Cell-free study of F plasmid partition provides evidence
for cargo transport by a diffusion-ratchet mechanism
Anthony G. Vecchiarelli, Ling Chin Hwang, and Kiyoshi Mizuuchi1
Laboratory of Molecular Biology, National Institute of Diabetes, and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892
Contributed by Kiyoshi Mizuuchi, February 12, 2013 (sent for review November 27, 2012)
Increasingly diverse types of cargo are being found to be segregated
and positioned by ParA-type ATPases. Several minimalistic systems
described in bacteria are self-organizing and are known to affect the
transport of plasmids, protein machineries, and chromosomal loci.
One well-studied model is the F plasmid partition system, SopABC.
In vivo, SopA ATPase forms dynamic patterns on the nucleoid in
the presence of the ATPase stimulator, SopB, which binds to the
sopC site on the plasmid, demarcating it as the cargo. To under-
stand the relationship between nucleoid patterning and plasmid
transport, we established a cell-free system to study plasmid par-
tition reactions in a DNA-carpeted flowcell. We observed deple-
tion zones of the partition ATPase on the DNA carpet
surrounding partition complexes. The findings favor a diffu-
sion-ratchet model for plasmid motion whereby partition com-
plexes create an ATPase concentration gradient and then climb
up this gradient toward higher concentrations of the ATPase.
Here, we report on the dynamic properties of the Sop system on
a DNA-carpet substrate, which further support the proposed
bacterial chromosome segregation|ParA ATPase|plasmid segregation|
spatial pattern organization|chromosome dynamics
genomic information for all life forms. In bacteria, this
fundamental process is poorly understood. Low-copy bacterial
genomes, including plasmids and chromosomes, encode active
partition (Par) systems to ensure stability. Par systems are
minimalistic in that only three dedicated components are re-
quired: a partition site on the DNA, a partition site-binding
protein, and a nucleoside triphosphatase (NTPase). Par sys-
tems have been classified according to the type of NTPase
involved: Walker-type (generically called “ParA”), actin-like,
or tubulin-like (reviewed in ref. 1). Reconstitution of purified
Par components of R1 plasmid in a cell-free system unveiled
the mechanism involving an actin-like ATPase, ParM, in which
elongating filaments of the ATPase push plasmids to opposite cell
poles (2). Tubulin-like GTPases also appear to function as a fila-
ment (3). However, all chromosome-based and most plasmid-based
systems use ParAs, and mounting evidence shows that ParA-like
ATPases also are responsible for transporting large protein ma-
chineries (reviewed in ref. 4). However, the underlying mechanism
for reactions of this category remains unresolved.
The Sop system (stability of plasmid) of F plasmid is one of the
first Par systems to be identified (5, 6) and is considered a para-
digm for the study of ParA-mediated DNA segregation. The
three plasmid-encoded system components are SopA (the ParA-
type ATPase), SopB (or ParB in other systems; i.e., the partition
site-binding protein), and sopC (or parS in other systems; i.e., the
cis-acting partition site on the plasmid). The first task of a parti-
tion system is to identify its DNA cargo. SopB accomplishes this
task by loading onto sopC and forming a partition complex, which
has been visualized in vivo by fluorescence microscopy as punc-
tuate foci (7, 8). The partition complex is believed to contain
a large number of SopB dimers, some bound specifically to sopC
and additional dimers bound near sopC (9–11).
roper DNA segregation ensures the faithful inheritance of
SopB-stimulated ATPase activity of SopA is critical to the
partition reaction and plasmid stability, but how ATP hydrolysis
drives plasmid movement is unknown. SopA has weak ATPase
activity that is mildly stimulated by SopB or nonspecific DNA
(nsDNA) (12). However, when SopB and nsDNA are combined,
synergistic stimulation is observed. These properties generally are
shared by other ParAs (reviewed in ref. 1). In vitro, several ParAs
also bind nsDNA, and this activity requires or is enhanced by ATP
(reviewed in ref. 4). A conserved basic patch of C-terminal resi-
dues has been implicated as the nsDNA-binding interface (13,
14), and mutation of SopA at this interface damages the ATP-
dependent nsDNA-binding activity in vitro and plasmid stability
in vivo (15).
In vivo, nsDNA mainly takes the form of the nucleoid. Several
fluorescent versions of plasmid and chromosomal ParAs display
dynamic patterns on the nucleoid (reviewed in ref. 4). Both
ATPase activity and the ability to interact with the cognate parti-
tion complex are essential for this dynamicpatterning. With either
capacity inactivated, dynamic patterning ceases. Overall, the evi-
dence suggests that the ParB-induced patterning by ParAs on the
nucleoid plays a key role in partition, but mechanistic insight is
limited. ParA patterns on the nucleoid have been interpreted as
filaments that pull the plasmid cargo (reviewed in ref. 1). We have
proposed an alternative diffusion-ratchet model in which the
partition complex stimulates the local release of nucleoid-bound
ParA, generating a ParA gradient that provides the motive force
for plasmid movement (16).
To gain further insight into Par-mediated cargo-transport
mechanisms, we reconstituted the P1 and F plasmid partition
reactions in a cell-free system, whereby an nsDNA-coated flowcell
surface (the DNA carpet) functioned as an artificial nucleoid (17).
internal reflection fluorescence microscopy (TIRFM). For both F
ParA-type partition systems self-organize and pattern the bac-
terial nucleoid to organize plasmids, chromosomes, and protein
machinery spatially. To study how protein patterns generate
cargo movement, we reconstituted and visualized the partition
system of F plasmid using a DNA-carpeted flowcell as an arti-
ficial nucleoid surface. We found that the partition proteins
could bridge plasmid to the DNA carpet dynamically and me-
diate plasmid motion. Our data favor a diffusion-ratchet
mechanism inherently different from classical motor protein or
actin/microtubule filament-based transport. We expect surface-
mediated patterning to become increasingly recognized as a
means of intracellular transport in all kingdoms of life.
Author contributions: A.G.V. and K.M. designed research; A.G.V. and L.C.H. performed
research; A.G.V. contributed new reagents/analytic tools; A.G.V. and L.C.H. analyzed data;
and A.G.V. and K.M. wrote the paper.
The authors declare no conflict of interest.
Freely available online through the PNAS open access option.
