Kennedy_et_al-2008-Molecular_Microbiology

Delayed activation of Xer recombination at dif by FtsK during
septum assembly in Escherichia coli
Sean P. Kennedy,1,2,3
Fabien Chevalier1,2,3
and
François-Xavier Barre1,2,3
*
1
CNRS, Centre de Génétique Moléculaire, UPR 2167,
91198 Gif-sur-Yvette, France.
2
Université Paris-Sud, 91405 Orsay, France.
3
Université Pierre et Marie Curie- Paris 6, 75005 Paris,
France.
Summary
The co-ordination and synchronization of DNA repli-
cation, chromosome partitioning and cell division in
bacteria are critical to survival. In Escherichia coli,
the septal protein FtsK links cell division and chro-
mosome segregation through its integral membrane
N-terminal and cytoplasmic C-terminal domains. FtsK
is responsible for promoting decatenation and
dimer resolution in the later stages of chromosome
segregation by activating recombination at dif
by the site-specific Xer recombinases. Here, we
formally demonstrate, using novel assay based on
real-time quantitative polymerase chain reaction, that
dif recombination depends not only on proteins
upstream of FtsK in the septum assembly pathway,
but also on the activity of downstream proteins. Work
in synchronized cell cultures further showed that
even though FtsK is recruited early to the septum, dif
recombination only occurs shortly before cell divi-
sion and this activity requires a closing septum. We
propose a model whereby septum localization and
concentration of FtsK co-ordinate its various roles in
chromosome segregation and cell division.
Introduction
During cell proliferation, it is imperative that DNA
synthesis, chromosome segregation and cell division
be co-ordinated to ensure the stable inheritance of the
genetic material. In eukaryotes, this is achieved by dis-
crete checkpoint mechanisms that delay certain steps
until others are completed. In contrast, there is no check-
point or temporal separation between cell cycle events in
bacteria, giving the impression of a lack of co-ordination.
For instance, formation of a division septum on only par-
tially segregated chromosomes occurs each time chromo-
some dimers are formed (see Bigot et al., 2007 for a
review), even though nucleoid occlusion should prevent
the initiation of division when DNA is present at midcell
(Wu and Errington, 2004; Bernhardt and de Boer, 2005).
Septum formation itself is a complex process that involves
the ordered recruitment of more than a dozen proteins
(Vicente et al., 2006). Early events in assembly corre-
spond to the recruitment and stabilization of the tubulin-
like FtsZ and actin-like FtsA proteins, but no visible
constriction at midcell (Aarsman et al., 2005). Constriction
occurs during the second stage of septum formation, after
the recruitment of proteins, such as FtsQ and the
transpeptidase FtsI. FtsK recruitment to the septum pro-
ceeds independently of FtsQ and FtsI (Wang and Lutken-
haus, 1998; Yu et al., 1998), but FtsQ and FtsI both
require FtsK for their successful targeting (Chen and
Beckwith, 2001). Thus, FtsK bridges early and late stages
of septum formation.
FtsK itself is required for chromosome dimer resolution
(CDR). Chromosome dimers form during replication of
circular chromosomes as a result of RecA-dependent
homologous recombination events (Fig. 1A). In Escheri-
chia coli, two tyrosine recombinases, XerC and XerD,
resolve dimers by adding a cross-over at a specific site
on the chromosome, dif. Recombination at dif is both
spatially and structurally controlled. Spatial proximity of
dif sites on a dimer, separated by 4.6 Mbp of DNA, is
ensured by FtsK, which functions as a homo-hexameric
DNA pump anchored in the septum (Aussel et al., 2002;
Saleh et al., 2005; Massey et al., 2006). The direction
towards which FtsK pumps DNA is dictated by specific
8 bp sequences, the KOPS, for FtsK orientating polar
sequences (Bigot et al., 2005; Levy et al., 2005; Bigot
et al., 2006; Sivanathan et al., 2006). As KOPS are
skewed from the origin of replication of the chromosome
towards dif, the dif sites carried by a dimer are brought
together at midcell (Fig. 1A). Structurally, dif sites carried
on a chromosome dimer are more recombinogenic than
sites carried by monomers (Barre et al., 2000; Perals
et al., 2001). This control is lost, however, when FtsK is
overproduced (Barre et al., 2000; Perals et al., 2001). It is
known that FtsK is required to activate Xer recombination
Accepted 8 March, 2008. *For correspondence. E-mail barre@cgm.
cnrs-gif.fr; Tel. (+33) 169823224; Fax (+33) 169823160.
Molecular Microbiology (2008) 68(4), 1018–1028 ᭿ doi:10.1111/j.1365-2958.2008.06212.x
First published online 10 April 2008
© 2008 The Authors
Journal compilation © 2008 Blackwell Publishing Ltd

at dif via a direct interaction with XerD and ATP hydrolysis
(Aussel et al., 2002; Yates et al., 2003; 2006; Massey
et al., 2004). This suggests that the structural control
exerted on dif recombination results from the restriction of
Xer recombination activation by FtsK to a time when
monomeric chromosomes are normally segregated away
from the septum (Fig. 1A). In this way, FtsK recruitment to
the septum and its requirement for proper septum assem-
bly would serve to spatially and temporally regulate
recombination at dif (Barre et al., 2000). In support of this
Fig. 1. A system for real-time analysis of
dimer resolution.
