Segrosome assembly at the pliable parH centromere.

Segrosome assembly at the pliable
parH centromere
Meiyi Wu1, Massimiliano Zampini1, Malte Bussiek2, Christian Hoischen3,
Stephan Diekmann3 and Finbarr Hayes1,*
1Faculty of Life Sciences and Manchester Interdisciplinary Biocentre, The University of Manchester,
131 Princess Street, Manchester M1 7DN, UK, 2Department of Genetics, University of Kassel,
Heinrich-Plett-Str. 40, D-34132 Kassel and 3Department of Molecular Biology, Leibniz Institute for Age
Research, Fritz-Lipmann-Institute, 07745 Jena, Germany
Received December 16, 2010; Revised January 28, 2011; Accepted February 14, 2011
ABSTRACT
The segrosome of multiresistance plasmid TP228
comprises ParF, which is a member of the ParA
ATPase superfamily, and the ParG ribbon–helix–
helix factor that assemble jointly on the parH
centromere. Here we demonstrate that the distinct-
ive parH site (�100-bp) consists of an array of de-
generate tetramer boxes interspersed by AT-rich
spacers. Although numerous consecutive AT-steps
are suggestive of inherent curvature, parH lacks
an intrinsic bend. Sequential deletion of parH tetra-
mers progressively reduced centromere function.
Nevertheless, the variant subsites could be
rearranged in different geometries that
accommodated centromere activity effectively re-
vealing that the site is highly elastic in vivo. ParG
cooperatively coated parH: proper centromere
binding necessitated the protein’s N-terminal
flexible tails which modulate the centromere
binding affinity of ParG. Interaction of the ParG
ribbon–helix–helix domain with major groove bases
in the tetramer boxes likely provides direct readout
of the centromere. In contrast, the AT-rich spacers
may be implicated in indirect readout that mediates
cooperativity between ParG dimers assembled on
adjacent boxes. ParF alone does not bind parH but
instead loads into the segrosome interactively with
ParG, thereby subtly altering centromere conform-
ation. Assembly of ParF into the complex requires
the N-terminal flexible tails in ParG that are con-
tacted by ParF.
INTRODUCTION
The transmission of genetic information from generation-
to-generation is a fundamental biological process that
must take place with high fidelity. The molecular events
that underpin accurate genome segregation in eucaryotes
are comparatively well-described (1). In contrast, under-
standing of the mechanism of procaryotic DNA segrega-
tion is more rudimentary. However, the compact genetic
modules that mediate the precise partitioning of plasmids
are highly informative systems in which to unravel this
process in precise detail (2).
Four distinct classes of plasmid segregation cassette
have been defined (3). The two most well-studied types
each comprise a pair of autoregulated genes and a
nearby centromere analogue. The first gene specifies an
ATPase that either possesses Walker box motifs (ParA)
or is an actin homologue (ParM), whereas the accom-
panying gene encodes a centromere binding factor
(CBF) (4–8). The CBF is a site-specific DNA binding
protein that loads onto the centromere to produce a nu-
cleoprotein complex of defined geometry (9–12). The
ATPase does not directly contact the centromere, but
instead interacts with the CBF to assemble the mature
segrosome. In the case of ParM, ATP-induced
filamentation from the segrosome propels each member
of a plasmid pair in opposite directions to achieve segre-
gation (13). ParA homologues also polymerize in response
to ATP binding, a process that is influenced by the CBF
and/or by DNA (14–21). The ParA filaments emanating
from the segrosome may drive plasmids towards the
cell poles, or retraction of the ParA polymers may draw
plasmids in opposite directions away from the
cytokinetic zone (4,15). Recent in vivo studies favour the
latter (22,23).
*To whom correspondence should be addressed. Tel: +44 161 3068934; Fax: +44 161 3065201; Email: finbarr.hayes@manchester.ac.uk
5082–5097 Nucleic Acids Research, 2011, Vol. 39, No. 12 Published online 4 March 2011
doi:10.1093/nar/gkr115
� The Author(s) 2011. Published by Oxford University Press.