1To whom correspondence should be addressed. E-mail: email@example.com.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
| Published online March 11, 2013www.pnas.org/cgi/doi/10.1073/pnas.1302745110
Sop and P1 Par, we found that partition complex clusters could
form zones on the surrounding DNA carpet in which the partition
ATPase was depleted. These depletion zones are a critical com-
ponent of the diffusion-ratchet model (16). However, after for-
mation of the ATPase depletion zone, most partition complex
clusters dissociated from the DNA carpet without extensive
In vivo, the P1 Par system seems to transition between mobile
Sop system, on the other hand, patterns the nucleoid via a dy-
namically oscillating behavior (19) that is similar to several other
ParA-mediatedpartition systems(reviewedin ref. 4). Inthis study,
we used our cell-free setup to focus on how SopA and SopB me-
diate the dynamic movement of F plasmid, as being more repre-
find evidence for SopA filament formation on the DNA carpet.
Instead, we observed many partition complex clusters exhibiting
dynamic behaviors, including lateral movements on the DNA
carpet without any detectable SopA filaments. Similar dynamics
also have been observed in the P1 Par system (17). Our results
demonstrate that the P1 and F systems use the same principles of
outcome—faithful inheritance of genomic information. However,
subtle differences in their respective biochemistries likely underlie
variations in the resulting patterns seen in vivo.
ATP-Dependent SopA Binding to the DNA Carpet.To study F plasmid
partition in a cell-free system, we created a biomimetic of the
nucleoid surface by carpeting the flowcell with sonicated DNA
fragments (∼500 bp) at high density (∼1,000 fragments/μm2;
Materials and Methods). TIRFM was used to visualize Sop pro-
teins and plasmids interacting with the DNA carpet and with each
other in real time.
We first characterized the interaction between SopA and
nsDNA by using a functional SopA protein fused at its C terminus
to GFP (Fig. S1 A and B). When infused into the flowcell with
ATP and Mg2+, SopA-GFP bound the DNA carpet with high af-
finity (Fig. 1A). As shown previously for native SopA (20), ATP
was required for DNA binding; ATPγS or ADP could not replace
ATP. Taking into account the fluorescence intensity of a single
to be (6.1 ± 1.1) × 104dimers/μm2(Materials and Methods).
We then looked at SopA-GFP release from the DNA carpet.
SopA-GFP was flowed onto the carpet with ATP to varying
densities. The solution then was switched to wash buffer (without
SopA-GFP and ATP), and fluorescence intensity was monitored
over time (Fig. 1B). With increasing initial SopA-GFP density on
the DNA carpet, the apparent dissociation rate slowed. This ef-
fect was caused mainly by the rebinding of protein molecules that
were released from the DNA carpet upstream of the observation
area within the flowcell during washing. To eliminate the influ-
ences of rebinding, the flowcell was imaged immediately adjacent
to the flow convergence point (SI Materials and Methods and Fig.
S2) and at low SopA-GFP density (∼0.5% of saturation). We
obtained an apparent dissociation rate of 1.9/min, which is similar
to the rate of ∼1.5/min obtained previously for P1 ParA release
from individual lambda DNA molecules (16).
Elimination of the rebinding artifact also was achieved by in-
cluding competitor DNA in the wash buffer (Fig. 1C). DNA in
the wash buffer blocks the dissociated SopA-GFP from rebinding
the DNA carpet. When DNA was added to the wash buffer, the
observed dissociation rate of SopA-GFP was 1.9/min regardless
of where the measurement was done in the flowcell. The ATP-
turnover rate of SopA in the presence of saturating DNA (0.01/min;
ref. 21) is ∼200-fold slower than the rate of SopA release from
DNA. Thus, DNA release is not obligatorily coupled to ATP
hydrolysis. We conclude that an ATP-bound state of SopA can
DNA is ATP specific. SopA-GFP (0.5 μM) was mixed with 1 mM of the in-
dicated nucleotide upstream of the flowcell and was infused onto the DNA
carpet at a rate of 20 μL/min. The fluorescence intensity of SopA-GFP that
bound the DNA carpet (normalized to the saturated carpet intensity) was
measured over time. (B) Kinetics of SopA disassembly from DNA. Using
ATP, SopA-GFP was bound to the DNA carpet as in A. Once SopA-GFP
binding reached the carpet densities indicated as a percentage of satura-
tion, flow was switched to buffer without SopA (t = 0), and the decrease in
fluorescence intensity was monitored over time. The dissociation curve
measured at 0.5% saturation (red) was fit to a single exponential decay
function to obtain an apparent dissociation rate of 1.9/min. (C) Cofactor
effects on the kinetics of SopA disassembly from DNA. SopA-GFP was
bound to the DNA carpet up to saturation. At t = 0, the flow was switched
to wash buffer with or without the indicated cofactors (2 μM SopB, 100 μg/
mL sonicated DNA, or 2 mM nucleotide). For B and C, the y axis is nor-
malized to the SopA-GFP intensity before buffer switch. (D) SopB inhibits
SopA binding to DNA. SopA-GFP (0.5 μM) was preincubated alone or with
SopB at the molar ratios indicated for 30 min at 23 °C. Then ATP (1 mM)
was added, and the sample was infused at 20 μL/min until SopA-GFP
binding to the DNA carpet reached a plateau. Flow then was stopped, and
steady-state SopA-GFP binding to the DNA carpet was measured. The
y axis is the steady-state SopA-GFP intensity normalized to the saturated
carpet intensity of SopA-GFP. Error bars represent the SD of at least three
independent experiments. (E) FRAP analysis of SopA-GFP exchange on the
DNA carpet. SopA-GFP (0.5 μM) was preincubated with or without stoi-
chiometric amounts of SopB for 30 min at 23 °C. ATP (1 mM) was added,
and the sample was infused into the flowcell at 20 μL/min. (Upper) In the
presence of SopB, flow was stopped once SopA-GFP binding to the DNA
carpet reached steady state. (Lower) Without SopB, flow was stopped once
SopA-GFP reached a DNA-carpet density similar to that reached with SopB
present. SopA-GFP on the DNA carpet then was photobleached, and re-
covery was measured over time. The data were fit to a single (+SopB) or
double (SopA alone) exponential function. The y axis is SopA-GFP intensity
normalized to the saturated carpet intensity of SopA-GFP. (F) Summary of
rate data. Also see Fig. S1 and Movie S1.
