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Fluorescent reporter plasmids for single-cell and bulk-level composition assays in E. faecalis
Kelsey M Hallinen1, Keanu A Guardiola-Flores1, Kevin B Wood1,2
1Department of Biophysics, University of Michigan, Ann Arbor, Michigan, United States of America.
Plos One
|May 6, 2020
Summary
Researchers developed new fluorescent reporters for studying Enterococcus faecalis (E. faecalis) bacterial populations. These tools enable tracking bacterial dynamics at single-cell and community levels, aiding in selection and competition experiments.
Area of Science:
- Microbiology
- Bacterial genetics
- Fluorescence microscopy
Background:
- Fluorescent reporters are crucial for monitoring bacterial population dynamics.
- Limited reporter options exist for Gram-positive bacteria like Enterococcus faecalis (E. faecalis).
Purpose of the Study:
- To expand the available fluorescent reporter toolkit for E. faecalis.
- To identify optimal reporters for bulk and single-cell level assays in E. faecalis.
Main Methods:
- Eight fluorescent reporters were integrated into a plasmid (pBSU101) for E. faecalis.
- Fluorescence spectra were measured in liquid cultures across growth phases.
- Spectral unmixing was used to analyze two-strain mixtures.
- Codon-optimized blue (BFP) and red (RFP) fluorescent proteins were incorporated.
Main Results:
- All constructs showed detectable fluorescence in E. faecalis cells and biofilms.
- Reporter signals varied with cell density and growth phase, with clear spectral signatures in later phases.
- Six reporter pairs enabled accurate population estimation in two-strain mixtures using spectral unmixing.
- Codon-optimized BFP and RFP enhanced fluorescence in growing cells.
Conclusions:
- Developed and validated novel fluorescent reporters for E. faecalis.
- Identified specific reporter pairs suitable for quantitative population analysis.
- Demonstrated the utility of codon optimization for improved reporter performance.
- Provided a foundation for fluorescence-based assays in E. faecalis selection and competition studies.
