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Toroidal nucleoids in Escherichia coli exposed to chloramphenicol
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Structural Biology Journal of Structural Biology 138 (2002) 199–206 www.academicpress.com
Toroidal nucleoids in Escherichia coli exposed to chloramphenicol Steven B. Zimmerman Laboratory of Molecular Biology, National Institutes of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Building 5, Room 329, Bethesda, MD 20892-0560, USA Received 26 February 2002, and in revised form 16 April 2002
Abstract The DNA of growing cells of Escherichia coli occurs in one or a few lobular bodies known as nucleoids. Upon exposure to chloramphenicol, the nucleoids assume compact, rounded forms (‘‘cm-nucleoids’’) that have been described as ring- or sphereshaped. Multiple views of single cells or spheroplasts, however, support a different, curved toroid shape for cm-nucleoids. The multiple views were obtained either by DNA fluorescence imaging as the cells or spheroplasts reoriented in liquid medium or by optical sectioning using phase-contrast or fluorescence imaging of immobilized cells. The curved toroid shape is consistent with electron microscope images of thin sections of chloramphenicol-treated cells. The relationship of this structure to active and inactive nucleoids and to the smaller toroidal forms made by in vitro DNA condensation is discussed. Published by Elsevier Science (USA). Keywords: Chloramphenicol; Escherichia coli; Nucleoid; Toroid
1. Introduction The genomic DNA of bacteria is largely restricted to one or a few discrete bodies per cell known as nucleoids (Drlica and Bendich, 2000; Woldringh and Nanninga, 1985; Woldringh and Odijk, 1999). The nucleoids within rapidly growing cells of Escherichia coli appear as variable, lobular arrays (‘‘normal nucleoids’’). Exposure of the cells to chloramphenicol causes a striking transformation of their nucleoids to more uniform, rounded images (‘‘cm-nucleoids;’’ Dworsky, 1974; Kellenberger, 1952; Morgan et al., 1967; Steinberg, 1952; Zusman et al., 1973). Many other treatments of cells which inhibit protein synthesis cause a similar rounding of their nucleoids, including exposure to aureomycin (Kellenberger and Ryter, 1956; Steinberg, 1952), terramycin (Steinberg, 1952), erythromycin (Dworsky, 1974), or puromycin or sodium azide (unpublished observations of the author), or growth without an essential amino acid (unpublished data cited in Hobot et al., 1985; Van Helvoort et al., 1998). The rounding of the nucleoids appears to result from a loss of cotranslational insertion linkages that joined the nucleoidal DNA to the cell envelope in growing E-mail address: [email protected]. 1047-8477/02/$ - see front matter. Published by Elsevier Science (USA). PII: S 1 0 4 7 - 8 4 7 7 ( 0 2 ) 0 0 0 3 6 - 9
bacteria (Woldringh et al., 1995). Loss of the linkages then allows the powerful condensing forces present in the cytoplasm, such as macromolecular crowding and ligand binding (Woldringh et al., 1995; Zimmerman and Murphy, 1996), to cause rearrangement of the nucleoids into the characteristic shapes associated with chloramphenicol exposure. This study presents a new structure to account for the characteristic images of cm-nucleoids which may also have application to the structures of normal nucleoids. Nucleoids of chloramphenicol-treated cells have been examined by light microscopy of whole cells and by electron microscopy of whole cells or thin sections of cells (Kellenberger, 1952; Morgan et al., 1967; Steinberg, 1952; Zusman et al., 1973). Three shapes of nucleoid images have been found, which will be referred to here as O, C, and fused shapes. 1.1. O-shaped nucleoid images Ring-shaped nucleoid images were noted by Kellenberger (1952) some 50 years ago and were further studied by Steinberg (1952). In later reports and reviews by Kellenberger and collaborators, the nucleoid structures responsible for the O shapes have been depicted as hollow spheres (e.g., Fig. 1, Kellenberger, 1960; Fig. 2C of
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Bohrmann et al., 1991; Robinow and Kellenberger, 1994). The material filling the center of the O-shaped image appeared to be similar to the cytoplasm which surrounded the nucleoid as seen by either light or electron microscopy, leading to suggestions that the central region of their suggested hollow-sphere structure contained a cytoplasmic invagination or inclusion (Kellenberger, 1960; Morgan et al., 1967; Woldringh and Nanninga, 1976; Zusman et al., 1973; Bohrmann et al., 1991).
2. Materials and methods 2.1. Materials Chloramphenicol, DAPI (40 ,6-diamidino-2-phenylindole), low gelling temperature agarose (Type VII), and crystallized chicken egg white lysozyme (EC 3.2.1.17) were purchased from Sigma Chemical, and formaldehyde (38% solution, containing 12% methanol) was purchased from Mallinckrodt.
1.2. C-shaped nucleoid images C-shaped nucleoid images of size similar to that of the O-shaped images were noted by Steinberg (1952) and by Zusman et al. (1973). Despite the inconsistency of Cshape images with a spherical model, alternative structural interpretations of the C- and O-shaped images have apparently not been presented. 1.3. Fused shape nucleoid images A small fraction of the cells treated with chloramphenicol develop fused nucleoid structures at the midcell position that suggest linked pairs of unresolved nucleoids stopped in the act of cell division. Rarer instances of fusions of three or more nucleoids also occur at scattered positions within the cell envelope (Kellenberger, 1952; Steinberg, 1952; Zusman et al., 1973), probably due to the progressive nucleoid coalescence that accompanies chloramphenicol treatment (Schaechter and Laing, 1961; Van Helvoort et al., 1996, 1998). 1.4. Interpretation and implications The present results indicate that the C and O shapes are both images of a single underlying structure, which is suggested to be a curved toroid, a smoothly deformed derivative of a ring torus. By this interpretation, the central ‘‘core’’ of O-shaped nucleoid images looks like the surrounding cytoplasm because it is the cytoplasm in which the toroids are immersed. The existence of intracellular toroidal nucleoids has important implications. First, such structures are a link to the extensive body of experimental and theoretical information on toroidal DNA condensates formed in vitro (Bloomfield, 1997; Hud and Downing, 2001). Second, the structure of cm-nucleoids is likely to provide insights into the largely undefined structure of the normal nucleoid from which it is directly derived. Finally, the underlying toroidal structure may help in understanding the remarkable stability of cm-nucleoids as shown by their resistance to urea denaturation (Murphy and Zimmerman, 2000) and to morphological change during fixation and dehydration for microscopy (Woldringh and Nanninga, 1976).
