Journal of Structural Biology 178 (2012) 260–269

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Journal of Structural Biology

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Characterization of Escherichia coli nucleoids released by osmotic shock

Anna S. Wegner a,b, Svetlana Alexeeva a,1, Theo Odijk b,c,⇑, Conrad L. Woldringh a,⇑
a
Swammerdam Institute for Life Sciences, BioCentrum Amsterdam, University of Amsterdam, Kruislaan 316, 1098 SM Amsterdam, The Netherlands
b
Section Theory of Complex Fluids, Kluyver Institute for Biotechnology, Delft University of Technology, Julianalaan 67, 2628 BC Delft, The Netherlands
c
Lorentz Institute for Theoretical Physics, Niels Bohrweg 2, 2333 CA Leiden, The Netherlands

a r t i c l e i n f o a b s t r a c t

Article history: Nucleoids were isolated by osmotic shock from Escherichia coli spheroplasts at relatively low salt concen-
Received 29 November 2011 trations and in the absence of detergents. Sucrose-protected cells, made osmotically sensitive by growth
Received in revised form 2 March 2012 in the presence of ampicillin or by digestion with low lysozyme concentrations (50–5 lg/ml), were
Accepted 3 March 2012
shocked by 100-fold dilution of the sucrose buffer. Liberated nucleoids stained with 40 ,6-diamidino-2-
Available online 6 April 2012
phenylindole dihydrochloride hydrate (DAPI), the dimeric cyanine dye TOTO-1, or fluorescent DNA-bind-
ing protein appeared as cloud-like structures, in the absence of phase contrast. Because UV-irradiation
Keywords:
disrupted the DAPI-stained nucleoids within 5–10 s, they were imaged by time-lapse microscopy with
Escherichia coli
Nucleoid
exposure times less than 2 s. The volume of nucleoids isolated from ampicillin- or low-lysozyme spher-
Supercoiling oplasts and minimally exposed to UV (<2 s) was on average 42 lm3. Lysozyme at concentrations above
Polymer physics 1 lg/ml in the lysate compacted the nucleoids. Treatment with protease E or K (20–200 lg/ml) and
Lysozyme-spheroplast sodium dodecyl sulfate (SDS; 0.001–0.01%) caused a twofold volume increase and showed a granular
Ampicillin-spheroplast nucleoid at the earliest UV-exposure; the expansion could be reversed with 50 lM ethidium bromide,
Osmotic shock but not with chloroquine. While DNase (1 lg/ml) caused a rapid disruption of the nucleoids, RNase
DAPI/UV-radiation damage (0.1–400 lg/ml) had no effect. DAPI-stained nucleoids treated with protease, SDS or DNase consisted
Protein cross-links
of granular substructures at the earliest exposure similar to UV-disrupted nucleoids obtained after pro-
Ethidium bromide
longed (>4 s) UV irradiation. We interpret the measured volume in terms of a physical model of the
nucleoid viewed as a branched DNA supercoil crosslinked by adhering proteins into a homogeneous
network.
Ó 2012 Elsevier Inc. All rights reserved.

1. Introduction
The bacterial nucleoid has been studied extensively both in situ
and as an isolated structure. Electron microscope observations of
thin sections showed a meshwork of aggregated DNA fibers in
well-delineated or dispersed regions in which the DNA must have
suffered structural changes due to fixation and dehydration (for
reviews see Robinow and Kellenberger, 1994; Woldringh and
Nanninga, 1985). Additional information was obtained from iso-
lated nucleoids prepared by the so-called cytochrome-c-spreading
technique, showing for the first time supercoiled, branched DNA
loops radiating out of a ‘‘spider-like’’ core (Delius and Worcel,
1974; Kavenoff and Bowen, 1976; Meijer et al., 1976). These struc-
tures have frequently been interpreted as resulting from protein
and RNA cross-links and have led to the so-called ‘‘rosette’’ model
⇑ Corresponding authors. Address: Faculty of Science, Swammerdam Institute for
Life Sciences, University of Amsterdam, Science Park 904 (Room C2. 267), 1098 XH
Amsterdam, The Netherlands (C.L. Woldringh).
E-mail addresses: odijktcf@orange.nl (T. Odijk), c.l.woldringh@uva.nl (C.L.
Woldringh).
1 (Delius and Worcel, 1974). The results supported the interpreta-
Current address: Laboratory of Food Microbiology, Wageningen University,
Bomenweg 2, 6703 HD Wageningen, The Netherlands.
for a folded chromosome (see for review Toro and Shapiro, 2010).
The folds or domains are considered to represent independent
supercoiled loops, but the nature of possible crosslinks that form
the supercoiling barriers and that determine the size of isolated
nucleoids has remained obscure.
When analyzing the various procedures historically used to re-
lease the bacterial nucleoid, we may discern two agents that seem
to have been important in determining its size and structure: (i)
the presence of detergents and (ii) the concentration of lysozyme
in protocols without detergent. Since the introduction of the first
protocol by Stonington and Pettijohn (1971), detergents (non-ionic
Brij-58 and anionic deoxycholate) at a high salt concentration (1 M
NaCl) or spermidine have been used in most studies (Worcel and
Burgi, 1972, 1974; Kornberg et al., 1974; Drlica and Worcel,
1975; Meijer et al., 1976; Materman and van Gool, 1978; Murphy
and Zimmerman, 2000; Foley et al., 2010). Depending on the lysis
temperature, the detergent-salt method produced membrane-at-
tached (at 10 °C) or membrane-free (at 25 °C) ‘‘particles’’ as ana-
lyzed by sedimentation through sucrose gradients (Worcel and
Burgi, 1974) or by ‘‘visualization’’ with the electron microscope
tion that the released nucleoids were intact and supercoiled as

