Genome-resolved Metagenomics Research Articles

Intimate microbe-water-mineral interactions mediate alkalization in the pyroxene-rich iron ore mines in Panxi area, Southwest China

In contrast to acid mine drainage, the microbial assembly and (bio)geochemical processes in alkaline mine conditions remain under-investigated. Here, microbe-water-mineral interactions were systematically investigated in two representative iron mines with alkaline conditions in the Panxi mining area, Southwest China. Compared to reference riverine samples less interfered by mining activities, the iron ore samples, composed of vanadium-titanium magnetite and pyroxene-rich bedrocks, exhibited elevated levels of Fe, HCl-extractable Fe(II), total sulfur, nitrate and sulfate, but lower total carbon (TC). Meanwhile, the mine drainage showed significantly higher sulfate, but lower TC concentrations than the riverine samples. Intriguingly, the Serpentinimonas spp., typically reported in serpentinites, prevailed in the microbial communities from the mine samples exhibiting higher pH. This suggests that the alkaline environments in Panxi mines result from serpentinization-like reactions. Enrichment of Thiobacillus spp. was observed in the mine-dwelling microbial communities, positively correlated with total sulfur, sulfate, nitrate, and Fe(II). Genome-resolved metagenomics suggested a chemoautotrophic lifestyle for the Thiobacillus species (e.g., carbon fixation, sulfur oxidation, and oxygen respiration), which may generate H+ and mitigate alkalization. This study provides valuable insights into progressive development of alkaline mine ecosystems and offers guidance for developing appropriate engineering strategies to restore the abandoned alkaline mines.

Metaproteogenomics resolution of a high-CO2 aquifer community reveals a complex cellular adaptation of groundwater Gracilibacteria to a host-dependent lifestyle

BackgroundBacteria of the candidate phyla radiation (CPR), constituting about 25% of the bacterial biodiversity, are characterized by small cell size and patchy genomes without complete key metabolic pathways, suggesting a symbiotic lifestyle. Gracilibacteria (BD1-5), which are part of the CPR branch, possess alternate coded genomes and have not yet been cultivated. The lifestyle of Gracilibacteria, their temporal dynamics, and activity in natural ecosystems, particularly in groundwater, has remained largely unexplored. Here, we aimed to investigate Gracilibacteria activity in situ and to discern their lifestyle based on expressed genes, using the metaproteogenome of Gracilibacteria as a function of time in the cold-water geyser Wallender Born in the Volcanic Eifel region in Germany.ResultsWe coupled genome-resolved metagenomics and metaproteomics to investigate a cold-water geyser microbial community enriched in Gracilibacteria across a 12-day time-series. Groundwater was collected and sequentially filtered to fraction CPR and other bacteria. Based on 725 Gbps of metagenomic data, 1129 different ribosomal protein S3 marker genes, and 751 high-quality genomes (123 population genomes after dereplication), we identified dominant bacteria belonging to Gallionellales and Gracilibacteria along with keystone microbes, which were low in genomic abundance but substantially contributing to proteomic abundance. Seven high-quality Gracilibacteria genomes showed typical limitations, such as limited amino acid or nucleotide synthesis, in their central metabolism but no co-occurrence with potential hosts. The genomes of these Gracilibacteria were encoded for a high number of proteins involved in cell to cell interaction, supporting the previously surmised host-dependent lifestyle, e.g., type IV and type II secretion system subunits, transporters, and features related to cell motility, which were also detected on protein level.ConclusionsWe here identified microbial keystone taxa in a high-CO2 aquifer, and revealed microbial dynamics of Gracilibacteria. Although Gracilibacteria in this ecosystem did not appear to target specific organisms in this ecosystem due to lack of co-occurrence despite enrichment on 0.2-µm filter fraction, we provide proteomic evidence for the complex machinery behind the host-dependent lifestyle of groundwater Gracilibacteria.38kgAxaMce3Yn9ghud_S2pVideo

Genome-resolved metagenomics revealed novel microbial taxa with ancient metabolism from macroscopic microbial mat structures inhabiting anoxic deep reefs of a Maldivian Blue Hole.

