FEMS Microbiology Letters 187 (2000) 1^7
www.fems-microbiology.org
MiniReview
Control of division gene expression in Escherichia coli
Susan J. Dewar *, Robert Dorazi
Department of Biological Sciences, Heriot Watt University, Edinburgh EH14 4AS, UK
Received 26 January 2000; received in revised form 7 March 2000; accepted 9 March 2000
Abstract
Duplication of the Escherichia coli bacterial cell culminates in the formation of a division septum that splits the progenitor cell into two
identical daughter cells. Invagination of the cell envelope is brought about by the co-ordinated interplay of a family of septation-specific
proteins that act locally at mid-cell at a specific time in the cell cycle. The majority of the genes known to be required for septum formation
are found within the large mra cluster located at 2 min on the E. coli genetic map (nucleotides 89 552^107 474). Examination of the controls
exerted on the mra operon shows that E. coli uses an extraordinary range of strategies to co-ordinate the expression of the cell division genes
with respect to each other and to the cell cycle. ß 2000 Federation of European Microbiological Societies. Published by Elsevier Science
B.V. All rights reserved.
Keywords : Cell division; Regulation; Fts protein; Transcriptional organization; Gene expression ; Escherichia coli
1. Introduction
The speed and e¤ciency with which bacterial cells can
replicate is unequalled. During rapid growth in rich medium, Escherichia coli cells can duplicate in a little over
20 min, which demands the seamless integration of general
metabolism with the dedicated biochemical and genetic
pathways that drive cell growth, chromosome replication
and ultimately, cell division. Cells in balanced growth
rarely get this wrong ; it is exceptional to ¢nd a DNAless cell, or one which is of greater or lesser mass than
the others, testament to the exquisite pro¢ciency of the
control networks that co-ordinate these events. While we
are still some way from understanding the intracellular
signals that transduce the physiological state of the cell
into cell growth, the global networks that harmonise patterns of gene expression in response to changing environmental conditions such as starvation (cS ) or heat shock
(c32 , cE ) are increasingly well understood. A number of
recent molecular studies have also shed light on the regulatory networks that ensure smooth progression through
the growth and division cycle. This review will examine the
diverse regulatory strategies, including a growth-rate-dependent mechanism, antisense RNAs and quorum-sensing
regulatory proteins, that control the synthesis and activity
* Corresponding author. Tel. : +44 (131) 451 3457 ;
Fax: +44 (131) 451 3009; E-mail : [email protected]
of the primary division genes located in the 2-minute mra
cluster, nucleotides 89 552^107 474.
2. Cell growth and division: the E. coli life cycle
Our current understanding of the E. coli cell cycle has
been greatly aided by the study of temperature-sensitive
growth and division mutants, while recent advances in
£uorescent microscopy have provided insights into the dynamic process of development in living bacterial cells. The
life cycle of E. coli appears straightforward. The bacterium
grows in length with little change in diameter, until it
reaches a critical size that is twice its original length.
Cell division is then initiated at mid-cell with the formation of a contractile ring, comprised largely of the tubulinlike protein, FtsZ. Evidence suggests that initiation of the
Z-ring is prompted by the activation of a single site at
mid-cell in response to an as yet uncharacterised cell cycle
signal. Although the factors that regulate the timing of
Z-ring assembly are not yet well understood, a series of
elegant molecular studies on the Min proteins has gone a
considerable way towards describing the mechanism by
which topological speci¢city in the placement of the ring
may be achieved (reviewed in [1]). The Z-ring provides a
framework onto which at least eight other septation proteins, including ZipA, FtsA, FtsQ, FtsL, FtsK, FtsI, FtsN,
and FtsW, are recruited.
Control of the intracellular concentration of the septa-
0378-1097 / 00 / $20.00 ß 2000 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.
