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

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 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.