Vol. 63, No. 2, 1975
BIOCHEMICAL
CYCLIC
AMP AND CCLICIN
C.R.
Harwood
Biological
is
October
for by
in
protein
with
of
TNJ,
Kent,
England
that its
which they
are
parent
of
B,
the
cya+
I
and
be
(1,2)
or
following of
adenyl
never
less.than
K colicins
necessary when
increased
measurements
Escherichia
lacks
m+
E,
AMP may
spontaneous
cultures
crp-1)
suggest
observed
Meynell
cyclic
However,
growing
PP78 (cya
strain
CT2
that
whether
(3,4).
mitomycin-C titres
G.G.
synthesis cyclic AMP.
suggest
synthesis
colicin
SYNTHESIS
31,1975
reports
colicin
RESEARCH COMMUNICATIONS
University
Kent
SUMMARY: Spontaneous largely independent of Several
and
Laboratory, Canterbury,
Received
AND BIOPHYSICAL
coli
cyclase half
of
K-12
and
CAMP
the
titres
receptor
strain.
Methods Bacteria. -e were
The
kindly
provided
thi m--
str-s
cya+
thi m-w
str-r
E
D-!_
to
either
resistant colicinogenic ColV
factors
Other
colicin
for
an Hfr
methods. 15 min),
assays
Ira its
and colicin to not
K-12,
(5).
Pastan
appears E,
prepare be
E.coli
derivative,
hence
tested
Each
B before
their
they
grown Cal+
are
is
not
HfrH
is
HfrH
strain
being
respective
because
1100
PP78,
strain
1~78,
and
Strain
Lac-.
K or
1100
was
made
with
derivatives. stably
host. Culture lacuna
on
of
Dr.
crp+
could by
strains
by
donors
inherited
121°
two
sonicated
Copyright o 1975 by Academic Press, Inc. All rights of reproduction in my form resewed.
media counts
(all
on
sterilized
chloroformed
samples
535
by
radial
by
heating
samples
end
diffusion
are
to
BIOCHEMICAL
Vol. 63, No. 2, 1975
described
elsewhere
on centrifuged
(6).
cells
AND BIOPHYSICAL
Total
bacterial
RESEARCH COMMUNICATIONS
protein
was estimated
(7). Results
In
cultures
containing
cells
become constant have
elapsed
overnight
at
for
making
for
the
in growing
strains
in the strain
4
YE broth
,l
.2
($ LFC) may not
10 or more generations cultures
without
shakan at
- 5 hr for
.3
-4
of grown
glucose,
37O
strain
These included
measurements.
0
$ colicin-
and PP78 were therefore
1100
or
the
experiments,
same medium,
1100,
counts
until
present
37O in 2 ml unshaken
first
or ColK-235,
cultures
In the of
l/10,000
3 hr
ColE2-P9
as measured by lacuna
(6,8).
co1+ derivatives
diluted
carrying
and
incubated
PP78, before the
optical
.5 .6 .7 OPTICAL DENJlTY
Figure 1. Relation between total bacterial protein/ml culture of strains 1100 and ~~78 and O.D. for Cal+ Co1r derivatives Co1 factors: B-1~98, A (lower and upper graphs, respectively). El-K30, l ; E2-P9, o ; Ia-CT&, q ; K-235,Dr .
536
;
Vol. 63, No. 2, 1975
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNlCATlONS
i
Log*,
Relation between log relative and log O.D. for ColK-235 in
Figure 2. /ml culture PP78 (-o-)r
density
(O.D.)
titrations,
at
colony
lacuna
counts
factors:
9).
(the
The two Colshowed obvious O.D.
protein
650
run, total and,
latter
are not
parental
larger
gave the and O.D.
bacterial
counts
differences
and formed
derivatives
OPTICAL
(Fig.
with
colonies.
protein,
feasible
with
and also strain
colicin
1100
ColB and Co11
their
The relation
537
Cal+ derivatives,
grew to a greater
Nevertheless,
same relationship 1).
colicin concentration strains 1100 (-•-) and
ColE and ColK factors,
strains, since
DENSITY
between
their total
between
Cal+ bacterial
colicin
titre
BIOCHEMICAL
Vol. 63, No. 2,1975
and O.D.
was then
The results 1. a plot
ColK'
for
can be summarized At
colony
of colicin 2.
determined
AND BIOPHYSKAL
At
cultures
counts titre
higher
slope
above
(Fig.
