鈴木 志野

ABSTRACT 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. In vitro 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. IMPORTANCE Introns 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.

Bacteria defend against hostile environments through a variety of molecular mechanisms, including ribosome hibernation. Previously, bacteria were shown to initiate ribosome hibernation by activating protective proteins known as hibernation factors. It was demonstrated that hibernation factors prevent ribosome degradation by nucleases, which allows bacteria to safely store their inactive ribosomes and survive under starvation or persistent stress. Because homologs of hibernation factors were found in diverse lineages of bacteria, it is currently assumed that the mechanism of ribosome hibernation is highly conserved across species. Here, we assess 46,015 complete bacterial genomes to reveal the principles underlying the origin and evolution of these essential proteins in bacterial cells. We find that hibernation factors emerged in ancient bacteria as relatively large proteins that then gradually reduced in size and have undergone complete extinction in over 10% of studied bacteria. We then demonstrate that the degeneration of ancient hibernation factors is often accompanied by "borrowing" hibernation factors from other species via gene transfers, de novo gene birth or fusion of truncated hibernation factors with fragments from other stress-response proteins. These findings reveal a unique evolutionary pathway in which bacteria respond to the reductive evolution of hibernation machinery by inventing novel hibernation mechanisms, thus restoring their capacity to survive starvation and stress. This model implies that most ribosome hibernation factors are yet to be discovered and predicts the organisms that rely on currently unknown hibernation mechanisms.

Abstract Ribosomes are essential for protein synthesis and require ribosome biogenesis factors (RBFs) for assembly. To uncover the evolutionary diversity of ribosome biogenesis, we analyzed over 30,000 bacterial genomes and revealed that Candidate Phyla Radiation (CPR), also known as the phylum Patescibacteria, characterized by reduced genomes and smaller ribosomes, has about half the average number of RBFs compared with non-CPR bacteria. Notably, key RBFs such as der, obgE, and rbfA, considered indispensable, are conserved in only around 20%–70% of CPR genomes. Since such repertoires were not observed in reduced genomes of other phyla, CPR presumably diverged early in bacterial evolution. We further confirmed that ribosomal structural changes correlate with reduced RBFs, evidencing co-evolution between RBFs and the ribosome. These findings suggest that ribosomal biogenesis is more flexible than recognized, and the small cell and genome sizes of CPR bacteria and their early divergence may influence the unusual repertoires of RBFs. Teaser Ribosome biogenesis in CPR bacteria was unexpectedly flexible, challenging traditional views of this essential process in evolution.

Serpentinization, a geochemical process found on modern and ancient Earth, provides an ultra-reducing environment that can support microbial methanogenesis and acetogenesis. Several groups of archaea, such as the order Methanocellales, are characterized by their ability to produce methane. Here, we generate metagenomic sequences from serpentinized springs in The Cedars, California, and construct a circularized metagenome-assembled genome of a Methanocellales archaeon, termed Met12, that lacks essential methanogenesis genes. The genome includes genes for an acetyl-CoA pathway, but lacks genes encoding methanogenesis enzymes such as methyl-coenzyme M reductase, heterodisulfide reductases and hydrogenases. In situ transcriptomic analyses reveal high expression of a multi-heme c-type cytochrome, and heterologous expression of this protein in a model bacterium demonstrates that it is capable of accepting electrons. Our results suggest that Met12, within the order Methanocellales, is not a methanogen but a CO2-reducing, electron-fueled acetogen without electron bifurcation.