three-dimensional imaging of the highly bent architecture of bdellovibrio bacteriovorus by using

JOURNAL OF BACTERIOLOGY, Apr. 2008, p. 2588–2596 Vol. 190, No. 70021-9193/08/$08.00�0 doi:10.1128/JB.01538-07

Three-Dimensional Imaging of the Highly Bent Architectureof Bdellovibrio bacteriovorus by Using

Cryo-Electron Tomography�†Mario J. Borgnia, Sriram Subramaniam, and Jacqueline L. S. Milne*

Laboratory of Cell Biology, Center for Cancer Research, National Cancer Institute, National Institutes ofHealth, Bethesda, Maryland 20892

Received 24 September 2007/Accepted 7 January 2008

Bdellovibrio bacteriovorus cells are small deltaproteobacterial cells that feed on other gram-negative bacteria,including human pathogens. Using cryo-electron tomography, we demonstrated that B. bacteriovorus cells arecapable of substantial flexibility and local deformation of the outer and inner membranes without loss of cellintegrity. These shape changes can occur in less than 2 min, and analysis of the internal architecture of highlybent cells showed that the overall distribution of molecular machines and the nucleoid is similar to that inmoderately bent cells. B. bacteriovorus cells appear to contain an extensive internal network of short and longfilamentous structures. We propose that rearrangements of these structures, in combination with the uniqueproperties of the cell envelope, may underlie the remarkable ability of B. bacteriovorus cells to find and enterbacterial prey.

Bacteria of the genus Bdellovibrio are highly motile preda-tors that reside in diverse aquatic and terrestrial environments,as well as in the mammalian digestive tract (28; for a review,see reference 33). They prey on a variety of gram-negativebacteria, including several human pathogens, and could be-come an important tool in the probiotic treatment of disease(32). Within this genus, Bdellovibrio bacteriovorus is the speciesthat has been best characterized biochemically and genetically(27). Investigations over the last four decades (20, 27, 33) havehelped elucidate several aspects of the complex life cycle ofthis organism, which includes a free-swimming or attack phaseand a prey-bound phase. Attack-phase B. bacteriovorus is pro-pelled by a single long sheathed flagellum and is capable ofattaining speeds up to 160 �m/s. The predator swims and turnsin apparently random directions until it finds and attaches to aprey cell. It then digests a region of the host cell outer mem-brane to make a small entry pore, penetrates into the hostperiplasm, and forms a growth chamber by reshaping and re-sealing the host outer membrane. The established parasitedepletes the host cytoplasm and undergoes growth to form asingle elongated spiral cell and segmented division to generatemultiple progeny, and it develops a flagellum to gain motility.Ultimately, the chamber ruptures, releasing the nascent bac-teria to reinitiate the life cycle.

Conventional electron microscopic (EM) studies have pro-vided valuable insights into morphological changes that occurduring the life cycle of B. bacteriovorus (1, 2, 6, 7). However,room temperature EM imaging typically requires specimen

preparation steps that include chemical fixation, removal of theaqueous phase by treatment with organic solvents, and embed-ding in plastic resins. These procedures can introduce artifacts,such as distortion of the cell shape, deterioration of membranestructures, and aggregation of soluble multiprotein complexes.Moreover, the contrast in the images originates primarily fromthe stain and not from the intrinsic density of target structures,limiting the resolution attainable by this approach. Atomicforce microscopic studies have also been used to probe thebacterial surface, but detailed internal structures are not visibleand the samples are also subjected to freeze-drying and stain-ing (24, 25). Cryo-EM and cryo-electron tomography are pow-erful alternatives for visualization of the global architecture ofbacterial cells that have been preserved in their native state (4,19, 29, 39). In this method, thin hydrated specimens are rapidlycooled to �180°C in liquid ethane to produce vitrified sampleswithout chemical fixation and dehydration. Further, the spec-imens can be imaged using minimal electron doses and withoutadditives to enhance contrast, so the resultant images closelyreflect native cellular conditions.

Here we used cryo-EM and cryo-electron tomography tovisualize attack-phase B. bacteriovorus and the spatial distribu-tion of key molecular complexes in situ. We found that eventhough this organism has internal architectural elements thatare very similar to those of other gram-negative bacteria, it canundergo dramatic changes in shape, as demonstrated by mold-ing of the bacterial shape to the topography of a carbon sub-strate in less than 1 to 2 min.