PII: S 0 3 7 8 - 1 0 9 7 ( 0 0 ) 0 0 1 2 2 - 1
FEMSLE 9357 17-5-00
Cyaan Magenta Geel Zwart
2
S.J. Dewar, R. Dorazi / FEMS Microbiology Letters 187 (2000) 1^7
tion-speci¢c proteins is of pivotal importance for cell division. Even small £uctuations in the levels of the essential
cell division proteins can severely disrupt cell growth [2^4].
Mutants that either cannot make, or make a thermolabile
form of any of the individual Fts proteins, are blocked in
division and grow as smooth or partially constricted ¢laments depending on the stage at which the gene product
acts [5]. In contrast, the modest overproduction of FtsZ
protein, which is rate-limiting for division, induces extra
divisions as evidenced by the production of minicells at
cell poles; while still higher levels of FtsZ block cell division. Indeed, the molar ratio of FtsA to FtsZ is critical for
correct cell growth, since elevating the level of one protein
at the expense of the other results in ¢lamentation, an
e¡ect that may be suppressed by the co-ordinate overexpression of the other [2,3].
Under normal conditions therefore, the concentration of
the division proteins must be maintained within de¢ned
limits to avoid the potentially damaging e¡ects of unbalanced protein synthesis. In view of this, it is not surprising
that the cell division genes are subject to complex regulation. The majority of the genes that are essential and speci¢c for cell division are located in the 2-min mra cluster.
Expression of these genes is regulated by an elaborate
arrangement of interwoven controls that ensures the division proteins are synthesised in appropriate amounts during the cell cycle.
3. Transcriptional control of the mra cluster: a plethora of
promoters
Even cursory examination of the arrangement of genes
within the mra cluster attests to their close association and
unconventional arrangement. Sixteen genes encoding proteins involved in cell division and/or envelope biosynthesis
are crowded together in a contiguous run spanning 17.9
kb. The genes are all read in the same direction, with
many of the ORFs overlapping or separated by only
very short gaps. The regulatory signals for individual
genes are routinely found in the neighbouring upstream
gene (Fig. 1). This unusual genetic arrangement yields a
set of overlapping mRNAs initiated from a series of upstream promoters. Transcription of ftsZ, which lies at the
distal end of the cluster, is directed from multiple promoters, some of which lie many hundreds of nucleotides
upstream of it. At least six ftsZ promoters lie within the
three genes immediately adjacent to it. Of these, two promoters lie within ddlB (ftsQ2p1p) and transcribe the
downstream genes, ftsQ, ftsA, ftsZ (and ¢nally envA which
precedes the single strong terminator at the end of the
cluster). A single promoter within ftsQ (ftsAp) transcribes
ftsA and ftsZ, while the three promoters within ftsA
(ftsZ4p3p2p) transcribe ftsZ [6^9]. EnvA has its own promoter located in the intergenic space between it and ftsZ
suggesting that, while ftsQ, ftsA and ftsZ may be co-ordi-
FEMSLE 9357 17-5-00
nately expressed, transcription of envA is likely to be distinct from that of its upstream neighbours. The absence of
transcriptional terminators within the cluster potentially
allows transcripts initiated at the mra promoter at its
proximal end to transcribe the ftsZ and envA genes
some 17 kb downstream. In keeping with this observation,
Dai and Lutkenhaus [10] reported that sequences at the
5P end of the cluster were required for full ftsZ expression.
They found that the prophage V16-2, which carries a 6-kb
fragment of DNA extending from the middle of ftsW to
just beyond envA, was unable to complement a null allele
of ftsZ, but that the more extensive chromosomal insert
carried on FP104 could. More recently, Fla«rdh et al. [11]
have used a P(ftsZ^lacZ) fusion introduced into the chromosomal locus to monitor transcription reaching ftsZ
from upstream promoters. They were able to show that
the insertion of strong transcriptional terminators into
ddlB (ddlB: :6) reduced transcription of ftsZ by twothirds, supporting the tenet that distant upstream elements
are required for its full expression. The nature of these
activating upstream sequences is not yet clear, but two
recent studies support the possibility of there being some
contribution from the mra promoter. Hara et al. [12] have
shown that Pmra is necessary for full expression of the
genes from mraZ to ftsW, while a subsequent study by
Mengin-Lecreulx et al. [13] showed that substitution of
Pmra by Plac causes a concomitant 30% reduction in the
levels of FtsZ protein. Several promoter-like sequences
have been described for both the ftsI^murD and the
orfC^ftsL regions [13^15] which, if shown to be active,
may also contribute to the expression of the downstream
genes.