1047-8477/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.jsb.2012.03.007
 A.S. Wegner et al. / Journal of Structural Biology 178 (2012) 260–269
addition of ethidium bromide caused them to relax at a concentra-
tion of 2 lg/ml or re-compact at higher concentrations (>4 lg/ml;
Worcel and Burgi, 1972). However, the hydrodynamic properties
determined from sedimentation in sucrose gradients showed large
variations (Hecht et al., 1977; Odijk, 2002). In part, these could be
caused by entanglement with envelope fragments (Meijer et al.,
1976; Materman and van Gool, 1978). The theoretical analysis
(Odijk, 2002) of the sedimentation data of nucleoids obtained by
the detergent method (Hecht et al., 1977) proves that their dimen-
sions are typically of the order of 0.5 lm (see also Hecht et al.,
1975). This is much smaller than the nucleoids obtained by the
osmotic shock method (Cunha et al., 2001b; this work). The impli-
cation is that we have to be careful in interpreting the response to
enzymatic treatments in the respective isolation methods.
Nucleoids released by the detergent method were found to be
sensitive to treatment with RNase (Worcel and Burgi, 1972; Petti-
john and Hecht, 1973) and proteases (Drlica and Worcel, 1975)
possibly indicating the existence of crosslinks. Moreover, deter-
gents can be expected to dissociate proteins from the DNA and
they could therefore influence subsequent structural transitions
upon enzyme treatments. To circumvent such problems, Sloof
et al. (1983) introduced an osmotic shock procedure without deter-
gents using protoplasts of Bacillus licheniformis. In a previous study
(Cunha et al. (2001a) we compared this method with the lysis with
detergents using the same Escherichia coli spheroplast suspension.
We observed that both procedures resulted in comparable sizes of
the isolated nucleoids visualized by fluorescence microscopy, in
spite of the large difference in salt concentrations (1 M NaCl in
the detergent method versus 10 mM NaCl in the osmotic shock
method).
In subsequent studies (Cunha et al., 2001b) we emphasized the
osmotic shock method and we determined the compaction of iso-
lated nucleoids by polyethyleneglycol (PEG) and measured the
dynamics of fluorescently labeled DNA regions in the isolated
structures (Cunha et al., 2005). We found that when released by os-
motic shock, the nucleoids expanded like a spring into cloud-like
structures with an average volume of about 30 lm3. Romantsov
et al. (2007) obtained nucleoids by osmotic shock in the shape of
rounded, delineated structures with a volume of about 18 lm3.
These nucleoid volumes are well below the theoretical value of
un-crosslinked nucleoids expanded by excluded volume effects
and estimated to be about ten times larger (Cunha et al., 2001b).
What causes this variation in size of isolated nucleoids and what
prevents their full expansion?
As lysozyme is known to be a basic protein (Kuehner et al.,
1999) it could well bind to DNA causing nucleoid compaction. In
this work we have therefore refined previous protocols and we
have prepared spheroplasts either in the absence of lysozyme,
though by growing cells in the presence of ampicillin, or by using
much lower concentrations of lysozyme (as low as 0.5–0.05 lg/
ml in the lysate). The goal of nucleoid isolation is to prepare nucle-
oids that are expanded versions of their counterparts in vivo. Ide-
ally, they should not be perturbed by the compounds used in the
isolation protocol. We hope that our new protocols are a further
step in realizing this ideal.
The present nucleoids obtained by osmotic shock and stained
with DAPI, appear to expand faster than previously described (Cun-
ha et al., 2001a,b). We have therefore measured their sizes from
images taken by time-lapse microscopy within 2 s of UV-exposure.
The cloudlike structures appear to be larger (42 lm3) and to fall
apart fast during UV-irradiation as a network of granules with
what appears to be Brownian motion superposed. A similar expan-
sion of granular substructures was observed after treatment with
protease, sodium dodecyl sulfate (SDS) or DNase. In contrast to
studies using the detergent method (Worcel and Burgi, 1972),
RNase appeared to have no effect. We show that our current meth-
261
od of preparing and imaging low-lysozyme or lysozyme-free
nucleoids is particularly suitable for establishing their dimensions.
These sizes will be discussed in terms of a physical model in which
the nucleoid is considered to be a branched DNA supercoil cross-
linked by adhering proteins into a homogeneous network (see
Fig. 5 and Appendix B for a description of this model).
2. Materials and methods
2.1. Strains and growth conditions
Strains E. coli K-12 MC4100 (laboratory strain LMC500) and
E. coli 500/pSACT11 were grown at 28 °C in glucose minimal med-
ium containing 6.33 g of K2HPO4.3H2O, 2.95 g of KH2PO4, 1.05 g of
(NH4)2SO4, 0.10 g of MgSO4.7H2O, 0.28 mg of FeSO4.7H2O, 7.1 mg
of Ca(NO3)2.4H2O, 4 mg of thiamine, 4 g of glucose and 50 mg of ly-
sine, per liter pH 7.0 at 28 °C (doubling time at 28 °C was about
85 min). NaCl was added to adjust the osmolarity of the medium
to 300 mosM (Micro-Osmometer, Advanced Instruments). Cell
growth was monitored at 450 nm with a spectrophotometer. Expo-
nential growth was maintained by periodical dilutions of the cul-
ture. For E. coli LMC500, we estimate the number of chromosome
equivalents per nucleoid to be about 1.58 (see e.g. Huls et al.,
1999). E. coli 500/pSACT11, grown in the presence of 100 lg/ml
ampicillin, was used so as to visualize nucleoids with the fluores-
cent fusion protein HupA-mRFP (red fluorescent protein) after
induction with 0.3 mM IPTG. Strain E. coli K-12 FH2973/pFHC2973
(obtained from F. Hansen, Biocentrum DTU, Denmark. See Nielsen
et al., 2006) was grown in the same medium with 0.5% glycerol as
carbon source and supplemented with 100 lg/ml ampicillin (dou-
bling time at 28 °C was about 120 min). When 0.58 M sucrose was
added to E. coli LMC500 growing in glucose-minimal medium (see
below) the doubling time at 28 °C increased to 120 min. When
indicated (as TY) the glucose minimal medium was supplemented
with 10 g of bactotryptone, 5 g of yeast extract, 5 g of NaCl and
15 mmol NaOH per liter giving a doubling time at 28 °C of about
60 min.
2.2. Plasmid construction
A HupA-mRFP protein fusion was constructed using standard
laboratory techniques (Miller, 1992). mRFP from pGEM-XbBg-
mRFPsv (Alexeeva et al., 2010) was used to create a mRFP fusion
to the C-terminus of HupA, amplified from chromosomal E. coli
MC4100 DNA. See Supplementary data (S2.1) for details.
2.3. Chemicals
DAPI was obtained from Sigma and was prepared by dissolving
500 lg/ml in distilled water with the help of sonication. TOTO-1
(dimeric cyanine dye) and FM4-64 were obtained from Molecular
Probes (Invitrogen), sodium dodecyl sulfate from Koch-Light Labo-
ratories and glutaraldehyde from BDH (UK). RNase A was pur-
chased from Sigma, bovine serum albumin (BSA) and protease E
from Calbiochem, protease K from Invitrogen, pancreatic Dnase I
from Roche Diagnostics.
2.4. Preparation of lysozyme-spheroplasts
Except for the application of a cold-shock (4 °C) to the cells be-
fore lysozyme addition, the protocol was that described essentially
by Cunha et al. (2005). In short: 2 ml of E. coli cells grown in glu-
cose minimal medium (Gmin) to an OD450 of 0.2 (5  107 cells
per ml) were centrifuged in an Eppendorf centrifuge for 5 min at
10,000 rpm. The cell pellets were resuspended in 475 ll of
