Lighting Up Clostridium Difficile: Reporting Gene Expression Using Fluorescent Lov Domains

Buckley, Anthony M.; Jukes, Caitlin; Candlish, Denise; Irvine, June; Spencer, Janice; Fagan, Robert P.; Roe, Andrew J.; Christie, John M.; Fairweather, Neil F.; Douce, Gill

doi:10.1038/srep23463

Cited by 57 publications

(69 citation statements)

References 38 publications

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“…It was also reported that the C. difficile Min system, which includes an E. coli -like MinE protein in addition to the Firmicutes -like DivIVA, is capable of oscillation when produced in B. subtilis , suggesting that C. difficile may exhibit features that integrate both systems (91). These advances were aided by new cell biological tools that now permit the use of fluorescence microscopy probes in anaerobes such as C. difficile (16, 112). Further variations on existing themes are also now emerging by studying non-model organisms.…”

Section: Introductionmentioning

confidence: 99%

Bacterial Cell Division: Nonmodels Poised to Take the Spotlight

Eswara

Ramamurthi

2017

Annu. Rev. Microbiol.

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The last three decades have witnessed an explosion in mechanistic details on how model bacterial organisms such as Escherichia coli, Bacillus subtilis, and Caulobacter crescentus undergo binary fission. These advances were possible by not only advances in microscopy that allowed cell biological questions to be answered, but also by the clever use of genetic manipulations in these systems in which specific hypothesis could be directly and easily tested. More recently, research using traditionally understudied organisms, or “non-model” systems, has revealed several alternate mechanistic strategies that bacteria use to divide and propagate. In this review, we will highlight these new findings and compare these strategies to cell division mechanisms elucidated in well-established model organisms.

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Section: Introductionmentioning

confidence: 99%

Bacterial Cell Division: Nonmodels Poised to Take the Spotlight

Eswara

Ramamurthi

2017

Annu. Rev. Microbiol.

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“…Anaerobic fluorescent proteins, like flavin-binding fluorescent proteins (FbFPs), which do not require oxygen for chromophore maturation (10), have been examined as possible effective reporters. For example, the FbFP phiLOV2.1 has been used as a reporter and fluorescent tag in Clostridioides difficile (formerly Clostridium difficile) (11). However, while FbFPs show strong fluorescence in Escherichia coli, in C. acetobutylicum, FbFPs like phiLOV2.1 and CreiLOV did not show fluorescence above background levels of the control (12).…”

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confidence: 99%

“…A central component of the Z ring is the tubulin homolog, FtsZ (16), a selfactivating GTPase widely conserved among prokaryotes (17,18). FtsZ was previously used to characterize the phiLOV fluorescent system in C. difficile (11). ZapA, one of several components of the Z ring assembly in model prokaryotes, interacts with FtsZ during Z ring assembly (19) to enhance the polymerization and stability of FtsZ (16,(19)(20)(21).…”

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confidence: 99%

A Strongly Fluorescing Anaerobic Reporter and Protein-Tagging System for Clostridium Organisms Based on the Fluorescence-Activating and Absorption-Shifting Tag Protein (FAST)

