Positive selection procedure for entrapment of insertion sequence elements in gram-negative bacteria.

Area of Science:

• Microbiology
• Molecular Biology
• Genetics

Background:

• Insertion sequence (IS) elements are mobile genetic components found in prokaryotic genomes.
• Efficient methods for isolating and characterizing IS elements in diverse bacterial species are crucial for understanding genome dynamics.
• Gram-negative bacteria present unique challenges for genetic manipulation and element isolation.

Purpose of the Study:

• To construct a versatile broad-host-range plasmid for the positive selection and isolation of IS elements in gram-negative bacteria.
• To demonstrate the utility of the constructed plasmid in identifying novel IS elements.
• To characterize a putative IS element isolated from Agrobacterium tumefaciens.

Main Methods:

• Construction of the broad-host-range plasmid pUCD800, incorporating the sacB gene from Bacillus subtilis.
• Utilizing the sacB gene for positive selection on sucrose-containing media, where its inactivation confers resistance.
• Transformation of gram-negative bacteria with pUCD800 and selection for growth on 5% sucrose medium.

Main Results:

• The plasmid pUCD800 enabled positive selection for IS element insertions that inactivate the sacB gene.
• A 1.4-kilobase putative IS element was successfully isolated from Agrobacterium tumefaciens NT1RE.
• The isolated IS element demonstrated characteristics suggesting it is unique to Agrobacterium strains.

Conclusions:

• The pUCD800 plasmid is an effective tool for the positive selection and isolation of IS elements in gram-negative bacteria.
• This approach facilitates the discovery of novel mobile genetic elements.
• The identified IS element represents a potentially new genetic component specific to Agrobacterium.