SopA-GFP interaction with the DNA carpet. (A) SopA binding to
Vecchiarelli et al.PNAS
| Published online March 11, 2013
bind nsDNA dynamically before ATP hydrolysis. The dissocia-
tion rate of SopA-GFP from DNA is increased significantly by
including SopB in the wash buffer (Fig. 1C). The rate of SopA
dissociation in the presence of 2 μM SopB in the wash buffer was
8/min. Adding ADP or ATP to the wash buffer had no significant
effect on SopA-GFP dissociation. The data suggest that SopB
can accelerate SopA dissociation from DNA.
SopB Mediates SopA Interaction with the DNA Carpet. To examine
SopB effects on the steady-state DNA-binding activity of SopA,
we preincubated SopA-GFP and ATP with varying concen-
trations of SopB. The SopA-GFP/SopB/ATP mixture was flowed
onto the DNA carpet until the SopA-GFP density reached
a steady state. When flow was stopped, the SopA-GFP density
decreased to a new steady state (Fig. 1D). With no SopB, SopA-
GFP (0.5 μM) quickly saturated the DNA carpet as above. With
equimolar or greater amounts of SopB, the SopA-GFP density
on the DNA carpet dropped to less than 2% of saturation (Fig.
1D, Inset). SopB (2 μM) reduced the steady-state binding of SopA
to the DNA carpet by more than 100-fold, whereas the same
concentration of SopB accelerated the dissociation of SopA from
DNA by only fourfold (Fig. 1F). Thus, SopB-stimulated dissoci-
ation of SopA alone cannot explain the dramatic reduction in
steady-state DNA binding by SopA in the presence of SopB. The
data suggest that SopB also reduces the effective association rate
of SopA-ATP and DNA.
after photobleaching (FRAP) analysis of SopA-GFP exchange on
the DNA carpet with and without SopB (Fig. 1E). When ATP-
bound SopA-GFP alone was infused, the recovery curve fit a dou-
ble exponential function with recovery rates of 4.6 ± 0.7/min (58%
of total fraction) and 0.6 ± 0.2/min (42% of total fraction), im-
a rate of 3.6 ± 0.2/min, implying that the slower-exchanging pop-
steady-state SopA binding to the DNA carpet in the presence of
SopB. Therefore, SopB also must inhibit SopA binding to the
DNA carpet from solution. Together, the findings suggest that
a direct interaction with SopB converts SopA-ATP in solution to
a non–DNA-binding state and/or prevents the generation of the
DNA-binding form of SopA.
with rapid exchange kinetics as measured by FRAP (Fig. S1).
SopA-GFP did not affect the steady-state level of SopB binding or
its exchange rate on the carpet significantly.
via a transient interaction and does not involve a stable SopA–
SopB complex that lasts long enough to slow the DNA-bound
P1 ParA–ParB interaction on the DNA carpet (17).
No SopA-GFP Filaments Were Found on the DNA Carpet. Several
studies have shown that ParAs form filaments in vitro under
varying conditions (reviewed in ref. 4). We searched for and did
not find any evidence suggesting that SopA bound the DNA
carpet as long filaments. First, SopA-GFP uniformly bound, ex-
changed, and dissociated from the DNA carpet without any
pattern that suggested filamentation (Movie S1). SopA-GFP did
form filament bundles on unpassivated glass or quartz surfaces,
where nonspecific protein binding and denaturation is possible,
subunits within a long filament, if formed, would be relatively
immobile compared with the subunits at the filament ends.
Therefore, if SopA-GFP bound the DNA carpet as a filament,
the majorityofSopA-GFPmoleculesshouldbe relativelystatic.
From this viewpoint, we examined the motion of individual
SopA-GFP molecules bound to the DNA carpet. By diluting
SopA-GFP 10,000-fold with unlabeled SopA, we resolved sin-
gle SopA-GFP molecules and studied their diffusion charac-
teristics at varying SopA densities on the DNA carpet by single-
particle tracking (Fig. 2A and Movie S2). At low SopA density
(∼1% of saturation), the majority of SopA-GFP particles (91%,
n = 183) were observed to diffuse randomly on the DNA carpet
for distances >200 nm before releasing or photobleaching (Fig.
2B). Most particles diffused across the DNA carpet for several
microns with an apparent diffusion coefficient of 0.85 ± 0.14
μm2·s−1(Fig. 2C and Fig. S3A). These data confirm that SopA-
GFP can hop intersegmentally from one DNA molecule to an-
other in a fashion that seems to be independent of other SopA-
GFP particles close by. This behavior was more apparent in the
presence of flow, where particle movement was visibly biased by
the flow direction (Fig. 2D and Fig. S3B).
The fraction of SopA molecules that exhibited restricted mo-
SopA onthe DNA carpet.Whencarpet bindingbySopA reached
saturation, the percentage of SopA-GFP particles whose trajec-
tories did not exceed 200 nm rose to 29% (n = 237) (Fig. 2 E
and F and Fig. S3C). However, even at saturation, the diffusion
properties of the majority of SopA molecules were not signifi-
cantly different from those at lower SopA densities. The increase
in the fraction of SopA-GFP molecules engaged in constrained
diffusion may reflect excluded volume effects (molecular crowd-
ing), weak cooperative binding to DNA, or both. However, the
SopA density on the DNA carpet that was necessary for this effect
nM) and SopA-GFP (50 pM) were mixed with 1 mM ATP and infused onto the DNA carpet. (Scale bar: 5 μm.) (B) SopA-GFP particle motion on the DNA carpet at
low SopA density. At ∼1% saturation of the DNA carpet by SopA, flow was stopped, and the diffusion of SopA-GFP particles (n = 100) was tracked (yellow
lines). (Scale bar: 5 μm.) (C) A typical SopA-GFP particle trajectory at 1% saturation. (Scale bar: 1 μm.) (D) An example of SopA-GFP particle motion influenced
by flow (10 μL/min). (Scale bar: 1 μm.) (E) SopA-GFP particle motion on the DNA carpet at 100% saturation. SopA-GFP particle diffusion was tracked as in B.
