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Synchronization of E. Coli K 12 by membrane selection
Vol. 41, No. 2, 1970
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
SYNCHRONIZATION OF -E. COLI K 12 BY MEMBRANE SELECTION Donald J. Cummings Department of Microbiology University of Colorado Medical Center Denver, Colorado
Received September 16, 1970
summary E. coli K 12 Hfr and F- strains were synchronized using the membrane The bacteria were deposited onto a Polyvic selec%aTchnique (1,2). (Polyvinyl chloride) Membrane and eluted with growth medium, About 20 percent of the Hfr strain and 7 to 9 percent of the F- strain remained bound to the membraneand at division, each bacterial strain yielded a culture of the youngest cells in an exponential population. Introduction Helmstetter
and Cununings(1) reported that cultures of --E. coli B/r
ctxld be synchrcnised using a membraneselection procedure. the bacteria were deposited on a millipore the bacterium; the filter mediumat 37V.
was inverted
filter
In this procedure
of pore size smaller than
and the cells eluted with
growth
Hithin 35 to 40 minutes, about 95 percent of the initial
populaticm of the cells were eluted from the filter 5 percent of the cells remained firmly
and discarded (2);
bound to the membraneand grcmth
of these cells persisted upon the continual addition of eluant. 95 percent of the newly divided cells eluted,
thus providing
At division,
a continuous
source of the youngest cells in an exponential population. The main advantage of this membranemethod was that no stress was applied to the exponential
population in order to achieve synchrony (1.3).
Consequently, investigations replication
had a direct
on the periodicity
of protein
synthesis and DNA
bearing on understanding the cell cycle (2,4,5,6). 471
BIOCHEMICAL
Vol. 41, No. 2, 1970
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
A disadvantage was that only -E. coli B and B/r strains were subject to this selection the effects
(1,2).
of the sex factor
could be studied, above.
Other bacteria,
and conjugaticn
2. &K
of g, coli
readily
cells when division
from the membrane, about half the
thus preventing
and eluted sporadically
the collection
This communication describes the applica-
tion of the membraneselectian
technique to -E. coli
was achieved using a polyvinyl
chloride membrane.
Materials Growth of Bacteria.
E. -- coli K 12 strains
grown in nutrient
AB 253 F- and AB 259 Hfr were
As before (1.2) the bacteria
A 'I-liter
culture
was inoculated with about
2 x 108 cells and grown for 12 to 14 hours at 37°C. culture were used to inoculate
titer
were filtered
Eluticn
to
5 x 107/ml
the synchrony apparatus (2) and the remain-
thrcugh a 0.6511 ME (Millipore
Corporation,
and 6 to 10 x 107/ml, respectively, filter
the bacteria were . (BDWP,293 mmdiameter, 0.6 w pore size, poly-
chloride membrane, Millipore
Corporaticn,
Bedford, Mass.)
wetted with 40 ml of 0.8 percent of Bacto-Tryptone
The filter
broth.
was begun with 2 1 of growth mediumand the eluant volume was
restored to 2 1 after
25 min. and 40 min.
40 to 50 ml/min. at 50 to 55 min. after first
of the
When the cell density of g. coli K 12 Hfr and F- had reached a
of 3
was first
Two liters
and used as the eluant (1.2).
deposited cn a Polyvic vinyl
were
broth and then adapted to minimal medium( a
modified C-salts medium(2)).
Synchrony.
K 12 in which synchrony
and Methods
supplied by Dr. A. L. Taylor.
Bedford, Mass.)
of the ycungest
with 5. -coli K 12 could be eliminated if a different
type of membranewere utilized.
ing 5-liters
with
occurred on the membrane. We considered the possibility
that this difficulty
first
synthesis
cm macromolecular
12 remained on the filter
K
time (unpublished results),
kindly
12 in which
did not bind to the membranein the manner described
Instead of eluting
population
especially
This yielded a flow rate of
commencingelution.
