Revealing microbiota characteristics and predicting flavor-producing sub-communities in Nongxiangxing baijiu pit mud through metagenomic analysis and metabolic modeling.

The microorganisms of the pit mud (PM) of Nongxiangxing baijiu (NXXB) have an important role in the synthesis of flavor substances, and they determine attributes and quality of baijiu. Herein, we utilize metagenomics and genome-scale metabolic models (GSMMs) to investigate the microbial composition, metabolic functions in PM microbiota, as well as to identify microorganisms and communities linked to flavor compounds. Metagenomic data revealed that the most prevalent assembly of bacteria and archaea was Proteiniphilum, Caproicibacterium, Petrimonas, Lactobacillus, Clostridium, Aminobacterium, Syntrophomonas, Methanobacterium, Methanoculleus, and Methanosarcina. The important enzymes ofPMwere in bothGH and GT familymetabolism. A total of 38 high-quality metagenome-assembled genomes (MAGs) were obtained, including those at the family level (n = 13), genus level (n = 17), and species level (n = 8). GSMMs of the 38 MAGs were then constructed. From the GSMMs, individual and community capabilities respectively were predicted to be able to produce 111 metabolites and 598 metabolites. Twenty-three predicted metabolites were consistent with the metabonomics detected flavors and served as targets. Twelve sub-community of were screened by cross-feeding of 38 GSMMs. Of them, Methanobacterium, Sphaerochaeta, Muricomes intestini, Methanobacteriaceae, Synergistaceae, and Caloramator were core microorganisms for targets in each sub-community. Overall, this study of metagenomic and target-community screening could help our understanding of the metabolite-microbiome association and further bioregulation of baijiu.

Archaea influence composition of endoscopically visible ileocolonic biofilms.

The gut microbiota has been implicated as a driver of irritable bowel syndrome (IBS) and inflammatory bowel disease (IBD). Recently we described, mucosal biofilms, signifying alterations in microbiota composition and bile acid (BA) metabolism in IBS and ulcerative colitis (UC). Luminal oxygen concentration is a key factor in the gastrointestinal (GI) ecosystem and might be increased in IBS and UC. Here we analyzed the role of archaea as a marker for hypoxia in mucosal biofilms and GI homeostasis. The effects of archaea on microbiome composition and metabolites were analyzed via amplicon sequencing and untargeted metabolomics in 154 stool samples of IBS-, UC-patients and controls. Mucosal biofilms were collected in a subset of patients and examined for their bacterial, fungal and archaeal composition. Absence of archaea, specifically Methanobrevibacter, correlated with disrupted GI homeostasis including decreased microbial diversity, overgrowth of facultative anaerobes and conjugated secondary BA. IBS-D/-M was associated with absence of archaea. Presence of Methanobrevibacter correlated with Oscillospiraceae and epithelial short chain fatty acid metabolism and decreased levels of Ruminococcus gnavus. Absence of fecal Methanobrevibacter may indicate a less hypoxic GI environment, reduced fatty acid oxidation, overgrowth of facultative anaerobes and disrupted BA deconjugation. Archaea and Ruminococcus gnavus could distinguish distinct subtypes of mucosal biofilms. Further research on the connection between archaea, mucosal biofilms and small intestinal bacterial overgrowth should be performed.

Metagenomic insight into taxonomic composition, environmental filtering and functional redundancy for shaping worldwide modern non-lithifying microbial mats.

Modern microbial mats are relictual communities mostly found in extreme environments worldwide. Despite their significance as representatives of the ancestral Earth and their important roles in biogeochemical cycling, research on microbial mats has largely been localized, focusing on site-specific descriptions and environmental change experiments. Here, we present a global comparative analysis of non-lithifying microbial mats, integrating environmental measurements with metagenomic data from 62 samples across eight sites, including two new samples from the recently discovered Archaean Domes from Cuatro Ciénegas, Mexico. Our results revealed a notable influence of environmental filtering on both taxonomic and functional compositions of microbial mats. Functional redundancy appears to confer resilience to mats, with essential metabolic pathways conserved across diverse and highly contrasting habitats. We identified six highly correlated clusters of taxa performing similar ecological functions, suggesting niche partitioning and functional specialization as key mechanisms shaping community structure. Our findings provide insights into the ecological principles governing microbial mats, and lay the foundation for future research elucidating the intricate interplay between environmental factors and microbial community dynamics.