MATERIALS AND METHODS

Bacterial strains and culture conditions. The host-dependent B. bacteriovorusstrain used in this study was HD100 (� DSM50701). Predatory B. bacteriovoruswas cocultured at 22 to 25°C with Escherichia coli strain RP3098, a mutant withall flagellar and chemotaxis proteins deleted (31). Liquid cocultures were shakenat 250 rpm. To prepare prey stock cultures, individual RP3098 colonies selectedfrom YPD agar plates (1.0% yeast extract, 2.0% peptone, 2.0% agar, 2.0%glucose; Teknova, Hollister, CA) were grown overnight at 37°C and 250 rpm in

* Corresponding author. Mailing address: Laboratory of Cell Biol-ogy, National Cancer Institute, National Institutes of Health, Building50, Room 4306, 50 South Drive, Bethesda, MD 20892. Phone: (301)594-2063. Fax: (301) 480-3834. E-mail: [email protected].

† Supplemental material for this article may be found at http://jb.asm.org/.

� Published ahead of print on 18 January 2008.

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3 ml of YT broth (1.6% tryptone, 1.0% yeast extract, 0.5% NaCl; Teknova,Hollister, CA) supplemented with 10 mM Tris-HCl (pH 7.5), harvested bycentrifugation (Eppendorf 5415 D microcentrifuge; 16,000 � g, 30 s, 23°C), andresuspended in 3 ml of NB500 medium (25 mM Na-HEPES, 2 mM CaCl2, 16mg/liter WL Difco nutrient broth [pH 7.6]). Clonal B. bacteriovorus isolates wereobtained initially and then at periodic intervals to prevent enrichment of spon-taneous host-independent variants, using a two-layer agar selection procedure.Liquid cultures passaged through 0.45-�m filters were diluted, mixed with 1optical density at 600 nm (OD600) unit of prey cells in 5 ml of 42°C top agar(0.7% agar in NB500), and plated onto 10 ml of NB500 medium containing 1.5%agar at 22°C in a 10-cm-diameter round petri dish. Plaques that appeared within1 week were transferred to 2 ml of NB500 medium containing �0.2 OD600 unitof an E. coli prey stock culture, most of which cleared within 24 h. Clearedcultures were filtered through 0.45-�m filters and passaged by 20-fold dilutioninto 2 ml of NB500 medium containing 0.2 OD600 unit of a prey culture. Main-tenance cultures were passaged at least biweekly.

Specimen preparation. Enriched B. bacteriovorus cultures were prepared forEM by adding prey cells at a final concentration of 1 to 2 OD600 units to anovernight maintenance culture and monitoring the preparation by optical mi-croscopy (magnification, �400) until most prey cells were cleared from theculture. The clearing times were variable, especially with high prey densities.Aliquots of cleared culture (1 ml) were centrifuged (Eppendorf 5415 D micro-

centrifuge; 5,000 � g, 30 s, 23°C) to remove the prey cells and immature cellstrapped in bdelloplasts. All culture transfers were done carefully to preventdisruption of the remaining bdelloplasts and to avoid the release of immaturecells. Supernatants were transferred to a fresh tube and examined with a lightmicroscope. Highly motile B. bacteriovorus was the dominant species in thisfraction. A 3- to 5-�l aliquot of the supernatant was deposited onto the carbonside of a holey carbon grid (Quantifoil MultiA; Micro Tools GmbH, Germany)prepared as indicated below and held with tweezers. After 1 min, the tweezersand grid were positioned in a plunge-freeze apparatus (Vitrobot; FEI Corp.,Oregon), blotted from both sides (2 to 10 s, 23°C, 90% chamber humidity), andrapidly vitrified in liquid ethane. To aid in the alignment of tomographic tiltseries, all holey carbon grids were glow discharged, incubated for 2 min with 3 �lof 15-nm protein A-gold conjugate (BBInternational, Cardiff, United Kingdom)on the carbon side, which was followed by two 30-s washes with MilliQ water, airdried, and glow discharged a second time immediately prior to application ofspecimens and plunge freezing.

