Group I introns in tRNA genes of Patescibacteria

Introns are generally considered rare in bacteria, yet they are frequently observed in Patescibacteria, which have highly reduced genomes. To systematically explore the diversity, roles, and evolution of introns in Patescibacteria, we first focused on the tRNA introns. Using 95 complete genomes, we identified tRNA^Asn and tRNA^Asp genes previously undetected by standard annotation tools due to group I introns inserted at an unusual position, 35/36, in the anticodon loop. <i>In vitro</i> splicing assays confirmed that these introns catalyze precise self-splicing, validating our computational approach. A large-scale survey of complete bacterial genomes revealed that intron insertions at position 35/36 are highly enriched in Patescibacteria but rare in other phyla. Subgroup classification indicated that 81% of all tRNA introns belong to the IC subgroup, whereas nearly all Patescibacteria introns were classified as IA. As most tRNA introns lack homing endonuclease genes, horizontal transfer appears limited. Comparative analysis across bacterial phyla showed that Patescibacteria and Cyanobacteriota exhibit the highest prevalence of group I introns (~40% of genomes). In contrast, group II introns, which require protein cofactors for activity, were more common in other bacteria, including Cyanobacteriota, but absent in Patescibacteria. Collectively, these findings suggest that Patescibacteria harbor introns with phylum-specific trends in abundance, structure, and evolutionary lineage. The coexistence of extensive genome reduction and persistent group I introns may reflect an adaptive strategy, where introns serve as efficient RNA-based regulatory elements, potentially substituting for complex protein-mediated systems.IMPORTANCEIntrons were traditionally thought to be rare in bacteria, yet their occurrence and diversity may have been underestimated. Here, we present the first comprehensive overview of group I and group II introns in Patescibacteria. While most introns are readily identified, group I introns inserted at position 35/36 within the anticodon loop often escape detection by standard annotation tools; through experimental verification, we demonstrate that these introns are accurately spliced despite their unusual insertion site. Notably, approximately 40% of genomes in both Patescibacteria and Cyanobacteriota harbor group I introns; however, while around 20% of Cyanobacteriota genomes also contain group II introns, none were detected in Patescibacteria. These results illustrate a previously overlooked phylogenetic distribution of group I and group II introns across the bacterial domain.