Ultrastructure of organohalide-respiring Dehalococcoidia revealed by cryo-electron tomography

2021 ◽  
Author(s):  
Danielle L. Sexton ◽  
Gao Chen ◽  
Fadime Kara Murdoch ◽  
Ameena Hashimi ◽  
Frank E. Löffler ◽  
...  
Keyword(s):  
Electron Tomography ◽  
Cell Envelope ◽  
Cell Structure ◽  
Lipid Vesicles ◽  
Cell Types ◽  
Reductive Dehalogenation ◽  
Outer Leaflet ◽  
New Findings ◽  
Cryo Electron Tomography ◽  
Cell Envelopes

Dehalococcoides mccartyi ( Dhc ) and Dehalogenimonas spp. ( Dhgm ) are members of the class Dehalococcoidia , phylum Chloroflexi, characterized by streamlined genomes and a strict requirement for organohalogens as electron acceptors. Here, we used cryo-electron tomography to reveal morphological and ultrastructural features of Dhc strain BAV1 and ‘ Candidatus Dehalogenimonas etheniformans’ strain GP cells at unprecedented resolution. Dhc cells were irregularly shaped discs (890 ± 110 nm long, 630 ± 110 nm wide and 130 ± 15 nm thick) with curved and straight sides that intersected at acute angles, whereas Dhgm cells appeared as slightly flattened cocci (760 ± 85 nm). The cell envelopes were composed of a cytoplasmic membrane (CM), a paracrystalline surface layer (S-layer) with hexagonal symmetry and ∼22 nm spacing between repeating units, and a layer of unknown composition separating the CM and the S-layer. Cell surface appendages were only detected in Dhc cells, whereas both cell types had bundled cytoskeletal filaments. Repetitive globular structures, ∼5 nm in diameter and ∼9 nm apart, were observed associated with the outer leaflet of the CM. We hypothesized that those represent organohalide respiration (OHR) complexes and estimated ∼30,000 copies per cell. In Dhgm cultures, extracellular lipid vesicles (20 - 110 nm in diameter) decorated with putative OHR complexes but lacking an S-layer were observed. The new findings expand our understanding of the unique cellular ultrastructure and biology of organohalide-respiring Dehalococcoidia . Importance: Dehalococcoidia respire organohalogen compounds and play relevant roles in bioremediation of groundwater, sediments and soils impacted with toxic chlorinated pollutants. Using advanced imaging tools, we have obtained 3-dimensional images at macromolecular resolution of whole Dehalococcoidia cells revealing their unique structural components. Our data detail the overall cellular shape, cell envelope architecture, cytoskeletal filaments, the likely localization of enzymatic complexes involved in reductive dehalogenation, and the structure of extracellular vesicles. The new findings expand our understanding of the cell structure-function relationship in Dehalococcoidia with implications for Dehalococcoidia biology and bioremediation.

scholarly journals Three-Dimensional Imaging of the Highly Bent Architecture of Bdellovibrio bacteriovorus by Using Cryo-Electron Tomography

2008 ◽  
Vol 190 (7) ◽  
pp. 2588-2596 ◽  
Author(s):  
Mario J. Borgnia ◽  
Sriram Subramaniam ◽  
Jacqueline L. S. Milne
Keyword(s):  
Electron Tomography ◽  
Cell Envelope ◽  
Three Dimensional ◽  
Local Deformation ◽  
Cell Integrity ◽  
Human Pathogens ◽  
Bdellovibrio Bacteriovorus ◽  
Internal Network ◽  
Cryo Electron Tomography ◽  
Shape Changes

ABSTRACT Bdellovibrio bacteriovorus cells are small deltaproteobacterial cells that feed on other gram-negative bacteria, including human pathogens. Using cryo-electron tomography, we demonstrated that B. bacteriovorus cells are capable of substantial flexibility and local deformation of the outer and inner membranes without loss of cell integrity. These shape changes can occur in less than 2 min, and analysis of the internal architecture of highly bent cells showed that the overall distribution of molecular machines and the nucleoid is similar to that in moderately bent cells. B. bacteriovorus cells appear to contain an extensive internal network of short and long filamentous structures. We propose that rearrangements of these structures, in combination with the unique properties of the cell envelope, may underlie the remarkable ability of B. bacteriovorus cells to find and enter bacterial prey.