Gene fusions have been used extensively to determine
the contribution of the promoters within the ddlB^ftsZ
locus (in the absence of the upstream sequences) to transcription of ftsZ [8,9,16]. By constructing fusions between
the ddlB^ftsQAZ sequence and lacZ on a V vector, it has
been shown that approximately 45% of transcription from
within this region is initiated from ftsQ2p1p, the two promoters that lie within ddlB, while ftsZ4p3p contribute a
further 37%. The remaining activity is accounted for from
ftsAp, 12% and ftsZ2p, 5% [8]. Several of these promoters
have been shown to be growth-rate-sensitive, and variations in mRNA levels between studies may feasibly be
caused by culture conditions generating a variety of
growth rates.
A distinctly di¡erent transcriptional pattern emerges
when the DNA sequence upstream of ddlB is included in
the analysis and transcription reaching ftsZ from these
upstream sequences is monitored from chromosomal fusions to the native ftsZ gene rather than from prophage
integrants or from plasmids. Using this system it becomes
apparent that during rapid growth the bulk of ftsZ transcripts ( s 66%) are initiated from sequences upstream of
ddlB [11]. The ftsQ2p1p promoters contribute only a fraction of their plasmid-encoded value at 15% and ftsZ4p3p
Cyaan Magenta Geel Zwart
S.J. Dewar, R. Dorazi / FEMS Microbiology Letters 187 (2000) 1^7
3
Fig. 1. The chromosomal arrangement of the 2-min mra cluster is shown on the upper diagram. The direction of transcription is indicated, as is the position of the single transcriptional terminator (T) that lies after the envA gene. The ¢ne structure of the ddlB^ftsZ region is detailed below. Open arrowheads indicate promoters, their designation is shown underneath, while the antisense transcript (stfZ) is shown by a ¢lled arrowhead. DnaA binding
sites are represented by ¢lled circles and the inverted repeat regulatory sequences within ftsQ, by inverted arrows. The letter E indicates the positions of
two RNase E processing sites. The method by which the promoters internal to the ddlB^ftsZ region are regulated is summarised in the table. A `+'
symbol indicates the promoter is activated under the given conditions, a `3' that it is insensitive to them. When more than one promoter may be subject to regulation, brackets denote the promoters included. The mraZ^envA region is drawn to scale.
are reduced to 12%. A minor contribution is made from
ftsAp and ftsZ2p whose levels are also reduced slightly to
4% and 2%, respectively. Despite the change in emphasis
on the importance of the upstream promoters for transcription of ftsZ, the multiple controls exerted over the
ftsZ proximal promoters suggest that they are of signi¢cant importance in determining the ¢nal levels of the cell
division proteins. They may modulate synthesis of the
proteins in response to particular intracellular signals or
environmental conditions or play a role in establishing the
correct relative ratio of division proteins. How might regulation of these promoters be achieved?
4. Cranking the speed: gearboxes and growth rate
independence
The rate of transcription of ftsZ varies with cell growth
so that it increases as the generation time lengthens
[6,17,18]. Since average cell size is larger in fast-growing
cells, the overall e¡ect of this growth-rate-regulated control is to maintain constant the in vivo levels of FtsZ
protein per cell cycle. Of the six promoters that contribute
FEMSLE 9357 17-5-00
to ftsZ transcription from within the ddlB^ftsZ region,
ftsQp1, ftsZ4p and 3p are inversely growth-rate-dependent
and increase in activity on entry into stationary phase
[8,17,18]. The regulation of ftsQ2p and 1p is of particular
interest, since these companion promoters account for almost half of ftsZ transcription. When cells are growing
quickly ftsQ2p is strongly induced, while ftsQ1p is induced
as the growth rate declines until it becomes predominant
at low growth rates [6].
