Chemoreceptors in Caulobacter crescentus: Trimers of Receptor Dimers in a Partially Ordered Hexagonally Packed Array

Chemoreceptor arrays are macromolecular complexes that formextended assemblies primarily at the poles of bacterial cellsand mediate chemotaxis signal transduction, ultimately controllingcellular motility. We have used cryo-electron tomography todetermine the spatial distribution and molecular architectureof signaling molecules that comprise chemoreceptor arrays inwild-type Caulobacter crescentus cells. We demonstrate thatchemoreceptors are organized as trimers of receptor dimers,forming partially ordered hexagonally packed arrays of signalingcomplexes in the cytoplasmic membrane. This novel organizationat the threshold between order and disorder suggests how chemoreceptorsand associated molecules are arranged in signaling assembliesto respond dynamically in the activation and adaptation stepsof bacterial chemotaxis.

INTRODUCTION

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INTRODUCTION

Two key multiprotein complexes are essential for mediating bacterialchemotaxis: the chemosensory apparatus, which receives and transmitsenvironmental cues, and the motility system that mediates cellularmovement (23). Methyl-accepting chemotaxis proteins (MCPs),or chemoreceptors, form the basis of the signaling apparatusand direct cell movement by regulating the histidine autokinaseCheA, which ultimately modulates the direction of flagellumrotation. Chemoreceptors and CheA form a ternary complex withthe adaptor protein CheW, which together provide a signalingscaffold that plays a crucial role in signal processing in bacterialchemotaxis (12, 13, 27). In the dimorphic gram-negative Caulobactercrescentus, the chemosensory apparatus and sole flagellum arelocalized to a single pole of the highly motile swarmer celltype (2, 7), while both complexes are completely absent fromnonmotile stalked cells (1, 10, 19).

We previously identified chemoreceptor arrays at the polar regionof intact wild-type Escherichia coli and Bdellovibrio bacteriovoruscells by using low-dose cryo-electron microscopy (5, 27). Inthese organisms, arrays were characterized in an edge-on orientationby striations orthogonal to the cytoplasmic membrane that correspondedto chemoreceptors, and by a line of density near the cell poles,which was designated as the signaling scaffold composed of CheAand CheW. The presence of both chemoreceptors and CheA withinthe arrays was confirmed by immuno-electron microscopy (27).Here, we extend these studies to another gram-negative bacteriumand describe the partially ordered hexagonal arrangement ofchemotaxis signaling complexes at the flagellated pole in Caulobacterswarmer cells.

MATERIALS AND METHODS

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MATERIALS AND METHODS

Bacterial strains and culture conditions. C. crescentus strain CB15N was cultured in peptone-yeast extract(PYE) medium (0.2% Bacto peptone, 0.1% yeast extract, 0.03%MgSO₄7H₂O, and 0.007% CaCl₂·2H₂O) (8) at 30°C and250 rpm.

Specimen preparation, data collection, and image analysis. Chemoreceptor arrays were observed in over 65 individual Caulobacterswarmer cells that were separated from the stalked cell populationby using a modified synchronization protocol. Briefly, a sampleof late-log-phase (optical density at 600 nm of less than $~$ 0.6)Caulobacter culture was divided into aliquots in Eppendorf tubesand centrifuged at 8,000 rpm for $~$ 1 min. Supernatants were discarded,and the cells were washed twice in M2 salts medium, pelleted,and resuspended in $~$ 700 µl of fresh M2 medium, and an equalvolume of Percoll (Sigma-Aldrich Co., St. Louis, MO) was added.Samples were centrifuged for 20 min at 10,500 rpm, and cellsseparated into an upper stalked cell band, which was discarded,and a second swarmer cell band that was collected and washedtwice in fresh PYE medium to remove residual Percoll prior tosample preparation. From these enriched motile populations,three-dimensional tomographic volumes, or tomograms, were generatedfor 12 swarmer cells, each exhibiting a single chemoreceptorarray and single polar flagellum. Caulobacter cells (5 µl)at an optical density at 600 nm of $~$ 0.5 nm were withdrawn directlyfrom the PYE medium, mixed with 10- or 15-nm gold particles,and placed on glow-discharged Quantifoil MultiA grids (MicroTools GmbH, Germany). The grids were blotted and plunge-frozenin liquid ethane maintained at ca. –180°C. For two-dimensionalcryo-projection images and cryo-electron tomography, grids containingplunge-frozen cells were placed in cartridges and loaded intothe cryo-transfer system of a Polara G2 microscope (FEI Corp.,Oregon). The microscope was equipped with a field emission gunoperating at 300 kV, and a 2Kx2K charge-coupled device cameraat the end of a GIF 2000 (Gatan, Inc., California) energy filteringsystem. Typically, low-dose tomographic tilt series (0.45 to0.75 e^–/Å² per image) were collected over an angularrange of ±70 ° in 1° intervals by using a lineartilt scheme at effective magnifications of x18,000 (pixel size,7.5 Å) and underfocus values ranging from 4 to 6 µm.Fiducial marker-based alignments were performed on full-resolutionimages, and three-dimensional reconstructions computed by weightedback projection of aligned images that were binned 4x4. Alignmentsand reconstructions were performed using the IMOD (15) softwarepackage, both IMOD and the Amira software suite were used forsegmentations, and density maps were rendered in Chimera (18).