Electron Tomography of Frozen-Hydrated Samples

2000 ◽  
Vol 6 (S2) ◽  
pp. 310-311
Author(s):  
M. Marko ◽  
C.-E. Hsieh ◽  
B.K. Rath ◽  
C.A. Mannella ◽  
B.F. McEwen
Keyword(s):  
Electron Tomography ◽  
Lipid Vesicles ◽  
Spindle Pole ◽  
Image Processing Techniques ◽  
Spindle Pole Bodies ◽  
Reconstructed Volume ◽  
Cryo Electron Tomography ◽  
Projection Images ◽  
Processing Techniques ◽  
Triad Junctions

Cryo-electron tomography offers a means of obtaining the 3-D ultrastructure of specimens that have not been chemically fixed or stained. The technique is still under development, but it has already been applied to several biological specimens including prokaryotic cells, centrioles, sperm axonemes, mitochondria, spindle-pole bodies, lipid vesicles, isolated skeletal-muscle triad junctions, chromatin fragments, actin bundles, and macromolecules.Electron tomography itself is a nearly mature technology. It is most often used with conventional plastic sections 100- 1000nm thick. The specimen is imaged in the TEM, using a tilt stage to record a series of projection images over a large angular range. Using image-processing techniques, the tilt images are back-projected to form a reconstructed volume of the specimen. The reconstruction is a 3-D array of pixels or volume elements (voxels), and can be sliced in any direction to form a series of 2-D images.

scholarly journals Evolutionary Remodeling of the Cell Envelope in Bacteria of the Planctomycetes Phylum

2020 ◽  
Vol 12 (9) ◽  
pp. 1528-1548
Author(s):  
Mayank Mahajan ◽  
Christian Seeger ◽  
Benjamin Yee ◽  
Siv G E Andersson
Keyword(s):  
Cell Wall ◽  
Electron Tomography ◽  
Cell Envelope ◽  
Outer Membrane Proteins ◽  
Cell Wall Proteins ◽  
Secretion Systems ◽  
Peptidoglycan Biosynthesis ◽  
Multiple Copies ◽  
Cellular Phenotypes ◽  
Cell Envelopes

Abstract Bacteria of the Planctomycetes phylum have many unique cellular features, such as extensive membrane invaginations and the ability to import macromolecules. These features raise intriguing questions about the composition of their cell envelopes. In this study, we have used microscopy, phylogenomics, and proteomics to examine the composition and evolution of cell envelope proteins in Tuwongella immobilis and other members of the Planctomycetes. Cryo-electron tomography data indicated a distance of 45 nm between the inner and outer membranes in T. immobilis. Consistent with the wide periplasmic space, our bioinformatics studies showed that the periplasmic segments of outer-membrane proteins in type II secretion systems are extended in bacteria of the order Planctomycetales. Homologs of two highly abundant cysteine-rich cell wall proteins in T. immobilis were identified in all members of the Planctomycetales, whereas genes for peptidoglycan biosynthesis and cell elongation have been lost in many members of this bacterial group. The cell wall proteins contain multiple copies of the YTV motif, which is the only domain that is conserved and unique to the Planctomycetales. Earlier diverging taxa in the Planctomycetes phylum contain genes for peptidoglycan biosynthesis but no homologs to the YTV cell wall proteins. The major remodeling of the cell envelope in the ancestor of the Planctomycetales coincided with the emergence of budding and other unique cellular phenotypes. The results have implications for hypotheses about the process whereby complex cellular features evolve in bacteria.

scholarly journals PilY1 and minor pilins form a complex priming the type IVa pilus in Myxococcus xanthus

