Development of a method for markerless gene deletion in Pseudomonas putida

Area of Science:

• Microbiology
• Molecular Biology
• Genetic Engineering

Background:

• Pseudomonas putida is a versatile bacterium with significant industrial and environmental applications.
• Efficient genetic manipulation tools are crucial for engineering P. putida strains for specific functions.
• Existing methods for gene deletion can be laborious and may leave unwanted markers.

Purpose of the Study:

• To develop a novel negative counterselection system for Pseudomonas putida.
• To facilitate markerless in-frame gene deletions in P. putida.
• To enable the disruption of specific genes, including those involved in surface adhesion and flagellar motility.

Main Methods:

• Development of a negative counterselection system utilizing uracil phosphoribosyltransferase (UPRTase).
• Construction of a P. putida strain resistant to 5-fluorouracil (5-FU).
• Design and utilization of vectors for targeted gene disruption (surface adhesion protein, flagellum operon, endonuclease genes).

Main Results:

• Successful implementation of the uracil phosphoribosyltransferase-based negative counterselection system.
• Efficient disruption of target genes, including surface adhesion protein, flagellum biosynthesis operon, and endonuclease genes.
• Generation of markerless, in-frame chromosomal deletions in P. putida.

Conclusions:

• The developed negative counterselection system is effective for Pseudomonas putida.
• This system allows for precise and scarless genetic modifications.
• The methodology facilitates advanced genetic engineering of P. putida for research and industrial purposes.