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At least 181 records · Page 10
All sourcesNASA NTRSDOE OSTI
DOE OSTI2025

Isolation and characterization of 24 phages infecting the plant growth-promoting rhizobacterium Klebsiella sp. M5al

Bacteriophages largely impact bacterial communities via lysis, gene transfer, and metabolic reprogramming and thus are increasingly thought to alter nutrient and energy cycling across many of Earth’s ecosystems. However, there are few model systems to mechanistically and quantitatively study phage-bacteria interactions, especially in soil systems. Here, we isolated, sequenced, and genomically characterized 24 novel phages infectingKlebsiellasp. M5al, a plant growth-promoting, nonencapsulated rhizosphere-associated bacterium, and compared many of their features against all 565 sequenced, dsDNAKlebsiellaphage genomes. Taxonomic analyses revealed that theseKlebsiellaphages belong to three known phage families (Autographiviridae,Drexlerviridae, andStraboviridae) and two newly proposed phage families (CandidatusMavericviridaeand Ca.Rivulusviridae). At the phage family level, we found that core genes were often phage-centric proteins, such as structural proteins for the phage head and tail and DNA packaging proteins. In contrast, genes involved in transcription, translation, or hypothetical proteins were commonly not shared or flexible genes. Ecologically, we assessed the phages’ ubiquity in recent large-scale metagenomic datasets, which revealed they were not widespread, as well as a possible direct role in reprogramming specific metabolisms during infection by screening their genomes for phage-encoded auxiliary metabolic genes (AMGs). Even though AMGs are common in the environmental literature, only one of our phage families,Straboviridae, contained AMGs, and the types of AMGs were correlated at the genus level. Host range phenotyping revealed the phages had a wide range of infectivity, infecting between 1–14 of our 22 bacterial strain panel that included pathogenicKlebsiellaandRaoultellastrains. This indicates that not all capsule-independent Klebsiella phages have broad host ranges. Together, these isolates, with corresponding genome, AMG, and host range analyses, help build theKlebsiellamodel system for studying phage-host interactions of rhizosphere-associated bacteria.

Science & Technology - Other Topics↗
NASA NTRS2002

Spatial distribution of total, ammonia-oxidizing, and denitrifying bacteria in biological wastewater treatment reactors for bioregenerative life support

Bioregenerative life support systems may be necessary for long-term space missions due to the high cost of lifting supplies and equipment into orbit. In this study, we investigated two biological wastewater treatment reactors designed to recover potable water for a spacefaring crew being tested at Johnson Space Center. The experiment (Lunar-Mars Life Support Test Project-Phase III) consisted of four crew members confined in a test chamber for 91 days. In order to recycle all water during the experiment, an immobilized cell bioreactor (ICB) was employed for organic carbon removal and a trickling filter bioreactor (TFB) was utilized for ammonia removal, followed by physical-chemical treatment. In this study, the spatial distribution of various microorganisms within each bioreactor was analyzed by using biofilm samples taken from four locations in the ICB and three locations in the TFB. Three target genes were used for characterization of bacteria: the 16S rRNA gene for the total bacterial community, the ammonia monooxygenase (amoA) gene for ammonia-oxidizing bacteria, and the nitrous oxide reductase (nosZ) gene for denitrifying bacteria. A combination of terminal restriction fragment length polymorphism (T-RFLP), sequence, and phylogenetic analyses indicated that the microbial community composition in the ICB and the TFB consisted mainly of Proteobacteria, low-G+C gram-positive bacteria, and a Cytophaga-Flexibacter-Bacteroides group. Fifty-seven novel 16S rRNA genes, 8 novel amoA genes, and 12 new nosZ genes were identified in this study. Temporal shifts in the species composition of total bacteria in both the ICB and the TFB and ammonia-oxidizing and denitrifying bacteria in the TFB were also detected when the biofilms were compared with the inocula after 91 days. This result suggests that specific microbial populations were either brought in by the crew or enriched in the reactors during the course of operation.