Cryo-EM and cryo-tomography. Specimens were transferred and imaged atliquid nitrogen temperatures using a Polara microscope (FEI Corp., Oregon)equipped with a field emission gun operating at 300 kV. Projection images wererecorded using a 2K by 2K charge-coupled device camera located at the end ofa postcolumn GIF 2000 (Gatan Inc., Pleasanton, CA) energy filter. Low-dosetomographic tilt series (1 to 2 e�/Å2 per image) were collected over an angular

FIG. 1. Intracellular structure and variation in the shape of B. bacteriovorus. (a) Low-dose, energy-filtered, projection EM images of vitrifiedcells allow detection of key intracellular components, as indicated by arrows. A, outer membrane; B, inner membrane; C, sheathed flagellum; D,rotor complex; E, chemotaxis receptor array; F, dense granule; G, nucleoid; H, macromolecular complexes; I, peptidoglycan layer; J, needlelikestructures at the anterior pole. (a to e) Examples illustrating the flexibility of B. bacteriovorus cells, which range from cells with moderate bends(a) to U-shaped bends (b) to cells distorted by virtue of intercellular contact (c) and cell edges flattened by contact with the carbon substrate. Thelarge black dots at the top and bottom right of the images are 15-nm gold particles deposited on the carbon film and used as fiducial markers toalign multiple images in a tilt series. (a and c to e) Scale bar � 200 nm. (b) Scale bar � 1 �m.

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range of �69° in 1.5° to 5° intervals (linear or Saxton collection schemes), usingan effective magnification of �18,000, �22,500, or �27,500 and underfocusvalues ranging from 5 to 15 �m. Gold fiducial alignment was performed forfull-resolution images. Three-dimensional (3D) reconstructions were obtainedby weighted back projection of aligned images that were binned 4�4. Tomo-graphic reconstructions obtained in this way were processed by 3D nonlinearanisotropic diffusion prior to segmentation. Alignment, reconstruction, denois-ing, and segmentation were all done using IMOD (18).

Elemental mapping. Electron energy loss spectroscopy (EELS) for elementalmapping was performed with an FEI Tecnai F20 S-Twin electron microscopewith a probe size of �1 nm, using previously described procedures (21). Hyper-spectral images were recorded using a Gatan energy filter (model 2001), incor-porating a cooled 1K by 1K charge-coupled device camera. For each pixel in thescanned image, spectral channel intensities were summed in the direction per-pendicular to the energy dispersion and were corrected for dark-current andchannel gain variations. Vitrified specimens of B. bacteriovorus cells were allowedto sublime in the vacuum of the microscope column for 12 h. Data were collectedfrom these dried samples after they were transferred to a room temperaturespecimen holder. To determine the presence of different elements in a specimen,a high-angle angular dark-field scanning transmission EM image was first ob-tained. EELS spectra of regions containing electron-dense granules showed thepresence of distinct phosphorus, oxygen, and calcium peaks higher than those forcontrol regions not containing the electron-dense granules. The relative propor-tions of various elements present were estimated using software in Digital-Micrograph 3.9.4 GMS1.4.4 and a Hartree-Slater model for estimating crosssections.

RESULTS

Representative projection images of frozen hydrated B. bac-teriovorus cells (Fig. 1) demonstrated the qualitative improve-ment obtained by use of rapid vitrification and cryo-EM imag-ing in a nearly native state compared to conventional EMimaging. The cellular architecture was well preserved, asshown by the integrity of the outer and inner membranes andthe considerable internal detail visible in the cytoplasm.

Attack-phase B. bacteriovorus cells are typically shaped likecurved rods with a length of 1.02 � 0.15 �m and a width of0.3 � 0.02 �m (means � standard deviations; n � 50). The twopoles of a cell are distinct; the posterior end containing theflagellum is rounder, whereas the anterior end, which is in-volved in prey penetration, is usually flatter. Single cells andclusters of stacked cells having different degrees and directionsof curvature, ranging from U- to comma- to S-shaped cells, canbe captured under physiologically relevant conditions in vitre-ous ice, as shown in the low-magnification image in Fig. 1b.Some cells bend so extensively that they exhibit apparent sur-face interactions between different membrane regions of thesame cell (Fig. 1c). Occasionally, constrictions in the cell widthwere observed at areas of contact with other cells (Fig. 1d) orwith the substrate (see Fig. 6). In rare cases, membrane exten-sions induced by contact with the carbon were found to occurboth along the length of the cell (Fig. 1e) and at the poles. Incontrast, these types of bends and distortions have not beenobserved in any of hundreds of tomographically imaged E. colicells examined (39; Cezar Khursigara and Sriram Subrama-niam, unpublished observations).