2.2. Cell growth, drug treatment, and spheroplast formation E. coli C600 cells were grown with shaking at 37 °C in LB medium (Sambrook et al., 1989). The fluorescent dye DAPI (0.5 lg/ml) was added to the initial growth medium kerlund et al., 1992). to label the DNA of the nucleoids (A Cells in exponential phase ðA600 nm ¼ 0:25Þ were treated with chloramphenicol (30 lg/ml) for 1 h under growth conditions. After the culture was chilled by swirling the growth flask in ice water, the cells were fixed by addition of 1/20 vol of 38% formaldehyde for >1 h at 0 °C. Normal cells were prepared similarly except that the chloramphenicol was omitted. Cells were washed twice by centrifugation and resuspension in LB medium containing 2% formaldehyde before use except where noted. Nucleoid shapes in unfixed chloramphenicol-treated cells were not obviously different than those fixed with formaldehyde. Cells for conversion to spheroplasts were grown, treated with chloramphenicol, and chilled as above; 28 ml of chilled culture was centrifuged and the cell pellet was resuspended in 500 ll of 100 mM NaCl–20% sucrose–10 mM TrisHCl buffer, pH 8.1. Egg white lysozyme (40 lg in 100 ll of 120 mM TrisHCl buffer, (pH 8.1)–50 mM sodium EDTA (pH 7)) was added; after 10 min at 37 °C, 33 ll of 38% formaldehyde was added and the sample was held at least 1 h at 0 °C before use. 2.3. Light microscopy Microscope images from samples in free solution. Cells were diluted in 45 or 95% glycerol or in growth medium containing 2% formaldehyde. The appearance of the nucleoids and cells was similar in all media. Samples of 5 ll of cell dilutions were applied to a glass slide, a coverglass was added, and observation by microscopy was begun immediately. Multiple images were collected from individual floating cells or spheroplasts as they reoriented in solution. A selected object was observed continuously, either by DAPI fluorescence under low-intensity light or by phase-contrast imaging. Focus of the object and its position within the field were readjusted as necessary; simultaneous fluorescence and phase-contrast images were taken at intervals. The intensity of the mercury arc light
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source for fluorescence was increased stepwise between groups of 5–10 pictures to counteract fading due to photobleaching. Images from a Zeiss Axioskop microscope (Model 20 with 100-W mercury arc; Plan-Apochromat 100 objective with numerical aperture ¼ 1:4) with phase-contrast and fluorescence optics (using Zeiss filter set 02 for DAPI images) were collected with a Snappy video capture device (Play) from a Panasonic color digital camera (Model GP-KR222). The automatic gain control of the camera was activated when a series of fluorescence images were being collected from a single object. A Sony color video monitor (Model PVM 14N2U) was used to facilitate focusing and sample positioning. Changes in brightness or contrast of images (Photoshop, version 5.0, Adobe) were applied equally to all members of serial images. Microscope images of samples embedded in agarose. Fixed cells were embedded in agarose by mixing a 5-ll aliquot on a slide with an equal volume of a solution of 2% low gelling temperature agarose–20 mM sodium diethylmalonate buffer, pH 7.1, that had been melted and cooled to 37 °C. A coverslip was immediately added and sealed with Vaspar (Murray et al., 1994). In order to collect optical sections of individual agarose-embedded cells, the fine focus adjustment of the microscope was supplemented with an expanded dial that allowed the manual advance of the objective by increments of 1/3 lm. Phase-contrast exposures for optical sectioning were made with the maximal light intensity provided by the standard visible light source of the microscope to enhance contrast with the nucleoids. 2.4. Electron microscopy Thin sections of cells treated with 30 lg/ml of chloramphenicol for 1 h were prepared and electron microscope images were obtained as before (Zimmerman and Murphy, 2001).
3. Results 3.1. Effect of chloramphenicol exposure on nucleoid shapes in cells of E. coli Addition of 30 lg/ml of chloramphenicol to an exponentially growing culture of E. coli caused cell growth to slow within a few minutes and to largely cease within 30 min. The effect was reversible; growth resumed in <1 h after removal of the drug (Morgan et al., 1967; unpublished results of the author). Addition of the chloramphenicol caused the irregularly shaped nucleoids of rapidly growing cells (Fig. 1C) to change to the O- and Cshaped images that are characteristic of chloramphenicol exposure (Figs. 1A and B). Most of the chloramphenicoltreated cells contained a single nucleoid. O-shaped nu-
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Fig. 1. Light microscopy of chloramphenicol-treated and control E. coli C600 cells. (A, B) Cells 60 min after addition of 30 lg/ml chloramphenicol. (C) Cells grown in the absence of chloramphenicol. Images are simultaneous phase-contrast and DNA fluorescence exposures with inverted color. Scale bar, 5 lm.
