Unique thylakoid membrane architecture of a unicellular N 2 -fixing cyanobacterium revealed by electron tomography

Cyanobacteria, descendants of the endosymbiont that gave rise to modern-day chloroplasts, are vital contributors to global biological energy conversion processes. A thorough understanding of the physiology of cyanobacteria requires detailed knowledge of these organisms at the level of cellular architecture and organization. In these prokaryotes, the large membrane protein complexes of the photosynthetic and respiratory electron transport chains function in the intracellular thylakoid membranes. Like plants, the architecture of the thylakoid membranes in cyanobacteria has direct impact on cellular bioenergetics, protein transport, and molecular trafficking. However, whole-cell thylakoid organization in cyanobacteria is not well understood. Here we present, by using electron tomography, an in-depth analysis of the architecture of the thylakoid membranes in a unicellular cyanobacterium, Cyanothece sp. ATCC 51142. Based on the results of three-dimensional tomographic reconstructions of near entire cells, we determined that the thylakoids in Cyanothece 51142 form a dense and complex network that extends throughout the entire cell. This thylakoid membrane network is formed from the branching and splitting of membranes and encloses a single lumenal space. The entire thylakoid network spirals as a peripheral ring of membranes around the cell, an organization that has not previously been described in a cyanobacterium. Within the thylakoid membrane network are areas of quasi-helical arrangement with similarities to the thylakoid membrane system in chloroplasts. This cyanobacterial thylakoid arrangement is an efficient means of packing a large volume of membranes in the cell while optimizing intracellular transport and trafficking.


INTRODUCTION
Cyanobacteria are widely accepted as the evolutionary precursors of the chloroplasts of plants and algae. Modern-day cyanobacteria are an environmentally significant and diverse group of microbial phototrophs, and a number of strains are used as model systems to study fundamental processes including photosynthesis, nitrogen fixation and carbon sequestration. However, a comprehensive understanding of cyanobacterial biology also requires detailed knowledge of cellular architecture, an area that has not been as thoroughly explored. Like chloroplasts, cyanobacteria are characterized by an internal complexity that includes differentiated membrane systems and compartmentation. Classified as gram-negative bacteria, cyanobacteria have an outer membrane and cytoplasmic membrane, and all cyanobacteria known to date, except Gloeobacter violaceus (Nakamura et al., 2003), have an internal system of thylakoid membranes in which the light reactions of photosynthesis and respiration occur.
Besides the thylakoid membranes, the cyanobacterial cell interior contains components such as carboxysomes, glycogen granules, cyanophycin granules, polyphosphate bodies, lipid bodies, and polyhydroxybutyrate (PHB) granules, depending on the strain and growth conditions (Allen, 1984). The arrangement, number, and associations of these components with each other and with the membrane systems remain largely uncharacterized at high resolution in many strains of cyanobacteria. However, recognition of this cellular complexity and interest in understanding the compartmentalization of enzymatic functions in cyanobacteria have led to studies such as those focusing on the components of the shell surrounding carboxysomes, a form of bacterial microcompartment in which the initial reactions of carbon fixation occur (Klein et al., 2009).
The architecture of thylakoid membranes in cyanobacteria is of particular interest because of their critical roles in housing the photosynthetic and respiratory complexes, maintaining a proton gradient for the production of ATP, and maximizing light capture.
Regarding the organization of the thylakoid membranes, a number of characterizations have been made (for a recent review, see (Nevo et al., 2009) 6 thylakoid membrane organization in cyanobacteria were based on random thin section electron micrographs and freeze-fracture studies, from which the overall thylakoid organization was extrapolated. In all cases, thylakoid membranes are found as pairs of bilayers that enclose the thylakoid lumenal space, where the pH is lower than in the surrounding cytoplasm during active photosynthetic and respiratory electron transport.
In a number of strains of cyanobacteria, thylakoid membranes appear to follow the shape of the cell envelope, forming multiple concentric membrane layers interior to the plasma membrane (Mullineaux, 1999). This type of organization is particularly evident in widely studied strains such as Synechocystis sp. PCC 6803, Synechococcus elongatus sp. PCC 7942 and some Prochlorococcus strains (reviewed in (Liberton and Pakrasi, 2008). In contrast, other strains, such as some of those in the genus Cyanothece, have thylakoid membranes that appear to be arranged in a radial pattern within the cell, like spokes of a wheel, but still parallel to each other (Porta et al., 2000).
Regardless of their overall arrangement, thylakoid membranes are typically separated from each other by a space sufficient for a double row of phycobilisomes, large lightharvesting antenna complexes, to fit between the membranes (Mullineaux, 1999), but have been reported with appressed regions in a few strains, including Prochloron (Giddings et al., 1980) and Acaryochloris marina (Marquardt et al., 2000;Chen et al., 2009).
The fact that thylakoids routinely appear in 2-D images as parallel membrane layers with no apparent connection between the layers is consistent with a model of arrangement in which the membranes enclose multiple independent lumenal spaces.
However, such an organization raises numerous important questions regarding the maintenance of a proton gradient in individual compartments and the translocation of cellular components between isolated lumenal spaces. analysis of a cyanobacterium has not been described until now.
To address these open issues in cyanobacterial cell biology, we began a study of the whole-cell architecture of the unicellular nitrogen-fixing cyanobacterium Cyanothece sp.
ATCC 51142 (hereafter Cyanothece 51142). By using serial electron tomography combined with montaging, we were able to reconstruct large volumes of cyanobacterial cells in three dimensions, the first report of such extensive reconstructions in an oxygenic photosynthetic prokaryote. In Cyanothece 51142, thylakoid membranes are radially arranged around a central cytoplasmic area. Models constructed from tomographic data showed that this band of radial thylakoids spirals around the cell periphery, an organization not previously seen in cyanobacteria. Our data show that this peripheral band of thylakoid membranes is a dense and complex interconnected network derived from the continuous branching and splitting of membranes.
Importantly, we show that the thylakoid membranes form one extensive system enclosing a single space, the thylakoid lumen. The extensive nature of the thylakoid membrane network and the intricate detail of its organization were not apparent in