Interpreting an Archaean paleoenvironment through 3D imagery of microbialites.

While stromatolites, and to a lesser extent thrombolites, have been extensively studied in order to unravel Precambrian (>539 Ma) biological evolution, studies of clastic-dominated microbially induced sedimentary structures (MISS) are relatively scarce. The lack of a consolidated record of clastic microbialites creates questions about how much (and what) information on depositional and taphonomic settings can be gleaned from these fossils. We used µCT scanning, a non-destructive X-ray-based 3D imaging method, to reconstruct morphologies of ancient MISS and mat textures in two previously described coastal Archaean samples from the ~3.48 Ga Dresser Formation, Pilbara, Western Australia. The aim of this study was to test the ability of µCT scanning to visualize and make 3D measurements that can be used to interpret the biotic-environmental interactions. Fossil MISS including mat laminae with carpet-like textures in one sample and mat rip-up chips in the second sample were investigated. Compiled δ13C and δ34S analyses of specimens from the Dresser Fm. are consistent with a taxonomically diverse community that could be capable of forming such MISS. 3D measurements of fossil microbial mat chips indicate significant biostabilization and suggest formation in flow velocities >25 cm s-1. Given the stratigraphic location of these chips in a low-flow lagoonal layer, we conclude that these chips formed due to tidal influence, as these assumed velocities are consistent with recent modeling of Archaean tides. The success of µCT scanning in documenting these microbialite features validates this technique both as a first step analysis for rare samples prior to the use of more destructive techniques and as a valuable tool for gaining insight into microbialite taphonomy.

15N-DNA stable isotope probing reveals niche differentiation of ammonia oxidizers in paddy soils.

Chemoautotrophic canonical ammonia oxidizers (ammonia-oxidizing archaea (AOA) and ammonia-oxidizing bacteria (AOB)) and complete ammonia oxidizers (comammox Nitrospira) are accountable for ammonia oxidation, which is a fundamental process of nitrification in terrestrial ecosystems. However, the relationship between autotrophic nitrification and the active nitrifying populations during 15N-urea incubation has not been totally clarified. The 15N-labeled DNA stable isotope probing (DNA-SIP) technique was utilized in order to study the response from the soil nitrification process and the active nitrifying populations, in both acidic and neutral paddy soils, to the application of urea. The presence of C2H2 almost completely inhibited NO3--N production, indicating that autotrophic ammonia oxidation was dominant in both paddy soils. 15N-DNA-SIP technology could effectively distinguish active nitrifying populations in both soils. The active ammonia oxidation groups in both soils were significantly different, AOA (NS (Nitrososphaerales)-Alpha, NS-Gamma, NS-Beta, NS-Delta, NS-Zeta and NT (Ca. Nitrosotaleales)-Alpha), and AOB (Nitrosospira) were functionally active in the acidic paddy soil, whereas comammox Nitrospira clade A and Nitrosospira AOB were functionally active in the neutral paddy soil. This study highlights the effective discriminative effect of 15N-DNA-SIP and niche differentiation of nitrifying populations in these paddy soils. KEY POINTS: • 15N-DNA-SIP technology could effectively distinguish active ammonia oxidizers. • Comammox Nitrospira clade A plays a lesser role than canonical ammonia oxidizers. • The active groups in the acidic and neutral paddy soils were significantly different.

PrimerEvalPy: a tool for in-silico evaluation of primers for targeting the microbiome.