cleoid images predominated in these cells; distinctly Cshaped nucleoid images were several-fold less frequent. 3.2. Multiple views of cm-nucleoids within cells or spheroplasts Interpretation of the O and C shapes depends whether they arise from the same or from different underlying structures. Multiple views collected from individual cmnucleoids have provided an answer to this question. If a single structure is responsible for both shapes, then individual nucleoids should present both C- and O-shaped images. Multiple images were acquired in two ways, namely, by taking multiple exposures of individual cells or spheroplasts undergoing reorientation in liquid media or by taking a series of pictures at different focal positions of individual cells embedded in agarose. Reorientation in liquid media. Examples of sequences of images collected from individual cells in liquid media are shown in Figs. 2A–C, E, and F. These simultaneous DNA fluorescence and phase-contrast exposures clearly showed the occurrence of both O- and C-shaped nucleoid images as an individual cell tumbled in solution. Both O- and Cshaped images were also seen by phase contrast alone (last two images in Figs. 2B and E). The images in series A of Fig. 2 were from an unfixed cell, indicating a lack of effect of the fixation procedure. The images of Fig. 2D were from a spheroplast; the similarity of the nucleoid in the spheroplast to those in the cells suggests that the space available to the nucleoid does not directly determine its shape.
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The orientation of shorter cells and spheroplasts was difficult to follow as they tumbled in solution, particularly during end-for-end rotations. However, two examples are shown in Fig. 2 where the orientation was followed through the entire sequence of pictures. Cell E, containing a single nucleoid, showed both nucleoid shapes. Cell F is an example from the small proportion of longer cells which contain two nucleoids after chloramphenicol exposure. The two nucleoids in such cells are produced at symmetrical positions in the same cytoplasm and, on that basis, might reasonably be expected to have the same structure. It is interesting, therefore, that the longer cells not infrequently contained one each of the O- and C-shaped nucleoids (as in the cell of Fig. 2F), supporting a common underlying structure for both nucleoid images. Optical sections. Examples of images collected as optical sections of immobilized single cells are shown in Fig. 3. The pictures in each vertical series were collected from a single cell at focusing positions separated by axial increments of 1/3 lm. Pictures near the middle of each series approximate a conventional ‘‘best focus’’ position for the cell as a whole, whereas pictures closer to the ends of each series are increasingly out of focus for the cell as a whole, but increasingly more in focus for the boundaries of the cell. The part of the specimen which is in focus at any one time is defined as its depth of field, estimated to be 0.4 lm under the present conditions based upon the discussion in Wallace et al. (2001). Given that the diameter of the chloramphenicol-treated cells was 1.2 lm and the apparent diameter of the O-shaped nucleoid images was 0.9– 1.0 lm, the individual images of Fig. 3 represent thick sections of the cells. The focusing series in Fig. 3 demonstrates that the nucleoids can be flat (cell A) or more rounded (cells B, C, and E). Whereas some cells showed a change in nucleoid shape between O and C shape as the focal plane was moved (cells C and E), some did not (cells A, B, and D). 3.3. Electron microscope images of sections of chloramphenicol-treated cells Electron microscope images of thin sections of the chloramphenicol-treated cell preparations used for light microscopy were similar to examples in the literature
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(Fig. 4; cf Zusman et al., 1973). The nucleoid voids in the thin sections were consistent with the O, C, and fused-nucleoid shapes in Figs. 1–3. 3.4. Structural models for the cm-nucleoid These results clearly demonstrate that a single nucleoid structure can give rise to both C- and O-shaped images. What is that underlying shape? The hollow sphere model of Kellenberger (see Section 1) is inconsistent with C-shaped images and will not be considered further. The O-shaped images immediately suggest a torus structure (Fig. 5). However, to obtain C-shaped images from an ring-shape structure requires that part of the boundary of the ring is lost from the image, presumably by off-center oblique optical sectioning of the nucleoid. Such sectioning is unlikely because it implies a largely ‘‘out of focus’’ image and because the Cshaped images should be accompanied by pairs of small circular regions resulting from sectioning at or near right angles to the plane of the torus. (See the animation of such oblique sectioning of a torus at http://mathworld.wolfram.com/ToricSection.html.) Although the torus model is unsatisfactory, a related structure that results from a smooth bending of the torus can readily account for both O and C shapes; we will refer to this modified structure as the ‘‘curved toroid’’ model (Fig. 5). The curved toroid model discussed here is based on the baseball seam curve (Thompson, 1998; see also R. B. Thompson, http://www.mathsoft.com/asolve/ baseball/baseball.html). In making the current model, the seam length of the original baseball seam curve has been decreased (relative to the size of the baseball) to make a more gradually curving toroid. As the seam length is decreased, the seam approaches the shape of a great circle, corresponding to the circular path of a conventional torus (‘‘belt ball,’’ Thompson, 1998). Hence, both the torus and the curved toroid models are shapes that fit onto a spherical surface (see Section 4). Projections of both models onto the viewing plane, i.e., the plane of the microscope slide, are compared in Fig. 6 over a range of inclinations to the viewing axis. The three columns of shapes for the curved toroid are for rotations of 0°, 45°, and 90° around the viewing axis. Note the absence of C shapes in projections from the torus model, and the abundance of C and flattened
b Fig. 2. Serial images of individual cells or spheroplasts undergoing reorientation in liquid media. Last two images in (B) and (E) are inverted phasecontrast exposures; other images are simultaneous phase-contrast and DNA fluorescence exposures with inverted color. Each series is a sequence of images from a single chloramphenicol-treated cell (A–C, E, F) or a spheroplast prepared from such a cell (D). The cell in (A) was unfixed; the cells or spheroplast in the other series were formaldehyde-fixed as described under Materials and methods. The orientation of cells in (E) and (F) was followed throughout the series and their images are aligned in the figure so as to preserve that orientation. Scale bar, 5 lm. Fig. 3. Serial images at different focal planes in chloramphenicol-treated cells. Chloramphenicol-treated cells were embedded in agarose. Images were obtained from individual cells at vertical intervals of 1/3 lm. The images of series A–C and the last image in D and E are inverted phase-contrast images. Other images in D and E are inverted images by simultaneous phase-contrast and DNA fluorescence. Scale bar, 5 lm.