(Scale bar: 5 μm.) (F) A SopA-GFP particle trajectory with reduced mobility at 100% saturation. (Scale bar: 1 μm.) See also Fig. S3 and Movie S2.
Single-particle tracking of SopA-GFP. (A) Total internal reflection fluorescence (TIRF) image of single SopA-GFP particles on the DNA carpet. SopA (500
| www.pnas.org/cgi/doi/10.1073/pnas.1302745110Vecchiarelli et al.
to be significant was at least two orders of magnitude greater than
that expected on the nucleoid in vivo. If one assumes that 1,000
SopA dimers exist inside an Escherichia coli cell with one chro-
mosome (22), ∼0.2% of the chromosomal DNA would be bound
by SopA. Also, nucleoid-associated proteins and transcription
factors dynamically occupy a significant fraction of the bacterial
chromosome and likely prevent SopA from forming stable higher-
order complexes on the nucleoid surface. Therefore, we conclude
that at physiologically relevant SopA/DNA densities only a minor
fraction of SopA molecules may engage in constrained diffusion,
whereas the majority of SopA can bind independently and hop
across DNA as dimers.
SopA Disassembly Is Required for Plasmid Movement on the DNA
Carpet. We have reported that in the reconstituted cell-free
system of the F-plasmid partition reaction SopB-bound sopC
plasmids were tethered transiently to the DNA carpet via DNA-
bound SopA-ATP (17). Here we studied how Sop protein con-
tent governs the dynamics of partition complexes on the DNA
carpet. A supercoiled pBR322::sopC plasmid (5 kb) labeled with
Alexa647 (sopC-647) was preincubated with SopA-GFP, SopB,
and ATP, and the mixture then was infused into the DNA-car-
peted flowcell for observation. Preincubation before infusion
results in the assembly of large partition complex clusters. After
infusion, flow was stopped, and imaging was continued (Fig. 3A
and Movie S3). SopA-GFP binding to the DNA carpet spiked
transiently and decreased to a steady-state level within a few
minutes (Fig. 3B). The plasmid clusters tethered to the DNA
carpet with colocalized SopA-GFP. SopA-GFP then dissociated
gradually before the plasmid clusters were released (Fig. 3C).
Upon release from the DNA carpet, these clusters still contained
a significant amount of SopA-GFP. Also, many of the larger
clusters showed a decrease in plasmid fluorescence, concomitant
with the decrease in SopA-GFP intensity, albeit at a slower rate.
The finding indicates that some plasmid copies are released from
the tethered cluster as SopA-GFP is released (Fig. 3D, foci 2 and
4). Interestingly, as SopA-GFP content decreased, most plasmid
foci displayed tethered particle motion before release from the
(Fig. 3E and Movie S4). We infer that an initial static plasmid
cluster is anchored to the DNA carpet with many tethers and that
the number of tethers decreases as SopA-GFP disassembles from
the complex, and the plasmid cluster wiggles on the few remaining
tethers. Once all tethers are lost, the plasmid cluster is released.
This progression confirms that SopB/sopC plasmid clusters are
tethered to the DNA carpet via their associated SopA-GFP.
This inference is supported further by covisualization of SopA-
GFP and SopB-Alexa647 (mixed 1:9 with unlabeled SopB) in the
presence of unlabeled sopC plasmid. The Sop proteins, plasmid,
and ATP were preincubated before infusion to form large plas-
mid clusters. SopA-GFP and SopB-Alexa647 formed large yel-
low foci that anchored transiently to the DNA carpet (Movie
S5). The tethered foci subsequently faded to red and released.
Quantification of the fluorescence intensities showed that both
proteins disassembled from the complex, but SopA dissociated
faster than SopB (Fig. 4A).
The details of the dynamics of individual plasmid clusters on
the DNA carpet varied. Some clusters bound and released the
carpet quickly; others stayed on longer. The SopA:SopB ratio of
a plasmid cluster appeared to influence both the rate of SopA
release and the dwell time of the cluster on the carpet. Those
exhibiting the fastest decline in SopA:SopB ratio were the first to
release from the carpet (Movie S6 and Fig. 4 B and C, foci 1 and
2). Foci that exhibited a slow decline in SopA:SopB ratio, or no
decline at all, stayed on longer (Fig. 4 B and C, foci 3 and 4). The
data suggest that excess SopB (over SopA) on the plasmid
interacts with SopA on the carpet to bolster bridging complexes,
and a prerequisite to plasmid movement is SopB-stimulated
removal of SopA from the bridge at a rate that exceeds the
Some Plasmid Clusters Exhibited Lateral Motion. As SopA-GFP
disassembled, some plasmid clusters moved laterally on the DNA
carpet before dissociating fully (Fig. 5A and Movie S7). The mo-
tion was random in the absence of flow. With flow, some plasmid
clusters rolled downstream while maintaining contact with the
was similar to that observed for other clusters at their time of
carpet dissociation (Fig. 3C), suggesting that new anchor-points
were being established at a rate similar to the rate at which old
anchor-points were released.
All plasmid clusters eventually dissociated into solution but
occasionally returned to rebind the carpet transiently (Fig. 5B
and Movie S7). These plasmid clusters paused intermittently on
the DNA carpet, sometimes rolling laterally for several microns
before falling back into solution. These revisiting clusters had
significantly less SopA-GFP content than the clusters at the time
of the initial dissociation from the DNA carpet. We believe this
rolling mode of plasmid motion, although only transiently ob-
served in our current setup, more closely reflects the in vivo
dynamics of the Sop system (Discussion).
We also assembled the P1 Par system in our cell-free system by
preincubating Alexa647-labeled parS plasmid with ParA-GFP,
ParB, and ATP. As with F Sop, the P1 Par system generated large
partition-complex clusters that released ParA-GFP over time, and
some complexes began traveling laterally on the DNA carpet be-
plasmid clusters split into smaller complexes. These traveling
partition complexes (17). We conclude that transient bridging
allowsthe partition complexes to roll on the DNA carpet, but their
dynamic movement and weak associations with the DNA carpet
The ParA family of ATPases can separate, transport, and position
DNA and large protein machineries inside bacterial cells. ParAs
that act on plasmids are key models for understanding how ATP-
driven patterning of biological surfaces leads to cargo transport.