262 A.S. Wegner et al. / Journal of Structural Biology 178 (2012) 260–269
sucrose-buffer consisting of 0.58 M sucrose, 10 mM Na2HPO4/
NaH2PO4-buffer pH 7.4 (hereafter written as NaPi), 10 mM EDTA
and 100 mM NaCl, at a temperature of 4 °C. After this cold-shock,
25 ll of a freshly dissolved lysozyme solution (1 mg/ml, or, as indi-
cated, 0.1 mg/ml) was added giving a final concentration of 50 or
5 lg/ml. Within 5–10 min of incubation at room temperature
(RT) a suspension of >95% spheroplasts was obtained. Osmotic
shock (see Section 2.6) was applied 10–15 min after lysozyme
addition. Additional details of the protocol are given in Supplemen-
tary data (S2.2).
2.5. Preparation of ampicillin spheroplasts
Ten milliliter of a suspension of E. coli cells growing exponen-
tially in glucose minimal medium (doubling time 90 min at
28 °C) were centrifuged and resuspended in 10 ml of glucose min-
imal medium containing 0.58 M sucrose. After 4 h of adaptation
and growth (doubling time 120 min), ampicillin (pI = 4.95; Hout
and Poole, 1969) was added to the culture at a final concentration
of 5 mg/ml. Microscopic inspection showed that in 60–90 min
most cells either displayed central bulges or had converted to
spheroplasts. The suspension (4 ml) was then centrifuged in an
Eppendorf centrifuge for 10 min at 2500 rpm and the pellet resus-
pended at RT in 2 ml of the sucrose-buffer, mentioned above.
Faster centrifugation resulted in breakage of spheroplasts and
expansion of prematurely liberated nucleoids. The ampicillin-
spheroplast suspension was kept at RT until osmotic shock.
2.6. Osmotic shock procedure
Osmotic shock of either lysozyme- or ampicillin-spheroplasts
was performed by adding 10 ll of spheroplast suspension to
990 ll of 10 mM NaPi buffer (pH 7.0) in an Eppendorf tube. This
represents a hypotonic shock by a 100-fold dilution of the sucrose
buffer giving a final concentration of 1 mM NaCl and of 0.5 or
0.05 lg/ml lysozyme in the lysate. After carefully inverting the
tube once, the lysate was kept at RT until staining and microscopic
preparation. Nucleoids obtained from either lysozyme- or ampicil-
lin-spheroplasts will be called ‘‘lysozyme-nucleoids’’ or ‘‘ampicil-
lin-nucleoids’’, respectively, throughout.
2.7. Microscopy
Lysates containing liberated nucleoids were stained with DAPI
at a final concentration of 0.1 lg/ml. For comparison, TOTO-1
was used at a final concentration of 2 lM. The stained suspensions
were either observed under a coverslip to observe both ghosts and
nucleoids or as a hanging-drop preparation to observe free-floating
nucleoids. Application of a cover slip could sometimes cause
adherence of nucleoids to the glass surface or nucleoid disruption
by liquid motion. For a hanging-drop preparation a coverslip with
20 ll of the lysate was inverted, placed over a hole in a metal ob-
ject slide and fixed with two stickers.
Nucleoid suspensions were photographed with a Coolsnap fx
charge-coupled device camera using an Olympus BX60 micro-
scope equipped with a 100 UplanFL 1.3 oil immersion phase
contrast lens. DAPI fluorescence images were taken with an Olym-
pus cube unit (U-MWU) with a single band-pass excitation filter
(330–385 nm) and a long-pass barrier filter (>420 nm). (The
absorption maximum of DAPI is at 358 nm, close to the near
UV-A line at 365 nm of the mercury vapor lamp.) For TOTO-1,
HupA-RFP or ethidium bromide (EtBr) fluorescence images, an
Olympus U-MNG cube unit was used with an excitation filter of
530–550 nm, a dichroic mirror of 570 nm, and a long-pass emis-
sion filter of >590 nm. TOTO-1 was sometimes also excited with
an Olympus cube unit U-MNB with a single band excitation filter
(470–490 nm), dichroic mirror of 500 nm, and a long-pass emis-
sion filter >515 nm.
The size of the isolated nucleoids was estimated as described
previously using the Object-Java software package developed in
this laboratory and the macro ‘‘Island-method’’ (Cunha et al.,
2001b). In short, an ‘‘index volume’’ of the nucleoid was calculated
from the area of a circular threshold that contained 50% of the total
fluorescence intensity within a sufficiently wide region of interest
(radius 100 pixels or 6.7 lm) around the nucleoid (see also http://
simon.bio.uva.nl/objectj/examples/Island/).
To minimize radiation damage of DAPI-stained nucleoids by
UV-excitation (Cunha et al., 2001b), the iris diaphragm of the mer-
cury lamp was closed to well within the field of view and the
preparation was scanned by hand with the UV-shutter remaining
open. As yet unexposed nucleoids coming into the field of view
were focused and automatically photographed at a 0.5 s time inter-
val. Sequences of 5–10 images were taken of every individual
nucleoid coming into the field of view. The first image that
appeared in focus, usually after 1–2 s of exposure, was selected
for volume measurement.
3. Results
3.1. Size and stability of liberated nucleoids
In protocols for nucleoid isolation using the detergent method,
the lysozyme concentration used to obtain spheroplasts was gen-
erally 400 lg/ml in the lysate (Worcel and Burgi, 1972). In our pre-
vious work applying the osmotic shock method, we used this
(Cunha et al., 2001a) or a somewhat lower lysozyme concentration
(300 lg/ml, resulting in 3 lg/ml after osmotic shock; Cunha et al.,
2001b). That different protocols can result in different properties of
spheroplasts and even prevent the liberation of nucleoids upon os-
motic shock is described in Supplementary data (Section S2.2;
Figs. S1 and S2).
It should be noted that different dilutions in the osmotic shock
procedure give rise to different final concentrations of NaCl and
lysozyme in the lysate. With its isoelectric point (pI) of 11.16
(Kuehner et al., 1999), lysozyme is a strongly charged molecule
carrying about nine positive charges under our conditions that
could compact the DNA like other multivalent ions. In Fig. 1 we
show how the size of liberated nucleoids changes with the final
concentration of the lysozyme in the lysate. The NaCl concentra-
tion in these lysates varied from 1 to 11 mM. Addition of 200–
1000 mM NaCl only resulted in a slight compaction, but increased
the stability of the DAPI-stained nucleoids (in the presence of 1 M
NaCl nucleoids were still intact after 20 s UV irradiation; results
not shown).
High lysozyme concentrations (>1 lg/ml) in the lysate not only
determined the size of the DAPI-stained nucleoids but also their
stability toward UV irradiation. In order to investigate this
influence of lysozyme on nucleoid stability further, we studied
lysozyme-free nucleoids obtained from ampicillin-generated
spheroplasts (see Section 2; ampicillin pI = 4.95; Hout and Poole,
1969). Snapshot images of such nucleoids usually had much larger
sizes (>200 lm3; see Table 1). These dimensions agree quite well
with the maximum size of 209 lm3 predicted for the uncrosslinked
nucleoid theoretically (see Appendix B). Apparently, these DAPI-
stained nucleoids were more labile under UV irradiation than lyso-
zyme-nucleoids, and the images were obtained in an incipient
stage of disruption by UV radiation (see below). We therefore pho-
tographed the free-floating nucleoids using time-lapse imaging in
which the first image could be obtained within 1 s of irradiation.
In the time sequences of Fig. 2, we discern that lysozyme-nucleoids
from 300 lg/ml lysozyme-spheroplasts are stable during at least
 A.S. Wegner et al. / Journal of Structural Biology 178 (2012) 260–269 263

