Download Direct interaction of FtsZ and MreB is required for septum synthesis

The EMBO Journal (2013) 32, 1953–1965
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THE
EMBO
JOURNAL
Direct interaction of FtsZ and MreB is required
for septum synthesis and cell division in
Escherichia coli
Andrew K Fenton and Kenn Gerdes*
Centre for Bacterial Cell Biology, Institute for Cell and Molecular
Biosciences, Baddiley-Clark Building, Medical School, Newcastle
University, Newcastle upon Tyne, UK
How bacteria coordinate cell growth with division is not
well understood. Bacterial cell elongation is controlled by
actin–MreB while cell division is governed by tubulin–
FtsZ. A ring-like structure containing FtsZ (the Z ring) at
mid-cell attracts other cell division proteins to form
the divisome, an essential protein assembly required for
septum synthesis and cell separation. The Z ring exists at
mid-cell during a major part of the cell cycle without
contracting. Here, we show that MreB and FtsZ of
Escherichia coli interact directly and that this interaction
is required for Z ring contraction. We further show that
the MreB–FtsZ interaction is required for transfer of cellwall biosynthetic enzymes from the lateral to the mature
divisome, allowing cells to synthesise the septum.
Our observations show that bacterial cell division is
coupled to cell elongation via a direct and essential
interaction between FtsZ and MreB.
The EMBO Journal (2013) 32, 1953–1965. doi:10.1038/
emboj.2013.129; Published online 11 June 2013
Subject Categories: cell & tissue architecture; microbiology &
pathogens
Keywords: cell division; cell elongation; FtsZ; MreB; septum
Introduction
Actin and tubulin homologues are essential for the coordination of internal processes in all cells. These common and
conserved proteins form structural elements that make up the
cytoskeleton, a dynamic protein network that orchestrates
many cellular processes, including protein recruitment and
trafficking, cell motility and cell division in both eukaryotic
and prokaryotic organisms (Erickson, 2007; Fletcher and
Mullins, 2010). To gain a deeper insight into the cellular
functions of cytoskeletons, it is important to understand
how activities of both actin and tubulin families of proteins
are coordinated. In eukaryotes, actin is regulated by tubulin
microtubules that carry protein factors able to alter
actin filament activities (Basu and Chang, 2007), while in
prokaryotes such interactions through additional protein
factors have not been described. The concept of a
*Corresponding author. Centre for Bacterial Cell Biology, Institute for
Cell and Molecular Biosciences, Baddiley-Clark Building, Medical
School, Newcastle University, Richardson Road, Newcastle
upon Tyne NE2 4AX, UK. Tel.: þ44 (0)191 2225318;
Fax: þ44 (0)191 2227424; E-mail: [email protected]
Received: 15 February 2013; accepted: 10 May 2013; published
online: 11 June 2013
& 2013 European Molecular Biology Organization
prokaryotic cytoskeleton arose from the discovery of two
essential proteins: FtsZ and MreB, which are homologues of
tubulin and actin capable of forming macro-molecular
structures (Lutkenhaus et al, 1980; Wachi et al, 1987;
Bork et al, 1992; De Boer et al, 1992). FtsZ and MreB are
essential for cell division and shape maintenance respectively
(Bi and Lutkenhaus, 1991; Jones et al, 2001). After a decade
of research on MreB and over two on FtsZ in many bacterial
species, it is clear that a general theme of organising cell
morphology via an essential underlying cytoskeletal structure
is an approach taken by most bacterial cells (Michie and
Löwe, 2006; Typas et al, 2012). However, it is uncertain how
the cytoskeleton interacts with the different components of
the cell to carry out this function.
MreB is widely conserved among rod-shaped bacteria and
interacts with the cell-wall elongation machinery through a
membrane-spanning complex comprising of MreC, MreD
and RodZ (Divakaruni et al, 2005; Kruse et al, 2005; Shiomi
et al, 2008; Bendezú et al, 2009; White et al, 2010). This
complex acts together with the peptidoglycan (PG) cell-wall
biosynthetic enzymes to produce lateral PG, thereby giving
bacterial rods their characteristic shapes. Pioneering work
suggested that MreB formed a continuous helical pattern on
the inside face of the cytoplasmic membrane (Jones et al,
2001). However, higher-resolution microscopy has indicated
that MreB forms short filamentous structures in vivo
(Domı́nguez-Escobar et al, 2011; Garner et al, 2011;
Van Teeffelen et al, 2011). Biochemical characterisation of
MreB has been difficult; although this family of proteins
can form filaments in vitro, their dynamics are far from
fully understood. However, solved crystal structures have
provided insights into the biochemical properties of MreB
including its establishment as a true actin homologue and
defining the MreB–RodZ interaction surface (Van den Ent
et al, 2001, 2010). MreB proteins from different species are
known to directly interact with the cytoplasmic membrane,
though the exact mechanism of binding varies between
species (Salje et al, 2011). In E. coli, MreB has an
N-terminal amphipathic helix that directly anchors MreB
filaments to the inner leaflet of the cytoplasmic membrane
(Salje et al, 2011).
FtsZ polymers, in conjunction with FtsA, ZapA, ZapB and
other condensation factors, form a ring-like macro-molecular
complex at mid-cell called the Z ring (De Boer, 2010).
The regulated assembly of this structure is responsible for
the spatial and temporal coordination of bacterial cell
division. Recruitment of additional cell division factors to
the Z ring occurs in an ordered mostly linear pattern, each
protein requiring the localisation of upstream factors for its
localisation forming a mature divisome complex (De Boer,
2010). Interestingly, the Z ring forms early in the cell cycle
and persists without contracting until late in the cell
cycle, indicating that maturation of the divisome occurs in
The EMBO Journal
VOL 32 | NO 13 | 2013 1953
MreB–FtsZ interaction is essential in E. coli
AK Fenton and K Gerdes
two steps (Aarsman et al, 2005); the first being Z ring
establishment, the second, downstream factor recruitment
and Z ring contraction. The signal or events that trigger Z ring
contraction are unknown.
When the divisome is complete, it provides the biochemical and mechanical activities required for septal
PG synthesis, septation and separation (De Boer, 2010). In
addition to cell division, the Z ring has an emerging
secondary role in pre-septal PG synthesis, prior to synthesis
of the septum (De Pedro et al, 1997; Aaron et al, 2007; Varma
et al, 2007). The enzymatic requirements for pre-septal PG
synthesis are not fully understood, although this process
requires ZipA and seems to depend on the activity of
penicillin-binding protein (PBP)2 rather than PBP3
(Wientjes and Nanninga, 1989; Varma and Young, 2009;
Potluri et al, 2012).
The mreB gene is essential in E. coli under conditions that
support rapid cell growth; however, it is possible to physiologically suppress the requirement for mreB by overexpression of FtsZ (Kruse et al, 2005; Bendezú and de Boer, 2008).
Cells conditionally suppressed in this way grow as irregular
spheres as they have lost the mechanism ensuring lateral PG
incorporation (Kruse et al, 2005). The mechanism for this
suppression is unclear, although it has been suggested that
overexpression of FtsZ allows formation of Z rings, which
will physically reach around the diameter of spherical cells
(Kruse et al, 2005). Alternatively, additional FtsZ could help
overcome membrane perturbation events brought about by
uncoupling of membrane biosynthesis rates with cell volume
in mreB mutants (Bendezú and De Boer, 2008).
