A novel putative genus phage CW39: implications for CRISPR-Cas9-based phage resistance in Streptomyces avermitilis.

Streptomyces avermitilis NRRL 8165 is an industrial model strain for the production of abamectin. Phage contamination during the fermentation process can lead to significant economic losses. At present, the known phages of S. avermitilis NRRL 8165 are scarce, making it difficult to meet the demands of research on pollution control mechanisms. Here, we isolated a novel phage, termed CW39, from soil samples. This phage exhibits a unique biological characteristic of solid-dependent infection. Transmission electron microscopy (TEM) revealed that the head measures approximately 65 nm in diameter, while the tail has a length of roughly 266 nm. Whole-genome sequencing revealed phage CW39 was 122,122 bp in genome size, exhibiting a GC content of 49.34%. Average nucleotide identity (ANI) analysis showed that phage CW39 shares only 64.4% nucleotide identity with its closest relative in the genus Samistivirus, which is significantly below the 70% genus-level classification threshold set forth by the International Committee on Taxonomy of Viruses (ICTV). Phylogenetic tree analysis further revealed that phage CW39 forms an independent monophyletic branch at the root. These findings indicate that phage CW39 likely represents a novel putative genus within the class Caudoviricetes. CRISPR-Cas9 plasmids targeting three key proteins of CW39 (the major capsid protein, head maturation protease, and portal protein) were separately transformed into S. avermitilis NRRL 8165, and all conferred enhanced phage resistance. This study not only enriches the Streptomyces phage resource library, but also provides a feasible strategy for using the CRISPR-Cas9 system to prevent and control phage contamination in industrial fermentation.