From single bacterial cell imaging towards <i>in vivo</i> single-molecule biochemistry studies

Endesfelder, Ulrike

doi:10.1042/ebc20190002

Cited by 21 publications

(12 citation statements)

References 99 publications

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“…Identification of conditions or additional regulators controlling FlmE and FlmR, or additional targets outside of flagella regulation like HeuR, might point to a role for these sRNAs beyond fine-tuning the biogenesis cascade to modulating flagellar morphology in response to different environments. This would expand the complexity of the system, whose temporal and spatial dynamics in individual bacteria might be disentangled by future studies with single-cell transcriptomics or imaging (Dar et al, 2021;Endesfelder, 2019;Homberger et al, 2022).…”

Section: Discussionmentioning

confidence: 99%

Interplay of two small RNAs fine-tunes hierarchical flagellar gene expression in the foodborne pathogenCampylobacter jejuni

König

Svensson

Sharma

2023

Preprint

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Like for many enteric bacteria, flagella are a crucial virulence factor for the foodborne pathogenCampylobacter jejuni, allowing the bacteria to move through the viscous mucus of the human intestine. Assembly of the complex flagellar machinery and filament requires hierarchical regulation via transcriptional control of each component. InC. jejuni, class I genes are transcribed from σ70-dependent promoters and class II/III genes with the help of the alternative sigma factors RpoN (σ54) and FliA (σ28). In contrast to transcriptional control, less is known about post-transcriptional regulation of flagellar biosynthesis cascades via small regulatory RNAs (sRNAs). Here, we characterized two sRNAs with opposing effects on the cascade that fine-tuneC. jejuniflagellar filament assembly and thereby impact motility. We demonstrate that the highly conservedCampylobactersRNA CJnc230 (FlmE, flagellar length and motility enhancer), encoded downstream of the flagellar hook structural protein FlgE, is dependent on RpoN and that RNase III processes CJnc230 from theflgEmRNA, while RNase Y and PNPase mature its 3' end. We identify mRNAs encoding a regulator of flagella-flagella interactions and the anti-σ28factor FlgM as direct targets of CJnc230 repression. Overexpression of CJnc230 de-represses FliA activity and upregulates class III flagellar genes, such as the major flagellinflaA, culminating in longer flagella and increased motility. In contrast, overexpression of the FliA-dependent sRNA CJnc170 (FlmR, flagellar length and motility repressor) reduces flagellar length and motility. Overall, our study demonstrates sRNA-mediated post-transcriptional regulation fine-tunesC. jejuniflagellar biosynthesis through balancing of the hierarchically expressed components.

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Section: Discussionmentioning

confidence: 99%

Interplay of two small RNAs fine-tunes hierarchical flagellar gene expression in the foodborne pathogenCampylobacter jejuni

König

Svensson

Sharma

2023

Preprint

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“…A number of reviews on the use of various CFM methods in the studies of bacteria at the subcellular level have been published. They were focused, in particular, on the structure of the cytoskeleton and nucleoid (Yao and Carballido-López 2014); the dynamics of transcription (Stracy and Kapanidis 2017) and translation (Gahlmann and Moerner 2014); the dynamics of protein-DNA interaction (Uphoff 2016); DNA replication and repair (Li et al 2018), the organization of secretion systems and intracellular compartmentalization (Schneider and Basler 2016); intracellular signaling and gene expression dynamics (Kentner and Sourjik 2010); spatial organization and life-style of pathogenic bacteria (Singh and Kenney 2021); mechanisms of action of antimicrobial compounds (Choi et al 2016); as well as technical aspects (Haas et al 2014) and prospects for using CFM in the study of intracellular biochemistry at the level of single molecules (Endesfelder 2019).…”

Section: Bacteriamentioning

confidence: 99%

Computerized fluorescence microscopy of microbial cells

Puchkov

2021

World J Microbiol Biotechnol

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The upgrading of fluorescence microscopy by the introduction of computer technologies has led to the creation of a new methodology, computerized fluorescence microscopy (CFM). CFM improves subjective visualization and combines it with objective quantitative analysis of the microscopic data. CFM has opened up two fundamentally new opportunities for studying microorganisms. The first is the quantitative measurement of the fluorescence parameters of the targeted fluorophores in association with certain structures of individual cells. The second is the expansion of the boundaries of visualization/resolution of intracellular components beyond the "diffraction limit" of light microscopy into the nanometer range. This enables to obtain unique information about the localization and dynamics of intracellular processes at the molecular level. The purpose of this review is to demonstrate the potential of CFM in the study of fundamental aspects of the structural and functional organization of microbial cells. The basics of computer processing and analysis of digital images are briefly described. The fluorescent molecules used in CFM with an emphasis on fluorescent proteins are characterized. The main methods of superresolution microscopy (nanoscopy) are presented. The capabilities of various CFM methods for exploring microbial cells at the subcellular level are illustrated by the examples of various studies on yeast and bacteria.

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“…While single molecules could previously only be studied in the controlled, but limited environment of in vitro assays, SMLM imaging allows us to measure their function in their native cellular environment. Consequently, SMLM imaging has become a powerful tool for studying cell biology (Endesfelder, 2019).…”

Section: Introductionmentioning

confidence: 99%

Establishing Live-Cell Single-Molecule Localization Microscopy Imaging and Single-Particle Tracking in the Archaeon Haloferax volcanii

et al. 2020

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In recent years, fluorescence microscopy techniques for the localization and tracking of single molecules in living cells have become well-established and are indispensable tools for the investigation of cellular biology and in vivo biochemistry of many bacterial and eukaryotic organisms. Nevertheless, these techniques are still not established for imaging archaea. Their establishment as a standard tool for the study of archaea will be a decisive milestone for the exploration of this branch of life and its unique biology. Here, we have developed a reliable protocol for the study of the archaeon Haloferax volcanii. We have generated an autofluorescence-free H. volcanii strain, evaluated several fluorescent proteins for their suitability to serve as single-molecule fluorescence markers and codon-optimized them to work under optimal H. volcanii cultivation conditions. We found that two of them, Dendra2Hfx and PAmCherry1Hfx, provide state-of-the-art single-molecule imaging. Our strategy is quantitative and allows dual-color imaging of two targets in the same field of view (FOV) as well as DNA co-staining. We present the first single-molecule localization microscopy (SMLM) images of the subcellular organization and dynamics of two crucial intracellular proteins in living H. volcanii cells, FtsZ1, which shows complex structures in the cell division ring, and RNA polymerase, which localizes around the periphery of the cellular DNA.This work should provide incentive to develop SMLM strategies for other archaeal organisms in the near future.

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From single bacterial cell imaging towards in vivo single-molecule biochemistry studies

Cited by 21 publications

References 99 publications

Interplay of two small RNAs fine-tunes hierarchical flagellar gene expression in the foodborne pathogenCampylobacter jejuni

Interplay of two small RNAs fine-tunes hierarchical flagellar gene expression in the foodborne pathogenCampylobacter jejuni

Computerized fluorescence microscopy of microbial cells

Establishing Live-Cell Single-Molecule Localization Microscopy Imaging and Single-Particle Tracking in the Archaeon Haloferax volcanii

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