Efficient RecABC-dependent, homologous recombination between coliphage lambda and plasmids requires a phage ninR...

Area of Science:

• Molecular Biology
• Genetics
• Microbial Genetics

Background:

• Phage lambda vectors are crucial tools in molecular biology for cloning and genetic manipulation.
• Homologous recombination is a fundamental process in DNA repair and genetic exchange.
• Previous studies indicated variability in phage-plasmid recombination efficiencies, but the underlying genetic factors were not fully elucidated.

Purpose of the Study:

• To identify and characterize the genetic determinant responsible for increased phage-plasmid homologous recombination frequencies mediated by the RecABC pathway.
• To map the identified gene within the phage lambda genome.
• To explain the observed inefficiencies in screening phage libraries using phage-plasmid recombination and to propose solutions.

Main Methods:

• Genetic mapping of the recombination determinant within the phage lambda genome.
• Comparative analysis of recombination frequencies in different phage lambda vectors.
• Characterization of the identified gene's function in recombination.

Main Results:

• A gene, designated rap (recombination adept with plasmid), located at ninG (orf 204) of phage lambda, was identified.
• The rap gene confers a 100-fold increase in recombinant frequencies for RecABC pathway-mediated, phage-plasmid homologous recombination.
• A similar recombination determinant was found in Charon 4A, located within phi 80-derived sequences.
• The absence of the Rap+ phenotype in certain lambda vectors was identified as the cause of inefficient library screening.

Conclusions:

• The rap gene is a key determinant for efficient phage-plasmid homologous recombination.
• Understanding the rap gene's location and function is critical for optimizing lambda vectors.
• The findings enable the rational design of improved lambda vectors for enhanced screening of phage libraries via homologous recombination.