Department of Molecular and Cellular Biology,
The Biological Laboratories, Harvard University, Cambridge,
Massachusetts 02138
SMC protein is required for chromosome condensation and for the
faithful segregation of daughter chromosomes in Bacillus
subtilis. The visualization of specific sites on the chromosome
showed that newly duplicated origin regions in growing cells of an
smc mutant were able to segregate from each other but that
the location of origin regions was frequently aberrant. In contrast,
the segregation of replication termini was impaired in smc
mutant cells. This analysis was extended to germinating spores of an
smc mutant. The results showed that during germination,
newly duplicated origins, but not termini, were able to separate from
each other in the absence of SMC. Also, DAPI
(4',6'-diamidino-2-phenylindole) staining revealed that chromosomes in
germinating spores were able to undergo partial or complete replication
but that the daughter chromosomes were blocked at a late stage in the
segregation process. These findings were confirmed by time-lapse
microscopy, which showed that after duplication in growing cells the
origin regions underwent rapid movement toward opposite poles of the
cell in the absence of SMC. This indicates that SMC is not a required
component of the mitotic motor that initially drives origins apart
after their duplication. It is also concluded that SMC is needed to
maintain the proper layout of the chromosome in the cell and that it
functions in the cell cycle after origin separation but prior to
complete segregation or replication of daughter chromosomes. It is
proposed here that chromosome segregation takes place in at least two
steps: an SMC-independent step in which origins move apart and a
subsequent SMC-dependent step in which newly duplicated chromosomes
condense and are thereby drawn apart.
 |
INTRODUCTION |
Bacteria face the challenge of
packaging a chromosome that is about 1 mm in contour length into a
structure called the nucleoid that is only about 1 µm across its long
axis. Yet the highly compacted chromosome in the nucleoid must be
capable of undergoing duplication into daughter chromosomes that can be
segregated to the progeny cells with high fidelity. Some insights into
the arrangement of the chromosome in the cell and its segregation have
come from recent advances in bacterial cytology, which have made it
possible to visualize individual sites on chromosomes in living and
fixed cells. These studies have shown that the replication origin
regions of the chromosome localize toward opposite poles of the cell, while the region of the replication terminus generally remains located
near the middle of the cell (5, 6, 17, 21, 30). Thus, the
chromosome appears to be folded in an ordered arrangement, with the
origin region located near one end of the nucleoid, the terminus near
the other end, and the quarter points on the chromosome located in
between (28). Time-lapse microscopy has revealed that the
movement of the origin regions toward the poles occurs rapidly and
abruptly during the cell cycle (5, 6, 25, 28, 29). Terminus
regions of the chromosome also separate from each other but not as
rapidly or as far apart as the origin regions (29). Thus,
chromosome segregation is a dynamic process in bacteria.
A key challenge is to understand how these chromosome movements are
choreographed. An important insight has come from the discovery in
bacteria of homologs of the eukaryotic SMC (structural maintenance of
chromosomes) proteins, a family of proteins that is now known to be
present in bacteria and archaea, as well as in eukaryotes (9,
26). In eukaryotes, these large proteins (>135 kDa) play a key
role in chromosome condensation and segregation during mitosis, as well
as in DNA recombination and transcriptional silencing. Also, eukaryotes
have multiple SMC proteins, multiple members of which assemble in
complexes, such as the condensin complex, which mediates chromosome
condensation, and the cohesion complex, which mediates the
cohesion of sister chromatids. In contrast, most bacteria for which a
complete sequence is known have a single smc gene.
In Bacillus subtilis, the smc gene is
indispensable for the maintenance of chromosome compaction and for
viability above 24°C (3, 7, 20). Escherichia
coli does not have an SMC homolog. Instead, it produces a protein
called MukB whose function is similar to that of SMC (22).