Several indirect observations have raised the possibility
that MreB plays a role in cell division. On a morphological
level, compromising MreB function in bacterial cells
gives both cell elongation and division phenotypes (Wachi
and Matsuhashi, 1989; Fenton et al, 2010). The most direct
evidence to suggest an involvement of MreB in division
comes from Immuno-Fluorescence Microscopy (IFM)
studies of Caulobacter crescentus, indicating that MreB
forms ring-like structures at mid-cell that colocalise with
the Z rings (Figge et al, 2004). In E. coli, MreB also
localises as bands or rings, often positioned at mid-cell in
pre-divisional cells (Vats and Rothfield, 2007; Vats et al,
2009). However, independent studies using IFM and GFPlabelled MreB did not observe ring-like MreB structures
(Karczmarek et al, 2007; Swulius and Jensen, 2012).
Moreover, different fusions between MreB and fluorescent
proteins yield different subcellular patterns (Swulius and
Jensen, 2012). Thus, the localisation pattern of MreB
relative to FtsZ has not been firmly established in E. coli.
Here, we report a novel role of MreB in bacterial cell
division using E. coli as the model organism. Microscopic
observations validate previous suggestions that MreB is
recruited to mid-cell and we comprehensively describe
MreB dynamics in living cells. We show that MreB is
recruited to the septum in virtually all cells via a direct
interaction with FtsZ. We identify a mutation in MreB that
removes the interaction with FtsZ and simultaneously blocks
cell division. Remarkably, a single amino-acid (aa) change in
FtsZ simultaneously restores the interaction with and
suppresses the division defect of the MreB variant. Using
fluorescently tagged cell-wall biosynthetic enzymes, we discovered that inhibition of cell division was correlated with
1954 The EMBO Journal VOL 32 | NO 13 | 2013
the lack of recruitment of PBPs 1B and 2 to the Z ring. Our
data support a model in which MreB delivers PBP1B and 2,
and perhaps additional factors to the Z ring, thereby generating a link between cell elongation and division in bacteria.
Results
MreB is recruited to the Z ring
To study MreB protein dynamics, we generated a functional
mYpet–MreB fusion protein (Supplementary Materials and
methods) and expressed it in wild-type (wt) E. coli MG1655
cells at a level that did not affect growth rate or cell morphology (Supplementary Figure S1A). These cells had B6% of
the total MreB pool labelled with mYpet (Supplementary
Figure S1B). Addition of mYpet–MreB in this way had no
impact on wt MreB protein levels and was therefore considered a phenotypically neutral cytoskeleton-labelling method
(Supplementary Figure S1B).
Our mYpet-labelling method revealed that MreB formed
ring-like structures in addition to the punctate pattern present
along the cell periphery (Figure 1A). The ring-like patterns
only appeared at mid-cell in cells undergoing division. MreB
structures at mid-cell colocalised with Z rings labelled with
an FtsZ–mCherry fusion protein (Figure 1B). These MreB
bands were present at all stages of cell invagination and were
never observed independently of Z rings, raising the possibility that Z rings recruit MreB (see Movie in Supplementary
Figure S9). Examining and scoring this colocalisation
revealed that 75% of Z rings had overlapping MreB bands
(449 Z rings scored, n ¼ 11).
To observe the localisation pattern of unlabelled native
MreB and compare it to the mYpet–MreB observations we
used IFM. E. coli cells were chemically fixed and polyclonal
anti-MreB antibodies used to detect localisation patterns (see
Supplementary Figure S1E for western blot). IFM revealed a
very similar punctuated MreB localisation pattern along
the cell periphery interrupted by MreB bands at mid-cell
(Supplementary Figure S1C). Ring-like IFM signals colocalised with 68% of FtsZ–mCherry-labelled Z rings
(Supplementary Figure S1D) and are thus consistent with
the mYpet–MreB labelling (117 Z rings scored, n ¼ 3).
We also studied a functional fluorescent MreB sandwich
fusion encoded by mreB–rfpSW, which had been successfully
introduced into the E. coli chromosome by allelic replacement
in strain FB76 (Bendezú et al, 2009). MreB–RFPsw showed
localisation patterns at mid-cell very similar to those described above (Supplementary Figures S1F and G).
Using temperature-sensitive mutant strains revealed that
mYpet–MreB could be recruited to Z rings consisting of only
FtsZ and other essential condensation factors, without the
additional downstream septation factors such as FtsA
(Supplementary Figure S2). These findings were consistent
with previous work; however, they did not reveal anything
about interaction dynamics (Vats and Rothfield, 2007).
Time-lapse fluorescent microscopy showing mYpet–MreB
behaviour revealed that MreB was rapidly recruited to newly
formed Z rings (within 2.5 min) and frequently remained
colocalised with Z rings throughout the entire cell division
period (see Movie in Supplementary Figure S9, stills from the
time lapse are shown in Figure 1C). Additionally in cells
showing no previous Z-ring–MreB colocalisation, MreB was
recruited to Z rings at the time of ring establishment
& 2013 European Molecular Biology Organization
MreB–FtsZ interaction is essential in E. coli
AK Fenton and K Gerdes
A
of cells (Supplementary Figure S10C). Imaged in this way,
virtually all newly formed Z rings showed initial MreB
colocalisation; colocalisation either persisted through cell
division or in a minority of cases dispersed away from
the closing septum.
Brightfield
mYpet-MreB
B
C
Brightfield
mYpet-MreB
FtsZ-mCherry
Brightfield
mYpet-MreB
FtsZ-mCherry
Merge
Merge
t = 00:00
t = 10:00
t = 22:30
Figure 1 MreB is recruited to the FtsZ ring. (A) Representative
bright field and fluorescent micrographs of MG1655 expressing
the mYpet–MreB fluorescent protein from pAKF106 (PBAD::mYpet–
mreBCD). Examples of both non-dividing and dividing cells at
different stages of cell division are shown. (B) Dual-label fluorescence micrographs of MG1655 expressing both the mYpet–MreB
fusion from pAKF106 and FtsZ–mCherry from pQW59 (Plac::
ftsZ–mCherry). Merged images reveal signals colocalised at midcell. Using this technique, MreBwt–FtsZ colocalisation frequency
was 75% (499 Z rings scored, n ¼ 11). (C) Selected images from a
time-lapse series following a single MG1655 cell expressing the
same fluorescent proteins shown in B. Arrows indicate the location
and colocalisation of major MreB foci and FtsZ rings. The full timelapse series and additional MreB–FtsZ colocalisation examples are
shown in Supplementary Figures S9 and S10. All cells were grown
in M9 glucose media with 0.05% arabinose inducer at 301C 3 h prior
to imaging (see Supplementary Figure S1A and B for growth curves
and western blot). Induction of the pQW59 plasmid was not
required to give Z rings labelled with FtsZ–mCherry. All images
shown are representative of at least three independent repeats.
Scale bars ¼ 2 mm.
(Supplementary Figure S10A). Spontaneous Z ring relocation
events also led to MreB signal migration to the new division
site (Supplementary Figure S10B). Finally, MreB bands were
seen to disperse away from established Z rings in a minority
& 2013 European Molecular Biology Organization
MreB interacts with FtsZ
Using a Bacterial Two-Hybrid (BTH) approach (Karimova
et al, 2005), we screened a series of known cell division
factors for interaction with two full-length MreB fusions
(T25–MreB and T18–MreB). This included all fusions
reported by Karimova et al (2005) and full-length FtsZ
labelled at both termini (Figure 2A). Initially, this approach
revealed a cryptic pattern of tagged MreB protein interaction
signals, some of which were strong (Figure 2A). The interaction pattern varied considerably between the two fusion
series (compare panels (i) and (ii) in Figure 2A), raising the
possibly that some of the signals were artefacts of the reporter
technique.