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Fig. 4. Electron microscopy of thin sections of chloramphenicol-treated E. coli C600 cells. Scale bar, 2 lm.
Fig. 5. Torus and curved toroid models. Three-dimensional rendering was done with the program Blender (version 2.03).
shapes in projections from the curved toroid model that are similar in shape to those observed in Figs. 1–4.
4. Discussion 4.1. A toroidal model for cm-nucleoids Multiple views of cm-nucleoids support an underlying, smoothly bent toroidal shape. A single shape is assumed here, but it is unclear whether there are a variety of related curved toroidal shapes. A naturally occurring toroid such as that proposed here is very unusual. Toroidal structures
Fig. 6. Projected views of torus and curved toroid models. In order to simulate their appearance as images in the microscope, the torus and curved toroid were projected upon the viewing plane (x, y plane) as a function of their angle of inclination to the viewing axis (z axis). For the curved toroid, three series are shown which differ by 45° increments of rotation around the z axis. Drawings were done with the program SigmaPlot 2001 for Windows (version 7.0, SSPS).
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have been suggested to occur in sperm cells based partially upon in vitro reconstitution experiments (Hud et al., 1993). Toroidal DNA arrangements have also been proposed to occur in bacteriophage, and related structures can be found in phage lysates (Klimenko et al., 1967). A sharply folded and compressed toroidal model has been proposed for bacteriophage DNA in which the ultimate shape and size of the folded toroid reflected the phage head’s shape and size (Hud, 1995). The more limited distortion of the torus shape that is suggested here may also be designed to allow nucleoids to fit within the diameter of the cell envelope. Toroidal DNA forms are well-known products of in vitro DNA condensation caused by multiply charged ligands (reviewed in Bloomfield, 1997). Although the cm-nucleoids and the in vitro forms appear to share a basically toroidal shape, they differ in many important respects. The toroids formed in vitro are much smaller than the intracellular form described here: O-shaped cmnucleoids are roughly 1 lm in diameter vs 0.2 lm or considerably less in diameter for in vitro products (Yoshikawa et al., 1999). In vitro condensates also typically contain many DNA molecules per toroid, rather than the one or two DNA molecules presumably present in the cm-nucleoids. The solution conditions and ligands are obviously very different in vitro and in vivo. It will be important to determine the bound ligands and supercoiling status of the DNA of cm-nucleoids to begin to understand the folding behavior of the DNA in these enormous structures. 4.2. DNA organization in cm-nucleoids The chromosomes of rapidly growing cells are complex, containing multiple origins and multiple, partially replicated daughter chromosomes (Løbner-Olesen and Kuempel, 1992; Helmstetter, 1996). Exposure of such cells to chloramphenicol is expected to allow continued DNA replication, but to inhibit the separation of completed chromosomes and the initiation of new cycles of replication (Donachie and Begg, 1989; Hiraga et al., 1990). The physical correspondence between the resulting chromosomes and the toroidal cm-nucleoids remains to be determined. Gene localizations within cm-nucleoids, such as were done by Niki et al. (2000) on normal nucleoids, may be helpful in evaluating these possibilities. Concentric winding of the DNA such as can occur in bacteriophage (Ceritelli et al., 1997) or in in vitro DNA condensates (Hud and Downing, 2001) seems unlikely in the toroidal cm-nucleoids. Such winding of a length of DNA many fold longer than the cell would seem to present formidable problems during formation of the characteristic cm-nucleoids or their reconversion to normal nucleoids upon removal of the chloramphenicol. Rather, the present results support a model in which chloramphenicol exposure or the other means of inhibi-
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ting protein synthesis causes the cellular chromosome to undergo a basically isometric compaction. Such compaction would presumably result from localized foldings which may, in turn, reflect variations in local states of supercoiling of the DNA as well as other factors. The curved toroid model may have advantages to the cell as a ‘‘temporary’’ storage mode for its relatively inactive DNA during bacteriostasis: 1. Uniform curvature. The underlying spherical shape of the baseball seam-based models provides a uniform path for the DNA to follow, allowing the necessary folding of the DNA to be accommodated in a globally uniform way. 2. Flexible length. The amount of DNA in individual cm-nucleoids may vary due to differences in replication or differing extents of coalescence. Significant differences can be accommodated by varying the length of the ‘‘seams’’ by changes in their closest approach in the baseball seam curve. These changes in seam length can occur without changing the size of the virtual sphere upon which it resides.Because of the above considerations, structures similar to the curved toroid may also be appropriate for other inactive nucleoid configurations, including those of stationary-phase cells. The small size of such cells, or of slowly growing cells in general, would require a modification of current methods. 4.3. Application to normal cell nucleoids Nucleoid shapes in rapidly growing cells are highly variable in appearance. Preliminary results using multiple views of nucleoids within individual normal cells, as done here with chloramphenicol-treated cells, suggest that a significant part of the variation is a result of imaging from different directions of relatively fewer actual structures.
Acknowledgments Comments on the manuscript by Gary Felsenfeld and Martin Gellert are appreciated. The suggestions of David Zimmerman for graphics presentation and preparation have been very useful.