ParAs bind nsDNA in vitro and colocalize with the nucleoid in
vivo (reviewed in ref. 4), but how this interaction plays a role in
the transport mechanism is a subject of considerable debate. Two
models have been proposed. First, filament-pulling models pro-
pose that ParA polymerizes into a continuous filament that
nonspecifically binds the nucleoid (23). When a filament end
encounters the plasmid, ParB stimulates ParA disassembly but
maintains contact with the depolymerizing end, which results in
plasmid pulling. As an alternative, we previously proposed a diffu-
sion-ratchet model (16) that does not invoke ParA filamentation.
Here, and in a companion paper (17), we made use of a cell-free
reaction system to try to distinguish between these two models. In
this study, we examined Sop protein dynamics and the motion of F
and P1 plasmid partition complexes on a DNA-carpeted flowcell
surface; a biomimetic of the nucleoid.
No SopA Filaments Were Detected. ParAs, including SopA, have
been reported to form filament-like helices in vivo and filament-
bundles in vitro, leading to models that resemble eukaryotic mi-
tosis. However, the conditions for in vitro polymerization vary
considerably among Par systems (reviewed in ref. 4). For SopA, in
vitro polymerization was stimulated by SopB and inhibited by
nsDNA (20). Because the majority of SopA colocalizes with the
nucleoid (15, 19), where DNA concentration is very high, it is
unlikely that SopA forms long, self-supporting filaments in vivo.
Vecchiarelli et al.PNAS
| Published online March 11, 2013
evenly as it was introduced with ATP into the flowcell, with no
bound SopA is inconsistent with long-lasting SopA filaments on
the DNA carpet. The exchange rate also was faster than the
for P1 ParA (17), indicating that DNA release is not obligatorily
coupled to ATP hydrolysis. Subunits of a hypothetical SopA fila-
ment are expected to be relatively stationary during their lifetime
within the filament. Instead, we found that the majority of in-
dividual SopA-GFP molecules hopped rapidly across DNA seg-
ments that made up the carpet. This finding agrees with previous
in vitro studies showing the rapid disassembly of SopA filaments
upon addition of nsDNA (20). We conclude that SopA does not
form nucleoid-associated filaments that persist long enough for
However, the possibility of limited SopA polymerization on DNA
still remains. Indeed, a fraction of carpet-bound SopA exchanged
However, this species was not observed in the presence of SopB.
DNA Bridging by SopA and SopB. When SopB was infused together
with SopA and ATP, SopB strongly inhibited SopA binding to the
DNA carpet. One simple explanation is that SopB-stimulated
ATP hydrolysis by SopA leads to its accelerated release from
DNA. However, SopA exchange on the DNA carpet was only
slightly faster when SopB was present. Together, the data indicate
that SopB inhibits the SopA–DNA interaction mainly by pre-
venting the accumulation of the DNA-binding form of SopA in
solution without triggering ATP hydrolysis. Carpet-bound SopB
exchanged quickly (t1/2= 2 s) and with no significant influence by
SopA, indicating that the SopA–SopB interaction on DNA is
transient but commits DNA-bound SopA to ATP hydrolysis.
When plasmid DNA was preincubated with SopA and SopB in
the presence of ATP, the proteins formed large plasmid clusters,
which anchored to the DNA carpet when infused into the
flowcell. When the sample contained SopA and SopB at ratios of
1:1 or lower, the steady-state SopA density on the carpet was
≤2% of saturation. Despite the low density, the plasmid was
anchored to the carpet soon after infusion, indicating that SopB
contributes to the formation of the DNA-bridging tether. Plas-
mid anchoring was followed by a decrease in plasmid-associated
SopA, and eventually the plasmid detached.
The bridging activity of SopA and SopB studied here has fea-
tures similar to the DNA-bridging complexes described for P1
and pSM19035 of Streptococcus pyogenes (14, 24). For all systems,
DNA bridging required both proteins and ATP, and bridge dis-
assembly was coupled to ParB-stimulated ATP hydrolysis. For P1,
the complex was proposed to bridge plasmid and nucleoid and
was referred to as the “nucleoid-adaptor complex” (NAC) (24).
For pSM19035, the bridging was proposed to play a role in
plasmid pairing as well as in forming an NAC-like complex (14).
Likewise, the large plasmid clusters studied here likely are con-
nected by the same protein-mediated contacts that tether plasmid
to the DNA carpet. We note that the interplasmid contacts within
a plasmid cluster generally persisted longer than the bridging
interactions between a plasmid cluster and the carpet, suggesting
of SopA-GFP (green) and sopC-647 (red). SopA-GFP (0.5 μM), SopB (1 μM),
and sopC-647 (0.1 nM) were preincubated together with 1 mM ATP for 15
min at 23 °C and were infused into the flowcell at 20 μL/min for 2 min before
flow stoppage and movie acquisition. A freeze-frame TIRF image is shown.
(Scale bar: 5 μm.) (B) SopA-GFP intensity was quantified at four different
regions of the DNA carpet where plasmid-clusters bound and released
within the time course indicated. (C) (Left) The average SopA-GFP intensity
Sop system effects on plasmid-cluster dynamics. (A) Covisualization
on the carpet was subtracted from the SopA-GFP intensities colocalized with
the four plasmid foci (y axis). (Right) Intensity also was converted to an es-
timated number of SopA-GFP dimers (y axis). (D) (Left) The sopC-647 in-
tensity was plotted over time to identify when the plasmid clusters bound
and released the DNA carpet (y axis). (Right) Intensity also was converted to
an estimated plasmid-copy number (y axis). (E) A time-lapse image series
displays the typical decrease in SopA-GFP and the wiggling motion that is
associated with large plasmid clusters before release. (Scale bar: 1 μm.) Also
see Movies S3 and S4.
| www.pnas.org/cgi/doi/10.1073/pnas.1302745110Vecchiarelli et al.
that the high SopB content of the cluster contributes to the sta-
bility of plasmid-plasmid association.
After plasmid clusters anchored to the carpet, SopA content
and, at a slower rate, SopB content decreased, typically within
a few minutes, resulting in a reduction of the A/B ratio. We be-
lieve SopA release is primarily the result of a SopB-stimulated
conformational change on SopA that eventually leads to ATP
hydrolysis. The decrease in SopB likely reflects the release of
plasmid copies from the cluster. We have shown that SopB
stimulates the release of SopA not only from the plasmid but also
from the surrounding DNA carpet (17). The development of
SopA (or ParA) depletion zones by a partition complex is one of
the central components of the diffusion-ratchet mechanism we
have proposed (16).