The BTH assay is known to be particularly sensitive
to detecting interactions between membrane-associated
proteins (Karimova et al, 2005). MreB of E. coli interacts
with the cell membrane via its N-terminal amphipathic helix.
Deletion of this helix effectively abolished the ability of MreB
to bind to the membrane (Salje et al, 2011). Therefore, we
repeated the interaction study with MreB variants devoid of
the N-terminal amphipathic helix (Figure 2A, MreBDN panels
of (i) and (ii)). A striking and similar effect was observed for
both interaction series; only the single interaction signals
between the soluble MreB and FtsZ were present in both
cases (Figure 2A). This result was consistent with previous
yeast two-hybrid studies suggesting a direct MreB–FtsZ interaction (Tan et al, 2011).
Using tap tagging, previous large-scale protein-interaction
studies in E. coli identified potential interaction partners for
FtsZ and MreB. Although other protein targets were found,
these studies identified a putative MreB–FtsZ interaction
(Butland et al, 2005; Hu et al, 2009). Here, we used in vivo
crosslinking to validate the MreB–FtsZ interaction suggested
by our BTH analysis. Wild-type cells expressing N-terminally
His-tagged MreB were treated with formaldehyde, a
chemical crosslinker with a very short spacer arm (2.3–
2.7 Å) (Ishikawa et al, 2006). Crosslinked complexes were
purified, dissociated and subjected to western blot analysis
(Figure 2D). This revealed that His–MreB was able to pull out
native FtsZ from wt cells, consistent with previous studies.
Thus, two independent methods support that MreB and FtsZ
interact.
MreBD285Adoes not interact with FtsZ and blocks cell
division
By alignment of diverse MreB sequences, we identified a
number of conserved residues located outside the five elements that form the canonical actin nucleotide-binding
pocket (Bork et al, 1992) and changed them to alanine.
These MreB variants were tested using the BTH assay. As
seen from Figure 2B, changing the aspartate at position 285 to
alanine severely reduced the interaction signal with FtsZ,
whereas the interaction signals with wt MreB, MreC and
RodZ were unchanged (Figure 2B and C). Using both
MreBD285A fusions in the BTH assay showed that self-binding
was maintained (Supplementary Figure S3A). Mapping of
The EMBO Journal
VOL 32 | NO 13 | 2013 1955
MreB–FtsZ interaction is essential in E. coli
AK Fenton and K Gerdes
+v
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–v
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Figure 2 MreB interacts directly with FtsZ and this interaction is abolished in the MreBD285A variant. (A) Bacterial two-hybrid (BTH) screen of
E. coli cell division factors against MreB. FtsZ has been labelled in both the N-terminus (N) and C-terminus (c). An MreB variant with a 9 aa
N-terminal truncation (DN) is also included in this screen. Both T25- and T18-fusion orientations are shown for each BTH combination (i) and
(ii). (B) BTH plate showing full-length T18-labelled MreB and MreBD285A against T25-labelled FtsZ (c) and known MreB interaction partners
essential for cell elongation. The BTH101 strain was used for all BTH cloning, a full list of plasmid vectors used for this screen
can be found in Supplementary Table SI. Cells were spotted onto NA plates containing ampicillin (50 mg/ml), kanamycin (25 mg/ml) and
X-gal (40 mg/ml). Plates were incubated for 48 h at 301C prior to imaging. (C) BTH b-galactosidase activity assays showing T18-labelled MreB
and D285A variant against T25-labelled FtsZ and MreBwt. Error bars ¼ s.d., n ¼ 4. In all cases, BTH ve control strains contained empty
pKNT25 and pUT18 vectors and þ ve control strains contained the pKT25-zip and pUT18C-zip fusions. (D) Western blots of His-tagged MreB
and MreBD285A formaldehyde crosslink and pull-down preparations, using both anti-FtsZ and anti-MreB primary antibodies. MC1000 cells
containing either pTK500 (Plac::His8–mreB) or pAKF126 (Plac::His8–mreBD285A) were grown to an OD600 of E0.3 without induction, crosslinked
with 1% formaldehyde and quenched after 10 min using 150 mM glycine. Resulting crosslinked complexes were affinity purified using the
MagneHisTM suspension, crosslinks were reversed by incubation at 951C for 1 h and complexes analysed. Equal volumes of protein complex
preparations, un-crosslinked His–MreB and His–MreBD285A preparations and purified His–MreB (200 ng) and native FtsZ (100 ng) proteins
were used as controls, n ¼ 4. MreB and FtsZ antibody affinity control western blots are shown in Supplementary Figure S3B.
Asp285 on the Thermotoga maritima MreB crystal structure
showed it to locate in subdomain IIA, near the predicted
proto-filament interface and away from the RodZ interface
(Van den Ent et al, 2001, 2010) (Supplementary Figure S3C).
Together, these results showed that MreBD285A could still
1956 The EMBO Journal VOL 32 | NO 13 | 2013
interact with the cell-wall elongation complex and that the
reduced BTH interaction with FtsZ was not caused by
reduced protein stability or misfolding.
To validate the BTH result, the MreBD285A variant
was included in the in vivo crosslinking assay (Figure 2D).
& 2013 European Molecular Biology Organization
MreB–FtsZ interaction is essential in E. coli
AK Fenton and K Gerdes
As seen, the MreBD285A –FtsZ interaction was severely
reduced (41000-fold). We conclude that the aspartate
at þ 285 in MreB is essential for the MreB–FtsZ interaction.
To assess the phenotype of the MreBD285A variant, we took
two complementary approaches. In the first, we used
an MreB depletion system to study the MreBD285A variant
phenotype in the absence of wt MreB (Kruse et al, 2005). The
depletion system utilises a DmreBCD strain complemented in
trans by a low-copy R1 plasmid carrying Pmre::mreBCD.
Depletion of the complementing plasmid was triggered by
induction of the anti-sense RNA copA (under Plac control),
which rapidly (within E10 min) blocks R1 plasmid
replication (Supplementary Figure S4A). Both the plasmid
and the MreBCD proteins were depleted by the natural turnover and dilution in growing E. coli populations with full loss
of MreB signal on western blots taking E1 h (Supplementary
Figure S4B). Complementation by MreBCD or MreBD285ACD
was provided by a third plasmid carrying the test genes under
control of the PBAD promoter (see Supplementary Figure S4A
for a diagram of the experimental setup).
As expected, the R1 (Pmre::mreBCD) plasmid complemented the mreBCD deletion. Moreover, the PBAD::mreBwtCD or
PBAD::mreBD285ACD plasmids had no effect on cell morphology or growth without induction (Figure 3A, left panels).
Also as expected, depletion of MreBCD without the replenishment of MreBCD from the test plasmid led to the formation of rounded spherical cells (Figure 3A, middle panels),
and rod-shape morphology was maintained by replacement
with wt MreBCD, with a minor increase in cell width
(Figure 3A, upper right panel). In contrast, cells expressing
the MreBD285ACD variant in the absence of wt MreBCD
exhibited extreme lengthening (Figure 3A, lower right
panel). These experiments demonstrated that MreBD285A
supported cell elongation but inhibited cell division. Also,
under all conditions tested, the MreBD285A variant was unable
to complement MreB depletion in liquid or on plates, and was
thus incompatible with long-term cell viability.
