Biochimie 83 (2001) 149−154

© 2001 Société française de biochimie et biologie moléculaire / Éditions scientifiques et médicales Elsevier SAS. All rights reserved.
S0300908401012457/REV

Isolation of the Escherichia coli nucleoid

Sónia Cunhaa,b, Theo Odijkb, Erhan Süleymanoglua, Conrad L. Woldringha*

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, 2600 GB Delft, the Netherlands
(Received 15 December 2000; accepted 18 January 2001)

Abstract — Numerous protocols for the isolation of bacterial nucleoids have been described based on treatment of cells with
sucrose-lysozyme-EDTA and subsequent lysis with detergents in the presence of counterions (e.g., NaCl, spermidine). Depending on
the lysis conditions both envelope-free and envelope-bound nucleoids could be obtained, often in the same lysate. To investigate the
mechanism(s) involved in compacting bacterial DNA in the living cell, we wished to isolate intact nucleoids in the absence of
detergents and high concentrations of counterions. Here, we compare the general lysis method using detergents with a procedure
involving osmotic shock of Escherichia coli spheroplasts that resulted in nucleoids free of envelope fragments. After staining the DNA
with DAPI (4’,6-diamidino-2-phenylindole) and cell lysis by either isolation procedure, free-floating nucleoids could be readily
visualized in fluorescence microscope preparations. The detergent-salt and the osmotic-shock nucleoids appeared as relatively compact
structures under the applied ionic conditions of 1 M and 10 mM, respectively. RNase treatment caused no dramatic changes in the size
of either nucleoid. © 2001 Société française de biochimie et biologie moléculaire / Éditions scientifiques et médicales Elsevier SAS
Escherichia coli / nucleoid isolation / cell lysis / osmotic shock / DNA compaction

1. Introduction envelope-free or envelope-bound nucleoids could be ob-

Ever since it was realized that DNA occurred as a
confined structure within the bacterial cell [1] one has
tried to isolate it. Isolation of this entity was expected to
provide information on the mechanism of folding of DNA
within the cytoplasm.
The visualized DNA has been called chromatin body
[2], nucleo- or DNA-plasm [3], or nucleoid [4]. The term
nucleoid or bacterial chromosome is generally used to
describe the packaged DNA structure in situ, including
associated protein and RNA components. Isolated nucle-
oids may have lost or gained certain associated compo-
nents during the lysis procedure. Ideally, an isolated
nucleoid should be as identical as possible to the intra-
cellular chromosome in vivo. Because in actively growing
cells the chromosome is involved in coupled transcription/
translation [5] and in replication, repair and recombina-
tion, isolated nucleoids could also comprise the compo-
nents involved in these processes.
In early isolation procedures (see for reviews [6, 7]) it
was experienced that the bacterial DNA spontaneously
unfolded upon cell lysis using lysozyme and detergents. In
most studies, therefore, counterions [8, 9] or spermidine
[10] were added to prevent this unfolding. In addition, it
was found that depending on the lysis conditions, either
*Correspondence and reprints.
E-mail address: woldringh@bio.uva.nl (C.L. Woldringh).
tained [6, 7].
To understand the presence of an in vivo phase sepa-
ration between DNA and cytoplasm as indicated by
microscopic observation (for review see [11]) and as
predicted on the basis of physical considerations [12, 13],
we wish to analyze nucleoid compaction in vitro using
crowding agents like polyethylene glycol (PEG 20 000) or
proteins. Because the use of detergents or high salt
conditions may induce the dissociation of DNA binding
proteins and may generate nucleoids attached to envelope
fragments and RNA (see below), we used an osmotic
shock method [14]. Here we compare the detergent-salt
(or detergent–spermidine) method with that of osmotic
shock for obtaining isolated nucleoids. We describe some
properties of both types of nucleoids stained with DAPI
(4’,6-diamidino-2-phenylindole) as visualized by fluores-
cence microscopy.
2. Nucleoid isolation by the detergent-salt
or detergent-spermidine method
2.1. Nucleoids and envelope fragments
Nucleoids isolated by the detergent method have been
given various names depending on the applied conditions.
For instance, ‘low- and high-lysozyme nucleoids’ are
obtained after incubation with 0.4 and 4 mg/mL lysozyme,
respectively [15]. It should be noted that lysozyme con-
150 Cunha et al.