from these observations that lysozyme compacts and stabilizes

Fig.1. Snapshot images of DAPI-stained nucleoids from E. coli LMC500 cells grown
in glucose-minimal medium prepared under a coverslip. The sucrose buffer
containing 300 lg/ml lysozyme was diluted 100- to 10-fold giving final lysozyme
concentrations in the lysate of 3 (A), 6 (B), 10 (C) and 30 lg/ml (D). Average index-
volumes of nucleoids (see also Table 1) decreased from 30 lm3 (A) to 10 lm3
(D). Magnification bar 2 lm.
4 s of UV irradiation (Fig. 2A), while ampicillin-nucleoids already
start to expand after 2.5 s of illumination (Fig. 2B). The latter also
applies to nucleoids obtained from 50 and 5 lg/ml lysozyme-
spheroplasts (0.5–0.05 lg/ml in the lysate; Fig. 2C), indicating that
a high lysozyme concentration in the lysate (3 lg/ml) protects
nucleoids against radiation damage.
It should be noted that TOTO-1-stained nucleoids pre-irradiated
with UV for 20 s in the absence of DAPI showed no expansion. This
suggests that DAPI renders the DNA much more sensitive to UV,
possibly causing direct strand breaks. Such destruction in the pres-
ence of DAPI has been described for UV irradiation of isolated
chinese hamster cell chromosomes (Buys et al., 1986).
Table 1 presents a quantitative overview of the nucleoid vol-
umes obtained in the various protocols. While snapshot images
of nucleoids from 300 lg/ml lysozyme-spheroplasts give volumes
of 25–27 lm3, ampicillin nucleoids, stained with either DAPI or
TOTO-1, were almost 10 larger. However, volumes of minimally
irradiated DAPI-nucleoids from time-lapse sequences of either
ampicillin-spheroplasts or low-lysozyme spheroplasts (50 lg/ml)
were repeatably in a range of 34–54 lm3 (Supplementary data,
Table S1), with an average of 42 lm3. As expected, addition of a fi-
nal concentration of 3 lg/ml lysozyme to ampicillin-nucleoids did
indeed cause a decrease in volume (row 6 in Table 1). We conclude
nucleoids upon their release by osmotic shock.
In Fig. 2B and C it can be seen that DAPI-stained nucleoids irra-
diated for 5 s or longer fall apart into granular substructures that
apparently show Brownian motion (cf. also Supplementary movie,
Fig. S3B). The earliest images of nucleoid sequences (<2 s UV expo-
sure) exhibited a more homogeneous structure, often consisting of
large lobular regions as shown in Fig. 3. We do not know whether
these lobules can be ascribed to replication and segregation stages
of the in situ nucleoid.
3.2. Treatment of nucleoids with protease, SDS, Rnase and DNase
Because lysozyme-nucleoids were smaller (30 lm3) than the-
oretically predicted (209 lm3; see Appendix B), the possible pres-
ence of protein cross-links was investigated by treating lysates
with protease E or K and SDS. High-lysozyme-nucleoids (300 lg/
ml; Fig. 2A) were found to be unaffected by incubation with 20
to 200 lg/ml protease E (pI  4) at 37 °C for 60 min. Addition of
the negatively charged BSA (pI = 4.72; Vilker et al., 1981) at the
same molarity showed no effect on nucleoid volumes (results not
shown).
To ascertain the activity of the protease, lysozyme-nucleoids
were isolated from E. coli LMC500/pSACT11 cells in which nucle-
oids are visualized by the DNA-binding protein HupA-RFP.
Although the HupA-RFP-fluorescence vanished within several min-
utes of enzyme incubation after addition of protease E, the nucle-
oids remained intact for an additional 60 min as became evident
from DAPI-stained preparations (results not shown). In contrast,
low-lysozyme- (50 lg/ml) and ampicillin-nucleoids did show a
doubling in volume after 30 min (Table 2), while the control nucle-
oids remained stable for at least 24 h (Table 2, row 2) and even
72 h (Table 1, row 7). Protease E-treated nucleoids could still be
compacted by addition of 20 lg/ml EtBr (row 12 in Table 2). Nucle-
oids treated with 20 lg/ml protease K (pI  4) for 150 min already
showed a granular structure within 2 s of UV-exposure, i.e. before
disruption by radiation damage (Fig. 4 A, B).
Compared to the protease treatment, the effect of addition of
SDS was dramatic. Even high-lysozyme-nucleoids (300 lg/ml)

Table 1
Comparison of index-volumes of nucleoids of E. coli MC4100 from independent growth and isolation experiments. Free-floating lysozyme- or ampicillin-nucleoids in hanging-
drop preparations were photographed by snapshot or time-lapse imaging and measured using the ObjectJ macro ‘‘Island method’’.