In the second approach, we compared ‘low level’ overexpression of MreB and MreBD285A, together with MreCD, in
wt cells. Overexpression was carefully controlled in these
experiments to not perturb cell viability, cell width or growth
rate (Supplementary Figures S3D and S4C, D). Interestingly,
such mild expression of MreBD285A led to a 45% increase in
average cell length, suggesting that cell division was inhibited
(Figure 3B). In some 17% of such cases, this inhibition
was very strong, resulting in cells greater than 18 mm in
length (Figure 3B(i) and Supplementary Figure S4D).
Statistical analysis showed that expression of MreBD285A but
not wt MreB significantly increased the average cell length
(Figure 3B(ii)). This supported the result from the depletion
study and gave us a tractable genetic system to investigate the
MreBD285A elongation phenotype.
MreBD285Ais recruited to MreB assemblies but not
to the Z ring
To investigate how the MreBD285A variant exerted its
dominant-negative effect in cells also expressing wt MreB,
we introduced the D285A allele into our mYpet–mreB
construct. In wt cells, mYpet–MreBD285A exhibited a typical
MreB localisation pattern with lateral patchy foci,
suggesting that MreBD285A was capable of entering MreB
assemblies (Figure 3C). Double labelling by expression of
& 2013 European Molecular Biology Organization
mYpet–MreBwt and mYpet–MreBD285A in FB76 (mreB–RFPsw)
showed that the fusion proteins colocalised, thereby confirming that the D285A variant entered MreB assemblies
(compare left and right panels of Figure 3D).
Next, we investigated the MreBD285A localisation pattern
relative to FtsZ using an FtsZ–mCherry fusion protein
and the same MreB-labelling strategy as shown in
Figure 1B. Statistical analysis revealed a significant reduction
in MreB–Z-ring recruitment from 75% for MreBwt to 28% for
the MreBD285A variant (414 Z rings, n ¼ 7, P40.01). Although
this represents a large drop in colocalisation, the 28% proportion of colocalisation exhibited by MreBD285A is probably
an overestimate of MreBD285A–Z-ring recruitment, as this
variant could be recruited indirectly to the Z ring through
its interaction with untagged wt MreB at the septum.
Mutations in ftsZ restore the FtsZ–MreB BTH interaction
signal
Using an error-prone variant of Pfu polymerase, Pfu(exo )
D473G, we PCR amplified the ftsZ open reading frame (ORF)
and used the BTH assay to identify mutations in ftsZ that reestablished the interaction with MreBD285A (Figure 4A,
Supplementary Figure S5A) (Biles and Connolly, 2004). This
screening generated five single aa changes in FtsZ that robustly
restored the MreBD285A–FtsZ interaction signal. All five FtsZ
variants interacted with wt FtsZ and all but one (FtsZP203Q)
could still bind wt MreB (summarised in Figure 4A). When
mapped onto the FtsZ crystal structure from Methanococcus
jannaschii and Staphylococcus aureus all five aa changes
clustered to a discrete patch on one face of FtsZ (Figure 4B).
To test the functionality of the FtsZ variants in the absence
of wt FtsZ, we used the FtsZ depletion strain VIP205 (Garrido
et al, 1993). Depletion of FtsZ reproducibly generated a E106
reduction in cell viability, which could be fully
complemented by inducing wt ftsZ in trans (Supplementary
Figure S5B). Three of the five FtsZ variants: P203R, T296A
and M302L fully complemented FtsZ depletion giving the
same CFU as the wt ftsZ control. One variant, D301N,
partially complemented the depletion giving a 100-fold drop
in CFU. Only FtsZP203Q did not complement FtsZ depletion
(Supplementary Figure S5B, summarised in Figure 4A).
Strikingly, FtsZP203Q was the variant that no longer gave a
positive-BTH interaction signal with MreBwt (Figure 4A,
Supplementary Figure S5A). When used in a complementation assay, the FtsZP203Q variant elongated cells, raising the
possibility that inhibition of cell division occurred through a
mechanism similar to that of MreBD285A -induced elongation
(Supplementary Figure S5D). Most importantly, the existence
of aa changes in FtsZ that restore the interaction with MreB
showed that the MreB–FtsZ interaction was direct.
FtsZP203Q rescues the MreBD285A phenotype
Expression of mreBD285A in wt cells led to cell elongation
through blockage of cell division (Figure 3A) and reduced the
number of cells in a growing culture compared to a control
culture expressing MreBwt (compare lines 1 and 3 in
Figure 4C, see also Supplementary Figure S5C). Taking
advantage of this reduction in viable counts, we tested the
FtsZ variants for rescue of the MreBD285A phenotype in the
FtsZ depletion strain VIP205. FtsZP203Q was a particularly
important variant to test as it was the only one that interacted
with MreBD285A but not with MreBwt (Figure 4A).
The EMBO Journal
VOL 32 | NO 13 | 2013 1957
MreB–FtsZ interaction is essential in E. coli
AK Fenton and K Gerdes
Un-induced
A
Depleted
Replaced
mreBCD
mreBD285ACD
Un-induced
B
Induced
*
20.00
(i)
18.00
(ii)
16.00
Cell length (μm)
mreBCD
14.00
12.00
10.00
o
8.00
o
6.00
4.00
mreBD285CD
2.00
0.00
_
+
Empty vector
control
_
+
mreBCD
Over expression
_
+
mreBD285ACD
Over expression
C
Brightfield
mYpet-MreB
Merge
Brightfield
mYpet-MreBD285A
Merge
D
Brightfield
mYpet-MreB
MreB-RFPSW
Brightfield
mYpet-MreBD285A
MreB-RFPSW
Figure 3 The MreBD285A variant inhibits cell division but not cell elongation. (A) Representative brightfield micrographs showing the MC1000
MreBCD depletion strain (MC1000 Dmre, pKG339, pTK554). Images show cells without MreB depletion ‘un-induced’, depleted of MreB
‘depleted’ or complemented with either mreBCD or mreBD285ACD expressed from pAKF128 (PBAD::mreBCD) or pAKF129 (PBAD::mreBD285ACD)
‘replaced’. Cells were grown in LB þ tetracycline (20 mM), 2 mM IPTG and 0.2% arabinose for 3 h at 301C prior to imaging. See Supplementary
Figure S4A for diagram of depletion system. (B) MC1000 cells containing pAKF128 or pAKF129. Cells were grown in M9 medium with
0.000,05% arabinose inducer at 301C for 5 h. (i) Representative bright field micrographs of cells with and without induction (ii) Box and
Whisker plot showing cell length measurements with mreBCD and mreBD285ACD ‘overexpression’ compared to an empty vector control
(pBAD24). Central line ¼ mean length, box ¼ ± s.d. and whiskers ¼ maximum/minimum lengths observed. *t-test ¼ P40.01, o ¼ no significant
difference between populations. 670 cells were sampled for each condition, n ¼ 3. For cell width measurements and a full series of control
images, see Supplementary Figure S4C and D. (C) Representative brightfield and fluorescent micrographs of MC1000 carrying pAKF131
(PBAD::mypet–mreBCD; left panel) or pAKF132 (PBAD::mypet–mreBD285ACD; right panel), n ¼ 3. Note the septal localisation of the mYpet–MreB
versus the punctate localisation pattern of mYpet–MreBD285A. (D) mYpet–MreB (pAKF131) and mYpet–MreBD285A (pAKF132) colocalise with
the MreB–RFPSW in the FB76 strain. Arrows have been used to highlight selected colocalising foci, n ¼ 3. Scale bars ¼ 5 mm
(A, B) and 2 mm (C, D).
