Bdellovibrio predation cycle characterized at nanometre-scale resolution with cryo-electron tomography.

Bdellovibrio bacteriovorus is a microbial predator that offers promise as a living antibiotic for its ability to kill Gram-negative bacteria, including human pathogens. Even after six decades of study, fundamental details of its predation cycle remain mysterious. Here we used cryo-electron tomography to comprehensively image the lifecycle of B.

Discovery of a Novel Inner Membrane-Associated Bacterial Structure Related to the Flagellar Type III Secretion System.

The bacterial flagellar type III secretion system (fT3SS) is a suite of membrane-embedded and cytoplasmic proteins responsible for building the flagellar motility machinery. Homologous nonflagellar (NF-T3SS) proteins form the injectisome machinery that bacteria use to deliver effector proteins into eukaryotic cells, and other family members were recently reported to be involved in the formation of membrane nanotubes. Here, we describe a novel, evolutionarily widespread, hat-shaped structure embedded in the inner membranes of bacteria, of yet-unidentified function, that is present in species containing fT3SS.

Novel transient cytoplasmic rings stabilize assembling bacterial flagellar motors.

The process by which bacterial cells build their intricate flagellar motility apparatuses has long fascinated scientists. Our understanding of this process comes mainly from studies of purified flagella from two species, Escherichia coli and Salmonella enterica. Here, we used electron cryo-tomography (cryo-ET) to image the assembly of the flagellar motor in situ in diverse Proteobacteria: Hylemonella gracilis, Helicobacter pylori, Campylobacter jejuni, Pseudomonas aeruginosa, Pseudomonas fluorescens, and Shewanella oneidensis.

: an Interactive Open-Access Microbiology Textbook.

Here, we describe a new open-access digital textbook for microbiology, (available at cellstructureatlas.org). The book addresses a fundamental gap in existing textbooks, namely, what bacterial and archaeal cells look like and how the macromolecular structures they contain give rise to their diverse and complex functions.

In situ imaging of bacterial outer membrane projections and associated protein complexes using electron cryo-tomography.

The ability to produce outer membrane projections in the form of tubular membrane extensions (MEs) and membrane vesicles (MVs) is a widespread phenomenon among diderm bacteria. Despite this, our knowledge of the ultrastructure of these extensions and their associated protein complexes remains limited. Here, we surveyed the ultrastructure and formation of MEs and MVs, and their associated protein complexes, in tens of thousands of electron cryo-tomograms of ~90 bacterial species that we have collected for various projects over the past 15 years (Jensen lab database), in addition to data generated in the Briegel lab.

A cryo-electron tomography workflow reveals protrusion-mediated shedding on injured plasma membrane.

Cryo-electron tomography (cryo-ET) provides structural context to molecular mechanisms underlying biological processes. Although straightforward to implement for studying stable macromolecular complexes, using it to locate short-lived structures and events can be impractical. A combination of live-cell microscopy, correlative light and electron microscopy, and cryo-ET will alleviate this issue.

Simulations of Proposed Mechanisms of FtsZ-Driven Cell Constriction.

To divide, bacteria must constrict their membranes against significant force from turgor pressure. A tubulin homolog, FtsZ, is thought to drive constriction, but how FtsZ filaments might generate constrictive force in the absence of motor proteins is not well understood. There are two predominant models in the field.

In situ imaging of the bacterial flagellar motor disassembly and assembly processes.

The self-assembly of cellular macromolecular machines such as the bacterial flagellar motor requires the spatio-temporal synchronization of gene expression with proper protein localization and association of dozens of protein components. In Salmonella and Escherichia coli, a sequential, outward assembly mechanism has been proposed for the flagellar motor starting from the inner membrane, with the addition of each new component stabilizing the previous one. However, very little is known about flagellar disassembly.

ETDB-Caltech: A blockchain-based distributed public database for electron tomography.

Three-dimensional electron microscopy techniques like electron tomography provide valuable insights into cellular structures, and present significant challenges for data storage and dissemination. Here we explored a novel method to publicly release more than 11,000 such datasets, more than 30 TB in total, collected by our group. Our method, based on a peer-to-peer file sharing network built around a blockchain ledger, offers a distributed solution to data storage.

Electron Cryotomography of Bacterial Secretion Systems.

In biology, function arises from form. For bacterial secretion systems, which often span two membranes, avidly bind to the cell wall, and contain hundreds of individual proteins, studying form is a daunting task, made possible by electron cryotomography (ECT). ECT is the highest-resolution imaging technique currently available to visualize unique objects inside cells, providing a three-dimensional view of the shapes and locations of large macromolecular complexes in their native environment.

Simulations suggest a constrictive force is required for Gram-negative bacterial cell division.

To divide, Gram-negative bacterial cells must remodel cell wall at the division site. It remains debated, however, whether this cell wall remodeling alone can drive membrane constriction, or if a constrictive force from the tubulin homolog FtsZ is required. Previously, we constructed software (REMODELER 1) to simulate cell wall remodeling during growth.