Growth mediuma Sphero- plastb Lysoz. Conc. Staining/photo- Index volume Number nucleoids Compare with Ref.
c
(lg/ml) graphyd lm3 (CV)e measuredf or Fig. (indep. exp’s)g
G-min Lys 300 DAPI/Snapshot 27 – Cunha et al.
G-min Lys 300 DAPI/Snapshot 25 (84) 50 Fig. 3A
G-min Amp – DAPI/Snapshot 253 (21) 12 –
G-min Amp – TOTO/Snapshot 215 (26) 26 –
Gmin + S Amp – DAPI/T-lapse after 72 h 42 (50) 127 Fig. 4-
41 (50) 24
G-min + S Amp +300h DAPI/T-lapse 25 (71) 10 –
Gmin + S Amp – DAPI/T-lapse 36 (45) 151 (4x)
G-min Lys 50 DAPI/T-lapse 44 (43) 180 Fig. 3B (6x)
LB + S Amp – DAPI/T-lapse 69 (42) 28 –
LB + S Amp – DAPI/T-lapse 89 (46) 32 –
G-min + S Amp – DAPI/T-lapse 41 (48) 34 Fig. 3C
G-min + S Amp – TOTO/T-lapse 45 (49) 16 –
a
G-min, glucose-minimal medium; LB, trypton-yeast extract medium;+S, medium supplemented with 0.58 M sucrose.
b
Spheroplast formation: Lys, lysozyme method; Amp, ampicillin method (see text).
c
Lysozyme concentration in incubation mixture; final concentration in lysate after osmotic shock is x 1/100.
d
See Section 2 for Snapshot or Time-lapse imaging.
e
Although volumes are given in lm3 it should be noted that it is an ‘‘index volume’’ (see Section 2) used to compare nucleoid volumes in the different experiments. The
radius of the region of interest (ROI) used in the macro ‘‘Island-method’’ was 100 pixels.
f
The relatively low numbers in individual experiments are due to the fact that from a time-lapse sequence of DAPI-stained, free-floating nucleoids only few nucleoids are
measurable (see Section 2).
g
Indep. exp’s: number of independent growth and lysis experiments that have been averaged.
h
Lysozyme added before shock; final concentration in lysate is 3 lg/ml.
264 A.S. Wegner et al. / Journal of Structural Biology 178 (2012) 260–269

Fig.2. Time-lapse sequences of DAPI-stained nucleoids in hanging-drop preparations, expanding during 4–6 s UV-exposure into a network of granules. In each panel the
upper number represents the time of irradiation in seconds; the lower number signifies the index volume as determined with the Island-method macro. (A) Nucleoid
liberated from a 300 lg/ml lysozyme-spheroplast. (B) Nucleoid from an ampicillin-spheroplast. The first image was captured while the microscope stage was still moving (see
Section 2). (C) Nucleoid from a 50 lg/ml lysozyme-spheroplast. Magnification bars 5 lm.

to spherical structures. Because of the fairly high isoelectric point

Fig.3. First images (UV-exposure less than 2 s) selected from time-lapse sequences
of DAPI-stained nucleoids obtained from ampicillin-spheroplasts of E. coli LMC500,
prepared in a hanging drop. At this early stage of exposure the nucleoid seems
composed of a few relatively large lobules that rapidly fall apart into a network of
granules (cf. Fig. 2). Average volume of nucleoids 42 lm3 (see Table 1). Magnifi-
cation bar 5 lm.
dispersed within several minutes upon addition of 0.01–0.5% SDS
at RT. Table 2 (last rows) shows how nucleoids that are first ex-
panded with 0.01% SDS, can become compacted again by the addi-
tion of 20 lg/ml EtBr. It should be noted that DAPI-stained
nucleoids treated with SDS showed the same expansion via granu-
lar substructures as observed for protease (Fig. 4B). As SDS can be
expected to denature and dissociate DNA-binding proteins, we as-
cribe the limited expansion of nucleoids to the release of protein
crosslinks. This is supported by the observation that fixation with
0.25% glutaraldehyde prevented the expansion of DAPI-stained
nucleoids by SDS (0.01%; 15 min), but not the subsequent disrup-
tion by UV-exposure (results not shown).
In studies using the detergent method, RNase was found to un-
fold the isolated nucleoids (Worcel and Burgi, 1972). Treatment of
the present nucleoids isolated by osmotic shock with RNase A con-
centrations varying from 0.1 to 400 lg/ml only showed a collapse
of RNase A (pI = 9.6; Tanford and Hauenstein, 1956), nucleoids
were shocked in 10 mM NaPi at pH 8.5 of the buffer to diminish
the positive charge of the RNase. Again, only collapsed nucleoids
were observed, which were similar to high-lysozyme nucleoids
(cf. Fig. 1D). Addition of the negatively charged BSA at the same
molarity showed no compaction (results not shown). On the
whole, we know that the nucleoids isolated by the two respective
methods are dramatically different in size (Odijk, 2002), so they are
not identical entities. Hence the different response to treatment by
RNase need not be surprising.
Nucleoids subjected to mechanical forces (e.g. by preparation
under a coverslip) can become disrupted into thread-like struc-
tures. Contrary to expectation, treatment with pancreatic DNase I
(pI = 4.7–5.3; Funakoshi et al., 1980) in the presence of 1 mM
Mg2+, did not result in such threads, but in nucleoids expanding
as a granular network (Fig. 4C), similar to those of protease
(Fig. 4B) or SDS treatments. This suggests that the expansion of
DAPI-stained nucleoids during prolonged UV irradiation (Fig. 2B,
C) likewise results from breaks in the DNA strands.
3.3. Treatment of nucleoids with chloroquine and EtBr
Romantsov et al. (2007) report that varying the concentration of
chloroquine (10–68 lM) caused changes in volume that reflected
the relaxation and positive supercoiling of their nucleoids. In our
system, adding chloroquine in a range of concentrations between
12.5 and 500 lM to DAPI-stained nucleoids did not show any sig-
nificant change in volume (Table 3). Chloroquine has been found to
 A.S. Wegner et al. / Journal of Structural Biology 178 (2012) 260–269 265

Table 2
Index-volumes of nucleoids of E. coli MC4100 treated with 200 lg/ml protease E and SDS (preparations and measurements as in Table 1).
(c)
Growth Sphero- Lysoz. Conc. Staining/ photo- Treatment Index volume Number nucleoids
medium(a) plast(b) (lg/ml) graphy(d) lm3 (CV)(e) measured(e)
Gmin Lys 50 DAPI/Tlapse Control 47 (51) 26
Gmin Lys 50 DAPI/Tlapse Control after 24 h 36 (47) 50
Gmin Lys 50 DAPI/Tlapse protE, 30’ 69 (45) 34
Gmin Lys 50 DAPI/Tlapse protE, 90’ 83 (95) 15
Gmin Amp - DAPI/Tlapse Control 37 (38) 62
Gmin Amp - DAPI/Tlapse protE, 30’ 75 (55) 38
Gmin Amp - DAPI/Tlapse protE, 24 h 195 (74) 7
Gmin Lys 50 TOTO/Snapshot Control 51 (27) 16
Gmin Lys 50 ‘‘ protE, 45’ 105 (60) 37
Gmin Lys 50 DAPI/Tlapse Control 41 (48) 40
Gmin Lys 50 DAPI/Tlapse protE, 30’ 87 (66) 12
Gmin Lys 50 Snapshot protE, 30’ + 20 lg/ml EtBr 19 (55) 31
Gmin Lys 5 DAPI/Tlapse Control 35 (60) 56
Gmin Lys 5 DAPI/Tlapse +0.01% SDS 119 (57) 46
Gmin Lys 5 DAPI/Tlapse +0.01% SDS + 20 lg/mlEtBr 24 (104) 20

