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Updated: Apr 19, 2026
Generation of Fluorescent Protein Fusions in Candida Species
Published on: March 4, 2017
Use of mCherry Red fluorescent protein for studies of protein localization and gene expression in Clostridium
Eric M Ransom1, Craig D Ellermeier1, David S Weiss2
1Department of Microbiology, University of Iowa, Iowa City, Iowa, USA.
Abstract:
Fluorescent proteins are powerful reporters in biology, but most require O2 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 CFPopt 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 CFPopt. 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 σ(v) and that induction by lysozyme is dose dependent and uniform across the population of lysozyme-treated cells.
Insights
Researchers developed mCherryOpt, a novel fluorescent protein for studying anaerobic bacteria like Clostridium difficile. This tool enhances protein localization and gene expression studies in this important pathogen.
Area of Science:
- Microbiology
- Molecular Biology
- Biotechnology
Background:
- Fluorescent proteins are vital biological reporters but typically require oxygen for maturation, limiting their use in anaerobic organisms.
- Clostridium difficile, a significant cause of hospital-acquired diarrhea, is a strict anaerobe, necessitating new research tools.
- Previous work showed codon-optimized cyan fluorescent protein (CFPopt) can be used in C. difficile with fixation.
Purpose of the Study:
- To develop and characterize a new codon-optimized fluorescent protein, mCherryOpt, for enhanced utility in anaerobic bacteria.
- To create and validate plasmids for protein localization and gene expression studies in Clostridium difficile using mCherryOpt.
Main Methods:
- Codon optimization of mCherry for low-GC content bacteria.
- Construction of mCherryOpt-based plasmids: pRAN473 (protein localization) and pDSW1728 (gene expression).
- Demonstration of protein localization (MldA, ZapA) and gene expression (pdaV operon) in C. difficile.
Main Results:
- mCherryOpt shows faster fluorescence acquisition and better signal-to-noise ratio than CFPopt.
- Septal localization of cell division proteins MldA and ZapA was successfully visualized.
- Expression of the pdaV operon was characterized, confirming dependence on sigma factor σ(v) and dose-dependent lysozyme induction.
Conclusions:
- mCherryOpt is a superior fluorescent reporter for anaerobic bacteria, offering improved performance over CFPopt.
- The developed plasmids (pRAN473, pDSW1728) are effective tools for studying protein localization and gene expression in C. difficile.
- This work provides valuable new methods for investigating the biology of Clostridium difficile.