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Structural Biology Journal of Structural Biology 138 (2002) 199–206 www.academicpress.com
Toroidal nucleoids in Escherichia coli exposed to chloramphenicol Steven B. Zimmerman Laboratory of Molecular Biology, National Institutes of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Building 5, Room 329, Bethesda, MD 20892-0560, USA Received 26 February 2002, and in revised form 16 April 2002
Abstract The DNA of growing cells of Escherichia coli occurs in one or a few lobular bodies known as nucleoids. Upon exposure to chloramphenicol, the nucleoids assume compact, rounded forms (‘‘cm-nucleoids’’) that have been described as ring- or sphereshaped. Multiple views of single cells or spheroplasts, however, support a different, curved toroid shape for cm-nucleoids. The multiple views were obtained either by DNA fluorescence imaging as the cells or spheroplasts reoriented in liquid medium or by optical sectioning using phase-contrast or fluorescence imaging of immobilized cells. The curved toroid shape is consistent with electron microscope images of thin sections of chloramphenicol-treated cells. The relationship of this structure to active and inactive nucleoids and to the smaller toroidal forms made by in vitro DNA condensation is discussed. Published by Elsevier Science (USA). Keywords: Chloramphenicol; Escherichia coli; Nucleoid; Toroid
1. Introduction The genomic DNA of bacteria is largely restricted to one or a few discrete bodies per cell known as nucleoids (Drlica and Bendich, 2000; Woldringh and Nanninga, 1985; Woldringh and Odijk, 1999). The nucleoids within rapidly growing cells of Escherichia coli appear as variable, lobular arrays (‘‘normal nucleoids’’). Exposure of the cells to chloramphenicol causes a striking transformation of their nucleoids to more uniform, rounded images (‘‘cm-nucleoids;’’ Dworsky, 1974; Kellenberger, 1952; Morgan et al., 1967; Steinberg, 1952; Zusman et al., 1973). Many other treatments of cells which inhibit protein synthesis cause a similar rounding of their nucleoids, including exposure to aureomycin (Kellenberger and Ryter, 1956; Steinberg, 1952), terramycin (Steinberg, 1952), erythromycin (Dworsky, 1974), or puromycin or sodium azide (unpublished observations of the author), or growth without an essential amino acid (unpublished data cited in Hobot et al., 1985; Van Helvoort et al., 1998). The rounding of the nucleoids appears to result from a loss of cotranslational insertion linkages that joined the nucleoidal DNA to the cell envelope in growing E-mail address: [email protected]. 1047-8477/02/$ - see front matter. Published by Elsevier Science (USA). PII: S 1 0 4 7 - 8 4 7 7 ( 0 2 ) 0 0 0 3 6 - 9
bacteria (Woldringh et al., 1995). Loss of the linkages then allows the powerful condensing forces present in the cytoplasm, such as macromolecular crowding and ligand binding (Woldringh et al., 1995; Zimmerman and Murphy, 1996), to cause rearrangement of the nucleoids into the characteristic shapes associated with chloramphenicol exposure. This study presents a new structure to account for the characteristic images of cm-nucleoids which may also have application to the structures of normal nucleoids. Nucleoids of chloramphenicol-treated cells have been examined by light microscopy of whole cells and by electron microscopy of whole cells or thin sections of cells (Kellenberger, 1952; Morgan et al., 1967; Steinberg, 1952; Zusman et al., 1973). Three shapes of nucleoid images have been found, which will be referred to here as O, C, and fused shapes. 1.1. O-shaped nucleoid images Ring-shaped nucleoid images were noted by Kellenberger (1952) some 50 years ago and were further studied by Steinberg (1952). In later reports and reviews by Kellenberger and collaborators, the nucleoid structures responsible for the O shapes have been depicted as hollow spheres (e.g., Fig. 1, Kellenberger, 1960; Fig. 2C of
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Bohrmann et al., 1991; Robinow and Kellenberger, 1994). The material filling the center of the O-shaped image appeared to be similar to the cytoplasm which surrounded the nucleoid as seen by either light or electron microscopy, leading to suggestions that the central region of their suggested hollow-sphere structure contained a cytoplasmic invagination or inclusion (Kellenberger, 1960; Morgan et al., 1967; Woldringh and Nanninga, 1976; Zusman et al., 1973; Bohrmann et al., 1991).
2. Materials and methods 2.1. Materials Chloramphenicol, DAPI (40 ,6-diamidino-2-phenylindole), low gelling temperature agarose (Type VII), and crystallized chicken egg white lysozyme (EC 3.2.1.17) were purchased from Sigma Chemical, and formaldehyde (38% solution, containing 12% methanol) was purchased from Mallinckrodt.
1.2. C-shaped nucleoid images C-shaped nucleoid images of size similar to that of the O-shaped images were noted by Steinberg (1952) and by Zusman et al. (1973). Despite the inconsistency of Cshape images with a spherical model, alternative structural interpretations of the C- and O-shaped images have apparently not been presented. 1.3. Fused shape nucleoid images A small fraction of the cells treated with chloramphenicol develop fused nucleoid structures at the midcell position that suggest linked pairs of unresolved nucleoids stopped in the act of cell division. Rarer instances of fusions of three or more nucleoids also occur at scattered positions within the cell envelope (Kellenberger, 1952; Steinberg, 1952; Zusman et al., 1973), probably due to the progressive nucleoid coalescence that accompanies chloramphenicol treatment (Schaechter and Laing, 1961; Van Helvoort et al., 1996, 1998). 1.4. Interpretation and implications The present results indicate that the C and O shapes are both images of a single underlying structure, which is suggested to be a curved toroid, a smoothly deformed derivative of a ring torus. By this interpretation, the central ‘‘core’’ of O-shaped nucleoid images looks like the surrounding cytoplasm because it is the cytoplasm in which the toroids are immersed. The existence of intracellular toroidal nucleoids has important implications. First, such structures are a link to the extensive body of experimental and theoretical information on toroidal DNA condensates formed in vitro (Bloomfield, 1997; Hud and Downing, 2001). Second, the structure of cm-nucleoids is likely to provide insights into the largely undefined structure of the normal nucleoid from which it is directly derived. Finally, the underlying toroidal structure may help in understanding the remarkable stability of cm-nucleoids as shown by their resistance to urea denaturation (Murphy and Zimmerman, 2000) and to morphological change during fixation and dehydration for microscopy (Woldringh and Nanninga, 1976).
