Cloning, expression, and mapping of the Aeromonas hydrophila aerolysin gene determinant in Escherichia coli K-12

Area of Science:

• Microbiology
• Molecular Biology
• Genetics

Background:

• Aeromonas hydrophila is a Gram-negative bacterium known for producing toxins.
• Aerolysin is a key virulence factor contributing to the pathogenicity of A. hydrophila.

Purpose of the Study:

• To identify and characterize the aerolysin gene (aer) from Aeromonas hydrophila AH2.
• To determine the DNA regions responsible for aerolysin expression and activity.
• To analyze the protein product of the aerolysin gene.

Main Methods:

• Screening of a cosmid gene library for hemolytic and cytotoxic activities.
• Plasmid construction and functional expression in Escherichia coli K-12.
• Deletion analysis and transposon mutagenesis.
• Protein detection using E. coli maxicells and immunoblotting.

Main Results:

• A 5.8-kilobase EcoRI fragment containing the aerolysin gene (aer) was identified.
• The functional aerolysin gene product was localized to a 1.4-kilobase region.
• Flanking DNA regions (aerB and aerC) were found to modulate aerolysin production and activity.
• The aerolysin gene product was identified as a 54,000-dalton protein.

Conclusions:

• The gene coding for aerolysin was designated aerA.
• Regulatory regions aerB and aerC influence aerolysin expression and activity.
• Understanding these genetic elements is crucial for studying A. hydrophila pathogenicity.