Versatile allelic replacement and self-excising integrative vectors for plasmid genome mutation and complementation

Area of Science:

• Microbiology
• Molecular Biology
• Genetics

Background:

• Multidrug-resistant plasmids carrying virulence genes are spreading among Gram-negative pathogens.
• Existing vector tools for studying plasmid-borne genes are insufficient for comprehensive analysis.
• Understanding plasmid-encoded virulence factors is crucial for combating infectious diseases.

Purpose of the Study:

• To develop and characterize novel self-excising integrative vectors for studying plasmid-borne genes in Gram-negative pathogens.
• To provide a tool for stable, single-copy complementation without antibiotic selection.
• To facilitate the functional assessment of plasmid-encoded genes and operons.

Main Methods:

• Development of self-excising integrative vectors with chromophore expression for detection.
• Allelic replacement strategies for generating precise plasmid and chromosomal mutations.
• Application of vectors for gene function assessment and operon transfer in pathogenic bacteria.

Main Results:

• Demonstrated the utility of self-excising integrative vectors for stable, single-copy complementation.
• Successfully generated markless point mutations and moved large operons using the developed vectors.
• Utilized chromophore expression for efficient detection of target gene modification and colony isolation.

Conclusions:

• The newly developed vectors are versatile tools for studying plasmid-encoded genes in medically important Gram-negative pathogens.
• These vectors enable functional genetic analysis, including complementation and gene/operon manipulation, without antibiotic pressure.
• The system simplifies screening procedures, accelerating research on plasmid-mediated virulence and resistance.