Intracellular arrangement and compartmentation in Cyanothece 51142 cells
Cyanothece 51142 is a unicellular diazotrophic cyanobacterium that is routinely grown under nitrogen-fixing conditions in a light regime of 12 h light/12 h dark, so that the cells perform photosynthesis during the light period and nitrogen fixation in the dark (Sherman et al., 1998). The periodic alterations in storage granule accumulation and degradation have been described: carbon is stored as glycogen during the light period and utilized during the dark, while nitrogen is stored as cyanophycin during the dark and utilized during the light period (Schneegurt et al., 1994;Li et al., 2001). In order to determine if these diurnal changes included alterations to the thylakoid membranes, we examined cells collected at time points throughout the diurnal cycle. We prepared Cyanothece 51142 cells for electron microscopy by high-pressure freezing followed by freeze-substitution and embedding in resin. Whole cells and details of intracellular features of cells harvested at timepoints L10 and D10 (after growth in 10 hours of light or dark, respectively) are shown in Figure 1. For this analysis, we examined thin sections of more than 250 cells from each time point, including cells that were harvested from independently grown cultures. From this in-depth analysis, we were able to thoroughly document intracellular features and changes that occurred during the diurnal cycle.
We found that, regardless of the time point during the diurnal cycle, Cyanothece 51142 cells display an intracellular arrangement that is fairly consistent between cells and cultures: a central cytoplasmic region containing ribosomes, carboxysomes, and polyphosphate bodies surrounded by a peripheral ring of thylakoid membranes ( Figure   1A and D). In the thylakoid membrane region, the accumulation and distribution of the numerous glycogen granules at time point L10 were observed ( Figure  During the dark period, cyanophycin is usually found near the plasma membrane as large round inclusions ( Figure 1E) approximately 200 nm in diameter. Cyanophycin granules are larger and more electron-dense than the more numerous and smaller lipid bodies ( Figure 1E). Cyanophycin granules have a homogeneous interior and can have a delimiting boundary in some preparations ( Figure 1E). Although glycogen granules, cyanophycin granules, and lipid bodies were all observed in close proximity with thylakoid membranes and the plasma membrane, direct continuity or connectivity between the thylakoid membranes or the plasma membrane and these inclusions could not be conclusively determined. In each of these tomograms, we found a similar cellular arrangement to that observed in thin sections and described above.
However, these tomograms encompassed only ~10% of the total cell volume. For a more complete 3-D analysis, we prepared serial thick sections of ~300 nm from L10 time point cells. We collected montaged (2x2) dual axis tilt series from seven serial sections and constructed tomograms of approximately 80% of the volume of one cell and approximately half the volume of two other cells, an analysis that included a total of ~2600 tomographic slices. These tomograms are the most complete reconstructions of an oxygenic photosynthetic cell to date, and provide a comprehensive insight into the whole cell organization of a cyanobacterium.
One cell was examined in detail and models were constructed using the IMOD software ( Figure 2). This cell was oriented along the long axis, and the tomographic reconstruction included the entire cell except for ~0.3 µm and ~0.6 µm at the beginning and end of the volume, respectively. Cyanothece 51142 cells are typically oblong, measuring ~3.5-4.5 µm long and ~2.5 µm wide; this cell measured 4.5 µm x 2.4 µm at its widest points. Figure  Membranes were also observed that deviated from the radial arrangement by forming loops (asterisk in Figure 2B), membranes concentric to the plasma membrane, or membranes that extended into the cell interior (asterisks in Figure 2C). The tomographic data confirmed that the cell was essentially divided into two regions: the thylakoid membrane region that contained glycogen granules, lipid bodies, some ribosomes, some polyphosphate bodies, and the central cytoplasmic region that included carboxysomes, the majority of the polyphosphate bodies, ribosomes, and presumably the nuclear material. We generated models that included many of these components to show their arrangements within the cell ( Figure 2E-G and Supplemental Video S1).
We expanded upon the analyses of thin sections and small-scale tomograms with a detailed examination of the organization of cellular components in their whole-cell context. Slice images of representative regions of the tomogram upon which our analyses were based are shown in Figure 2H-M. Ribosomes were very numerous, and located largely within the central cytoplasmic region, although many were also found between thylakoid membranes and near the plasma membrane. Glycogen granules were numerous and found between thylakoid membranes; their electron density varied throughout the depth of each serial thick section, appearing more electron transparent in optical sections near the surface of a serial section ( . Lipid bodies varied slightly in size and shape, from ~60 nm to ~100 nm, and were almost always found in the near vicinity of thylakoid membranes, specifically often found near the tips or edges of the membranes ( Figure 2K). However, direct association or continuity between lipid bodies and thylakoids or the plasma membrane could not be conclusively determined. Our large volume tomographic reconstructions showed nearly the entire thylakoid membrane system in Cyanothece 51142 cells, so that the whole-cell membrane organization could be visualized in this organism for the first time. We modeled the thylakoid membranes in approximately half the cell in order to understand the underlying architecture, and thylakoid membranes in the portion of the cell that was not modeled appeared to be arranged in a comparable manner (Figure 2). The thylakoid membranes occupied a considerable portion of the cell volume: using IMOD software, we calculated the volume of the modeled portion of the thylakoids to be 0.79 µm 3 , or about 15 % of the volume of that half of the cell.