BACKGROUND: The selection of primer pairs in sequencing-based research can greatly influence the results, highlighting the need for a tool capable of analysing their performance in-silico prior to the sequencing process. We therefore propose PrimerEvalPy, a Python-based package designed to test the performance of any primer or primer pair against any sequencing database. The package calculates a coverage metric and returns the amplicon sequences found, along with information such as their average start and end positions. It also allows the analysis of coverage for different taxonomic levels. RESULTS: As a case study, PrimerEvalPy was used to test the most commonly used primers in the literature against two oral 16S rRNA gene databases containing bacteria and archaea. The results showed that the most commonly used primer pairs in the oral cavity did not match those with the highest coverage. The best performing primer pairs were found for the detection of oral bacteria and archaea. CONCLUSIONS: This demonstrates the importance of a coverage analysis tool such as PrimerEvalPy to find the best primer pairs for specific niches. The software is available under the MIT licence at https://gitlab.citius.usc.es/lara.vazquez/PrimerEvalPy .

AnnoView enables large-scale analysis, comparison, and visualization of microbial gene neighborhoods.

The analysis and comparison of gene neighborhoods is a powerful approach for exploring microbial genome structure, function, and evolution. Although numerous tools exist for genome visualization and comparison, genome exploration across large genomic databases or user-generated datasets remains a challenge. Here, we introduce AnnoView, a web server designed for interactive exploration of gene neighborhoods across the bacterial and archaeal tree of life. Our server offers users the ability to identify, compare, and visualize gene neighborhoods of interest from 30 238 bacterial genomes and 1672 archaeal genomes, through integration with the comprehensive Genome Taxonomy Database and AnnoTree databases. Identified gene neighborhoods can be visualized using pre-computed functional annotations from different sources such as KEGG, Pfam and TIGRFAM, or clustered based on similarity. Alternatively, users can upload and explore their own custom genomic datasets in GBK, GFF or CSV format, or use AnnoView as a genome browser for relatively small genomes (e.g. viruses and plasmids). Ultimately, we anticipate that AnnoView will catalyze biological discovery by enabling user-friendly search, comparison, and visualization of genomic data. AnnoView is available at http://annoview.uwaterloo.ca.

Long-term chemical and organic fertilization induces distinct variations of microbial associations but unanimous elevation of soil multifunctionality.

Intricate microbial associations contribute greatly to the multiple functions (multifunctionality) of natural ecosystems. However, the relationship between microbial associations and soil multifunctionality (SMF) in artificial ecosystems, particularly in agricultural ecosystem with frequent fertilization, remains unclear. In this study, based on a 28-year paddy field experiment, high-throughput sequencing and networks analysis was performed to investigate changes in soil microbial (archaea, bacteria, fungi, and protists) associations and how these changes correlate with SMF under long-term fertilization. Compared to no fertilization (CK), both chemical fertilization with N, P, and K (CF) and chemical fertilization plus rice straw retention (CFR) treatments showed significantly higher soil nutrient content, grain yield, microbial abundance, and SMF. With the exception of archaeal diversity, the CF treatment exhibited the lowest bacterial, fungal, and protist diversity, and the simplest microbial co-occurrence network. In contrast, the CFR treatment had the lowest archaeal diversity, but the highest bacterial, fungal, and protist diversity. Moreover, the CFR treatment exhibited the most complex microbial co-occurrence network with the highest number of nodes, edges, and interkingdom edges. These results highlight that both chemical fertilization with and without straw retention caused high ecosystem multifunctionality while changing microbial association oppositely. Furthermore, these results indicate that rice straw retention contributes to the development of the soil microbiome and ensures the sustainability of high-level ecosystem multifunctionality.

Metabolic potential of Nitrososphaera-associated clades.