1958 The EMBO Journal VOL 32 | NO 13 | 2013
& 2013 European Molecular Biology Organization
MreB–FtsZ interaction is essential in E. coli
AK Fenton and K Gerdes
Cells were depleted of FtsZwt and complemented by
induction of ftsZ from a plasmid (pAKF135-Ptet::ftsZ, or
pAKF137-Ptet::ftsZP203Q) induced at a level that did not
seriously alter cell morphology. Expression of MreB or
MreBD285A was induced in these strains from pAKF128
(PBAD::mreBCD) or pAKF129 (PBAD::mreBD285ACD). After 8 h
of growth in M9 media, cells were plated on solid medium
containing antibiotics, IPTG (to replenish wt FtsZ), but
A
without induction of the plasmid-borne ftsZ and mreBCD
genes (Figure 4C, Supplementary Figure S5C).
Expression of FtsZP203Q plus MreBwt reduced viable
counts four-fold (Figure 4C, compare MreBwt expression
with FtsZ and FtsZP203Q complementation of VIP205).
Critically, in cells expressing MreBD285A the pattern was
reversed: FtsZP203Q expression increased viable counts by
nearly 200-fold compared to FtsZwt (Figure 4C, compare
MreB
binding
MreBD285A
binding
FtsZ
binding
ΔftsZ
complementation
FtsZ
++
–
++
++
FtsZP203R
++
+
++
++
FtsZP203Q
–
+
+
–
FtsZT296A
++
+
++
++
FtsZD301N
++
+
++
+
FtsZM302L
++
+
++
++
B (i)
(ii)
C
Strain
mreB allele
used for
overexpression
ftsZ allele
used for VIP205
complementation
Average CFU/ml after
MreB overexpression
Corrected fold
complementation
VIP205/pAKF128/pAKF135
MreBWT
FtsZWT
3 ×106
1
VIP205/pAKF128/pAKF137
MreBWT
FtsZP203Q
7 ×105
0.22
VIP205/pAKF129/pAKF135
MreBD285A
FtsZWT
4 ×104
1
VIP205/pAKF129/pAKF137
MreBD285A
FtsZP203Q
8.4 ×106
193
Figure 4 Single aa changes in FtsZ restore functional interaction with MreBD285A. (A) Table summarising the phenotypes of ftsZ point mutants
identified in the ftsZ–mreBD285A PCR mutagenesis screen. Bacterial two-hybrid (BTH) scores represent signal strength from BTH101 double
transformations, plated on NA plates with selective antibiotics and X-gal (see Supplementary Figure S5A for images of plates); þ þ ¼ strong
binding signal, þ ¼ weaker binding signal, ¼ no detectable signal. Details of plasmids used in these assays can be found in Supplementary
Table SI. ‘DftsZ complementation scores’ are representative of CFU measurements shown in Supplementary Figure S5B; þ þ ¼ full
(comparable to ftsZwt complementation control), þ ¼ partial (100 drop in CFU compared to ftsZwt control) and ¼ no complementation
(same CFU as ve empty vector contol). Complementation was assayed using the VIP205 (MC1061: Ptac::ftsZ) strain complementing with
Ptet::ftsZ (pAKF135) constructs containing the identified point mutations (pAKF136-140), the full list of plasmids can be found in
Supplementary Table SI. Note that only the FtsZP203R variant has no detectable MreB binding and cannot complement the VIP205 ftsZ
depletion strain. (B) Structural model of an FtsZ dimer of M. jannaschii (i) and S. aureus (ii) (Oliva et al, 2004; Matsui et al, 2012) showing the
positions of the aas in FtsZ (purple), changes to which can restore the interaction with MreBD285A. The two monomers of the FtsZ dimer are
shown in different shades of orange. GTP molecules are shown in blue. Identification of the equivalent aa positions in E. coli FtsZ were based
on a sequence alignment of FtsZ (not shown). (C) FtsZP203Q can rescue MreBD285A expression. E. coli VIP205 (Ptac::ftsZ) cells were grown
without IPTG inducer giving FtsZ depletion. This was complemented by expression of FtsZ from pAKF135 (Ptet::ftsZ) or pAKF137
(Ptet::ftsZP203Q) induced using 20 ng/ml chlorotetracycline. A FtsZ depletion system was used in this assay as overexpression of ftsZ can
lead to cell elongation and thus CFU reduction. Strains contained either pAKF128 (PBAD::mreBCD) or pAKF129 (PBAD::mreBD285ACD) induced
with 0.005% arabinose for controlled MreB overexpression. Cells were grown in M9 media for 8 h, serially diluted 1/10 and used for
enumeration on NA plates with antibiotics and 30 mM IPTG, but without induction of the plasmid-borne ftsZ and mreBCD genes. These plates
gave viable counts reflecting the effect of the mreB/ftsZ-mutant combinations in the liquid media. CFU measurements are an average of three
independent repeats (n ¼ 3). Note that the expression of MreBD285A reduced CFU as cells elongate (compare CFU/ml when MreBwt or
MreBD285A is overexpressed and the FtsZwt allele is used for VIP205 complementation). Using FtsZP203Q for VIP205 complementation in cells
overexpressing MreBwt reduced cell viability four-fold (see corrected fold complementation). In contrast, FtsZP203Q expression in VIP205 cells
also expressing MreBD285A yielded a near 200-fold higher CFU (in growing cultures). Images of serially diluted plates are shown in
Supplementary Figure S5C along with plate images showing MreBD285A complementation for all the FtsZ variants identified in this study.
& 2013 European Molecular Biology Organization
The EMBO Journal
VOL 32 | NO 13 | 2013 1959
MreB–FtsZ interaction is essential in E. coli
AK Fenton and K Gerdes
FtsZwt or FtsZP203Q rescue of MreBD285A expression).
Therefore, this assay demonstrated that the division-inhibition effect of MreBD285A could be counteracted by FtsZP203Q
but not by FtsZwt.
Cells elongated by MreBD285A contain regularly spaced
‘locked’ Z rings
To investigate the elongation phenotype of cells expressing
MreBD285A, we determined localisation patterns of fluorescently labelled cellular components. FtsZ–mCherry labelling
revealed that Z rings were spaced regularly along the lengths
of cells elongated by MreBD285A expression (Figure 5A,
Supplementary Figure S7A). DNA staining using
Hoescht showed that these Z rings formed between
segregated nucleoids. Thus, cell elongation was not due to
dysfunctional chromosome segregation (Supplementary
Figures S6A and B). Together, these observations suggested
that Z ring formation and positioning were unaffected in
elongated cells, but raised the question of what protein
activities were absent in the ‘locked’ Z rings.
Locked Z rings do not support septal synthesis of
peptidoglycan
To investigate the locked Z rings, we used a recently
developed method that specifically labels sites that are
actively synthesising PG. This technique exploits the
tolerance of bacterial PG biosynthetic enzymes to accommodate D-amino acids with diverse sizes into PG. Addition of a
fluorescent tag to these aas leads to progressive labelling of
the cell wall over time, through a combination of growth and
turnover (Kuru et al, 2012). In E. coli wt cells, pulse labelling
the cell wall with the fluorescent D-aa HADA gives a very
strong incorporation signal at Z rings, weaker incorporation
at the cell periphery and very little incorporation at the
relatively inert cell poles (described in Kuru et al (2012)
and shown in Figure 5B (i)). Control cells treated with the
FtsI inhibitor aztreonam show multiple sites of HADA incorporation colocalising with Z rings along their lengths
(Supplementary Figure S6C). In contrast, in elongated
MreBD285A-expressing cells, the cell-wall regions colocalising
with the locked Z rings failed to incorporate HADA without
affecting incorporation elsewhere (Figure 5B (ii)). This result
showed that the locked Z rings did not actively synthesise PG,
while the elongation enzymes remained functional.