Table I. Comparison of lysis-protocols for the isolation of nucleoids using either the detergent-salt or the osmotic shock method.
Procedure steps Detergent-salt Osmotic shock
Incubation buffer 10 mM Tris, pH 8, 10 mM EDTA, 100 mM NaCl, 10 mM Na phosphate, 10 mM EDTA,
20% sucrose 100 mM NaCl, 0.8 M sucrose (∼30%)
Lysozyme concentration ‘low’: 0.4 mg/mL; ‘high’: 4 mg/mL 0.4 mg/mL
Incubation time and temperature 40 s at 0 °C 30 min at 20 °C
Treatment ‘low salt’ or ‘high salt’ 0.5% Brij58, 0.2% DOC, 5 mM spermidine or 1 M (no detergent), osmotic shock by 100 ×
NaCl, lysate after 3 min at 10°C dilution in 10 mM NaCl

centrations above 0.8 mg/mL can lead to artificial and
secondary associations between DNA and envelope rem-
nants as emphasized by Silberstein and Inouye [16]. Also,
‘high-salt nucleoids’ [8] and ‘low-salt spermidine nucle-
oids’ [10] can be distinguished.
As an example of the detergent-spermidine method we
consider a relatively recent protocol given by Murphy and
Zimmerman [15]. The various steps of their procedure
will be discussed briefly below (see summary in the first
column of table I).
In the first step, intact cells (figure 1A) are plasmolyzed
in sucrose buffer containing lysozyme and EDTA to obtain
detergent-sensitive cells with a partly digested peptidogly-
can layer (figure 1B). In the second step, the cells are
treated with detergents such as Brij, deoxycholate and/or
Sarcosyl to disrupt the plasma membrane and allow
extrusion of the DNA (see hypothetical schemes in figure
1C, D).
It should be noted that in all protocols the lysis of E.
coli cells includes lysozyme to digest the peptidoglycan
layer. An exception is the protocol of Hinnebusch and
Bendich [17] in which E. coli cells are lysed within
agarose blocks by incubation in buffer containing only
sodium dodecyl sulfate (SDS) and proteinase K. The
ultrastructural changes occurring in E. coli cells during
SDS-lysis have been reported previously [18].
Murphy and Zimmerman [15, 19] used high-lysozyme-
spermidine nucleoids (table I). These nucleoids have a
much higher protein and RNA content than the high-salt
nucleoids. Cells lysed under the high-salt condition, usu-
ally 1 M NaCl, can either give rise to envelope-bound
(figure 1C) or envelope-free nucleoids (figure 1D), de-
pending on the temperature of lysis [20], which may vary
the degree of disruption of the peptidoglycan layer. The
‘low-salt spermidine nucleoids’ [15] are presumably al-
ways attached to membranous fragments derived from the
cell envelope (see figure 9 in [19]).
As discussed by Materman and Van Gool [21] cell lysis
has to proceed at higher temperatures (20–25 °C) to obtain
the release of the nucleoid purportedly through a single
gap in the cell envelope (figure 1D; see also [20]). Less or
more extensive digestion of the peptidoglycan layer may
lead to the extrusion of the DNA through many gaps in the
still rod-shaped remnant of the envelope (figure 1C) or to
trapping of vesicular envelope fragments, respectively. In
both cases the ‘binding’ of the nucleoid to envelope
fragments is probably due to entanglement rather than to
specific DNA-envelope attachment sites, as frequently
suggested in early studies (e.g., [22]).
2.2. Nucleoids and RNA
Numerous reports have described the occurrence of
indirect attachments of DNA to the plasma membrane via
coupled transcription, translation and translocation of
membrane proteins [25, 26]. These observations led to the
formulation of the transcription/translation-mediated seg-
regation model [27] or the transertion model [28] in which
the nascent RNA functions as an expansion factor shaping
the nucleoids into rod-like structures and moving them
during segregation. This idea of transcription/translation-
mediated expansion of the nucleoid was based on electron
microscope observations showing that treatment with
rifampin [22] or chloramphenicol [29] causes compaction
of the nucleoid. This suggested that nascent RNA repre-