NASA Discipline Life Support Systems↗
DOE OSTI2023

The algal microbiome protects Desmodesmus intermedius from high light and temperature stress

The mutualistic impacts of bacterial communities on algal growth and biomass accumulation are well documented, but it is unknown how temperature and light stress influence these mutualistic interactions. Here, we generated a bacteria-free (axenic) culture of the green alga Desmodesmus intermedius C046 and compared its growth and yield in the laboratory to its native xenic counterpart - featuring a microbiome that has been maintained with its algal host for at least 5 years – under unstressed and high light, high temperature stress conditions. We then added exogenous microbiomes from high light marine environments to the axenic culture to quantify the contribution of these newly generated microbial consortia in improving D. intermedius growth, yield, and resilience to high light and heat. The native microbiome increased growth and biomass accumulation, under both unstressed and stressed conditions. Three of the five newly derived microbiomes had increased growth compared to the axenic and negligible, decreased, or increased growth impacts compared to the xenic culture. Stress conditions led to decreased algal exudation, larger algal cells when grown alone, but smaller algal cells with an associated microbiome. In conclusion, our results suggest that the algal microbiome plays a role in microalgal response to heat and light stress, and efforts for screening new algal strains for high biomass productivity under those stress conditions should include a strategy for microbiome optimization.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Energy efficiency and biological interactions define the core microbiome of deep oligotrophic groundwater

While oligotrophic deep groundwaters host active microbes attuned to the low-end of the bioenergetics spectrum, the ecological constraints on microbial niches in these ecosystems and their consequences for microbiome convergence are unknown. Here, we provide a genome-resolved, integrated omics analysis comparing archaeal and bacterial communities in disconnected fracture fluids of the Fennoscandian Shield in Europe. Leveraging a dataset that combines metagenomes, single cell genomes, and metatranscriptomes, we show that groundwaters flowing in similar lithologies offer fixed niches that are occupied by a common core microbiome. Functional expression analysis highlights that these deep groundwater ecosystems foster diverse, yet cooperative communities adapted to this setting. We suggest that these communities stimulate cooperation by expression of functions related to ecological traits, such as aggregate or biofilm formation, while alleviating the burden on microorganisms producing compounds or functions that provide a collective benefit by facilitating reciprocal promiscuous metabolic partnerships with other members of the community. We hypothesize that an episodic lifestyle enabled by reversible bacteriostatic functions ensures the subsistence of the oligotrophic deep groundwater microbiome.

54 ENVIRONMENTAL SCIENCES↗
DOE OSTI2024

Experimental warming accelerates positive soil priming in a temperate grassland ecosystem

Abstract Unravelling biosphere feedback mechanisms is crucial for predicting the impacts of global warming. Soil priming, an effect of fresh plant-derived carbon (C) on native soil organic carbon (SOC) decomposition, is a key feedback mechanism that could release large amounts of soil C into the atmosphere. However, the impacts of climate warming on soil priming remain elusive. Here, we show that experimental warming accelerates soil priming by 12.7% in a temperate grassland. Warming alters bacterial communities, with 38% of unique active phylotypes detected under warming. The functional genes essential for soil C decomposition are also stimulated, which could be linked to priming effects. We incorporate lab-derived information into an ecosystem model showing that model parameter uncertainty can be reduced by 32–37%. Model simulations from 2010 to 2016 indicate an increase in soil C decomposition under warming, with a 9.1% rise in priming-induced CO 2 emissions. If our findings can be generalized to other ecosystems over an extended period of time, soil priming could play an important role in terrestrial C cycle feedbacks and climate change.

