Journal article
Use of mCherry Red fluorescent protein for studies of protein localization and gene expression in Clostridium difficile
Applied and environmental microbiology, Vol.81(5), pp.1652-1660
03/2015
DOI: 10.1128/AEM.03446-14
PMCID: PMC4325159
PMID: 25527559
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.
Details
- Title: Subtitle
- Use of mCherry Red fluorescent protein for studies of protein localization and gene expression in Clostridium difficile
- Creators
- Eric M Ransom - Department of Microbiology, University of Iowa, Iowa City, Iowa, USACraig D Ellermeier - Department of Microbiology, University of Iowa, Iowa City, Iowa, USADavid S Weiss - Department of Microbiology, University of Iowa, Iowa City, Iowa, USA david-weiss@uiowa.edu
- Resource Type
- Journal article
- Publication Details
- Applied and environmental microbiology, Vol.81(5), pp.1652-1660
- Publisher
- United States
- DOI
- 10.1128/AEM.03446-14
- PMID
- 25527559
- PMCID
- PMC4325159
- ISSN
- 0099-2240
- eISSN
- 1098-5336
- Grant note
- AI-087834 / NIAID NIH HHS R01 AI087834 / NIAID NIH HHS P51 OD011132 / NIH HHS R01 GM083975 / NIGMS NIH HHS GM-083975 / NIGMS NIH HHS
- Language
- English
- Date published
- 03/2015
- Academic Unit
- Microbiology and Immunology
- Record Identifier
- 9984001137402771
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