sents an expansion factor counteracting the compaction
force exerted through macromolecular crowding [11, 12].
However, in the case of isolated high-salt or
-spermidine nucleoids it is the same nascent RNA which
can now be considered to represent a compaction or
stabilizing factor, as RNAse treatment of these nucleoids
causes their unfolding as indicated by sucrose-gradient
analyses [23, 24]. Thus, when considering this behavior of
isolated nucleoids with respect to RNA there seems to
occur a reversal in the role of nascent RNA concerning
compaction of the nucleoid.
3. Nucleoid isolation by osmotic shock
In the osmotic-shock method ( table I, second column)
cells are incubated in a sucrose-containing buffer with
lysozyme and EDTA until all cells have converted to
spheroplasts (figure 2B) or, in the case of Gram-positive
cells, to protoplasts [14]. In our hands, a 100-fold dilution
of the suspension in 10 mM NaCl resulted in empty,
Isolation of E. coli nucleoids
Figure 1. Schematic representation of the hypothetical behavior
of cell structures during detergent-salt lysis (see also table I, first
column). A. The intact cell is represented by the cell wall (outer
membrane plus peptidoglycan layer), the plasma membrane,
soluble proteins, polyribosomes and the nucleoid. The DNA is
drawn as branched plectonemic supercoils, compacted in the cell
center by depletion forces (molecular crowding; see [11]). The
polyribosomes extend from the DNA to the plasma membrane.
B. Plasmolyzed cell with a partly digested peptidoglycan layer
obtained by incubation in a sucrose-lysozyme-EDTA buffer. C.
Detergent lysis at low temperature (0–10 °C). The plasma
membrane is largely dissolved and the polyribosomes have
dissociated causing strands of nascent RNA (drawn as short,
thick lines; RNA strands remain connected to the DNA via RNA
polymerase) to entangle with DNA segments. Most DNA bind-
ing proteins have also dissociated. Free loops of supercoiled
DNA are expelled through several gaps in the cell envelope
resulting in an ‘envelope-bound nucleoid’. D. Detergent lysis at
high temperature (20–25 °C). Similar to C except that the
nucleoid is expelled through a single gap in the cell envelope,
resulting in an ‘envelope-free nucleoid’.
spherical ghosts (as visible in phase contrast) and in
expanded nucleoids resembling globules with a radius of
2 to 3 µm. We suspect that the nucleoids are released
through a single gap; otherwise they would remain en-
tangled with the ghost as in the hypothetical figure 1C. In
a theoretical analysis, Odijk [30] calculated that liberated
nucleoids would swell within 10 min to a radius of 9 µm.
Taking branching of supercoils into consideration, pre-
151
Figure 2. Schematic representation of the hypothetical behavior
of cell structures during lysis by osmotic shock (see table I,
second column). A. Intact cell as in figure 1A. B. Prolonged
incubation in a sucrose-lysozyme-EDTA buffer results in the
conversion of a plasmolyzed cell (figure 1B) into an osmotically
sensitive spheroplast. C. 100-fold dilution of the spheroplast
suspension results in the liberation of the nucleoid from a large,
spherical envelope ghost. It is not known to what extent the
polyribosomes do remain intact.
liminary calculations show that the average radius of
gyration would be about 3 µm. The swelling does not
seem to continue as all nucleoids in the lysate have similar
sizes after standing for 4 h at room temperature. The
nucleoids we measure thus appear smaller than our
theoretical estimates. We hope to explain this discrepancy
in future work.
The complexes liberated by osmotic shock can be
described as ‘low-lysozyme low-salt’ nucleoids. Prelimi-
nar ethidium-bromide titration experiments indicated that
the nucleoids are supercoiled. By what constraints the size
of the isolated nucleoids is maintained is presently un-
known, but it is unlikely caused by RNA entanglements as
152 Cunha et al.