54 ENVIRONMENTAL SCIENCES↗
DOE OSTI2025

Phage-based delivery of CRISPR-associated transposases for targeted bacterial editing

Phage λ, a well-characterized temperate phage, has been recently leveraged for bacterial genome editing by selectively delivering base editors into targeted bacterial species. We extend this concept by engineering phage λ to deliver CRISPR-guided transposases, accomplishing large insertions and targeted gene disruptions. To achieve this, we engineered phage λ using homologous recombination paired with Cas13a-based counterselection for precise phage modifications. Initially, we established the utility of Cas13a in phage λ by conducting minimal recoding edits, deletions, and insertions. Subsequently, we scaled up the engineering to embed the comprehensive DNA-editing CRISPR-Cas transposase (DART) system within the phage genome, creating λ-DART phages. These modified λ-DART phages were then employed to infectEscherichia coli, generating CRISPR RNA-guided transposition events in the host genome. Applying our engineered λ-DART phages to monocultures and a mixed bacterial community comprising three genera led to efficient, precise, and specific gene knockouts and insertions in the targetedE. colicells, achieving editing efficiencies surpassing 50% of the population. This research enhances phage-mediated genome editing by enabling efficient in situ gene integrations in bacteria, offering an avenue for further application in microbial community contexts. This scalable method enables flexible microbial genome editing in situ to manipulate the function and composition of diverse ecosystems.

Science & Technology - Other Topics↗
DOE OSTI2020

Ecological and genomic analyses of candidate phylum WPS-2 bacteria in an unvegetated soil

Members of the bacterial candidate phylum WPS-2 (or Eremiobacterota) are abundant in several dry, bare soil environments. In a bare soil deposited by an extinct iron-sulfur spring, we found that WPS-2 comprised up to 24% of the bacterial community and up to 108 cells per g of soil based on 16S rRNA gene sequencing and quantification. A single genus-level cluster (Ca. Rubrimentiphilum) predominated in bare soils but was less abundant in adjacent forest. Nearly complete genomes of Ca. Rubrimentiphilum were recovered as single amplified genomes (SAGs) and metagenome-assembled genomes (MAGs). Surprisingly, given the abundance of WPS-2 in bare soils, the genomes did not indicate any capacity for autotrophy, phototrophy, or trace gas metabolism. Instead, they suggest a predominantly aerobic organoheterotrophic lifestyle, perhaps based on scavenging amino acids, nucleotides, and complex oligopeptides, along with lithotrophic capacity on thiosulfate. Network analyses of the entire community showed that some species of Chloroflexi, Actinobacteria, and candidate phylum AD3 (or Dormibacterota) co-occurred with Ca. Rubrimentiphilum and may represent ecological or metabolic partners. We propose that Ca. Rubrimentiphilum act as efficient heterotrophic scavengers. Combined with previous studies, these data suggest that the phylum WPS-2 includes bacteria with diverse metabolic capabilities.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Association between algal productivity and phycosphere composition in an outdoor Chlorella sorokiniana reactor based on multiple longitudinal analyses

Summary Microalgae as a biofuel source are of great interest. Bacterial phycosphere inhabitants of algal cultures are hypothesized to contribute to productivity. In this study, the bacterial composition of the Chlorella sorokiniana phycosphere was determined over several production cycles in different growing seasons by 16S rRNA gene sequencing and identification. The diversity of the phycosphere increased with time during each individual reactor run, based on Faith’s phylogenetic diversity metric versus days post‐inoculation ( R = 0.66, P < 0.001). During summer months, Vampirovibrio chlorellavorus , an obligate predatory bacterium, was prevalent. Bacterial sequences assigned to the Rhizobiales, Betaproteobacteriales and Chitinophagales were positively associated with algal biomass productivity. Applications of the general biocide, benzalkonium chloride, to a subset of experiments intended to abate V. chlorellavorus appeared to temporarily suppress phycosphere bacterial growth, however, there was no relationship between those bacterial taxa suppressed by benzalkonium chloride and their association with algal productivity, based on multinomial model correlations. Algal health was approximated using a model‐based metric, or the ‘Health Index’ that indicated a robust, positive relationship between C. sorokiniana fitness and presence of members belonging to the Burholderiaceae and Allorhizobium–Neorhizobium–Pararhizobium–Rhizobium clade. Bacterial community composition was linked to the efficiency of microalgal biomass production and algal health.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Identification of key bacterial populations affecting early embryonic development in cattle uterus