Just before release from the DNA carpet, the plasmid clusters
were held by a small number of anchor points and engaged in
tethered particle motion. During this period, some complexes
exhibited a “stepping” motion, whereby old anchor points were
released while new anchor points were established. Some clusters
stayed in this random-walk mode for some time before detaching
the diffusion-ratchet model, but it may reflect only one possible
mode of plasmid motion in vivo.
Partition System Dynamics in Vivo. Real-time in vivo cytology of
Par systems has provided a foundation on which to build
mechanistic models. Fluorescence microscopy of both the F Sop
and pB171 Par systems found that the plasmid chases nucleoid-
bound ParA at its movement front, leaving a region of the nu-
cleoid devoid of ParA in the wake of plasmid movement (19,
23). Concurrently, ParA reassociates with the nucleoid away
from the plasmid. When only one partition complex is present in
the cell, the plasmid focus is relatively mobile. However, a de-
crease in mobility is observed when ParA appears to colocalize
with the plasmid. Covisualization of P1 ParA and a plasmid
carrying the P1 par locus clearly show two distinct populations of
ParA: one colocalized with immobile plasmids and another
dispersed on the nucleoid (18). Plasmid-associated P1 ParA foci
disappeared from time to time, and this disappearance was
coupled to plasmid movement. Chromosomal ParAs also have
been observed as two discrete populations in vivo. Both ParA
from Caulobacter crescentus and ParAI from Vibrio cholerae
form foci that colocalize with their ParB-parS complexes during
periods of relative immobility (25, 26). In both cases, ParA
disappears from the mobile ParB-parS complex during segre-
gation, and the nucleoid-bound population of ParA redistributes
in response to ParB-parS motion. Our cell-free system recapit-
ulated the immobile phase of the in vivo system dynamics as well
as transition to the mobile phase. However, as discussed below, we
have reproduced the mobile phase only partially.
Comparing F and P1 System Dynamics. In vivo, P1 partition com-
plexes cycle between immobile and mobile phases, whereas an F
partition complex oscillates continuously from nucleoid pole to
pole without extended pauses (19). Therefore, our finding that
the Sop system also could anchor plasmid to the DNA carpet was
unexpected. When measuring the ATPase content of partition
complexes at the point of complex release from the DNA carpet,
the SopA content (25 ± 8 dimers per plasmid copy) was signif-
icantly higher than the ParA content (6 ± 3 dimers per plasmid
copy) (17). This result indicates that a smaller fraction of SopA
than of P1 ParA is capable of bridging the F partition complex to
the DNA carpet. Both systems generated ATPase depletion
zones on the DNA carpet around anchored partition complexes
(17). However, the zones were more robust and easier to detect
for the P1 system. The “premature” release of F partition
complexes from the DNA carpet could explain the weaker de-
pletion zones. We believe that development of the robust SopA
region of the DNA carpet where a plasmid cluster bound and released was measured for SopA-GFP (green) and SopB-Alexa647 (red) intensities over time.
Fluorescence on the DNA carpet was subtracted. The y axis is intensity converted to the number of SopA or SopB dimers (Materials and Methods). (Lower) Change
in the SopA:SopB molar ratio is displayed for the duration in which the plasmid cluster was associated with the DNA carpet. (B) Freeze-frame image shows four
plasmid clusters. (Scale bar: 2 μm.) (C) Effects of the SopA:SopB ratio on the dwell time of the clusters in B, quantified as in A. Also see Movies S5 and S6.
Effects of theSopA:SopB ratio on plasmid-cluster dynamics. (A) (Upper) Quantification of SopA and SopB dimers associated with plasmid-clusters. A
Vecchiarelli et al.PNAS
| Published online March 11, 2013
depletion zones necessary for plasmid movement in vivo requires
spatial confinement of the partition complex (see below).
After carpet detachment, partition complex clusters were ca-
pable of interacting transiently with SopA-bound carpet, which
we detected as short visits on the DNA carpet. These complexes
had a low SopA content and often rolled across the DNA carpet
for a few microns before dissociating into solution. Although
rolling was observed only transiently with our current setup, it
resembled Sop system dynamics in vivo. We found that some P1
partition complexes also revisited the DNA carpet after disso-
ciation. However, the ATPase depletion zones did not develop
around these traveling clusters, presumably because of the lack
of persistent interaction with the DNA carpet, and the motion
appeared random. Therefore, under the experimental conditions
used, the behavior of the F Sop and P1 Par systems was similar.
We conclude that both systems use the same diffusion-ratchet
mechanism, but subtle biochemical differences between ParA-
mediated systems could produce variations in the nucleoid-
patterning dynamics observed in vivo. These differences may
include the in vivo concentrations of Par proteins, the quanti-
tative differences of a variety of kinetic parameters as we have
observed (ref. 17 and this study), and possible crosstalk between
the system and the in vivo environment.
Spatial Confinement. We believe the plasmids did not roll per-
sistently along the DNA carpet, as they do on the nucleoid in
vivo, because our flowcell lacks a key requirement of the diffu-
sion-ratchet mechanism, namely, spatial confinement. Inside
bacterial cells, the gap between the nucleoid and inner mem-
brane is narrow (27). As a result, cargo with a large excluded
volume, such as a plasmid partition complex, cannot diffuse
quickly away from or through the nucleoid. Spatial confinement
of the partition complex would provide a persistent interaction
with the nucleoid surface and an uninterrupted generation of the
SopA depletion zone. This depletion would lead to the contin-
uous and directionally biased Brownian motion of the partition
complex as it searches for higher SopA concentrations on the
nucleoid. The perpetual cycle of chasing and redistributing the
ATPase would result in the oscillation of a single partition
complex. Multiple partition complexes would repulse one an-
other because of the ATPase depletion zone that develops on the
nucleoid in between them. The 25-μm-thick flowcell we currently
use does not provide the spatial confinement necessary for the
continual motion observed in vivo. We currently are exploring
several methods to provide sufficient confinement to our cell-
free system because we believe it is one of the key parameters for
persistent and directional transport of cargo.