Figure 3. Nucleoids liberated from the same spheroplast suspension of E. coli MC1000 by the two methods summarized in table I
and photographed as free-floating complexes in a 10 µm thick liquid layer (free-floating nucleoids are distinguished from glass-surface
attached structures because they show Brownian movement). The E. coli cells were grown in glucose minimal medium (see insets of
a phase contrast image of a living cell with its DAPI-stained nucleoid by fluorescence microscopy. DAPI was added to the growth
medium at a final concentration of 1 µg/mL about 1 h before harvesting the cells). A. Detergent-salt nucleoids (in 1 M NaCl). B.
Osmotic-shock nucleoids (in 10 mM NaCl). C. Detergent-salt nucleoids (from A) treated with 10 µg/mL Rnase for 30 min at room
temperature. D. Osmotic-shock nucleoids (from B) likewise treated with Rnase. Images were acquired using a Princeton cooled CCD
camera mounted on an Olympus BH-2 fluorescence microscope equipped with a 100 × SPlan PL phase-contrast objective.
Magnification bar, 5 µm.

suggested for the detergent-salt nucleoids (see figure 1C,
D). Sloof et al. [14] reported that nucleoids isolated from
Bacillus licheniformis protoplasts by osmotic shock are
unaffected by treatment with 10 µg/mL RNAse as ana-
lyzed by sucrose-density centrifugation. Because of the
low salt concentration used in this procedure, ribosomes
are not expected to dissociate from the mRNA. Conse-
quently, they may still persist in the form of polyribo-
somes and no artificial stabilization of the nucleoid by
DNA-RNA associations is to be expected.
4. Microscopic observation of isolated nucleoids
Nucleoids isolated from DAPI-stained cells can readily
be observed by fluorescence microscopy. Both methods of
isolation (table I) resulted in globular, sometimes elon-
gated complexes that did not aggregate. They showed
similar sizes when prepared in a liquid layer of about 10
µm thick and photographed as free-floating complexes
(figure 3A, B). However, upon excitation of the DAPI
fluorochrome (using a dichroic filter cube containing a
band pass filter with transmission between 300–400 nm),
the nucleoids desintegrated rapidly and disappeared
within about 10 s. The nucleoids seemed to ‘break apart’
by movement of granular and threadlike structures, both
barely visible.
The preparation procedure applied in figure 3 differs
from the usual method which involves an air-drying step
[15, 31]. We omitted this step as it may change the
nucleoid environment and subjects the complexes to a
large surface tension.
Isolation of E. coli nucleoids
Murphy and Zimmerman [19] described the stabilizing
effect of PEG8000 (added at concentrations of 4 to 12%)
on detergent-spermidine nucleoids denatured by exposure
to slightly elevated temperatures or to increased salt
concentrations. In contrast, our microscopic observations
on free-floating nucleoids, isolated from glucose-grown E.
coli cells by either method, showed a compaction upon
addition of PEG 20 000. A gradual decrease of nucleoid
volume from about 25 to less than 2 µm3 was observed
with increasing PEG 20 000 concentrations of 2 to 14%
(to be published elsewhere).
It should be noted that the nucleoids prepared from the
same spheroplast suspension by either the detergent-salt
(figure 3A) or the osmotic-shock (figure 3B) method are of
comparable size in spite of a large difference in ionic
strength: 1 M and 10 mM NaCl, respectively. A significant
reduction in the size of the globules obtained by osmotic
shock was observed when the NaCl concentration was
increased to 200 mM.
When treated with 10 µg/mL RNase, nucleoids isolated