A. Chromosome dimer formation and
resolution in E. coli. The top row of cells
depicts cell division when no dimer is formed.
The bottom row shows dimer formation,
caused by RecA-mediated homologous
recombination during replication. Spatial
control of the dif system is shown as
recombination does not proceed when the
sites are not colocalized. The loading of FtsK
and the activation of its DNA translocation
activity result in the colocalization of dif sites
and the activation of the XerCD
recombinases. A single cross-over between
adjacent dif sites yields the resolution of the
dimers to monomers.
B. Our system to measure recombination
uses duplicated dif sites in direct repeats
ﬂanking a kanamycin resistance (KanR
)
cassette. Recombination at dif to resolve a
chromosome dimer, formed as described in
the previous panel, can initiate in three
distinct ways in this modiﬁed system: I, II and
III. Products from I and II result in products
that can be measured using our qPCR
system.
C. Resolution of dimers can be monitored by
qPCR on a chromosomal substrate containing
two direct repeats of dif. Here, reaction II from
B is illustrated. The resolved product can be
measured by disappearance of the
intervening cassette (upper drawing, primers
X/Y) and the consequent appearance of a
single dif product (lower drawing, primers
X/Z). Genomic DNA is digested with HindIII
to prevent contamination by larger products
encompassing the cassettes when using
primers X/Z.
RecA
XerCD
A
dif
oriC
primers
FtsK
* HindIII
I
II III
I-a I-b III-bIII-a
I
II
III
X Y Z
Z Y X
X Y ZY
Z X
B
XerCD
C
*
*
* *
Kan
R
Kan
R
II
Chromosome
Dimer
II-a II-b
FtsK
Loads
DNA
Pumped
Temporal control of chromosome dimer resolution 1019
© 2008 The Authors
Journal compilation © 2008 Blackwell Publishing Ltd, Molecular Microbiology, 68, 1018–1028

model, it was shown, using a density shift assay, that
inhibition of FtsI activity by cephalexin treatment, which is
downstream of FtsK in the septum assembly pathway,
decreased the amount of sister chromatid exchanges
detected at dif (Steiner and Kuempel, 1998a). This could
reflect a delay in Xer recombination activation by FtsK
with respect to its septal recruitment. However, optimal
conditions for a density shift assay require relatively high
levels of recombination and this technique requires that
cells be grown for an additional two generations after the
addition of cephalexin. This left open the possibility that
disruption of the septum by prolonged cephalexin treat-
ment might simply lower the frequency of sister chromatid
exchanges below the threshold of detection. Indeed, the
transpeptidase activity of FtsI is required, directly or indi-
rectly, for the assembly of nascent division sites and
thereby for future assembly of FtsZ rings (Pogliano et al.,
1997), and cephalexin treatment results in a defect in
targeting of FtsK to the septum in extended time points
(Yu et al., 1998). As a consequence of these points, the
reason for the potential delay in Xer recombination acti-
vation observed after cephalexin treatment remained
elusive, as did the exact role played by FtsK in this
regulation.
In light of this, we decided to re-investigate the mecha-
nisms that restrict FtsK activity during the cell cycle using
highly sensitive real-time procedures. To this aim, we
developed a novel assay to measure XerCD recombina-
tion between chromosomal dif sites. Using this assay, we
formally demonstrate that dif recombination occurs shortly
before, or commensurate with, septum closure. The most
likely explanation for this is that FtsK activity is delayed
with respect to its septal recruitment. Based on our obser-
vation that FtsK activity is not primarily controlled at the
transcriptional level and that its overproduction abolishes
the spatial and structural control over Xer recombination
at dif (Barre et al., 2000; Perals et al., 2001; Aussel et al.,
2002; Massey et al., 2004), we propose a model whereby
active hexameric complexes of FtsK cannot form in the
cell before septum invagination because of the low cellu-
lar concentration of the protein and its low constant of
association. As a consequence, dif sites would not be
brought together and XerCD would not be activated until
septum invagination has commenced.
Results
Monitoring recombination at dif in real time
To study the timing of Xer recombination at dif and its
importance in the overall structural control of CDR, we
required a method of monitoring dif recombination in real-
time while not disrupting its normal regulation. Whereas E.
coli normally harbours a single dif site per chromosome
which serves to resolve dimers (Fig. 1A), we use a strain
carrying two directly repeated dif site flanking a gene
coding for antibiotic resistance (Fig. 1B). Such a cassette
has been shown to allow for the successful analysis of dif
recombination in vivo (Perals et al., 2000). Resolution of
dimers in this strain can be initiated in three ways (I, II and
III, Fig. 1B), two of them resulting in the excision of the
cassette in at least one of the two sister chromosomes
(I and II, Fig. 1B). Plating assays to measure loss of the
resistance cassette show excision to occur at a rate of 12%
per cell per generation in normal cells. This corresponds to
the reported rate of chromosome dimer formation (Steiner
and Kuempel, 1998b; Perals et al., 2000). Importantly,
cassette excision drops to 0.7% per cell per generation in
recA cells, which do not form chromosome dimers (Perals
et al., 2000). This result demonstrates that excision of the
cassette is structurally controlled despite the close proxim-
ity of the dif sites flanking the cassette (Fig. 1B).