Soil ammonia-oxidizing archaea (AOA) play a crucial role in converting ammonia to nitrite, thereby mobilizing reactive nitrogen species into their soluble form, with a significant impact on nitrogen losses from terrestrial soils. Yet, our knowledge regarding their diversity and functions remains limited. In this study, we reconstructed 97 high-quality AOA metagenome-assembled genomes (MAGs) from 180 soil samples collected in Central Germany during 2014-2019 summers. These MAGs were affiliated with the order Nitrososphaerales and clustered into four family-level clades (NS-α/γ/δ/ε). Among these MAGs, 75 belonged to the most abundant but least understood δ-clade. Within the δ-clade, the amoA genes in three MAGs from neutral soils showed a 99.5% similarity to the fosmid clone 54d9, which has served as representative of the δ-clade for the past two decades since even today no cultivated representatives are available. Seventy-two MAGs constituted a distinct δ sub-clade, and their abundance and expression activity were more than twice that of other MAGs in slightly acidic soils. Unlike the less abundant clades (α, γ, and ε), the δ-MAGs possessed multiple highly expressed intracellular and extracellular carbohydrate-active enzymes responsible for carbohydrate binding (CBM32) and degradation (GH5), along with highly expressed genes involved in ammonia oxidation. Together, these results suggest metabolic versatility of uncultured soil AOA and a potential mixotrophic or chemolithoheterotrophic lifestyle among 54d9-like AOA.

Dynamic evolution of the mTHF gene family associated with primary metabolism across life.

BACKGROUND: The folate cycle of one-carbon (C1) metabolism, which plays a central role in the biosynthesis of nucleotides and amino acids, demonstrates the significance of metabolic adaptation. We investigated the evolutionary history of the methylenetetrahydrofolate dehydrogenase (mTHF) gene family, one of the main drivers of the folate cycle, across life. RESULTS: Through comparative genomic and phylogenetic analyses, we found that several lineages of Archaea lacked domains vital for folate cycle function such as the mTHF catalytic and NAD(P)-binding domains of FolD. Within eukaryotes, the mTHF gene family diversified rapidly. For example, several duplications have been observed in lineages including the Amoebozoa, Opisthokonta, and Viridiplantae. In a common ancestor of Opisthokonta, FolD and FTHFS underwent fusion giving rise to the gene MTHFD1, possessing the domains of both genes. CONCLUSIONS: Our evolutionary reconstruction of the mTHF gene family associated with a primary metabolic pathway reveals dynamic evolution, including gene birth-and-death, gene fusion, and potential horizontal gene transfer events and/or amino acid convergence.

Origin and modern microbial ecology of secondary mineral deposits in Lehman Caves, Great Basin National Park, NV, USA.

Lehman Caves is an extensively decorated high desert cave that represents one of the main tourist attractions in Great Basin National Park, Nevada. Although traditionally considered a water table cave, recent studies identified abundant speleogenetic features consistent with a hypogenic and, potentially, sulfuric acid origin. Here, we characterized white mineral deposits in the Gypsum Annex (GA) passage to determine whether these secondary deposits represent biogenic minerals formed during sulfuric acid corrosion and explored microbial communities associated with these and other mineral deposits throughout the cave. Powder X-ray diffraction (pXRD), scanning electron microscopy with electron dispersive spectroscopy (SEM-EDS), and electron microprobe analyses (EPMA) showed that, while most white mineral deposits from the GA contain gypsum, they also contain abundant calcite, silica, and other phases. Gypsum and carbonate-associated sulfate isotopic values of these deposits are variable, with δ34SV-CDT between +9.7‰ and +26.1‰, and do not reflect depleted values typically associated with replacement gypsum formed during sulfuric acid speleogenesis. Petrographic observations show that the sulfates likely co-precipitated with carbonate and SiO2 phases. Taken together, these data suggest that the deposits resulted from later-stage meteoric events and not during an initial episode of sulfuric acid speleogenesis. Most sedimentary and mineral deposits in Lehman Caves have very low microbial biomass, with the exception of select areas along the main tour route that have been impacted by tourist traffic. High-throughput 16S rRNA gene amplicon sequencing showed that microbial communities in GA sediments are distinct from those in other parts of the cave. The microbial communities that inhabit these oligotrophic secondary mineral deposits include OTUs related to known ammonia-oxidizing Nitrosococcales and Thaumarchaeota, as well as common soil taxa such as Acidobacteriota and Proteobacteria. This study reveals microbial and mineralogical diversity in a previously understudied cave and expands our understanding of the geomicrobiology of desert hypogene cave systems.