Blue holes are vertical water-filled openings in carbonate rock that exhibit complex morphology, ecology, and water chemistry. In this study, macroscopic microbial mat structures found in complete anoxic conditions in the Faanu Mudugau Blue Hole (Maldives) were studied by metagenomic methods. Such communities have likely been evolutionary isolated from the surrounding marine environment for more than 10,000 years since the Blue Hole formation during the last Ice Age. A total of 48 high-quality metagenome-assembled genomes (MAGs) were recovered, predominantly composed of the phyla Chloroflexota, Proteobacteria and Desulfobacterota. None of these MAGs have been classified to species level (<95% ANI), suggesting the discovery of several new microbial taxa. In particular, MAGs belonging to novel bacterial genera within the order Dehalococcoidales accounted for 20% of the macroscopic mat community. Genome-resolved metabolic analysis of this dominant microbial fraction revealed a mixotrophic lifestyle based on energy conservation via fermentation, hydrogen metabolism and anaerobic CO2 fixation through the Wood-Ljungdahl pathway. Interestingly, these bacteria showed a high proportion of ancestral genes in their genomes providing intriguing perspectives on mechanisms driving microbial evolution in this peculiar environment. Overall, our results provide new knowledge for understanding microbial life under extreme conditions in blue hole environments.

Assimilatory sulphate reduction by acidogenesis: The key to prevent H2S formation during food and green waste composting for sustainable urbanization

Food and green waste composting is a nature-based process for sustainable management of municipal organic waste. Although widely used for these organic wastes, composting is hindered by rapid organic acidification and malodor emission, particularly hydrogen sulphide (H2S). Using genome-resolved metagenomics, this study aimed to decipher the underlying mechanisms of sulphate reduction to H2S by acidogenesis during food and green waste composting. Results showed that both volatile fatty acids (VFAs) and H2S were significantly produced within the first week of composting. Further analysis identified that bacteria (e.g. Lactobacillus) with acid-producing genes (ackA and LDH) were key accomplices to assimilatory sulphate reduction for producing H2S by providing VFAs, especially acetic and lactic acids, as a carbon source. Such sulphate reduction could be directly induced by bacteria (e.g. Bacillus, and Ureibacillus) with intact assimilatory sulphate-reducing genes (cysNC, cysH, and cysJ/I). Nevertheless, individual bacteria (e.g. Paenibacillus and Acetobacter) without any sulphate-reducing genes could collaborate with their partners carrying complemented genes to complete sulphate reduction toward H2S. Furthermore, VFAs production could acidify composting substrates to abiotically aggravate the conversion of sulphur ions (e.g. S2- and HS-) to H2S. Nevertheless, alkaline additives suppressed these mechanisms to prevent both acidogenesis and H2S production, thereby, reducing the global acidification and human toxicity potentials of food and green waste composting.

Hyperexpansion of genetic diversity and metabolic capacity of extremophilic bacteria and archaea in ancient Andean lake sediments

BackgroundThe Andean Altiplano hosts a repertoire of high-altitude lakes with harsh conditions for life. These lakes are undergoing a process of desiccation caused by the current climate, leaving terraces exposed to extreme atmospheric conditions and serving as analogs to Martian paleolake basins. Microbiomes in Altiplano lake terraces have been poorly studied, enclosing uncultured lineages and a great opportunity to understand environmental adaptation and the limits of life on Earth. Here we examine the microbial diversity and function in ancient sediments (10.3–11 kyr BP (before present)) from a terrace profile of Laguna Lejía, a sulfur- and metal/metalloid-rich saline lake in the Chilean Altiplano. We also evaluate the physical and chemical changes of the lake over time by studying the mineralogy and geochemistry of the terrace profile.ResultsThe mineralogy and geochemistry of the terrace profile revealed large water level fluctuations in the lake, scarcity of organic carbon, and high concentration of SO42--S, Na, Cl and Mg. Lipid biomarker analysis indicated the presence of aquatic/terrestrial plant remnants preserved in the ancient sediments, and genome-resolved metagenomics unveiled a diverse prokaryotic community with still active microorganisms based on in silico growth predictions. We reconstructed 591 bacterial and archaeal metagenome-assembled genomes (MAGs), of which 98.8% belonged to previously unreported species. The most abundant and widespread metabolisms among MAGs were the reduction and oxidation of S, N, As, and halogenated compounds, as well as aerobic CO oxidation, possibly as a key metabolic trait in the organic carbon-depleted sediments. The broad redox and CO2 fixation pathways among phylogenetically distant bacteria and archaea extended the knowledge of metabolic capacities to previously unknown taxa. For instance, we identified genomic potential for dissimilatory sulfate reduction in Bacteroidota and α- and γ-Proteobacteria, predicted an enzyme for ammonia oxidation in a novel Actinobacteriota, and predicted enzymes of the Calvin–Benson–Bassham cycle in Planctomycetota, Gemmatimonadota, and Nanoarchaeota.ConclusionsThe high number of novel bacterial and archaeal MAGs in the Laguna Lejía indicates the wide prokaryotic diversity discovered. In addition, the detection of genes in unexpected taxonomic groups has significant implications for the expansion of microorganisms involved in the biogeochemical cycles of carbon, nitrogen, and sulfur.EPcqnK6xUD66cr6MUL2q3UVideo