FtsQ2p is regulated by a quorum-sensing mechanism
e¡ected through SdiA, a member of the LuxR family of
transcriptional activators ([19], and see below) while
ftsQ1p, which belongs to a family of `gearbox' promoters,
is positively regulated by the stationary growth phase sigma factor cS [20,21]. The growth rate dependence of an
ftsQp1: :lacZ fusion is abolished in a strain in which the
rpoS gene is insertionally inactivated. The e¡ect of the
gearbox is to ensure the production of constant amounts
of product, in this case FtsQ, FtsA and to some degree
FtsZ, per cell at any growth rate [22]. Gearbox promoters
share sequence homology in their promoter regions ;
ftsQ1p has strong sequence homology with the stationary-phase-activated bolA promoter (bolA expression
Cyaan Magenta Geel Zwart
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S.J. Dewar, R. Dorazi / FEMS Microbiology Letters 187 (2000) 1^7
causes cells to become smaller and more rounded as they
move into stationary phase). The promoters include a
short AT-rich region (a UP element) thought to be activated by the carboxy-terminal domain of the K-subunit of
RNA polymerase [23,24]. The housekeeping promoter
ftsQ2p is only transcribed by EcD , whereas ftsQ1p is recognised by RNA polymerase sigma factors cD and cS ,
with EcS being favoured on entry into stationary phase
[25]. When the relative contributions of ftsZ2p3p4p are
analysed at di¡erent growth rates, 4p, 3p and 2p also
show an inverse growth rate dependence, as well as moderate induction in stationary phase [17,18]. In contrast
with ftsQ1p, in vitro studies show that ftsZ2p3p4p are
preferentially transcribed by EcD rather than by EcS , so
although they exhibit a growth-phase-dependent mode of
expression, it must be achieved through an uncharacterised mechanism that is distinct from the RpoS-dependent
gearbox system.
There have been several reports suggesting that transcription of ftsZ is periodic in the cell cycle, with transcription taking place just before cell division [17,26^28].
Garrido et al. [28] used RT-PCR to show that the abundance of ftsZ mRNA oscillates periodically during the cell
cycle, becoming maximal coincident with the initiation of
DNA replication. A second study, also using cells
synchronised by membrane elution, showed that ftsZ expression is maximal around the middle of the cell cycle
and minimal at the time of cell division [29]. It had been
suggested that DnaA protein might provide a link between
the control of initiation and cell division, although cell
division is not triggered by chromosome replication per
se [30]. Transcription of ftsZ from an ftsAp^ftsZ4p3p^
lacZ fusion on a V phage was shown to increase at the
non-permissive temperature in a dnaA46 host, potentially
mediated by the three DnaA boxes located within the ftsQ
and ftsA genes [31]. However, re-examination of the role
of DnaA in the regulation of ftsZ concluded that the effects described earlier were likely to result from physiological changes and were not speci¢c to DnaA [28,32]. In a
strain in which ftsZ is separated from its natural chromosomal regulatory signals and is instead inducible from the
tac promoter by the addition of exogenous IPTG, ftsZ
loses its periodic expression and is expressed continuously
throughout the cell cycle [4,28]. Surprisingly, cells induced
to overproduce FtsZ by 40% above wild-type levels are
una¡ected in cell growth, suggesting that additional factors ensure the correct timing of cell division. How the
periodic expression of division genes is achieved remains
unclear.