Cryo-electron tomographic methods (34) allow visualizationof structure in the third dimension and significantly extend theinformation that can be obtained from projection images suchas those shown in Fig. 1. An example of the improvement invisualization of cellular detail is shown in Fig. 2, in which thetype of information obtained from a two-dimensional projec-tion (Fig. 2a) is compared with the type of information ob-

FIG. 2. Cellular interior revealed by cryo-electron tomography. (a) Pro-jection image recorded as part of a tilt series for a specimen of plunge-frozenB. bacteriovorus cells. (b) Central 8-nm slice through the reconstructed 3Dvolume obtained from the same tilt series. (c) 3D rendering of key cellularstructures segmented from the tomographic volume. The flagellum, innerand outer membranes, and filamentlike structures in the cytoplasm are green,putative ribosomes are red, and dense granules are blue. The inset in panel ashows a projection image with periodic, 26-Å-spaced striations in the nucleoidregion, consistent with the expected packing arrangement of DNA. The insetin panel b shows the anterior end of the cell, including structures (indicatedby arrowheads) that protrude outward and may be relevant for making con-tact with prey. (a and b) Scale bars � 150 nm. (Panel a inset) Scale bar � 10nm. (Panel b inset) Scale bar � 25 nm.

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tained from 3D tomographic analysis (Fig. 2b and 2c) of thesame cell. The space between the inner and outer membranes,measured using tomographic analysis, is �25 nm along thebody of the cell and increases to �40 nm at the anterior pole.The prominent granules in the cytoplasm appear to be quasi-spherical and have different diameters. In some cases, a thinboundary layer of density is discernible at the interface be-tween the granules and the cytoplasm. EELS demonstratedthat the prominent dense granules are enriched in phosphorus,oxygen, and calcium compared to other regions of the samecell or regions corresponding to the grid, both of which havedistinct elemental profiles (Fig. 3). These granules have nopreferential cellular localization, and there is no obvious cor-relation between granule size and cell length (Fig. 4).

The elemental composition of the granules suggests thatthey are similar to the acidocalcisomes of other cells (12) butnot to the carbon-enriched, phosphorus-depleted bodies seenin Caulobacter crescentus, which are thought to consist of bu-tyric acid in association with electron-dense salts (9). B. bac-teriovorus cells are both highly enriched in cellular polyphos-phates compared to E. coli (3) and contain inwardly directedphosphate transporters, as well as the genes encoding the keyenzymes required for polyphosphate metabolism, includingpolyphosphate kinase 1 and several polyphosphate phospho-hydrolases (27). Whether any of these enzymes localize at theouter edges of the dense granules remains to be determined,but the dense granules likely serve as critical energy reserves toensure survival under starvation conditions (5), among otherpotential functions attributed to polyphosphates (17).

Detailed inspection of the interior of the cell revealed sev-eral other features whose probable identities can be surmisedbased on their shape, size, and location. In the outer reaches of

FIG. 3. Elemental analysis of Bdellovibrio dense granules. (a) Projection image of a cell recorded using scanning transmission EM. Circles 1,2, and 3 indicate the centers of square regions (20 by 20 nm) analyzed by EELS to determine the elemental composition of the dense granules,the surrounding nucleoid, and the extracellular medium, respectively. (b) EELS spectra of the three regions, indicating that the dense granules areenriched in phosphorus, oxygen, and calcium. See Materials and Methods for additional details.

FIG. 4. Distribution of the locations and sizes of the dense gran-ules along the length of the cell. (a) The data show that the densegranules are not present at specific positions relative to the pole ofthe cell and can be various sizes. (b) Correlation between the size ofthe dense granules and the size of the cell, expressed in granulesize/cell length, indicating that there is no obvious correlation be-tween the size of the cells and the overall size of the granules.