Cells elongated by MreBD285A display mislocalisation
of PBPs
To gain insight into the mechanism of MreBD285A-mediated
cell elongation through inhibition of Z ring-dependent PG
biosynthesis, we used a combination of fluorescent protein
tagging and time-lapse microscopy. We focused on the localisation patterns of the PBP enzymes: PBP1A, PBP1B, PBP2
and PBP3, all of which are important for cell-wall and septum
synthesis.
In wt cells, PBP1A is usually localised to the cell membrane
and is thought to principally function in cell elongation
(Vollmer and Bertsche, 2008; Banzhaf et al, 2012). In cells
expressing MreBD285A, PBP1A–mCherry formed discrete foci
at the cell periphery in both elongated and dividing
cells, consistent with the expected pattern (Supplementary
Figure S6D compared to Supplementary Figure S6E). In timelapse imaging, these foci migrated along the long axis of
1960 The EMBO Journal VOL 32 | NO 13 | 2013
elongating cells, similar to the behaviour of MreB (compare
movies of Supplementary Figure S11A and Supplementary
Figure S6D and E). Thus the localisation pattern of PBP1A–
mCherry, which functions mainly in cell elongation (Vollmer
and Bertsche, 2008), was not seriously affected by MreBD285A.
Both PBP1B and PBP2 are involved in cell elongation, but
have putative roles in cell division (Den Blaauwen et al, 2003;
Bertsche et al, 2006; Banzhaf et al, 2012; van der Ploeg et al,
2013). In wt cells both proteins localise as stable foci at
the dividing septum, in addition to migrating peripheral
foci along the cell length, reminiscent of MreB localisation
(Figures 5C and D—red arrows in control cells). Discriminating between these two behaviours was technically
challenging; here septum-associated foci were defined as
those that stayed stationary at the cell periphery for more
than three frames (7.5 min) and had a ‘partner’ focus on the
opposing side of the membrane (Supplementary Figure S11
and stills in Supplementary Figures S7B and C). In MreBD285A
elongated cells, such stable septum-like behaviours of PBP1B
and PBP2 were not observed, with both proteins forming only
longitudinally migrating foci (Supplementary Figure S11 and
stills in Figure 5C and D). In contrast, using a PBP3-specific
cell division inhibitor resulted in stationary PBP1B and PBP2
bands or ring-like foci in elongated cells (Supplementary
Figures S8B and C). This suggested that both PBP1B and
PBP2 were not successfully recruited to the locked Z rings
present in MreBD285A elongated cells.
PBP3 is the penultimate essential factor known to be
recruited to the Z ring (De Boer, 2010). Thus if PBP3 is
present at the septa, it can be considered a marker for the
localisation of all upstream cell division proteins (De Boer,
2010). In wt cells, PBP3 formed bands or rings at mid-cell
immediately prior to cell division and remained associated
with the constricting ring (Figure 5D, red arrows). In elongated MreBD285A cells, PBP3 rings were also detected, suggesting that all upstream septal proteins were present in these
locked structures (Figure 5E, white arrows). Over time, the
PBP3 rings dispersed instead of contracting (Supplementary
Figure S11).
The presence of PBP3 in locked Z rings raised the possibility that these structures contained all the essential cell
division factors. To investigate this, we studied the localisation pattern of a FtsN–mCherry fusion, which is the last
essential cell division factor (De Boer, 2010). In wt cells,
FtsN–mCherry is recruited immediately prior to septation
(Figure 5F—red arrows in control cells, S7E). Expression of
this fusion in MreBD285A elongated cells gave bands or
rings of FtsN, similar to those observed for PBP3
(Figure 5F, white arrows).
In summary, our results demonstrated that locked
Z rings recruited PBP3 and FtsN but failed to recruit PBP1B
or PBP2. These observations indicated that the FtsZ–MreB
interaction functions to deliver PBP1B and PBP2 to the Z ring.
Discussion
The discovery of a conserved bacterial cytoskeleton consisting of tubulin–FtsZ and actin–MreB was relatively recent and
no mechanisms for coordination of these factors have been
described—that is—each element has until recently been
considered largely independent of the other. Here we show
that, in E. coli, MreB interacts directly with FtsZ and that the
& 2013 European Molecular Biology Organization
MreB–FtsZ interaction is essential in E. coli
AK Fenton and K Gerdes
A
FtsZ–
mCherry
B
(i)
(ii)
FtsZ–
mCherry
HADA
C
PBP1B–
mCherry
D
PBP2–
mCherry
E
PBP3–
mCherry
F
FtsN–
mCherry
Figure 5 PBPs are mislocalised and septal PG synthesis is inhibited in MreBD285A-expressing cells. MC1000 cells elongated by expressing
MreBD285A from pAKF129 (PBAD::mreBD285ACD) containing a second plasmid expressing GFP-tagged division and elongation factors:
(A, B) FtsZ–mCherry (pAKF133, PBAD::ftsZ–mCherry), (C) PBP1B–mCherry (pAKF145, Plac::mrcB–mCherry), (D) PBP2–mCherry (pAKF146,
Plac::mrdA–mCherry), (E) PBP3–mCherry (pAKF147, Plac::ftsI–mCherry) and (F) FtsN–mCherry (pAKF150, Plac::mCherry-ftsN). In most cases
cells were grown in M9 media for 5 h at 301C with 0.000,05% arabinose. For expression from pAKF146, cells were grown in M9 glucose with
0.1% arabinose inducer. Empty vector and MreB overexpression control images are shown in Supplementary Figure S7; contrasting elongated
cells treated with PBP3-sepcific inhibitors are shown in Supplementary Figure S8. Elongated cells contain multiple evenly spaced Z rings (A).
Fixed HADA fluorescent D-aa-labelled cells (B). MC1000 pAKF133 cells containing empty pBAD24 vector (i) or elongated through MreBD285ACD
expression from pAKF129 (ii) were incubated with 1 mM HADA for 30 s, cells were fixed using 70% ethanol and washed in PBS before
microscopy. Note HADA incorporation at mature Z rings in (i—red arrows) are absent from elongated MreBD285A cells (ii—blue arrows), see
Supplementary Figure S6C for FtsI inhibitor controls. Note FtsZ, PBP1B, PBP2, PBP3 and FtsN are all recruited to the septum during normal cell
division—red arrows (Supplementary Figure S7). In MreBD285A elongated cells, PBP3 and FtsN are recruited to inhibit septa (E, F—white
arrows), whereas PBP1B and PBP2 are not (D, E). The behaviours of tagged PBP–mCherry fusions are shown in Supplementary Figure S11.
Images are representative of at least four independent repeats. Scale bar ¼ 2 mm.
interaction is essential for Z ring constriction, divisome
maturation and PG septal synthesis.
