Systematic mapping of bacteriophage gene essentiality with HIDEN-SEQ

Key Points:

• The study details comprehensive protocols for preparing culture media, handling bacterial strains, isolating uropathogenic E. coli, culturing bacteriophages, and constructing plasmids and phage mutants, including the use of CRISPR–Cas systems for genetic manipulation.
• HIDEN-SEQ libraries were constructed for phages T4, Bas37, and Bas54 using mariner transposon mutagenesis combined with anti-CRISPR gene insertion and Cas13a-based counterselection to generate saturated mutant pools for functional genomic analysis.
• Competitive fitness assays were performed by infecting host bacteria with phage mutant libraries at low multiplicity of infection, followed by genomic DNA extraction and sequencing to assess gene essentiality under various conditions, including defense system activation and different growth media.
• Sequencing data from HIDEN-SEQ libraries were processed with quality control, read trimming, subsampling, and transposon insertion site mapping; gene essentiality was classified using a custom pipeline integrating insertion saturation, positional bias, and read counts to categorize genes into essentiality tiers.
• Additional analyses included conditional essentiality testing via resampling statistics, genome sequencing and assembly of new clinical E. coli isolates and phage mutants, and structural prediction of candidate antidefense genes using bioinformatics tools such as AlphaFold and HHpred.