A novel Queuovirinae lineage of Pseudomonas aeruginosa phages encode dPreQ0 DNA modifications with a single GA motif that provide restriction and CRISPR Cas9 protection in vitro.

Deazaguanine DNA modifications are widespread in phages, particularly in those with pathogenic hosts. Pseudomonas phage iggy substitutes ∼16.5% of its genomic 2'-deoxyguanosine (G) with dPreQ0, and the iggy deazaguanine transglycosylase (DpdA) is unique in having a strict GA target motif, not observed previously. The iggy PreQ0 modification is shown to provide protection against both restriction endonucleases and Cas9 (when present in PAM), thus expanding our understanding of the deazaguanine modification system, its potential, and diversity. Phage iggy represents a new genus of Pseudomonas phages within the Queuovirinae subfamily; which have very little in common with other published phage genomes in terms of nucleotide similarity (<10%) and common proteins (<2%). Interestingly, shared similarity is concentrated in dpdA and preQ0 biosynthesis genes. TEM imaging confirmed a siphovirus morphology with a prolate icosahedral head and a non-contractile flexible tail with one long central tail spike. The observed protective effect of the deazaguanine modification on the iggy DNA may contribute to its broad within-species host range. Phage iggy was isolated on Pseudomonas aeruginosa PAO1, but also infects PDO300, PAK, PA14, as well as 10 of 27 tested environmental isolates and 13 of 20 tested clinical isolates of P. aeruginosa from patients with cystic fibrosis.

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© The Author(s) 2023. Published by Oxford University Press on behalf of Nucleic Acids Research.