New quantitative methods for measuring plasmid loss rates reveal unexpected stability

Area of Science:

• Microbiology
• Molecular Biology
• Genetics

Background:

• Plasmid loss rate measurements are crucial for understanding plasmid stabilization.
• Conventional assays often overestimate plasmid loss rates due to growth rate differences between plasmid-free and plasmid-containing cells.
• Existing methods to correct for growth differences are susceptible to experimental errors.

Purpose of the Study:

• To develop and validate new experimental methods for accurately measuring inherent plasmid loss rates.
• To re-evaluate plasmid loss frequencies for common plasmids like R1 and pSC101 derivatives.
• To investigate potential unknown stabilization mechanisms in plasmids.

Main Methods:

• Mathematical analysis demonstrating how growth rate errors impact loss rate estimations.
• Development of a high-throughput microscopy assay for rapid screening of plasmid-free cells (tens of minutes).
• Modification of a counterselection-based assay, inspired by the Luria-Delbrück test, to separate plasmid loss from growth effects.

Main Results:

• Both novel methods yielded concordant results, indicating significantly lower plasmid loss rates than previously reported.
• The par⁻ version of mini-R1 showed an observed loss rate of approximately 10⁻³ per cell per generation.
• The measured loss rate for par⁻ mini-R1 appears lower than expected based on known R1 stabilization mechanisms.

Conclusions:

• Plasmid loss rates are likely much lower than estimated by conventional methods.
• The low observed loss rate for par⁻ mini-R1 suggests the existence of uncharacterized plasmid stabilization mechanisms.
• Further research is needed to elucidate these novel stabilization strategies, potentially involving copy number control or improved partitioning.