Uncharacterized Bacterial Structures Revealed by Electron Cryotomography.
ABSTRACT: Electron cryotomography (ECT) can reveal the native structure and arrangement of macromolecular complexes inside intact cells. This technique has greatly advanced our understanding of the ultrastructure of bacterial cells. We now view bacteria as structurally complex assemblies of macromolecular machines rather than as undifferentiated bags of enzymes. To date, our group has applied ECT to nearly 90 different bacterial species, collecting more than 15,000 cryotomograms. In addition to known structures, we have observed, to our knowledge, several uncharacterized features in these tomograms. Some are completely novel structures; others expand the features or species range of known structure types. Here, we present a survey of these uncharacterized bacterial structures in the hopes of accelerating their identification and study, and furthering our understanding of the structural complexity of bacterial cells.IMPORTANCE Bacteria are more structurally complex than is commonly appreciated. Here we present a survey of previously uncharacterized structures that we observed in bacterial cells by electron cryotomography, structures that will initiate new lines of research investigating their identities and roles.
Project description:Bacterial nanowires have garnered recent interest as a proposed extracellular electron transfer (EET) pathway that links the bacterial electron transport chain to solid-phase electron acceptors away from the cell. Recent studies showed that Shewanella oneidensis MR-1 produces outer membrane (OM) and periplasmic extensions that contain EET components and hinted at their possible role as bacterial nanowires. However, their fine structure and distribution of cytochrome electron carriers under native conditions remained unclear, making it difficult to evaluate the potential electron transport (ET) mechanism along OM extensions. Here, we report high-resolution images of S. oneidensis OM extensions, using electron cryotomography (ECT). We developed a robust method for fluorescence light microscopy imaging of OM extension growth on electron microscopy grids and used correlative light and electron microscopy to identify and image the same structures by ECT. Our results reveal that S. oneidensis OM extensions are dynamic chains of interconnected outer membrane vesicles (OMVs) with variable dimensions, curvature, and extent of tubulation. Junction densities that potentially stabilize OMV chains are seen between neighboring vesicles in cryotomograms. By comparing wild type and a cytochrome gene deletion mutant, our ECT results provide the likely positions and packing of periplasmic and outer membrane proteins consistent with cytochromes. Based on the observed cytochrome packing density, we propose a plausible ET path along the OM extensions involving a combination of direct hopping and cytochrome diffusion. A mean-field calculation, informed by the observed ECT cytochrome density, supports this proposal by revealing ET rates on par with a fully packed cytochrome network.
Project description:Electron cryotomography (ECT) is an emerging technology that allows thin samples such as small bacterial cells to be imaged in 3D in a nearly native state to 'molecular' (approximately 4nm) resolution. As such, ECT is beginning to deliver long-awaited insight into the positions and structures of cytoskeletal filaments, cell wall elements, motility machines, chemoreceptor arrays, internal compartments, and other ultrastructures. Here we briefly explain ECT, review its recent contributions to microbiology, and conclude with a discussion of future prospects.
Project description:BACKGROUND:Cells are powered by a large set of macromolecular complexes, which work together in a crowded environment. The in situ mechanisms of these complexes are unclear because their 3D distribution, organization, and interactions are largely unknown. Electron cryotomography (cryo-ET) can address these knowledge gaps because it produces cryotomograms-3D images that reveal biological structure at ?4-nm resolution. Cryo-ET uses no fixation, dehydration, staining, or plastic embedment, so cellular features are visualized in a life-like, frozen-hydrated state. To study chromatin and mitotic machinery in situ, we subjected yeast cells to genetic and chemical perturbations, cryosectioned them, and then imaged the cells by cryo-ET. FINDINGS:Here we share >1,000 cryo-ET raw datasets of cryosectioned budding yeast Saccharomyces cerevisiaecollected as part of previously published studies. These data will be valuable to cell biologists who are interested in the nanoscale organization of yeasts and of eukaryotic cells in general. All the unpublished tilt series and a subset of corresponding cryotomograms have been deposited in the EMPIAR resource for the community to use freely. To improve tilt series discoverability, we have uploaded metadata and preliminary notes to publicly accessible Google Sheets, EMPIAR, and GigaDB. CONCLUSIONS:Cellular cryo-ET data can be mined to obtain new cell-biological, structural, and 3D statistical insights in situ. These data contain structures not visible in traditional electron-microscopy data. Template matching and subtomogram averaging of known macromolecular complexes can reveal their 3D distributions and low-resolution structures. Furthermore, these data can serve as testbeds for high-throughput image-analysis pipelines, as training sets for feature-recognition software, for feasibility analysis when planning new structural-cell-biology projects, and as practice data for students.
Project description:Electron cryotomography (ECT) enables intact cells to be visualized in 3D in an essentially native state to 'macromolecular' (?4 nm) resolution, revealing the basic architectures of complete nanomachines and their arrangements in situ. Since its inception, ECT has advanced our understanding of many aspects of prokaryotic cell biology, from morphogenesis to subcellular compartmentalization and from metabolism to complex interspecies interactions. In this Review, we highlight how ECT has provided structural and mechanistic insights into the physiology of bacteria and archaea and discuss prospects for the future.