Despite the lack of sequence similarity, MukB bears strong structural
similarity to SMCs: both proteins are composed of an N-terminal domain
containing an ATPase motif, two central coiled coil domains that are
connected by a flexible linker sequence, and a globular C-terminal
domain (14). Based on its structural similarity to kinesin,
MukB was proposed to be a force-generating motor for chromosome
segregation (18, 22). Also, MukB and SMC form antiparallel
homodimers (19), such that the N-terminal domain of one
molecule would interact with the C terminus of its partner and vice
versa. For both types of proteins, it has been shown that the
C-terminal domain, which contains a helix-turn-helix motif, is
essential for binding to DNA (1, 24) and for the association
of SMC with the chromosome (7). B. subtilis SMC
was shown to have a higher affinity for single-stranded DNA than for
double-stranded DNA (8), although in yeast the opposite is
the case (1). A model for how SMC could cause chromosomes to
compact by the introduction of supercoils has been proposed (12,
13).
I wished to investigate the role of SMC in the arrangement of the
chromosome in the cell and the movement and segregation of the origin
and terminus regions. For this purpose, I took advantage of a
previously devised procedure for visualizing specific regions on the
chromosome in living cells based on the use of green fluorescent protein (GFP)-LacI to decorate a cassette of the lactose operon operator that had been inserted into the chromosome. Using this system
in combination with time-lapse microscopy, it was discovered that newly
duplicated origin regions move rapidly apart toward opposite poles of
the cell in the complete absence of gene for smc, although
the position of origin regions in mutant cells was aberrant. On the
other hand, segregation of the terminus region was impaired in an
smc mutant strain. A two-step model for chromosome segregation is proposed in which origin proximal regions are separated in an SMC-independent manner but the final separation of daughter chromosomes is mediated by SMC.
| |
MATERIALS AND METHODS |
Media and bacterial strains.
Cells were grown in rich medium
(Luria-Bertani) or in S750 defined minimal medium (10).
Strain PG63 (lacO cassette at origin region, expression of
GFP-LacI under control of pveg,
smc::kan) was created by transformation
of AT63 (pveg-gfp-lacI [Mlsr] pAT12
[lacO cassette, Cmr] integrated at the
spo0J locus [359°]) (28, 29) with chromosomal DNA from strain PG
388 (smc::kan
[7]), selecting for chloramphenicol (Cm, 5 µg/ml),
Mls (erythromycin and lincomycin, 5 and 25 µg/ml, respectively), and
kanamycin (Kan, 5 µg/ml) at 23°C. Similarly, strain PG6
(smc::kan, GFP-LacI, lacO
cassette at terminus region) was created by transformation of AT62
(pveg-gfp-lacI [Mlsr] pAT12 [lacO
cassette, Cmr] integrated at 180°) (28) with
chromosomal DNA from PG388, selecting for Cmr,
Mlsr, and Kanr. Disruption of the
smc gene was verified by PCR and Western blot.
Purification and germination of B. subtilis
spores.
Spores of B. subtilis were purified and
germinated by the procedure of Callister and Wake (4), with
the following modifications. In order to avoid purification of spores
that accumulated suppressors of smc (a frequent event),
cells were streaked out on DSM plates and incubated at room temperature
for 5 days. Cells with a deletion of smc create spores only
at low frequency (3), 0.5% in contrast to 82% for
wild-type cells. Suppressor colonies (smc colonies are
small, smooth, and rounded, in contrast to more wrinkled and much
larger colonies from the wild type) were excised from the plates, which
were flooded with 1 M KCl-0.5 M NaCl. Cells were washed twice in
distilled water (dH2O) and incubated in 10 mM Tris buffer
(pH 7.5) containing 2 mg of lysozyme per ml for 60 min at 37°C,
followed by successive washes in 1 M NaCl, H2O, 0.05% sodium dodecyl sulfate, and 50 mM Tris-10 mM EDTA (pH 7.5) and three
times with distilled water. Purified spores were stored at 4°C or at
80°C in dH2O-5% glycerol.
