FEDS: a Novel Fluorescence-Based High-Throughput Method for Measuring DNA Supercoiling In Vivo

Area of Science:

• Molecular Biology
• Genetics
• Microbiology

Background:

• DNA supercoiling is crucial for essential cellular processes like transcription and replication.
• Existing methods for measuring DNA supercoiling in vivo are time-consuming and lack single-cell resolution.

Purpose of the Study:

• To develop a high-throughput method for measuring bacterial DNA supercoiling in vivo at the single-cell level.
• To investigate the heterogeneity of DNA supercoiling within bacterial populations.
• To identify novel regulators of DNA supercoiling.

Main Methods:

• Developed Fluorescent Evaluation of DNA Supercoiling (FEDS), a novel method utilizing a dual-fluorescent reporter plasmid.
• FEDS employs a supercoiling-sensitive promoter driving GFP expression and a constitutive promoter driving RFP expression for normalization.
• Applied FEDS to analyze single-cell DNA supercoiling in bacterial populations.

Main Results:

• FEDS enables high-throughput, single-cell measurement of bacterial DNA supercoiling in vivo.
• Revealed significant single-cell heterogeneity in DNA supercoiling, challenging previous assumptions of homogeneous population shifts.
• Identified a regulatory feedback loop where increased DNA supercoiling represses a gene that decreases supercoiling.

Conclusions:

• FEDS is a powerful tool for dissecting DNA supercoiling dynamics at the single-cell level.
• Bacterial populations exhibit complex, heterogeneous DNA supercoiling patterns.
• Discovered a novel regulatory mechanism influencing DNA supercoiling in Salmonella enterica.