RecT Recombinase Expression Enables Efficient Gene Editing in Enterococcus spp

Area of Science:

• Microbiology
• Genetics
• Molecular Biology

Background:

• Enterococcus faecium is a versatile bacterium with both beneficial commensal roles and pathogenic, antibiotic-resistant strains.
• Current gene-editing methods for E. faecium are inefficient, hindering functional and mechanistic studies.
• Understanding the genetic basis of E. faecium's diverse activities is crucial due to its public health significance.

Purpose of the Study:

• To develop improved gene-editing technologies for Enterococcus species.
• To enhance the efficiency of recombineering in both commensal and antibiotic-resistant E. faecium strains.
• To facilitate genetic studies for characterizing E. faecium functions and mechanisms.

Main Methods:

• Expression of Enterococcus faecium RecT recombinase to improve recombineering efficiency.
• Combined use of RecT, CRISPR-Cas9, and guide RNAs (gRNAs) for scarless single-stranded DNA recombineering.
• Utilized RecT for chromosomal insertions of DNA templates for gene deletion mutants.
• Employed CRISPR-Cas9-mediated recombineering to knock out sortase A genes.

Main Results:

• RecT expression significantly boosted recombineering efficiency in various Enterococcus species.
• CRISPR-Cas9-RecT system enabled highly efficient scarless gene editing in E. faecium.
• RecT facilitated efficient chromosomal DNA insertions for generating gene deletion mutants.
• Successful knockout of sortase A genes demonstrated the utility of the developed methods.

Conclusions:

• RecT-mediated recombineering provides a rapid and efficient method for genetic manipulation in E. faecium.
• These improved methods will accelerate functional and mechanistic studies of Enterococcus species.
• The developed techniques address limitations in current genetic engineering approaches for E. faecium.