BIOCHEMICAL
Vol.71,No.1,1976
BACTERIOPHAGE-P22
AND BIOPHYSICAL
DERIVATIVE
'Katsutoshi The
Public
of
May
HIGH
DENSITY
Health,
Microbiology,
The
Shiroganedai,
Minato-ku,
Tokyo Received
WITH
Mise
Department
of
RESEARCH COMMUNICATIONS
108,
Institute
Japan.
15,1976 Summary
Bacteriophage P22Cm21 was differentiated from the original phage P22. The buoyant of phage P22Cm21 was higher than that of 0.007 g per cm3. The possible biological this higher density is discussed.
in some characters density in CsCl solution phage P22 by as much as implication involved in
Introduction P22Cm were
phages,
isolated
plaque-forming from
Salmonella
typhimurium
ization
at
phages
can
high be
(1). on
usually
The
other
the
indicator 1 - 10
found
among
Mise,
and
Copyright All rights
into
obtained
of
P22Cml
wild-type
P22
strains.
The high,
ml.
of
I an
P22Cm),
Cm, chloramphenicol; plaque-forming
0 1976 by Academic Press, Inc. of reproduction in any form reserved.
312
this
of
very II
tiny
lysates
I
phage
plaques was
P22Cm21
makes
PFU per
differences
not
(Group
so be
II
(Suzuki
and
we describe
resistance
ml. on
could
microscope report,
P22Cm
I P22Cm, Group 11 10
than
Cmr, unit.
PlCm,
physiological Group
makes Group
In
the
more
electron
These
titer
No structual
(Group under
II,
and
on PlCm-lysogenic $ CC& marker by lysogen-
by
designated
observations).
used: PFU,
groups
very
P22
infection.
these,
titer
PFU per
phages
grown
single
Group The
between
the
two
was
designated
x 10'
unpublished
$ Abbreviations chloramphenicol;
upon
One
strains.
lysate
transduced
Salmonella
group,
high,
P22Cm)
and
classified
plaques
lysates
phage
frequencies
characteristics small
a P22
recombinants
that
to
Vol.71,No.1,1976
phage
BIOCHEMICAL
P22Cm21
original
phage
Bacterial as
shows
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
higher
density
Materials
and
in
CsCl
solution
than
the
P22.
strains,
described
bacteriophages,
in
Transduction
Methods
the
previous
generally
media paper
followed
and
chemicals
used
were
(1).
the
method
of
Lennox
(2).
CsCl density-gradiens centrifugation. A O.l-ml aliquot of a P22Cm lysate (about 5 x 10: PFU per ml) and the same amount of a P22 PFU per ml) were suspended in 5.2 ml of lysate (about 2 x 10 CsCl-0.1 M Tris buffer, pH 7.0. The refractive index of the solution was 1.3834. After centrifugation for 18 hr at 30,000 rpm and 5oC in Beckman SW50.1 rotor, about 55 fractions were collected. Each fraction was assayed for plaque-forming activities and for abilities to transduce the Cm marker. Plaque formers in a P22Cm lysate and those in a P22 lysate were differentiated from the form of plaques on the indicator strain LT2 4415. The former made small while the latter did ringed plaques. plaques, Sucrose density-gradient centrifugation. A 0.1 ml tion containing P22Cm21 (3 x 106 PFU per ml) and per ml) were layered on a 5.0-ml gradient of 5 standard saline citrate. After centrifugation of 28 min at 20,000 rpm and 15'C, 77 fractions were the botton of a tube and assayed for plaque-forming and Cmr-transducing activities.
of phage sflluP22 (1 x 10 PFU 20 % sucrose in the gradient for collected from activities
Results In
spite
phages
had
ed
to
the
P22Cm21 The
of several
made
and
Group
I phage
temperature
a P22Cm21
particles
are
For
or
P22Cm1,
P22Cm21
the
phages.
phage
Phage
suggest
more
of
1).
rate
were
period of
higher
Group
a Salmonella -
those
much may
latent
on
than
(Fig.
results
defective
plaques
the
longer
which
of tiny
and
at
activities,
characteristics
very
size
smaller
of
plaque-forming
characteristics
burst
These
their
P22
than
was
possibility
somewhat
P22 that
different
P22Cm
less
relat-
example,
phage
strain
4415.
P22Cm21
were
much
a representative
inactivated
phage
or
II
or the from
at P22Cml
a high (Fig.