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/
by-nc/2.5), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

The tri-partite segrosome of multiresistance plasmid
TP228 comprises the ParA homologue, ParF, and the
ParG CBF which assemble on the parH centromere
(Figure 2). ATP binding promotes the polymerization of
ParF into dynamic, extensive multistranded filaments that
are implicated in segregation (15). The dimeric ParG
protein comprises C-terminal regions that interlock into
a ribbon–helix–helix (RHH) fold linked to a pair of
flexible N-terminal extensions (24). The folded region
harbours the major determinants for dimerization, for
binding to the parH centromere and to the OF operator
site, as well as for ParF interaction (18,24–26). The ParG
mobile tails are also multifunctional. First, arginine
fingers stabilize the transition state during nucleotide hy-
drolysis by their partner proteins. The ParG N-terminal
tail includes an arginine finger-like motif that stimulates
ATP hydrolysis by ParF (18). This stimulation may be a
crucial aspect of the cycle of ParF polymerization and
depolymerization during segregation. Second, ParF poly-
merization is stimulated by the ParG flexible tail (18). The
tails either may reorganize or stabilize ParF filaments by
tethering ParF monomers within a single protofilament or
aligned protofilaments. Alternatively, ParG might cluster
at points of polymer growth or disassembly (15,18). In this
sense, ParG may play a role similar to formins and related
factors that influence the elongation and disassembly of
actin filaments in eucaryotes, or may be analogous to
microtubule-associated proteins that modulate tubulin
dynamics (19). Third, ParG binds to the OF operator
during transcriptional repression of the parFG genes
(25). OF comprises eight degenerate 50-ACTC-30 boxes
arranged in a combination of direct and inverted orienta-
tion (26). Each tetramer motif recruits one ParG dimer,
implying that the fully bound operator is cooperatively
coated by up to eight dimers. The OF operator apparently
has evolved with subsites that bind ParG dissimilarly to
produce a nucleoprotein complex fine-tuned for optimal
interaction with the transcription machinery (26). A tran-
sient b-strand element in the ParG mobile tail associates
with the protein’s folded RHH domain thereby further
modulating the binding of the protein to the operator
(25). The mechanism by which this interaction between
flexible and folded domains affects DNA binding is
elusive.
CBFs have heterogeneous primary sequences that cor-
relate with the diversity in plasmid centromere organiza-
tion (27). The precise loading of each CBF onto its
cognate centromere is a vital early step that is crucial for
correct segrosome assembly and the subsequent cascade of
events during partitioning. Here, the interaction of ParG
with the distinctive parH centromere is dissected: parH is a
complex multisubsite locus that nevertheless can accom-
modate a variety of synthetic subsite re-arrangements for
accurate segregation. Both direct and indirect readout of
parH potentially are required for correct coating of the
centromere by ParG emphasizing that an intricate set of
interactions mediate the loading of the protein onto the
site. The centromere binding specificity of ParG is
enhanced by the protein’s flexible N-terminal tails which
also are necessary for recruitment of ParF to the mature
segrosome.
MATERIALS AND METHODS
Strains, plasmids and molecular biology procedures
Plasmids were propagated and analysed using Escherichia
coli DH5a (28). Strain BL21 (Novagen) was employed for
protein overproduction and plasmid partition assays were
performed in the polA strain BR825 (29). Recombinant
plasmids for overexpression of the parF and parG genes
were described previously (30). ParG derivatives with 9, 19
or 30 amino acid deletions of the N-terminal tail were
produced from plasmids constructed elsewhere (25). The
partition probe vector pFH450 is a derivative of the
bi-replicon plasmid pALA136 (31,32). Plasmid pFH547
comprises the parFGH region cloned in pFH450 (33).
Plasmid pMW20 was constructed in two steps. First, a
promoter-less parFG cassette was amplified from
pFH547, digested with SacI–XbaI, and inserted between
the same sites in the arabinose-inducible expression vector
pBAD30 (34) to generate plasmid pMW19. The
arabinose-inducible parFG cassette then was amplified
from pMW19, digested with XhoI, and inserted in the
same site in pFH450 to produce pMW20. Derivatives of
parH possessing a full complement of 50-ACTC-30 boxes,
but with one or more rearrangements (Figure 1), were
constructed by inserting double-stranded oligonucleotides
carrying the appropriate sequences and with EcoRV–NsiI
compatible ends between the same sites in pMW20.
Derivatives of parH bearing deletions of 50-ACTC-30
boxes were constructed first by amplifying the appropriate
regions from pFH547, cleaving the PCR products with
BamHI–XhoI, and inserting between the same sites in
pFH450. The arabinose-inducible parFG cassette from
pMW19 was then inserted as an XhoI fragment in the
same orientation in each case. The nucleotide sequences
of the inserts in all plasmid constructs were verified. DNA
cloning and other molecular biology procedures followed
standard protocols.
Plasmid segregation assays
Segregation assays were performed using pFH450 or
pMW20 derivatives that replicate at low copy number in
strain BR825 as detailed elsewhere (33). Briefly, the
relevant plasmid-bearing strains were grown for �25 gen-
erations without chloramphenicol selective pressure.
