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Journal of Bacteriology, January 2006, p. 805-808, Vol. 188, No. 2
0021-9193/06/$08.00+0     doi:10.1128/JB.188.2.805-808.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Intact Carboxysomes in a Cyanobacterial Cell Visualized by Hilbert Differential Contrast Transmission Electron Microscopy

Yasuko Kaneko,¹ Radostin Danev,² Kuniaki Nagayama,² and Hitoshi Nakamoto^3*

Department of Regulation Biology, Saitama University, Saitama, 338-8570, Japan,¹ Okazaki Institute for Integrative Bioscience, National Institutes of Natural Sciences, Myodaiji-cho, Okazaki 444-8787, Japan,² Department of Biochemistry and Molecular Biology, Saitama University, Saitama, 338-8570, Japan³

Received 23 June 2005/ Accepted 27 October 2005

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Carboxysomes in rapidly frozen ice-embedded whole cells of the cyanobacterium Synechococcus sp. strain PCC 7942 were visualized by the recently developed Hilbert differential contrast transmission electron microscope. Structural details of carboxysomes were especially clearly visualized in the ruptured cells. The novel electron microscopy exhibited the paracrystalline arrays of molecules of the enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase in the carboxysomes in much better contrast than conventional transmission electron microscopy with ultrathin sections of cells. The carboxysome was surrounded by a 5- to 6-nm-thick monolayer shell which consisted of orderly arrays of globular particles.

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All cyanobacteria examined so far and many, but not all, chemoautotrophs contain polyhedral inclusion bodies that are bound by a unilamellar protein shell or coat (16). In thin sections, these bodies usually appear to be 90 to 500 nm in diameter and are most often observed as regular hexagons (16).

The enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) within these bodies appears to be packed into paracrystalline arrays, at least under some conditions (9, 15), but it is not clear whether such a well-defined molecular assembly of enzyme molecules is of physiological significance or merely a preparation artifact (1). The ordered arrangement may be disturbed during cell rupture and purification; on the other hand, it may sometimes be created as a drying artifact during preparation of the negative staining (2).

The ultimate goal of biological electron microscopy is to visualize ultrastructures in cells as they are in the living state. Rapidly frozen ice-embedded cells provide the most realistic images since they are free from artifacts introduced by lengthy sample preparation processes. However, detailed structures could not be visualized in them by conventional transmission electron microscopy (TEM) because of low contrast. The low contrast of nonstained biological samples can be overcome through the use of phase-contrast methods: that is, techniques using phase plates, which are frequently applied in visible light microscopy. Due to the fundamental issue of the electric charge imparted to phase plates by electron collisions (13), however, these methods have not yet been applied to TEM. Recently, the charging issue has been successfully resolved (7, 8) and novel forms of electron microscopy have been introduced: Zernike phase contrast (4) and Hilbert differential contrast (HDC) (6, 12). Among these methods, HDC, of which the experimental schematics are shown in Fig. 1A, is particularly useful because of its high contrast. Like differential interference contrast used in light microscopy, HDC can display topographical features of images through the effect of a phase plate inserted in the back focal plane of the objective lens (Fig. 1A). The -phase plate covering the half-plane of the aperture (Fig. 1B) converts the phase retardation by transparent objects to amplitude contrast. Image formation theory recognizes this conversion as the conversion of the image modulation function, the so-called contrast transfer function (CTF), from the sine functional modulation unique to conventional TEM (Fig. 1C) to the cosine functional modulation (Fig. 1C) (6, 13). Due to the intense low-frequency components recoverable with the cosine CTF, HDC can provide high contrast. In contrast to Zernike phase contrast (4, 5), which also carries the cosine CTF, the HDC CTF is an odd function characterized by a big jump at the frequency origin (6). This odd nature is transferred to the point spread function (PSF) of HDC, which has an antisymmetric profile. The antisymmetric double peak, which stands in contradistinction to the symmetry of the conventional PSF, is responsible for the differential contrast (6). The HDC-TEM has allowed us to visualize detailed ultrastructures of ice-embedded whole cells of the cyanobacterium Synechococcus sp. strain PCC 7942 (11). Thus, the HDC-TEM provides novel opportunities to examine subcellular structures in the living state. In the present study, the obtained HDC-TEM images of carboxysomes were carefully analyzed and compared with conventional TEM images of ultrathin sections.