Thylakoid membranes in
In slice images of the cell, we noted that the thylakoid membranes were oriented at different angles, depending on the position in the volume, and analysis of the tomogram showed that the angle of the membranes gradually changed when progressing through the volume ( Figure 3A-C ). Models showed that the thylakoids formed a dense band of membranes that spiraled around the cell ( Figure 3D-G), which accounted for the continually changing angle of the membranes seen in the tomogram. This organization is particularly evident when a portion of the thylakoids is rendered in a contrasting color and the model is rotated (yellow vs green, Figure 3D-G). We examined tomograms generated from portions of other cells and found that this spiral ring of thylakoids was a general pattern in all cells examined. Evidently, this spiral organization represents the typical architecture of thylakoid membranes in Cyanothece 51142, one that has not been previously observed in any cyanobacterium.
Compared to other cyanobacteria in which thylakoid membrane organization has been examined in detail, Cyanothece 51142 cells contain comparatively more thylakoid membrane layers in addition to this spiral architecture. We sought to understand how this dense spiral band of membranes is constructed from its individual membrane components. In order to show how individual thylakoid membranes fit into this spiral pattern, we modeled a single thylakoid and its neighboring membranes ( Figure 3H-J).
This membrane (designated by the yellow arrowhead in Figure 3D and H) formed a long ribbon that extended for approximately 1500 nm before branching ( Figure 3I).
Another branch occurred within about 600 nm ( Figure 3J). In the small region modeled

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in Figure 3H-J, four branch points were present within this group of membranes. The thylakoids in Cyanothece 51142 are thus not separate membranes that spiral around the cell, but rather components that form a larger system.

