Functional analysis of the holin-like proteins of mycobacteriophage Ms6

Area of Science:

• Microbiology
• Molecular Biology
• Virology

Background:

• The Ms6 bacteriophage, a double-stranded DNA virus, possesses a lysis module with an endolysin (LysA) and a class II holin-like protein (Gp4).
• Additional lysis proteins, Gp1, LysB, and Gp5, are encoded within the Ms6 lysis module.
• A second putative holin, Gp5, with a single transmembrane domain, was identified at the end of the lytic operon.

Purpose of the Study:

• To investigate the function of Ms6 lysis proteins, particularly Gp4 and Gp5, in the phage lysis process.
• To determine the roles of Gp4 and Gp5 in controlling the timing of lysis during mycobacteriophage Ms6 infection.

Main Methods:

• Analysis of amino acid sequences to identify protein domains (e.g., SAR, TMD).
• Bacterial lysis assays using pairwise combinations of endolysin and holin proteins in Escherichia coli.
• Construction and analysis of Ms6 deletion mutants (Ms6(Δgp4), Ms6(Δgp5)).
• One-step growth curves and single-burst-size experiments to assess phage viability and lysis timing.
• Protein cross-linking experiments to study Gp4 and Gp5 interactions.

Main Results:

• Neither Gp4 nor Gp5 alone triggered lysis with LysA in E. coli.
• Ms6 mutants lacking Gp4 (Ms6(Δgp4)) exhibited accelerated lysis, while mutants lacking Gp5 (Ms6(Δgp5)) showed delayed lysis.
• Cross-linking experiments confirmed that Ms6 Gp4 and Gp5 can oligomerize and interact with each other.
• The gene products of gp4 and gp5 are nonessential for phage viability but influence lysis timing.

Conclusions:

• The Ms6 lysis module involves more than the canonical endolysin-holin system.
• Both Gp4 and Gp5 function as holins, contributing to the regulation of lysis timing.
• The coordinated action of Gp4 and Gp5 is crucial for achieving programmed lysis timing in Ms6 infection.