Shino Suzuki

ABSTRACT Patescibacteria is a bacterial phylum with small genomes, frequent loss of essential genes, and the presence of introns. While many aspects of Patescibacteria remain enigmatic, an intriguing feature is the widespread occurrence of introns within their compact genomes. To better understand the diversity, roles, and evolution of bacterial introns, we focused on tRNA introns and analyzed Patescibacteria complete genome. Notably, 20% of these genomes lacked at least one tRNA gene for a canonical amino acid, primarily tRNA^Asn and tRNA^Asp, whereas other tRNA genes were readily detected. This observation led us to conduct further analyses, resulting in the discovery of a novel group I intron insertion site at position 35/36 within the anticodon loop that likely prevented detection by conventional annotation tools. Splicing assays demonstrated that these bacterial introns are catalytically active and capable of self-splicing. To assess the broader distribution of this insertion site across bacteria, we analyzed 4,934 bacterial genomes and identified 269 group I introns within tRNA genes across 14 phyla. Nearly 70% of introns at position 35/36 originate from Patescibacteria, indicating that this feature is largely confined to the phylum. Subgroup classification showed that 79% of all tRNA introns belonged to the IC subgroup, whereas almost all Patescibacteria introns were assigned to IA, suggesting a distinct evolutionary origin. As most tRNA introns lacked homing endonuclease genes, horizontal transfer appears limited. Collectively, these findings advance our understanding of the phylogenetic distribution and evolutionary history of bacterial group I introns in tRNAs, with particular emphasis on Patescibacteria. IMPORTANCE Group I introns in bacterial tRNA genes were previously known only in a limited number of phyla. Our study expands this knowledge by identifying a novel insertion position in tRNA genes of phylum Patescibacteria and mapping their phylogenetic distribution across bacterial lineages. Our result revealed that group I introns inserted in tRNA genes differed in subgroups between Patescibacteria and other bacteria, highlighting the evolutionary uniqueness of introns of Patescibacteria. Additionally, we found that group I introns are maintained in 43% of bacterial phyla, with tRNA insertions being the most common. Our findings highlight that even in complete genomes, the presence of group I introns can hinder the detection of all 20 canonical tRNA genes by conventional tRNA annotation tools. This study illustrates the overlooked phylogenetic distribution of group I introns across the bacterial domain.

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.

<jats:p>Planetary protection is a guiding principle aiming to prevent microbial contamination of the solar system by spacecraft (forward contamination) and extraterrestrial contamination of the Earth (backward contamination). Bioburden reduction on spacecraft, including cruise and landing systems, is required to prevent microbial contamination from Earth during space exploration missions. Several sterilization methods are available; however, selecting appropriate methods is essential to eliminate a broad spectrum of microorganisms without damaging spacecraft components during manufacturing and assembly. Here, we compared the effects of different bioburden reduction techniques, including dry heat, UV light, isopropyl alcohol (IPA), hydrogen peroxide (H<jats:sub>2</jats:sub>O<jats:sub>2</jats:sub>), vaporized hydrogen peroxide (VHP), and oxygen and argon plasma on microorganisms with different resistance capacities. These microorganisms included <jats:italic>Bacillus atrophaeus</jats:italic> spores and <jats:italic>Aspergillus niger</jats:italic> spores, <jats:italic>Deinococcus radiodurans</jats:italic>, and <jats:italic>Brevundimonas diminuta</jats:italic>, all important microorganisms for considering planetary protection. <jats:italic>Bacillus atrophaeus</jats:italic> spores showed the highest resistance to dry heat but could be reliably sterilized (i.e., under detection limit) through extended time or increased temperature. <jats:italic>Aspergillus niger</jats:italic> spores and <jats:italic>D. radiodurans</jats:italic> were highly resistant to UV light. Seventy percent of IPA and 7.5% of H<jats:sub>2</jats:sub>O<jats:sub>2</jats:sub> treatments effectively sterilized <jats:italic>D. radiodurans</jats:italic> and <jats:italic>B. diminuta</jats:italic> but showed no immediate bactericidal effect against <jats:italic>B. atrophaeus</jats:italic> spores. IPA immediately sterilized <jats:italic>A. niger</jats:italic> spores, but H<jats:sub>2</jats:sub>O<jats:sub>2</jats:sub> did not. During VHP treatment under reduced pressure, viable <jats:italic>B. atrophaeus</jats:italic> spores and <jats:italic>A. niger</jats:italic> spores were quickly reduced by approximately two log orders. Oxygen plasma sterilized <jats:italic>D. radiodurans</jats:italic> but did not eliminate <jats:italic>B. atrophaeus</jats:italic> spores. In contrast, argon plasma sterilized <jats:italic>B. atrophaeus</jats:italic> but not <jats:italic>D. radiodurans</jats:italic>. Therefore, dry heat could be used for heat-resistant component bioburden reduction, and VHP or plasma for non-heat-resistant components in bulk bioburden reduction. Furthermore, IPA, H<jats:sub>2</jats:sub>O<jats:sub>2</jats:sub>, or UV could be used for additional surface bioburden reduction during assembly and testing. The systemic comparison of sterilization efficiencies under identical experimental conditions in this study provides basic criteria for determining which sterilization techniques should be selected during bioburden reduction for forward planetary protection.</jats:p>