2.2. Cell growth, drug treatment, and spheroplast formation E. coli C600 cells were grown with shaking at 37 °C in LB medium (Sambrook et al., 1989). The fluorescent dye DAPI (0.5 lg/ml) was added to the initial growth medium kerlund et al., 1992). to label the DNA of the nucleoids (A Cells in exponential phase ðA600 nm ¼ 0:25Þ were treated with chloramphenicol (30 lg/ml) for 1 h under growth conditions. After the culture was chilled by swirling the growth flask in ice water, the cells were fixed by addition of 1/20 vol of 38% formaldehyde for >1 h at 0 °C. Normal cells were prepared similarly except that the chloramphenicol was omitted. Cells were washed twice by centrifugation and resuspension in LB medium containing 2% formaldehyde before use except where noted. Nucleoid shapes in unfixed chloramphenicol-treated cells were not obviously different than those fixed with formaldehyde. Cells for conversion to spheroplasts were grown, treated with chloramphenicol, and chilled as above; 28 ml of chilled culture was centrifuged and the cell pellet was resuspended in 500 ll of 100 mM NaCl–20% sucrose–10 mM TrisHCl buffer, pH 8.1. Egg white lysozyme (40 lg in 100 ll of 120 mM TrisHCl buffer, (pH 8.1)–50 mM sodium EDTA (pH 7)) was added; after 10 min at 37 °C, 33 ll of 38% formaldehyde was added and the sample was held at least 1 h at 0 °C before use. 2.3. Light microscopy Microscope images from samples in free solution. Cells were diluted in 45 or 95% glycerol or in growth medium containing 2% formaldehyde. The appearance of the nucleoids and cells was similar in all media. Samples of 5 ll of cell dilutions were applied to a glass slide, a coverglass was added, and observation by microscopy was begun immediately. Multiple images were collected from individual floating cells or spheroplasts as they reoriented in solution. A selected object was observed continuously, either by DAPI fluorescence under low-intensity light or by phase-contrast imaging. Focus of the object and its position within the field were readjusted as necessary; simultaneous fluorescence and phase-contrast images were taken at intervals. The intensity of the mercury arc light
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source for fluorescence was increased stepwise between groups of 5–10 pictures to counteract fading due to photobleaching. Images from a Zeiss Axioskop microscope (Model 20 with 100-W mercury arc; Plan-Apochromat 100 objective with numerical aperture ¼ 1:4) with phase-contrast and fluorescence optics (using Zeiss filter set 02 for DAPI images) were collected with a Snappy video capture device (Play) from a Panasonic color digital camera (Model GP-KR222). The automatic gain control of the camera was activated when a series of fluorescence images were being collected from a single object. A Sony color video monitor (Model PVM 14N2U) was used to facilitate focusing and sample positioning. Changes in brightness or contrast of images (Photoshop, version 5.0, Adobe) were applied equally to all members of serial images. Microscope images of samples embedded in agarose. Fixed cells were embedded in agarose by mixing a 5-ll aliquot on a slide with an equal volume of a solution of 2% low gelling temperature agarose–20 mM sodium diethylmalonate buffer, pH 7.1, that had been melted and cooled to 37 °C. A coverslip was immediately added and sealed with Vaspar (Murray et al., 1994). In order to collect optical sections of individual agarose-embedded cells, the fine focus adjustment of the microscope was supplemented with an expanded dial that allowed the manual advance of the objective by increments of 1/3 lm. Phase-contrast exposures for optical sectioning were made with the maximal light intensity provided by the standard visible light source of the microscope to enhance contrast with the nucleoids. 2.4. Electron microscopy Thin sections of cells treated with 30 lg/ml of chloramphenicol for 1 h were prepared and electron microscope images were obtained as before (Zimmerman and Murphy, 2001).
3. Results 3.1. Effect of chloramphenicol exposure on nucleoid shapes in cells of E. coli Addition of 30 lg/ml of chloramphenicol to an exponentially growing culture of E. coli caused cell growth to slow within a few minutes and to largely cease within 30 min. The effect was reversible; growth resumed in <1 h after removal of the drug (Morgan et al., 1967; unpublished results of the author). Addition of the chloramphenicol caused the irregularly shaped nucleoids of rapidly growing cells (Fig. 1C) to change to the O- and Cshaped images that are characteristic of chloramphenicol exposure (Figs. 1A and B). Most of the chloramphenicoltreated cells contained a single nucleoid. O-shaped nu-
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Fig. 1. Light microscopy of chloramphenicol-treated and control E. coli C600 cells. (A, B) Cells 60 min after addition of 30 lg/ml chloramphenicol. (C) Cells grown in the absence of chloramphenicol. Images are simultaneous phase-contrast and DNA fluorescence exposures with inverted color. Scale bar, 5 lm.