Alignment of C. crescentus and Escherichia coli C-terminal amino acid sequences. For each chemoreceptor sequence, the N-terminal amino acid residuesthat proceeded from the start of the HAMP domain (as determinedby domain assignments in the MiST database [22]) were deletedto facilitate the alignment of the C-terminal portions of eachchemoreceptor. The remaining cytoplasmic residues from eachchemoreceptor sequence were aligned, not only to illustratethe known sequence conservation between chemoreceptors of differentspecies but also to demonstrate that several Caulobacter chemoreceptorscontain a significantly increased number of C-terminal residues(see Fig. 2C, MCPs A, C, E, J, and N). Sequences were alignedby using the clustawl alignments in a CLC sequence viewer (CLCbio, Cambridge, MA).

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FIG. 2. Measurements of chemoreceptor arrays from Caulobacter cells. Chemoreceptor arrays were manually segmented from tomographic volumes of the polar regions of intact frozen-hydrated Caulobacter cells by using the Amira visualization software suite, and array dimensions were measured. (A) Schematic representation of the polar region of a Caulobacter cell demonstrating measurements of array length, width, and elliptical area (area = L/2 x W/2 x ${pi}$ ). (B) Schematic representation of a chemosensory array with chemoreceptors indicated in red and signaling molecules indicated in blue. The schematic shows the dimensions of the total array height (Ht), the periplasmic array height (Hp), and the cytoplasmic array height (Hc) that were measured from two-dimensional projection images and three-dimensional tomograms. (C) Sequence comparison of the cytoplasmic amino acids residues of chemoreceptors from E. coli (red) and Caulobacter (blue).

Tomographic averaging. Although the packing arrangements of molecules correspondingto the scaffolding portion of the chemoreceptor array were prominentlyobserved directly from tomographic slices, densities correspondingto chemoreceptors were less well ordered. Thus, to further probethe molecular architecture of Caulobacter chemoreceptor arrays,we combined three-dimensional alignment procedures similar tothose used in single particle analysis with image averagingmethods comparable to those used in two-dimensional electroncrystallography. From the tomographic volumes described above,1,200 subvolumes (50 x 50 x 150 in pixels; 7.5 Å/pixel)were extracted from the ordered regions of the Caulobacter tomogramsto create a stack of subvolumes. The subvolumes were classifiedby using the local-maximum clustering method (25), and the majorclusters were averaged to create an initial template for furtheralignment and averaging. The subvolumes were aligned againstthe initial template by using the grid-threading Monte Carlosearching algorithm (26). The aligned subvolumes were then averagedwith a weight factor depending on the correlation coefficientbetween a subvolume and the template:

Formula
Here, w(i) is the weight of subvolume i, and C_iis the correlation between it and the template. C and ${delta}$ C arethe average and the standard deviation of correlation coefficientsof all subvolumes, respectively. a and b are two parametersthat define the dependence of the weight on the correlationcoefficient distribution. In the present study we used a = 2and b = 0. This weighted average enhanced the contribution of"like" images while reducing the contribution of "nonlike" images.Using the averaged volumes as templates, further alignment andweighted averaging were performed until no significant changewas observed in the alignments and average results. The atomicmodels of trimeric E. coli chemoreceptors was adapted from dimericreceptor models (14, 24) and were manually placed into the densitymap and locally fit using the Chimera (18) software package.