For germination of spores, a culture with an optical density (OD) of
0.6 to 0.8 was washed in H2O and resuspended in 0.5% glucose containing S750 medium supplemented with 0.3% aspartate, 0.1%
glutamate, 0.025% Casamino Acids, 0.05% yeast extract, and 0.025%
sodium citrate. After a heat treatment at 70°C for 30 min, spores
were centrifuged, resuspended in medium, and incubated at 30°C for 30 min, followed by incubation at 23°C. The 30-min incubation at 30°C
had no effect on the germination of smc cells, but it
increased the germination efficiency and synchronicity considerably. As
a control for the possibility that the sporulation protocol selected
for mutations that suppressed the smc mutant defect,
germinated spores were grown to mid-exponential phase and resuspended
in sporulation medium. After incubation for 5 days, spore-forming
efficiency was tested by heat treatment (30-min incubation at 80°C
and plating onto rich medium) and was found to be similar (0.4 to 2%)
to that observed for the first round of sporulation.
Microscopy.
Microscopy was performed as described in Webb et
al. (29). For the acquisition of single pictures, all
strains were grown at 23°C to mid-exponential phase, and aliquots
were observed on an Olympus BX60 microscope. Discrete fluorescent foci
could be seen in 40 to 70% of the smc mutant cells (PG63
and PG6) compared to 80 to 90% of the cells in the case of the wild
type. I attribute this difference in the efficiency with which foci
could be detected to the observation that smc mutant cells
are more difficult to flatten out on slides than are wild-type cells.
For time-lapse analysis, cells were harvested in early stationary phase
and resuspended in fresh medium (at an OD of ca. 0.25 at 600 nm).
Cultures were incubated at 23°C for 1.5 to 3 h, and aliquots
were placed on a microscope slide with a pad of agarose containing
growth medium. A coverslip was placed on the cells, excess agarose was
cut away, and images were acquired every 4 min. Measurement of the
position of origins in the cells were determined using Metamorph 3.0 software.
Immunofluorescence microscopy.
Antibodies against FtsZ
protein were affinity purified according to standard methods (kind gift
of J. Kemp, Harvard University). Then, 0.1-ml aliquots of
mid-exponential-phase cells were diluted into 1.9 ml of
methanol and fixed for 10 min at room temperature. After
centrifugation, the cell pellet was dried. Immunofluorescence microscopy was performed according to the method of Pogliano et al.
(23).
| |
RESULTS |
Abnormal chromosome arrangement in cells lacking SMC.
In
previous work, fusions of GFP to either LacI or Spo0J have been used to
visualize the location of the replication origin in living cells.
However, as noted by Britton et al. (3), the presence of an
smc mutation interferes with the visualization of
fluorescent foci from Spo0J-GFP. Likewise, Moriya et al.
(20) have shown that the position of Spo0J foci is perturbed
in smc mutant cells using immunofluorescence. An attractive
explanation for this, as suggested by Britton et al. (3), is
that Spo0J binds to multiple, widely separated sites in the origin
region (16) and that the binding of the partition protein to
the origin region involves the formation of a higher-order structure in
a manner that is dependent on SMC. In the case of GFP-LacI, however, visualization of the origin region is achieved by use of a
lacO cassette, which consists of multiple copies of the
binding site for the lactose operon repressor arranged in tandem and
inserted at a single site in the chromosome. The binding of GFP-LacI
that is expressed in the cell to lacO sequences results in a
fluorescent focus that can be visualized in living cells. Hence,
fluorescent foci from GFP-LacI are not expected to be dependent
upon a higher-order chromosome structure. I therefore reasoned
that it should be possible to visualize GFP-LacI foci in cells lacking
SMC and hence that the fluorescent fusion protein could be used to
investigate the effect of an smc null mutation on the
number, location, and movement of replication origin regions. To do
this, an smc insertion-deletion mutation
(smc388) was introduced by transformation (see Materials and Methods) into strain AT63 (29), which harbors a
GFP-LacI-producing construct and tandem copies of lacO
near the origin of replication (359°). The presence of
the smc388 mutation in one such transformant (strain PG63) was confirmed both from its phenotype (it was temperature sensitive and defective in chromosome condensation and segregation [3, 7, 20]) and directly by means of the PCR.
The numbers and positions of the origin regions of the
chromosomes in cells of strains AT63 and PG63 growing at 23°C
(doubling times of 50 and 185 min, respectively) were analyzed
using fluorescence microscopy (Fig. 1).
