Examination of Enterococcus faecalis Toxin-Antitoxin System Toxin Fst Function Utilizing a Pheromone-Inducible...

Area of Science:

• Microbiology
• Molecular Biology
• Genetics

Background:

• Enterococcus faecalis is a significant nosocomial pathogen and a model organism for Gram-positive cocci.
• Existing genetic tools for E. faecalis have limitations in regulated gene expression, often showing high background expression or using problematic inducers.

Purpose of the Study:

• To construct and characterize a novel expression vector for E. faecalis with tightly regulated gene expression.
• To demonstrate the utility of this vector for studying toxin-antitoxin (TA) systems.

Main Methods:

• Construction of the pCIE expression vector utilizing the Pq pheromone-responsive promoter from plasmid pCF10.
• Demonstration of tight repression, sensitivity to low pheromone levels, and a wide dynamic range of the Pq promoter.
• Application of the vector to control expression of toxic peptides from two E. faecalis TA loci: par_pAD1 and par-EF0409.

Main Results:

• The Pq promoter exhibits tight repression and responds to nanogram quantities of peptide pheromone, offering a large dynamic range.
• The vector allowed for the controlled expression of toxic peptides from both plasmid and chromosomal TA loci.
• Differences in toxin regulation and effects were observed between the two studied TA systems.

Conclusions:

• The pCIE vector provides a valuable tool for regulated gene expression in E. faecalis.
• This system overcomes limitations of previous vectors, enabling precise study of TA systems and other genes.
• The vector facilitates deeper investigation into E. faecalis physiology and the function of par TA systems.