2).
constituents those
of
a P22
particle. To P22Cm21
demonstrate was
submitted
this
possibility, to
CsCl
each density-gradient
313
of
phages
P22Cml
centrifugation
and in
Vol. 71, No. 1,1976
A
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
P22 P22Cm21 P22Cml
*a
A
0
0
Time of incubahon (min)
reference
phage
Cmr-transducing
phage
lysate
P22Cm1,
than
that Phage
gradient. phage
the
with
density
same of
phage
P22Cm21 As
P22Cm21
results;
shown
P22 was in
sedimented
of
phage was
Fig.
3,
the phage
P22.
P22Cm21 higher
the
of
- 0.008
centrifuged 5,
the
slightly
in
314
of
contrast
phage
to
of
with
cm3.
the
5 - 20
phage
obtain
P22Cm21
g per
than
in
either
that
times
the
ones
particles,
plaque-forming faster
density
In
than
four
density
the
plaque-forming
experiments
by 0.006
then
Fig.
as
that of
the
in
well
plaque-forming,
We repeated the
of phages P22, P22Cml used was Salmonella typhi-
As shown as
agreed
or
4).
stently
P22.
particles
transducing (Fig.
curves strain
Heat inactivation of phages P22, P22Cml and Phages were diluted loo-fold in prewarmed 0.01 buffer, pH 7.4, and incubated at 73.513. Left, ) and P22Cml( o ); right, P22( o ) and P22Cm21 Strain 4415 was used as the indicator for titraplaque-forming particles.
to
a P22Cml
30 60 90120
Time of incubation (min)
0
Fig 1. One step growth and P22Cm21. The host murium 4415. Fig 2. P22Cm21. M Tris P22( o ( 0 ). of tion
30 60 90120
consiwas
higher
% sucrose
particles those
P22
of
of phage
P22.
Vol.71,No.1,1976
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Q
Fraction
number
Fig 3. CsCl equilibrium density-gradient centrifugation of P22Cml lysate. A mixture of P22Cml with P22 was centrifuged in CsCl solution as described in Materials and Methods. The total number of fractions was 57. ( 1 Plaque-forming activity of P22Cml; ( o ), Cmr-traisdicing activity of P22Cml; ( A ), plaque-forming activity of P22. One unit on the ordiiate corresponds to 5 x 10 PFU per ml for ( A ) and PFU per ml for ( o ), 2 x 10 5 x lo4 transductants per ml for ( l ). Fig 4. CsCl equilibrium density-gradient centrifugation of P22Cm21 lysate. The total number of fractions was 55. ( 0 ), Plaque-forming activity of P22Cm21; ( 0 ), Cmr-transducing activity of P22Cm21; ( h ), plaqueforming activity f P22. One unit on the ordinaie corresponds to 1 x 10 9 PFU per ~1 for ( o ), 5 x 10 PFU per ml for ( A ) and 1 x 10 transductants per ml for ( 0 1.
The
Cmr-transducing
phage
P22Cm21
particles
and
co-sedimented
in
the
the
plaque-forming
gradient
(data
ones not
of
shown).
Discussion Our
experiments
density P22. is
of If
the
dependent
phage
presented P22Cm21
difference upon
here was
in the
length
clearly
higher
density of
315
than between
DNA alone,
indicated that
of
that wild-type
the phage
phages
P22Cm21
and
phage
P22Cm21
would
P22
BIOCHEMICAL
Vol.71,No.1,1976
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
1
Fig 5. Sucrose-gradient centrifugation lysate. The total number of fraction Plaque-forming activity of P22Cm21; forming activity of P22.
be
expected
formula
to of
Phage
Weigle
P22
by
the
"headful"
of
the
particle
contained
in
that
,the
and unless
DNA eer--
se
Unlike have
been
coli
system
different
et
al
is
known
to
of
all
from
P22
DNA's all
among
7, that
the
viral
the
the
amount
From
the
basis
into
the
length
of
is
P22
the
of
the
particles
8).
The
amount
the
original
the
the
we
DNA
can can
the
particle
also
the
density
be
expect
same, of
the
phages.
workers
316
in are
or
particle
DNA that
model,
same
system,
various
of
this
proteins
by
in
which
6).