Three separate MreB-labelling approaches showed that
MreB colocalised with Z rings, in addition to adopting its
previously established punctate localisation pattern. Unlike
the patterns reported in Vats and Rothfield (2007), these ringlike patterns only appeared at mid-cell and in cells
& 2013 European Molecular Biology Organization
undergoing division. Time-lapse microscopy revealed that
MreB was recruited early to the Z ring and remained
colocalised throughout ring constriction. Our time lapses
indicated that most if not all Z rings recruited MreB at the
time of formation.
A single aa change in MreB (aspartate þ 285 to alanine)
simultaneously reduced MreB recruitment to the Z ring
The EMBO Journal
VOL 32 | NO 13 | 2013 1961
MreB–FtsZ interaction is essential in E. coli
AK Fenton and K Gerdes
(1)
FtsZ
Z ring
Z ring + cell division factors = mature divisome
FtsA
FtsK
FtsQ
ZipA
FtsB
FtsW
FtsL
FtsI
(PBP3)
(2)
MreB
FtsN
PBP1B
MreC
RodZ
MreD
PBP2
RodA
‘Elongation’ complex
PBP1A
PBP1B
MreB–FtsZ interaction delivers PBP1B
and PBP2 to the septum
PBP2
(3)
Septal-PG biosynthesis
complex
Figure 6 Model of the interaction network of cell elongation with cell division factors in E. coli. (1) FtsZ polymers at mid-cell assemble into the
Z ring. (2) The direct interaction between MreB and FtsZ (this study) allows the downstream transfer of PBP1B and PBP2 into the mature
divisome (3) (hatched arrow). This provides all the biosynthetic enzyme activities required for septal PG synthesis. Ordered assembly of the 10
essential cell division proteins was adapted from De Boer (2010). Binding structure of the elongation complex information was adapted from
Kruse et al (2005); Bendezú et al (2009); White et al (2010); Banzhaf et al (2012). FtsI-, FtsN- and PBP1B-binding information was adapted from
Müller et al (2007). FtsI-, PBP2-binding information was taken from Van der Ploeg et al (2013).
and inhibited cell division. In cells devoid of wt MreB,
inhibition of division by MreBD285A was severe, showing
that recruitment of MreB to the Z ring was essential.
MreBD285A was still capable of binding the known
MreB-interacting partners MreC and RodZ, and supported
cell elongation in an MreB depletion strain. Thus,
MreBD285A was specifically defective in supporting cell division. The aspartate at position 285 in MreB is conserved
among evolutionarily distant bacteria, raising the possibility
that interactions through this residue could be a conserved
phenomenon.
Crosslinking experiments showed that FtsZ and MreB
interacted in vivo and that this interaction was abolished in
MreBD285A. Single aa changes in FtsZ restored the interaction
with MreBD285A, yielding strong support of a direct interaction. The aa changes in FtsZ were not located to the protofilament interface, GTP-binding site, or C-terminal FtsA/
ZipA-binding regions, suggesting that they would not disrupt
the formation of functional Z rings (Ma and Margolin, 1999;
Oliva et al, 2004; Matsui et al, 2012). One of the FtsZ variants
(P203Q) had simultaneously lost its capability to complement
depletion of wt FtsZ and its interaction with wt MreB but,
strikingly, counteracted cell division inhibition exerted by
ectopic expression of MreBD285A. This shows that
the FtsZP203Q variant, although lethal when expressed as
the sole copy of FtsZ, could support cell division in the
right context. This lends further strong support to the
finding that the MreB–FtsZ interaction is direct and essential.
Cells elongated by ectopic expression of MreBD285A formed
regularly spaced, ‘locked’ Z rings, segregated their nucleoids
and formed mature divisomes containing PBP3 and FtsN.
However, these mature Z rings did not incorporate the
fluorescent D-aa HADA, showing that no septal PG biosynthesis had taken place (see Figure 5B(ii) compared to
Supplementary Figure S6C). PBP3 and FtsN are recruited
late to the divisome, just before the onset of division
(Addinall et al, 1997; De Boer, 2010) (Figure 6). Strikingly,
even though the locked Z rings contained PBP3 and FtsN they
did not recruit PBP1B and PBP2. This result was somewhat
unexpected since PBP1B interacts directly with PBP3 (and
FtsN) (Bertsche et al, 2006; Müller et al, 2007) and PBP2
seems to interact with PBP3 in vivo (Van der Ploeg et al,
2013). FtsN is the last known essential cell division protein
recruited to the divisome (Müller et al, 2007; de Boer, 2010).
1962 The EMBO Journal VOL 32 | NO 13 | 2013
Since FtsN has been proposed to function as a trigger
of septation (Corbin et al, 2004; Gerding et al, 2009;
Lutkenhaus, 2009), it was interesting to learn that locked Z
rings contained FtsN, yet still did not divide. Taken together,
our results show that PBP1B, PBP2 and possibly additional
factors must be recruited to the divisome by MreB for cell
division to occur. Our results therefore provide a mechanism
for recruitment of essential PBP enzymes to the divisome
from the elongation complex, namely PBP1B and/or
PBP2 (Figure 6).
Based on these observations, we propose a model for the
elongation to division transition of cell-wall biosynthesis. In
the model, a proportion of the MreB pool is recruited to the
Z ring to deliver protein factors from the cell-wall elongation complex (Figure 6). This recruitment depends on
the MreB–FtsZ interaction that mediates transfer of protein
factors between elongation and divisome complexes, in
particular PBP2 and PBP1B identified in this study.
However, other factors, such as MurG and MraY, are known
to be present in both the elongation and divisome complexes
(Aaron et al, 2007; Mohammadi et al, 2007). Further studies
are required to determine if they are also recruited to the Z
ring by an MreB-dependent mechanism.
MreB is a conditionally essential protein: during rapid
growth, the cells become spherical, inflate and lyse.
However, during slow growth, the cells propagate as spheres
(Bendezú and De Boer, 2008). Thus our observations raise
the question of how the spherical cells are able to divide in
the absence of MreB that is clearly needed for division of wt
cells. Spherical cells cannot synthesise lateral cell wall due to
the lack of MreB; thus their cell wall is synthesised though the
FtsZ-dependent septal and pre-septal cell-wall synthesis
machinery (Varma and Young, 2009). Since, in the absence
of MreB, there is no lateral cell wall, we infer that PBP1B and
2 by default are associated with the septal and/or pre-septal
synthesis machinery, thus allowing the enzymes to perform
their essential function. Alternatively, the absence of MreB
changes the cell-wall machinery so dramatically that
the requirement for PBP1B and/or 2 is bypassed.
This study has shown that MreB plays an essential role in
cell division of E. coli under rapid growth conditions; previous work revealed that FtsZ plays a role in cell elongation
through pre-septal synthesis (De Pedro et al, 1997).
These observations expanded upon the established roles of
& 2013 European Molecular Biology Organization
MreB–FtsZ interaction is essential in E. coli
AK Fenton and K Gerdes
both proteins and their associated protein complexes.
The understanding that MreB and FtsZ interact during
cell division to coordinate cell elongation with division opens
a new window to study how cell division is controlled in
bacteria. We suggest that the PBP1B and PBP2 proteins play
essential roles in septal and/or pre-septal PG synthesis,
respectively. It could be argued that MreB may not be
involved in pre-septal PG synthesis because spherical cells of
an mreB deletion indeed synthesised pre-septal PG (Potluri et al,
2012). However, as discussed above, the round cell morphology
may bypass the requirement of MreB to deliver PBP 1B and 2 to
the divisome, thereby resolving any such apparent paradox.