See notes of a–e Table 1.

show decreased binding affinity to DNA at higher salt concentra-

tions (Kwakye-Berko and Meshnick, 1989). Because our shockbuf-
fer contained 1 mM NaCl and 10 mM sodium phosphate versus
10 mM NaCl in the procedure of Romantsov et al. (2007), ionic
strength cannot explain the discrepancy. Because chloroquine has
also been reported to become degraded by UV irradiation (Karim
et al., 1994), experiments using DAPI-stained nucleoids were re-
peated with TOTO-1 staining, but failed to show volume changes,
except at the highest concentration (500 lM; Table 3, row 7).
In contrast, addition of EtBr did show relaxation (0.5 lg/ml) and
compaction (20 lg/ml) of liberated nucleoids (Table 3, row 11 and
12), suggesting that the insensitivity to chloroquine is not the
result of the presence of nicks in the DNA.

4. Discussion

Bacterial nucleoids released by osmotic shock and stained with

DAPI were observed by fluorescence microscopy to be cloud-like
structures, sensitive to radiation damage. To characterize the effect
of lysozyme, the nucleoids were isolated either in the absence of
lysozyme (ampicillin-nucleoids) or at low lysozyme concentrations
(0.5–0.05 lg/ml in the lysate). With our standard protocol the
majority of the nucleoid mass turns out to be released from the
spheroplast, but always remains attached to its ghost. The liber-
ated nucleoids have volumes about 400 larger than those
in vivo and usually show two or more lobules (Fig. 3) that may
reflect the state of replication and segregation in the cell.
Paradoxically, under conditions that cause the peptidoglycan
layer to be well digested, the osmotic shock procedure does not re-
sult in nucleoid release, but in so-called balloons (Fig. S1 and S2).
These represent expanded but delineated structures that were
insensitive to UV exposure. We think that within these structures
the swollen nucleoid is still restricted by the outer membrane.
Within the context of our standard protocol, however, such struc-
tures are never obtained.
There are significant differences between the observations of
Romantsov et al. (2007) and ours. First, they used a radically differ-
ent protocol for the osmotic shock of their spheroplasts by includ-
ing 0.025% formaldehyde in their shock buffer; they also
incorporate a much higher concentration of 8 lg/ml lysozyme in
their lysate. Their nucleoids are smaller (18 lm3), appear to be
much more delineated than ours, and do not have the cloud-like
appearance of the nucleoids here described. Second, they observed
an effect of chloroquine on nucleoid volume, whereas we only
Fig.4. Images selected from time-lapse sequences (0.5 s interval) of DAPI-stained
nucleoids obtained from 5 lg/ml lysozyme-spheroplasts of E. coli LMC500, prepared
in a hanging drop. (A) Time sequence of untreated control nucleoid. Average index
volume 57 lm3. Magnification bar 5 lm. (B) Time sequence of nucleoid treated
with 20 lg/ml proteinase K for 150 min. Average index volume 103 lm3 (see
Table 2). Magnification bar 5 lm. (C) Image selected from a time sequence of
nucleoids treated with 1 lg/ml DNase and 1 mM MgCl2 for 3 min, taken after 1.5 s
exposure. Note the granular substructure.
found an effect with EtBr. It has been reported that chloroquine
binds less efficiently to DNA than quinacrine or EtBr (Jones et al.,
1979) and this may bear on the disparity. Further experiments
are required to elucidate these differences and to establish
whether the ‘‘structural units’’ as characterized by fluorescence
correlation spectroscopy (Romantsov et al., 2007) are similar to
the granular substructures described here. (These ‘‘units’’
ultimately derive only from the osmotic pressure because a wave
vector dependence was absent in their experiments.)
Zimmerman (2006a,b) reported on the compaction and degra-
dation of nucleoids prepared by the so-called polylysine-spermi-
dine procedure, a modification of the detergent method. Because
this lysis procedure causes associations between nucleoids and
cytoplasmic and envelope cell remnants, the final structures
266 A.S. Wegner et al. / Journal of Structural Biology 178 (2012) 260–269

Table 3
Index volumes of nucleoids treated with chloroquine or EtBr.

Growth Sphero- Lysoz. Conc.(c) Staining/photo- graphy(d) Treatment Index volume Number nucleoids
medium(a) plast(b) (lg/ml) lm3 (CV)(e) measured(e)
Gmin + S Amp - Dapi/Timelapse Control 58 (42) 20
Gmin + S Amp - Dapi/Timelapse 25 lM Chqu 52 (54) 28
Gmin + S Amp - Dapi/Timelapse 100 lM Chqu 54 (59) 53
Gmin Lys 5 DAPI/Tlapse Control 35 (60) 56
Gmin Lys 5 TOTO/Snapshot 12.5 lM Chqu 52 (41) 44
Gmin Lys 5 TOTO/Snapshot 50 lM Chqu 42 (59) 27
Gmin Lys 5 TOTO/Snapshot 500 lM Chqu (258 lg/ml) 39 (57) 29
Gmin Lys 50 DAPI/Tlapse 250 lM Chqu 32 (72) 34
Gmin Lys 50 DAPI/Tlapse 500 lM Chqu (258 lg/ml) 16 (72) 23
Gmin Lys 50 DAPI/Tlapse Control 46 (27) 37
Gmin Lys 50 EtBr/Snapshot 0.5 lg/mlEtBr 82 (48) 45
Gmin Lys 50 DAPI/Snapshot 20 lg/mlEtBr + DAPI 7 (68) 23
Gmin Lys 50 EtBr/Snapshot 100 lg/mlEtBr 1 (85) 21
Gmin Lys 50 DAPI/Tlapse Control 41 (48) 40
Gmin Lys 50 EtBr/Snapshot 2 lg/ml EtBr 46 (68) 13
Gmin Lys 50 EtBr/Snapshot 20 lg/ml EtBr (50 lM) 18 (45) 27
Gmin Lys 50 EtBr/Snapshot 100 lg/ml EtBr (250 lM) 1 (98) 20

See notes of a–e Table 1.

