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

Abstract

Chemoreceptor arrays are macromolecular complexes that form extended assemblies primarily at the poles of bacterial cells and mediate chemotaxis signal transduction, ultimately controlling cellular motility. We have used cryo-electron tomography to determine the spatial distribution and molecular architecture of signaling molecules that comprise chemoreceptor arrays in wild-type Caulobacter crescentus cells. We demonstrate that chemoreceptors are organized as trimers of receptor dimers, forming partially ordered hexagonally packed arrays of signaling complexes in the cytoplasmic membrane. This novel organization at the threshold between order and disorder suggests how chemoreceptors and associated molecules are arranged in signaling assemblies to respond dynamically in the activation and adaptation steps of bacterial chemotaxis.Two key multiprotein complexes are essential for mediating bacterial chemotaxis: the chemosensory apparatus, which receives and transmits environmental cues, and the motility system that mediates cellular movement (23). Methyl-accepting chemotaxis proteins (MCPs), or chemoreceptors, form the basis of the signaling apparatus and direct cell movement by regulating the histidine autokinase CheA, which ultimately modulates the direction of flagellum rotation. Chemoreceptors and CheA form a ternary complex with the adaptor protein CheW, which together provide a signaling scaffold that plays a crucial role in signal processing in bacterial chemotaxis (12,13,27). In the dimorphic gram-negative Caulobacter crescentus, the chemosensory apparatus and sole flagellum are localized to a single pole of the highly motile swarmer cell type (2, 7), while both complexes are completely absent from nonmotile stalked cells (1,10,19).We previously identified chemoreceptor arrays at the polar region of intact wild-type Escherichia coli and Bdellovibrio bacteriovorus cells by using low-dose cryo-electron microscopy (5, 27). In these organisms, arrays were characterized in an edge-on orientation by striations orthogonal to the cytoplasmic membrane that corresponded to chemoreceptors, and by a line of density near the cell poles, which was designated as the signaling scaffold composed of CheA and CheW. The presence of both chemoreceptors and CheA within the arrays was confirmed by immuno-electron microscopy (27). Here, we extend these studies to another gram-negative bacterium and describe the partially ordered hexagonal arrangement of chemotaxis signaling complexes at the flagellated pole in Caulobacter swarmer cells. MATERIALS AND METHODSBacterial 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% MgSO 4 ⅐ 7H 2 O, and 0.007% CaCl 2 ⅐ 2H 2 O) (8) at 30°C and 250 rpm. Specimen preparation, data collection, and image analysis. Chemoreceptor arrays were observed in over 65 individual Caulobacter swarmer cells that were separated from the stalked cell population by using a modified synchronization protocol. Brief...

Universal architecture of bacterial chemoreceptor arrays

“…These observations support the notion that the major architectural contacts occur near the signaling sub-domain of chemoreceptors (4). Although the basic arrangement of all of the arrays was clearly hexagonal, none of the arrays were perfectly regular, supporting the idea that the degree of local order could reflect activation and/or regulation (22). The size of the arrays, and thus the estimated number of receptors, varied by an order of magnitude (from Ϸ1,200 in M. magneticum to Ϸ14,400 in C. jejuni; Table 1), without obvious correlation to the cell size or bacterial taxonomy.…”

“…This structure, combined with pulsed ESR and crystallographic studies of a CheA-CheW dimer, led to a third ''hedgerows of dimers'' model for the architecture of chemoreceptor arrays (20). Finally, through direct imaging of intact Caulobacter crescentus cells, we (21) and others (22) showed that the chemoreceptors in that organism are arranged in a hexagonal lattice whose 12-nm spacing suggested that trimers of receptor dimers occupied each threefold symmetric vertex. Whereas the MCPs of E. coli and 16 of the 18 MCPs of C. crescentus belong to the same signaling domain class (36H), those from T. maritima belong to a different class (44H) (3).…”

Molecular architecture of chemoreceptor arrays revealed by cryoelectron tomography of Escherichia coli minicells

“…2) of receptor arrays was determined by subvolume analysis. The structure is similar in overall appearance to the structures reported for two known receptor arrays (25,26), yet it reveals crucial previously undescribed details. In a transverse section parallel to the cytoplasmic membrane, our image shows a hexagonal lattice with 13.2-nm spacing ( Fig.…”

“…The interactions of some of the key components have been characterized by intensive studies using X-ray crystallography, NMR, ESR spectroscopy, chemical interaction mapping, and disulfide cross-linking (11,16,(21)(22)(23)(24). Cryoelectron tomography (cryo-ET) has revealed the most detailed view of intact bacterial receptor arrays currently available (3,(25)(26)(27). The 12-nm hexagonal lattice demonstrated by these studies suggests that it is probably a universally conserved pattern (3).…”

“…The 12-nm hexagonal lattice demonstrated by these studies suggests that it is probably a universally conserved pattern (3). The averaged density of the arrays in Caulobacter cells suggests that six MCP trimers are arranged 7.5 nm apart in a hexagonally packed lattice (26). Cryo-ET was also used to visualize the receptor arrays in E. coli (27).…”