Temperature-dependent coliphage induces distinct temporal bacterial morphological dynamics during infection

ABSTRACT Phage infection undergoes a series of physiological transitions, holding crucial information about phage replication dynamics and potential phage-derived antimicrobials. Although phage-induced cytological changes have been used to infer phage-hijacking mechanisms, current approaches are limited by the lack of comprehensive single-cell morphological analysis and insufficient resolution of temporal dynamics, particularly for phages displaying short latent periods, thereby hindering systematic characterization of morphological transitions throughout the infection cycle. Here, we characterized a newly identified coliphage with a genome of 53 kbp, Tiny, which exhibits an unusually long latent period, making it an ideal candidate for resolving temporal morphological transitions. Tiny exhibits both temperature- and host-dependent killing profiles against diverse Escherichia coli strains, including ATCC 25922, uropathogenic E. coli , and avian pathogenic E. coli . While its replication efficiency is temperature-dependent, showing enhanced productivity at lower temperatures, the duration of its adsorption and latent period is largely host-dependent. Depending on the bacterial strain, Tiny exhibits either a prolonged latent period in slow-adsorbing strains or a rapid one in fast-adsorbing strains, regardless of infection temperature, suggesting phage-host compatibility. Single-cell bacterial cytological profiling of Tiny-infected ATCC 25922 cells revealed that Tiny progressively induces distinct bacterial morphological transitions throughout its lytic cycle, suggesting sequential interference with host physiology as part of its replication cycle prior to cell lysis. This work establishes a broadly applicable framework for dissecting lytic phage biology with high temporal resolution and lays the foundation for future integrative omics studies aimed at understanding how phages sequentially modulate their bacterial hosts. IMPORTANCE Antibiotics trigger unique patterns of morphological changes in bacteria, and these compound-specific signatures provide a basis for determining mechanisms of action in antibiotic discovery. By the same concept, phage-induced morphological changes can reveal key insights into phage replication dynamics and guide the identification of phage-derived antimicrobials. However, the complexity of phage biology and the variability of phage-host interactions pose challenges in interpreting these phenotypic outcomes. Here, we employed a phage-host pair that exhibits an unusually prolonged latent duration as a model to establish a broadly applicable framework for dissecting lytic phage biology with high temporal resolution. Through single-cell bacterial morphological analyses, this approach captures dynamic infection processes inducing morphological transitions across the phage replication cycle. This work provides a phenotypic analysis pipeline to advance our understanding of phage-host interactions and lays the foundation for future integrative omics studies to elucidate how phages sequentially modulate their bacterial hosts.