Biochimie 83 (2001) 149−154 © 2001 Société française de biochimie et biologie moléculaire / Éditions scientifiques et médicales Elsevier SAS.

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Isolation of the Escherichia coli nucleoid
Sónia Cunhaa,b, Theo Odijkb, Erhan Süleymanoglua, Conrad L. Woldringha*
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
a

1. Introduction 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 nucleoids may have lost or gained certain associated components during the lysis procedure. Ideally, an isolated nucleoid should be as identical as possible to the intracellular chromosome in vivo. Because in actively growing cells the chromosome is involved in coupled transcription/ translation [5] and in replication, repair and recombination, isolated nucleoids could also comprise the components 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).

envelope-free or envelope-bound nucleoids could be obtained [6, 7]. To understand the presence of an in vivo phase separation 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 fluorescence 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-

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

centrations above 0.8 mg/mL can lead to artificial and secondary associations between DNA and envelope remnants as emphasized by Silberstein and Inouye [16]. Also, ‘high-salt nucleoids’ [8] and ‘low-salt spermidine nucleoids’ [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 peptidoglycan 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-lysozymespermidine 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, usually 1 M NaCl, can either give rise to envelope-bound (figure 1C) or envelope-free nucleoids (figure 1D), depending 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 always 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 segregation 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/translationmediated 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 represents 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,

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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 binding 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’.

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.

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 entangled 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-

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. Preliminar ethidium-bromide titration experiments indicated that the nucleoids are supercoiled. By what constraints the size of the isolated nucleoids is maintained is presently unknown, but it is unlikely caused by RNA entanglements as

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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 analyzed by sucrose-density centrifugation. Because of the low salt concentration used in this procedure, ribosomes are not expected to dissociate from the mRNA. Consequently, they may still persist in the form of polyribosomes 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 elongated 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 bacterial nucleoids in an expanded but stable structure that can readily be visualized by fluorescence microscopy as freefloating globules. So far, the behavior of nucleoids towards changes in the concentration of counterions or towards RNase treatment have been analyzed as changes in sedimentation profiles in sucrose gradient centrifugation. 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 liberated by osmotic shock and treated with increasing PEG 20 000 or protein concentrations. From these measurements we hope to calculate the force necessary to constrain the bacterial nucleoid within its in vivo volume and thereby explain the existence of a phase separation between 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 Scientific Research (NWO, ALW-FOM).

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