Streett

Kalis

Papoutsakis

2019

Appl Environ Microbiol

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Visualizing protein localization and characterizing gene expression activity in live Clostridium cells is limited for lack of a real-time, highly fluorescent, oxygen-independent reporter system. Enzymatic reporter systems have been used successfully for many years with Clostridium spp.; however, these assays do not allow for real-time analysis of gene expression activity with flow cytometry or for visualizing protein localization through fusion proteins. Commonly used fluorescent reporter proteins require oxygen for chromophore maturation and cannot be used for most strictly anaerobic Clostridium organisms. Here we show that the fluorescenceactivating and absorption-shifting tag protein (FAST), when associated with the fluorogenic ligand 4-hydroxy-3-methylbenzylidene-rhodanine (HMBR; now commercially available) and other commercially available ligands, is highly fluorescent in Clostridium acetobutylicum under anaerobic conditions. Using flow cytometry and a fluorescence microplate reader, we demonstrated FAST as a reporter system by employing the promoters of the C. acetobutylicum thiolase (thl), acetoacetate decarboxylase (adc), and phosphotransbutyrylase (ptb) metabolic genes, as well as a mutant P thl and modified ribosome binding site (RBS) versions of P adc and P ptb . Flow cytometrybased sorting was efficient and fast in sorting FAST-expressing cells, and positively and negatively sorted cells could be effectively recultured. FAST was also used to tag and examine protein localization of the predicted cell division FtsZ partner protein, ZapA, to visualize the divisome localization in live C. acetobutylicum cells. Our findings suggest that FAST can be used to further investigate Clostridium divisomes and more broadly the localization and expression levels of other proteins in Clostridium organisms, thus enabling cell biology studies with these organisms. IMPORTANCE FAST in association with the fluorogenic ligand HMBR is characterized as a successful, highly fluorescent reporter system in C. acetobutylicum. FAST can be used to distinguish between promoters in live cells using flow cytometry or a fluorescence microplate reader and can be used to tag and examine protein localization in live, anaerobically grown cells. Given that FAST is highly fluorescent under anaerobic conditions, it can be used in several applications of this and likely many Clostridium organisms and other strict anaerobes, including studies involving cell sorting, sporulation dynamics, and population characterization in pure as well as mixed cultures, such as those in various native or synthetic microbiomes and syntrophic cultures. Citation Streett HE, Kalis KM, Papoutsakis ET. 2019. A strongly fluorescing anaerobic reporter and protein-tagging system for Clostridium organisms based on the fluorescenceactivating and absorption-shifting tag protein (FAST). Appl Environ Microbiol 85:e00622-19. by guest http://aem.asm.org/ Downloaded from RESULTS Expression of FAST off the native and modified C. acetobutylicum thiolase promoter in E. coli and C...

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“…The idea of reformatting natural photoreceptors was used to transform small light, oxygen, and voltage (LOV) sensing domains -a class of blue-light photoreceptors found in plants, algae and bacteria -into cyan-green fluorescent proteins [11][12][13] . LOV proteins associate FbFPs proved to surpass GFP-like fluorescent proteins for reporting on protein expression in absence of oxygen in bacteria 14,17,18 , fungi 19 and mammalian cells 20 . FbFPs allowed in particular the study of host-pathogen interactions under physiologically relevant anaerobic conditions 21,22 .…”

Section: Flavin-binding Cyan-green Fluorescent Proteinsmentioning

confidence: 99%

CHAPTER 3. The Glowing Panoply of Fluorogen-based Markers for Advanced Bioimaging

Gautier¹

2018

Optogenetics

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Biological Sciences rely nowadays extensively on imaging tools to decipher the complexity of living organisms. Fluorescence microscopy allows the study of biological processes with an unprecedented temporal and spatial resolution. The revolution in fluorescence imaging has been the development of a large toolbox of fluorescent proteins able to reveal the abundance, position and dynamics of proteins. The fluorescence toolbox has been recently expanded with innovative reporters enabling to visualize proteins, and other biomolecules such as RNA, in new ways. These innovative reporters are bipartite systems composed of a genetically encoded tag forming a fluorescent complex with a small organic fluorogenic chromophore (also called fluorogens). This chapter is a user-oriented presentation of some of the most mature fluorogen-based markers available to biologists.

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Lighting Up Clostridium Difficile: Reporting Gene Expression Using Fluorescent Lov Domains

Cited by 57 publications

References 38 publications

Bacterial Cell Division: Nonmodels Poised to Take the Spotlight

Bacterial Cell Division: Nonmodels Poised to Take the Spotlight

A Strongly Fluorescing Anaerobic Reporter and Protein-Tagging System for Clostridium Organisms Based on the Fluorescence-Activating and Absorption-Shifting Tag Protein (FAST)

CHAPTER 3. The Glowing Panoply of Fluorogen-based Markers for Advanced Bioimaging

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