Plasmid retention was then determined by replica plating
colonies to agar medium with and without the antibiotic.
The values presented are the means of at least three inde-
pendent tests with typical standard deviations (SDs) of
�10%.
Protein production and purification
The hexahistidine-tagged ParF and ParG proteins were
overproduced and purified by Ni2+ affinity chromatog-
raphy as described previously (30).
Gel retardation assays
DNA fragments for gel retardation assays were PCR
products amplified from appropriate plasmid templates
using one primer bearing a 50 biotin label and a second
unlabelled primer, or were generated by annealing
Nucleic Acids Research, 2011, Vol. 39, No. 12 5083

complementary primers one of which was 50 biotinylated.
Purification of the fragments and conditions for retard-
ation assays were outlined in detail previously (26).
Briefly, biotinylated DNA (2 nM) was incubated for
20min at 25�C in binding buffer [10mM Tris–HCl, pH
7.5, 50mM KCl, 1mM dithiothreitol, 5mM MgCl2,
0.05mg/ml poly(dI–dC)] with the ParG concentrations
shown in figure legends. Reaction mixtures were
electrophoresed on 10% polyacrylamide gels in 0.5�
Tris–borate–EDTA (TBE) buffer for 30–90min at 80V
at 22�C. DNA was transferred by capillary action to posi-
tively charged nylon membranes (Roche), and the
transferred DNA fragments were immobilized by UV
crosslinking. The biotin end-labelled DNA was detected
using the LightShift chemiluminescent EMSA kit
(Pierce) (30).
DNase I footprinting
The preparation and purification of biotinylated PCR
products, conditions for DNase I footprinting reactions
with ParG, denaturing gel electrophoresis, and detection
of biotinylated DNA followed procedures described in
detail recently (26).
Atomic force microscopy and data analysis
For sample preparation in atomic force microscopy
(AFM), freshly cleaved mica was functionalized with
poly-L-lysine (PL) to support secure immobilization of
DNA (35). The mica disc was incubated with 30 ml of a
10 mg/ml aqueous PL solution for 30 s, subsequently
washed with 4ml of Millipore water and dried under a
nitrogen stream. A DNA fragment encompassing the
parH region was amplified using pFH547 as template.
Protein–DNA binding reactions (20 ml) included the
purified PCR fragment (2.5 nM) and ParG (1–3 mM) in
binding buffer (10mM Tris–HCl, 50mM KCl, 1mM
EDTA, pH 7.5). After 15–30min, the mixture was
diluted 30-fold in binding buffer and 30 ml of this
dilution were immediately placed on the PL-mica. After
60 s of incubation, the mica was rinsed carefully with 2ml
of Millipore water and allowed to dry under a gentle
nitrogen stream. Measurements were performed with an
Figure 1. Deletion and mutational analysis of the parH centromere. The segregation probe vector pMW20 is illustrated at the top. This plasmid
replicates at medium copy number via the pMB1 ori. Replication switches to a low copy number via the P1 replicon in a polA mutant, but the
plasmid is segregationally unstable in this background. The parFG genes are expressed from an arabinose-inducible promoter (PBAD) instead of from
their native regulatory sequences. The distribution and orientation of the variant 50-ACTC-30 motifs in the parH-OF region are indicated by open
arrowheads. The segments cloned in pMW20 and tested for segregation activity are shown. Tetramer boxes that were inverted from their normal
orientation are denoted with filled arrowheads. The 5- and 11-bp insertions in the parH+5 and parH+11 sites are indicated by filled bars. Segregational
stability assays were conducted as outlined in the text. The relative centromere activities (RCA) associated with the sites are also shown compared to
the activity conferred by the intact parH-OF region. The pMW20 vector without parH-OF had an RCA of 0.31. The values presented are the means
of at least three independent tests with typical standard deviations of �10%.
5084 Nucleic Acids Research, 2011, Vol. 39, No. 12

AFM instrument as described (36) and FESP tips (Veeco)
in tapping mode. Fields of 1� 1 mm were scanned at line
rates of 1–2Hz and resolution of 512� 512 pixels. AFM
images were plane corrected with SPIP software (Image
Metrology, Denmark) and saved in bitmap format for
further analysis with ImageJ software (version 1.41o,
NIH, USA). The images were scaled to 2048� 2048
pixels using bilinear pixel interpolation. The entire
contours of the DNA molecules were then traced using
the freehand line option and saved as xy-coordinates.