We adapted this assay for real-time studies by measur-
ing the accumulation of cells with excised cassettes
(CEC) throughout a time-course by quantitative poly-
merase chain reaction (qPCR). Three primer pairs were
used in order to insure maximum sensitivity in the assay
(Fig. 1C and Experimental procedures); Primers X and Y
yield a 246 bp product on non-recombined molecules
which retain the multiple dif sites. Primers X and Z,
conversely, yield a 214 bp product if Xer recombination
resulted in excision of the cassette (Fig. 1C). In this way,
we are able to measure direct conversion of dif sites from
non-recombined to recombined states. As there is only
1 kb of sequence between the two dif sites, it is possible
for a larger PCR product, of 1515 bp between X and Z
primers, to be amplified. Therefore, the purified genomic
DNA was first digested with the restriction enzyme
HindIII to prevent any product from forming from a
non-recombined template (Fig. 1C). Agarose gels and
denaturing profiles confirm that there is no measurable
contamination from the long XZ product in our reactions
(data not shown). A final set of primers was designed to
yield a 255 bp from a region 1 kb upstream of dif as a
control for both reaction efficiency and to measure and
normalize chromosome copy number. Importantly, in all
reported reactions, the sum of non-recombined and
recombined products was equal to that measured in the
control reaction. Efficiency and accuracy of our qPCR-
based analysis is exemplified by the ability to detect
extremely low amounts of recombination at dif (< 0.1%
CEC), and the accuracy of the system overall.
Stable propagation of the cassette and induction
of its excision
Normal Xer recombination at dif prevents stable mainte-
nance of the cassette in the wild-type strain. We needed
1020 S. P. Kennedy, F. Chevalier and F.-X. Barre ᭿
© 2008 The Authors

therefore to propagate the cassette in cells in which Xer
recombination was inducible. Because of the extremely
low concentration of the Xer recombinases required for
CDR, the XerCD proteins themselves make poor targets
for induction and regulation (F.X. Barre, unpublished).
Instead, we decided to regulate Xer recombination by
working in cells where wild-type FtsK is inducible. As
the N-terminal domain of FtsK is normally essential for
cell division (Begg et al., 1995; Diez et al., 1997; Draper
et al., 1998; Liu et al., 1998; Geissler and Margolin,
2005; Bernard et al., 2007; Goehring et al., 2007), cells
carried chromosomal ftsK alleles defective in Xer recom-
bination activation as a result of either a deletion of the
C-terminal domain (Bigot et al., 2004) or through a
K997A substitution (Barre et al., 2000) which abolishes
ATP hydrolysis.
Our initial attempts with a high-copy FtsK expression
vector revealed chromosomal recombination at dif even
in the absence of inducer (data not shown). These
results, in addition to other published reports alluding
to the importance of the cellular concentration of FtsK
(Barre et al., 2000; Perals et al., 2001), lead us to
suspect leaky expression and to move to a pSC101-
derived low-copy expression system. Additionally, the
chromosomal TTG start and the weak Shine Dalgarno
sequence TGGAGA of the chromosomal ftsK gene
were conserved on the plasmid. On this construct, in
the absence of glucose, < 1% CEC was measured
after overnight growth in FtsKcat
(Table 1) carrying a
C-terminal truncation of FtsK and a dif cassette (Fig. 2
and 0 h and data not shown). Induction of FtsK protein
in this strain, by the addition of 0.2% arabinose, restored
a normal phenotype to the cells, and a frequency of
10.76% of CEC per generation was determined geneti-
cally. Finally, a time-course experiment conﬁrmed that
cassette excision begins between 2 and 3 h after arabi-
nose induction (Fig. 2).
Structural control after ectopic production of FtsK
It had been previously observed that FtsK production from
an ectopic promoter could alter both the spatial and the
structural control of Xer recombination (Barre et al., 2000;
Perals et al., 2001). Conservation of the structural control
in our system was conﬁrmed by comparing the frequency
of cassette excision in normal and recA strains (Fig. 2). In
FtsKcat
cells, the proportion of CEC reached 41.71% after
6 h against 5.27% in its recA derivative. This corresponds
Table 1. List of strains.
