Efficient markerless gene deletions in Pseudomonas protegens Pf-5 using a upp-based counterselective system

Area of Science:

• Microbiology
• Molecular Biology
• Genetics

Background:

• Pseudomonas protegens Pf-5 is a biocontrol agent.
• Efficient methods for gene deletion are crucial for understanding gene function and genome engineering.

Purpose of the Study:

• To develop a counterselective system for markerless gene deletions in P. protegens Pf-5.
• To enable efficient genetic manipulation of P. protegens Pf-5.

Main Methods:

• Development of a uracil phosphoribosyltransferase (upp)-based counterselective system.
• Utilized a suicide plasmid (pJQ200UPP) for gene deletions.
• Created a 5-fluorouracil (5-FU) resistant strain (Pf5139) for counterselection.

Main Results:

• Successfully achieved markerless deletion of the upp gene.
• Performed markerless gene deletions of the Gac/Rsm system components.
• Successfully deleted a 17 kb DNA fragment with a deletion ratio of 20-50%.
• Constructed a strain with triple gene deletions.
• No correlation observed between deleted fragment length and deletion ratio.

Conclusions:

• The upp-based counterselective system is efficient and valuable for markerless gene deletions in P. protegens Pf-5.
• This system has significant potential for gene function studies and genome reduction in Pseudomonas species.