The traced contours were also marked in the images in
order to pinpoint two xy-coordinates that define the
region along each contour which was occupied by ParG.
To this end, the points at which the height begins to
increase relative to the free DNA were identified using
the height threshold tool and their xy-coordinates were
defined using the point selection tool. To compare the
regions occupied by protein with the putative binding
sites in the DNA fragments, the saved xy-coordinates
were characterized by their distances from the DNA
terminus which is nearest the protein complex. These dis-
tances, as well as total DNA lengths, were determined by
summing the distances between the successive coordinates
of the entire contours using Excel.
Bending analysis
Plasmid DNA carrying parH, OF and the 50-end of parF
was digested with various restriction endonucleases
producing different fragments for curvature analysis.
DNA was mixed with loading dye and analysed on
equilibrated native polyacrylamide gels as outlined previ-
ously (37). Gels were pre-run for �3 h until current and
temperature remained constant. Electrophoresis was
carried out in 1� TBE at 150V (8mA) for �4 h (migration
distance of bromphenol blue �14 cm) at 4 or 23�C. Gels
were stained for 30min in an aqueous solution of ethidium
bromide (1mg/l), followed by rinsing in water prior to
documentation. The 1 kb Plus DNA ladder (Invitrogen)
and the 1 kb DNA ladder (New England Biolabs) served
as marker fragments together with plasmid fragments re-
sulting from restriction digests running in the same lane as
the parH fragment. Migration of all fragments was
determined for each gel and calibration curves were
plotted using the marker fragments (logarithm of
number of base pairs versus distance migrated). The
apparent sizes in the acrylamide gel relative to the calibra-
tion curve were determined.
RESULTS
Defining and dissecting the parH centromere in vivo
The OF operator upstream of parFG comprises eight
variant 50-ACTC-30 motifs arranged in a combination of
direct and inverted orientation (Figure 1). The tetramer
boxes are separated regularly by 4-bp AT-rich sequences.
A single ParG dimer loads onto each 50-ACTC-30 box,
suggesting that the operator is cooperatively coated by
as many as eight dimers during transcriptional repression
of parFG (26). Inspection of the region further upstream
of OF revealed a second cluster of 12 degenerate
50-ACTC-30 motifs (parH), also separated by AT-rich
spacers (Figure 1). One of the boxes is inverted
compared to the remainder. These 12 50-ACTC-30 boxes
are embedded in a set of longer repeats that were noted
previously in this region and which were originally
proposed as the putative centromere locus at which
segrosome assembly occurs (4,30).
As repeat motifs are characteristic of plasmid centro-
meres (4), attempts were made to support the partitioning
of a segregationally unstable test plasmid harbouring the
full complement of 20 50-ACTC-30 boxes, i.e. parH-OF,
when the ParFG proteins were provided in trans from a
compatible plasmid. The proteins were produced either
from genes under the control of the native parFG expres-
sion signals or from a lactose-inducible promoter.
Expression of parFG from a variably inducible arabinose
promoter was also tested. In addition, selected synthetic
promoters with strengths from weak to high (38) were
trialled. None of these approaches elicited improved seg-
regation of the vector possessing the complete set of
tetramer boxes compared to the same plasmid lacking
the repeat sequences. As an alternative strategy, the
parFG genes were inserted in the segregation probe
vector, pFH450 (31), under the control of an
arabinose-inducible promoter (PBAD; 34). This manipula-
tion produced plasmid pMW20 that entirely lacks any of
the natural regulatory sequences upstream of parFG
(Figure 1). With arabinose induction, the plasmid was
maintained at a frequency of 18±3% during non-
selective growth for approximately 25 generations in an
E. coli polA mutant in which the plasmid replicates using
the low copy number P1 replicon. However, insertion of
the parH-OF region 50 of the arabinose promoter
improved retention to 59±16%, a value close to that
observed with the intact partition cassette (33). Similar
retention values were obtained when the parH-OF region
was cloned elsewhere downstream of parFG in pMW20
(data not shown). Thus, the parH-OF region exhibits
ectopic, centromere-like activity when located in cis to
parFG. One advantage in expressing parFG from PBAD is
that any contribution of OF to centromere activity can be
examined independently of its regulatory functions. The
conditions required for in trans activity of the ParFG
proteins at parH-OF have yet to be defined: the appropri-
ate intracellular protein concentrations and/or the
temporal pattern of parFG expression that is required to
support centromere activity in trans may be difficult to
replicate artificially. Alternatively, a DNA topological
requirement or a positional effect of the genes and/or
centromere may influence segregation activity. These
possibilities require further investigation.