Abstract Superovulation is an important animal breeding biotechnology, while the quality of embryos obtained from superovulation is unstable in cattle. The relationship between the microorganisms in the cattle uterus and embryo qualities was determined to identify the key bacterial populations affecting early embryonic development. A total of 10 Xia Nan cows underwent superovulation, we collected cervical mucus and flush samples to investigated by 16S rDNA sequencing. Results showed that there were abundant microorganisms in cervical mucus, but no obvious relationship with the quality of embryos. The clustering results of flush samples were consistent with the grouping of embryo quality. Proteobacteria accounted for more than 95% of the total bacterial community in group A with the best embryo quality (qualified embryo ratio above 0.8), and as embryo quality decreased, the Proteobacteria proportion also decreased. In contrast to the proportion of Proteobacteria, the proportions of Firmicutes and Bacteroidetes significantly increased as embryo quality decreased. For group C with the worst embryo quality, the proportions of Firmicutes and Bacteroidetes increased to 4.7 times and 12.3 times of group A, respectively. These results showed that the quantities and proportions of Firmicutes and Bacteroidetes may be related to early embryonic development in cattle.

Lyu, Chenchen↗
DOE OSTI2022

Generating Ectropis obliqua axenic larvae in support of functional microbiome studies

Abstract Ectropis obliqua Prout (Lepidoptera: Geometridae) is a serious leaf‐eating pest in tea gardens throughout China, with rich bacterial communities in the larval gut. Few studies have investigated whether these bacteria play an important role in growth, development, and adaptability of E. obliqua . Axenic larval models are a useful tool for the study of interactions between gut bacteria and host insects. At present, artificial diet and antibiotics are typically used to remove larval gut bacteria for study; however, this method may change the natural food of insects and the habitat provided by their host plants, and cannot completely eliminate insect gut bacteria. Therefore, in order to establish an axenic insect feeding model, we first investigated the effects of egg age, different disinfectant concentration, and treatment time on the eggs of E. obliqua through orthogonal experiments. Next, we fed E. obliqua larvae tissue‐cultured tea seedlings under aseptic conditions. Thus, we successfully generated a feeding model of gut axenic larvae in the laboratory. Based on it, we then compared the mortality of axenic and non‐axenic E. obliqua larvae infected with the entomopathogenic fungus Beauveria bassiana (Balsamo) Vuill. These results demonstrated the feasibility of this axenic larval model and that B. bassiana is a broad‐spectrum entomogenous fungus with a toxic effect on E. obliqua .

Su, Qingqing↗
DOE OSTI2022

Hyphae move matter and microbes to mineral microsites: Integrating the hyphosphere into conceptual models of soil organic matter stabilization

Associations between soil minerals and microbially derived organic matter (often referred to as mineral-associated organic matter or MAOM) form a large pool of slowly cycling carbon (C). Here, the rhizosphere, soil immediately adjacent to roots, is thought to control the spatial extent of MAOM formation because it is the dominant entry point of new C inputs to soil. However, emphasis on the rhizosphere implicitly assumes that microbial redistribution of C into bulk (non-rhizosphere) soils is minimal. We question this assumption, arguing that because of extensive fungal exploration and rapid hyphal turnover, fungal redistribution of soil C from the rhizosphere to bulk soil minerals is common, and encourages MAOM formation. First, we summarize published estimates of fungal hyphal length density and turnover rates and demonstrate that fungal C inputs are high throughout the rhizosphere–bulk soil continuum. Second, because colonization of hyphal surfaces is a common dispersal mechanism for soil bacteria, we argue that hyphal exploration allows for the non-random colonization of mineral surfaces by hyphae-associated taxa. Third, these bacterial communities and their fungal hosts determine the chemical form of organic matter deposited on colonized mineral surfaces. Collectively, our analysis demonstrates that omission of the hyphosphere from conceptual models of soil C flow overlooks key mechanisms for MAOM formation in bulk soils. Moving forward, there is a clear need for spatially explicit, quantitative research characterizing the environmental drivers of hyphal exploration and hyphosphere community composition across systems, as these are important controls over the rate and organic chemistry of C deposited on minerals.

