Chemoreceptor arrays are supramolecular transmembrane machines of unknown structure that allow bacteria to sense their surroundings and respond by chemotaxis. We have combined X-ray crystallography of purified proteins with electron cryotomography of native arrays inside cells to reveal the arrangement of the component transmembrane receptors, histidine kinases (CheA) and CheW coupling proteins. Trimers of receptor dimers lie at the vertices of a hexagonal lattice in a "two-facing-two" configuration surrounding a ring of alternating CheA regulatory domains (P5) and CheW couplers. Whereas the CheA kinase domains (P4) project downward below the ring, the CheA dimerization domains (P3) link neighboring rings to form an extended, stable array. This highly interconnected protein architecture underlies the remarkable sensitivity and cooperative nature of transmembrane signaling in bacterial chemotaxis.protein structure | hybrid methods | two-component systems C hemotactic bacteria sense their surrounding conditions through an array of transmembrane chemoreceptors (methylaccepting chemotaxis proteins, or MCPs), which are found with histidine kinases (CheA) and couplers (CheW) in polar clusters (1-3) and along the sides of cells (4, 5). Repellents and attractants bind to the periplasmic domains of the MCPs either directly (6, 7) or via periplasmic binding proteins (8). The status of the binding domain is transmitted along the length of the receptors through the transmembrane region, across one or more HAMP (histidine kinases, adenyl cyclases, MCPs, and some phosphatases) domain (s), and down the coiled-coil cytoplasmic signaling domain where they ultimately regulate the activity of the histidine kinase CheA located at the receptors' cytoplasmic tips (1-3, 9). CheA is a large, five-domain (P1-P5) protein. P1 contains the substrate histidine, P2 is the docking site for the response regulator CheY, P3 is the dimerization domain, P4 binds ATP and is the kinase, and P5 binds CheW. P1, P2, and P3 are connected to each other by flexible linkers (1, 2). Crystal structures of all domains from Thermotoga maritima CheA are already available (10-13).In the model system Escherichia coli, the addition of attractants or removal of repellents results in kinase inactivation, causing the flagella to rotate counterclockwise. In that case, the multiple flagella form one large bundle that propels the cells smoothly forward and the cells "run." In contrast, addition of repellents or removal of attractants activates CheA, which autophosphorylates and then transfers the phosphoryl group to the second messenger CheY, which in turn binds to the flagellar motors and changes the direction of flagellar rotation to clockwise (CW). This switch results in disassembly of the flagellar bundle and causes the cells to "tumble" (14). CheA also regulates the activity of the receptor-modifying enzyme CheB (a methylesterase), which together with CheR (a methyltransferase) controls the methylation state of residues in the MCP adaptation region (1). Methylation...

SUMMARY Cell polarization is an integral part of many unrelated bacterial processes. How intrinsic cell polarization is achieved is poorly understood. Here, we provide evidence that Caulobacter crescentus uses a multimeric pole-organizing factor (PopZ) that serves as a hub to concurrently achieve several polarizing functions. During chromosome segregation, polar PopZ captures the ParB•ori complex and thereby anchors sister chromosomes at opposite poles. This step is essential for stabilizing bipolar gradients of a cell division inhibitor and setting up division near midcell. PopZ also affects polar stalk morphogenesis and mediates the polar localization of the morphogenetic and cell cycle signaling proteins CckA and DivJ. Polar accumulation of PopZ, which is central to its polarizing activity, can be achieved independently of division and does not appear to be dictated by the pole curvature. Instead, evidence suggests that localization of PopZ largely relies on PopZ multimerization in chromosome-free regions, consistent with a self-organizing mechanism.

Chemoreceptors are key components of the high-performance signal transduction system that controls bacterial chemotaxis. Chemoreceptors are typically localized in a cluster at the cell pole, where interactions among the receptors in the cluster are thought to contribute to the high sensitivity, wide dynamic range, and precise adaptation of the signaling system. Previous structural and genomic studies have produced conflicting models, however, for the arrangement of the chemoreceptors in the clusters. Using whole-cell electron cryo-tomography, here we show that chemoreceptors of different classes and in many different species representing several major bacterial phyla are all arranged into a highly conserved, 12-nm hexagonal array consistent with the proposed ''trimer of dimers'' organization. The various observed lengths of the receptors confirm current models for the methylation, flexible bundle, signaling, and linker sub-domains in vivo. Our results suggest that the basic mechanism and function of receptor clustering is universal among bacterial species and was thus conserved during evolution.bacterial ultrastructure ͉ chemotaxis ͉ electron cryo-tomography

Filament-forming cytoskeletal proteins are key organizers of all cells. Bacterial homologs of the major eukaryotic cytoskeletal families have now been discovered, but studies suggest that yet more cytoskeletal proteins remain to be identified. Here we demonstrate that the metabolic enzyme CTP Synthase (CtpS) forms filaments in Caulobacter crescentus. These filaments are bifunctional and regulate Caulobacter curvature independently of CtpS catalytic activity. The morphogenic role of CtpS requires its functional interaction with the intermediate filament crescentin. Interestingly, the E. coli CtpS homolog also forms filaments both in vivo and in vitro, suggesting that CtpS polymerization may be widely conserved. E. coli CtpS can replace the enzymatic and morphogenic functions of Caulobacter CtpS, indicating that Caulobacter has adapted a conserved filament-forming protein for a secondary role. These results implicate CtpS as a novel bifunctional member of the bacterial cytoskeleton and suggest that localization and polymerization may be important properties of metabolic enzymes.