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the cytoplasm are scattered dense macromolecular complexes,likely to be ribosomes. These complexes are not present in thecentral region of the cell, which is diffuse and has a differenttexture than the rest of the cytoplasm. Based on its centrallocation, we believe that this region corresponds to the nucle-oid. Further, two-dimensional images of this region in othercells revealed a stacked pattern with a periodicity of �26 Åconsistent with higher-order structures of DNA (Fig. 2a, inset),such as those previously observed in thin vitreous sections of

Deinococcus radiodurans (13). Distinct structures were alsovisible on the outside of the cell. In particular, protrudingdense structures perpendicular to the outer membrane wereobserved at the anterior pole of the cell (Fig. 2b, inset). It ispossible that these structures correspond to the structuresthought to be responsible for prey attachment or penetration(27). At the other end of the cell, the base of the flagellum isclearly visible, and tomographic imaging showed that the mem-brane-embedded rotor complex is always offset from the lon-

FIG. 5. Visualization of the motility and chemotaxis machinery of B. bacteriovorus. (a) (Top left panel) Eight-nanometer slice extracted froma tomographic volume in an orientation coplanar with the axis of the rotor complex. Sections of the individual elements of the ring are visible. (Topright panel) Schematic diagram of the tomogram corresponding to the slice shown in the top left panel showing components expected to be foundin a single rotor complex. (Bottom panels) Four orthogonal slices across the rotor complex shown in the top left panel at places indicated by thetick marks. The density resulting from the flagellum is visible as a small dark ring in the fourth bottom panel and is white in the schematic diagramin the top right panel; a faintly visible density corresponding to the flagellar sheath is green in the top right panel. The density from the flagellumis also visible at the center of the third bottom panel along with contributions from the density indicated by yellow in the top right panel and likelyresults from the P-ring. The much darker density in the outermost regions of the third panel is due to the outer membrane, and the level is roughlythe same as the level of density indicated by yellow in the top right panel. The densities in the first and second panels indicated by red, blue, andorange in the schematic diagram likely correspond to contributions from the C-ring, the MS-ring, and the MotAB complex, respectively. (b)Eight-nanometer tomographic slice through the center of the same cell near the flagellar pole, showing the spatial arrangement of the chemotaxisreceptor array (visible as a band in the cytoplasm [dark line of density inside the inner membrane]) relative to the pole. Scale bar � 50 nm.



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gitudinal axis of the cell (see Movies S1, S2, and 3 in thesupplemental material). Remarkably, the major components ofthe rotor complex are discernible even at the extremely lowsignal-to-noise ratios corresponding to the 3D density map fora single complex. The nearby polar chemotaxis receptor array(39) is also evident in tomograms of individual cells (Fig. 5).

While most cells were completely embedded in vitreous ice,the interaction with occasional thin layers of carbon extendingfrom the rim or narrow strips separating two closely spacedholes resulted in cell membranes bending around the carbonfilm (Fig. 6). These cells presented an opportunity to examinethe interaction with the substrate. Tomographic reconstructionallowed us to determine that both the inner and outer mem-branes wrap around the substrate without any apparent loss ofcellular integrity. Comparison of highly bent and moderatelybent cells revealed no discernible differences in the cellular

features noted above, including the nucleoid, which remainedundistorted and closely followed the curvature of the bend (seeMovies S2 and S3 in the supplemental material). There wasalso no appreciable difference in the �25-nm spacing betweenthe inner and outer membranes at regions with the greatestcurvature relative to other areas of the cell (Fig. 6c, inset).

Another interesting structural feature of the B. bacteriovoruscellular interior is the extended network of internal filaments(Fig. 7; see Movie S1 in the supplemental material). Somefilaments are parallel to the longitudinal axis of the cell (Fig.7a), while others are distributed in the transverse direction(Fig. 7b) or are bundled in clusters perpendicular to the mem-brane plane and arranged in a periodic pattern with a spacingof �12 nm (Fig. 7c). These filaments appear to be similar tothose previously reported for C. crescentus (4), and althoughtheir identity is not known at present, they are highly sugges-

a

e f g h

b c d

FIG. 6. Cryo-electron tomography of vitrified B. bacteriovorus cells shaped around the edges of the carbon film-vitreous ice interface. Panelsa to d and panels e to h show two examples. (a and e) Low-dose projection images; (b, c, f, and g) 6-nm-thick tomographic slices from differentdepths of the cellular tomograms; (d and h) segmented rendering of portions of the bent cells, showing the wrapping of the cells around the edgeof the carbon film. Scale bars � 200 nm. The inset in panel c shows that despite extensive changes in curvature, the spacing between the inner andouter membranes is maintained along the length of the bacterium.

FIG. 7. Visualization of cytoskeletal elements in the cytoplasm of B. bacteriovorus: tomographic slices showing the presence of bundles offilaments oriented parallel (a and b) and transverse (indicated by the circle) (c) to the plane of the plasma membrane. Scale bars � 100 nm.