54 ENVIRONMENTAL SCIENCES↗
DOE OSTI2022

Assessment of rhizosphere bacterial diversity and composition in a metal hyperaccumulator (Boehmeria nivea) and a nonaccumulator (Artemisia annua) in an antimony mine

Abstract Aims Heavy metal hyperaccumulators are widely used in mining restoration due to their ability to accumulate and transport heavy metals, compared to nonaccumulators. Rhizosphere bacteria in metal hyperaccumulators play a key role in the uptake of heavy metals from soil; however, assessments of the differences of rhizosphere bacteria between metal hyperaccumulators and nonaccumulator are scarce. Methods and results To understand the difference of bacterial composition between hyperaccumulator and nonaccumulator in rhizosphere, the diversity and composition of rhizosphere bacteria in a metal hyperaccumulator (Boehmeria nivea) and a nonaccumulator (Artemisia annua) grown in the same field in Xikuangshan were evaluated using Illumina Miseq high-throughput sequencing technology. Boehmeria nivea and A. annua had 3926 overlapping OTUs, 19,736 and 17,579 unique OTUs, respectively. Boehmeria nivea had lower Chao1 index, Shannon index and Pielou index than A. annua. The dominant phyla and genera of rhizosphere bacteria in B. nivea and A. annua were similar, but some rhizosphere bacterial communities with heavy metal remediation ability mainly appeared in the rhizosphere of the hyperaccumulator. Compared to A. annua, B. nivea showed a significantly higher relative abundance of rhizosphere bacteria, such as Acidobacteria and Bacteroidete at the phylum level and RB41 at the genus level. Some specific rhizosphere bacteria with the ability to bind metal, such as Leifsonia and Kibdelosporangium, were only found in the rhizosphere of B. nivea. Conclusion Results indicated that B. nivea, as a metal hyperaccumulator, has a key function in governing metal-resistant rhizosphere bacteria in response to antimony compound pollution stress. Significance and Impact of Study Understanding the diversity of rhizosphere bacteria between hyperaccumulators and nonaccumulators is beneficial to formulate strategies to improve metal uptake efficiency by selecting specific plant species and rhizosphere bacteria grown on polluted soil.

Lin, Yuxiang↗
DOE OSTI2023

Herbivorous Fish Microbiome Adaptations to Sulfated Dietary Polysaccharides

Marine herbivorous fish that feed primarily on macroalgae, such as those from the genus Kyphosus, are essential for maintaining coral health and abundance on tropical reefs. Here, deep metagenomic sequencing and assembly of gut compartment-specific samples from three sympatric, macroalgivorous Hawaiian kyphosid species have been used to connect host gut microbial taxa with predicted protein functional capacities likely to contribute to efficient macroalgal digestion. Bacterial community compositions, algal dietary sources, and predicted enzyme functionalities were analyzed in parallel for 16 metagenomes spanning the mid- and hindgut digestive regions of wild-caught fishes. Gene colocalization patterns of expanded carbohydrate (CAZy) and sulfatase (SulfAtlas) digestive enzyme families on assembled contigs were used to identify likely polysaccharide utilization locus associations and to visualize potential cooperative networks of extracellularly exported proteins targeting complex sulfated polysaccharides. These insights into the gut microbiota of herbivorous marine fish and their functional capabilities improve our understanding of the enzymes and microorganisms involved in digesting complex macroalgal sulfated polysaccharides.