Strain Genotype Reference
FC1 AB1157, ftsK100 K997A (ATP mutant) CmR
This study
FC2 (FtsKATP-
) FC1, dif-Kn-dif, KnR
, CmR
This study
FC313 (FtsKcat
) AB1157, ftsK1, dif-Kn-dif, CmR
Perals et al. (2000)
FC324 (RecA) FC313, recA56 srl::Tn10, CmR
, TetR
Perals et al. (2000)
FC3 (FtsIts
) FC2, leuO::Tn10, ftsI23ts
, KnR
, CmR
, TetR
This study
FC4 (FtsZts
) FC2, leuO::Tn10, ftsZ84ts
, KnR
, CmR
, TetR
This study
FC5 (FtsAts
) FC2, leuO::Tn10, ftsA12ts
, KnR
, CmR
, TetR
This study
FC6 (FtsQts
) FC2, leuO::Tn10, ftsQ1ts
, KnR
, CmR
, TetR
This study
SK20 (DnaAts
) AB1157, ftsK1, dif-Kn-dif, dnaA46ts
, tna::Tn10, TetR
, KnR
, CmR
This study
AB1157 thr-1 leu-6 thi-1 lacY1 galK2 ara-14 xyl-5 mtl-1 proA2 hi-4 argE3 str-31
tsx-33 supE44 rec+
Laboratory strain
CAG12095 leuO3051::Tn10, TetR
Singer et al. (1989; Riley et al., 2006)
WM1115 TX3772 leuO::Tn10 ftsA12ts
, TetR
Geissler et al. (2003)
FC7 AB1157, leuO::Tn10 ftsA12ts
, TetR
This study
WM2193 leuO::Tn10, TOE1 (ftsQ1ts
), TetR
Begg et al. (1980)
FC8 AB1157, leuO::Tn10, TOE1 (ftsQ1ts
), TetR
This study
ALO2343 MG1655 dnaA46ts
, tna::Tn10, TetR
Boye et al. (2000)
GC3443 AB1157(lJFL100), tonA, leu+
, ftsZ84ts
Robin et al. (1990)
GC3446 AB1157(lJFL100), tonA, leu+
, ftsI23ts
Robin et al. (1990)
FtsK and recA
0.0
0.5
1.0
1.5
2.0
2.5
3.0
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0 1 2 3 4 5 6
FtsK %CEC - qPCR
recA / FtsK %CEC - qPCR
OD600
Hours after WT ftsK induction
%CellswithExcisedCassette(CEC)
FtsK growth
recA / FtsK growth
cat
cat
cat
cat
cat
Fig. 2. Cassette excision upon ectopic FtsK production in FtsK
and FtsK/recA strains. FtsK is induced at 0 h, and excision of the
dif-kan-dif cassette is monitored (bars) along with cell growth (lines)
for both a strain carrying a C-terminal deletion of FtsK and a recA
double mutant. Cumulative growth is measured at OD600 on the left
y-axis. The per cent of CEC is reported on the right y-axis.
© 2008 The Authors

to a mean frequency of excision per cell per generation of
10.52% and 1.10% respectively.
FtsZ-ring assembly is required for dif recombination
Earlier data by Kuempel’s group pointed to the necessity
of FtsZ-ring formation for Xer recombination at dif (Steiner
and Kuempel, 1998a). As an additional control for the
accuracy of our assay, we constructed mutant strains
conﬁrming that the lack of FtsK localization at the septum
led to a block of Xer recombination. We chose
temperature-sensitive alleles of ftsZ and ftsA which arrive
at an early stage in septum formation and are required for
the septal localization of FtsK (Wang and Lutkenhaus,
1998; Yu et al., 1998; Dorazi and Dewar, 2000). Deletion
of the C-terminal domain of FtsK affects cell division at the
level of septum formation as well as dimer resolution (Diez
et al., 1997; Bigot et al., 2004), which might complicate
data interpretation. We therefore decided to perform these
experiments in FtsKATP-
cells, which carry a chromosomal
copy of FtsK defective in ATP hydrolysis along with the
previously described dif cassette. No cell division defects
other than those linked to chromosome dimer formation
were observed in this FtsK mutant (Bigot et al., 2004). In
this context, the efficiency of Xer recombination at dif
decreased to 5.29% per cell per generation, as measured
genetically, possibly because inactive C-terminal domains
poison the activity of FtsK hexamers (Massey et al.,
2006). Further, we used a YFP C-terminal fusion of FtsK
(FtsK–YFP) to monitor its localization in the cell and its
arrival at the septum. This led to an additional decrease in
the rate of cassette excision to 3.43% per cell per gen-
eration, as measured genetically, likely because the
fusion affects the interaction of the C-terminal tail of FtsK
with KOPS or XerD (Barre et al., 2000; Yates et al., 2003;
2006). Nevertheless, this rate was sufficient for our
experiments given the high sensitivity of the qPCR assay.
Shifting ftsZ84ts
and ftsA12ts
derivatives of FtsKATP-
cells to
42°C almost immediately blocked Xer recombination at
dif, which correlated with a loss of septal localization of
FtsK–YFP (Fig. 3B and C). In contrast, shifting the control
strain FtsKATP-
cells, carrying wild-type alleles of ftsZ and
ftsA, to 42°C did not affect localization of FtsK–YFP to the
septum or cassette excision. In this case, we measured a
mean excision rate of 3.56% per cell per generation
(Fig. 3A).