If spatial confinement can sustain a SopA depletion zone in
vivo, how does the Sop system maintain its ability to anchor the
partition complex to the nucleoid and cluster plasmid copies?
Interplasmid bridging may be a general prerequisite for partition
to sort out the appropriate partners to be separated, not unlike
sister chromatid cohesion and homolog pairing in mitosis and
meiosis. In the context of plasmid partition, this sorting process
may be needed when several plasmid copies occupy a single
nucleoid. If too many plasmid copies formed independent par-
tition complexes with accompanying ATPase depletion zones,
these zones would merge, and the segregation mechanism that is
based on the ATPase distribution gradient would break down. A
corollary to this effect would be a weakened drive for the plasmid
copies to move away from each other. It is attractive to speculate
that this mechanism leads to interplasmid bridging, which
reduces the number of cargo units to a level needed for efficient
segregation and positioning.
Many biomolecular reactions rely on the internal dynamics of
a single, large, molecular machine. In contrast, the system studied
the geometrical settings of the cell provide boundaries for the
system components. The reaction steps that generate the overall
system dynamics occur as parallel and repeated cycles of assembly
and disassembly of many local complexes. When combined as
that this type of biomolecular-patterning and cargo-transport sys-
tem exists in all kingdoms of life and shares many of the reaction
principles identified here. We study a handful of examples that are
composed of a relatively small number of components, including
theMinD-MinE systemthatpatternsthe membraneto localizethe
bacterial divisome (28), the DNA-target search system used in
transposition target immunity (29), and the P1 and F plasmid
complex pattern-mediated transport mechanisms.
carpet. (A) Trajectories (white lines) of F plasmid clusters on the DNA carpet
up to the point of the first detachment. Images show the initial position of
the plasmid clusters immediately before lateral movement. (B) Trajectories
(white lines) of the F plasmid clusters during a brief revisit to the DNA carpet
and moving laterally. (C) As in B, except the P1 Par system was observed.
Split trajectories show when a P1 plasmid cluster splits into smaller com-
plexes. SopA- or ParA-GFP is green, and sopC- or parS-plasmid is red. For all
images, flow was stopped before movie acquisition. (Scale bar: 1 μm.) Also
see Fig. S4 and Movies S7 and S8.
Examples of F and P1 plasmid clusters moving laterally on the DNA
| www.pnas.org/cgi/doi/10.1073/pnas.1302745110Vecchiarelli et al.
Materials and Methods Download full-text
Strains, Plasmids, Media, and Growth Conditions. The construction details of
strains and plasmids used in this study are given in SI Materials and Methods.
Protein Expression and Purification. Hexahistidine-tagged SopA, SopA-GFP,
and SopB proteins were purified by Ni-NTA affinity chromatography and
subsequent ion exchange chromatography and/or gel filtration (SI Materials
and Methods). Details on SopB labeling with Alexa Fluor 647 are given in SI
Materials and Methods.
Flowcell Preparation. Lipid bilayer and NeutrAvidin coating of the flowcell
was performed as previously described (29). Sonicated salmon sperm DNA,
biotinylated at both ends, was attached to the flowcell surface at high
density as described (17) with minor modifications (SI Materials and Meth-
ods). The DNA fragments used to make the carpet were 500 ± 250 bp in
length. Using an estimated DNA-binding length of 8 bp per SopA dimer (30),
we calculated there are ∼60 dimers per DNA fragment at saturation. There-
fore, the density of the DNA carpet is estimated to be 1,000 fragments/μm2.
TIRFM Setup. The prism-type TIRFM setup using an Eclipse TE2000E micro-
scope (Nikon) with a PlanApo 60× NA = 1.40 oil-immersed objective and
magnifier setting at 1.5× was essentially as described (28), with minor
modifications (SI Materials and Methods). FRAP experiments were carried
out as described (28). The power of the 488-nm laser was sufficient for
bleaching SopA-GFP and also bleached SopB-Alexa647 to a lesser degree.
Microfluidics and Sample Handling for TIRFM. Unless stated otherwise, all
experiments were performed in Sop buffer: 50 mM Hepes (pH 7.5), 100 mM
KCl, 10% (vol/vol) glycerol, 5 mM MgCl2, 2 mM DTT, 0.1 mg/mL α-casein, 0.6
mg/mL ascorbic acid. Phosphoenolpyruvate (2 mM) (Sigma) and pyruvate
kinase (10 μg/mL) (Sigma) were added also for ATP regeneration.
connected to a syringe containing SopA-GFP, and the other inlet was con-
nected to a syringe containing wash buffer (as specified). The Y-patterned
flow channel was imaged at the point of flow convergence to minimize the
effect of protein rebinding to the DNA carpet during measurements of the
dissociation rate (SI Materials and Methods).
Partition-reaction components were preincubated before infusion and
were loaded into a 300-μL loop connected to a polyether ether ketone
Rheodyne injection valve. A syringe with Sop buffer was connected upstream
of the valve to push the sample into the flowcell. Tubing downstream of the
valve was connected to the flowcell inlet (dead volume = 20 μL). For most
experiments, the sample was infused into the flowcell at 20 μL/min for 2 min,
and flow was stopped for data acquisition.
Materials and conditions used for the P1 Par system experiments were as
described in ref. 17.
Single-Particle Tracking. SopA-GFP (50 pM) was incubated with 0.5 μM SopA
and 1 mM ATP in Sop buffer at 23 °C for 15 min. The sample then was in-
fused into the flowcell at 10 μL/min to the specified SopA densities on the
carpet, based on the amount of SopA-GFP known to be required to saturate
the DNA carpet. Before acquisition, background fluorescence was photo-
bleached using 1.4 mW laser power for 10 s. Streaming movies were ac-
quired at 10 Hz for 500 frames (50 s) with illumination and acquisition
parameters as described (SI Materials and Methods). Data analysis details are
given in SI Materials and Methods.
Estimating Protein Concentration and Plasmid Copy Number on the DNA
Carpet. The average fluorescence intensity of single labeled molecules was
measured and used to calculate the number of fluorescent molecules within
a region of interest. Details are given in SI Materials and Methods.