Septa were visualized using Normarski differential interference
contrast, which visualizes septa as subtle demarcations between cells
(Fig. 1A, B, and C). A high proportion of PG63 cells (ranging from 15 to 35% in three experiments) showed a higher-than-normal number
of origin regions. Unlike the parent strain (AT63), in which only one,
two, or four foci were generally visible (Fig. 1A), some
smc mutant cells exhibited three (Fig. 1B) and as many as
six (data not shown) foci. Cells mutant for smc are known to produce anucleate cells at high frequency (3, 7, 20). Yet,
the overall DNA to protein ratio for mutant cells is similar to that of
wild-type cells (20). The results indicate that the presence
of anucleate cells (15 to 19% under these growth conditions) might be
compensated for to some extent by the presence of other cells that have
a higher than normal number of chromosomal origin regions and thus DNA.
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FIG. 1.
Fluorescence microscopy of B. subtilis cells
producing GFP-LacI and carrying tandem copies of lacO near
the origin (A and B) or terminus (C and D) of replication. (A) AT63
(wild type). (B) PG63 (smc::kan). (C)
AT62 (wild type). (D) PG6 (smc::kan).
The white lines indicate septa, which were visualized by differential
interference contrast (Nomarski) microscopy. The scale bar (thick white
line) represents 2 µm. Cells were grown in rich medium at 23°C, and
images were collected during the mid-exponential phase of growth.
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The positions of the fluorescent foci was determined by measuring the
distance of each focus from the nearest pole of the cell using
Metamorph software and were plotted as a function of cell length.
Figure 2A and B present
the results for wild-type cells; for simplicity, only the data for
cells displaying one (Fig. 2A), two (Fig. 2A), or four (Fig. 2B) foci
are shown. As can be seen in the figure, cells with two foci were
generally smaller than cells with four foci. In the case of cells with
two fluorescent foci, the signals tended to be located toward opposite poles of the cell (open and filled circles in Fig. 2A) (28, 30). Sometimes cells with two foci were observed in which the foci were close to the mid-cell position or in the same half of the
cell (dotted line indicates mid-cell point; Fig. 2A). This was only
observed for small cells and, on this basis and on the basis of
previous observations made by time-lapse microscopy, I presume that
these represent young cells in which the origin region had undergone
duplication but that the resulting daughter origins have not yet
migrated toward opposite poles of the cell. In the case of wild-type
cells with four foci, the signals tended to be evenly spaced across the
cell, with two foci located near opposite poles (filled circles and
open squares) and the other two foci (open circles and filled squares
in Fig. 2B) in between. These positions correspond to polar positions
in the future daughter cells.
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FIG. 2.
Distance of fluorescent foci from the cell pole as a
function of cell size. (A and B) AT63 (GFP-LacI, lacO
cassette at 359°). (C and D) PG63
(smc::kan, GFP-LacI, lacO
cassette at 359°). Cells were grown in rich medium at 23°C, and
images were obtained during the mid-exponential phase of growth. Panels
A and C show cells with one ( ) or two ( and  ) fluorescent
foci; panels B and D show cells with four foci (, ,  , and  ,
with "" being the closest and "" being the farthest focus
from the pole chosen for measurements). The measurements in panels C
and D do not include very large cells, in which origin signals tended
to be even more random than in small cells.
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In the case of smc mutant cells, a strikingly different
pattern of origin positions was observed (Fig. 2C and D). First, many smc mutant cells had fluorescent foci located very close to
one or both cell poles, significantly closer than was observed in wild-type cells. Second, the location of origins was more irregular than was observed for the wild type. Whereas in wild-type cells with
two fluorescent foci the origins tended to localize toward opposite
ends of the cell in a symmetric pattern (Fig. 2A), in mutant cells one
origin was often very close to a pole but the other origin was located
in a more or less random position (Fig. 2C). In contrast to wild-type
cells with four foci, in which two origins were found in each half of
the cell (Fig. 2B), in mutant cells three or four of the origins were
often found in the same half of the cell (Fig. 2D).
Terminal regions of the chromosomes fail to separate in
smc null cells.