The Cyanothece 51142 thylakoid membrane network is extensively branched and contains areas with a quasi-helical organization
In thin section electron micrographs of Cyanothece 51142 cells, the thylakoid membranes appeared as separate sacs enclosing a lumenal space (Figure 1 and (Schneegurt et al., 1994;Schneegurt et al., 2000;Li et al., 2001)). In our tomograms, most of the thylakoid membranes in Cyanothece 51142 were parallel to each other, separated by approximately 50-60 nm, and did not form stacks or appressed areas.
Tomograms showed that, rather than forming individual sheets, the membranes were in fact interconnected, and the areas where the membranes coalesced could be clearly visualized (Figures 3 and 4). We examined the distribution of branching, splitting, or number and distribution of the interconnections were sufficient to show that the thylakoid membrane network forms an extensive system inclosing a single lumenal space.
While branching and splitting of membranes could be widely spaced, in some regions such interconnections appeared close together, or in clusters ( Figure 4A-B). We examined these regions in detail in order to determine if these membranes formed a specific organization, as has been observed in the plant chloroplast thylakoid  (Figure 3-4). Since we could find no evidence for physical continuity with the plasma membrane, and no structures resembling transport vesicles were observed, a possibility for thylakoid membrane biogenesis is the formation or growth of additional membrane from existing thylakoids. Such occurrences have been proposed for chloroplast thylakoids (Brangeon and Mustardy, 1979). The means of lipid delivery to the thylakoid membrane to facilitate this expansion remains unknown, however, but might involve the abundant lipid bodies observed in this study (Figure 2).
In higher plant chloroplasts, thylakoid membranes have a distinct architecture, forming an intricate network of stacks of flattened or appressed lamellae, the grana, that are connected by unstacked stroma thylakoids that traverse the chloroplast stroma matrix.
The foremost model of grana-stroma organization describes stroma thylakoids as righthanded helices that wind around grana stacks (Mustardy and Garab, 2003). In contrast, cyanobacterial thylakoid membranes typically do not form grana stacks and stroma thylakoids. A cyanobacterium or cyanobacterial ancestor is widely recognized as the progenitor of chloroplasts, and it is logical that some aspects of the modern-day chloroplast morphology evolved from the endosymbiont and may be maintained in present-day cyanobacteria. However, evidence of a morphological link between chloroplasts and cyanobacteria has previously been elusive. Our finding of rudimentary areas of helical arrangement within the thylakoid membrane system in Cyanothece 51142 may be such a link, which furthermore suggests that some aspects of chloroplast morphology originated in the cyanobacterial ancestor and were not a new invention in chloroplasts. This cyanobacteria-chloroplast similarity implies that homologous machinery to form and maintain helical arrangement was present in the prokaryotic ancestor and persists to some degree in at least one present-day cyanobacterium.
The model of thylakoid membrane architecture in cyanobacteria that emerges from our work is one that is much more similar to the thylakoid system in plant chloroplasts than has been previously described. Rather than simple layers bridged by connecting

Bacterial cell growth conditions
Cyanothece sp. ATCC 51142 cells were grown in liquid ASP2 medium without added nitrogen under 12 h light/12 h dark conditions under 30 µmol photons m -2 s -1 white light and at 30°C for 5-6 days (Stöckel et al., 2008).

Sample preparation for electron microscopy
Cells for transmission electron microscopy were ultra-rapidly frozen by high-pressure freezing. 100 ml culture aliquots were centrifuged, the cell pellet was resuspended in a small volume, pipetted into planchettes with 100-200 mm-deep wells, and frozen in a

Electron microscopy and tilt series acquisition
For conventional electron microscopy, thin sections (~80 nm) were cut from high pressure-frozen cells and digital images were viewed and collected using a LEO 912 transmission electron microscope operating at 120 kV and a ProScan digital camera.
For intermediate voltage electron microscopy, thick sections, or serial thick sections (250-350 nm), were cut and tilt series were acquired using a FEI Tecnai TF30 microscope operating at 300 kV. Images were collected at X15,000 from +60° to -60° at 1° intervals about two orthogonal axes using a Gatan digital camera.

Tomogram construction and modeling
Images were aligned using gold fiducial markers (15 nm). Single axis tomograms were constructed (from montaged or single images) using eTomo software (a component of 3DMOD), and the single axis tomograms were combined into a dual-axis tomogram.