FtsI transpeptidase activity is required for dif
recombination
The dependence of dif recombination on FtsZ and FtsA
suggested that FtsK needs to be anchored at the
septum to be active. If this was the only requirement,
inactivation of proteins downstream of FtsK in the cell
division assembly pathway should not affect dif recom-
bination. However, early experiments using a density
shift assay indicated that this might not be so (Steiner
and Kuempel, 1998a). This hypothesis was checked
by monitoring cassette excision in ftsQ1ts
and ftsI23ts
FtsKATP-
cells. In both cases, a shift to the non-
permissive temperature immediately abolished Xer
recombination at dif, even though FtsK–YFP remained
localized to the septum up to several hours after the
shift (Fig. 3D and E).
As FtsQ and FtsI both interact with FtsK (Chen and
Beckwith, 2001; Di Lallo et al., 2003; Karimova et al.,
2005), it was possible that inactivation of their thermosen-
sitive alleles at 42°C prevented dif recombination by an
undetectable decrease in the stability of FtsK complexes
at the septum. We therefore decided to monitor Xer
recombination at dif in normal cells before and after treat-
ment with cephalexin, which blocks the transpeptidase
activity of FtsI, but does not affect the overall organization
of the septum over short periods of time (Pogliano et al.,
1997). Addition of cephalexin to FtsKATP-
cells again
immediately stopped Xer recombination at dif, without
affecting FtsK–YFP localization (Fig. 3F).
Finally, we recorded the position of FtsK at the septum
and the level of recombination up to 7 h after the shift to
the non-permissive temperature or the addition of ceph-
alexin. We were thus able to conﬁrm the continued
localization at the septum of FtsK coupled with the lack
of recombination up to that time (Fig. 3 and data not
shown).
dif recombination is concomitant with septum closure
We decided to further investigate if Xer recombination at
dif correlated with FtsK-recruitment to the septum or with
septum closure by monitoring these events in a culture
of synchronized cells. To this aim, we introduced the
dnaA46ts
allele, which is non-functional at 42°C but can
be reactivated upon a return to 30°C (von Freiesleben
et al., 2000), in FtsKcat
cells. Synchronization can then
be achieved when cells are grown in minimal media,
which also avoids multiple rounds of replication and
increases the total duration of a complete cell cycle.
FtsK–YFP production was induced at the beginning of
the time-course. The proportion of CEC reached 1.27%
during the 4 h of initial growth at 30°C (Fig. 4A). FACS
analysis on rifampicin and cephalexin-treated cells
showed that approximately 60% of cell synchronization
were achieved during the 2.5 h incubation at 42°C,
which corresponds to their approximate generation time
(data not shown). Indeed, the number of cells with FtsK–
YFP rings decreased from approximately 15.4% at
4:00 h to less than 2.2% at 6:30 h, indicating that most
cells had completed rounds of cell division (Fig. 4B).
© 2008 The Authors

Concomitantly, the proportion of CEC increased to
4.01% (Fig. 4A). Upon release to the permissive tem-
perature, we observed very little recombination at dif up
to the 9:15 h time point, even though the level of FtsK at
the septum increased to a peak close to 50% at this
time (Fig. 4B). In the next 15 min period, however, we
observed a sharp increase in CEC from 6.12% to
9.12%. The burst of recombination correlated with a
decrease in the proportion of cells showing FtsK–YFP
foci from 45.9% to 28.4% (Fig. 4B). In addition, we
observed a 2.1-fold increase in colony-forming units
(cfu) on plates at the 9:15 h and 9:30 h time points (from
2.27 ¥ 107
to 4.84 ¥ 107
), and microscopically measured
a decrease in the mean length of cells from an average
of 7.25 to 3.62 mm between these same points, indicat-
ing that cell division had occurred.
To exclude that part of the delay in Xer recombination
observed in the time-course experiment we performed on
synchronized cells was due to the use of a modiﬁed
version of FtsK with a C-terminal YFP tag, we repeated
*
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ftsQ(Ts)
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ftsK
Growth (OD)
%CEC - qPCR
42°C (non-permissive temp.)
A
D
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%CEC - qPCR
42°C
ftsA(Ts)
*
C
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Growth (OD)
%CEC - qPCR
42°C
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Cephalexin
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CumulativeGrowthasMeasuredbyOD600andCorrectedforDilutions
Hours Hours
Growth (OD)
%CEC - qPCR
42°C
Growth (OD)
%CEC - qPCR
42°C
Growth (OD)
%CEC - qPCR
%CellswithExcisedCassettes(CEC)
Fig. 3. dif recombination in temperature-sensitive division protein mutants: Growth and recombination were measured for the control strain
carrying no Ts mutation (A), Ts alleles of proteins recruited before FtsK, FtsZ (B) and FtsA (C), or after FtsK, FtsQ (D) and FtsI (E), and after
cephalexin treatment (F). Photos show FtsK–YFP microscopy at the indicated (*) time point. Growth in these graphs is measured at OD600
(lines). The cultured are continually diluted to support logarithmic growth, but total growth is reported on the left y-axis as total growth,
corrected for dilution. The per cent of CEC is reported on the right y-axis (bars).