09 BIOMASS FUELS↗
DOE OSTI2025

Two novel Patescibacteria: Phycocordibacter aenigmaticus gen. nov. sp. nov. and Minusculum obligatum gen. nov. sp. nov., both associated with microalgae optimized for carbon dioxide sequestration from flue gas

The functional roles of bacterial symbionts associated with microalgae remain understudied despite the importance of microalgae in biotechnology and environmental microbiology. 16S rRNA gene sequencing was conducted to analyze bacterial communities associated with two microalgae optimized for growth with flue gas containing 5%–10% CO 2 . Two dominant bacteria with no taxonomic classification beyond the class level (Paceibacteria) were discovered repeatedly in the most productive algal cultures. Long-read metagenomic sequencing was conducted to yield high-quality metagenomes, from which two novel species were discovered under the Seqcode (seqco.de/r:ywe1blo2), Phycocordibacter aenigmaticus gen. nov. sp. nov. and Minusculum obligatum gen. nov. sp. nov. The genus Phycocordibacter gen. nov. was proposed as the nomenclatural type of the family Phycocordibacteraceae fam. nov. and the order Phycocordibacterales ord. nov. Both bacteria possessed features typical of Patescibacteria such as reduced genomes (<800 kbp), lack of complete glycolysis and tricarboxylic acid (TCA) cycle pathways, and inability to synthesize amino acids. Instead, they rely on the reductive pentose phosphate pathway (Calvin cycle) for essential biosynthesis and redox balance. P. aenigmaticus may also rely on elemental sulfur oxidation (sdo), partial nitrite reduction (nirK), and sulfur-related amino acid metabolism (SAMe → SAH). Both bacteria were found in high relative abundance in cultures of Tetradesmus obliquus HTB1 (freshwater) and Nannochloropsis oceanica IMET1 (marine), suggesting a tight association with microalgae in various environments. The absence of full metabolic pathways for energy production suggests extreme metabolic limitations and obligate symbiosis, most likely with other bacteria associated with the microalgae.

54 ENVIRONMENTAL SCIENCES↗
DOE OSTI2025

A bloom of a single bacterium shapes the microbiome during outdoor diatom cultivation collapse

Algae-dominated ecosystems are fundamentally influenced by their microbiome. We lack information on the identity and function of bacteria that specialize in consuming algal-derived dissolved organic matter in high algal density ecosystems such as outdoor algal ponds used for biofuel production. Here, we describe the metagenomic and metaproteomic signatures of a single bacterial strain that bloomed during a population-wide crash of the diatom, Phaeodactylum tricornutum, grown in outdoor ponds. 16S rRNA gene data indicated that a single Kordia sp. strain (family Flavobacteriaceae) contributed up to 93% of the bacterial community during P. tricornutum demise. Kordia sp. expressed proteins linked to microbial antagonism and biopolymer breakdown, which likely contributed to its dominance over other microbial taxa during diatom demise. Analysis of accompanying downstream microbiota (primarily of the Rhodobacteraceae family) provided evidence that cross-feeding may be a pathway supporting microbial diversity during diatom demise. In situ and laboratory data with a different strain suggested that Kordia was a primary degrader of biopolymers during algal demise, and co-occurring Rhodobacteraceae exploited degradation molecules for carbon. An analysis of 30 Rhodobacteraceae metagenome assembled genomes suggested that algal pond Rhodobacteraceae commonly harbored pathways to use diverse carbon and energy sources, including carbon monoxide, which may have contributed to the prevalence of this taxonomic group within the ponds. These observations further constrain the roles of functionally distinct heterotrophic bacteria in algal microbiomes, demonstrating how a single dominant bacterium, specialized in processing senescing or dead algal biomass, shapes the microbial community of outdoor algal biofuel ponds.

Kordia↗
DOE OSTI2026

Multidimensional scaling informed by F -statistic: Visualizing grouped microbiome data with inference

