Flavin-based fluorescent proteins: emerging paradigms in biological imaging

Mukherjee, Arnab; Schroeder, Charles M.

doi:10.1016/j.copbio.2014.07.010

Cited by 80 publications

(71 citation statements)

References 41 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…In addition, the requirement for O 2 precludes use of mCherryOpt for studies of dynamic protein localization in live C. difficile cells, although this should not be a problem in anaerobes that tolerate transient exposure to air (44). Some alternative fluorescent labels currently under development are smaller and do not require O 2 , so they may overcome these limitations (45,46). The limitations of mCherryOpt notwithstanding, our findings suggest it will prove to be a useful addition to the C. difficile molecular biology toolbox.…”

Section: Discussionmentioning

confidence: 99%

Use of mCherry Red Fluorescent Protein for Studies of Protein Localization and Gene Expression in Clostridium difficile

Ransom

Ellermeier

Weiss

2015

Appl Environ Microbiol

105

123

View full text Add to dashboard Cite

Fluorescent proteins are powerful reporters in biology, but most require O 2 for chromophore maturation, making them inherently difficult to use in anaerobic bacteria. Clostridium difficile, a strict anaerobe with a genomic GC content of only 29%, is the leading cause of hospital-acquired diarrhea in developed countries, and new methods for studying this pathogen are sorely needed. We recently demonstrated that a cyan fluorescent protein called CFP opt that has been codon optimized for production in low-GC bacteria can be used to study protein localization in C. difficile provided the cells are fixed prior to exposure to air. We describe here a codon-optimized variant of mCherry (mCherryOpt) that exhibits faster acquisition of fluorescence and a better signal-to-noise ratio than CFP opt . We utilized mCherryOpt to construct plasmids for studying protein localization (pRAN473) and gene expression (pDSW1728) in C. difficile. Plasmid pRAN473 is an mCherryOpt fusion vector with a tetracycline-inducible promoter. To document its biological utility, we demonstrated septal localization of two cell division proteins, MldA and ZapA. Plasmid pDSW1728 is designed for cloning a promoter of interest upstream of mCherryOpt. As proof of principle, we studied the expression of the pdaV operon, which is required for lysozyme resistance. In confirmation and extension of previous reports, we found that expression of the pdaV operon requires the alternative sigma factor v and that induction by lysozyme is dose dependent and uniform across the population of lysozyme-treated cells.C lostridium difficile is a low-GC, spore-forming bacterium that is burdening the health care systems of developed countries (1-3). While genetic techniques to study C. difficile are becoming increasingly available, the repertoire of tools remains limited. This is due in part to the strictly anaerobic environment required to grow C. difficile.Green fluorescent protein (GFP) can be produced in cells grown anaerobically, but it is unable to fluoresce because chromophore maturation requires O 2 for dehydration reactions that introduce double bonds into amino acids (4). Nevertheless, GFP produced in an anaerobic environment can mature and fluoresce upon subsequent exposure to air (4, 5). We recently took advantage of this observation to show that GFP can be used to localize cell division proteins in anaerobically grown Escherichia coli (6). Similarly, we showed that a derivative of cyan fluorescent protein named CFP opt (because it has been codon optimized for low-GC bacteria) can be used to localize cell division proteins in anaerobically grown C. difficile (6). In both organisms, it was necessary to fix cells anaerobically to preserve their architecture and then expose them to air overnight to allow chromophore maturation, which required many hours. Fixation was necessary in the case of E. coli to ensure that the localization observed reflected anaerobic conditions rather than subsequent adaptation to air. In the case of C. difficile, fixation was necessary b...

show abstract

Section: Discussionmentioning

confidence: 99%

Use of mCherry Red Fluorescent Protein for Studies of Protein Localization and Gene Expression in Clostridium difficile

Ransom

Ellermeier

Weiss

2015

Appl Environ Microbiol

105

123

View full text Add to dashboard Cite

show abstract

“…Replacement of this cysteine within the LOV domain abolishes its photochemical activity, improving the fluorescent properties of the protein [12,13]. Mutation of this invariant cysteine is therefore a prerequisite for the generation LOV-based FPs [14 ].…”

Section: Lov-domain Function and Fluorescencementioning

confidence: 99%

LOV-based reporters for fluorescence imaging

Buckley

Petersen

Roe

et al. 2015

Current Opinion in Chemical Biology

107

102

View full text Add to dashboard Cite

Chromophore-binding domains from plant and bacterial photoreceptor proteins have recently gathered increasing attention as new sources of genetically encoded fluorescent proteins (FPs). In particular, FPs based on the flavin-binding LOV (light, oxygen, or voltage sensing) domain offer advantages over green fluorescent protein (GFP) owing to their smaller size, pH and thermal stability, utility under anaerobic conditions and their ability to generate reactive oxygen species. This review focuses on the potential applications of this emerging class of fluorescent reporters, discusses the advantages and limitations of LOV-based FPs, whilst offering insights regarding the further development of this technology for bioimaging and photodynamic therapy.

show abstract

“…So far, there has only been a flavin-based fluorescent protein derived from Pseudomonas putida that works under anaerobic conditions [100]. This has been used to characterize two endogeneous promoters of C. cellulolyticum [101].…”

Section: Genetic Partsmentioning

confidence: 99%

Gas fermentation: cellular engineering possibilities and scale up

2017

View full text Add to dashboard Cite

Low carbon fuels and chemicals can be sourced from renewable materials such as biomass or from industrial and municipal waste streams. Gasification of these materials allows all of the carbon to become available for product generation, a clear advantage over partial biomass conversion into fermentable sugars. Gasification results into a synthesis stream (syngas) containing carbon monoxide (CO), carbon dioxide (CO2), hydrogen (H2) and nitrogen (N2). Autotrophy–the ability to fix carbon such as CO2 is present in all domains of life but photosynthesis alone is not keeping up with anthropogenic CO2 output. One strategy is to curtail the gaseous atmospheric release by developing waste and syngas conversion technologies. Historically microorganisms have contributed to major, albeit slow, atmospheric composition changes. The current status and future potential of anaerobic gas-fermenting bacteria with special focus on acetogens are the focus of this review.

show abstract

Flavin-based fluorescent proteins: emerging paradigms in biological imaging

Cited by 80 publications

References 41 publications

Use of mCherry Red Fluorescent Protein for Studies of Protein Localization and Gene Expression in Clostridium difficile

Use of mCherry Red Fluorescent Protein for Studies of Protein Localization and Gene Expression in Clostridium difficile

LOV-based reporters for fluorescence imaging

Gas fermentation: cellular engineering possibilities and scale up

Contact Info

Product

Resources

About