Materials and methods
Strains and growth media
For details of all strains and plasmids used in this study see
Supplementary Table SI. All microscopic observations and growth
curves were carried out using M9 growth media (Na2HPO4 0.6 g/l,
KH2PO4 0.3 g/l, NaCl 0.05 g/l, NH4Cl 0.1 g/l, 0.2% w/v casamino
acids, 0.001% w/v thiamine, 1 mM CaCl2, 1 mM MgS04, pH ¼ 7.4)
made fresh from autoclaved components. Glycerol (0.5% v/v) was
typically used as a carbon source; however, glucose (0.4% w/v)
was occasionally used where indicated.
Epi-fluorescence microscopy
Epi-flourescence microscopy was carried out using an Olympus 1X71
inverted microscope through a 100X Zeiss Plan-NEOFLUAR oil objective (NA 1.3), held at 301C using an environmental chamber.
For mYpet–MreB detection, MG1655 pAKF106 (PBAD::mYpet–
mreB) stationary-phase cultures were diluted 1/100 into 25 ml M9
glucose media with 0.05% arabinose in a 125 ml conical flask. Under
these conditions, induction of the PBAD promoter was minimal and
did not affect growth rate or cell morphology (Supplementary
Figures S1A and B). Resulting mixtures were incubated for 3 h at
301C, typically giving OD600 values between 0.29 and 0.33. Culture
samples were spotted onto pre-set 1% M9 glucose agarose pads,
which had been pre-equilibrated to 301C and imaged immediately.
The same conditions were used for the MreB/FtsZ double-labelling
strain: MG1655, pAKF106, pQW59 (Plac::ftsZ–mCherry) and all
microscopy involving the FB76 (mreB–RFPSW) strain.
IFM was carried out using a standard protocol, the full details of
which are given as Supplementary Data. Cells were fixed in 1%
formaldehyde and 0.1% glutaraldehyde. MreB localisation was
detected using rabbit polyclonal anti-MreB antibodies first reported
in Kruse et al (2003), an antibody specificity western blot is shown
in Supplementary Figure S1E. FITC-conjugated anti-rabbit-goat IgG
antibody (Sigma-Aldrich) was used for secondary antibody binding
and fluorescent detection.
Full details of the microscopic apparatus and software used for
image acquisition, media recipes, IFM methods and details of the
mYpet–MreB tag development can be found in the Supplementary
Materials and methods.
D285A
‘overexpression’ microscopy
MreB
Native mreB and mreBD285A was ‘overexpressed’ from plasmids
pAKF128 and pAKF129 in MC1000 using empty vector (pBAD24)
as a control. MC1000 (Dlac, DaraD) was used for microscopic
studies as it allows fine control of expression from Plac and PBAD
promoters. Stationary-phase cultures were diluted 1/100 into 25 ml
M9 media with 0.000,05% (33.3 mM) arabinose. Resulting mixtures
were incubated shaking at 301C for 5 h giving OD600 values between
0.3 and 0.4. Cells were imaged and their dimensions measured
using the MATLAB plug-in MicrobeTracker (Sliusarenko et al,
2012). The experiment was repeated in triplicate with at least 200
cells measured per repeat. A 670 cell sample cutoff was chosen as
this equalled the smallest sample size collected.
For detection of PBP-fluorescent protein tags in mreBD285Aexpressing cells the same protocol was used; except in the case of
pAKF146 (Plac::mrdA–mCherry) where M9 glucose media with
0.1% arabinose inducer was used to repress expression. For
chromosomal DNA staining 1 mg/ml Hoechst 33342 (Invitrogen)
was used to stain cells immediately prior to imaging.
& 2013 European Molecular Biology Organization
BTH analysis
Plasmids used for BTH analysis (Karimova et al, 2005) were cotransformed into BTH101 (cya-99). Five microlitre of stationaryphase culture of representative transformants was spotted
onto nutrient agar (NA) plates containing selective antibiotics
and 40 mg/ml 5-bromo-4-chloro-3-indolyl-b-D-galactopyranoside
(X-gal). After incubation at 301C for 24–48h, plates were scanned
on an Epson perfection V700 photo flatbed scanner. Measurements
of b-galactosidase activity in liquid media were performed on 1 ml
aliquots of growth-phase cultures as described in Miller (1972).
MreB–FtsZ in vivo formaldehyde crosslinking
E. coli MC100 cultures expressing His-tagged MreB from pTK500 or
His–MreBD285A from pAKF126 were crosslinked using 1% formaldehyde for 10 min and quenched using 150 mM glycine.
Crosslinked His–MreB complexes were purified under denaturing
conditions as described in Ishikawa et al (2006). For full details see
Supplementary Materials and methods. Equal volumes of
protein were loaded onto SDS-PAGE gels and individual proteins
identified by western blot using anti-FtsZ and anti-MreB antibodies
(both used at a 1/10 000 dilution) (Kruse et al, 2003; Galli and
Gerdes, 2012).
BTH ftsZ point mutagenesis screen
An error-prone PCR using a modified Pfu (exo ) D473G polymerase
and primers: AKF_FtsZ_pKNT25_F and AKF_FtsZ_pKNT25_R were
used to amplify the ftsZ ORF from pKNT25–ftsZ exactly as described
in Biles and Connolly (2004). PCR products were digested with both
HindIII and BamHI, ligated into pKNT25–ftsZ (cut with the same
enzymes) and transformed into BTH101-containing pUT18–
mreBD285A. Candidates that consistently gave blue cultures on NA
plates containing X-gal (40 mg/ml) were selected and both plasmids
re-isolated. Re-isolated pKNT25–ftsZ-mutant plasmids were cotransformed into BTH101 strains with either pUT18–ftsZ,
pUT18C–mreB or pUT18C–mreBD285A to confirm the ‘re-binding’
phenotype and check protein variants could still bind FtsZ
(Supplementary Figure S5A) before being sequenced. Primer sequences are given as Supplementary Data.
Other methods
Purification of native FtsZ protein was carried out exactly as
described in Galli and Gerdes (2012). Affinity purification of Histagged MreB was carried out using a HisTrap HP column (GE
Healthcare). MreB depletion microscopy was carried out as
described in Kruse et al (2005). Cell-wall HADA staining was
carried out as described in Kuru et al (2012). In all cases, the full
protocols are given as Supplementary Data.
Supplementary data
Supplementary data are available at The EMBO Journal Online
(http://www.embojournal.org).
Acknowledgements
We would like to thank Waldemar Vollmer and Romain Mercier for
critical reading of the manuscript; Bernard Connolly for the
generous gift of Pfu (exo-) D473G error-prone polymerase; Erkin
Kuru, Yves Brun and Michael VanNieuwenhze for the gift of the
HADA fluorescent aas; and Elisa Galli for purified FtsZ protein. We
would also like to thank and acknowledge Chen Chen and Qing
Wang who conducted essential preliminary work on this project.
CC generated the mreB point mutant library and conducted
preliminary work on mreB mutant-cell division phenotypes. QW
engineered the pQW59 vector and the functional Ypet–linker–MreB
fusion, which was further modified by AKF and used in this study.
Author Contributions: AKF carried out the majority of experiments, designed the experimental programme and co-authored the
manuscript. KG designed parts of the experimental programme and
co-authored the manuscript. This work was supported by the
BBSRC and European Commission DIVINOCELL program.
Conflict of interest
The authors declare that they have no conflict of interest.
The EMBO Journal
VOL 32 | NO 13 | 2013 1963
MreB–FtsZ interaction is essential in E. coli
AK Fenton and K Gerdes
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