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Anke Treuner-Lange ◽  
Yi-Wei Chang ◽  
Timo Glatter ◽  
Marco Herfurth ◽  
Steffi Lindow ◽  
...  
Keyword(s):  
Cell Surface ◽  
Bacterial Cell ◽  
Cell Biology ◽  
Electron Tomography ◽  
Cell Envelope ◽  
Myxococcus Xanthus ◽  
Core Proteins ◽  
Complex Functions ◽  
Cryo Electron Tomography ◽  
Conserved Core

Abstract Type IVa pili are ubiquitous and versatile bacterial cell surface filaments that undergo cycles of extension, adhesion and retraction powered by the cell-envelope spanning type IVa pilus machine (T4aPM). The overall architecture of the T4aPM and the location of 10 conserved core proteins within this architecture have been elucidated. Here, using genetics, cell biology, proteomics and cryo-electron tomography, we demonstrate that the PilY1 protein and four minor pilins, which are widely conserved in T4aP systems, are essential for pilus extension in Myxococcus xanthus and form a complex that is an integral part of the T4aPM. Moreover, these proteins are part of the extended pilus. Our data support a model whereby the PilY1/minor pilin complex functions as a priming complex in T4aPM for pilus extension, a tip complex in the extended pilus for adhesion, and a cork for terminating retraction to maintain a priming complex for the next round of extension.

scholarly journals Cryo-Electron Tomography Elucidates the Molecular Architecture of Treponema pallidum, the Syphilis Spirochete

2009 ◽  
Vol 191 (24) ◽  
pp. 7566-7580 ◽  
Author(s):  
Jacques Izard ◽  
Christian Renken ◽  
Chyong-Ere Hsieh ◽  
Daniel C. Desrosiers ◽  
Star Dunham-Ems ◽  
...  
Keyword(s):  
Electron Tomography ◽  
Cell Envelope ◽  
Treponema Pallidum ◽  
Basal Bodies ◽  
Image Modeling ◽  
Molecular Architecture ◽  
Periplasmic Space ◽  
Membrane Biology ◽  
Cytoplasmic Filaments ◽  
Cryo Electron Tomography

ABSTRACT Cryo-electron tomography (CET) was used to examine the native cellular organization of Treponema pallidum, the syphilis spirochete. T. pallidum cells appeared to form flat waves, did not contain an outer coat and, except for bulges over the basal bodies and widening in the vicinity of flagellar filaments, displayed a uniform periplasmic space. Although the outer membrane (OM) generally was smooth in contour, OM extrusions and blebs frequently were observed, highlighting the structure's fluidity and lack of attachment to underlying periplasmic constituents. Cytoplasmic filaments converged from their attachment points opposite the basal bodies to form arrays that ran roughly parallel to the flagellar filaments along the inner surface of the cytoplasmic membrane (CM). Motile treponemes stably attached to rabbit epithelial cells predominantly via their tips. CET revealed that T. pallidum cell ends have a complex morphology and assume at least four distinct morphotypes. Images of dividing treponemes and organisms shedding cell envelope-derived blebs provided evidence for the spirochete's complex membrane biology. In the regions without flagellar filaments, peptidoglycan (PG) was visualized as a thin layer that divided the periplasmic space into zones of higher and lower electron densities adjacent to the CM and OM, respectively. Flagellar filaments were observed overlying the PG layer, while image modeling placed the PG-basal body contact site in the vicinity of the stator-P-collar junction. Bioinformatics and homology modeling indicated that the MotB proteins of T. pallidum, Treponema denticola, and Borrelia burgdorferi have membrane topologies and PG binding sites highly similar to those of their well-characterized Escherichia coli and Helicobacter pylori orthologs. Collectively, our results help to clarify fundamental differences in cell envelope ultrastructure between spirochetes and gram-negative bacteria. They also confirm that PG stabilizes the flagellar motor and enable us to propose that in most spirochetes motility results from rotation of the flagellar filaments against the PG.