resemble lysed cells rather than released nucleoids (see Fig. 11 in
Murphy and Zimmerman, 2000). Their conclusions about possible
cross-links are difficult to compare with our results on nucleoid
stability and expansion.
Our results do conform well to the observations of Sloof et al.
(1983), whose method was the basis for our procedure in the
beginning stages of this work. Although they had a high final con-
centration of 5 lg/ml lysozyme in the lysate, their results agree
with ours in that their nucleoids were sensitive to treatment with
proteinase K but insensitive to RNase. They also studied the re-
sponse of their nucleoids to increasing concentrations of EtBr
(elaborating on the work of Worcel and Burgi, 1972). In an analysis
by sucrose gradient centrifugation and electron microscopy, Sloof
et al., 1983 found a relaxation with 0.5 lg/ml EtBr but a condensa-
tion after surface-spreading in the presence of 100 lg/ml EtBr,
similar to our fluorescent nucleoids (Table 3).
The observation that removal of proteins causes about a dou-
bling in the size of the nucleoids (Table 2; Fig. 4A and B) suggests
that protein cross-links play a role in the limited expansion of the
nucleoids upon their release by osmotic shock. Such protein-DNA
interactions are in accordance with the theoretical inference that
a large number of cross-links must be present in isolated nucleoids
(Cunha et al., 2001b).
The present low-lysozyme and lysozyme-free nucleoids were
shown to become compacted by final lysozyme concentrations in
the lysate greater than 1 lg/ml (Fig. 2). We think that lysozyme
may not only compact but also stabilize a nucleoid both against
UV exposure and treatment with protease. The presence of lyso-
zyme in many previous studies using the detergent method (e.g.
Drlica and Worcel, 1975) could also explain the varying results
concerning nucleoid stability obtained as a function of enzyme, salt
and temperature (see for reviews Pettijohn, 1982; Pettijohn and
Sinden, 1985).
We have argued here that our improved protocol for nucleoid
isolation also leads to a more quantitative assessment of nucleoid
sizes than in previous experiments. It is then expedient to discuss
these dimensions in terms of a physical model of the bacterial
nucleoid as presented in earlier work (Odijk, 1998, 2000, 2002;
Cunha et al., 2001b). The nucleoid is viewed as a branched plecto-
nemic supercoil with adhering proteins which are assumed to in-
duce crosslinks between two sections of double-stranded DNA
(Fig. 5). The supercoil interacts with itself via the excluded-volume
effect (and with cytoplasmic proteins, in the case in vivo) and is
perturbed by thermal motion. For convenience, we present an out-
line of the statistical physical model in Appendix B (the equations
referred to below are stated there). The typical index volume Vui of
the uncrosslinked nucleoid, i.e. the DNA supercoil if it were free
from proteins, is there argued to be about 209 lm3 which is in ac-
cord with the maximum sizes achievable in our studies (see Tables
1 and 2), as we have already pointed out above.
Both ampicillin and low-lysozyme nucleoids have an average
index volume Vni of about 42 lm3 if the irradiation is minimal.
What does this small size imply? According to Eq. (4), crosslinking
purportedly collapses the supercoil in part; statistical arguments
show that the smaller index volume Vni is a measure of the typical
number of plectonemic segments N H between two crosslinks. The
number of crosslinks N s =N H is predicted to be about 25. This turns
out to be of the order of magnitude of the number of ‘‘domains’’
established in strand nicking experiments (Sinden and Pettijohn,
1981; Worcel and Burgi, 1972; Drlica et al., 1978). It would be
interesting to monitor Vni as a function of the degree of nicking
to see if this correspondence is really causal. In our osmotic com-
paction experiment a decade ago, we studied the interaction of iso-
lated E. coli nucleoids with polyethylene glycol in order to
determine the free energy of a nucleoid as a function of its size
(Cunha et al., 2001b). A scaling formalism then also establishes
the number of crosslinks which is an order of magnitude greater
than that found here on the basis of Eq. (4). At this stage, the dis-
crepency is not so worrisome since numerical coefficients are lack-
ing in both scaling pictures (the one on crosslinks proposed in
Cunha et al., 2001b and the other outlined in Appendix B). Another
quantity of interest is RH ’ 0:67 lm predicted by Eq. (2). This
scale, the distance between crosslinks, is of the order of the
wavelength of light. It may be difficult to monitor the correlation
structure of the nucleoid by fluorescence microscopy (see Supple-
mentary movie, Fig. S3).
As we add protease E to ampicillin nucleoids, the index volume
increases from 37 to 75 lm3 after half an hour (see Table 2). Eq (4)
then yields N s =N H ’ 4, so the number of crosslinks decreases quite
fast. Ultimately, after a day, the typical index volume of the nucle-
oid has increased to about 195 lm3. Assuming that no nicks have
been introduced, this value suggests that all protein appears to
have been removed from the supercoil (compare with the theoret-
ical prediction ’209 lm3 for the uncrosslinked superhelix quoted
above).
Finally, the specific linking difference r is thought to saturate to
about 0.15 in absolute magnitude (Pruss, 1985). Accepting this, we
compute a supercoil diameter Ds of about 7 nm from the theory of
Ubbink and Odijk (1999) at an ionic strength of about 0.005 M (this
is an estimate of the ionic strength of our saline phosphate buffer).
 A.S. Wegner et al. / Journal of Structural Biology 178 (2012) 260–269 267

Fig.5. Schematic representation of the E. coli chromosome and of liberated nucleoids. (A) A 5 min-segment of the chromosome (total 100 min) is drawn as a randomly
branched chain of 200 Kuhn segments (which is a fractal). Possible crosslinks by DNA-binding proteins (blue circles) are indicated by double arrows. The plectonemic coil has
a diameter Ds. (B) Representation of the branched supercoil crosslinked by proteins (blue doublets) with an index volume of 42 lm3. Removal of protein crosslinks causes an
expansion (right panel) to a theoretical index volume of 209 lm3 (see text Appendix B). (C) Liberated nucleoids, crosslinked (left panel) and uncrosslinked (right panel), as
observed under the microscope; compare with Figs. 3 and 4 C, respectively.