Genome-resolved metagenomics reveals the nitrifiers enrichment and species succession in activated sludge under extremely low dissolved oxygen

Nitrification, a process carried out by aerobic microorganisms that oxidizes ammonia to nitrate via nitrite, is an indispensable step in wastewater nitrogen removal. To facilitate energy and carbon savings, applying low dissolved oxygen (DO) is suggested to shortcut the conventional biological nitrogen removal pathway, however, the impact of low DO on nitrifying communities within activated sludge is not fully understood. This study used genome-resolved metagenomics to compare nitrifying communities under extremely low- and high-DO. Two bioreactors were parallelly operated to perform nitrification and DO was respectively provided by limited gas-liquid mass transfer from the atmosphere (AN reactor, DO < 0.1 mg/L) and by sufficient aeration (AE reactor, DO > 5.0 mg/L). Low DO was thought to limit nitrifiers growth; however, we demonstrated that complete nitrification could still be achieved under the extremely low-DO conditions, but with no nitrite accumulation observed. Kinetic analysis showed that after long-term exposure to low DO, nitrifiers had a higher oxygen affinity constant and could maintain a relatively high nitrification rate, particularly at low levels of DO (<0.2 mg/L). Community-level gene analysis indicated that low DO promoted enrichment of nitrifiers (the genera Nitrosomonas and Nitrospira, increased by 2.3- to 4.3-fold), and also harbored with 2.3 to 5.3 times higher of nitrification functional genes. Moreover, 46 high-quality (>90 % completeness and <5 % contamination) with 3 most abundant medium-quality metagenome-assembled genomes (MAGs) were retrieved using binning methods. Genome-level phylogenetic analysis revealed the species succession within nitrifying populations. Surprisingly, compared to DO-rich conditions, low-DO conditions were found to efficiently suppressed the ordinary heterotrophic microorganisms (e.g., the families Anaerolineales, Phycisphaerales, and Chitinophagales), but selected for the specific candidate denitrifiers (within phylum Bacteroidota). This study provides new microbial insights to demonstrate that low-DO favors the enrichment of autotrophic nitrifiers over heterotrophs with species-level successions, which would facilitate the optimization of energy and carbon management in wastewater treatment.

Sampling fish gut microbiota - A genome-resolved metagenomic approach.

Despite a surge in microbiota-focused studies in teleosts, few have reported functional data on whole metagenomes as it has proven difficult to extract high biomass microbial DNA from fish intestinal samples. The zebrafish is a promising model organism in functional microbiota research, yet studies on the functional landscape of the zebrafish gut microbiota through shotgun based metagenomics remain scarce. Thus, a consensus on an appropriate sampling method accurately representing the zebrafish gut microbiota, or any fish species is lacking. Addressing this, we systematically tested four methods of sampling the zebrafish gut microbiota: collection of faeces from the tank, the whole gut, intestinal content, and the application of ventral pressure to facilitate extrusion of gut material. Additionally, we included water samples as an environmental control to address the potential influence of the environmental microbiota on each sample type. To compare these sampling methods, we employed a combination of genome-resolved metagenomics and 16S metabarcoding techniques. We observed differences among sample types on all levels including sampling, bioinformatic processing, metagenome co-assemblies, generation of metagenome-assembled genomes (MAGs), functional potential, MAG coverage, and population level microdiversity. Comparison to the environmental control highlighted the potential impact of the environmental contamination on data interpretation. While all sample types tested are informative about the zebrafish gut microbiota, the results show that optimal sample type for studying fish microbiomes depends on the specific objectives of the study, and here we provide a guide on what factors to consider for designing functional metagenome-based studies on teleost microbiomes.