As parH and OF both comprise arrays of 50-ACTC-30
boxes, the independent efficacies of the two regions in
centromere function were tested in pMW20. The parH
region displayed centromere activity that was indistin-
guishable from that of the complete parH-OF region
(Figure 1). The operator locus alone also was an effective
centromere, albeit with slightly less activity than parH: OF
may have dual roles in transcriptional repression of parFG
(26) and in centromere function. As the activity of parH
was not enhanced appreciably by OF, the centromeric
Nucleic Acids Research, 2011, Vol. 39, No. 12 5085

properties of parH alone were characterized further.
Progressive deletion of pairs of 50-ACTC-30 boxes from
parH was accompanied by concomitant gradual reduc-
tions in the retention levels of the test plasmids
(Figure 1). Notably, plasmids bearing only four (parH9–
12) or six (parH7–12) tetramer boxes were maintained at
approximately half the frequency conferred by full-length
parH.
The parH and OF sites display comparable centromere
activities, but the arrangements of the 50-ACTC-30 motifs
in the two loci differ markedly: parH comprises eleven
direct repeats and one inverted repeat (Figure 1),
whereas OF consists of three direct repeats interspersed
with five inverted boxes (26). To assess further the malle-
ability of these sequences for partition activity, synthetic
parH centromeres with reconfigured 50-ACTC-30 boxes
were cloned in pMW20 and tested in segregation assays
(Figure 1). A site in which all of the tetramer boxes were
oriented similarly was a proficient centromere (parHDIR).
Inversion of the six rightward motifs or the five leftmost
boxes of parH caused only modest decreases in segrega-
tion activity (parHRinv and parHLinv, respectively). A site
in which alternating tetramers in parH were inverted from
their canonical orientation was a less effective centromere,
although remained partially functional (parHALT).
Insertion of one-half helical turn at the centre of parH
only modestly affected centromere activity (parH+5),
whereas insertion of a complete turn (parH+11) reduced
centromere function more severely. Thus, a variety of
natural and artificial 50-ACTC-30 box dispositions are
viable for parH centromere action, although a minimum
of eight repeats is required for efficient activity. Moreover,
altering the relative helical positions of the two halves of
parH was well-tolerated whereas maintaining these pos-
itions, but increasing the distance between the halves,
was more deleterious.
Discrete binding of ParG to parH and OF in vitro
The ParG protein binds to the OF operator to achieve
transcriptional repression of the parFG genes (25,26).
DNase I footprinting in vitro of the parH centromere
region revealed that ParG also protects the entire set of
12 50-ACTC-30 boxes from digestion on both DNA
strands (Figure 2). The AT-rich spacers separating
certain boxes were slightly less well protected, most obvi-
ously on the top strand. The DNA fragments used in these
reactions harboured both parH and OF so as to ascertain
whether ParG interacted continuously or discontinuously
with the two loci: the zones of protection were separated
by a �5-bp window that remained fully accessible to
DNase I (Figure 2). These data correlate with recent ob-
servations that delimited the extent of ParG interaction
with OF (26).
As a second strategy to probe the interaction of ParG
with parH, a fragment possessing the centromere (365 bp)
was bound by ParG and visualized by AFM (Figure 3A).
The images showed protein binding as discrete extended
foci. The measured total DNA length was 117±5nm
which was somewhat shorter (�5%) than the 124 nm
expected for B-form DNA of this length. Shortening to
this extent has been observed previously by AFM of
naked DNA (35,39), so the reduced parH fragment
lengths observed here do not necessarily reflect
ParG-induced compaction. Therefore, the approximate
DNA helical rise was 0.32 nm/bp for these surface prep-
arations. To examine if the visible ParG binding
Figure 2. DNase I footprinting of ParG at the parH centromere and adjacent OF operator. The distribution and orientation of the variant
50-ACTC-30 motifs in the parH-OF region are indicated by open arrowheads. The bent arrow indicates the putative parFG promoter (25,26).
Footprinting reactions were performed as outlined in the ‘Materials and Methods’ section using PCR fragments biotinylated at the 50-ends of
either top or bottom strands. ParG concentrations (mM monomer, left to right): 0, 0.05, 0.1, 0.2, 0.6, 1.0 and 1.5. A+G, Maxam–Gilbert sequencing
reactions. The relative dispositions on the top and bottom strands of the parH region that are protected from DNase I digestion are shown in the
bottom panel. The 50-ACTC-30 motifs are boxed.
5086 Nucleic Acids Research, 2011, Vol. 39, No. 12