The last duplex base-pair of the phage lambda chromosome. Involvement in packaging, ejection and routing of lambda...

Area of Science:

• Molecular Biology
• Virology
• Genetics

Background:

• The cosN site is crucial for bacteriophage lambda DNA packaging by the terminase enzyme.
• Mutations in cosN typically correlate with defects in DNA packaging and cleavage.
• Specific cosN mutations can be lethal despite moderate effects on cleavage and packaging.

Purpose of the Study:

• To investigate the unusual lethality of a specific cosN mutation at the rightmost base-pair (-1 position).
• To characterize the effects of the cosN-1T mutation on lambda DNA packaging, cleavage, and infectivity.
• To elucidate the role of the rightmost base-pair in DNA injection and subsequent chromosome fate.

Main Methods:

• Genetic and molecular analysis of bacteriophage lambda mutants.
• Assessment of DNA packaging efficiency and cohesive end formation.
• Analysis of DNA injection and chromosome cyclization in infected host cells.
• Calcium-ion mediated transformation assays.

Main Results:

• The cosN-1T mutation, a G.C to T.A transversion at position -1, reduced lambda particle yield fivefold, rendering particles non-infectious.
• Particles with the cosN-1T mutation exhibited normal morphology and contained chromosomes with intact cohesive ends, but were defective in DNA injection (only 25% ejected DNA).
• Ejected chromosomes from cosN-1T particles failed to cyclize, unlike wild-type chromosomes, and the mutation did not affect Ca2+-mediated transformation.

Conclusions:

• The rightmost base-pair of the lambda chromosome is critical for efficient DNA injection into host cells.
• The cosN-1T mutation specifically disrupts DNA ejection and/or subsequent chromosome cyclization, independent of DNA packaging or cleavage.
• Specific DNA:protein interactions involving the rightmost base-pair are essential for successful viral DNA delivery and infection.