Persistent pseudolysogeny of bacteriophage phiSASD1 in Streptomyces avermitilis and its extrachromosomal plasmid-like DNA form

ABSTRACT Pseudolysogeny represents a distinct phage-host interaction mode that differs from the classical lytic and lysogenic cycles, yet it remains poorly characterized in filamentous actinomycetes. In this study, bacteriophage phiSASD1 infecting Streptomyces avermitilis was investigated. Three phiSASD1-carrying strains exhibiting pseudolysogenic characteristics were obtained through a plaque survival assay combined with PCR verification. Physiological characterization revealed that these strains exhibited stable resistance to superinfection by the homologous phage, while SAPL-1 continuously released high titers of infectious phage particles (up to 4.62 × 10⁷ PFU/mL) and maintained substantial mycelial biomass during the tested cultivation period. Adsorption assays showed that the representative strain SAPL-1 displayed markedly reduced adsorption capacity for phiSASD1, and mitomycin C treatment did not induce significant phage production, suggesting an atypical infection pattern distinct from classical integrative lysogeny. Southern blot analysis showed that the phiSASD1 genome was present in both total DNA and plasmid DNA fractions, with restriction patterns consistent with those of the free phage genome. Pulsed-field gel electrophoresis further supported the presence of an extrachromosomal phage-derived DNA molecule, while transmission electron microscopy revealed population heterogeneity, including intact and lysing cells. Functional assessment of the putative recombinase-associated region did not detect chromosomal integration under the tested conditions. Collectively, these results support a persistent pseudolysogenic association in which phiSASD1 is maintained as an extrachromosomal plasmid-like DNA form. This study provides new insights into alternative survival strategies of actinobacteriophages and provides a basis for further mechanistic studies and for phage monitoring and control in industrial fermentation systems. IMPORTANCE To our knowledge, this study provides systematic evidence that bacteriophage phiSASD1 can establish a persistent pseudolysogenic association with the industrially important actinomycete Streptomyces avermitilis , while its genome persists outside the host chromosome as an extrachromosomal plasmid-like DNA element. These findings extend the classical lytic-lysogenic framework by illustrating a persistent phage-host coexistence mode characterized by non-integrated phage DNA maintenance, continuous production of infectious phage particles, and population heterogeneity. The study therefore provides a basis for further investigation of the molecular mechanisms underlying phage persistence and phage-host coexistence and may inform phage monitoring and contamination control strategies in industrial fermentation systems.