40 minutes, about 80 percent of the Hfr strain 472
Within the
and 91 to 93 percent
Vol. 41, No. 2, 1970
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
eluted from the membrane. Samples for analysis of synchrcny
of F- strain were collected
at 50 to 55 min. which corresponded to the generation times
of these organisms. This time of collecticn to yield Counting.
has been previously
shown (1.2)
optinmm synchrony. Cell number and sine distribution
were determined using a
Coulter Counter Model B, a 35 u aperture tube and a Model J Automatic Particle Size Distributicn
Analyzer (1,2,6). Results and Discussion
The synchrony achieved for -E. coli
SYNCHRONIZATION
I2 Hfr and F' is illustrated
K
OF
E.COLI
I
I
1
I
20
40
60
a0
MINUTES
in
K12
I
I
100
120
INCUBATION
Figure 1. Synchronization ofE. coli K 12. For the Hfr strain, 2-l at 5 x lOy/mlwere deposited cm tEe membraneyielding a synchrcnous populatiq with a cell density of about 1 x 107/m1. For the T strain, 2-l at 1 x 10 /ml were used yielding a synchronous pcpulaticm with a cell density of 6 x 106/ml. 473
Vol. 41, No. 2, 1970
Fig. 1.
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
As expected, for about the first
in cell number occurred;
the bacteria
40 minutes of growth, little
then doubled in number during the next
20 minutes and synchrcny persisted for at least two generations.
SIZE
OISTRISUllOH
DURING
IELATIVE
increase
SYNC~EONOUS
CUSIC
In general,
GROWTH
MlCllONS
of the synchronously dividing populations shown Figure 2. Size distribution in Fig, 1. Note that at 52 and 53 minutes, both strains displayed a broad size distribution and that the I- strain size distribution was biphasic. The vertical dotted lines indicate the midpoint of the exponential size distributions. 474
Vol.
41, No.
BIOCHEMICAL
2, 1970
these results
agreed with
those
AND
obtained
Sampling the Hfr strain
was complicated
action
self
of the pili
property
(71,
may also account
membrane; the Hfr yielded 7 to 9 percent minutes
it
the
in cell
This
titers,
bound to the Polyvic bound whereas
that
only
the first
40
in synchrony the degree of
number is only one criterion
collectian,
cells
again were smaller, as expected.
time
for
In Fig. 2,
the selected cells were distributed sine distribution.
The
and after one generation time, the The size distribution
of the
ane generaticn time (52 min.) was, in fact,
About half the cells were in the
synchrony.
change in cell size (2.6).
increased in size with
biphasic.
size range and the other half in
smallest
This could only occur in a synchronously dividing
the larger range.
B/r,
in the F- strain.
cells
K 12 F- after
at higher
of the cells
range of the exponential
smallest
--E. coli
due to the inter-
number of cells
1) and is an indication
can be noted that just after
within
occurred
with
in the number of cells
must be accompanied by a periodic
This
that
COMMUNICATIONS
remained on the membrane after
This is evident
increases
by the fact
about 20 percent
achieved is best
Periodic
(1.2)
for the greater
under the same conditionscfig. synchrony
RESEARCH
previously
agglutination
of the F- cells
of elutian.
BIOPHYSICAL
popula-
tion, The results presented here indicated that -E. coli K 12 can be synchronized by the membraneselection subject to better alteration alterations
technique.
Whether or not F- strains
synchrony than Hfr strains is not clear.
of the minimal growth mediumcould yield
better
are intrinsically
It may be that synchrony and such
are being investigated. Ackncmledgements
The work reported here was supported by a grant from the U. S. Public Health Service AI-06472. References 1.
Helmstetter,
608 (1964).
C.
E.
and Cummings,D.
475
J.,
Biochim.
Biophys. Acta, 82,
Vol. 41, No. 2.1970
2. 3. b, 5. 6. 7.
BIOCHEMICAL
AND BIOPt-lYSfCAL RESEARCH COMMUNICATIONS
Cummings,D. J,, Biochim. Biophys. Acta, E, 41, (1965). Maaloe, 0., and Kjeldgaard, "Control of Macromolecular Synthesis," H. A. Benjamin, Inc., New York (1966). Helmstetter, C. E., and Cooper, S., J. Mol. Biol. 3l, 507 (1968). Clark, D. J., and Maaloe, O., J. Mol. Biol., 23, 99 (1967). Helmstetter, C. E., J. Mol. Biol., 2, 417 (1967). Brinton, C. C. Jr., Trans. N.Y. Acad. Sci., 27, 1003 (1965).
