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 article info Article history: Received 29 November 2011 Received in revised form 2 March 2012 Accepted 3 March 2012 Available online 6 April 2012 Keywords: Escherichia coli Nucleoid Supercoiling Polymer physics Lysozyme-spheroplast Ampicillin-spheroplast Osmotic shock DAPI/UV-radiation damage Protein cross-links Ethidium bromide abstract Nucleoids were isolated by osmotic shock from Escherichia coli spheroplasts at relatively low salt concen- trations and in the absence of detergents. Sucrose-protected cells, made osmotically sensitive by growth in the presence of ampicillin or by digestion with low lysozyme concentrations (50–5 lg/ml), were shocked by 100-fold dilution of the sucrose buffer. Liberated nucleoids stained with 4 0 ,6-diamidino-2- 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 disrupted the DAPI-stained nucleoids within 5–10 s, they were imaged by time-lapse microscopy with exposure times less than 2 s. The volume of nucleoids isolated from ampicillin- or low-lysozyme spher- oplasts and minimally exposed to UV (<2 s) was on average 42 lm 3 . Lysozyme at concentrations above 1 lg/ml in the lysate compacted the nucleoids. Treatment with protease E or K (20–200 lg/ml) and sodium dodecyl sulfate (SDS; 0.001–0.01%) caused a twofold volume increase and showed a granular nucleoid at the earliest UV-exposure; the expansion could be reversed with 50 lM ethidium bromide, but not with chloroquine. While DNase (1 lg/ml) caused a rapid disruption of the nucleoids, RNase (0.1–400 lg/ml) had no effect. DAPI-stained nucleoids treated with protease, SDS or DNase consisted of granular substructures at the earliest exposure similar to UV-disrupted nucleoids obtained after pro- 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 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 (Delius and Worcel, 1974). The results supported the interpreta- 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 ⇑ 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 Current address: Laboratory of Food Microbiology, Wageningen University, Bomenweg 2, 6703 HD Wageningen, The Netherlands. Journal of Structural Biology 178 (2012) 260–269 Contents lists available at SciVerse ScienceDirect Journal of Structural Biology journal homepage: www.elsevier.com/locate/yjsbi