH
autoclaved
and stable
at 4°C.
15 minutes,
in the yeast
with
of either
crystalline
or papain
to acknowledge
Moreover,
II). factor(s)
0.1
to
16 psi)
of the temperature
and identification
wish
spermine
thermophily
of yeast
beef
extract
(lysogenic)
activity
hydrolysis
to yeast
did not maintain
1961),
by addition
in media
autolsate
greater
dialysate
The
and Mr.
yeast
that
peptone
studied
Spermine
in subcultures
It is not precipitable
hydrolyzed
we have
with
in many
factor(s)
proteoae
(1961)
and Sobotka,
possible
or yeast
may be present
Temperature ,
Baker
megatherium
extract
inactivation,
in combination
supernatant , B.
by Tabor
temperature.
is made
supernatant
plus
temperature.
(Sie,
organisms
thermophile
heat
at high
extract
finding
at high
of mesophiles
mesophilic
acid
from
of mesophiles
As shown
of beef
of the recent
M) stabilized.DNA
the growth
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
by the addition
In view
autolyeate. lo-?
BIOCHEMICAL
mg. /ml.
does
not alter
of the factor(s)
the technical
P. Shield
208
autolysate pepsin,
its effectiveness. is underway.
assistance
of Mrs.
H. Harvey
BIOCHEMICAL
Vol. 6, No. 3,196l
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
TABLE II GROW!N OF MESOpHllJC BACTERIA AT 37%
AMI AT s5.C. IN 2 PERCENT YXAST AU'IULYSATE l’¶IAT WASTRJUTEII Ih’ VARIOUS WAYS, AND IN DIFEEREXT BASA.L MEDIA
T
hmrtll 1erium
of B. subtilia
w
Treatment
Base
6 days
15 hour8
WC. 6 daya
15 hour8
H90
0.04
1.42
0.20
1.55
B.P.l
0.10
1.49
0.9
1.20
R.T.M:
0.66
1.10
0.89
1.10
W
0.70
1.n
0.98
2.45
B.P.
0.85
1.39
1.20
2.50
R.T.M.
1.52
1.49
1.58
2.60
*a0
0.10
1.40
0.48
1.70
B.P.
al4
1.3h
0.48
0.80
R.T.Y.
0.62
2.20
1.12
1.9
W
0.42
0.80
0.38
1.20
B.P.
0.12
1.u)
0.42
1.20
R.TY.
1.02
1.99
L42
1.35
None
Acid hydralyzed and neutralized
Concentrated dialyeate (48 hour8 against 10 volumes of H,O under continuous stirring at L’ C.)
Concentrated dialpate, acid hydrolyzed and neutralized
ssc.
37-c.
37%
l Soof extract + protease pePton z Regular thenophile medium. References Baker,
H.,
Sobotka,
H.,
Hutner, S.H., Baker, H., Lockwood, S., Sanders,
Maas, Sie, Tabor,
W. K. , and Davis, E.H.
C.,
Baker,
H. t Biochem.
H.,
and Hutner,
S.H.,
J. Gen. Microbiol.9, 485 (1953F
Aaronson, S., Nathan, H.A., Rodriguez, E., M., and Petersen, R. A., J. Protosool 4,259 (1957). B. D.,
Proc.
Sobotka,
Biophys.
Natl.
H. ) Nature
Res.
209
Comm.
Acad.
Sci. 2,785
(1961). 4,228
In press.
(1961).
(1952).
BIOCHEMICAL
FACTORS
Department
INDUCING
MESOPHILIC
Received Organisms range Davis,
been
1952;
mesophilic
to grow shown
to require
was
Brooklyn,
and Sobotka,
culture
of several
able
AT
55”C.*
New
York
New Jersey
to grow
Further
as well
studies
when
factor(s)
of mesophilic
yeast
revealed
(Maas that
from
supported
of
(Albimi
medium several
and
a strain
autolysate
that
fluid
which
biothermal
shown
to the culture
as the supernatant
contained
strains
added
their
supplements
we have
at 55°C.
was
than
growth
Recently,
York)
1961).
also
other
City,
higher
additional
1957).
New
preparations
bacterial
at temperatures
et al.,
bacteria
mercial
H. Baker of Medicine, Jersey
College
Hutner
Laboratory,
TO GROW
8, 1961
able
have
Baker
Hall
September
BACTERIA
E. H. C. Sie and H. Sobotka The Mount Sinai Hospital, New York,
of Chemistry,
Seton
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
(Sie, other
com-
a thermophilic grwoth
at 55°C.
bacteria.