P22-Salmonella
reported (3,
by
of
changed the
in
(5,
of
constituents
DNA on
encapusilated
(4),
head
densities
in
be
determined
a phage
are
8 % excess
(3).
mechanism
the the
5 to
DNA is
length
that
contain
of P22Cm21 was 77. ( o ), ( 4 ), plaque-
of
in
several
density
the
lambda-Escherichia
DNA in phage
these
lambda.
mutants
mutants For
is
example,
BIOCHEMICAL
Vol.71,No.1,1976
phage
lambda
lambda are
well
the
higher
of
DNA or
particles.
be
contains
p4 contains
Further
that
b2
the fully
18 % less
9 % excess
explained
by
the
experiments density lower
of
phage
content
of
case
DNA than
DNA.
action
are
Preliminary latter
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
now
The of
in
protein
more
wild
type,
herein
is
due
with
probable,
endonuclease.
to
determine
to
the
the
phage the
whether
higher in
isolated though
while
described
lambda-specific
constituents
experiments is
results
progress
P22Cm21
the
content phage DNA suggest
former
can
not
denied. Acknowledgments
We are grateful to Drs bara for their kind advice ed by a grant provided by Government (no. 057367).
H. Ikeda, S. and criticism. the Ministry
Kanegasaki and Y. This study was of Education, The
SakakisupportJapanese
References 1. 2. 3. 4. 5. 6. 7. 8.
Mise, K. (1976) Virology, in press. Lennox, E. S. (1955) Virology 1, 190 - 206. Weigle, J., Meselson, M. and Paigen, K. (1959) J. Mol. Biol. 1, 379 - 386. Streisinger, G., Emrich, J. and Stahl, M. M. (1967) Proc. Nat. Acad. Sci. USA. 57, 292 - 295. Rhoades, M., MacHattic L. A. and Thomas, C. A. Jr. (1968) J. Mol. Biol. 37, 21 - 40. Tye, B-K.,Chan, R. K. and Botstein, D. (1974) J. Mol. Biol. 85, 485 - 500. Kellenberger, G., Zichichi, M. L. and Weigle, J. (1961) J. Mol. Biol. 3, 399 - 408. Weil, J., Cunningham, R., Martin III, R., Mitchell, E. and Bolling, B. (1972) Virology 50, 373 - 380.
317
Vol.71,No.1,1976
BACTERIOPHAGE-P22
AND BIOPHYSICAL
DERIVATIVE
'Katsutoshi The
Public
of
May
HIGH
DENSITY
Health,
Microbiology,
The
Shiroganedai,
Minato-ku,
Tokyo Received
WITH
Mise
Department
of
RESEARCH COMMUNICATIONS
108,
Institute
Japan.
15,1976 Summary
Bacteriophage P22Cm21 was differentiated from the original phage P22. The buoyant of phage P22Cm21 was higher than that of 0.007 g per cm3. The possible biological this higher density is discussed.
in some characters density in CsCl solution phage P22 by as much as implication involved in
Introduction P22Cm were
phages,
isolated
plaque-forming from
Salmonella
typhimurium
ization
at
phages
can
high be
(1). on
usually
The
other
the
indicator 1 - 10
found
among
Mise,
and
Copyright All rights
into
obtained
of
P22Cml
wild-type
P22
strains.
The high,
ml.
of
I an
P22Cm),
Cm, chloramphenicol; plaque-forming
0 1976 by Academic Press, Inc. of reproduction in any form reserved.
312
this
of
very II
tiny
lysates
I
phage
plaques was
P22Cm21
makes
PFU per
differences
not
(Group
so be
II
(Suzuki
and
we describe
resistance
ml. on
could
microscope report,
P22Cm
I P22Cm, Group 11 10
than
Cmr, unit.
PlCm,
physiological Group
makes Group
In
the
more
electron
These
titer
No structual
(Group under
II,
and
on PlCm-lysogenic $ CC& marker by lysogen-
by
designated
observations).
used: PFU,
groups
very
P22
infection.
these,
titer
PFU per
phages
grown
single
Group The
between
the
two
was
designated
x 10'
unpublished
$ Abbreviations chloramphenicol;
upon
One
strains.
lysate
transduced
Salmonella
group,
high,
P22Cm)
and
classified
plaques
lysates
phage
frequencies
characteristics small
a P22
recombinants
that
to
Vol.71,No.1,1976
phage
BIOCHEMICAL
P22Cm21
original
phage
Bacterial as
shows
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
higher
density
Materials
and
in
CsCl
solution
than
the
P22.
strains,
described
bacteriophages,
in
Transduction
Methods
the
previous
generally
media paper
followed
and
chemicals
used
were
(1).
the
method
of
Lennox
(2).