In slow-growing cells, the terminus
regions are generally located near the middle of the cell (28,
30) (Fig. 1C) and, following duplication, rapidly move apart
(29). Thus, large cells usually have two fluorescent signals
located near the quarter points of the cell just prior to cell division
(data not shown). To analyze the position of termini in smc
mutant cells, strain AT62 carrying the lacO cassette at the
terminus region (181°) was transformed with chromosomal DNA from
PG388 to create strain PG6. Whereas termini in wild-type cells
(AT62) were generally located at the mid-cell position (Fig. 1C),
fluorescent signals from the terminus in PG6 cells were often found
near the cell pole (Fig. 1D). Importantly, and contrary to chromosomal
origins which were distributed throughout the filament in the mutant
cells (Fig. 1B), terminus signals were often clustered at one site in the cell (Fig. 1D, arrows). This was observed in about 60% of the
cells and especially in large cells or filaments. I interpret these
observations to indicate that the absence of SMC causes a defect in
replication through the terminus or a defect in the segregation of
newly duplicated terminus regions.
Germinating spores of an smc mutant are defective in
chromosome segregation.
A complication in studying the role of SMC
in chromosome segregation during growth is the heterogeneity of the
cells both with respect to the stage of the cell cycle and the number
of replication origin regions (ranging up to six; see above). For this
reason the effect of an smc mutation was examined during outgrowth of germinated spores, since only a single chromosome is
packaged into the spore and spores can be germinated with partial synchrony by a regimen involving heat treatment followed by suspension in germination medium. Accordingly, spores from strains PY79, AT63,
PG388, PG63, and PG6 were purified. After heat treatment at 70°C
for 30 min, the spores were suspended in germination medium and allowed
to germinate at 30°C. After 30 min, outgrowth was allowed to
proceed at room temperature (~23°C).
Spores from the smc mutant strains underwent germination and
the initial stage of outgrowth (cell elongation) normally, as judged by
the conversion of phase-bright spores to phase-dark cells during the
30-min period of germination and the ensuing elongation of cells
emerging from the germinated spores during the first 150 min of the
outgrowth period (Fig. 3). However, by 210 min, when most of the cells from the wild-type spores started to
segregate their chromosomes, as seen by the bilobed appearance of
nucleoids (Fig. 3, arrow), cells from the mutant spores still contained
one nucleoid. In the wild type, clear separation of nucleoids commenced
at about 270 min (arrow), and by 330 min all of the wild-type cells had
two segregated nucleoids. In contrast, >95% of the smc
mutant cells (PG388 and PG63) had only one nucleoid mass at 330 min,
although cell elongation was comparable to that of the wild type.
Occasionally, some mutant cells were observed that had two nucleoid
masses, but these nucleoids frequently had an abnormal morphology (330 min, arrow). After germination, the mutant cells continued to grow and
entered exponential-phase growth but with a much longer doubling time
(240 min) than for the wild type (70 min).
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FIG. 3.
Time course of germinating spores from strain PY79 (wild
type) and PG388 (smc::kan). Samples
of cells at the indicated times after the start of germination were
fixed and stained with DAPI prior to image acquisition. DAPI images of
purified spores are not shown since spores show a strong blue
autofluorescence. The scale bars (thick white lines) represent 2 µm.
The arrows indicate partially (210-min time point) and completely (270- and 330-min time points) separated nucleoids.
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One possibility for the failure of chromosome segregation to take place
in germinated spores of the smc mutant could be a defect in
the initiation of replication. The lacO cassette decorated with GFP-LacI was used to investigate this issue because it provided a
means to visualize origin regions. Because maturation of GFP is a slow
process, it was necessary to modify the procedure described above by
suspending the germinating cells in a buffer (T-base) lacking nutrients
for 1.5 h following 150 min in germination medium in order to
obtain bright fluorescent foci. Following this procedure, two separated
origin regions could be seen in all wild-type (Fig. 4A) and smc mutant cells (Fig.
