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Microscopic methods for distinguishing among three cell types in TOL plasmid-carrying Pseudomonas putida cultures
1Department of Environmental Engineering and Sciences, Clemson University, SC 29634-0919, USA.
Abstract:
Microscopic methods were developed that enable the sensitive quantification of different cell types that are generated by plasmid instability processes when Pseudomonas putida PaW164 (X+), which carries a TOL plasmid (pWW0-164), is grown in chemostat culture. Cells that have lost the structural TOL genes (X-) or the entire TOL plasmid (X0) can be quantified in a background of 6000 X+ cells using catechol agarose miniplates. X0 cells can be quantified in a background of 3500 X+ or X- cells using carbenicillin agarose miniplates. These methods represent significant improvements in sensitivity over conventional plating methods.
Insights
New microscopic methods precisely quantify Pseudomonas putida cell types affected by plasmid instability. These sensitive techniques improve the detection of cells with lost TOL genes or plasmids in bacterial cultures.
Area of Science:
- Microbiology
- Molecular Biology
- Biotechnology
Background:
- Plasmid instability in Pseudomonas putida (PaW164) affects bacterial populations during chemostat culture.
- Understanding these instabilities is crucial for maintaining desired bacterial traits and functions.
Purpose of the Study:
- To develop sensitive microscopic quantification methods for bacterial cell types arising from plasmid instability.
- To accurately enumerate cells with TOL gene loss (X-) or complete TOL plasmid loss (X0) in Pseudomonas putida cultures.
Main Methods:
- Development of novel microscopic quantification techniques using catechol and carbenicillin agarose miniplates.
- Application of these methods to detect and quantify specific cell populations within a mixed bacterial background.
Main Results:
- Quantification of X- cells (TOL gene loss) in a background of 6000 X+ cells (wild-type) using catechol miniplates.
- Quantification of X0 cells (plasmid loss) in a background of 3500 X+ or X- cells using carbenicillin miniplates.
- Demonstrated significant improvements in sensitivity compared to conventional plating methods.
Conclusions:
- The developed microscopic methods offer highly sensitive and specific quantification of bacterial cells affected by plasmid instability.
- These techniques provide valuable tools for studying plasmid dynamics and managing bacterial populations in research and industrial applications.