cleoid images predominated in these cells; distinctly Cshaped nucleoid images were several-fold less frequent. 3.2. Multiple views of cm-nucleoids within cells or spheroplasts Interpretation of the O and C shapes depends whether they arise from the same or from different underlying structures. Multiple views collected from individual cmnucleoids have provided an answer to this question. If a single structure is responsible for both shapes, then individual nucleoids should present both C- and O-shaped images. Multiple images were acquired in two ways, namely, by taking multiple exposures of individual cells or spheroplasts undergoing reorientation in liquid media or by taking a series of pictures at different focal positions of individual cells embedded in agarose. Reorientation in liquid media. Examples of sequences of images collected from individual cells in liquid media are shown in Figs. 2A–C, E, and F. These simultaneous DNA fluorescence and phase-contrast exposures clearly showed the occurrence of both O- and C-shaped nucleoid images as an individual cell tumbled in solution. Both O- and Cshaped images were also seen by phase contrast alone (last two images in Figs. 2B and E). The images in series A of Fig. 2 were from an unfixed cell, indicating a lack of effect of the fixation procedure. The images of Fig. 2D were from a spheroplast; the similarity of the nucleoid in the spheroplast to those in the cells suggests that the space available to the nucleoid does not directly determine its shape.
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The orientation of shorter cells and spheroplasts was difficult to follow as they tumbled in solution, particularly during end-for-end rotations. However, two examples are shown in Fig. 2 where the orientation was followed through the entire sequence of pictures. Cell E, containing a single nucleoid, showed both nucleoid shapes. Cell F is an example from the small proportion of longer cells which contain two nucleoids after chloramphenicol exposure. The two nucleoids in such cells are produced at symmetrical positions in the same cytoplasm and, on that basis, might reasonably be expected to have the same structure. It is interesting, therefore, that the longer cells not infrequently contained one each of the O- and C-shaped nucleoids (as in the cell of Fig. 2F), supporting a common underlying structure for both nucleoid images. Optical sections. Examples of images collected as optical sections of immobilized single cells are shown in Fig. 3. The pictures in each vertical series were collected from a single cell at focusing positions separated by axial increments of 1/3 lm. Pictures near the middle of each series approximate a conventional ‘‘best focus’’ position for the cell as a whole, whereas pictures closer to the ends of each series are increasingly out of focus for the cell as a whole, but increasingly more in focus for the boundaries of the cell. The part of the specimen which is in focus at any one time is defined as its depth of field, estimated to be 0.4 lm under the present conditions based upon the discussion in Wallace et al. (2001). Given that the diameter of the chloramphenicol-treated cells was 1.2 lm and the apparent diameter of the O-shaped nucleoid images was 0.9– 1.0 lm, the individual images of Fig. 3 represent thick sections of the cells. The focusing series in Fig. 3 demonstrates that the nucleoids can be flat (cell A) or more rounded (cells B, C, and E). Whereas some cells showed a change in nucleoid shape between O and C shape as the focal plane was moved (cells C and E), some did not (cells A, B, and D). 3.3. Electron microscope images of sections of chloramphenicol-treated cells Electron microscope images of thin sections of the chloramphenicol-treated cell preparations used for light microscopy were similar to examples in the literature
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(Fig. 4; cf Zusman et al., 1973). The nucleoid voids in the thin sections were consistent with the O, C, and fused-nucleoid shapes in Figs. 1–3. 3.4. Structural models for the cm-nucleoid These results clearly demonstrate that a single nucleoid structure can give rise to both C- and O-shaped images. What is that underlying shape? The hollow sphere model of Kellenberger (see Section 1) is inconsistent with C-shaped images and will not be considered further. The O-shaped images immediately suggest a torus structure (Fig. 5). However, to obtain C-shaped images from an ring-shape structure requires that part of the boundary of the ring is lost from the image, presumably by off-center oblique optical sectioning of the nucleoid. Such sectioning is unlikely because it implies a largely ‘‘out of focus’’ image and because the Cshaped images should be accompanied by pairs of small circular regions resulting from sectioning at or near right angles to the plane of the torus. (See the animation of such oblique sectioning of a torus at http://mathworld.wolfram.com/ToricSection.html.) Although the torus model is unsatisfactory, a related structure that results from a smooth bending of the torus can readily account for both O and C shapes; we will refer to this modified structure as the ‘‘curved toroid’’ model (Fig. 5). The curved toroid model discussed here is based on the baseball seam curve (Thompson, 1998; see also R. B. Thompson, http://www.mathsoft.com/asolve/ baseball/baseball.html). In making the current model, the seam length of the original baseball seam curve has been decreased (relative to the size of the baseball) to make a more gradually curving toroid. As the seam length is decreased, the seam approaches the shape of a great circle, corresponding to the circular path of a conventional torus (‘‘belt ball,’’ Thompson, 1998). Hence, both the torus and the curved toroid models are shapes that fit onto a spherical surface (see Section 4). Projections of both models onto the viewing plane, i.e., the plane of the microscope slide, are compared in Fig. 6 over a range of inclinations to the viewing axis. The three columns of shapes for the curved toroid are for rotations of 0°, 45°, and 90° around the viewing axis. Note the absence of C shapes in projections from the torus model, and the abundance of C and flattened
b Fig. 2. Serial images of individual cells or spheroplasts undergoing reorientation in liquid media. Last two images in (B) and (E) are inverted phasecontrast exposures; other images are simultaneous phase-contrast and DNA fluorescence exposures with inverted color. Each series is a sequence of images from a single chloramphenicol-treated cell (A–C, E, F) or a spheroplast prepared from such a cell (D). The cell in (A) was unfixed; the cells or spheroplast in the other series were formaldehyde-fixed as described under Materials and methods. The orientation of cells in (E) and (F) was followed throughout the series and their images are aligned in the figure so as to preserve that orientation. Scale bar, 5 lm. Fig. 3. Serial images at different focal planes in chloramphenicol-treated cells. Chloramphenicol-treated cells were embedded in agarose. Images were obtained from individual cells at vertical intervals of 1/3 lm. The images of series A–C and the last image in D and E are inverted phase-contrast images. Other images in D and E are inverted images by simultaneous phase-contrast and DNA fluorescence. Scale bar, 5 lm.
