Isolation, Engineering, and Ecology of Temperate Phages in the Human Gut

Isolation, Engineering, and Ecology of Temperate Phages in the Human Gut

The human gut contains a large and dynamic microbial ecosystem that includes numerous bacteriophages. Temperate phages capable of lysogeny play an important role in shaping bacterial diversity, facilitating gene exchange, and maintaining ecological stability. Recent metagenomic advances have revealed immense viral diversity within the gut microbiome; however, the functional behavior and induction dynamics of prophages remain poorly understood. About 90% of gut bacteria have prophages, yet the triggers and conditions that cause the transition from the lysogenic to the lytic cycles are not fully characterized. It is necessary to understand these dynamics to elucidate the phage-driven modification of host microbial populations and their potential roles in human health and disease.

This study aimed to identify inducible prophages in diverse human gut bacterial isolates and to determine how environmental stimuli, polylysogeny, and host genetics influence prophage induction and stability.

A total of 252 gut bacterial isolates representing five major phyla were analyzed for prophage induction under eight experimental conditions that mimic gut and stress environments. These involved exposure to standard growth medium, mitomycin C, hydrogen peroxide, Stevia extract, and nutrient depletion. Viral DNA was isolated from supernatants of 433 and sequenced using high-throughput sequencing to detect phage genomes.

Inducible prophages were found by sequence alignment and comparative analyses to define unique viral species. Statistical tests, including Fisher’s exact test, Kendall’s rank correlation, Wilcoxon rank-sum test, and ANOVA, were applied to investigate the associations between polylysogeny, induction conditions, and host genetic factors. Long-read sequencing was performed to detect prophage integration sites in the selected Bacteroidota caccae isolates carrying identical prophages but exhibiting different integration patterns.

About 18% of predicted prophase-like elements were detected in 94% of the isolates. They were experimentally inducible and resulted in the identification of 125 inducible prophages representing 63 unique phage species across 73 isolates.

Polylysogeny, the presence of multiple prophages within a single bacterial host, was particularly prominent in Bacteroidota species, with 68% of lysogens containing multiple inducible prophages. Polylysogenic isolates exhibited higher prophage activation under different conditions compared to single lysogens. This suggests that co-resident prophages may promote simultaneous induction and decrease lysogenic stability.

Differential induction was examined in five identical Bacteroidota caccae isolates, which carry the same two prophages: ΦWilby and ΦPomma. Despite sharing high genomic identity (99% ANI), the isolates revealed distinct induction preferences. ΦWilby was preferentially induced in standard medium (P = 0.006), while hydrogen peroxide exposure equalized induction levels between the two (P = 0.9). Long-read sequencing showed that ΦWilby consistently integrated into a tRNA gene via site-specific recombination, whereas ΦPomma is a transposable prophage and inserted randomly at four different genomic locations. These findings highlight integration site variability as a key determinant of differential induction in genetically identical hosts.

Overall, this study demonstrated that while prophages are prevalent among gut bacteria, only a subset actively undergoes lytic induction in response to experimental or environmental stimuli. Induction is highly context-dependent and influenced by factors like growth conditions, co-resident prophage interactions, and integration site specificity. The strong link between polylysogeny and induction frequency supports models of interprophage communication and anti-repressor cross-reactivity. Additionally, host genome architecture, specifically the prophage insertion site, plays a major role in shaping induction dynamics. Many prophages appear non-inducible and show reduced structural and lytic gene content. This indicates potential prophage domestication or degeneration. The validated collection of phage-host pairs generated in this study provides a valuable resource for future studies in synthetic biology, microbiome engineering, and phage therapy.

Reference: Dahlman S, Avellaneda-Franco L, Rutten EL, et al. Isolation, engineering and ecology of temperate phages from the human gut. Nature. 2025. doi:10.1038/s41586-025-09614-7

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