Oscillating behavior of <i>Clostridium difficile</i> Min proteins in <i>Bacillus subtilis</i>

Makroczyová, Jana; Jamroškovič, Ján; Krascsenitsová, Eva; Labajová, Naďa; Barák, Imrich

doi:10.1002/mbo3.337

Cited by 11 publications

(10 citation statements)

References 65 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The Clostridium divisome has not been well characterized due to the limitation of traditional FPs for viewing anaerobic cell division (40). It has been shown that the cell divisionassociated Min homolog in anaerobic Clostridioides difficile shows an oscillating pattern when tagged with yellow fluorescent protein (YFP) and expressed in B. subtilis (41). It was also shown that the DivIVA and MinD proteins show direct interaction in both C. difficile and Clostridium beijerinckii (40).…”

Section: Discussionmentioning

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

View full text Add to dashboard Cite

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...

show abstract

Section: Discussionmentioning

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

View full text Add to dashboard Cite

show abstract

“…subtilis , C . difficile MinD and MinE also performed pole-to-pole oscillations and inhibited spore formation [199]. Furthermore, DivIVa and MinD from C .…”

Section: Differences and Similarities To Other Para-type Atpasesmentioning

confidence: 99%

The E. coli MinCDE system in the regulation of protein patterns and gradients

Ramm

Heermann

Schwille

2019

Cell. Mol. Life Sci.

108

View full text Add to dashboard Cite

Molecular self-organziation, also regarded as pattern formation, is crucial for the correct distribution of cellular content. The processes leading to spatiotemporal patterns often involve a multitude of molecules interacting in complex networks, so that only very few cellular pattern-forming systems can be regarded as well understood. Due to its compositional simplicity, the Escherichia coli MinCDE system has, thus, become a paradigm for protein pattern formation. This biological reaction diffusion system spatiotemporally positions the division machinery in E. coli and is closely related to ParA-type ATPases involved in most aspects of spatiotemporal organization in bacteria. The ATPase MinD and the ATPase-activating protein MinE self-organize on the membrane as a reaction matrix. In vivo, these two proteins typically oscillate from pole-to-pole, while in vitro they can form a variety of distinct patterns. MinC is a passenger protein supposedly operating as a downstream cue of the system, coupling it to the division machinery. The MinCDE system has helped to extract not only the principles underlying intracellular patterns, but also how they are shaped by cellular boundaries. Moreover, it serves as a model to investigate how patterns can confer information through specific and non-specific interactions with other molecules. Here, we review how the three Min proteins self-organize to form patterns, their response to geometric boundaries, and how these patterns can in turn induce patterns of other molecules, focusing primarily on experimental approaches and developments.

show abstract

“…For example, a new operon containing mid-cell associated probable division genes has been reported that is specific for obligate anaerobe Clostridium difficile (113). 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).…”

Section: Introductionmentioning

confidence: 99%

Bacterial Cell Division: Nonmodels Poised to Take the Spotlight

Eswara

Ramamurthi

2017

Annu. Rev. Microbiol.

View full text Add to dashboard Cite

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.

show abstract

Oscillating behavior of Clostridium difficile Min proteins in Bacillus subtilis

Cited by 11 publications

References 65 publications

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

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

The E. coli MinCDE system in the regulation of protein patterns and gradients

Bacterial Cell Division: Nonmodels Poised to Take the Spotlight

Contact Info

Product

Resources

About