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.

In order to prevent celestial bodies (other than the Earth) from microbial contamination by spacecraft (forward contamination) and the Earth and its biosphere from extraterrestrial contamination (backward contamination), each planetary mission need to meet requirements of planetary protection. At Japan Aerospace Exploration Agency (JAXA), several missions to Mars or Enceladus have been lately proposed and studied. In order to improve the feasibility of these missions, planetary protection technologies with regard to bioburden control should be established. In this work, we introduce our planetary protection activities, such as the development of bioburden-controlled environments, identification of bioburden contamination risk through flight hardware series of events, bioburden reduction, bioburden assays, and so forth.   We also report planetary protection implementation activities for Martian Moons eXploration (MMX) missions. MMX was classified as Category III for Mars due to its orbiter operation around the planet, and as Category II to the Phobos because of the planned landing on Phobos and flyby of Deimos during the outbound leg. In addition, MMX was classified as Category V with Unrestricted Earth Return (UER) for the inbound leg. In accordance with the planetary protection requirements, we have been carrying out (1) impact and contamination probability analyses, (2) contamination control, (3) controlled sample collection (including regolith scattering effects), (4) impact sterilization tests, (5) radiation sterilization test, etc.

<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>

Abstract Developing a cleanroom and clean chambers (CCs) for Hayabusa2 returned samples has been discussed with the committee for Hayabusa2 sample curation facility since 2015. One major difference from the specifications of the CCs used for Itokawa samples is that a part of samples was decided to be handled and preserved in vacuum to avoid terrestrial nitrogen contamination with organics or unknown materials that might easily react with the samples. Thus, the CCs for Hayabusa2 samples were divided into two CCs for vacuum processes and three CCs for purified nitrogen conditions. The cleanroom was built in summer 2017, while the CCs were installed in the summer of 2018. After the installation of the CCs, instruments for initial descriptions, sample containers, handling tools for powder and particle samples, and jigs to assist handling samples were developed in parallel with functional checks and repeated rehearsals between the fall of 2018 and the fall of 2020. The curatorial works on Hayabusa2-retuned samples were conducted as previously planned. Simultaneously, contaminations and influences of inorganics, organics, microbial, and magnetic constructs have been assessed to evaluate their potential effects on the analysis of the returned samples. Additionally, the tools used to touch samples directly have been demagnetized to avoid sample magnetization during their handling and the tool magnetization was measured before and after their usages. The series of developments and experiences from the curatorial works of Hayabusa2-returned samples represent valuable implications for future sample return missions. Graphical Abstract

Abstract Serpentinization of ultramafic rocks provides molecular hydrogen (H₂) that can support lithotrophic metabolism of microorganisms, but also poses extremely challenging conditions, including hyperalkalinity and limited electron acceptor availability. Investigation of two serpentinization-active systems reveals that conventional H₂-/CO₂-dependent homoacetogenesis is thermodynamically unfavorable in situ due to picomolar CO₂ levels. Through metagenomics and thermodynamics, we discover unique taxa capable of metabolism adapted to the habitat. This included a novel deep-branching phylum, “Ca. Lithacetigenota”, that exclusively inhabits serpentinite-hosted systems and harbors genes encoding alternative modes of H₂-utilizing lithotrophy. Rather than CO₂, these putative metabolisms utilize reduced carbon compounds detected in situ presumably serpentinization-derived: formate and glycine. The former employs a partial homoacetogenesis pathway and the latter a distinct pathway mediated by a rare selenoprotein—the glycine reductase. A survey of microbiomes shows that glycine reductases are diverse and nearly ubiquitous in serpentinite-hosted environments. “Ca. Lithacetigenota” glycine reductases represent a basal lineage, suggesting that catabolic glycine reduction is an ancient bacterial innovation by Terrabacteria for gaining energy from geogenic H₂ even under hyperalkaline, CO₂-poor conditions. Unique non-CO₂-reducing metabolisms presented here shed light on potential strategies that extremophiles may employ for overcoming a crucial obstacle in serpentinization-associated environments, features potentially relevant to primordial lithotrophy in early Earth.