3),~suggesting
than
against
usually
the $ LFC was also
seven different
increased
inoreasing
that
111
the
slope
(Fig.
2),
the
the rate
I
I -I-O
for
ColE*
presumably
ColB and ColI of
oolicin
I
I
I
and because in
cultures
synthesis
II
- 0.2),
1.
of
No such increase
I
by
i
-0-5 Log,
OPTICAL
Relation between log relative Figure 3. /ml culture and log O.D. for ColIa-CT4 in and PP78 (-00).
538
= 0.1
(O.D.
had a slope
(6,8). with
II -1.5
108/ml
O.D.
counts,
1 was apparent
Co1 factors.
as follows:
less
colony
RESEARCH COMMUNlCATlONS
DEWITV’
colioin strains
concentration 1100
(-•-)
Vol. 63, No. 2, 1975
these
BIOCHEMICAL
cultures
The colicin
3. strain
was constant
(ColB-K98,
Fig.
When titres
4.
produced
by Cal+ 1100,
about
two-fold
(e.g.
little
affected
in growth titres
of
Fig.
previous
reports
CAMP system
might
that
with
ColE2+,
and absent
(g-3
CQ+,
neither
first
presence report carrying further
include
isolated
after
2). than
was no more than
with
only
lacuna
of
ir
colicin
colicin
colonies
The
related) which
cannot
of
strains (&
were tested
in the
(1).
on a cya+
be assessed
a
The second strain
without
by a grant
from the
Medical
Research
References
2. 3. 4.
crp),
(6,8).
This work was supported Council to G.G.M.
1.
5336
and CA-7900
24 hr incubation (2)
titres.
were smaller
derivatives
counts
which
the differences
have been non-specific.
measurements
is
of
to be
such differences
ColEl+, PP78
appears
in view
zones around
strain
ColE2 or ColEl, data
and,
made colicin-resistant,
of glucose gives
well
inhibition
to which
Fig.
therefore
and PP78,
1100
One reported
ColE2-P9,
3).
by the
do not
ColEla-16,
the difference
of
equal
were:
by Cal+ PP78 were less
synthesis
as were observed
O.D.
(COOK-235:
colicin
strains
of
1100
(ColB-K77,
produced
RESEARCH COMMUNICATIONS
by Cal+ derivatives
strain
or greater
those
Spontaneous
of
less
3);
range
produced
those
ColEl-K30);
ColIa-CT4:
in this
titres
~P78 compared to
AND BIOPHYSICAL
L., Kingsbury, D.T. and Helinski, D.R. (1973). J. Bact. 114, 577 - 591. Durkacz, B.W., Kennedy, C.K. and Sherratt, D.J. (1974). J. Bact. 117, 940 - 946. Nakazawa, A., and Tamada, T. (1972). Biochem. Biophys. Res. COmmM. 46, 1004 - 1010. Nakazawa, A., and Tamada, T. (1972). Biochem. Biophys. Res. commun. 42, 977 - 983. Katz,
539
Vol. 63, No. 2, 1975
5. 6. 7. 8. 9.
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
Crombrugghe, B. de, Chen, B., Gottesman, M. and Pastan, I. (1971). Nature, Lond. 230, 37 - 44. Hardy, K.G., Harwood, C.R. and Meynell, G.G. (1974). Molec. Gen. Genetics, m, 313 - 331. Lowry, O.H., Roseburgh, N.J., Farr, A.L. and Randal, R.J. J. Biol. Chem. a, (1951). 265 - 275. zti;51K_cj3znd Meynell, G.G. (1972). Genet. Res., Camb. f&y, (1973)
K.G., ieynell Molec. Gen.
&ez;Fiis,
540
Dowman, J.E. and Spratt, 125, 217 - 230.