© 2008 The Authors

this experiment using an unmodified version of FstK
(Fig. S1). As expected, the level of recombination was
higher than with the FtsK–YFP derivative, but we
observed the same burst of recombination between the
9:15 h and 9:30 h time points. In addition, we checked
that addition of cephalexin upon return to the permissive
temperature prevented recombination by the FtsK–YFP
derivative (Fig. S2). Taken together, these data provide
good evidence that dif recombination occurs shortly
before or concomitantly with septum closure.
Discussion
An integral player in both septum formation and CDR,
FtsK, is unique in its ability to link these two seemingly
independent processes in bacteria. Therefore, defining
the precise activation point of FtsK during chromosome
partitioning and cell division is critical to our understand-
ing of the co-ordination between these two processes.
Quantitative polymerase chain reaction monitoring of
Xer recombination at dif
We decided to investigate the timing of Xer recombination
at dif in real time by monitoring the excision of a dif
cassette, at the normal position of dif, using the highly
sensitive qPCR technique. In order to answer questions of
septum structure, we decided to monitor the recruitment
of FtsK at the septum using a YFP-tagged derivative of
the protein and fluorescent microcopy.
Using time-course experiments, we demonstrated that
we could regulate the expression of FtsK and follow the
accumulation of cells which had undergone recombina-
tion to resolve chromosome dimers (CEC) in real time.
Importantly, we showed a 10-fold reduction in a recA
mutant, which does not generate dimers. The recA data
yield further evidence that we have succeeded in main-
taining the normal structural control over the system that
restricts dif recombination to chromosomal dimers. Taken
together, these results confirm the applicability of our
system to study these specific events.
The timing of Xer recombination at dif
The use of individual temperature-sensitive mutants in
septal proteins that are normally recruited either before or
after FtsK allowed us to halt septum formation at pre-
defined steps and look for effects of recombination at dif.
The fact that FtsZts
and FtsAts
strains show no recombi-
nation and no recruitment of FtsK at the septum serves to
highlight the accuracy and fidelity of our qPCR analysis
with respect to true physiological conditions (Fig. 3B and
C). More interestingly, YFP-tagged FtsK was still present
at the septum in FtsQts
and FtsIts
strains up to 10 h after
the shift to the non-permissive temperature even though
Xer recombination activation was immediately arrested
(Fig. 3D and E).
To explore this hypothesis further, we made use of
DnaAts
-synchronized cultures in which both the precise
time of recruitment of FtsK–YFP and that of CDR recom-
bination at the dif site could be measured. In these experi-
ments, we consistently observed FtsK recruited to the
septum prior to any significant increase in dif-cassette
excision. This provided additional confirmation that the
late activation of Xer recombination by FtsK is specifically
linked to cellular division. Finally, our results showed that
the bulk of dif recombination occurs within 15 min prior to
cell division. Thus, the truly novel inference can be made
that no individual septal protein or enzymatic step up to
and including FtsI is required for FtsK activation. Instead,
it appears likely that FtsK and CDR requires a properly
functioning septum in its entirety.
4:00
9:15 9:30 10:00
6:30
B
0 2 4 6 8 10
0.0
0.5
1.0
1.5
2.0
2.5
0
2
4
6
8
10
12Growth (OD)
%CEC - qPCR
42°C
A
15.4% 2.2%
45.9%
1 3 5 7 9
28.4% 17.6%
*
*
*
*
*
*
3.00% recombination
0.220 OD of Growth
2.74% recombination
0.613 OD of Growth
OD600
Hours
%CEC
9:00
30.7%
Fig. 4. Observation of FtsK arrival and dif recombination in
synchronized cells.
A. Time-course experiment using dnaAts
cells. Synchronization of
the culture occurs at 42°C in the pink-shaded region. Cumulative
growth is measured at OD600 on the left y-axis (lines). The per cent
of CEC is reported on the right y-axis (bars).
B. Representative fields of cells showing FtsK–YFP fluorescence
microscopy at the indicated (*) time points. The percentage of cells
with visible FtsK foci is reported along with the time point for
each field.
© 2008 The Authors

Delayed activity of FtsK during septum formation
Our results formally demonstrate that activation of Xer
recombination at dif by FtsK is a very late process, which
depends on the final steps of septum assembly and
closure. Three possibilities exist to explain this result. First,
the activity of FtsK could start as soon as it arrives at the
septum, but Xer recombination activation would occur later
because FtsK must first bring the dif sites together, i.e. the
time required to ensure the spatial proximity of the dif sites
would explain the delay observed in Xer recombination.
