J Bacteriol, May 1998, p. 2564-2567, Vol. 180, No. 9
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Division Planes Alternate in Spherical Cells of
Escherichia coli
K. J.
Begg and
W. D.
Donachie*
Institute of Cell and Molecular Biology,
University of Edinburgh, Edinburgh EH9 3JR, Scotland
Received 26 November 1997/Accepted 25 February 1998
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ABSTRACT |
In the spherical cells of Escherichia coli rodA
mutants, division is initiated at a single point, from which a furrow
extends progressively around the cell. Using "giant" rodA
ftsA cells, we confirmed that each new division furrow is
initiated at the midpoint of the previous division plane and runs
perpendicular to it.
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TEXT |
Normal Escherichia coli
cells are rods that double in length and then divide across the shorter
axis to give two equal cells. If division is prevented, cells grow to
form elongated, multichromosomal cylinders (filaments). When filaments
divide, a septum forms between each pair of segregated chromosomes to
produce cells of normal length, each with a single chromosome, and the
septal planes are parallel to one another along the length of the long
rod (4, 8).
The cylindrical shape of E. coli cells depends on the
presence of two membrane proteins, RodA and PBP 2 (15, 16, 20, 21). If either is inactivated, E. coli cells become
spherical. If the physical separation of round sister cells is
prevented by immobilizing the cells on agar, they give the appearance
of dividing in alternating planes at 90° to one another (9,
13). Recently it has been suggested that this appearance might be
an artifact due to cells rotating in place on the agar after each division (7). In order to test this explanation, we have
made very large spherical cells that divide rapidly into smaller
progeny cells without moving or bending. We show here that these
"giant" coccal E. coli cells do indeed divide along
planes alternating at right angles to one another and that the third
division is probably in the same plane as the first.
E. coli K-12 strain KJB24 was constructed by cotransducing a
rodA(Am) mutation (5) and a linked Tn5
into the suppressor-free (Sup0) strain W3110.
Kanamycin-resistant transductants were screened individually for the
spherical cell phenotype. These transductants showed extensive lysis in
rich medium but were stable in minimal medium. KJB24 is a spontaneous
variant that grows well in both media (the reason for the increased
stability is now known and will be reported elsewhere). In order to
produce even larger cells, a temperature-sensitive division mutant,
KJB28 [leu::Tn10 ftsA.12(Ts) rodA(Am) Sup0], was constructed by transducing
KJB24 with P1 prepared on strain TKF12
(leu::Tn10 ftsA.12) and selecting
tetracycline-resistant, temperature-sensitive transductants.
The division furrow is initiated at a single point and grows as an
arc around the cell.
Previous observations (2, 9, 13)
have suggested that the division of spherical cells is asymmetrical,
beginning on one side of the cell only. Since this fact is important to
our argument, we have shown this for a KJB28 cell, grown for 90 min without division at 42°C before being placed on a nutrient broth (NB)
agar surface at 30°C (Fig. 1). This
cell had a volume (~27 µm3) equal to that of about 20 average rod-shaped (W3110) or 6 coccal (KJB24) cells growing in this
medium. Division begins as a localized deformation of the cell surface
at a single point, from which a furrow extends and deepens until the
two ends meet. The two surfaces of the division furrow are separate
from the outset and the newly formed divided surfaces appear to be
almost flat, so that two hemispherical sister cells of equal size are
produced.
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FIG. 1.
Single cell of strain KJB28 [rodA(Am)
ftsA.12(Ts) Sup0] dividing on NB agar at
30°C, following 90 min of growth at 42°C without division.
Photographs were taken at 10-min intervals as indicated by numbers next
to the frames.
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[Note: KJB28 cells do not form partial constrictions at 42°C, unlike
mutants carrying ftsA(Ts) and rodA(Ts) alleles
(3). This difference appears to be because
rodA(Ts) cells are rods at 30°C and retain their poles for
several generations at 42°C, whereas rodA(Am) cells never
have poles (unpublished observations)].
Cell division takes place in alternating planes in rodA
cells.
Figure 2 shows successive
divisions of a giant KJB28 cell (volume, ~20 µm3 at
time zero) on NB agar at the permissive temperature. The formation of
an almost-flat plane on the newly divided side of each sister cell
makes it clear that the cells do not bend or rotate during division.
The figure also clearly shows that new division furrows start in the
middle of the previous division plane and run at 90° to that plane.
This pattern was seen in every KJB28 cell examined. Each cell also
elongates slightly between divisions, at 90° to the direction of
elongation of its mother cell. Similar alternation of the axis of cell
elongation of each division has been reported previously for cells of
Neisseria gonorrhoeae (24).
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FIG. 2.
Successive divisions of a KJB28 cell on NB agar at
30°C, following 90 min of growth at 42°C without division.
Photographs were taken at 10-min intervals as indicated by numbers next
to the frames.