RESULTS AND DISCUSSION

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RESULTS AND DISCUSSION

Receptor arrays were observed at the flagellated pole in bothedge-on (Fig. 1A) and face-on (Fig. 1B) orientations (also seethe movies in the supplemental material). Measurements fromtomographic volumes of Caulobacter cells (see Fig. 2A and Bfor nomenclature) revealed that chemoreceptor arrays were generallycircular or elliptical in shape, with mean dimensions of 170.5± 25.2 nm in length, along the longitudinal axis of thecell, and 96.0 ± 28.8 nm wide, corresponding to an averageelliptical area of 13,087.2 ± 4,618.8 nm2 (n = 12). Therelative consistency of Caulobacter array size is in markedcontrast to the larger variability we have observed in E. coli(27). In all cells examined, arrays were positioned on the convexside of the cell pole, and the spatial relationship betweenthe chemoreceptor array and the single polar flagellum, as measuredfrom the flagellum rotors to the approximate center of the chemoreceptorarrays, was relatively constant, with a mean distance of 290.8± 36.5 nm. The overall height of the arrays measuredfrom the chemoreceptor density observed in the periplasm tothe cytoplasmic edge of the signaling scaffold was 46.7 ±0.9 nm, suggesting that chemoreceptors in Caulobacter are longerthan those in E. coli (24). This increase is consistent withsequence comparisons of chemoreceptors from both organisms,which indicate that a significant proportion of Caulobacterchemoreceptors contain an increased number of amino acid residuesin their C-terminal region than those found in E. coli (Fig.2C). Arrays imaged in the edge-on orientation also displayeda second layer of density ca. 8 to 10 nm below the cytoplasmicmembrane (Fig. 1A, inset), which roughly corresponds to thepredicted interaction sites for CheR and CheB, enzymes involvedin regulating chemoreceptor methylation (12).

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FIG. 1. Direct visualization of Caulobacter chemoreceptor arrays. (A) Tomographic slice ( $~$ 5 nm) generated from the polar region of an intact Caulobacter swarmer cell demonstrating a continuous surface layer (SL), outer membrane (OM), cytoplasmic membrane (CM), and cytoplasmic filaments (F). (Inset) An expanded view of the array in an edge-on orientation highlighting the chemoreceptors and signaling scaffold (white arrowhead) and additional cytoplasmic density below the cytoplasmic membrane at the proposed CheR/B interaction sites (black arrowhead). (B) In the face-on orientation, chemoreceptor arrays are characterized by a distinct, partially ordered pattern $~$ 32 nm below the cytoplasmic membrane that corresponds to the signaling scaffold. (Inset) The power spectrum of this region clearly demonstrates the $~$ 12-nm hexagonal spacing. The scale bars in panels A and B are 100 nm; the panel A inset scale bar is 50 nm.

When imaged in the face-on orientation, Caulobacter chemoreceptorarrays displayed an approximately hexagonal packing arrangementwithin the elliptical boundaries of the array (Fig. 1B), withan apparent periodicity of $~$ 12 nm (Fig. 1B, inset). Using thedensities at the vertex of each lattice point, we carried outthree-dimensional averaging from this single cell tomogram todetermine the structure of the repeating signaling unit andthe general architecture of the Caulobacter chemoreceptor array.The averaged density of the idealized hexagonal packing allowedvisualization of the density contributions of individual repeatingunits in top and vertical sectional views (Fig. 3A and B). Atrimer of receptor dimers was modeled easily into each repeatingunit (see Materials and Methods). In this packing arrangement,six chemoreceptor trimers (representing 18 individual dimers)were arranged $~$ 7.5 nm apart and, based on this organization,we estimate that an average Caulobacter chemoreceptor arraywould contain $~$ 490 trimers, or $~$ 1470 chemoreceptor homodimers,fewer than the estimated number of receptors in an average E.coli cell (12, 16, 27). Cross-sectional slices at differentheights in the density map (as indicated in Fig. 3B) indicatethat interactions may occur between sets of trimers at differentpoints along the cytoplasmic region of the chemoreceptors (Fig.3C and D), suggesting that interactions at the trimer-trimerinterface may also be involved in the formation of the hexagonalunits. Interestingly, a cross-sectional view at a height correspondingto the cytoplasmic end of the chemoreceptor array (Fig. 3E)demonstrated additional density at every alternate lattice point,corresponding to the likely position of the scaffold of signalingmolecules (CheA and CheW) associated with the receptors. Thedensity map is not at a resolution sufficient to determine theorientation or stoichiometery of these signaling molecules,and therefore this density is shown schematically (Fig. 3B and E,gold circles). Based on a molecular interpretation where theextra density was assigned to signaling molecules, the architectureof the hexagonal chemoreceptor array unit resulted in an averageof three CheA dimers for six "trimers-of-dimers" for a perfectand fully occupied lattice. This is different from the biochemicallydetermined stoichiometries for the E. coli chemotaxis receptorassembly, where one receptor trimer-of-dimers is thought tointeract with one CheA dimer and two CheW molecules (16). Wespeculate that one possible reason for this difference may bethat different numbers of signaling proteins may be involvedin array formation; the Caulobacter genome codes for 18 MCPs,2 CheAs and 3 CheWs, versus the 4 MCPs and single copies ofCheA and CheW in E. coli. Our findings also strongly supportthe growing numbers of in vivo (3, 20, 21) and in vitro (4)experiments, which suggest that trimers of chemoreceptor dimers,along with CheA and CheW, form a core signaling unit. Althoughsymmetrization and averaging of the density map allowed forthe determination of the overall packing of the chemoreceptorarray, a closer inspection of the raw density map revealed thelocal disorder in array packing (Fig. 4A). Specifically, thedensity maps corresponding to hexagonal units were observedto be discontinuous, occasionally missing vertices and variableintertrimer spacing (Fig. 4B). Thus, our studies demonstratethat within the context of an approximate hexagonal lattice,chemoreceptors display local disorder.