Deciphering microbial metabolic interactions and their implications for community dynamics in acid mine drainage sediments

The microbially-mediated reduction processes have potential for the bioremediation of acid mine drainage (AMD), which represents a worldwide environment problem. However, we know little about the microbial interactions in anaerobic AMD sediments. Here we utilized genome-resolved metagenomics to uncover the nature of cooperative and competitive metabolic interactions in 90 AMD sediments across Southern China. Our analyses recovered well-represented prokaryotic communities through the reconstruction of 2625 population genomes. Functional analyses of these genomes revealed extensive metabolic handoffs which occurred more frequently in nitrogen metabolism than in sulfur metabolism, as well as stable functional redundancy across sediments resulting from populations with low genomic relatedness. Genome-scale metabolic modeling showed that metabolic competition promoted microbial co-occurrence relationships, suggesting that community assembly was dominated by habitat filtering in sediments. Notably, communities colonizing more extreme conditions tended to be highly competitive, which was typically accompanied with increased network complexity but decreased stability of the microbiome. Finally, our results demonstrated that heterotrophic Thermoplasmatota associated with ferric iron and sulfate reduction contributed most to the elevated levels of competition. Our study shed light on the cooperative and competitive metabolisms of microbiome in the hazardous AMD sediments, which may provide preliminary clues for the AMD bioremediation in the future.

Non-redundant metagenome-assembled genomes of activated sludge reactors at different disturbances and scales

Metagenome-assembled genomes (MAGs) are microbial genomes reconstructed from metagenomic data and can be assigned to known taxa or lead to uncovering novel ones. MAGs can provide insights into how microbes interact with the environment. Here, we performed genome-resolved metagenomics on sequencing data from four studies using sequencing batch reactors at microcosm (~25 mL) and mesocosm (~4 L) scales inoculated with sludge from full-scale wastewater treatment plants. These studies investigated how microbial communities in such plants respond to two environmental disturbances: the presence of toxic 3-chloroaniline and changes in organic loading rate. We report 839 non-redundant MAGs with at least 50% completeness and 10% contamination (MIMAG medium-quality criteria). From these, 399 are of putative high-quality, while sixty-seven meet the MIMAG high-quality criteria. MAGs in this catalogue represent the microbial communities in sixty-eight laboratory-scale reactors used for the disturbance experiments, and in the full-scale wastewater treatment plant which provided the source sludge. This dataset can aid meta-studies aimed at understanding the responses of microbial communities to disturbances, particularly as ecosystems confront rapid environmental changes.

Source-to-tap investigation of the occurrence of nontuberculous mycobacteria in a full-scale chloraminated drinking water system.

Nontuberculous mycobacteria (NTM) in drinking water are a significant public health concern. However, an incomplete understanding of the factors that influence the occurrence of NTM in drinking water limits our ability to characterize risk and prevent infection. This study sought to evaluate the influence of season and water treatment, distribution, and stagnation on NTM in drinking water. Samples were collected source-to-tap in a full-scale, chloraminated drinking water system approximately monthly from December 2019 to November 2020. NTM were characterized using culture-dependent (plate culture with matrix-assisted laser desorption ionization-time-of-flight mass spectrometry [MALDI-TOF MS] isolate analysis) and culture-independent methods (quantitative PCR and genome-resolved metagenomics). Sampling locations included source waters, three locations within the treatment plant, and five buildings receiving water from the distribution system. Building plumbing samples consisted of first draw, 5-min flush, and full flush cold-water samples. As the study took place during the COVID-19 pandemic, the influence of reduced water usage in three of the five buildings was also investigated. The highest NTM densities source-to-tap were found in the summer first draw building water samples (107 gene copies/L), which also had the lowest monochloramine concentrations. Flushing was found to be effective for reducing NTM and restoring disinfectant residuals, though flush times necessary to improve water quality varied by building. Clinically relevant NTM species, including Mycobacterium avium, were recovered via plate culture, with increased occurrence observed in buildings with higher water age. Four of five NTM metagenome-assembled genomes were identified to the species level and matched identified isolates.IMPORTANCENTM infections are increasing in prevalence, difficult to treat, and associated with high morbidity and mortality rates. Our lack of understanding of the factors that influence NTM occurrence in drinking water limits our ability to prevent infections, accurately characterize risk, and focus remediation efforts. In this study, we comprehensively evaluated NTM in a full-scale drinking water system, showing that various steps in treatment and distribution influence NTM presence. Stagnant building water contained the highest NTM densities source-to-tap and was associated with low disinfectant residuals. We illustrated the differences in NTM detection and characterization obtained from culture-based and culture-independent methods, highlighting the complementarity between these approaches. We demonstrated that focusing NTM mitigation efforts in building plumbing systems, which have the highest NTM densities source-to-tap, has potential for immediate positive effects. We also identified steps during treatment that increase NTM levels, which provides beneficial information for utilities seeking to reduce NTM in finished water.

