BiFCROS: A Low-Background Fluorescence Repressor Operator System for Labeling of Genomic Loci

Milbredt, Sarah; Waldminghaus, Torsten

doi:10.1534/g3.117.040782

Cited by 4 publications

(3 citation statements)

References 49 publications

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“…The separation of DNA regions after their duplication during DNA replication (segregation) has been studied extensively using fluorescent repressor/operator (FROS) systems, or ParB/parS systems [10]. Repeats of specific DNA sequences are inserted at a single site on the chromosome whose segregation dynamics are to be investigated, and a specific binding protein (a transcriptional repressor or ParB protein) are expressed as fluorescent protein fusion to visualize the position of the binding cassette within the cell.…”

Section: B Subtilis Shows Growth Arrest When Subjected To Blue Lightmentioning

confidence: 99%

Bacterial cell growth is arrested by violet and blue, but not yellow light excitation during fluorescence microscopy

Najjar

Teeseling

Mayer

et al. 2020

BMC Mol and Cell Biol

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Background Fluorescence microscopy is a powerful tool in cell biology, especially for the study of dynamic processes. Intensive irradiation of bacteria with UV, blue and violet light has been shown to be able to kill cells, but very little information is available on the effect of blue or violet light during live-cell imaging. Results We show here that in the model bacterium Bacillus subtilis chromosome segregation and cell growth are rapidly halted by standard violet (405 nm) and blue light (CFP) (445–457 nm) excitation, whereas they are largely unaffected by green light (YFP). The stress sigma factor σB and the blue-light receptor YtvA are not involved in growth arrest. Using synchronized B. subtilis cells, we show that the use of blue light for fluorescence microscopy likely induces non-specific toxic effects, rather than a specific cell cycle arrest. Escherichia coli and Caulobacter crescentus cells also stop to grow after 15 one-second exposures to blue light (CFP), but continue growth when imaged under similar conditions in the YFP channel. In the case of E. coli, YFP excitation slows growth relative to white light excitation, whereas CFP excitation leads to cell death in a majority of cells. Thus, even mild violet/blue light excitation interferes with bacterial growth. Analyzing the dose-dependent effects of violet light in B. subtilis, we show that short exposures to low-intensity violet light allow for continued cell growth, while longer exposures do not. Conclusions Our experiments show that care must be taken in the design of live-cell imaging experiments in that violet or blue excitation effects must be closely controlled during and after imaging. Violet excitation during sptPALM or other imaging studies involving photoactivation has a threshold, below which little effects can be seen, but above which a sharp transition into cell death occurs. YFP imaging proves to be better suited for time-lapse studies, especially when cell cycle or cell growth parameters are to be examined.

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Section: B Subtilis Shows Growth Arrest When Subjected To Blue Lightmentioning

confidence: 99%

Bacterial cell growth is arrested by violet and blue, but not yellow light excitation during fluorescence microscopy

Najjar

Teeseling

Mayer

et al. 2020

BMC Mol and Cell Biol

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“…1Bvi). This technique (called Bimolecular Fluorescence Complementation [BiFC] or split FP) has over the years improved the detection of protein-protein interactions and is finding its way to other in vivo applications (66)(67)(68)(69)(70)(71)(72). It is important to note here that the maturation of the split FP, a process that starts when the complementary fragments associate, is equivalent to that of the intact fluorescent protein (73).…”

Section: A Fluorescent Toolbox For Bacterial Vivisection Fluorescent ...mentioning

confidence: 99%

“…Recently, the signal-to-noise ratio of this method was further improved by the development of a FROS system consisting of two different repressor-operator modules combined with Bimolecular Fluorescence Complementation (BiFC) (67,69). This new system uses two different repressor proteins (CI and Gal4), each fused to one subfragment of a "split" mVenus molecule, and enables the fluorescence signal to be reconstituted on a hybrid array constructed by combining the cognate operator sites of both repressors in an alternating fashion (71).…”

Section: Nucleic Acidsmentioning

confidence: 99%

Bacterial Vivisection: How Fluorescence-Based Imaging Techniques Shed a Light on the Inner Workings of Bacteria

Cambré

Aertsen

2020

Microbiol Mol Biol Rev

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SUMMARY The rise in fluorescence-based imaging techniques over the past 3 decades has improved the ability of researchers to scrutinize live cell biology at increased spatial and temporal resolution. In microbiology, these real-time vivisections structurally changed the view on the bacterial cell away from the “watery bag of enzymes” paradigm toward the perspective that these organisms are as complex as their eukaryotic counterparts. Capitalizing on the enormous potential of (time-lapse) fluorescence microscopy and the ever-extending pallet of corresponding probes, initial breakthroughs were made in unraveling the localization of proteins and monitoring real-time gene expression. However, later it became clear that the potential of this technique extends much further, paving the way for a focus-shift from observing single events within bacterial cells or populations to obtaining a more global picture at the intra- and intercellular level. In this review, we outline the current state of the art in fluorescence-based vivisection of bacteria and provide an overview of important case studies to exemplify how to use or combine different strategies to gain detailed information on the cell’s physiology. The manuscript therefore consists of two separate (but interconnected) parts that can be read and consulted individually. The first part focuses on the fluorescent probe pallet and provides a perspective on modern methodologies for microscopy using these tools. The second section of the review takes the reader on a tour through the bacterial cell from cytoplasm to outer shell, describing strategies and methods to highlight architectural features and overall dynamics within cells.

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Observing DNA in live cells

Weiss

Naor

Shechtman

2018

Biochm. Soc. Trans.

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The structural organization and dynamics of DNA are known to be of paramount importance in countless cellular processes, but capturing these events poses a unique challenge. Fluorescence microscopy is well suited for these live-cell investigations, but requires attaching fluorescent labels to the species under investigation. Over the past several decades, a suite of techniques have been developed for labeling and imaging DNA, each with various advantages and drawbacks. Here, we provide an overview of the labeling and imaging tools currently available for visualizing DNA in live cells, and discuss their suitability for various applications.

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BiFCROS: A Low-Background Fluorescence Repressor Operator System for Labeling of Genomic Loci

Cited by 4 publications

References 49 publications

Bacterial cell growth is arrested by violet and blue, but not yellow light excitation during fluorescence microscopy

Bacterial cell growth is arrested by violet and blue, but not yellow light excitation during fluorescence microscopy

Bacterial Vivisection: How Fluorescence-Based Imaging Techniques Shed a Light on the Inner Workings of Bacteria

Observing DNA in live cells

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