A set of powerful negative selection systems for unmodified Enterobacteriaceae

Area of Science:

• Microbiology
• Bacterial Genetics
• Molecular Biology

Background:

• Genetic manipulation is crucial for understanding bacterial pathogenesis.
• Existing genetic tools are often limited to laboratory strains, hindering research on clinical isolates.
• Developing broadly applicable genetic systems for diverse bacterial strains remains a challenge.

Purpose of the Study:

• To design and validate a modular and general negative selection strategy for genetic engineering in clinical bacterial isolates.
• To achieve high selection stringency in Escherichia coli and Salmonella without strain-specific optimization.
• To enable genetic transfer and manipulation in arbitrary bacterial strains, including clinical isolates.

Main Methods:

• Development of a negative selection system based on inducible toxins.
• Testing the system's efficacy and stringency in clinical isolates of Escherichia coli and Salmonella.
• Application of the system for allele transfer via phage-mediated generalized transduction and direct transfer between strains.

Main Results:

• The designed negative selection system demonstrated high stringency in clinical Escherichia coli and Salmonella isolates.
• The system outperformed previously reported negative selection systems in unmodified E. coli strains.
• The high stringency facilitated negative selection in phage-mediated transduction and allele transfer between arbitrary strains without requiring phage.

Conclusions:

• The modular negative selection strategy offers a powerful and generalizable tool for bacterial genetics.
• This system overcomes limitations of existing tools, enabling definitive genetic experiments in both laboratory and clinical bacterial isolates.
• The design holds potential for extension to other bacterial species within the Enterobacteriaceae family and beyond.