4B, arrows, 81 cells counted). These findings are consistent with the
idea that smc is not needed for the initiation of
replication during germination or the partial separation of newly
duplicated origin regions. Conversely, when spores of PG6, which
carries the lacO cassette at the terminus, were germinated
for 240 min and resuspended in T-base, only a single focus was
detectable (Fig. 4D, arrows, 54 cells counted), in contrast to
wild-type cells that had two well-separated foci. Further, many of the
wild-type cells had undergone cell division (Fig. 4C, white bar). These
results support a function for SMC in the complete separation or
replication of daughter chromosomes.
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FIG. 4.
Fluorescence and Nomarski DIC images of germinating
spores of cells producing GFP-LacI and carrying tandem copies of
lacO. Images were collected 1.5 h after resuspension in
salt buffer. Panels A and C show wild-type cells (AT63 and AT62,
respectively) and panels B and D show the smc mutant cells
(PG63 and PG6, respectively) containing the lacO cassette
near the origin (359°; A and B) or near the terminus (180°; C and
D). The scale bars (thick white lines) represent 2 µm, the arrows
indicate the position of fluorescent foci, and the white bars show the
position of the septa.
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SMC is not required for the rapid separation of chromosomal
origins.
One of the striking findings of this investigation is
that origin regions can and do localize toward the cell poles in the absence of SMC. This observation suggests that the "mitotic" motor that drives newly duplicated origins apart does not depend on SMC. To
test this possibility directly, time-lapse microscopy was used to
monitor origin movement in smc mutant cells. Cells of strain
PG63 were grown until early stationary phase and resuspended in fresh
minimal medium. Previous time-lapse microscopy experiments have shown
that in wild-type cells the origin regions separate from each other
abruptly during the cell cycle. Because smc mutant cells
grow with a long doubling time (4 h at 25°C), it was necessary to
observe a large number of cells in order to catch those that were at
the stage of origin separation. Of 115 cells studied, 9 were observed
in which origin separation was taking place. In all cases, the origin
regions were observed to move apart in a dynamic fashion, similar to
that observed in wild-type cells. The results of four such experiments
are shown in Fig. 5, with the
fluorescence signals shown in the left-hand column and the corresponding Nomarski DIC images in the right-hand column. In the
first time-lapse sequence, a fluorescent focus (labeled with white
lines) undergoes duplication into two foci between min 4 and 12. The
two daughter foci then move apart during min 16 to 24. The black bar in
the 24-min image indicates the position of a newly formed septum. A
similar pattern of origin movement is seen in the second sequence. In
the third sequence, a fluorescent focus located somewhat near the pole
undergoes duplication with one of the daughter foci moving to the
mid-cell position and the other remaining relatively stationary.
Conversely, a focus located at the mid-cell position in the
fourth sequence undergoes duplication near the cell middle, and the
resulting daughter foci move apart toward opposite poles of the cell.
Both the pattern of separation of newly duplicated foci from a
polar position and from a mid-cell position have been observed
previously in wild-type cells (29).
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FIG. 5.
Time-lapse microscopy. Cells from the smc
mutant PG63, which produces GFP-LacI and contains the lacO
cassette at 359°, were grown on agarose pads containing S750 medium
at room temperature. The left-hand columns show fluorescence from GFP,
and the right-hand columns show Nomarski DIC images. The numbers
indicate the time in minutes at which the images were acquired. The
scale bar (thick white line) represents 2 µm. The white lines
indicate the position of fluorescent foci; the black lines indicate the
emergence of a septum.
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Upon quantifying the results for all nine time-lapse sequences, I
find that fluorescent foci moved apart an average distance of 1.25 µm
(standard deviation, 0.11 µm) over a period of 8 to 12 min in
smc mutant cells compared to 1.4 µm in wild-type cells (29). The average velocity during the period of abrupt
movement was calculated to be 0.125 µm/min in the mutant cells
compared to the previously reported value of 0.17 µm/min in wild-type
cells. I conclude that the separation of newly duplicated origins does not depend on SMC and that the extent and velocity of movement in cells
lacking SMC is close to that observed in wild-type cells.