Methods To obtain
the supernatant,
B-194,
was
grown
defined
medium
hydrolysate.
aeration et al.,
used
as inoculum.
at 50,000
to one liter
r. p. m.
To test
for 5 ml.
work
The
of test
ml.
portions
Fernbach
flasks;
suspension
was
is supported
G-17492, 205
1 percent
of a 16 hour
casein
culture
by a refrigerated
autoclaved factor(s)
consisting
by grant
with
in a chemically
of the thermophikmedium
clarified was
stearothermophilus
7-9 hours
one ml.
of temperature
medium,
for
supplemented
and the supernatant
the activity
Bacillus
at 55°C.
1953),
Three-hundred-fifty
added
*his
without (Baker
were
natant,
the thermophile,
the same
National
centrifuge day.
in the thermophile
of supernatant
and base, Science
was
superpH 6. 5,was
Foundation.
Vol. 6, No. 3‘1961 added for
BIOCHEMICAL
to 10 ml.
30 minutes
water
Fernbach
flasks,
at 16 psi.
They
suspension
were
carried
medium
et. were
were
1953).
Strains
maintained
Bacillus
strains
alvei,
bacteria
agar
7068;
B.
megatherium,
B.
subtilis,
B.
sphaericus,
coli
9637.
58;
medium
autolysate, philes 0.5 plus
7-hour
at 55°C. percent
proteose
a combination
equalled
or surpassed
B.
is directly
addition
of thermophile
M.
B.
(Baker study
in four
In yeast
extract
at 55°C.
with
when (Table
I).
thermo-
Unlike
yeast
the growth beef
good overnight
Growth
the growth of yeast
206
I).
of meso-
extract
or thermophile peptone,
RA-91,
regular
percent
to the concentration
concentrations
sphaericus,
(Table
incubated
70544;
- B. megatherium
but not with
0.5
842;
and Rscherichia
and B.
autolysate
increases
sphaericus,
did not support
and proteose
media,
B.
strains
7067;
B. macerans,
B. polymyxa,
Allen);
peptone
supplemented
proportional
at low
less;
supernatant
supernatant
medium
as
study:
7064;
(lysogenic);
subtilis
thermophile
that
probe
in this
B. cereus’,
observed
and proteose
In the same
at 55”C.,
Dr.
extract
of beef
6 days.
pronounced
was
less;
being
units
the present
included
subtilis-B2
from
peptone.
required
most
(obtained
without
for
basal
density
a red-sensitive
were 604;
B.
or thermophile
or beef
in optical
megatherium
7067;
subtilis-Ba
autolysate
B.
growth
Assays
and Discussion
B. brevis,
subtilis
RA-91
B.
yeast
phile
B.
at 55°C. and inoculated
selected
bacteria
7056;
At 55°C.)
(lysogenic); with
662;
of a distilled
slants.
of mesophilic
(ATCC)
recorded with
and autoclaved
one drop
uninoculated
equipped
Results Thirteen
was
of mesophilic
on nutrient
with
caps,
and incubated
with
Growth
Densichron,
aluminum
inoculated
Flasks
as controls.
with
bacteria,
out in triplicate.
by a Welch
al.,
covered
of the mesophilic
served
measured
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
supernatant growth
at 55°C.
at 37°C.)
of these
four
of yeast
autolyeate
response.
The
autolysate
and
growth
mesophiles,
effect
(i. e. 1 percent);
alone; is growth
Vol. 6, No. 3,196l
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
TABLEI
r
GIiOWTIiOFMFZ3OPHILICBACTERIAAT 37' C. Am 55%. IN THEHMOPHILE SIJP%,NATAEJT AhD IN YEASTAUTOLYSATE f
Growth1 of
7 hr. supenatant (v/v) (in percent)
J+ eubtllisa
lh beef extract l protease peptone 0
;iliS zt
B. subt TAe
37’c. 15 hrs.
0.08
0.42
0.10
0.26
1.x)
0.28
0.95
0.17
1.18
0.66
1.28
I 0.59
1.07
0.68
0.95
0.85
1.43
0.44
1.27
0.80
1.22
0.47
1.10
0.18
1.37
o.l.4
0.94
0.12
1.28
Ob33
1.44
0.43
1.66
0.15
1.60
0.28
1.63
0.73
1.56
0.86
1.60
0.52
2.35
1.25
1.78
1.lJ.l
1.55
0.00
0.003
0.00
0.003
0.00
1.75
0.00
0.003
0.9
0.68
0.60
2.10
0.44
2.80
0.55
1.91
0.66
1.20
0.82
2.29
0.62
3.30
0.75
2.13
1.21
2.30
1.21
2.71
1.76
3.40
1.28
2.&S
0.10
0.22
0.08
0.26
0.08
0.42
0.10
0.26
0.38
1.60
0.24
1.22
0.61
2.00
0.47
0.52
lLJ.4
1.70
1.94
1.33
0.10
0.26
10
0.25
25
50
b
.08
0.26
Yeast autolysati
In thermophil medium 0
Inbeefextract + Pro*oaepeptone
' Growth was recorded in optical density units. 2 A vitamin B, requiring strain. 3 Good growth (O.D. above 1,s) wz.s observed after
207
48 hours of incubation.