Project description:While electron cryotomography (ECT) provides "molecular" resolution, three-dimensional images of unique biological specimens, sample crowdedness, and/or resolution limitations can make it difficult to identify specific macromolecular components. Here we used a 1.4 nm Nanogold cluster specifically attached to the Fc fragment of IgG to monitor its interaction with the neonatal Fc receptor (FcRn), a membrane-bound receptor that transports IgG across cells in acidic intracellular vesicles. ECT was used to image complexes formed by Nanogold-labeled Fc bound to FcRn attached to the outer surface of synthetic liposomes. In the resulting three-dimensional reconstructions, 1.4 nm Nanogold particles were distributed predominantly along the interfaces where 2:1 FcRn-Fc complexes bridged adjacent lipid bilayers. These results demonstrate that the 1.4 nm Nanogold cluster is visible in tomograms of typically thick samples (approximately 250 nm) recorded with defocuses appropriate for large macromolecules and is thus an effective marker.
Project description:Type IV secretion systems (T4SSs) are large macromolecular machines that translocate protein and DNA and are involved in the pathogenesis of multiple human diseases. Here, using electron cryotomography (ECT), we report the in situ structure of the Dot/Icm type IVB secretion system (T4BSS) utilized by the human pathogen Legionella pneumophila This is the first structure of a type IVB secretion system, and also the first structure of any T4SS in situ While the Dot/Icm system shares almost no sequence similarity with type IVA secretion systems (T4ASSs), its overall structure is seen here to be remarkably similar to previously reported T4ASS structures (those encoded by the R388 plasmid in Escherichia coli and the cag pathogenicity island in Helicobacter pylori). This structural similarity suggests shared aspects of mechanism. However, compared to the negative-stain reconstruction of the purified T4ASS from the R388 plasmid, the L. pneumophila Dot/Icm system is approximately twice as long and wide and exhibits several additional large densities, reflecting type-specific elaborations and potentially better structural preservation in situ.
Project description:The endosomal-sorting complex required for transport (ESCRT) is evolutionarily conserved from Archaea to eukaryotes. The complex drives membrane scission events in a range of processes, including cytokinesis in Metazoa and some Archaea. CdvA is the protein in Archaea that recruits ESCRT-III to the membrane. Using electron cryotomography (ECT), we find that CdvA polymerizes into helical filaments wrapped around liposomes. ESCRT-III proteins are responsible for the cinching of membranes and have been shown to assemble into helical tubes in vitro, but here we show that they also can form nested tubes and nested cones, which reveal surprisingly numerous and versatile contacts. To observe the ESCRT-CdvA complex in a physiological context, we used ECT to image the archaeon Sulfolobus acidocaldarius and observed a distinct protein belt at the leading edge of constriction furrows in dividing cells. The known dimensions of ESCRT-III proteins constrain their possible orientations within each of these structures and point to the involvement of spiraling filaments in membrane scission.
Project description:How rod-shaped bacteria form and maintain their shape is an important question in bacterial cell biology. Results from fluorescent light microscopy have led many to believe that the actin homolog MreB and a number of other proteins form long helical filaments along the inner membrane of the cell. Here we show using electron cryotomography of six different rod-shaped bacterial species, at macromolecular resolution, that no long (> 80 nm) helical filaments exist near or along either surface of the inner membrane. We also use correlated cryo-fluorescent light microscopy (cryo-fLM) and electron cryo-tomography (ECT) to identify cytoplasmic bundles of MreB, showing that MreB filaments are detectable by ECT. In light of these results, the structure and function of MreB must be reconsidered: instead of acting as a large, rigid scaffold that localizes cell-wall synthetic machinery, moving MreB complexes may apply tension to growing peptidoglycan strands to ensure their orderly, linear insertion.
Project description:Chlamydiae comprise important pathogenic and symbiotic bacteria that alternate between morphologically and physiologically different life stages during their developmental cycle. Using electron cryotomography, we characterize the ultrastructure of the developmental stages of three environmental chlamydiae: Parachlamydia acanthamoebae, Protochlamydia amoebophila and Simkania negevensis. We show that chemical fixation and dehydration alter the cell shape of Parachlamydia and that the crescent body is not a developmental stage, but an artefact of conventional electron microscopy. We further reveal type III secretion systems of environmental chlamydiae at macromolecular resolution and find support for a chlamydial needle-tip protein. Imaging bacteria inside their host cells by cryotomography for the first time, we observe marked differences in inclusion morphology and development as well as host organelle recruitment between the three chlamydial organisms, with Simkania inclusions being tightly enveloped by the host endoplasmic reticulum. The study demonstrates the power of electron cryotomography to reveal structural details of bacteria-host interactions that are not accessible using traditional methods.
Project description:Members of the Gcn5-related N-acetyltransferase (GNAT) superfamily catalyze the acetylation of a wide range of small molecule and protein substrates. Due to their abundance in all kingdoms of life and diversity of their functions, they are implicated in many aspects of eukaryotic and prokaryotic physiology. Although numerous GNATs have been identified thus far, many remain structurally and functionally uncharacterized. The elucidation of their structures and functions is critical for broadening our knowledge of this diverse and important superfamily. In this work, we present the structural and kinetic analyses of two previously uncharacterized bacterial acetyltransferases - SACOL1063 from Staphylococcus aureus strain COL and CD1211 from Clostridium difficile strain 630. Our structures of SACOL1063 show substantial flexibility of a loop that is likely responsible for substrate recognition and binding compared to structures of other homologs. In the CoA complex structure, we found two CoA molecules bound in both the canonical AcCoA/CoA-binding site and the acceptor-substrate-binding site. Our work also provides initial clues regarding the substrate specificity of these two enzymes; however, their native function(s) remain unknown. We found both proteins act as N- rather than O-acetyltransferases and preferentially acetylate l-threonine. The combination of structural and kinetic analyses of these two previously uncharacterized GNATs provides fundamental knowledge and a framework on which future studies can be built to elucidate their native functions.