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FIG. 1. (A) Schematics of HDC-TEM. (B) A half-plane phase plate is shown. It is set exactly at the back focal plane, where usually an aperture is situated. (C) Schematics of the CTF of HDC.

Synechococcus sp. strain PCC 7942 cells were cultured at 30°C under a light intensity of 20 µE/m²/s with 16-h light and 8-h dark cycles on BG-11 plates containing 1.5% (wt/vol) agar and 0.3% (wt/vol) sodium thiosulfate (14).

For preparation of ice-embedded cyanobacteria, the cells were collected in 0.2 M sucrose solution by centrifugation and dropped on a copper grid coated with carbon film. In log-phase growth of some gram-negative bacteria, turgor is known to be in the range of 0.5 M (3). Thus, 0.2 M sucrose may not have significant effect on altering the interior of the cyanobacterial cells. Holthuijzen et al. (10) stored carboxysomes in 20% (wt/wt) sucrose. After removing excess liquid carefully with the tip of a filter paper, the sample was frozen rapidly in liquid ethane using a Leica rapid freezing device (Leica EM CPC system). During the process of removing water, a number of cells were ruptured accidentally, although most of the cells remained intact and were covered by a thin layer of ice. The grid with ice-embedded cells was transferred to the specimen chamber of the TEM by a cryo-transfer system. The specimen chamber was cooled with liquid helium to reduce specimen damage caused by the electron dose. For observation, a JEOL JEM-3100FFC electron microscope with the HDC phase plate inserted into the back focal plane of the objective lens was operated at 300 kV.

For conventional chemical fixation, the cells were fixed in 2% glutaraldehyde in 0.05 M potassium phosphate buffer (pH 7.0) for 2 h at room temperature and in a refrigerator overnight. After rinsing in the buffer, the cells were postfixed with 2% OsO₄ in the buffer for 2 h at room temperature. They were then dehydrated in an acetone series and embedded in Spurr's resin. Ultrathin sections (silver-gold) were cut with a diamond knife on a Sorvall MT2-B ultramicrotome. After staining with uranyl acetate and lead citrate, the sections were observed with a Hitachi H-7500 TEM at an accelerating voltage of 100 kV.

Ice-embedded whole cells of the cyanobacterium Synechococcus sp. strain PCC 7942 were observed by HDC-TEM and conventional TEM for comparison. In contrast to the obscure image obtained by conventional TEM of the same ice-embedded cyanobacterial cell (Fig. 2A), detailed ultrastructure inside the cell was visualized with high contrast by HDC-TEM (Fig. 2B). The cells were surrounded by smooth cell walls and packed with various particles, filaments, and membranous structures (Fig. 2B). Among these, carboxysomes were easily recognized because of their characteristic polyhedral shape and size (Fig. 2B). Occasionally the paracrystalline arrangement of particles could be recognized (Fig. 2B inset, enlargement of the square). These must be RuBisCO molecules in a carboxysome. The detailed structure of carboxysomes was even more clearly observed in accidentally ruptured cells (Fig. 2C, arrows pointing to carboxysomes). The rupture of the cell was possibly induced when excess water was removed by filter paper in the instant prior to freezing. The partial leakage of cellular material probably lowered the density of the cytoplasm, making it more suitable for visualization of structural details by HDC-TEM. It seems that with thick whole cells containing dense cytoplasm, differential contrast between cytoplasm and other structures is more difficult to obtain. Examples of carboxysomes observed in this way are presented in Fig. 3A to H. We conclude that the paracrystalline arrangement is the configuration of RuBisCO in carboxysomes in vivo.

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FIG. 2. (A) Conventional TEM image of an ice-embedded whole cyanobacterial cell. (Square) Carboxysome (enlarged in inset). (B) HDC-TEM image of the same whole ice-embedded cyanobacterium. (Square) Carboxysome with paracrystalline RuBisCO molecules (enlarged in inset). (C) HDC-TEM image of ice-embedded cyanobacterium ruptured in the instant before freezing. Arrows, carboxysomes. Bars, 100 nm.

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FIG. 3. (A to H) Three-hundred-kilovolt HDC-TEM images of carboxysomes in ruptured ice-embedded cyanobacterial cells. Arrowheads, carboxysome shell consisting of arrays of globular structures; thick arrows, regularly arranged structures at intervals of about 12 nm; thin arrows, regularly arranged structures at intervals of 6 nm. An asterisk indicates the portion of carboxysome which does not exhibit the regular arrangement of molecules. Bar, 100 nm.

