The time course of morphological changes during lysis of Escherichia coli cells was examined with respect to an undisturbed release of nucleoids. The addition of detergents to plasmolyzed, osmotic sensitive cells resulted in the immediate reversal of plasmolysis followed by the appearance of rod-shaped ghost cells without any detectable spheroplast formation. Electron microscopic examination of the rod-shaped ghost cells revealed a zonal gap in the cell envelope, allowing the free release of

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... nucleoid. Due to the high ionic strength, a suitable cell lysis was shown to require higher incubation temperatures. However, in the absence of an appropriate control this may result in the sphering and vesiculation of ghost cell envelopes and even the unfolding of released nucleoids. To avoid this unfavorable consequence of lysis at high temperatures, a microscopic examination on the course of rod-shaped ghost formation is suggested. In a recent review on the nucleoid of Escherichia coli, Pettijohn emphasized the empirical nature of the methods used in the isolation of bacterial nucleoids. He described their successful isolation as a compromise between two extreme conditions of cell lysis resulting either in unfolded chromosomes or in nucleoids that remain trapped in the partially disrupted cell envelope (8). From the sedimentation analysis of purified nucleoids these extremes were characterized either as membrane-associated or membranefree but more or less unfolded complexes (9, 15). The proportion of both types of nucleoids was shown to depend on the temperature of cell lysis, the former being predominant at 0°C (9, 15). Although initially the association of the nucleoid with the cell membrane was considered to be specific (15), it was subsequently shown to result from a nonspecific binding of the nucleoid to the cell envelope (7). Since these data indicated that a variation in the sedimentation characteristics of isolated nucleoids may be traced back to their release from the cell during lysis, Korch et al. (5) examined the lysis requirements to reach the compromise of successful nucleoid isolation. Using a method for sedimentation analysis of crude lysates, they concluded that an efficient release of nucleoids requires a careful control of cell lysis (5). In this respect we studied the cytological changes occurring in lysing cells, trying to visualize directly the requirements for successful nucleoid release. MATERIALS AND MErHODS Bacterial strain and growth conditions. E. coli NF 87, D-10 metB argA relA1, a strain obtained from N. Fiil (University of Copenhagen) and closely related to the one used by Stonington and Pettijohn (12) , was grown in M9 minimal salts medium (16) supplemented with 0.2% glucose, 50 pug of methionine per ml, and 50 ug of arginine per ml. Lysis procedure. Exponentially growing cells were quickly pelieted at 5,000 x g (00C) and resuspended to a concentration of approximately 109 cells/ml. Cell lysis was carried out by detergent treatment of plasmolyzed, osmotic sensitive cells as described by Stonington and Pettijohn (12). However, to avoid interference with glutaraldehyde fixation, tris(hydroxymethyl)aminomethane buffer was replaced by triethanolamine (TEA) (1), and for similar reasons sodium citrate was used rather than ethylenediaminetetraacetate. To improve the electron microscopic visualization of nucleoid release, attempts were made to trap the nucleoid in the neighborhood of the cell from which it emerged by enmeshment of the cells in 1% agar before lysis. However, under these conditions cell lysis proceeded very slowly and was only successful at incubation temperatures equal to or higher than 200C. Morphokinetic analysis. At 1-min time intervals after the addition of detergents, 0.3-ml samples were quickly removed from the incubation mixture, and the absorbance at 660 nm was measured immediately in a Beckman-25 spectrophotometer. At the same time intervals, samples were also fixed with 2% (vol/vol) formaldehyde in 1 M NaCl and rapidly scanned in a Zeiss GFL phase-contrast microscope to obtain a representative series of micrographs. These micrographs allowed a reproducible quantitative reconstruction of the gross morphological changes observed in the cells during the course of cell lysis. Electron microscopic analysis. Samples were either withdrawn directly from the lysate or derived from the supernatant after a low-speed centrifugation of the lysate (5,000 x g, 5 min, 0°C). They were subsequently prefixed with 1% glutaraldehyde for 10 min 878 on May 4, 2020 by guest