476
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
SYNCHRONIZATION OF -E. COLI K 12 BY MEMBRANE SELECTION Donald J. Cummings Department of Microbiology University of Colorado Medical Center Denver, Colorado
Received September 16, 1970
summary E. coli K 12 Hfr and F- strains were synchronized using the membrane The bacteria were deposited onto a Polyvic selec%aTchnique (1,2). (Polyvinyl chloride) Membrane and eluted with growth medium, About 20 percent of the Hfr strain and 7 to 9 percent of the F- strain remained bound to the membraneand at division, each bacterial strain yielded a culture of the youngest cells in an exponential population. Introduction Helmstetter
and Cununings(1) reported that cultures of --E. coli B/r
ctxld be synchrcnised using a membraneselection procedure. the bacteria were deposited on a millipore the bacterium; the filter mediumat 37V.
was inverted
filter
In this procedure
of pore size smaller than
and the cells eluted with
growth
Hithin 35 to 40 minutes, about 95 percent of the initial
populaticm of the cells were eluted from the filter 5 percent of the cells remained firmly
and discarded (2);
bound to the membraneand grcmth
of these cells persisted upon the continual addition of eluant. 95 percent of the newly divided cells eluted,
thus providing
At division,
a continuous
source of the youngest cells in an exponential population. The main advantage of this membranemethod was that no stress was applied to the exponential
population in order to achieve synchrony (1.3).
Consequently, investigations replication
had a direct
on the periodicity
of protein
synthesis and DNA
bearing on understanding the cell cycle (2,4,5,6). 471
BIOCHEMICAL
Vol. 41, No. 2, 1970
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
A disadvantage was that only -E. coli B and B/r strains were subject to this selection the effects
(1,2).
of the sex factor
could be studied, above.
Other bacteria,
and conjugaticn
2. &K
of g, coli
readily
cells when division
from the membrane, about half the
thus preventing
and eluted sporadically
the collection
This communication describes the applica-
tion of the membraneselectian
technique to -E. coli
was achieved using a polyvinyl
chloride membrane.
Materials Growth of Bacteria.
E. -- coli K 12 strains
grown in nutrient
AB 253 F- and AB 259 Hfr were
As before (1.2) the bacteria
A 'I-liter
culture
was inoculated with about
2 x 108 cells and grown for 12 to 14 hours at 37°C. culture were used to inoculate
titer
were filtered
Eluticn
to
5 x 107/ml
the synchrony apparatus (2) and the remain-
thrcugh a 0.6511 ME (Millipore
Corporation,
and 6 to 10 x 107/ml, respectively, filter
the bacteria were . (BDWP,293 mmdiameter, 0.6 w pore size, poly-
chloride membrane, Millipore
Corporaticn,
Bedford, Mass.)
wetted with 40 ml of 0.8 percent of Bacto-Tryptone
The filter
broth.
was begun with 2 1 of growth mediumand the eluant volume was
restored to 2 1 after
25 min. and 40 min.
40 to 50 ml/min. at 50 to 55 min. after first
of the
When the cell density of g. coli K 12 Hfr and F- had reached a
of 3
was first
Two liters
and used as the eluant (1.2).
deposited cn a Polyvic vinyl
were
broth and then adapted to minimal medium( a
modified C-salts medium(2)).
Synchrony.
K 12 in which synchrony
and Methods
supplied by Dr. A. L. Taylor.
Bedford, Mass.)
of the ycungest
with 5. -coli K 12 could be eliminated if a different
type of membranewere utilized.
ing 5-liters
with
occurred on the membrane. We considered the possibility
that this difficulty
first
synthesis
cm macromolecular
12 remained on the filter
K
time (unpublished results),
kindly
12 in which
did not bind to the membranein the manner described
Instead of eluting
population
especially
This yielded a flow rate of
commencingelution.