CsCl density-gradiens centrifugation. A O.l-ml aliquot of a P22Cm lysate (about 5 x 10: PFU per ml) and the same amount of a P22 PFU per ml) were suspended in 5.2 ml of lysate (about 2 x 10 CsCl-0.1 M Tris buffer, pH 7.0. The refractive index of the solution was 1.3834. After centrifugation for 18 hr at 30,000 rpm and 5oC in Beckman SW50.1 rotor, about 55 fractions were collected. Each fraction was assayed for plaque-forming activities and for abilities to transduce the Cm marker. Plaque formers in a P22Cm lysate and those in a P22 lysate were differentiated from the form of plaques on the indicator strain LT2 4415. The former made small while the latter did ringed plaques. plaques, Sucrose density-gradient centrifugation. A 0.1 ml tion containing P22Cm21 (3 x 106 PFU per ml) and per ml) were layered on a 5.0-ml gradient of 5 standard saline citrate. After centrifugation of 28 min at 20,000 rpm and 15'C, 77 fractions were the botton of a tube and assayed for plaque-forming and Cmr-transducing activities.
of phage sflluP22 (1 x 10 PFU 20 % sucrose in the gradient for collected from activities
Results In
spite
phages
had
ed
to
the
P22Cm21 The
of several
made
and
Group
I phage
temperature
a P22Cm21
particles
are
For
or
P22Cm1,
P22Cm21
the
phages.
phage
Phage
suggest
more
of
1).
rate
were
period of
higher
Group
a Salmonella -
those
much may
latent
on
than
(Fig.
results
defective
plaques
the
longer
which
of tiny
and
at
activities,
characteristics
very
size
smaller
of
plaque-forming
characteristics
burst
These
their
P22
than
was
possibility
somewhat
P22 that
different
P22Cm
less
relat-
example,
phage
strain
4415.
P22Cm21
were
much
a representative
inactivated
phage
or
II
or the from
at P22Cml
a high (Fig.
2).
constituents those
of
a P22
particle. To P22Cm21
demonstrate was
submitted
this
possibility, to
CsCl
each density-gradient
313
of
phages
P22Cml
centrifugation
and in
Vol. 71, No. 1,1976
A
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
P22 P22Cm21 P22Cml
*a
A
0
0
Time of incubahon (min)
reference
phage
Cmr-transducing
phage
lysate
P22Cm1,
than
that Phage
gradient. phage
the
with
density
same of
phage
P22Cm21 As
P22Cm21
results;
shown
P22 was in
sedimented
of
phage was
Fig.
3,
the phage
P22.
P22Cm21 higher
the
of
- 0.008
centrifuged 5,
the
slightly
in
314
of
contrast
phage
to
of
with
cm3.
the
5 - 20
phage
obtain
P22Cm21
g per
than
in
either
that
times
the
ones
particles,
plaque-forming faster
density
In
than
four
density
the
plaque-forming
experiments
by 0.006
then
Fig.
as
that of
the
in
well
plaque-forming,
We repeated the
of phages P22, P22Cml used was Salmonella typhi-
As shown as
agreed
or
4).
stently
P22.
particles
transducing (Fig.
curves strain
Heat inactivation of phages P22, P22Cml and Phages were diluted loo-fold in prewarmed 0.01 buffer, pH 7.4, and incubated at 73.513. Left, ) and P22Cml( o ); right, P22( o ) and P22Cm21 Strain 4415 was used as the indicator for titraplaque-forming particles.
to
a P22Cml
30 60 90120
Time of incubation (min)
0
Fig 1. One step growth and P22Cm21. The host murium 4415. Fig 2. P22Cm21. M Tris P22( o ( 0 ). of tion
30 60 90120
consiwas
higher
% sucrose
particles those
P22
of
of phage
P22.