Abstract Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) and poly(butylene succinate-co-adipate) (PBSA) are typical biodegradable polyesters; however, their biodegradability in the ocean differs substantially. Herein, we focused on functional genes correlated with biodegradation in ocean environments using multi-meta-omics approaches to identify the microbial groups and esterase enzymes correlated with biodegradation. Within the PHBV plastispheres, five Gammaproteobacteria were abundant, several of which encoded over 10 different types of extracellular poly(3-hydroxybutyrate (PHB) depolymerases that are highly expressed in the ocean. Within PBSA plastispheres, ecosystems of microbes formed on plastics, only two species of Gammaproteobacteria genomes were highly abundant and expressed: one for hydrolyzing PBSA and the other for consuming cleaved monomers. The high diversity of degrading microorganisms and enzymes could be related to the stable biodegradability of PHBV, while the low biodiversity of PBSA-degraders and necessity of symbiotic relationships likely characterize the instability of the marine biodegradability of PBSA. These results provide fundamental knowledge for the development of biodegradable marine plastics.

The Committee on Space Research (COSPAR) Sample Safety Assessment Framework (SSAF) has been developed by a COSPAR appointed Working Group. The objective of the sample safety assessment would be to evaluate whether samples returned from Mars could be harmful for Earth's systems (e.g., environment, biosphere, geochemical cycles). During the Working Group's deliberations, it became clear that a comprehensive assessment to predict the effects of introducing life in new environments or ecologies is difficult and practically impossible, even for terrestrial life and certainly more so for unknown extraterrestrial life. To manage expectations, the scope of the SSAF was adjusted to evaluate only whether the presence of martian life can be excluded in samples returned from Mars. If the presence of martian life cannot be excluded, a Hold & Critical Review must be established to evaluate the risk management measures and decide on the next steps. The SSAF starts from a positive hypothesis (there is martian life in the samples), which is complementary to the null-hypothesis (there is no martian life in the samples) typically used for science. Testing the positive hypothesis includes four elements: (1) Bayesian statistics, (2) subsampling strategy, (3) test sequence, and (4) decision criteria. The test sequence capability covers self-replicating and non-self-replicating biology and biologically active molecules. Most of the investigations associated with the SSAF would need to be carried out within biological containment. The SSAF is described in sufficient detail to support planning activities for a Sample Receiving Facility (SRF) and for preparing science announcements, while at the same time acknowledging that further work is required before a detailed Sample Safety Assessment Protocol (SSAP) can be developed. The three major open issues to be addressed to optimize and implement the SSAF are (1) setting a value for the level of assurance to effectively exclude the presence of martian life in the samples, (2) carrying out an analogue test program, and (3) acquiring relevant contamination knowledge from all Mars Sample Return (MSR) flight and ground elements. Although the SSAF was developed specifically for assessing samples from Mars in the context of the currently planned NASA-ESA MSR Campaign, this framework and the basic safety approach are applicable to any other Mars sample return mission concept, with minor adjustments in the execution part related to the specific nature of the samples to be returned. The SSAF is also considered a sound basis for other COSPAR Planetary Protection Category V, restricted Earth return missions beyond Mars. It is anticipated that the SSAF will be subject to future review by the various MSR stakeholders.