40 minutes, about 80 percent of the Hfr strain 472
Within the
and 91 to 93 percent
Vol. 41, No. 2, 1970
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
eluted from the membrane. Samples for analysis of synchrcny
of F- strain were collected
at 50 to 55 min. which corresponded to the generation times
of these organisms. This time of collecticn to yield Counting.
has been previously
shown (1.2)
optinmm synchrony. Cell number and sine distribution
were determined using a
Coulter Counter Model B, a 35 u aperture tube and a Model J Automatic Particle Size Distributicn
Analyzer (1,2,6). Results and Discussion
The synchrony achieved for -E. coli
SYNCHRONIZATION
I2 Hfr and F' is illustrated
K
OF
E.COLI
I
I
1
I
20
40
60
a0
MINUTES
in
K12
I
I
100
120
INCUBATION
Figure 1. Synchronization ofE. coli K 12. For the Hfr strain, 2-l at 5 x lOy/mlwere deposited cm tEe membraneyielding a synchrcnous populatiq with a cell density of about 1 x 107/m1. For the T strain, 2-l at 1 x 10 /ml were used yielding a synchronous pcpulaticm with a cell density of 6 x 106/ml. 473
Vol. 41, No. 2, 1970
Fig. 1.
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
As expected, for about the first
in cell number occurred;
the bacteria
40 minutes of growth, little
then doubled in number during the next
20 minutes and synchrcny persisted for at least two generations.
SIZE
OISTRISUllOH
DURING
IELATIVE
increase
SYNC~EONOUS
CUSIC
In general,
GROWTH
MlCllONS
of the synchronously dividing populations shown Figure 2. Size distribution in Fig, 1. Note that at 52 and 53 minutes, both strains displayed a broad size distribution and that the I- strain size distribution was biphasic. The vertical dotted lines indicate the midpoint of the exponential size distributions. 474
Vol.
41, No.
BIOCHEMICAL
2, 1970
these results
agreed with
those
AND
obtained
Sampling the Hfr strain
was complicated
action
self
of the pili
property
(71,
may also account
membrane; the Hfr yielded 7 to 9 percent minutes
it
the
in cell
This
titers,
bound to the Polyvic bound whereas
that
only
the first
40
in synchrony the degree of
number is only one criterion
collectian,
cells
again were smaller, as expected.
time
for
In Fig. 2,
the selected cells were distributed sine distribution.
The
and after one generation time, the The size distribution
of the
ane generaticn time (52 min.) was, in fact,
About half the cells were in the
synchrony.
change in cell size (2.6).
increased in size with
biphasic.
size range and the other half in
smallest
This could only occur in a synchronously dividing
the larger range.
B/r,
in the F- strain.
cells
K 12 F- after
at higher
of the cells
range of the exponential
smallest
--E. coli
due to the inter-
number of cells
1) and is an indication
can be noted that just after
within
occurred
with
in the number of cells
must be accompanied by a periodic
This
that
COMMUNICATIONS
remained on the membrane after
This is evident
increases
by the fact
about 20 percent
achieved is best
Periodic
(1.2)
for the greater
under the same conditionscfig. synchrony
RESEARCH
previously
agglutination
of the F- cells
of elutian.
BIOPHYSICAL
popula-
tion, The results presented here indicated that -E. coli K 12 can be synchronized by the membraneselection subject to better alteration alterations
technique.
Whether or not F- strains
synchrony than Hfr strains is not clear.
of the minimal growth mediumcould yield
better
are intrinsically
It may be that synchrony and such
are being investigated. Ackncmledgements
The work reported here was supported by a grant from the U. S. Public Health Service AI-06472. References 1.
Helmstetter,
608 (1964).
C.
E.
and Cummings,D.
475
J.,
Biochim.
Biophys. Acta, 82,
Vol. 41, No. 2.1970
2. 3. b, 5. 6. 7.
BIOCHEMICAL
AND BIOPt-lYSfCAL RESEARCH COMMUNICATIONS
Cummings,D. J,, Biochim. Biophys. Acta, E, 41, (1965). Maaloe, 0., and Kjeldgaard, "Control of Macromolecular Synthesis," H. A. Benjamin, Inc., New York (1966). Helmstetter, C. E., and Cooper, S., J. Mol. Biol. 3l, 507 (1968). Clark, D. J., and Maaloe, O., J. Mol. Biol., 23, 99 (1967). Helmstetter, C. E., J. Mol. Biol., 2, 417 (1967). Brinton, C. C. Jr., Trans. N.Y. Acad. Sci., 27, 1003 (1965).
476