This does not appear to be the case as we did not observe
recombination even up to 7 h after the shift to 42°C or after
the addition of cephalexin in the FtsQts
and FtsIts
experi-
ments (Fig. 3 and data not shown). This time frame would
leave ample time for FtsK to load on DNA and track until it
encountered the dif cassette. If this explanation had been
true, Xer recombination should have been observed. A
second possibility exists that FtsK retains the capacity to
function as soon as it arrives at the septum, but another
septal protein is required to perform a switch in the activity
of FtsK to enable Xer recombination activation. We refute
this possibility because FtsK works outside the septum in
Xer recombination (Barre et al., 2000; Aussel et al., 2002;
Massey et al., 2004; Bigot et al., 2006). The third and final
explanation is that the activity of FtsK is restricted to the
latest stages of septation. Thus, our results highlight the
fact that FtsK activity in CDR is delayed with respect to its
septal recruitment.
Interdependence of chromosome segregation
and cell division
The delayed activation of FtsK reveals the high degree of
co-ordination between chromosome segregation and cell
division in bacteria, a feature that was masked by the
absence of a strict temporal separation. Indeed, FtsK is at
the centre of an integration loop between cell division
and chromosome segregation (Fig. 5A): late chromosome
segregation events, such as CDR or decatenation
(Grainge et al., 2007), require the activation of FtsK by
septum closure (this work); septum closure depends on
the septal recruitment of FtsQ and FtsI by FtsK (Chen and
Beckwith, 2001); FtsK recruitment depends on FtsZ-ring
formation and its stabilization by FtsA (Wang and Lutken-
haus, 1998; Yu et al., 1998); FtsZ-ring formation requires
that the bulk of the chromosomes be segregated away
from midcell (Bernhardt and de Boer, 2005).
A concentration-dependent model for the
spatio-temporal restriction of FtsK activity
What restricts FtsK activity to the late stages of septation?
The upstream region of ftsK contains two promoters. One
is constitutive, the other regulated by SOS (Wang and
Lutkenhaus, 1998; Dorazi and Dewar, 2000). This com-
plex regulation raised the possibility that the activity of
FtsK during the cell cycle could be controlled at the tran-
scriptional level. For instance, only at a late stage of
septum formation could sufficiently high amounts of FtsK
be produced. However, our observation that a low level of
ectopic production of FtsK did not affect the spatial and
structural control of FtsK, or the requirement for septum
formation, contradicts this hypothesis (Figs 2 and 3).
Based on the observation that FtsK activity is not pri-
marily controlled at the transcriptional level (this work) and
that its overexpression abolishes the spatial and structural
control over Xer recombination at dif (Barre et al., 2000;
Perals et al., 2001; Aussel et al., 2002), we propose a
model whereby active hexameric complexes of FtsK
cannot form in the cell before septum invagination
because of the low cellular concentration of the protein
and its low association constant. As a consequence, dif
sites would not be brought together and XerCD would not
be activated until septum invagination has commenced
(Fig. 5B). This model is supported by results showing that
high concentrations of FtsK are required in vitro in order to
promote hexamerization (Massey et al., 2006), and by the
failure to observe any self-interactions of FtsK in two-
hybrid experiments (Di Lallo et al., 2003).
Similarities and differences with the SpoIIIE
system of Bacillus
Work in Bacillus subtilis has demonstrated that SpoIIIE,
the FtsK homologue in this organism, is also a late player
in chromosome partitioning during sporulation (Wu and
Errington, 1994; Wu et al., 1995; Bath et al., 2000).
High Local Concentration
and Hexamerization
FtsK
Bulk Chr.
Segregation
Late Chr.
Segregation
Septum
Invagination
+
Closure
FtsZ / FtsA Ring
FtsQ / FtsI Recruitme
nt
C
-terminal ActivationCDR / Decatenation
Cell
Division
Chromosome
Segregation
A
B
dif
dif
dif
dif
Monomeric and
Inactive FtsK
Fig. 5. A model for FtsK function and regulation.
A. Integration of chromosome segregation and cell division.
B. Model for concentration-dependent control of FtsK activity. An
increase in local concentration of FtsK in the closing septum allows
for hexamerization and activation of DNA pumping and
XerCD-activating properties.
© 2008 The Authors

Indeed, the N-terminal portion of SpoIIIE is thought to play
a specific role in the formation of a pore in an otherwise
closed septum through which the genomic DNA is
pumped (Burton et al., 2007). The pore-forming model is
supported by data showing the inability of sigma factors to
diffuse between the spore and mother cell after the
arrival of SpoIIIE (Wu and Errington, 1997). In addition,
the N-terminal domain of SpoIIIE has been implicated
in septum closure and spore engulfment (Sharp and
Pogliano, 1999; Liu et al., 2006). Although no data
currently support pore-forming multimerization of the
N-terminal region of FtsK, the similarity between the time
and activity in SpoIIIE and FtsK opens possibility for future
work (Barre, 2007; Becker and Pogliano, 2007).
