Revealing bacterial cell biology using cryo-electron tomography

Khanna, K. N.; Villa, Elizabeth

doi:10.1016/j.sbi.2022.102419

Cited by 18 publications

(14 citation statements)

References 66 publications

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“…Imaging whole bacteria provides cellular context to subcellular features, allowing rapid screening of multiple bacteria at different stages of growth and capturing rare cellular events which may be obscured by sample thickness or removed by thinning in conventional cryo-EM studies ( 31 ).…”

Section: Resultsmentioning

confidence: 99%

Liquid phase electron microscopy of bacterial ultrastructure

Caffrey,

Pedrazo-Tardajos,

Liberti

et al. 2024

Preprint

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Recent advances in liquid phase scanning transmission electron microscopy (LP-STEM) have enabled the study of dynamic biological processes at nanometre resolutions, paving the way for live-cell imaging using electron microscopy. However, this technique is often hampered by the inherent thickness of whole cell samples and damage from electron beam irradiation. These restrictions degrade image quality and resolution, impeding biological interpretation. Here we detail the use of graphene encapsulation, STEM, and energy-dispersive X-ray (EDX) spectroscopy methods to mitigate these issues, providing unprecedented levels of intracellular detail in aqueous specimens. We demonstrate the feasibility of our approach using a radiation resistant, gram-positive bacterium, Deinococcus radiodurans, chosen specifically for its tolerance to the highly oxidative environments created by electron irradiation. This work shows the potential of LP-STEM to examine internal cellular structures in thick biological samples and identify key ultrastructure in these samples using a variety of imaging techniques.

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Section: Resultsmentioning

confidence: 99%

Liquid phase electron microscopy of bacterial ultrastructure

Caffrey,

Pedrazo-Tardajos,

Liberti

et al. 2024

Preprint

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“…However, there is still much research to be done to elucidate how this complex protein machinery works and visualize their structures and interactions in a natural context inside the plant cells. An innovative technique to solve problems such as the protein complex cannot be purified intact, or the function of the protein complex is lost outer the cells is the in situ observation of these protein complexes using the cryo-electron tomography ( Oikonomou and Jensen, 2017 ; Khanna and Villa, 2022 ; Saibil, 2022 ). With these novel technical approaches, researchers will have the ability to identify the heterogenous protein communities that could constitute the metabolon involved in AsA metabolism in plant cells.…”

Section: Biochemical Strategies For Regulation Of Asa Biosynthesis1mentioning

confidence: 99%

Genetic and biochemical strategies for regulation of L-ascorbic acid biosynthesis in plants through the L-galactose pathway

Castro

Castro²,

Cobos³

2023

Front. Plant Sci.

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Vitamin C (L-ascorbic acid, AsA) is an essential compound with pleiotropic functions in many organisms. Since its isolation in the last century, AsA has attracted the attention of the scientific community, allowing the discovery of the L-galactose pathway, which is the main pathway for AsA biosynthesis in plants. Thus, the aim of this review is to analyze the genetic and biochemical strategies employed by plant cells for regulating AsA biosynthesis through the L-galactose pathway. In this pathway, participates eight enzymes encoded by the genes PMI, PMM, GMP, GME, GGP, GPP, GDH, and GLDH. All these genes and their encoded enzymes have been well characterized, demonstrating their participation in AsA biosynthesis. Also, have described some genetic and biochemical strategies that allow its regulation. The genetic strategy includes regulation at transcriptional and post-transcriptional levels. In the first one, it was demonstrated that the expression levels of the genes correlate directly with AsA content in the tissues/organs of the plants. Also, it was proved that these genes are light-induced because they have light-responsive promoter motifs (e.g., ATC, I-box, GT1 motif, etc.). In addition, were identified some transcription factors that function as activators (e.g., SlICE1, AtERF98, SlHZ24, etc.) or inactivators (e.g., SlL1L4, ABI4, SlNYYA10) regulate the transcription of these genes. In the second one, it was proved that some genes have alternative splicing events and could be a mechanism to control AsA biosynthesis. Also, it was demonstrated that a conserved cis-acting upstream open reading frame (5’-uORF) located in the 5’-untranslated region of the GGP gene induces its post-transcriptional repression. Among the biochemical strategies discovered is the control of the enzyme levels (usually by decreasing their quantities), control of the enzyme catalytic activity (by increasing or decreasing its activity), feedback inhibition of some enzymes (GME and GGP), subcellular compartmentation of AsA, the metabolon assembly of the enzymes, and control of AsA biosynthesis by electron flow. Together, the construction of this basic knowledge has been establishing the foundations for generating genetically improved varieties of fruits and vegetables enriched with AsA, commonly used in animal and human feed.

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“…Even in the absence of a real cytoplasmic compartmentalization, the bacterial cytoplasmic organization is ensured by constructing proteins and nucleic acids scaffolds that form, through liquid-liquid phase separation (LLPS), local dynamic membrane-less functional condensates that can enrich specific nucleic acid and protein components (45,46). In addition, there are bacterial cytoskeletal protein filaments, involved in various processes, including cell elongation, cell division (such as the treadmilling protein, tubulin-homolog, FtsZ), chromosomal and plasmid segregation, and cell motility (47). The structure of the bacterial cytoplasm allows for temporal and spatial localization of proteins ("check points") involved in growth cycle progression, maintaining a "cell memory" ensuring the right topological distributions required for division planes, as it occurs in eukaryotic cells (48).…”

Section: The Envelope-associated Protein-rich Peripheral Cytoplasmmentioning

confidence: 99%

Bacterial Subcellular Architecture, Structural Epistasis, and Antibiotic Resistance

Baquero¹,

Martínez²,

Sanchez³

et al. 2023

Preprint

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Epistasis refers to how genetic interactions between some genetic loci affect phenotypes and fitness. In this study, we propose the concept “structural epistasis” to emphasize the role of the variable physical interactions between molecules located at particular spaces inside the bacterial cell in the emergence of novel phenotypes. The architecture of the bacterial cell (typically a gram-negative), which consists of concentrical layers of membranes, particles, and molecules with differing con-figurations and densities (from the outer membrane to the nucleoid) determines and is at its turn determined by the cell´s shape and size, depending on the growth phases, and exposure to toxic conditions, stress responses, and the bacterial environment. Antibiotics change the bacterial cell’s internal molecular topology, producing unexpected interactions among molecule. In contrast, changes in shape and size might alter antibiotic action. The mechanisms of antibiotic resistance (and their vectors, as mobile genetic elements) also influence molecular connectivity in the bacterial cell and can produce unexpected phenotypes, influencing the action of other antimicrobial agents.

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Revealing bacterial cell biology using cryo-electron tomography

Cited by 18 publications

References 66 publications

Liquid phase electron microscopy of bacterial ultrastructure

Liquid phase electron microscopy of bacterial ultrastructure

Genetic and biochemical strategies for regulation of L-ascorbic acid biosynthesis in plants through the L-galactose pathway

Bacterial Subcellular Architecture, Structural Epistasis, and Antibiotic Resistance

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