Differences in archaeal diversity and potential ecological functions between saline and hypersaline lakes on Qinghai-Tibet Plateau were driven by multiple environmental and non-environmental factors beyond the salinity.

BACKGROUND: Saline lakes are home to various archaea that play special and crucial roles in the global biogeochemical cycle. The Qinghai-Tibet Plateau hosts a large number of lakes with diverse salinity ranging from 0.1 to over 400 g/L, harboring complex and diverse archaea. To the best of our knowledge, the formation mechanisms and potential ecological roles of archaea in Qinghai-Tibetan Plateau saline lakes remain largely unknown. RESULTS: Using High-throughput Illumina sequencing, we uncovered the vastly distinct archaea communities between two typical saline lakes with significant salinity differences on the Qinghai Tibet Plateau (Qinghai saline lake and Chaka hypersaline lake) and suggested archaea played different important roles in methanogenesis-related and nitrate reduction-related functions of these two lakes, respectively. Rather than the individual effect of salinity, the composite effect of salinity with diverse environmental parameters (e.g., temperature, chlorophyll a, total nitrogen, and total phosphorus) dominated the explanation of the variations in archaeal community structure in different habitats. Based on the network analysis, we further found the correlations between dominant archaeal OTUs were tight but significantly different between the two habitats, implying that archaeal interactions may also largely determine the shape of archaeal communities. CONCLUSION: The present study improved our understanding of the structure and function of archaea in different saline lakes on the Qinghai-Tibet Plateau and provided a new perspective on the mechanisms underlying shaping their communities.

Mechanisms of secondary biogenic coalbed methane formation in bituminous coal seams: a joint experimental and multi-omics study.

Coal seam microbes, as endogenous drivers of secondary biogenic gas production in coal seams, might be related to methane production in coal seams. In this study, we carried out anaerobic indoor culture experiments of microorganisms from three different depths of bituminous coal seams in Huainan mining area, and revealed the secondary biogas generation mechanism of bituminous coal seams by using the combined analysis of macro-genome and metabolism multi-omics. The results showed that the cumulative mass molar concentrations (Molality) of biomethane production increased with the increase of the coal seam depth in two consecutive cycles. At the genus level, there were significant differences in the bacterial and archaeal community structures corresponding to the three coal seams 1#, 6#, and 9#(p < 0.05). The volatile matter of air-dry basis (Vad) of coal was significantly correlated with differences in genus-level composition of bacteria and archaea, with correlations of R bacterial = 0.368 and R archaeal = 0.463, respectively. Functional gene analysis showed that the relative abundance of methanogenesis increased by 42% before and after anaerobic fermentation cultivation. Meanwhile, a total of 11 classes of carbon metabolism homologues closely related to methanogenesis were detected in the liquid metabolites of coal bed microbes after 60 days of incubation. Finally, the fatty acid, amino acid and carbohydrate synergistic methanogenic metabolic pathway was reconstructed based on the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. The expression level of mcrA gene within the metabolic pathway of the 1# deep coal sample was significantly higher than that of the other two groups (p < 0.05 for significance), and the efficient expression of mcrA gene at the end of the methanogenic pathway promoted the conversion of bituminous coal organic matter to methane. Therefore, coal matrix compositions may be the key factors causing diversity in microbial community and metabolic function, which might be related to the different methane content in different coal seams.

Impact of diversified cropping systems and fertilization strategies on soil microbial abundance and functional potentials for nitrogen cycling.

