Engineering the Pseudomonas aeruginosa virus-isolation host viPAO1 reveals how host genetic barriers shape recoverable phage diversity

Technical Summary: Engineering the Pseudomonas aeruginosa virus-isolation host viPAO1

Problem Statement
While metagenomic sequencing has revealed an immense reservoir of viral diversity, experimentally isolated phage collections capture only a small, biased fraction of this diversity. This disparity is particularly acute for temperate phages, which are often predicted in bacterial genomes but rarely validated as infectious particles. A primary contributor to this gap is the host genotype itself. Bacterial isolation hosts possess intracellular defense systems (e.g., restriction-modification, abortive infection systems) and resident prophages that act as biological filters, preventing productive infection by otherwise compatible phages. Consequently, conventional isolation workflows using standard laboratory strains (like P. aeruginosa PAO1 or MPAO1) may systematically exclude diverse phage lineages that cannot overcome these specific host barriers.

Methodology
To address this, the authors engineered a series of progressively permissive P. aeruginosa hosts derived from the MPAO1 strain.

1. Host Engineering: Using DefenseFinder, PADLOC, and PHASTEST, the authors identified candidate defense systems (Gabija, Retron-I-B, Type I restriction-modification, Helicase-DUF2290) and resident prophages (Pf4, a CTX-like prophage, and Pf6) in MPAO1. They sequentially deleted these elements via two-step allelic exchange, culminating in the construction of viPAO1 (virus-isolation PAO1), a strain lacking all identified intracellular defenses and resident prophages.
2. Susceptibility Profiling: The authors challenged the parental strain, intermediate deletion mutants, and viPAO1 with panels of lytic and temperate phages using liquid infection assays. Phage susceptibility was quantified by measuring the area under the bacterial growth curve (AUC) post-infection.
3. Comparative Isolation Experiments:
   • Induced Lysates: Prophages were induced from four clinical P. aeruginosa isolates using mitomycin C. The resulting lysates were enriched and plated on six host backgrounds: MPAO1, viPAO1, and their respective T4P- and LPS-receptor mutant derivatives.
   • Environmental Samples: Four environmental samples (wastewater and biogas slurry) were similarly enriched on the six host backgrounds.
   • Sequencing: Plaque-derived lysates were pooled by host background and subjected to long-read Oxford Nanopore sequencing. Recovered sequences were assembled and curated as phage contigs or genomes.
4. Large-Scale Collection Generation: The authors screened 1,090 clinical P. aeruginosa isolates for inducible temperate phages using viPAO1 and related permissive hosts (dPAO1, dPAO1 ΔpilZ). Recovered phages were plaque-purified, and stable lysogens were generated for whole-genome sequencing to define prophage boundaries and insertion sites.
5. Taxonomic Analysis: Recovered phages and contigs were analyzed using vConTACT3 gene-sharing networks to compare their diversity against publicly available P. aeruginosa reference phages.

Key Results

• Gradient of Permissiveness: Sequential removal of intracellular barriers created a measurable gradient of host permissiveness. The deletion of Gabija and the Type I restriction-modification system produced the most significant increases in phage susceptibility, identifying them as major barriers in the MPAO1 background.
• Increased Recovery and Diversity:
  • In comparative isolation experiments using induced lysates, removing intracellular barriers (viPAO1 vs. MPAO1) resulted in a 3.5-fold increase in the recovery of distinct temperate phage genomes (from 2 genomes/2 genera to 7 genomes/6 genera).
  • In environmental enrichments, viPAO1 recovered 16 phage contigs spanning three genera, compared to only one contig recovered by MPAO1.
  • Receptor modification (T4P and LPS mutants) altered the composition of recovered phage subsets but did not increase the overall recovery count beyond that achieved by removing intracellular barriers.
• Broad Taxonomic Expansion: The large-scale screening of 1,090 clinical isolates yielded a curated collection of 69 unique, individually purified temperate phages. This collection spans 24 predicted genera (including 15 putative novel genera) and seven families (including one putative novel family).
• Network Distribution: Gene-sharing network analysis demonstrated that the recovered phages were broadly distributed across the P. aeruginosa phage diversity space, occupying regions not represented in existing reference databases. This confirms that host engineering accesses genomically distinct and previously under-sampled phage groups.

Significance and Claims
The paper establishes that host genotype is a critical determinant of experimentally recoverable phage diversity. The authors claim that:

1. Host Engineering as a Strategy: Engineering permissive hosts by removing intracellular defense systems and resident prophages is a practical and effective strategy for reducing isolation bias and expanding access to under-sampled regions of cultured phage diversity.
2. Mechanistic Insight: The study identifies specific barriers (notably Gabija and Type I restriction-modification) that limit phage recovery in P. aeruginosa, supporting a multilayered model of host-phage interactions where different barriers restrict different phage subsets.
3. Resource Generation: The study provides a valuable resource in the form of the viPAO1 strain and a curated collection of 69 temperate phages. These resources enable the conversion of predicted prophage regions from metagenomic data into experimentally tractable, individually available phage isolates.
4. Complementary Approach: The authors position host engineering as a necessary complement to environmental sampling and methodological innovation (e.g., droplet-based isolation) for uncovering hidden phage diversity, arguing that sampling source alone is insufficient to capture the full diversity of phages associated with a bacterial species.

The study concludes that while no single host can capture the entirety of natural phage diversity, reducing host-associated barriers significantly enhances the breadth of recoverable phage lineages, thereby improving the representativeness of cultured phage collections.