| |
DISCUSSION |
This report helps to clarify the role of SMC protein in the cell
cycle of B. subtilis. The principal finding is that newly duplicated chromosomal origins undergo rapid and abrupt
separation from each other in the absence of SMC, the
dynamics of movement being similar to that observed in wild-type cells
(29). I conclude from this that SMC is not needed in the
initial stages of chromosome segregation. Lemon and Grossman have
speculated that DNA replication is the driving force for chromosome
separation in bacteria (15). Based on their observation that
DNA polymerase localizes near the middle of the cell in B. subtilis, these workers propose a factory model in which the DNA
polymerase remains stationary and the DNA template is threaded
through the replication complex. The demonstration that SMC is
not required for origin separation is consistent with this model in
that the findings show that SMC is not the motor for moving origin
regions toward the cell poles.
A second principal contribution of the present work was the
demonstration of the aberrant placement of replication origins in cells
lacking SMC. Compared to the bipolar placement of origins observed in
wild-type cells, origins in mutant cells exhibited a somewhat random
distribution. For example, in contrast to the symmetric distribution of
origins found in wild-type cells, mutant cells were frequently observed
in which origins were asymmetrically distributed to one half of the
cell. Also, consistent with the idea that chromosomes are decondensed
in the absence of SMC (3, 7, 20), origins were often found
at a more extreme polar position in mutant cells than is observed in
wild-type cells. Aberrant positioning of replication origins was
unlikely to be due to an effect of the smc mutation on
placement of the division septum since the use of
immunofluorescence microscopy showed that Z-rings were generally not
mispositioned in mutant cells, although their number was
substantially reduced (data not shown). A similar role for SMC in the
proper placement of the chromosome has recently been reported for
Caulobacter crescentus. In wild-type cells of this aquatic
bacterium, origins are generally found at one or both poles of the
cell, but in cells lacking SMC origins are observed at intermediate
positions (11). It is concluded that proper folding of the
chromosome and its proper arrangement within the cell depends on SMC.
I have also discovered a role for SMC in the terminal stage of
chromosomal replication or separation. This was seen most clearly in
DAPI (4',6'-diamidino-2-phenylindole)-stained images of germinating spores of a mutant lacking the smc gene. During germination
such mutant spores underwent complete or partial chromosome
duplication, but the two daughter chromosomes did not completely
separate from each other. The results with GFP-LacI were consistent
with this interpretation. As noted above, fluorescent foci
corresponding to chromosomal origins in either growing cells or
germinating spores of an smc mutant were found to resolve
into two foci, which then moved apart from each other. In contrast,
foci corresponding to the terminal region frequently did not separate
or even resolve into two foci. This indicates either that replication
did not proceed across the terminus or that the terminal regions did
undergo replication but were blocked in subsequently moving apart from each other. In toto, the findings indicate that SMC functions after the
initiation of replication but before chromosomal termini are segregated.
In previous work, immunolocalization images were obtained suggesting
that SMC is not only associated with the chromosome but also with the
cell poles, especially in young cells (7). Since SMC is not
needed for newly duplicated origins to move apart, I speculate that SMC
does not become associated with the chromosome until after the
initiation of replication or after origins move apart and approach the
cell poles. Like its homologs in yeast (2), SMC might
bind preferentially to particular sites at scattered locations around
chromosome and introduce supercoils in flanking DNA, thereby causing
the chromosome to condense. This condensation could facilitate
nucleoid segregation and the final separation of the terminal regions
of the chromosome.
The work was performed in the laboratory of Richard Losick, whom
I thank for help in writing the manuscript. I also thank R. Britton for
helpful comments.
P.L.G. was a postdoctoral fellow of the Deutsche
Forschungsgemeinschaft. The work in Richard Losick's laboratory was
supported by grant GM15868.
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Akhmedov, A. T.,
C. Frei,
M. Tsai-Pflugfelder,
B. Kemper,
S. M. Gasser, and R. Jessberger.
1998.
Structural maintenance of chromosomes protein C-terminal domains bind preferentially to DNA with secondary structure.
J. Biol. Chem.
273:24088-24094[Abstract/Free Full Text].
|
| 2.
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Abstract
Introduction
