Structural Insights into Lactococcal Siphophage p2 Baseplate Activation Mechanism

Area of Science:

• Microbiology
• Structural Biology
• Virology

Background:

• Virulent phages infecting *Lactococcus lactis* (L. lactis) pose a significant threat to fermented dairy product quality.
• Skunavirus genus phages are frequently isolated from cheese environments, with phage p2 serving as a model siphophage.
• Phage p2's baseplate, crucial for host recognition, exhibits distinct rested and activated conformations, with activation dependent on Ca2+.

Purpose of the Study:

• To determine the structure of the activated phage p2 baseplate directly within the virion.
• To investigate the structural components and activation mechanism of the phage p2 baseplate.
• To elucidate the phage p2 infection pathway, particularly the interplay between baseplate activation and tail tip protein function.

Main Methods:

• Negative staining electron microscopy (EM) was employed to visualize the phage p2 virion.
• Nanobodies targeting the baseplate were used to stabilize and resolve its structure.
• X-ray crystallography data of the baseplate was used for comparison.

Main Results:

• The activated conformation of the phage p2 baseplate was successfully visualized directly in the virion, consistent with crystal structures.
• A second distal tail (Dit) hexamer was identified as a component of the baseplate, exhibiting a distinct topology.
• An uncoupling between baseplate activation and tail tip protein (Tal) opening was observed, suggesting a more intricate infection process.

Conclusions:

• The study provides the first direct visualization of the activated phage p2 baseplate in its native virion context.
• The discovery of a second Dit hexamer and the uncoupled activation/opening mechanism reveal novel aspects of phage-p2-host interaction.
• These findings necessitate a re-evaluation of the phage p2 infection mechanism, highlighting its complexity.