Chromosomal integration of phage lambda by means of a DNA insertion element

Area of Science:

• Bacteriophage genetics
• Molecular biology
• Microbial genetics

Background:

• Bacteriophage lambda is a well-studied model organism for lysogeny and integration.
• Transposon Tn9 confers chloramphenicol resistance and contains insertion sequence 1 (IS1) elements.
• Lysogenization involves prophage integration into the host chromosome.

Purpose of the Study:

• To investigate the lysogenization and excision capabilities of a modified bacteriophage lambda (lambdacam112).
• To determine the integration sites and excision mechanisms of lambdacam112 in Escherichia coli.
• To elucidate the role of insertion sequence 1 (IS1) in lambda integration and excision.

Main Methods:

• Lysogenization experiments with Escherichia coli K-12 using lambdacam112.
• Analysis of prophage integration sites (e.g., lacY, malB, attB).
• Induction experiments to study prophage excision efficiency.
• Heteroduplex analysis to identify DNA elements at integration junctions.
• Reversion assays to assess IS1-mediated excision activity.

Main Results:

• Lambdacam112 lysogenizes Escherichia coli at chromosomal locations other than attB, such as lacY and malB.
• Prophage excision occurs from various sites with differing efficiencies.
• Insertion sequence 1 (IS1) elements from Tn9 are found at the viral-chromosomal DNA junctions.
• Lambdacam112 exhibits excision activity dependent on IS1 elements.
• Prophage derepression and lambdacam112 infection enhance excision and reversion frequencies.

Conclusions:

• The IS1 termini of Tn9 can serve as alternative integration sites for lambda, replacing the attP site.
• Lambdacam112 encodes an excision activity that recognizes IS1 elements.
• Excision events are catalyzed by an IS1-encoded protein regulated by lambda repressor and N gene.