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

The bacterial flagellum is one of nature's most amazing and well-studied nanomachines. Its cell-wall-anchored motor uses chemical energy to rotate a microns-long filament and propel the bacterium towards nutrients and away from toxins. While much is known about flagellar motors from certain model organisms, their diversity across the bacterial kingdom is less well characterized, allowing the occasional misrepresentation of the motor as an invariant, ideal machine. Here, we present an electron cryotomographical survey of flagellar motor architectures throughout the Bacteria. While a conserved structural core was observed in all 11 bacteria imaged, surprisingly novel and divergent structures as well as different symmetries were observed surrounding the core. Correlating the motor structures with the presence and absence of particular motor genes in each organism suggested the locations of five proteins involved in the export apparatus including FliI, whose position below the C-ring was confirmed by imaging a deletion strain. The combination of conserved and specially-adapted structures seen here sheds light on how this complex protein nanomachine has evolved to meet the needs of different species.

The stress-bearing component of the bacterial cell wall-a multigigadalton bag-like molecule called the sacculus-is synthesized from peptidoglycan. Whereas the chemical composition and the 3-dimensional structure of the peptidoglycan subunit (in at least one conformation) are known, the architecture of the assembled sacculus is not. Four decades' worth of biochemical and electron microscopy experiments have resulted in two leading 3-D peptidoglycan models: ''Layered'' and ''Scaffold'', in which the glycan strands are parallel and perpendicular to the cell surface, respectively. Here we resolved the basic architecture of purified, frozenhydrated sacculi through electron cryotomography. In the Gramnegative sacculus, a single layer of glycans lie parallel to the cell surface, roughly perpendicular to the long axis of the cell, encircling the cell in a disorganized hoop-like fashion.cell wall ͉ Cryo-EM ͉ sacculus ͉ tomography ͉ cell shape T he organization of peptidoglycan within the sacculus is a longstanding question in microbiology. Although the sacculus completely envelopes the cell, protecting it from osmotic and mechanical lysis, it is also porous, and thus sieves nutrients and small proteins. Sacculi are constantly remodeled, but they are robust enough that they can be purified from cells and still retain their original shape. Early electron microscope images of purified sacculi were difficult to interpret because the images were 2-D projections through heavy metal-stained sacculi (1, 2). Nevertheless the slender appearance of peptidoglycan in thinsectioned cells (3) and the gaps perpendicular to the polar axis produced by a peptidoglycan-specific endopeptidase (4) lead to the (Circumferential) Layered model (Fig. 1A ) in which the glycan strands lie in the plane of the cell wall and wrap around the cell. The alternative Scaffold model (5) (Fig. 1B) was proposed to fit biochemical data such as pore size and crosslinkage. Various arguments have been made in favor of (6) and against (7) the Scaffold model, but the debate intensified after a recent NMR structure of a synthetic peptidoglycan subunit dimer was interpreted to support the Scaffold model (8). To gain further insight into this issue, we imaged unstained sacculi in 3-D by electron cryotomography (9). Results and DiscussionElectron Cryotomography of Purified Sacculi. Sacculi isolated from C. crescentus strain CB15N and E. coli strains MG1655 and XL-10 were vitrified across holey carbon grids in thin (Ͻ 100 nm) ice. Whereas the gossamer sacculi were barely discernable in the low-dose, 2-D projection images, they were clearly visible in the 3-D tomograms and resembled flattened cells [ Fig. 2 and supporting information (SI) Movie S1 and Movie S2]. In stark contrast to intact cells, the sacculi were flexible and in some positions had radii of curvature less than 20 nm, highlighting how cell shape is not determined by the ''rigidity'' of the sacculus, but rather the cytosolic turgor (osmotic pressure) pushing the cytoplasmic membrane outward against the sacc...

Electron cryotomography (ECT) produces three-dimensional images of cells in a near-native state at macromolecular resolution, but identifying structures of interest can be challenging. Here we describe a correlated "cryo-PALM"-ECT method for localizing objects within cryotomograms to beyond the diffraction limit of the light microscope, and use it to identify multiple and new conformations of the dynamic type VI secretion system in the crowded interior of Myxococcus xanthus.