Compared to carboxysomes prepared by conventional chemical fixation, resin embedding, and ultrathin sectioning (Fig. 4), structural details could be visualized with much higher contrast by HDC-TEM. The chemical fixation protocols applied in this study may not be the best protocols currently known; however, for this paper, the best four ultrathin-sectioned images were selected from among hundreds of carboxysome images obtained. All of the images by HDC-TEM were far clearer, devoid of artifacts, and obtained without the lengthy specimen preparation process, which is unavoidable with conventional chemical fixation, resin embedding, and ultrathin sectioning. Whole carboxysomes observed by HDC-TEM showed more sharp-edged polygonal shapes than those in plastic-embedded sectioned cells. The blunt appearance in the sectioned carboxysomes may be an artifact which is inevitable as long as observation is made with conventional thin sections. Individual RuBisCO holoenzyme molecules and the semicrystalline arrangements of the molecules were clearly discernible in the frozen whole carboxysomes by HDC-TEM. We have observed 35 carboxysomes in partially ruptured cells by HDC-TEM, and all of them exhibited similar semicrystalline arrangements. An ordered array of 10- to 12-nm-diameter doughnut-shaped particles with a dark center hole or depression with the appearance of RuBisCO molecules was detected in the carboxysomes. Similar particles were also detected in the sectioned specimens, although they were barely discernible (Fig. 4). RuBisCO molecules were arranged regularly at the peripheral region of the carboxysomes; however, at the central area, the regular arrangement was not observed (Fig. 3H, asterisk). This tendency was also vaguely observable in the ultrathin sections, especially in Fig. 4C and D. The regular arrangement of molecules can be seen at the periphery of the sections of carboxysomes, and not in the central area. There were two distinct patterns to the arrangements: one consisting of narrow lines of approximately 6-nm intervals (Fig. 3H, thin arrows), the other consisting of wider lines of approximately 12-nm intervals (Fig. 3H, thick arrows). This may indicate the orientation of the regularly arranged molecules. Considering the deep focal depth obtained by 300-kV HDC-TEM, the image obtained should be the projection image of the whole specimen. It appears that the highest contrast could be obtained when similar structures were arranged in the same direction, especially in the direction of image projection. In smaller carboxysomes, the regular arrangement was not as distinct as in the larger ones (compare Fig. 3A with Fig. 3H).

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FIG. 4. (A to D) One-hundred-kilovolt conventional TEM images of carboxysomes in ultrathin sections of chemically fixed and resin-embedded cyanobacterial cells. RuBisCO molecules are vaguely recognized in a paracrystalline arrangement. The shell appears partially as disrupted lines. Bar, 100 nm.

Carboxysomes were surrounded by an approximately 5- to 6-nm-thick shell (Fig. 3). This is thicker than the value of 3 to 4 nm reported in the literature (1). This apparent difference may be caused by the chemical fixation process. It is well known that chemically fixed, dehydrated, and resin-embedded specimens are shrunken. Alternatively, the globular structures visualized by HDC-TEM may appear slightly larger because of the nature of its imaging mechanism. Shively et al. (15) have observed a beaded substructure in a carboxysome shell of Thiobacillus neapolitanus. In the HDC-TEM images, the outer shell surrounding the body was always recognizable as linear arrays of globular structures (arrowheads in Fig. 3F), whereas the shell is very difficult to recognize by conventional electron microscopy with ultrathin-sectioned specimens.

HDC-TEM with its extraordinary high contrast for unfixed, undehydrated, and unstained whole cyanobacterial cells will be a powerful tool for elucidating in vivo carboxysomes and other subcellular structures.

 
This work was partially supported by the Ministry of Education, Science, Sports and Culture, Grant-in-Aid for Creative Scientific Research 13GS0016 to K.N., and Grant-in-Aid for Scientific Research on Priority Area (A) (no. 17053003) and Scientific Research (C) (no. 16570028) to H.N.

We are grateful to Andreas Holzenburg for discussion.

 
* Corresponding author. Mailing address: Department of Biochemistry and Molecular Biology, Saitama University, 255 Shimo-Okubo, Sakura-ku, Saitama 338-8570, Japan. Phone: 81-48-858-3403. Fax: 81-48-858-3384. E-mail: nakamoto{at}post.saitama-u.ac.jp.

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Journal of Bacteriology, January 2006, p. 805-808, Vol. 188, No. 2
0021-9193/06/$08.00+0     doi:10.1128/JB.188.2.805-808.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

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