Terminase Subunits from the Pseudomonas-Phage E217.

Area of Science:

• Virology
• Molecular Biology
• Biochemistry

Background:

• Pseudomonas phages are vital for phage therapy against P. aeruginosa.
• Understanding phage DNA packaging mechanisms, particularly terminase subunits, is crucial but limited.

Purpose of the Study:

• To investigate the terminase large (TerL) and small (TerS) subunits of the Pseudomonas phage E217.
• To elucidate the structural and functional characteristics of these subunits involved in DNA packaging.

Main Methods:

• Gene identification and sequence analysis of E217 TerL and TerS.
• X-ray crystallography of the TerL C-terminal domain.
• Cryo-electron microscopy (cryo-EM) reconstruction of the E217 TerS decamer.
• Site-directed mutagenesis of TerL active site residues.
• Analysis of phage growth and burst size upon TerS overexpression.

Main Results:

• E217 TerL exhibits a two-domain structure with an RNase H-like nuclease domain containing two magnesium ions.
• Mutations affecting magnesium coordination in TerL impaired phage growth, suggesting its importance.
• The E217 TerS forms a unique ring-like decamer, differing from known nonameric structures.
• E217 TerS possesses DNA-binding motifs and a central channel, with overexpression inhibiting phage production.

Conclusions:

• The study expands the molecular understanding of terminase subunits in Pseudomonas phages relevant to phage therapy.
• Structural and functional insights into E217 TerL and TerS provide a basis for future phage engineering and therapeutic applications.