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tive of the structures formed in vitro by bacterial actin-likeprotein homologs (for a review, see reference 23).

DISCUSSION

The name Bdellovibrio reflects two characteristics of thegenus: (i) the behavior of the cells resembles that of a feedingleech (bdello) because the cells usually attach by one of thepoles to the side of the prey and (ii) the shape of the cell bodyis approximately the shape of a comma (vibrio). However, themost striking feature of attack-phase B. bacteriovorus cells ob-served with a light microscope is their motility (33). Swimmingcells resemble elongated wiggling rods, but precise determina-tion of their shape and its influence on internal cellular archi-tecture is difficult using light microscopy since the cells are verysmall, move rapidly through the field of focus, and turnabruptly in random directions. Adherent cells appear to beprimarily comma shaped, but detailed morphological charac-terization is hindered by the limited resolution of light micros-copy. Conventional EM pictures have revealed pronounceddeviations from the comma shape (1, 2, 6, 7). However, the cellmembranes and internal structures of these cells are prone todamage resulting from fixation, desiccation, and staining pro-cedures, which precludes visualization of detailed cellular ar-chitecture for the range of observed shapes in the population.The analysis that we report here of intact frozen hydrated cellsusing cryo-EM and cryo-electron tomography indicated thatthe cells can bend with extreme curvature without suffering anyobvious structural damage. Cell bending is likely to occur priorto rather than during vitrification because the rapid rate ofcooling (�105°C/s) precludes formation of crystalline ice and isthus expected to block changes in cellular shape, which cannotoccur more rapidly than the rate of ice crystal formation.

Although B. bacteriovorus reproduces by multiple sequentialsegmentation, the bent shapes that we observed likely do notrepresent cells that were prematurely released from bdello-plasts. First, cells released prior to division would be expectedto show evidence of segmentation, which was not apparent inany of the hundreds of cellular images that we recorded. Sec-ond, a population of prematurely released cells is expected tobe heterogeneous in terms of length, contrary to the narrowdistribution that we observed in a given culture. Third, earlystudies on B. bacteriovorus cell division by use of conventionalEM also showed that the flagellum starts to develop during celldivision and continues to mature after septation has been com-pleted (7), but here all B. bacteriovorus cells that were bent alsohad fully developed flagella. Finally, the nucleoid occupies arelatively large and well-defined volume in the cell, which mustnevertheless be highly compact, because the size of the B.bacteriovorus genome (3.78 Mb) is similar to the size of thegenome of the much larger organism E. coli (27). In dividingcells, genetic material is expected to double in size and to besegregated to the daughter cells. Several projection images ofbent cells and the 3D tomographic reconstruction of an L-shaped cell shown in Fig. 6a show a single uninterrupted vol-ume in which ribosomes are not observed; this is again consis-tent with the presence of a single nucleoid instead of the twoseparate volumes that would be consistent with segregation ofgenetic material prior to segmentation. Thus, we concluded

that the vast majority of cells observed in our cultures must bemature, attack-phase B. bacteriovorus cells.

The next question concerns whether the shape of individualcells in a culture is fixed or changes dynamically before cells arelocked into a particular shape by vitrification. Our results in-dicate that cells could alter both their overall curvature and theshape of their membrane upon interaction with the substratewithin the 2 min that elapsed from deposition of the sample onthe holey carbon grid to freezing (Fig. 1). Importantly, thesechanges in shape occurred without compromising the integrityof the membrane, suggesting that an underlying active mech-anism mediates the changes, although the possibility that thereare simpler alternative mechanisms that cause changes in ad-hesion of outer membrane components cannot be excluded.Control of the degree of curvature could provide the cell with amechanism to modulate rotation about its longitudinal axis and,combined with the off-axis location of the flagellum, may enablea motile B. bacteriovorus cell to modify its speed and direction.Flexibility may also play a fundamental role during prey penetra-tion, as B. bacteriovorus enters the host periplasm very rapidlythrough a restricted pore (2), possibly using a mechanism involv-ing twitching mobility mediated by type IV pili (14). An intriguingpossibility is that the protrusions observed at the anterior pole(Fig. 2, inset) may represent these type IV pili. To our knowledge,dynamic shape variations of the kind that we are proposing herehave generally not been observed in prokaryotes, although it hasbeen suggested that alterations in filament length may be involvedin cellular motility and alterations in cell shape in the cell wall-freeorganism Spiroplasma (19).

