For this study, a strain of C. jejuni was chosen for its medical relevance, as it was originally isolated from the feces of a human infant diagnosed with GBS. Cells were grown to logarithmic phase in BHI medium under microaerophilic conditions then first observed under a light microscope with 1000-fold magnification as rod shaped and motile. They swam rapidly in straight lines through the view field of the microscope without tumbling or changes in direction.
Separately, the logarithmic cells were prepared for electron microscopy by the addition of colloidal gold fiducial markers and then flash frozen on carbon grids containing 2 μm holes. Grids were first viewed using transmission electron cryomicroscopy (cTEM) at 3000× magnification, as cell shapes are best characterized at this level (Fig. 1A–G). Cells vary in length with many spanning across multiple holes in the grid. They are slender with a gentle, spirillum shape and in the typical bacteria the flagella are clear at both poles with granules strongly visible. Flagella are extremely long and cannot be fully traced, as many flagella overlay in the frame of view. Some parts of cells on the carbon show signs of slight deformations consistent with the bacterium having some flexibility; in fact, a few cells show bends likely caused by lying across holes on the grid (Fig. 1E).
Figure 1. Low magnification cTEM images of Campylobacter jejuni on carbon grids. For scale, the diameter of the holes is 2 μm. (A–G) A survey of C. jejuni cells. At this magnification overall shape and some intracellular features are visible. (E) shows a particularly long cell where a hole on the grid likely forces a bend. (G) The lines define the length and width measurements. (H) Bar charts illustrating the maxima and minima (circles), first and third quartiles (beginning and end of rectangle), and medians (gap) of length and width measured in 100 C. jejuni. To determine the average width five measurements were performed in each cell.
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A noticeable feature of these preparations was that many of the bacteria were organized into groups on the grid (Fig. 1A–D). Typically, these were tight associations along the length of the cell in a side-by-side fashion. This required a tight fit with the cells matching their helical turns suggesting that the periodicity of the helical turn is tightly controlled. Upon closer inspection, tomograms for these groupings (see below) do not reveal any direct interactions between cells; however, the width of the periplasmic space appears more irregular when two cells are in proximity. This behavior appears to be a characteristic of Campylobacter, as it has not been observed in previous studies from a sizable and diverse set of other bacteria (Chen, 2011).
Using the cTEM images, a statistical analysis of 100 C. jejuni was performed. Typical C. jejuni have a single flagellum and flagellar motor at each pole. Campylobacter jejuni with a single flagella are likely to have undergone recent cell division. The length, defined as the distance along a path through the middle of a bacterium (Fig. 1G), ranges between 1.4 and 7.01 μm with a median of 2.00 μm, first and third quartiles of 1.72 and 2.44 μm, respectively (Fig. 1H). It is likely that longer cells are in the process of cell division. Width was measured as the average of five independent measurements per cell of the diameter of the outer membrane (Fig. 1G) and varies between 0.255 and 0.409 μm with a median of 0.346 μm, first and third quartiles 0.331 and 0.360 μm, respectively (Fig. 1H).
To explore the finer ultrastructure of C. jejuni, full 3D cryotomograms at higher magnification were recorded. Tilt series covering 120° of perspective were acquired of select cells within the same samples used for cTEM. Predominantly, cells with their polar region fully within the holes of the grid were chosen. Consequently, whole cells examined tended to be shorter; however, in comparison to polar features of longer cells there were no obvious differences.
Because of surface tension in the thin fluid layer in which the cells are suspended on the grid, flexible cells can become flattened (Tocheva et al. 2010). For C. jejuni, the cross-sections exhibit an almost circular shape (Fig 2A), perhaps because of the relatively small width or robust structure, justifying the width measurements reported above. The cross-sections also illustrate how the well-understood “missing wedge” of tilt limitations attenuate resolution along the z-axis, obscuring thin features such as membranes when they lie in the xy plane (Fig. 2A) (Gan and Jensen 2012).
Figure 2. Ultrastructure of Campylobacter jejuni by ECT. In A–C and E and F, duplicate images are highlighted on the right: outer membrane (OM, gold), inner membrane (IM, light blue), chemoreceptors (CR, purple), ribosomes (Rib, blue spheres), storage granules (SG, orange), and nucleoid (Nuc). The scale bar in A is 100 nm (also corresponds to [B–G]). (A) Two tomographic slices through a C. jejuni cell oriented to highlight how the chemoreceptor arrays form a complete collar (see dotted line in [B] for approximate location). The cells, due to their slender nature, experience only minor distortion during sample preparation. The arrows indicate the direction of the electron beam along which resolution is attenuated. (B) Tomographic slice of a C. jejuni cell. (C) A cartoon highlighting the various features of (B). Note the central and polar ribosome exclusion zones (REZ). In this figure, the variability in the thickness of the periplasm can also be seen. (D) 3D model of C. jejuni based on the full tomograms from (B). The two membranes, flagella, storage granules, and ribosomes are highlighted. Ribosomes were manually identified in the tomogram. The left model has two fully developed poles, with flagella and polar REZs. (E–G) Similar to (B–D) for a C. jejuni cell with a single flagellum on one pole that maintains the common polar features. The opposing pole is missing the typical features, perhaps the result of a recent cell division.
