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Abstract Trillions of microorganisms, collectively known as the microbiome, inhabit our bodies with the gut microbiome being of particular interest in biomedical research. Bacteriophages, the dominant virome constituents, can utilize suppressor tRNAs to switch to alternative genetic codes (e.g., the UAG stop-codon is reassigned to glutamine) while infecting hosts with the standard bacterial code. However, what triggers this switch and how the bacteriophage manipulates its host is poorly understood. Here, we report the discovery of a subgroup of minimal hepatitis delta virus (HDV)-like ribozymes – theta ribozymes – potentially involved in the code switch leading to the expression of recoded lysis and structural phage genes. We demonstrate their HDV-like self-scission behavior in vitro and find them in an unreported context often located with their cleavage site adjacent to tRNAs, indicating a role in viral tRNA maturation and/or regulation. Every fifth associated tRNA is a suppressor tRNA, further strengthening our hypothesis. The vast abundance of tRNA-associated theta ribozymes – we provide 1753 unique examples – highlights the importance of small ribozymes as an alternative to large enzymes that usually process tRNA 3’-ends. Our discovery expands the short list of biological functions of small HDV-like ribozymes and introduces a previously unknown player likely involved in the code switch of certain recoded gut bacteriophages.

RNA, widely recognized as an information-carrier molecule, is capable of catalyzing essential biological processes through ribozymes. Despite their ubiquity, specific functions in a biological context and phenotypes based on the ribozymes' activity are often unknown. Here, we present the discovery of a subgroup of minimal HDV-like ribozymes, which reside 3' to viral tRNAs and appear to cleave the 3'-trailers of viral premature tRNA transcripts. This proposed tRNA-processing function is unprecedented for any ribozymes, thus, we designate this subgroup as theta ribozymes. Most theta ribozymes were identified in Caudoviricetes bacteriophages, the main constituent (>90%) of the mammalian gut virome. Intriguingly, our findings further suggest the involvement of theta ribozymes in the transition of certain bacteriophages between distinct genetic codes, thus possibly contributing to the phage lysis trigger. Our discovery expands the limited repertoire of biological functions attributed to HDV-like ribozymes and provides insights into the fascinating world of RNA catalysis.

The bacterial SAR324 cluster is ubiquitous and abundant in the ocean, especially around hydrothermal vents and in the deep sea, where it can account for up to 30% of the whole bacterial community. According to a new taxonomy generated using multiple universal protein-coding genes (instead of the previously used 16S rRNA single gene marker), the former Deltaproteobacteria cluster SAR324 has been classified since 2018 as its own phylum. Yet, very little is known about its phylogeny and metabolic potential. We downloaded all publicly available SAR324 genomes (65) from all natural environments and reconstructed 18 new genomes using publicly available oceanic metagenomic data and unpublished data from the waters underneath the Ross Ice Shelf. We calculated a global SAR324 phylogenetic tree and identified six clusters (namely 1A, 1B, 2A, 2B, 2C and 2D) within this clade. Genome annotation and metatranscriptome read mapping showed that SAR324 clades possess a flexible array of genes suited for survival in various environments. Clades 2A and 2C are mostly present in the surface mesopelagic layers of global oceans, while clade 2D dominates in deeper regions. Our results show that SAR324 has a very versatile and broad metabolic potential, including many heterotrophic, but also autotrophic pathways. While one surface water associated clade (2A) seems to use proteorhodopsin to gain energy from solar radiation, some deep-sea genomes from clade 2D contain the complete Calvin–Benson–Bassham cycle gene repertoire to fix carbon. This, in addition to a variety of other genes and pathways for both oxic (e.g., dimethylsulfoniopropionate degradation) and anoxic (e.g., dissimilatory sulfate reduction, anaerobic benzoate degradation) conditions, can help explain the ubiquitous presence of SAR324 in aquatic habitats.