From Eqs (1) and (4), we know that Vn scales as Ds3/5 – the degree of
crosslinking is assumed to be independent of the degree of
supercoiling – so the index volume Vni is predicted to reach a
minimum value of about 13 lm3 (the original diameter is 52 nm,
see Appendix B). This estimate agrees with our measurements
performed on lysozyme nucleoids treated with ethidium bromide
at a concentratioin of 20 lg/ml (see Table 3). At a very much higher
concentration of 100 lg/ml, the nucleoid presumably collapses
onto itself.
The DAPI-stained nucleoids appeared very sensitive to UV irra-
diation and rapidly fell apart (within 5 s; Figs. 2 and 4) as a net-
work of granular substructures. We could discern temporal
fluctuations at short scales superposed on these structures. We be-
lieve these could be a signature of Brownian motion, but this
would have to be proved within a model. The granules could also
be observed after TOTO-1-staining, especially with nucleoids from
ampicillin-spheroplasts and when irradiated with the 470–490 nm
excitation filter (see also Fig. S3B). Presently, we do not know the
significance of the sub-resolution granules that can be observed
in all preparations, i.e. also after protease, SDS or DNase
treatments.
If the nucleoid does indeed behave like a rubbery gel, the inho-
mogeneities at the short scale R⁄ would be expected but, again, a
detailed theory would need to be correlated with experiment.
We hope to investigate this in future work.
Although we do not understand the nature of the granules
(Figs. 2 and 4 and Fig. S3) appearing in expanding nucleoids under
virtually all conditions, our observations suggest that the isolated
nucleoid is better described as a homogeneous, core-less DNA net-
work rather than a fanciful ‘‘rosette’’ consisting of individual DNA
loops radiating from a central core (Pettijohn, 1982; Toro and
Shapiro, 2010). With our improved isolation protocol, nucleoids
are now more amenable to quantitative studies such as the influ-
ence of specific DNA-binding proteins (like H-NS; ms. in prepara-
tion) and the application of microfluidic devices or optical
tweezers for further characterization of their physical properties.
Acknowledgments
The authors thank Karl Drlica for suggestions on the manu-
script, Flemming Hansen for giving strain FH2973, Tanneke den
Blaauwen and Rogier Stuger for discussions, Norbert Vischer,
268 A.S. Wegner et al. / Journal of Structural Biology 178 (2012) 260–269
Ronald Breedijk and Jolanda Verheul for technical help, Sonia
Cunha for preliminary experiments and Mark Hink for making
the movie (Supplementary data, Fig. S3B). This work was sup-
ported by the Dutch NWO program ‘‘From Molecule to Cell’’, grant
805-47.032-P.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.jsb.2012.03.007.
Appendix B
B.1. Physical model of the nucleoid
We here summarize several features of a physical model of the
nucleoid as formulated by one of us (Th.O.). The chromosomal DNA
helix of E. coli has a contour length of 1.6 mm and is a closed circu-
lar twisted curve. Under the usual conditions, the interwound helix
is a plectoneme of specific linking difference r = 0.06. However,
there are proteins associated with the DNA helix which relieve
some of the twisting tension and the effective value of r has been
estimated to be reff ’ 0.025 (Bliska and Cozzarelli, 1987). In the
physical model of the nucleoid, the latter variable is assumed to
be distributed uniformly along the chain.
In a concrete model of plectonemic DNA, electrostatic energy
arising from the self-energy of the charged DNA is balanced
against the bending and twisting energies of the DNA helix to-
gether with the configurational entropy (Ubbink and Odijk,
1999). The diameter Ds and effective contour length Ls of the
supercoil may be computed as a function of r and the ionic
strength I (the diameter is defined in Fig. 5). In polymer theory
the notion of statistical segments is often introduced (called Kuhn
segments). In the case at hand, the supercoil is viewed as consist-
ing of Ns segments of length As (Cunha et al., 2001b). But the
supercoil is branched (see Fig. 5) with a branching density
Ks = n3/Ns where n3 is the number of branches presumed to be tri-
functional (Vologodskii and Cozzarelli, 1996). The radius of gyra-
tion of the uncrosslinked supercoil may be written as (Odijk,
2002; Cunha et al., 2001b)
1=10 1=5 4=5 1=2
Ru ¼ 0:399N 1=2
s Ks Ds As  bN s
where we have introduced a length parameter b (note that there is
a misprint in Eq. (1) of Odijk (2002)). Eq. (1) represents the largest
size the isolated bacterial chromosome could adopt, at least theo-
retically. Superficially, the half power dependence on Ns in Eq. (1)
might lead one to think that the uncrosslinked chromosome is
actually a Gaussian random coil but it is not: under ideal condi-
tions the size would scale as N 1=4
s but the segments are nonover-
lapping because they have a nonzero diameter Ds and thus
interact via an excluded-volume effect. The exponent ½ of the
expanded branched coil stems from the theory of branched poly-
mers (Daoud and Joanny, 1981; Marko and Siggia, 1994; Cui and
Chen, 1996).
It has often been hypothesized that the bacterial nucleoid could
consist of ‘‘domains’’ that may act independently of each other
(Worcel and Burgi, 1972; Pettijohn and Hecht, 1973; Sinden and
Pettijohn, 1981; Pavitt and Higgins, 1993; Higgins et al., 1996).
The supercoil is then supposed to be crosslinked at certain posi-
tions which are impediments to supercoil diffusion (see Fig. 5).
In the scaling analysis of Odijk (2002), an uncrosslinked blob of
radius RH containing N H segments is introduced
 1=2
1=2 N
R ¼ bN  ¼ Ru
Ns
At large scales, the nucleoid is viewed as a rubbery gel in which
the crosslinks are distributed homogeneously. Hence, for the con-
centration of segments we must impose c ’ Nw/ R3H tbnd N s =R3n
where Rn is the radius of gyration of the crosslinked nucleoid. This
implies the relations
R3n
R ¼ ð3Þ
R2u
 6  2
Rn Vn
N ¼ Ns ¼ Ns ð4Þ
Ru Vu
Here, the volume of the nucleoid is Vn = 4pR3n =3 when it is cross-
linked and Vu = 4pR3u =3 when it is not. We note the strong sixth
power law in Eq. (4). In practice, the size of the nucleoid Rni has
been monitored by us at 50% threshold intensity. In Cunha et al.
(2001b), we have argued that Rn = 1.13 Rni. Moreover, in practice
also, the amount of DNA per nucleoid is not that contained in
one chromosome but more, namely, 1.58 chromosome equivalents
for E. coli LMC500 in the present study (valid for growth in a glu-
cose-minimal medium at 28 °C with Td = 85 min, C = 70 min and
D’ = 19 min (Huls et al., 1999)). The ionic strength of the 0.01 M
NaPi buffer is taken to be about 0.005 M at pH = 7. Then, the diam-
eter of the plectoneme is about 52 nm at reff = -0.025 as predicted
by theory (Ubbink and Odijk, 1999; we derive Ds by interpolation
within the tables presented there). The variables Ns, As and Ks
are estimated to be 4000, 0.158 lm and 0.71, respectively, (Cunha
et al., 2001b). Eq. (1) then yields Ru = 3.31 lm so that the index vol-
ume of the uncrosslinked nucleoid is predicted to be Vui = 209 lm3
(i.e. corrected for the number of chromosome equivalents and the
50% threshold factor).
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