Bacteriophage genome-wide transposon mutagenesis

Area of Science:

• Bacteriophage genomics
• Microbial genetics
• Molecular biology

Background:

• Bacteriophage genomes are gene-dense, often containing genes with unknown functions.
• Large lytic phages require unbiased functional genomics approaches to understand their genetic makeup.

Purpose of the Study:

• To develop and apply a novel pooled sequencing approach, phage transposon mutagenesis and sequencing (phage TnSeq), for large bacteriophage functional genomics.
• To identify fitness-conferring and dispensable genes in the Pseudomonas aeruginosa jumbo phage ΦKZ.

Main Methods:

• Development of phage TnSeq using the mariner transposase for pooled sequencing.
• Application of phage TnSeq to the jumbo phage ΦKZ (280,334 bp; 371 predicted genes).
• Extension of phage TnSeq to a base-modified phage.

Main Results:

• Approximately 110 genes in phage ΦKZ were identified as fitness-conferring, including known and novel essential genes.
• Around 261 non-essential genes were identified, including some structural proteins (capsid, tail) crucial for fitness.
• Phage TnSeq sensitively assigned quantitative fitness contributions for nearly all genes in parallel.

Conclusions:

• Phage TnSeq is a scalable technology for identifying essential phage genes and generating knockouts in non-essential genes.
• This method provides a comprehensive understanding of gene function and fitness contributions across the phage genome.
• Phage TnSeq is applicable to various phage types, including base-modified phages.