scholarly journals In Situ Visualization of the pKM101-Encoded Type IV Secretion System Reveals a Highly Symmetric ATPase Energy Center

mBio ◽  
2021 ◽  
Author(s):  
Pratick Khara ◽  
Liqiang Song ◽  
Peter J. Christie ◽  
Bo Hu
Keyword(s):  
Electron Tomography ◽  
Cell Envelope ◽  
Type Iv Secretion ◽  
Secretion Systems ◽  
Type Iv ◽  
Cryo Electron Tomography ◽  
Plasmid Pkm101 ◽  
Energy Center ◽  
Bacterial Type

Bacterial type IV secretion systems (T4SSs) play central roles in antibiotic resistance spread and virulence. By cryo-electron tomography (CryoET), we solved the structure of the plasmid pKM101-encoded T4SS in the native context of the bacterial cell envelope.

scholarly journals 3D-Visualization of Amyloid-β Oligomer and Fibril Interactions with Lipid Membranes by Cryo-Electron Tomography

2020 ◽  
Author(s):  
Yao Tian ◽  
Ruina Liang ◽  
Amit Kumar ◽  
Piotr Szwedziak ◽  
John H. Viles
Keyword(s):  
Lipid Bilayers ◽  
Molecular Mechanisms ◽  
Lipid Membrane ◽  
Electron Tomography ◽  
3D Visualization ◽  
Membrane Integrity ◽  
Amyloid Β ◽  
Lipid Vesicles ◽  
Aβ Oligomers ◽  
Cryo Electron Tomography

ABSTRACTAmyloid-β (Aβ) monomers assemble into mature fibrils via a range of metastable oligomeric and protofibrillar intermediates. These Aβ assemblies have been shown to bind to lipid bilayers. This can disrupt membrane integrity and cause a loss of cellular homeostasis, that triggers a cascade of events leading to Alzheimer’s disease. However, molecular mechanisms of Aβ cytotoxicity and how the different assembly forms interact with the membrane remain enigmatic. Here we use cryo-electron tomography (cryoET) to obtain three-dimensional nano-scale images of various Aβ assembly types and their interaction with liposomes. Aβ oligomers bind extensively to the lipid vesicles, inserting and carpeting the upper-leaflet of the bilayer. Furthermore, curvilinear protofibrils also insert into the bilayer, orthogonally to the membrane surface. Aβ oligomers concentrate at the interface of vesicles and form a network of Aβ-linked liposomes. While crucially, monomeric and fibrillar Aβ have relatively little impact on the membrane. Changes to lipid membrane composition highlights a significant role for GM1-ganglioside in promoting Aβ-membrane interactions. The different effects of Aβ assembly forms observed align with the highlighted cytotoxicity reported for Aβ oligomers. The wide-scale incorporation of Aβ oligomers and curvilinear protofibrils into the lipid bilayer suggests a mechanism by which membrane integrity is lost.

scholarly journals Ultrastructure of exospore formation in Streptomyces revealed by cryo-electron tomography

2020 ◽  
Author(s):  
Danielle L. Sexton ◽  
Elitza I. Tocheva
Keyword(s):  
Environmental Conditions ◽  
Electron Tomography ◽  
Cell Envelope ◽  
Model Organism ◽  
Structural Complexity ◽  
Spore Coat ◽  
Stages Of Growth ◽  
Cryo Electron Tomography ◽  
Streptomyces Albus ◽  
Adverse Environmental Conditions

AbstractMany bacteria form spores in response to adverse environmental conditions. Several sporulation pathways have evolved independently and occur through distinctive mechanisms. Here, using cryo-electron tomography (cryo-ET), we examine all stages of growth and exospore formation in the model organism Streptomyces albus. Our data reveal the native ultrastructure of vegetative hyphae, including the polarisome and filaments of ParA or FilP. In addition, we observed septal junctions in vegetative septa, likely involved in effector translocation between neighbouring cells. During sporulation, the cell envelope undergoes dramatic remodeling, including the formation of a spore cortex and two protective proteinaceous layers. Mature spores reveal the presence of a continuous spore coat and an irregular rodlet sheet. Together, these results provide an unprecedented examination of the ultrastructure in Streptomyces and further our understanding of the structural complexity of exospore formation.

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