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A few smaller cells were caught entirely suspended within a hole in the carbon grid (Figs. 1A, 2B). This enabled the description of the key morphological features from C. jejuni in the context of the entire cell. Using the tomogram of Figure 2B, a three-dimensional model was created based on the features seen in individual slices. A second cell is also modeled that lacks polar features at one end, likely because of a recent cell division (Fig. 2C).
The periplasm, bisected by the peptidoglycan, is characterized by a low contrast relative to the cytoplasm due to the lower density of macromolecules (Fig. 3B). At the poles, the periplasm has a dramatically irregular width due to the presence of the flagellar motor (Fig. 3). Elsewhere the width of the periplasm is less variable (Fig. 2B), although the outer membrane exhibits wrinkles not seen in the smooth inner membrane. Periplasmic width variation has been noted before; in cTEM of Escherichia coli and Pseudomonas aeroginosa the periplasmic width varies up to 10% (Matias et al. 2003), although some of the variation is attributable to distortions caused by sample preparation.
Figure 3. Polar architecture. (A, B) Tomographic slices and cartoons of two Campylobacter jejuni poles. Colors and labels are as above, with the addition of the flagellar motor (green) and flagellar hook and filament labeled. The scale bar in A is 100 nm and applies to (A–C). (C) (left) Tomographic slice providing a top view of a chemoreceptor array. The black box shows the region used to calculate the Fourier transform (right) demonstrating the hexagonal lattice. (D) (left) Slice through a subtomogram average of the C. jejuni flagellar motor showing its unusual periplasmic structure. (right) Isosurface of same subtomogram average. Membranes (same color code as in Figure 2), FliI (red), FlhAC (green), and as yet unidentified components (orange), scale bar is 25 nm.
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The two polar flagella provide the motility that allows C. jejuni to swim in the directed fashion seen by light microscopy. Gene deletions that disrupt the flagella are unable to colonize in host models demonstrating the importance of motility in pathogenicity (Nachamkin et al. 1998; Guerry 2007). The flagella are composed of a hook, junction, filament, and cap as previously noted (Galkin et al. 2008; Hendrixson 2008), consistent with the features seen in the tomograms (Fig. 3A). Viewed in the wider field of cTEM, the flagella are typically much longer than the body of the bacterium (Fig. 1F and G) with the filament measuring ~24 nm in thickness. Only a minority of the cells observed have a single flagellum suggesting that the flagellum and motor assemble quickly after cell division. The presence of locomotion machinery at each pole leads to open questions about how they work synergistically. Tumbling and swimming are well described in organisms such as E. coli; however, this model needs modification to be applicable to amphitrichous bacteria. During the limited observations made here by light microscopy, none of the C. jejuni tumbled during the time that they were within the field of vision. Based on current literature, it remains unclear how bipolar flagella coordinate to produce directed motility.
One of the features distinguishing C. jejuni from other species that have been visualized by ECT is that the flagella emerge from crater-like structures (best seen in Fig. 3). This unusual polar structure was first noted in negative stain where it was simply described as a “concave depression” (Pead 1979). In the tomograms, it is clear that the structure is the result of a thickening of the periplasm to accommodate the large flagellar motor (Fig. 3A and B). A protein ring on the periplasmic side of the outer membrane likely contributes to this (Chen et al. 2011).
Rod-shaped bacteria with flagella typically assemble chemoreceptor arrays near their poles. In C. jejuni the arrays are large structures that enclose the pole just inside the inner membrane (Fig. 2B). Cross-sections through the full cone of chemoreceptors appear as pronounced lines in tomographic slices (Figs. 2A and B, 3A and B). Fourier transforms clearly demonstrate the expected 12-nm hexagonal lattice (Fig. 3C) (Briegel, 2009). As reported previously, the cones start near the edge of the flagellar motor and extend ~200 nm along the length of the cell resulting in a surface area of ~75 nm2 per array corresponding to roughly 7500 receptors (Briegel et al. 2009). How these pairs of large receptor arrays communicate during chemotaxis is unknown. As in other organisms, we presume they communicate to the flagellar motors via the diffusing signaling protein CheY. The large, conical chemoreceptor array immediately adjacent to and surrounding the motor may then provide a focused and rapid signal. The small size of C. jejuni makes it likely that the two arrays (at opposing poles) experience the same concentrations of chemotactic molecules; however, it remains to be determined how these responses would be coordinated. Of note, the polar region enclosed by the chemoreceptor array excludes large molecules (see below), and may affect signal diffusion.