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Fig. 4. Electron microscopy of thin sections of chloramphenicol-treated E. coli C600 cells. Scale bar, 2 lm.
Fig. 5. Torus and curved toroid models. Three-dimensional rendering was done with the program Blender (version 2.03).
shapes in projections from the curved toroid model that are similar in shape to those observed in Figs. 1–4.
4. Discussion 4.1. A toroidal model for cm-nucleoids Multiple views of cm-nucleoids support an underlying, smoothly bent toroidal shape. A single shape is assumed here, but it is unclear whether there are a variety of related curved toroidal shapes. A naturally occurring toroid such as that proposed here is very unusual. Toroidal structures
Fig. 6. Projected views of torus and curved toroid models. In order to simulate their appearance as images in the microscope, the torus and curved toroid were projected upon the viewing plane (x, y plane) as a function of their angle of inclination to the viewing axis (z axis). For the curved toroid, three series are shown which differ by 45° increments of rotation around the z axis. Drawings were done with the program SigmaPlot 2001 for Windows (version 7.0, SSPS).
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have been suggested to occur in sperm cells based partially upon in vitro reconstitution experiments (Hud et al., 1993). Toroidal DNA arrangements have also been proposed to occur in bacteriophage, and related structures can be found in phage lysates (Klimenko et al., 1967). A sharply folded and compressed toroidal model has been proposed for bacteriophage DNA in which the ultimate shape and size of the folded toroid reflected the phage head’s shape and size (Hud, 1995). The more limited distortion of the torus shape that is suggested here may also be designed to allow nucleoids to fit within the diameter of the cell envelope. Toroidal DNA forms are well-known products of in vitro DNA condensation caused by multiply charged ligands (reviewed in Bloomfield, 1997). Although the cm-nucleoids and the in vitro forms appear to share a basically toroidal shape, they differ in many important respects. The toroids formed in vitro are much smaller than the intracellular form described here: O-shaped cmnucleoids are roughly 1 lm in diameter vs 0.2 lm or considerably less in diameter for in vitro products (Yoshikawa et al., 1999). In vitro condensates also typically contain many DNA molecules per toroid, rather than the one or two DNA molecules presumably present in the cm-nucleoids. The solution conditions and ligands are obviously very different in vitro and in vivo. It will be important to determine the bound ligands and supercoiling status of the DNA of cm-nucleoids to begin to understand the folding behavior of the DNA in these enormous structures. 4.2. DNA organization in cm-nucleoids The chromosomes of rapidly growing cells are complex, containing multiple origins and multiple, partially replicated daughter chromosomes (Løbner-Olesen and Kuempel, 1992; Helmstetter, 1996). Exposure of such cells to chloramphenicol is expected to allow continued DNA replication, but to inhibit the separation of completed chromosomes and the initiation of new cycles of replication (Donachie and Begg, 1989; Hiraga et al., 1990). The physical correspondence between the resulting chromosomes and the toroidal cm-nucleoids remains to be determined. Gene localizations within cm-nucleoids, such as were done by Niki et al. (2000) on normal nucleoids, may be helpful in evaluating these possibilities. Concentric winding of the DNA such as can occur in bacteriophage (Ceritelli et al., 1997) or in in vitro DNA condensates (Hud and Downing, 2001) seems unlikely in the toroidal cm-nucleoids. Such winding of a length of DNA many fold longer than the cell would seem to present formidable problems during formation of the characteristic cm-nucleoids or their reconversion to normal nucleoids upon removal of the chloramphenicol. Rather, the present results support a model in which chloramphenicol exposure or the other means of inhibi-
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ting protein synthesis causes the cellular chromosome to undergo a basically isometric compaction. Such compaction would presumably result from localized foldings which may, in turn, reflect variations in local states of supercoiling of the DNA as well as other factors. The curved toroid model may have advantages to the cell as a ‘‘temporary’’ storage mode for its relatively inactive DNA during bacteriostasis: 1. Uniform curvature. The underlying spherical shape of the baseball seam-based models provides a uniform path for the DNA to follow, allowing the necessary folding of the DNA to be accommodated in a globally uniform way. 2. Flexible length. The amount of DNA in individual cm-nucleoids may vary due to differences in replication or differing extents of coalescence. Significant differences can be accommodated by varying the length of the ‘‘seams’’ by changes in their closest approach in the baseball seam curve. These changes in seam length can occur without changing the size of the virtual sphere upon which it resides.Because of the above considerations, structures similar to the curved toroid may also be appropriate for other inactive nucleoid configurations, including those of stationary-phase cells. The small size of such cells, or of slowly growing cells in general, would require a modification of current methods. 4.3. Application to normal cell nucleoids Nucleoid shapes in rapidly growing cells are highly variable in appearance. Preliminary results using multiple views of nucleoids within individual normal cells, as done here with chloramphenicol-treated cells, suggest that a significant part of the variation is a result of imaging from different directions of relatively fewer actual structures.
Acknowledgments Comments on the manuscript by Gary Felsenfeld and Martin Gellert are appreciated. The suggestions of David Zimmerman for graphics presentation and preparation have been very useful.
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