Three highly alkaliphilic bacterial strains designated as A1^T, H1^T and B1^T were isolated from two highly alkaline springs at The Cedars, a terrestrial serpentinizing site. Cells from all strains were motile, Gram-negative and rod-shaped. Strains A1^T, H1^T and B1^T were mesophilic (optimum, 30 °C), highly alkaliphilic (optimum, pH 11) and facultatively autotrophic. Major cellular fatty acids were saturated and monounsaturated hexadecenoic and octadecanoic acids. The genome size of strains A1^T, H1^T and B1^T was 2 574 013, 2 475 906 and 2 623 236 bp, and the G+C content was 66.0, 66.2 and 66.1 mol%, respectively. Analysis of the 16S rRNA genes showed the highest similarity to the genera <italic> <named-content content-type="genus"> <ext-link xmlns:xlink="http://www.w3.org/1999/xlink" ext-link-type="uri" xlink:href="http://doi.org/10.1601/nm.9349" xlink:type="simple">Malikia</ext-link> </named-content> </italic> (95.1–96.4 %), <italic> <named-content content-type="genus"> <ext-link xmlns:xlink="http://www.w3.org/1999/xlink" ext-link-type="uri" xlink:href="http://doi.org/10.1601/nm.1817" xlink:type="simple">Macromonas</ext-link> </named-content> </italic> (93.0–93.6 %) and <italic> <named-content content-type="genus"> <ext-link xmlns:xlink="http://www.w3.org/1999/xlink" ext-link-type="uri" xlink:href="http://doi.org/10.1601/nm.1807" xlink:type="simple">Hydrogenophaga</ext-link> </named-content> </italic> (93.0–96.6 %) in the family <italic> <named-content content-type="family"> <ext-link xmlns:xlink="http://www.w3.org/1999/xlink" ext-link-type="uri" xlink:href="http://doi.org/10.1601/nm.1773" xlink:type="simple">Comamonadaceae</ext-link> </named-content> </italic>. Phylogenetic analysis based on 16S rRNA gene and phylogenomic analysis based on core gene sequences revealed that the isolated strains diverged from the related species, forming a distinct branch. Average amino acid identity values of strains A1^T, H1^T and B1^T against the genomes of related members in this family were below 67 %, which is below the suggested threshold for genera boundaries. Average nucleotide identity by <sc>blast</sc> values and digital DNA–DNA hybridization among the three strains were below 92.0 and 46.6 % respectively, which are below the suggested thresholds for species boundaries. Based on phylogenetic, genomic and phenotypic characterization, we propose <italic>Serpentinimonas</italic> gen. nov., <italic>Serpentinimonas raichei</italic> sp. nov. (type strain A1^T=NBRC 111848^T=DSM 103917^T), <italic>Serpentinimonas barnesii</italic> sp. nov. (type strain H1^T= NBRC 111849^T=DSM 103920^T) and <italic>Serpentinimonas maccroryi</italic> sp. nov. (type strain B1^T=NBRC 111850^T=DSM 103919^T) belonging to the family <italic> <named-content content-type="family"> <ext-link xmlns:xlink="http://www.w3.org/1999/xlink" ext-link-type="uri" xlink:href="http://doi.org/10.1601/nm.1773" xlink:type="simple">Comamonadaceae</ext-link> </named-content> </italic>. We have designated <italic>Serpentinimonas raichei</italic> the type species for the genus because it is the dominant species in The Cedars springs.

Microbial involvement in the pathogenesis have been suggested in both antineutrophil cytoplasmic antibody-associated vasculitis (AAV) and sarcoidosis, both of which have lung involvement. However, exhaustive research to assess the bacteria in the lung in AAV and in sarcoidosis have not been performed. We sought to elucidate the distinct dysbiotic lung microbiota between AAV and sarcoidosis. We used 16S rRNA gene high-throughput sequencing to obtain the bacterial community composition of bronchoalveolar lavage fluid (BALF) in patients with AAV (n = 16) compared to patients with sarcoidosis (n = 21). The patients had not undergone therapy with immunosuppressive medication when their BALF was acquired. No difference was observed in α-diversity between patients with AAV and patients with sarcoidosis when using all the detected taxa. We defined the taxa of the oral cavity by using the data of oral microbiota of healthy individuals from the Human Microbiome Project (HMP). The analysis using only oral taxa made the difference in α-diversity between AAV and sarcoidosis clearer compared with those using all the detected taxa. Besides, the analysis using detected taxa except for oral taxa also made the difference in α-diversity between AAV and sarcoidosis clearer compared with those using all the detected taxa. A linear negative relationship between the α-diversity and Birmingham vasculitis activity score (BVAS) was detected in the AAV group. The observed p-value for the effect of the disease groups on the ß-diversity was small while the effect of other factors including sex and smoking status did not have small p-values. By excluding oral taxa from all the detected taxa, we found a cluster mainly consisted of sarcoidosis patients which was characterized with microbial community monopolized by Erythrobacteraceae family. Our results suggested the importance of considering the influence of oral microbiota in evaluating lung microbiota.

<p>Scientific ocean drilling over the past half-century has significantly expanded our knowledge of life, ocean, and Earth. The discovery of the spatially vast deep-subseafloor biosphere, a milestone scientific achievement, has extended the planetary habitability of life on Earth. To date, multiple lines of evidence form the core samples have demonstrated that a remarkable amount of physiologically unknown microbial life is present deep beneath the ocean floor where water supply and energy substrates are severely limited. Although subseafloor microbial ecosystems generally function extremely slowly, the consequence of long-term activity on geological timescales may play significant ecological roles in global biogeochemical carbon and other elemental cycles.</p>