ACKNOWLEDGMENTS. We thank Vassili Ivanov for help with the microscope
setup, Yong-Woon Han for helpful suggestions regarding the study, and
Barbara Funnell for the P1 Par proteins. This work was supported by the
intramural research fund for the National Institute of Diabetes, and Diges-
tive and Kidney Diseases, National Institutes of Health, US Department of
Health and Human Services (K.M.). A.G.V. and L.C.H. were recipients of
a Nancy Nossal Fellowship.
1. Gerdes K, Howard M, Szardenings F (2010) Pushing and pulling in prokaryotic DNA
segregation. Cell 141(6):927–942.
2. Garner EC, Campbell CS, Weibel DB, Mullins RD (2007) Reconstitution of DNA
segregation driven by assembly of a prokaryotic actin homolog. Science 315(5816):
3. Larsen RA, et al. (2007) Treadmilling of a prokaryotic tubulin-like protein, TubZ,
required for plasmid stability in Bacillus thuringiensis. Genes Dev 21(11):1340–1352.
4. Vecchiarelli AG, Mizuuchi K, Funnell BE (2012) Surfing biological surfaces: Exploiting
the nucleoid for partition and transport in bacteria. Mol Microbiol 86(3):513–523.
5. Austin SJ, Abeles AL (1983) Partition of unit-copy miniplasmids to daughter cells. I. P1
and F miniplasmids contain discrete, interchangeable sequences sufficient to promote
equipartition. J Mol Biol 169(2):353–372.
6. Ogura T, Hiraga S (1983) Partition mechanism of F plasmid: Two plasmid gene-
encoded products and a cis-acting region are involved in partition. Cell 32(2):351–360.
7. Hirano M, et al. (1998) Autoregulation of the partition genes of the mini-F plasmid
and the intracellular localization of their products in Escherichia coli. Mol Gen Genet
8. Lim GE, Derman AI, Pogliano J (2005) Bacterial DNA segregation by dynamic SopA
polymers. Proc Natl Acad Sci USA 102(49):17658–17663.
9. Adachi S, Hori K, Hiraga S (2006) Subcellular positioning of F plasmid mediated by
dynamic localization of SopA and SopB. J Mol Biol 356(4):850–863.
10. Lynch AS, Wang JC (1995) SopB protein-mediated silencing of genes linked to the
sopC locus of Escherichia coli F plasmid. Proc Natl Acad Sci USA 92(6):1896–1900.
11. Pillet F, Sanchez A, Lane D, Anton Leberre V, Bouet JY (2011) Centromere binding
specificity in assembly of the F plasmid partition complex. Nucleic Acids Res 39(17):
12. Watanabe E, Wachi M, Yamasaki M, Nagai K (1992) ATPase activity of SopA, a protein
essential for active partitioning of F plasmid. Mol Gen Genet 234(3):346–352.
13. Hester CM, Lutkenhaus J (2007) Soj (ParA) DNA binding is mediated by conserved
arginines and is essential for plasmid segregation. Proc Natl Acad Sci USA 104(51):
14. Soberón NE, Lioy VS, Pratto F, Volante A, Alonso JC (2011) Molecular anatomy of the
Streptococcus pyogenes pSM19035 partition and segrosome complexes. Nucleic Acids
15. Castaing JP, Bouet JY, Lane D (2008) F plasmid partition depends on interaction of
SopA with non-specific DNA. Mol Microbiol 70(4):1000–1011.
16. Vecchiarelli AG, et al. (2010) ATP control of dynamic P1 ParA-DNA interactions: A key
role for the nucleoid in plasmid partition. Mol Microbiol 78(1):78–91.
17. Hwang LC, et al. (2013) ParA-mediated plasmid partition driven by protein pattern
self-organization. EMBO J, 10.1038/emboj.2013.34.
18. Hatano T, Niki H (2010) Partitioning of P1 plasmids by gradual distribution of the
ATPase ParA. Mol Microbiol 78(5):1182–1198.
19. Hatano T, Yamaichi Y, Niki H (2007) Oscillating focus of SopA associated with
filamentous structure guides partitioning of F plasmid. Mol Microbiol 64(5):
20. Bouet JY, Ah-Seng Y, Benmeradi N, Lane D (2007) Polymerization of SopA partition
ATPase: Regulation by DNA binding and SopB. Mol Microbiol 63(2):468–481.
21. Ah-Seng Y, Lopez F, Pasta F, Lane D, Bouet JY (2009) Dual role of DNA in regulating
ATP hydrolysis by the SopA partition protein. J Biol Chem 284(44):30067–30075.
22. Bouet JY, Rech J, Egloff S, Biek DP, Lane D (2005) Probing plasmid partition with
centromere-based incompatibility. Mol Microbiol 55(2):511–525.
23. Ringgaard S, van Zon J, Howard M, Gerdes K (2009) Movement and equipositioning
of plasmids by ParA filament disassembly. Proc Natl Acad Sci USA 106(46):
24. Havey JC, Vecchiarelli AG, Funnell BE (2012) ATP-regulated interactions between P1
ParA, ParB and non-specific DNA that are stabilized by the plasmid partition site,
parS. Nucleic Acids Res 40(2):801–812.
25. Fogel MA, Waldor MK (2006) A dynamic, mitotic-like mechanism for bacterial
chromosome segregation. Genes Dev 20(23):3269–3282.
26. Ptacin JL, et al. (2010) A spindle-like apparatus guides bacterial chromosome
segregation. Nat Cell Biol 12(8):791–798.
27. Mika JT, van den Bogaart G, Veenhoff L, Krasnikov V, Poolman B (2010) Molecular
sieving properties of the cytoplasm of Escherichia coli and consequences of osmotic
stress. Mol Microbiol 77(1):200–207.
28. Ivanov V, Mizuuchi K (2010) Multiple modes of interconverting dynamic pattern
formation by bacterial cell division proteins. Proc Natl Acad Sci USA 107(18):
29. Han YW, Mizuuchi K (2010) Phage Mu transposition immunity: Protein pattern
formation along DNA by a diffusion-ratchet mechanism. Mol Cell 39(1):48–58.
30. Hui MP, et al. (2010) ParA2, a Vibrio cholerae chromosome partitioning protein, forms
left-handed helical filaments on DNA. Proc Natl Acad Sci USA 107(10):4590–4595.
Vecchiarelli et al.PNAS
| Published online March 11, 2013