Early antitermination in the atypical coliphage mEp021 mediated by the Gp17 protein

Area of Science:

• Bacteriophage biology
• Molecular genetics
• Microbial genetics

Background:

• Coliphage mEp021 is a non-lambdoid phage requiring host factor Nus for its life cycle.
• It possesses a unique immunity repressor and an Nλ-like antiterminator protein, Gp17.

Purpose of the Study:

• To investigate the function of the Gp17 antiterminator protein and its interaction with nut sites in coliphage mEp021.
• To understand the role of host factor Nus in mEp021 replication and Gp17 activity.

Main Methods:

• Analysis of plasmid constructs with nut sites, transcription terminators, and GFP reporter genes.
• Infection assays using wild-type and mutant mEp021 phages in various host Nus mutants.
• Site-directed mutagenesis of the Gp17 arginine-rich motif (ARM).

Main Results:

• Gp17 expression is crucial for high reporter gene fluorescence, indicating antitermination activity at nut sites.
• Mutations in Gp17's ARM abolish its antiterminator function.
• Transcripts downstream of terminators are produced only when Gp17 is expressed.
• Overexpression of Gp17 partially restores mEp021 production in Nus-mutant hosts, unlike phage lambda.

Conclusions:

• Coliphage mEp021 Gp17 functions as an antiterminator, enabling read-through transcription at multiple nut sites, including a distant nutR2 site.
• The phage exhibits partial independence from host factor Nus for replication when Gp17 is overexpressed.
• Gp17's mechanism differs from canonical lambdoid N proteins, particularly in its interaction with host Nus factors.