Diversified cropping systems and fertilization strategies were proposed to enhance the abundance and diversity of the soil microbiome, thereby stabilizing their beneficial services for maintaining soil fertility and supporting plant growth. Here, we assessed across three different long-term field experiments in Europe (Netherlands, Belgium, Northern Germany) whether diversified cropping systems and fertilization strategies also affect their functional gene abundance. Soil DNA was analyzed by quantitative PCR for quantifying bacteria, archaea and fungi as well as functional genes related to nitrogen (N) transformations; including bacterial and archaeal nitrification (amoA-bac,arch), three steps of the denitrification process (nirK, nirS and nosZ-cladeI,II) and N2 assimilation (nifH), respectively. Crop diversification and fertilization strategies generally enhanced soil total carbon (C), N and microbial abundance, but with variation between sites. Overall effects of diversified cropping systems and fertilization strategies on functional genes were much stronger than on the abundance of bacteria, archaea and fungi. The legume-based cropping systems showed great potential not only in stimulating the growth of N-fixing microorganisms but also in boosting downstream functional potentials for N cycling. The sorghum-based intercropping system suppressed soil ammonia oxidizing prokaryotes. N fertilization reduced the abundance of nitrifiers and denitrifiers except for ammonia-oxidizing bacteria, while the application of the synthetic nitrification inhibitor DMPP combined with mineral N reduced growth of both ammonia-oxidizing bacteria and archaea. In conclusion, this study demonstrates a strong impact of diversified agricultural practices on the soil microbiome and their functional potentials mediating N transformations.

Simultaneous methane mitigation and nitrogen removal by denitrifying anaerobic methane oxidation in lake sediments.

Methane (CH4) is a potent greenhouse gas, with lake ecosystems significantly contributing to its global emissions. Denitrifying anaerobic methane oxidation (DAMO) process, mediated by NC10 bacteria and ANME-2d archaea, links global carbon and nitrogen cycles. However, their potential roles in mitigating methane emissions and removing nitrogen from lake ecosystems remain unclear. This study explored the spatial variations in activities of nitrite- and nitrate-DAMO and their functional microbes in Changdanghu Lake sediments (Jiangsu Province, China). The results showed that although the average abundance of ANME-2d archaea (5.0 × 106 copies g-1) was significantly higher than that of NC10 bacteria (2.1 × 106 copies g-1), the average potential rates of nitrite-DAMO (4.59 nmol 13CO2 g-1 d-1) and nitrate-DAMO (5.01 nmol 13CO2 g-1 d-1) showed no significant difference across all sampling sites. It is estimated that nitrite- and nitrate-DAMO consumed approximately 6.46 and 7.05 mg CH4 m-2 d-1, respectively, which accordingly achieved 15.07-24.95 mg m-2 d-1 nitrogen removal from the studied lake sediments. Statistical analyses found that nitrite- and nitrate-DAMO activities were both significantly related to sediment nitrate contents and ANME-2d archaeal abundance. In addition, NC10 bacterial and ANME-2d archaeal community compositions showed significant correlations with sediment organic carbon content and water depth. Overall, this study underscores the dual roles of nitrite- and nitrate-DAMO processes in CH4 mitigation and nitrogen elimination and their key environmental impact factors (sediment organic carbon and inorganic nitrogen contents, and water depth) in shallow lake, enhancing the understanding of carbon and nitrogen cycles in freshwater aquatic ecosystems.

Analysis of nearly 3000 archaeal genomes from terrestrial geothermal springs sheds light on interconnected biogeochemical processes.