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FIG. 3. Averaged packing arrangement of trimeric receptors in Caulobacter chemoreceptor arrays. (A) Averaged density map of a chemoreceptor array demonstrating idealized hexagonal packing, with trimeric chemoreceptors model structures (cyan and magenta) docked into a single hexagonal unit. (B) Edge-on perspective of the array showing one-half of the hexagonal unit described in panel A, with continuous receptor densities emanating from the signaling scaffold. The gold circles represent the extra density attributed to molecules within the signaling scaffold and have been placed surrounding the single hexagonal unit in which chemoreceptor trimer models have been docked. The three white dashed lines represent the heights at which cross-sectional cuts were made in the density map to obtain panels C through E, from top to bottom, respectively. (C and D) Cross-sectional views through the array at the receptor level demonstrate the trimer-of-dimer organization of chemoreceptors and suggest multiple interactions between adjacent trimers. (E) Cross-sectional view from below the level of chemoreceptor shows the extra density directly below every other receptor trimer (magenta). The scale bars in panels A and B are 20 and 10 nm, respectively, and the scale bars in panels C to E are 5 nm.

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FIG. 4. Partially ordered organization of chemoreceptor trimers-of-dimers in Caulobacter cells. (A) Tomographic slice ( $~$ 2.5 nm) of a subregion of the same chemoreceptor array presented in Fig. 1B. (B) Schematic representation of the in-plane clustering of the signaling scaffold and (C) model derived from a subset of the tomographic data in panels A and B demonstrating the organization of chemoreceptors trimers-of-dimers that combine with signaling molecules to form a quasihexagonal signaling array in the cytoplasmic membrane. The scale bars in panels A and B are 50 nm.

Quantitative models, based primarily on experimental responsemeasurements derived from E. coli cells, describe the high-performancefeatures of bacterial chemotaxis and predict that chemoreceptorsform allosteric arrays with as many as several dozen signalingunits acting cooperatively via clustered neighborhoods of stronglycoupled receptors (9, 11, 12, 17). The model we present herefor native Caulobacter chemoreceptor arrays confirms this expectationand demonstrates how chemoreceptors can achieve local clusteringwhile positioned in the context of a partially ordered, hexagonallypacked lattice (Fig. 4C). We propose that this disorder allowsfor a dynamic organization of the array, which potentially accommodatesdiversity in chemoreceptor scaffold architecture. The loosepacking, taken together with the $~$ 9-nm-wide central openingsin the hexagonal unit, may also provide a mechanism for theaccess of cytoplasmic enzymes such as CheR and CheB that arenecessary for chemoreceptor modification.

A recent study by Briegel et al. described the organizationof Caulobacter chemoreceptor arrays as an ordered and continuoushexagonal lattice, based on the analysis of tomograms generatedfrom edge-on views and with imposed hexagonal symmetry (6).In contrast, our results from both edge-on and face-on tomogramsshow that intrinsic disorder exists in the packing of chemoreceptorswithin the array (Fig. 4), which is averaged out when hexagonalsymmetry is enforced (Fig. 3). The lack of continuous orderis likely to be critical for chemotaxis signaling, since thistype of flexibility may allow chemoreceptor arrays to uniquelyadapt to changing chemical environments and might provide amechanism for the incorporation of newly synthesized array componentswhile maintaining the overall hexagonal packing arrangement.Our tomographic studies thus provide a new understanding ofboth the general architecture of Caulobacter chemoreceptor arraysand insights into the specific hexagonal units that comprisethe array. The essence of the structural organization of thiselegant macromolecular sensory apparatus appears to be a lateralorganizational scheme that maintains order on a global scalecoupled with local variability to facilitate fine-tuning ofthe chemotaxis response.

ACKNOWLEDGMENTS

We thank Z. Gitai for kindly providing C. crescentus strainCB15N and helpful advice, E. Sockett for insightful comments,and E. Tyler and A. Hoofring for expert assistance with preparationof the figures.

This study was supported by funds from the intramural programof the National Cancer Institute to S.S.

C.M.K. and S.S. designed the experiments; C.M.K. and X.W. performedthe experiments; C.M.K., X.W., and S.S. analyzed the data; andC.M.K. and S.S. wrote the manuscript.

FOOTNOTES

* Corresponding author. Mailing address: Laboratory of Cell Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bldg. 50, Rm. 4306, Bethesda, MD 20892. Phone: (301) 594-2062. Fax: (301) 480-3834. E-mail: ss1{at}nih.gov

Published ahead of print on 8 August 2008.

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

REFERENCES

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