5. Beyond mRNA: processing and post-transcriptional
controls
The Fts proteins are present in widely varying amounts,
despite the fact that the genes are all members of the same
FEMSLE 9357 17-5-00
transcriptional unit: e.g. the ratios of the proteins are in
the order of FtsZ (5000^20 000 [33,34]) :FtsA (50^200
[35]):PBP3 (100 [36,37]):FtsQ (25 [38]):FtsL (20^40 [14]).
In part, this di¡erential expression is due to the di¡erent
numbers and strengths of the internal promoters described
above, but it is also controlled through processing (and
decay) of the polycistronic transcripts and in the relative
rates of translation of the division proteins.
A potentially important element in the regulation of
gene expression is the stability of mRNA, and control of
the rate of mRNA decay is one means by which cells can
reduce the rate of biosynthesis of proteins during times
of slow growth [39]. The decay rate of transcripts does
vary, thus contributing to the di¡erential expression of
ftsZ:ftsA. Two major in vivo RNase E processing sites
have been identi¢ed [9], one within the ftsA coding sequence (this was originally designated ftsZp1 [6] but actually corresponds to the RNase E1/E2 cleavage sites) and
a second in the ftsA^Z intergenic region. Cleavage by
RNase E destabilises ftsA and possibly ftsQ mRNA, altering the ratio of ftsA to ftsZ mRNA approximately ¢vefold, and thereby contributing to the di¡erent levels of
expression of ftsA to ftsZ observed. It has also been reported that cells lacking the host factor-I (HF-I, which is
encoded by the hfq gene at 95 min on the E. coli chromosome) produce minicells at high frequency in stationary
phase and in poor medium [40]. The Hfq protein is
thought to regulate ompA mRNA stability by modulating
RNase E-dependent cleavage of its mRNA and it has been
suggested that Hfq may regulate the stability of ftsZ
mRNA in a similar manner; hfq: :cat mutants exhibit
two- to three-fold higher FtsZ protein levels than are observed in exponential phase cells. The increase in FtsZ
protein re£ects an increase in ftsZ mRNA abundance
and suggests that Hfq acts as a negative regulator of
ftsZ in cell division. In addition to the transcriptional
controls discussed above, there are also di¡erences in
translational e¤ciencies of ftsQ, ftsA and ftsZ from the
polycistronic transcripts. FtsQ and FtsA messages are
translated at low level (FtsQ 6 FtsA) from separate ribosome binding sites (RBS) on a common mRNA while
FtsZ is translated at a higher level [41]. The low rates of
synthesis of FtsQ and FtsA result from the low binding
a¤nity of RNA polymerase to their ribosome binding
sequences. Replacing the upstream sequences with the
RBS and initial few codons of an e¤ciently translated
gene, gene 10 of phage T7, resulted in the production of
up to 25-fold more protein [41].
A short coding gap exists between the stop codon of
ftsA and the start codon of ftsZ. The presence of this
region in high copy blocks cell division at elevated temperature [42]. It has been suggested that this may result from
the production of an antisense RNA (stfZ), or even a
small inhibitor protein from the sequence, although the
molecular basis of the inhibition remains unclear. There
is a precedent for the action of an antisense RNA in po-
Cyaan Magenta Geel Zwart
S.J. Dewar, R. Dorazi / FEMS Microbiology Letters 187 (2000) 1^7
tentiating production of FtsZ protein, since the mRNA
product of the dicF gene inhibits the synthesis of FtsZ in
a similar fashion [43]. DicF, thought to be part of the
lamboid phage relic Kim, encodes a 53-nucleotide
mRNA that blocks cell division by binding to, and inhibiting translation of, ftsZ mRNA. The function of the dicA
operon, of which dicF is a part, may have been to stop cell
division during lytic multiplication of the phage; it has no
discernible physiological role in the cell under normal
growth conditions [44]. If this is the case, then the partial
complementarity of dicF to the RBS region of ftsZ mRNA
may mimic the action of a natural division inhibitor such
as stfZ. Since transcription of an inhibitor like stfZ would
speci¢cally inhibit FtsZ production, controlling its transcription might provide a route to control the timing of
cell division. However, as with some of the earlier control
mechanisms, the main problem is in identifying ``how the
regulators are themselves regulated in relation to the cell's
periodic needs for division proteins'' [45].