Terrestrial geothermal springs are physicochemically diverse and host abundant populations of Archaea. However, the diversity, functionality, and geological influences of these Archaea are not well understood. Here we explore the genomic diversity of Archaea in 152 metagenomes from 48 geothermal springs in Tengchong, China, collected from 2016 to 2021. Our dataset is comprised of 2949 archaeal metagenome-assembled genomes spanning 12 phyla and 392 newly identified species, which increases the known species diversity of Archaea by ~48.6%. The structures and potential functions of the archaeal communities are strongly influenced by temperature and pH, with high-temperature acidic and alkaline springs favoring archaeal abundance over Bacteria. Genome-resolved metagenomics and metatranscriptomics provide insights into the potential ecological niches of these Archaea and their potential roles in carbon, sulfur, nitrogen, and hydrogen metabolism. Furthermore, our findings illustrate the interplay of competition and cooperation among Archaea in biogeochemical cycles, possibly arising from overlapping functional niches and metabolic handoffs. Taken together, our study expands the genomic diversity of Archaea inhabiting geothermal springs and provides a foundation for more incisive study of biogeochemical processes mediated by Archaea in geothermal ecosystems.

Insight into the microbial community of denitrification process using different solid carbon sources: Not only bacteria.

There is a lack of understanding about the bacterial, fungal and archaeal communities' composition of solid-phase denitrification (SPD) systems. We investigated four SPD systems with different carbon sources by analyzing microbial gene sequences based on operational taxonomic unit (OTU) and amplicon sequence variant (ASV). The results showed that the corncob-polyvinyl alcohol sodium alginate-polycaprolactone (CPSP, 0.86±0.04 mg NO3--N/(g·day)) and corncob (0.85±0.06 mg NO3--N/(g·day)) had better denitrification efficiency than polycaprolactone (PCL, 0.29±0.11 mg NO3--N/(g·day)) and polyvinyl alcohol-sodium alginate (PVA-SA, 0.24±0.07 mg NO3--N/(g·day)). The bacterial, fungal and archaeal microbial composition was significantly different among carbon source types such as Proteobacteria in PCL (OTU: 83.72%, ASV: 82.49%) and Rozellomycota in PVA-SA (OTU: 71.99%, ASV: 81.30%). ASV methods can read more microbial units than that of OTU and exhibit higher alpha diversity and classify some species that had not been identified by OTU such as Nanoarchaeota phylum, unclassified_ f_ Xanthobacteraceae genus, etc., indicating ASV may be more conducive to understand SPD microbial communities. The co-occurring network showed some correlation between the bacteria fungi and archaea species, indicating different species may collaborate in SPD systems. Similar KEGG function prediction results were obtained in two bioinformatic methods generally and some fungi and archaea functions should not be ignored in SPD systems. These results may be beneficial for understanding microbial communities in SPD systems.

Exploring the landscape of symbiotic diversity and distribution in unicellular ciliated protists.

BACKGROUND: The eukaryotic-bacterial symbiotic system plays an important role in various physiological, developmental, and evolutionary processes. However, our current understanding is largely limited to multicellular eukaryotes without adequate consideration of diverse unicellular protists, including ciliates. RESULTS: To investigate the bacterial profiles associated with unicellular organisms, we collected 246 ciliate samples spanning the entire Ciliophora phylum and conducted single-cell based metagenome sequencing. This effort has yielded the most extensive collection of bacteria linked to unicellular protists to date. From this dataset, we identified 883 bacterial species capable of cohabiting with ciliates, unveiling the genomes of 116 novel bacterial cohabitants along with 7 novel archaeal cohabitants. Highlighting the intimate relationship between ciliates and their cohabitants, our study unveiled that over 90% of ciliates coexist with bacteria, with individual hosts fostering symbiotic relationships with multiple bacteria concurrently, resulting in the observation of seven distinct symbiotic patterns among bacteria. Our exploration of symbiotic mechanisms revealed the impact of host digestion on the intracellular diversity of cohabitants. Additionally, we identified the presence of eukaryotic-like proteins in bacteria as a potential contributing factor to their resistance against host digestion, thereby expanding their potential host range. CONCLUSIONS: As the first large-scale analysis of prokaryotic associations with ciliate protists, this study provides a valuable resource for future research on eukaryotic-bacterial symbioses. Video Abstract.