Generation of Genetic Tools for Gauging Multiple-Gene Expression at the Single-Cell Level

Area of Science:

• Microbiology
• Molecular Biology
• Genetics

Background:

• Key microbial processes exhibit heterogeneous expression in single bacterial cells.
• Limited molecular tools hinder real-time, multi-gene expression analysis at the single-cell level, impeding understanding of phenotypic heterogeneity.
• Phenotypic heterogeneity is crucial in bacterial processes like competence, sporulation, and persistence.

Purpose of the Study:

• To develop and validate a genetic tool for gauging multiple-gene expression at the single-cell level in *Pseudomonas aeruginosa*.
• To investigate phenotypic heterogeneity using the novel tool.
• To provide a cost-effective method for studying gene expression in bacterial populations.

Main Methods:

• Generation of the pRGC (promoter-probe vector for transcriptional fusions) tool with three distinct fluorescent reporter genes (mCherry, GFP, CFP).
• Characterization and validation of pRGC using constitutive and iron-regulated promoters in *P. aeruginosa*.
• Development of pRGC variants for chromosomal integration or broader bacterial genus application.

Main Results:

• The pRGC tool successfully discriminated three fluorescence signals in single *P. aeruginosa* cells without complex image processing.
• Validated the tool's efficacy for analyzing gene expression of both constitutive and iron-regulated promoters.
• Generated variants suitable for long-term studies and application in diverse bacterial genera.

Conclusions:

• The pRGC genetic tool enables easy, rapid, and cost-effective investigation of multiple-gene expression at the single-cell level.
• Facilitates deeper understanding of microbial phenotypic heterogeneity in environmental and pathogenic bacteria.
• The proof-of-concept study demonstrated its utility in analyzing iron uptake and storage gene expression in *P. aeruginosa*.