6. Protein regulators of mra transcription
There are con£icting reports on the autoregulation of
genes at the mra locus. Transcription from the promoters
within ftsQ and ftsA has been shown to be derepressed in
cells lacking FtsA, leading to the suggestion that FtsA
may act as a direct repressor of one or both of these
promoters [17], although this was later challenged by Robin et al. [27]. Dai and Lutkenhaus [10] established that
ftsZ is not autoregulated within the ftsA^Z intergenic region, an observation that has been extended to include the
entire mra cluster [43]. Transcription from the ftsAp promoter within ftsQ has been shown to be in£uenced by
upstream sequences that form an inverted repeat [7]; however, while these sequences may constitute a target for a
protein regulator, no speci¢c binding proteins interacting
with them have yet been described. The product of the
unlinked sdiA gene up-regulates the ftsQ2p promoter
and its overexpression results in a small increase in the
production of FtsZ protein [46]. However, sdiA itself is
down-regulated by a signalling molecule that is released
into the medium during E. coli cell growth, suggesting
that cell-to-cell signalling may play a role in regulating
division gene expression [47]. SdiA is not essential, because
deletion of the gene has no obvious deleterious e¡ect on
cell growth or division. The cellular alarmone guanosine
tetraphosphate (ppGpp) has also been implicated as a possible transcriptional e¡ector of ftsZ expression during the
cell cycle (reviewed in [48]). Elevated levels of ppGpp suppress the ¢lamentation of an ftsZ84 strain at high temperature by increasing cellular FtsZ levels. It has been suggested that the down-regulation of upstream promoters by
ppGpp may allow a concomitant increase in transcription
from occluded promoters lying closer to ftsZ [49]. However, the mechanism may be rather more complex since
FEMSLE 9357 17-5-00
5
Navarro et al. [50] were unable to discern any direct e¡ect
of ppGpp level on any of the ftsQAZ promoters.
The product of another unlinked gene, mreB, represses
transcription of ftsI [51]. In the absence of this regulator,
cells become spherical as a result of overproduction of
PBP3 and possibly the genes adjacent to ftsI. Evidence
has also been presented to show that transcription from
an ftsQ^ftsA^lacZ fusion is induced ¢ve- to six-fold by
overproduction of the rcsB gene product [52]. The rcsB
gene was identi¢ed as a suppressor of the ftsZ84 division
mutant, the suppression being e¡ected by increased transcription from ftsAp to an unknown cellular stimulus [53].
RcsB is part of a two-component system that is involved
in the activation of capsular synthesis and the biosynthesis
of colanic acid, the connection between these pathways
and cell division remains to be elucidated.
7. Concluding remarks
The cell division proteins are required exclusively for the
biosynthesis of the septum, and as such are required at
only very speci¢c times in the cell cycle. The elaborate
regulation of these proteins has not yet been fully de¢ned
and we are still some way from understanding how their
activities are controlled within the cycle. Pre-existing proteins may be activated in response to an as yet uncharacterised cellular e¡ector; or alternatively, transcription
(and/or translation) of the division genes may be periodic.
Clearly, a complex regulatory network co-ordinates expression of the division genes within the mra operon,
but it is not entirely apparent why this should be so. A
simple reason might be that some redundancy has evolved
within the regulatory mechanisms and this provides a bu¡er to protect what is an essential cellular process from the
deleterious e¡ects of mutation. Alternatively, they may
provide a variety of targets that respond to regulation
by intra- or extracellular signals, allowing the modulation
of cell growth in response to other cellular processes. It
seems likely that the coming decade will uncover yet more
unanticipated aspects governing their control. One of the
most exciting challenges will be to develop methods to
monitor these events in real time, as integral aspects of
the dynamic processes of cell growth and division.
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