The presence and absence of periplasmic rings in bacterial flagellar motors correlates with stator type.

The bacterial flagellar motor, a cell-envelope-embedded macromolecular machine that functions as a cellular propeller, exhibits significant structural variability between species. Different torque-generating stator modules allow motors to operate in different pH, salt or viscosity levels. How such diversity evolved is unknown.

Distinguishing signal from autofluorescence in cryogenic correlated light and electron microscopy of mammalian cells.

In cryogenic correlated light and electron microscopy (cryo-CLEM), frozen targets of interest are identified and located on EM grids by fluorescence microscopy and then imaged at higher resolution by cryo-EM. Whilst working with these methods, we discovered that a variety of mammalian cells exhibit strong punctate autofluorescence when imaged under cryogenic conditions (80 K). Autofluorescence originated from multilamellar bodies (MLBs) and secretory granules.

Morphology of the archaellar motor and associated cytoplasmic cone in .

Archaeal swimming motility is driven by archaella: rotary motors attached to long extracellular filaments. The structure of these motors, and particularly how they are anchored in the absence of a peptidoglycan cell wall, is unknown. Here, we use electron cryotomography to visualize the archaellar basal body in KOD1.

Uncharacterized Bacterial Structures Revealed by Electron Cryotomography.

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.

Short FtsZ filaments can drive asymmetric cell envelope constriction at the onset of bacterial cytokinesis.

FtsZ, the bacterial homologue of eukaryotic tubulin, plays a central role in cell division in nearly all bacteria and many archaea. It forms filaments under the cytoplasmic membrane at the division site where, together with other proteins it recruits, it drives peptidoglycan synthesis and constricts the cell. Despite extensive study, the arrangement of FtsZ filaments and their role in division continue to be debated.

Cellular Electron Cryotomography: Toward Structural Biology In Situ.

Electron cryotomography (ECT) provides three-dimensional views of macromolecular complexes inside cells in a native frozen-hydrated state. Over the last two decades, ECT has revealed the ultrastructure of cells in unprecedented detail. It has also allowed us to visualize the structures of macromolecular machines in their native context inside intact cells.

The CDK-APC/C Oscillator Predominantly Entrains Periodic Cell-Cycle Transcription.

Throughout cell-cycle progression, the expression of multiple transcripts oscillate, and whether these are under the centralized control of the CDK-APC/C proteins or can be driven by a de-centralized transcription factor (TF) cascade is a fundamental question for understanding cell-cycle regulation. In budding yeast, we find that the transcription of nearly all genes, as assessed by RNA-seq or fluorescence microscopy in single cells, is dictated by CDK-APC/C. Three exceptional genes are transcribed in a pulsatile pattern in a variety of CDK-APC/C arrests.

A new view into prokaryotic cell biology from electron cryotomography.

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.

The Caltech Tomography Database and Automatic Processing Pipeline.

Here we describe the Caltech Tomography Database and automatic image processing pipeline, designed to process, store, display, and distribute electron tomographic data including tilt-series, sample information, data collection parameters, 3D reconstructions, correlated light microscope images, snapshots, segmentations, movies, and other associated files. Tilt-series are typically uploaded automatically during collection to a user's "Inbox" and processed automatically, but can also be entered and processed in batches via scripts or file-by-file through an internet interface. As with the video website YouTube, each tilt-series is represented on the browsing page with a link to the full record, a thumbnail image and a video icon that delivers a movie of the tomogram in a pop-out window.

Structural conservation of chemotaxis machinery across Archaea and Bacteria.

Chemotaxis allows cells to sense and respond to their environment. In Bacteria, stimuli are detected by arrays of chemoreceptors that relay the signal to a two-component regulatory system. These arrays take the form of highly stereotyped super-lattices comprising hexagonally packed trimers-of-receptor-dimers networked by rings of histidine kinase and coupling proteins.

Structure of bacterial cytoplasmic chemoreceptor arrays and implications for chemotactic signaling.

Most motile bacteria sense and respond to their environment through a transmembrane chemoreceptor array whose structure and function have been well-studied, but many species also contain an additional cluster of chemoreceptors in their cytoplasm. Although the cytoplasmic cluster is essential for normal chemotaxis in some organisms, its structure and function remain unknown. Here we use electron cryotomography to image the cytoplasmic chemoreceptor cluster in Rhodobacter sphaeroides and Vibrio cholerae.

New insights into bacterial chemoreceptor array structure and assembly from electron cryotomography.

Bacterial chemoreceptors cluster in highly ordered, cooperative, extended arrays with a conserved architecture, but the principles that govern array assembly remain unclear. Here we show images of cellular arrays as well as selected chemoreceptor complexes reconstituted in vitro that reveal new principles of array structure and assembly. First, in every case, receptors clustered in a trimers-of-dimers configuration, suggesting this is a highly favored fundamental building block.