Vol. 6, No. 3,196l is also
increased
growth
previously
tone.
No growth
occurs
or thermophilic At 55°C. grow
in liver
factor(s)
autoclaving.
showed
much
Enzymatic and its trypein,
plus
beef
at 37X.
chymotrypsin isolation
The
authors
extract
(10e4
its effect
to
on the
of
autolysate
or by
and proteoee
containing
no yeast
and proteose and B.
hydrolysate,
autolysate
peptone.
subtilis,
suggesting
pep-
7067,
that
can also
the temperature
substances.
in the yeast
autolysate
by 2 volumes (1 N HCl, (Table
is dialyzable of EtOH
autoclaved
and stable
at 4°C.
15 minutes,
in the yeast
with
of either
crystalline
or papain
to acknowledge
Moreover,
II). factor(s)
0.1
to
16 psi)
of the temperature
and identification
wish
spermine
thermophily
of yeast
beef
extract
(lysogenic)
activity
hydrolysis
to yeast
did not maintain
1961),
by addition
in media
autolsate
greater
dialysate
The
and Mr.
yeast
that
peptone
studied
Spermine
in subcultures
It is not precipitable
hydrolyzed
we have
with
in many
factor(s)
proteoae
(1961)
and Sobotka,
possible
or yeast
may be present
Temperature ,
Baker
megatherium
extract
inactivation,
in combination
supernatant , B.
by Tabor
temperature.
is made
supernatant
plus
temperature.
(Sie,
organisms
thermophile
heat
at high
extract
finding
at high
of mesophiles
mesophilic
acid
from
of mesophiles
As shown
of beef
of the recent
M) stabilized.DNA
the growth
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
by the addition
In view
autolyeate. lo-?
BIOCHEMICAL
mg. /ml.
does
not alter
of the factor(s)
the technical
P. Shield
208
autolysate pepsin,
its effectiveness. is underway.
assistance
of Mrs.
H. Harvey
BIOCHEMICAL
Vol. 6, No. 3,196l
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
TABLE II GROW!N OF MESOpHllJC BACTERIA AT 37%
AMI AT s5.C. IN 2 PERCENT YXAST AU'IULYSATE l’¶IAT WASTRJUTEII Ih’ VARIOUS WAYS, AND IN DIFEEREXT BASA.L MEDIA
T
hmrtll 1erium
of B. subtilia
w
Treatment
Base
6 days
15 hour8
WC. 6 daya
15 hour8
H90
0.04
1.42
0.20
1.55
B.P.l
0.10
1.49
0.9
1.20
R.T.M:
0.66
1.10
0.89
1.10
W
0.70
1.n
0.98
2.45
B.P.
0.85
1.39
1.20
2.50
R.T.M.
1.52
1.49
1.58
2.60
*a0
0.10
1.40
0.48
1.70
B.P.
al4
1.3h
0.48
0.80
R.T.Y.
0.62
2.20
1.12
1.9
W
0.42
0.80
0.38
1.20
B.P.
0.12
1.u)
0.42
1.20
R.TY.
1.02
1.99
L42
1.35
None
Acid hydralyzed and neutralized
Concentrated dialyeate (48 hour8 against 10 volumes of H,O under continuous stirring at L’ C.)
Concentrated dialpate, acid hydrolyzed and neutralized
ssc.
37-c.
37%
l Soof extract + protease pePton z Regular thenophile medium. References Baker,
H.,
Sobotka,
H.,
Hutner, S.H., Baker, H., Lockwood, S., Sanders,
Maas, Sie, Tabor,
W. K. , and Davis, E.H.
C.,
Baker,
H. t Biochem.
H.,
and Hutner,
S.H.,
J. Gen. Microbiol.9, 485 (1953F
Aaronson, S., Nathan, H.A., Rodriguez, E., M., and Petersen, R. A., J. Protosool 4,259 (1957). B. D.,
Proc.
Sobotka,
Biophys.
Natl.
H. ) Nature
Res.
209
Comm.
Acad.
Sci. 2,785
(1961). 4,228
In press.
(1961).
(1952).