Bacterial cells are filled with ribosomes. In E. coli there are ~27,000 ribosomes/μm3 (Bakshi et al. 2012). This is consistent with the density we estimate for C. jejuni based on a manual count of ribosomes (~25,000 ribosomes/μm3) (Fig. 2). In C. jejuni, they are typically found throughout the entire cytoplasm except for the nucleoid region where they are absent, creating a REZ (Fig. 2B and C). Surprisingly, a second REZ was also noted at the poles within the chemoreceptor array cones (Figs. 2B and C, 3A and B). This polar REZ is reminiscent of structures seen previously for an overexpression mutant of PopZ, a protein thought to anchor chromosomal origins to the pole, in Caulobacter crescentus (Ebersbach et al. 2008). Presumably a dense aggregate of PopZ prevents diffusion of ribosomes into this area. The C. jejuni polar REZ does not require an additional membrane nor does its genome encode for a PopZ homologue. The comparison to the PopZ mutant suggests that diffusion of large molecules into the polar REZ is prevented by a protein meshwork or aggregate, although other complex structures, such as glycans, cannot be ruled out. The role and nature of this novel REZ are major questions that await elucidation.
Most of the examined C. jejuni, all from exponentially growing cultures, had one or two granules with diameters between 25 and 70 nm often at the periphery of the polar REZ (Figs. 1, 2B and C, 3A and B). While this positioning likely has functional implications, its physical cause may simply be exclusion from the polar REZ and nucleoid (Beeby et al. 2012). Based on appearance (compared with previous studies), the presence of polyphosphate synthesis genes and the absence of genes encoding microcompartments (Jorda et al. 2013), we suggest that these granules are polyphosphate storage granules (also known as acidocalcisomes) (Comolli et al. 2006; Beeby et al. 2012; Tocheva et al. 2013). Polyphosphate storage granules are electron dense (visible as darker particles in tomograms) due to the high concentrations of phosphate complexed by counter ions such as magnesium and calcium. Organisms utilize polyphosphate to regenerate nucleotide triphosphates (Kuroda et al. 1997); therefore, polyphosphate storage granules are regarded as energy storage granules. In eukaryotes, polyphosphate storage granules are found in membrane bound organelles and a similar architecture has been suggested in bacteria (Docampo et al. 2005). Other types of membrane-encapsulated particles have been clearly visualized by ECT (Komeili 2006); however, in C. jejuni the polyphosphate storage granules are not enclosed by membranes. The presence of polyphosphate storage granules close to the polar REZ boundary may suggest an additional involvement in motility. In several human pathogens, polyphosphate synthesis genes are linked to virulence and are thought to play a role in stress responses and survival during stationary phase (Rao and Kornberg 1996). For these organisms, deletions of ppk genes, required for polyphosphate generation and utilization, results in bacteria with no apparent phenotypes in rich liquid media; however, there is a significant loss in motility on solid media (Rashid and Kornberg 2000). In C. jejuni, knockout mutants of ppk1 and ppk2 are similar, with impaired pathogenesis and stress responses, particularly in cell invasion, a process presumably dependent on motility (Candon et al. 2007; Gangaiah et al. 2009). This suggests a possible link to the polar location.
A detailed understanding of the ultrastructure and life cycle of C. jejuni will be useful in the search for new treatment strategies to combat this pathogen. This report presents a critical missing component, providing new insights into the complex chemoreceptor arrays, the presence of polyphosphate storage granules, and the surprising polar REZ. Each of these components likely plays a crucial role in C. jejuni infections. The large chemoreceptor arrays that form a cap at the poles may allow the organism to sense environmental changes quickly. The putative protein aggregate that forms the polar REZ may contribute to swift signal transduction by keeping the cytoplasmic molecules involved in this process localized to the poles. The unusually large motor and flagellum allow C. jejuni to penetrate the viscous mucous layer and ultimately invade host intestinal epithelia. Polyphosphate storage granules likely provide energy storage needed to transition between environments such as the nutrient-poor latent stage experienced between hosts. Further understanding of the features described here may open the road to novel methods to keep this pathogen out of our food chain.