B.G.
BIOCHEMICAL
CYCLIC
AMP AND CCLICIN
C.R.
Harwood
Biological
is
October
for by
in
protein
with
of
TNJ,
Kent,
England
that its
which they
are
parent
of
B,
the
cya+
I
and
be
(1,2)
or
following of
adenyl
never
less.than
K colicins
necessary when
increased
measurements
Escherichia
lacks
m+
E,
AMP may
spontaneous
cultures
crp-1)
suggest
observed
Meynell
cyclic
However,
growing
PP78 (cya
strain
CT2
that
whether
(3,4).
mitomycin-C titres
G.G.
synthesis cyclic AMP.
suggest
synthesis
colicin
SYNTHESIS
31,1975
reports
colicin
RESEARCH COMMUNICATIONS
University
Kent
SUMMARY: Spontaneous largely independent of Several
and
Laboratory, Canterbury,
Received
AND BIOPHYSICAL
coli
cyclase half
of
K-12
and
CAMP
the
titres
receptor
strain.
Methods Bacteria. -e were
The
kindly
provided
thi m--
str-s
cya+
thi m-w
str-r
E
D-!_
to
either
resistant colicinogenic ColV
factors
Other
colicin
for
an Hfr
methods. 15 min),
assays
Ira its
and colicin to not
K-12,
(5).
Pastan
appears E,
prepare be
E.coli
derivative,
hence
tested
Each
B before
their
they
grown Cal+
are
is
not
HfrH
is
HfrH
strain
being
respective
because
1100
PP78,
strain
1~78,
and
Strain
Lac-.
K or
1100
was
made
with
derivatives. stably
host. Culture lacuna
on
of
Dr.
crp+
could by
strains
by
donors
inherited
121°
two
sonicated
Copyright o 1975 by Academic Press, Inc. All rights of reproduction in my form resewed.
media counts
(all
on
sterilized
chloroformed
samples
535
by
radial
by
heating
samples
end
diffusion
are
to
BIOCHEMICAL
Vol. 63, No. 2, 1975
described
elsewhere
on centrifuged
(6).
cells
AND BIOPHYSICAL
Total
bacterial
RESEARCH COMMUNICATIONS
protein
was estimated
(7). Results
In
cultures
containing
cells
become constant have
elapsed
overnight
at
for
making
for
the
in growing
strains
in the strain
4
YE broth
,l
.2
($ LFC) may not
10 or more generations cultures
without
shakan at
- 5 hr for
.3
-4
of grown
glucose,
37O
strain
These included
measurements.
0
$ colicin-
and PP78 were therefore
1100
or
the
experiments,
same medium,
1100,
counts
until
present
37O in 2 ml unshaken
first
or ColK-235,
cultures
In the of
l/10,000
3 hr
ColE2-P9
as measured by lacuna
(6,8).
co1+ derivatives
diluted
carrying
and
incubated
PP78, before the
optical
.5 .6 .7 OPTICAL DENJlTY
Figure 1. Relation between total bacterial protein/ml culture of strains 1100 and ~~78 and O.D. for Cal+ Co1r derivatives Co1 factors: B-1~98, A (lower and upper graphs, respectively). El-K30, l ; E2-P9, o ; Ia-CT&, q ; K-235,Dr .
536
;
Vol. 63, No. 2, 1975
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNlCATlONS
i
Log*,
Relation between log relative and log O.D. for ColK-235 in
Figure 2. /ml culture PP78 (-o-)r
density
(O.D.)
titrations,
at
colony
lacuna
counts
factors:
9).
(the
The two Colshowed obvious O.D.
protein
650
run, total and,
latter
are not
parental
larger
gave the and O.D.
bacterial
counts
differences
and formed
derivatives
OPTICAL
(Fig.
with
colonies.
protein,
feasible
with
and also strain
colicin
1100
ColB and Co11
their
The relation
537
Cal+ derivatives,
grew to a greater
Nevertheless,
same relationship 1).
colicin concentration strains 1100 (-•-) and
ColE and ColK factors,
strains, since
DENSITY
between
their total
between
Cal+ bacterial
colicin
titre
BIOCHEMICAL
Vol. 63, No. 2,1975
and O.D.
was then
The results 1. a plot
ColK'
for
can be summarized At
colony
of colicin 2.
determined
AND BIOPHYSKAL
At
cultures
counts titre
higher
slope
above
(Fig.