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The joint activities of the RodA and PBP 2 proteins somehow ensure that
a cell has a cylindrical shape, grows by elongation, divides after each
replication of its single chromosome, and has division planes that run
at right angles to its long axis. Therefore, these may properly be
considered to be cell cycle proteins because, in their absence, cells
switch to a "default" cycle in which cells are spherical, large,
and multichromosomal; in which divisions take place by asymmetric
growth of division furrows; and in which division planes alternate by
90°. Like "natural" cocci (6, 10-14, 17-19, 22),
rodA mutants are large, multichromosomal cells but rapidly
segregate mutations into pure clones (1). We think that such
obligate homozygosity is likely to stem from a fixed spatial
relationship between chromosomes and division planes in the cell
(9).
Figure 2 also shows that the third division of a spherical cell
(observable at 150 min) starts in the center of the second division
plane, i.e., parallel to the plane of the first division. Divisions
therefore occur in only two dimensions, as in, for example, Lampropedia and Micrococcus, rather than in three
dimensions, as in Sarcina. It is interesting to speculate
about what distinguishes these two different patterns of division, and
whether yet another morphogene in E. coli restricts the
division of rodA cells to two dimensions.
A recent paper has challenged the conclusion that KJB24 cells divide in
alternating planes (7). KJB24 cells grown in a viscous
solution of Methocel formed chains and irregular clumps of cells, from
which it was concluded that division planes had in fact been parallel
to one another, as they are in rod-shaped cells. The irregular clumps
were dismissed as being due to the movement of cells in situ after
division, so that their presumed poles came to face in irregular
directions. While we agree that rotation of cells in the viscous liquid
may indeed have taken place, we could argue that this might well have
caused the formation of the chains of cells, even though divisions
actually took place in alternating planes. Figure
3B shows the way in which we think that
chains of cells form in Methocel (compared to on agar). As has been
shown conclusively (2, 3, 9; also this paper), division furrows in spherical E. coli cells begin at a
single point and proceed around the cell in an ever-deepening arc. If the sister cells are free to move during this process then the deepening furrow opens out the pair of cells so that they rotate through 90° until they are attached only at the point where the furrow has completed its traverse (as we see when cells are observed dividing in liquid medium). Initiation of the next set of furrows in
each daughter cell therefore takes place approximately parallel to the
first division plane, as shown in Fig. 3B. (If the second set of
furrows had actually been across the same cell axis, then the cells
would not have formed a chain, but rather a sort of daisy.) We think
that by ignoring the fact that division furrows are asymmetric in
spherical cells, it was not realized that pairs of dividing cells will
open out and rotate about their point of attachment, unless they are
immobilized on agar.
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FIG. 3.
Proposed explanation for the appearance of chains of
KJB24 cells after growth in Methocel. (A) On NB agar, cells are
prevented from moving as they divide so that alternating planes of cell
division give rise to tetrads. (B) In Methocel, cells are held less
firmly in place so that as the division furrow progresses from one
side, the cells fall apart. New division furrows are initiated in the
middle of the completed division planes and appear to be parallel or
nearly parallel to the first division plane. The process repeats to
give a kinked chain of cells (or, sometimes, an irregular clump).
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Our results show that in spherical cells, the division furrow begins
from a single point on the cell surface, and it has been shown that
this is also the case for FtsZ assembly (2). One of the most
interesting questions arising from this work is, therefore, that of
what provides the focus for FtsZ assembly at this single site on the
cell membrane. A clue to the answer may be given by the observation
that the point of origin of each new furrow is the midpoint of the
plane of the previous division: perhaps some elements of the previous
division machinery remain at this point and provide a nucleus for the
assembly of the next division arc. This idea will be the subject of a
separate communication.
Finally, it is interesting to compare the behavior of rodA E. coli cells with that of other kinds of spherical-celled bacteria. There are both gram-positive and gram-negative coccal species, e.g.,
species of the gram-positive genera Streptococcus,
Staphylococcus, Micrococcus,
Lampropedia, and Sarcina and species of the
gram-negative genus Neisseria. Not all of these show the
same pattern of division. Streptococcus spp. form long
chains of small spherical cells, i.e., successive division planes are
parallel to one another as in rod-shaped bacteria, whereas the other
cocci divide successively in different planes.
Staphylococcus and Sarcina divide in three planes
which alternate by 90°, forming cubical packets of eight cells. (The
Staphylococcus cells are not attached to one another except
by single junctions and therefore do not remain together in an orderly
way [23], but the Sarcina cells form very
regular packets.) Lampropedia cells divide successively in
only two planes, producing flat sheets of regularly arranged cells.
Micrococcus radiodurans characteristically forms only pairs
of cells that separate before their next division, but the planes of
the nascent septa can be seen in stained preparations (17).
The first two planes are at right angles (forming a tetrad) but the
third set of septa form at an intermediate angle. Our observations
indicate that successive septal orientations in giant rodA
ftsA cells (Fig. 2) most closely resemble those of
Lampropedia.
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ACKNOWLEDGMENTS |
This work was supported by a Programme Grant from the Medical
Research Council (United Kingdom).
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FOOTNOTES |
*
Corresponding author. Mailing address: I.C.M.B., Darwin
Building, King's Buildings, Mayfield Rd., Edinburgh EH9 3JR, Scotland. Phone: 0131 650 5354. Fax: 0131 650 8650. E-mail:
William.Donachie{at}ed.ac.uk.
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J Bacteriol, May 1998, p. 2564-2567, Vol. 180, No. 9
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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