The results of this work and the parallels that can be
drawn to the homologous system in Bacillus confirm a
need to advance our understanding of the function of the
N-terminal domain of FtsK. The fact that overproduction of
the FtsZAQ or FtsN septal proteins and mutations, such
as FtsA*, can compensate for the deletion of FtsK high-
light further parallels with SpoIIIE which itself is not
required in Bacillus for vegetative growth (Geissler and
Margolin, 2005; Bernard et al., 2007; Goehring et al.,
2007). Thus, it will be important to determine if the
transmembrane domain of FtsK carries important sig-
nals required for septum assembly, FtsK recruitment or
FtsK hexamerization. Also of great interest is the 600-
amino-acid ‘linker’ region that connects the transmem-
brane domain, required for septum formation, with the
C-terminal domain responsible for DNA pumping and
CDR. Although it is required for full activity of FtsK (Bigot
et al., 2004), we lack understanding with regard to what
properties contribute to its function and what contributions
it makes to the timing of FtsK hexamerization/activity.
Experimental procedures
Strains, growth and plasmid construction
All strains used in this study are AB1157 derivatives and
are described in Table 1. FtsK and FtsK–YFP low-copy ex-
pression vectors are derived from pSC101 and carry a
spectinomycin-resistance gene. 75 mg ml-1
spectinomycin
was used to maintain these vectors. Leaky expression was
prevented by conserving the wild-type low-affinity ribosome
binding site and the low-affinity TTG initiator codon of ftsK.
0.2% arabinose was used for FtsK induction of cultures.
Cultures were grown in Lennox Luria–Bertani with the follow-
ing exceptions: the ftsA12ts
was grown with reduced salt
(2.5 g l-1
) and the dnaA46ts
strain was grown in M9 minimal
media (Sambrook and Russell, 2001) supplemented with 1%
casamino acids. All cultures were inoculated into media pre-
warmed to 30°C and grown at this temperature until a shift
to the non-permissive temperature is indicated. For the
dnaA46ts
strain, the culture is returned to 30°C after synchro-
nization at the non-permissive temperature. Cultures were
continually diluted with pre-warmed fresh media to maintain
logarithmic growth at all times. Growth was measured at
600 nm. As cultures were diluted with fresh media to maintain
logarithmic growth, we report cumulative growth which mea-
sured the total increase in OD throughout the experiment,
correcting for any dilutions.
Sample preparation and genomic DNA extraction
During each time-course experiment, samples were taken by
snap-freezing 1.5 ml of cell culture in an eppendorf tube
using liquid nitrogen. Genomic DNA was purified using a
modified version of Murray and Thompson for bacteria
(Murray and Thompson, 1980). Briefly, samples were thawed
at 58°C in the presence of SDS and proteinase K to prevent
any further growth or recombination. After incubation with
CTAB (Sigma H9151), samples were extracted with phe-
nol : chloroform, treated with RNase and finally precipitated
with ethanol.
Quantitative polymerase chain reaction method
Genomic DNA samples were prepared in such a way as to
achieve the same DNA concentration of each individual
sample. HindIII restriction digests were set up with using a final
7.5 ng ml-1
DNA concentrations. The primers used for qPCR
were as follows: forward (X), 5′-TGACCGCCAACGACTGGAT
TC; reverse (Y), 5′-TTAATCGCGGCCTCGAGCAAG; reverse
(Z), 5′-GCGACAGACACTGCGCTCTTAG; control forward,
5′-CCGAAAGGAATAACGCCGACGAC; control reverse,
5′-ATCCTCCTGTGGCAAGCGAAG.
Monitoring cassette excision
The mean frequency of cassette excision per cell per gen-
eration between two time points a and b equals 1 - exp{[ln2/
ln(Nb/Na)] ¥ ln[(1 - CECb)/(1 - CECa)]}, where N stands for
the number of cells and CEC for the proportion of CEC. CEC
was determined genetically, as previously reported (Perals
et al., 2000), or by qPCR on a LightCycler 2.0 (Roche Scien-
tific; Nutley, NJ). The ratio of Nb to Na was determined from
the increase in cfu for the genetic approach or from the
growth in OD for the qPCR approach.
Microscopy
A DM6000-B Microscope (Leica; Wetzlar, Germany) coupled
to a Coolsnap HQ CCD camera (Photometrics; Tucson, AZ)
was used to image cells which were collected and fixed in a
solution of 5% paraformaldehyde/0.6% gluteraldehyde/
1¥ PBS. Cells were observed on slides overlaid with an
agarose pad. The program ImageJ (Abramoff et al., 2004)
was used for counting and size measurements.
Acknowledgements
We thank F. Cornet, S. Pichoff, J. Lutkenhaus, W. Margolin,
A. Løbner-Olesen, D.Joseleau-Petit and the NBRP (NIG,
Japan) for the kind gift of strains, and B. Michel for critical
reading. Work in the Barre laboratory is supported by EMBO
YIP, CNRS ATIP+, ANR BLANC, FRM and Région Ile de
France Grants.
© 2008 The Authors

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Supplementary material
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Please note: Blackwell Publishing is not responsible for the
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