Multidimensional scaling (MDS) is a widely used dimensionality reduction technique in microbial ecology data analysis that captures the multivariate structure of the data while preserving pairwise distances between samples. While improvements in MDS have enhanced the ability to reveal group-specific data patterns, these MDS-based methods require prior assumptions for inference, limiting their application in general microbiome analysis. Here, in this study, we introduce a new MDS-based ordination method, “F-informed MDS,” which configures the data distribution based on the F-statistic, the ratio of dispersion between groups sharing common and different characteristics. Using semisynthetic datasets, we demonstrate that the proposed method is robust to hyperparameter selection while maintaining statistical significance throughout the ordination process. Various quality metrics for evaluating dimensionality reduction confirm that F-informed MDS is comparable to state-of-the-art methods in preserving both local and global data structures. Its application to a diatom-associated bacterial community suggests the role of this new method in interpreting the community’s response to the host. Our approach offers a well-founded refinement of MDS that aligns with statistical test results, which can be beneficial for broader multidimensional data analyses in microbiology and ecology. This new visualization tool can be incorporated into standard microbiome data analyses.

Biological and medical sciences↗
DOE OSTI2026

Soil viral production count, respiration, and amplicon data

This study aimed to quantify rates of viral production in aridisol soil under conditions as close to natural field soil as possible given the perturbations necessary to manipulate viral abundances. Viruses were removed from soil, then added back to virus-depleted soil to control the initial viral abundances at either 100% (field_abund) or 10% (reduced_abund) of measured field viral abundance to generate treatments with field-relevant and reduced viral infection pressure, respectively. Replicates of batch incubation jars were harvested every 8 hours for 48 hours to enumerate bacteria and viruses by microscopy (n=5) and profile bacterial community composition by 16S rRNA amplicon sequencing (n=3).

Zimmerman, Amy [Pacific Northwest National Laborat↗
DOE OSTI2020

Single-Cell Genomics of Novel Actinobacteria With the Wood–Ljungdahl Pathway Discovered in a Serpentinizing System

Serpentinite-hosted systems represent modern-day analogs of early Earth environments. In these systems, water-rock interactions generate highly alkaline and reducing fluids that can contain hydrogen, methane, and low-molecular-weight hydrocarbons-potent reductants capable of fueling microbial metabolism. In this study, we investigated the microbiota of Hakuba Happo hot springs (~50°C; pH~10.5–11), located in Nagano (Japan), which are impacted by the serpentinization process. Analysis of the 16S rRNA gene amplicon sequences revealed that the bacterial community comprises Nitrospirae (47%), “Parcubacteria” (19%), Deinococcus-Thermus (16%), and Actinobacteria (9%), among others. Notably, only 57 amplicon sequence variants (ASV) were detected, and fifteen of these accounted for 90% of the amplicons. Among the abundant ASVs, an early-branching, uncultivated actinobacterial clade identified as RBG-16-55-12 in the SILVA database was detected. Ten single-cell genomes (average pairwise nucleotide identity: 0.98–1.00; estimated completeness: 33–93%; estimated genome size: ~2.3 Mb) that affiliated with this clade were obtained. Taxonomic classification using single copy genes indicates that the genomes belong to the actinobacterial class-level clade UBA1414 in the Genome Taxonomy Database. Based on metabolic pathway predictions, these actinobacteria are anaerobes, capable of glycolysis, dissimilatory nitrate reduction and CO 2 fixation via the Wood–Ljungdahl (WL) pathway. Several other genomes within UBA1414 and two related class-level clades also encode the WL pathway, which has not yet been reported for the Actinobacteria phylum. For the Hakuba actinobacterium, the energy metabolism related to the WL pathway is likely supported by a combination of the Rnf complex, group 3b and 3d [NiFe]-hydrogenases, [FeFe]-hydrogenases, and V-type (H + /Na + pump) ATPase. The genomes also harbor a form IV ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO) complex, also known as a RubisCO-like protein, and contain signatures of interactions with viruses, including clustered regularly interspaced short palindromic repeat (CRISPR) regions and several phage integrases. This is the first report and detailed genome analysis of a bacterium within the Actinobacteria phylum capable of utilizing the WL pathway. The Hakuba actinobacterium is a member of the clade UBA1414/RBG-16-55-12, formerly within the group “OPB41.” We propose to name this bacterium ‘Candidatus Hakubanella thermoalkaliphilus.'

59 BASIC BIOLOGICAL SCIENCES↗