The ability of B. bacteriovorus to undergo alterations inshape along its length leads to the pleomorphic shape di-versity seen in culture and during entry into prey cells. Thisis in contrast to findings for other bacteria, which show thattypically, well-defined shapes, such as rods, cocci, or spirals,occur within a population of a particular species (38). Bac-terial morphology appears to be maintained by the orderedinsertion of the peptidoglycan, using preexisiting strands astemplates, and regulated peptidoglycan bond turnover (36),which is higher in zones of active growth and cell divisionand does not occur in the polar area (10, 15). The exactmechanisms of spatially regulated peptidoglycan synthesisare uncertain. They are likely to involve not only the peni-cillin binding enzymes involved in cell wall synthetic com-plexes but also the cytoskeletal elements MreB and FtsZand cell shape-determining proteins, such as MreC andRodA, which may position the peptidoglycan synthesis ma-chinery in the cell (8, 11, 36). Only in certain instances, suchas the imposition of external templates, genetic mutation, orstarvation, is the normal shape typically disrupted (11, 38).Intracellular turgor pressure in bacteria, estimated to be ashigh as 3 to 5 atm (16), could also play a key role in main-taining established shapes by keeping the peptidoglycanlayer in a more extended conformation. Interaction of outermembrane lipoproteins and membrane proteins with thepeptidoglycan layer and the presence of complexes that spanthe inner and outer membranes or relatively weak electro-static interactions may also stabilize cell shape, although therelative contributions may vary among species (37).

The mechanisms which B. bacteriovorus employs to estab-lish and maintain its vibrioid shape likely parallel those of



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other bacteria since B. bacteriovorus has a peptidoglycancomposition similar to that of other bacteria (35) and pos-sesses penicillin-like binding proteins (26, 27) and cytoskel-etal elements homologous to MreB, MreC, RodA, FtsZ, andMltA (27), which are known to modulate shape in otherbacteria (8, 30, 38), as discussed above. The discovery offiber-like structures in the cytoplasm in the bent regions ofthe cell suggests that these proteins may also have unantic-ipated roles in mediating the observed relatively rapidchanges in shape coupled with changes in membrane curva-ture. In addition to the cytoskeletal components, we alsopropose that there must be a high degree of plasticity of theinner and outer membranes and possibly the peptidoglycanlayer in order for these bacteria to achieve rapid changes inshape along the cell surface. At least in the case of theU-shaped cells and the cells that curve around carbon film,such changes may require rapid expansion of peptidoglycanin one region coupled with degradation or contraction inanother region. The bending occurs along the longitudinalaxis of the cell, consistent with atomic force microscopicmeasurements that show that the primary elasticity followsthe longitudinal axis in isolated sacculi from E. coli (37).Our findings indicate that the density profiles of inner andouter monolayers of the B. bacteriovorus outer membraneare comparable (see Fig. S1 in the supplemental material).This is in contrast to findings reported by workers in ourlaboratory and by other workers for the corresponding E.coli outer membrane, in which the outer monolayer hasbeen observed to be denser than the inner monolayer (22,39). This morphological difference suggests that there maybe biochemical differences between the membranes of B.bacteriovorus and the membranes of other bacteria. B. bac-teriovorus spheroplasts have been reported to be more re-sistant to osmotic shock than E. coli spheroplasts (35), themembranes are sensitive to detergent extraction (35), andcells are more susceptible to sonication or freeze-thaw treat-ment (1). Further, cells released from a bdelloplast haveunusual levels of peptidoglycan turnover (35), supportingthe idea that the B. bacteriovorus cell envelope may have aunique composition and/or that there may be unique regu-lation of its murein layer and its interactions with the innerand outer cell membranes. The electron tomographic stud-ies that we describe here provided a foundation for furtherunderstanding the structural origins of the unique biology ofB. bacteriovorus cells and for assessing the connections be-tween cellular shape, cytoskeletal organization, and mem-brane structure.

ACKNOWLEDGMENTS

We thank Elizabeth Sockett, University of Nottingham, Notting-ham, United Kingdom, for providing strain HD100 and Y. C. Wang ofFEI Company, Hillsboro, OR, for assistance with EELS analysis.

This work was supported by funds from the Center for CancerResearch, National Cancer Institute, National Institutes of Health, toJ.L.S.M. and S.S.

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three-dimensional imaging of the highly bent architecture of bdellovibrio bacteriovorus by using

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