3),~suggesting
than
against
usually
the $ LFC was also
seven different
increased
inoreasing
that
111
the
slope
(Fig.
2),
the
the rate
I
I -I-O
for
ColE*
presumably
ColB and ColI of
oolicin
I
I
I
and because in
cultures
synthesis
II
- 0.2),
1.
of
No such increase
I
by
i
-0-5 Log,
OPTICAL
Relation between log relative Figure 3. /ml culture and log O.D. for ColIa-CT4 in and PP78 (-00).
538
= 0.1
(O.D.
had a slope
(6,8). with
II -1.5
108/ml
O.D.
counts,
1 was apparent
Co1 factors.
as follows:
less
colony
RESEARCH COMMUNlCATlONS
DEWITV’
colioin strains
concentration 1100
(-•-)
Vol. 63, No. 2, 1975
these
BIOCHEMICAL
cultures
The colicin
3. strain
was constant
(ColB-K98,
Fig.
When titres
4.
produced
by Cal+ 1100,
about
two-fold
(e.g.
little
affected
in growth titres
of
Fig.
previous
reports
CAMP system
might
that
with
ColE2+,
and absent
(g-3
CQ+,
neither
first
presence report carrying further
include
isolated
after
2). than
was no more than
with
only
lacuna
of
ir
colicin
colicin
colonies
The
related) which
cannot
of
strains (&
were tested
in the
(1).
on a cya+
be assessed
a
The second strain
without
by a grant
from the
Medical
Research
References
2. 3. 4.
crp),
(6,8).
This work was supported Council to G.G.M.
1.
5336
and CA-7900
24 hr incubation (2)
titres.
were smaller
derivatives
counts
which
the differences
have been non-specific.
measurements
is
of
to be
such differences
ColEl+, PP78
appears
in view
zones around
strain
ColE2 or ColEl, data
and,
made colicin-resistant,
of glucose gives
well
inhibition
to which
Fig.
therefore
and PP78,
1100
One reported
ColE2-P9,
3).
by the
do not
ColEla-16,
the difference
of
equal
were:
by Cal+ PP78 were less
synthesis
as were observed
O.D.
(COOK-235:
colicin
strains
of
1100
(ColB-K77,
produced
RESEARCH COMMUNICATIONS
by Cal+ derivatives
strain
or greater
those
Spontaneous
of
less
3);
range
produced
those
ColEl-K30);
ColIa-CT4:
in this
titres
~P78 compared to
AND BIOPHYSICAL
L., Kingsbury, D.T. and Helinski, D.R. (1973). J. Bact. 114, 577 - 591. Durkacz, B.W., Kennedy, C.K. and Sherratt, D.J. (1974). J. Bact. 117, 940 - 946. Nakazawa, A., and Tamada, T. (1972). Biochem. Biophys. Res. COmmM. 46, 1004 - 1010. Nakazawa, A., and Tamada, T. (1972). Biochem. Biophys. Res. commun. 42, 977 - 983. Katz,
539
Vol. 63, No. 2, 1975
5. 6. 7. 8. 9.
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
Crombrugghe, B. de, Chen, B., Gottesman, M. and Pastan, I. (1971). Nature, Lond. 230, 37 - 44. Hardy, K.G., Harwood, C.R. and Meynell, G.G. (1974). Molec. Gen. Genetics, m, 313 - 331. Lowry, O.H., Roseburgh, N.J., Farr, A.L. and Randal, R.J. J. Biol. Chem. a, (1951). 265 - 275. zti;51K_cj3znd Meynell, G.G. (1972). Genet. Res., Camb. f&y, (1973)
K.G., ieynell Molec. Gen.
&ez;Fiis,
540
Dowman, J.E. and Spratt, 125, 217 - 230.
B.G.