by either method appeared to maintain their size (figure
3C, D), although the detergent-salt nucleoids always
contained a faint border of granules and threads and
seemed to be more labile upon UV irradiation during
microscopy. The microscopic observations thus show that
isolated nucleoids, under the conditions studied so far, do
not undergo dramatic or abrupt changes in size.
5. Concluding remarks
Osmotic shock of E. coli spheroplasts liberates bacte-
rial nucleoids in an expanded but stable structure that can
readily be visualized by fluorescence microscopy as free-
floating globules. So far, the behavior of nucleoids to-
wards changes in the concentration of counterions or
towards RNase treatment have been analyzed as changes
in sedimentation profiles in sucrose gradient centrifuga-
tion. However, these changes are difficult to relate to the
actual structure and size of the nucleoids [32].
Therefore, we are presently applying image cytometry
[33] to measure volume distributions of nucleoids liber-
ated by osmotic shock and treated with increasing PEG
20 000 or protein concentrations. From these measure-
ments we hope to calculate the force necessary to con-
strain the bacterial nucleoid within its in vivo volume and
thereby explain the existence of a phase separation be-
tween nucleoid and cytoplasm.
Acknowledgments
We thank Mirjam Aarsman, Peter Huls and Evelien Pas for
technical advice and August van Gool and Nanne Nanninga for
comments on the manuscript. This work was supported by the
Physical Biology Program of the Dutch Organization for Scien-
tific Research (NWO, ALW-FOM).
153
References
[1] Mason D.J., Powelson D.M., Nuclear division as observed in live
bacteria by a new technique, J. Bacteriol. 71 (1956) 474–479.
[2] Robinow C.F., The chromatin bodies of bacteria, Bacteriol. Rev.
20 (1956) 207–242.
[3] Kellenberger E., Ryter A., Séchaud J., Electron microscope study
of DNA-containing plasms. II. Vegetative and mature phage DNA
as compared with normal bacterial nucleoids in different physio-
logical states, J. Biophys. Biochem Cytol. 4 (1958) 671–678.
[4] Ryter A., Kellenberger E., Birch-Andersen A., Malløe O., Etude au
microscope électronique de plasmas contenantde l’acide déoxyri-
bonucléique. I. Les nucléoides des bactéries en croissance active,
Z. Naturf. 13B (1958) 597–605.
[5] Miller O.L., Hamkalo B.A., Thomas C.A., Visualization of bacte-
rial genes in action, Science 169 (1970) 392–395.
[6] Hirschbein L., Guillen N., Characterization, assay, and use of
isolated bacterial nucleoids, Methods Biochem. Anal. 28 (1982)
297–328.
[7] Pettijohn D.E., Sinden R.R., Structure of the isolated nucleoid, in:
Nanninga N. (Ed.), Molecular cytology of Escherichia coli,
Academic Press, London, New York, 1985, pp. 199–227.
[8] Stonington G.O., Pettijohn D.E., The folded genome of Escheri-
chia coli isolated in a protein-DNA-RNA complex, Proc. Natl.
Acad. Sci. USA 68 (1971) 6–9.
[9] Worcel A., Burgi E., On the structure of the folded chromosome of
E. coli, J. Mol. Biol. 71 (1972) 127–147.
[10] Kornberg T., Lockwood A., Worcel A., Replication of the Escheri-
chia coli chromosome with a soluble enzyme system, Proc. Natl.
Acad. Sci. USA 71 (1974) 3189–3193.
[11] Woldringh C.L., Odijk T., Structure of DNA within the bacterial
cell: physics and physiology, in: Charlebois R.L. (Ed.), Organiza-
tion of the prokaryotic genome. Chapter 10, American Society for
Microbiology, Washington, D.C., 1999, pp. 171–187.
[12] Zimmerman S.B., Murphy L.D., Macromolecular crowding and
the mandatory condensation of DNA in bacteria, FEBS Lett. 390