Vol.71,No.1,1976
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Q
Fraction
number
Fig 3. CsCl equilibrium density-gradient centrifugation of P22Cml lysate. A mixture of P22Cml with P22 was centrifuged in CsCl solution as described in Materials and Methods. The total number of fractions was 57. ( 1 Plaque-forming activity of P22Cml; ( o ), Cmr-traisdicing activity of P22Cml; ( A ), plaque-forming activity of P22. One unit on the ordiiate corresponds to 5 x 10 PFU per ml for ( A ) and PFU per ml for ( o ), 2 x 10 5 x lo4 transductants per ml for ( l ). Fig 4. CsCl equilibrium density-gradient centrifugation of P22Cm21 lysate. The total number of fractions was 55. ( 0 ), Plaque-forming activity of P22Cm21; ( 0 ), Cmr-transducing activity of P22Cm21; ( h ), plaqueforming activity f P22. One unit on the ordinaie corresponds to 1 x 10 9 PFU per ~1 for ( o ), 5 x 10 PFU per ml for ( A ) and 1 x 10 transductants per ml for ( 0 1.
The
Cmr-transducing
phage
P22Cm21
particles
and
co-sedimented
in
the
the
plaque-forming
gradient
(data
ones not
of
shown).
Discussion Our
experiments
density P22. is
of If
the
dependent
phage
presented P22Cm21
difference upon
here was
in the
length
clearly
higher
density of
315
than between
DNA alone,
indicated that
of
that wild-type
the phage
phages
P22Cm21
and
phage
P22Cm21
would
P22
BIOCHEMICAL
Vol.71,No.1,1976
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
1
Fig 5. Sucrose-gradient centrifugation lysate. The total number of fraction Plaque-forming activity of P22Cm21; forming activity of P22.
be
expected
formula
to of
Phage
Weigle
P22
by
the
"headful"
of
the
particle
contained
in
that
,the
and unless
DNA eer--
se
Unlike have
been
coli
system
different
et
al
is
known
to
of
all
from
P22
DNA's all
among
7, that
the
viral
the
the
amount
From
the
basis
into
the
length
of
is
P22
the
of
the
particles
8).
The
amount
the
original
the
the
we
DNA
can can
the
particle
also
the
density
be
expect
same, of
the
phages.
workers
316
in are
or
particle
DNA that
model,
same
system,
various
of
this
proteins
by
in
which
6).
P22-Salmonella
reported (3,
by
of
changed the
in
(5,
of
constituents
DNA on
encapusilated
(4),
head
densities
in
be
determined
a phage
are
8 % excess
(3).
mechanism
the the
5 to
DNA is
length
that
contain
of P22Cm21 was 77. ( o ), ( 4 ), plaque-
of
in
several
density
the
lambda-Escherichia
DNA in phage
these
lambda.
mutants
mutants For
is
example,
BIOCHEMICAL
Vol.71,No.1,1976
phage
lambda
lambda are
well
the
higher
of
DNA or
particles.
be
contains
p4 contains
Further
that
b2
the fully
18 % less
9 % excess
explained
by
the
experiments density lower
of
phage
content
of
case
DNA than
DNA.
action
are
Preliminary latter
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
now
The of
in
protein
more
wild
type,
herein
is
due
with
probable,
endonuclease.
to
determine
to
the
the
phage the
whether
higher in
isolated though
while
described
lambda-specific
constituents
experiments is
results
progress
P22Cm21
the
content phage DNA suggest
former
can
not
denied. Acknowledgments
We are grateful to Drs bara for their kind advice ed by a grant provided by Government (no. 057367).
H. Ikeda, S. and criticism. the Ministry
Kanegasaki and Y. This study was of Education, The
SakakisupportJapanese
References 1. 2. 3. 4. 5. 6. 7. 8.
Mise, K. (1976) Virology, in press. Lennox, E. S. (1955) Virology 1, 190 - 206. Weigle, J., Meselson, M. and Paigen, K. (1959) J. Mol. Biol. 1, 379 - 386. Streisinger, G., Emrich, J. and Stahl, M. M. (1967) Proc. Nat. Acad. Sci. USA. 57, 292 - 295. Rhoades, M., MacHattic L. A. and Thomas, C. A. Jr. (1968) J. Mol. Biol. 37, 21 - 40. Tye, B-K.,Chan, R. K. and Botstein, D. (1974) J. Mol. Biol. 85, 485 - 500. Kellenberger, G., Zichichi, M. L. and Weigle, J. (1961) J. Mol. Biol. 3, 399 - 408. Weil, J., Cunningham, R., Martin III, R., Mitchell, E. and Bolling, B. (1972) Virology 50, 373 - 380.
317