(1996) 245–248.
[13] Odijk T., Osmotic compaction of supercoiled DNA into a bacterial
nucleoid, Biophys. Chem. 73 (1998) 23–30.
[14] Sloof P., Maagdelijn A., Boswinkel E., Folding of prokaryotic
DNA: isolation and characterization of nucleoids from Bacillus
licheniformis, J. Mol. Biol. 163 (1983) 277–297.
[15] Murphy L.D., Zimmerman S.B., Isolation and characterization of
spermidine nucleoids from Escherichia coli, J. Struct. Biol. 119
(1997) 321–335.
[16] Silberstein S., Inouye M., The effect of lysozyme on DNA-
membrane association in Escherichia coli, Biochim. Biophys. Acta
366 (1974) 149–158.
[17] Hinnebusch J., Bendich A.J., The bacterial nucleoid visualized by
fluorescence microscopy of cells lysed within agarose: comparison
of Escherichia coli and spirochetes of the genus Borrelia, J. Bac-
teriol. 179 (1997) 2228–2237.
[18] Woldringh C.L., van Iterson W., Effects of treatment with sodium
dodecyl sulfate on the ultrastructure of Escherichia coli, J. Bacte-
riol. 111 (1972) 801–813.
[19] Murphy L.D., Zimmerman S.B., Stabilization of compact spermi-
dine nucleoids from Escherichia coli under crowded conditions:
Implications for in vivo nucleoid structure, J. Struct. Biol. 119
(1997) 336–346.
[20] Meijer M., de Jong M.A., Woldringh C.L., Nanninga N., Factors
affecting the release of folded chromosomes from Escherichia
coli, Eur. J. Biochem. 63 (1976) 469–475.
[21] Materman E.C., Van Gool A.P., Nucleoid release from Escherichia
coli cells, J. Bacteriol. 133 (1978) 878–883.
[22] Dworsky P., Schaechter M., Effect of rifampin on the structure and
membrane attachment of the nucleoid of Escherichia coli, J. Bac-
teriol. 116 (1973) 1364–1374.
154
[23] Pettijohn D.E., Hecht R., RNA molecules bound to the folded
bacterial genome stabilize DNA folds and segregate domains of
supercoiling, Cold Spring Harbor Symp. Quant. Biol. 38 (1973)
31–42.
[24] Murphy L.D., Zimmerman S.B., Multiple restraints to the unfold-
ing of spermidine nucleoids from Escherichia coli, J. Struct. Biol.
132 (2000) 46–62.
[25] Kleppe K., ÕvrebÕ S., Lossius I., The bacterial nucleoid, J. Gen.
Microbiol. 112 (1979) 1–13.
[26] Vos-Scheperkeuter G.H., Witholt B., Co-translational insertion of
envelope proteins: theoretical considerations and implications,
Ann. Microbiol. (Inst. Pasteur) 133A (1982) 129–138.
[27] Woldringh C.L., Jensen P.R., Westerhoff H.V., Structure and
partitioning of bacterial DNA: determined by a balance of com-
paction and expansion forces? FEMS Microbiol. Lett. 131 (1995)
235–242.
[28] Norris V., Hypothesis: chromosome separation in Escherichia coli
involves autocatalytic gene expression, translation and membrane-
domain formation, Mol. Microbiol. 16 (1995) 1051–1057.
Cunha et al.
[29] Morgan C., Rosenkranz H.S., Carr H.S., Rose H.M., Electron
microscopy of chloramphenicol-treated Escherichia coli, J. Bac-
teriol. 93 (1967) 1987–2002.
[30] Odijk T., Dynamics of the expanding DNA nucleoid released from
a bacterial cell, Physica A 277 (2000) 62–70.
[31] Weitao T., Nordstroem K., Dasgupta S., Mutual suppression of
mukB and seqA phenotypes might arise from their opposing
influences on the Escherichia coli nucleoid structure, Mol. Micro-
biol. 34 (1999) 157–168.
[32] Murphy L.D., Zimmerman S.B., A dilatancy assay for nucleoid
denaturation: the centrifugation-dependent clumping of denatured
spermidine nucleoids, Anal. Biochem. 266 (1999) 16–22.
[33] Vischer N.O.E., Huls P.G., Ghauharali R.I., Brakenhoff G.J.,
Nanninga N., Woldringh C.L., Image cytometric method for
quantifying the relative amount of DNA in bacterial nucleoids
using Escherichia coli, J. Microsc. 196 (1999) 61–68.