Evaluating urine volume and host depletion methods to enable genome-resolved metagenomics of the urobiome.

The gut microbiome has emerged as a clear player in health and disease, in part by mediating host response to environment and lifestyle. The urobiome (microbiota of the urinary tract) likely functions similarly. However, efforts to characterize the urobiome and assess its functional potential have been limited due to technical challenges including low microbial biomass and high host cell shedding in urine. Here, to begin addressing these challenges, we evaluate urine sample volume (100 ml - 5 mL), and host DNA depletion methods and their effects on urobiome profiles in healthy dogs, which are a robust large animal model for the human urobiome. We collected urine from seven dogs and fractionated samples into aliquots. One set of samples was spiked with host (canine) cells to model a biologically relevant host cell burden in urine. Samples then underwent DNA extraction followed by 16S rRNA gene and shotgun metagenomic sequencing. We then assembled metagenome assembled genomes (MAGs) and compared microbial composition and diversity across groups. We tested six methods of DNA extraction: QIAamp BiOstic Bacteremia (no host depletion), QIAamp DNA Microbiome, Molzym MolYsis, NEBNext Microbiome DNA Enrichment, Zymo HostZERO, and Propidium Monoazide. In relation to urine sample volume, 3 3.0 mL resulted in the most consistent urobiome profiling. In relation to host depletion, individual (dog) but not extraction method drove overall differences in microbial composition. DNA Microbiome yielded the greatest microbial diversity in 16S rRNA sequencing data and shotgun metagenomic sequencing data, and maximized MAG recovery while effectively depleting host DNA in host-spiked urine samples. As proof-of-principle, we then mined MAGs for core metabolic functions and environmental chemical metabolism. We identified long chain alkane utilization in two of the urine MAGs. Long chain alkanes are common pollutants that result from industrial combustion processes and end up in urine. This is the first study, to our knowledge, to demonstrate environmental chemical degradation potential in urine microbes through genome-resolved metagenomics. These findings provide guidelines for studying the urobiome in relation to sample volume and host depletion, and lay the foundation for future evaluation of urobiome function in relation to health and disease.

Ecological associations distribution modelling of marine plankton at a global scale.

Marine plankton communities form intricate networks of interacting organisms at the base of the food chain, and play a central role in regulating ocean biogeochemical cycles and climate. However, predicting plankton community shifts in response to climate change remains challenging. While species distribution models are valuable tools for predicting changes in species biogeography under climate change scenarios, they generally overlook the key role of biotic interactions, which can significantly shape ecological processes and ecosystem responses. Here, we introduce a novel statistical framework, association distribution modelling (ADM), designed to model and predict ecological associations distribution in space and time. Applied on a Tara Oceans genome-resolved metagenomics dataset, the present-day biogeography of ADM-inferred marine plankton associations revealed four major biogeographic biomes organized along a latitudinal gradient. We predicted the evolution of these biome-specific communities in response to a climate change scenario, highlighting differential responses to environmental change. Finally, we explored the functional potential of impacted plankton communities, focusing on carbon fixation, outlining the predicted evolution of its geographical distribution and implications for ecosystem function.This article is part of the theme issue 'Connected interactions: enriching food web research by spatial and social interactions'.

Environmental activity-based protein profiling for function-driven enzyme discovery from natural communities

BackgroundMicrobial communities are important drivers of global biogeochemical cycles, xenobiotic detoxification, as well as organic matter decomposition. Their major metabolic role in ecosystem functioning is ensured by a unique set of enzymes, providing a tremendous yet mostly hidden enzymatic potential. Exploring this enzymatic repertoire is therefore not only relevant for a better understanding of how microorganisms function in their natural environment, and thus for ecological research, but further turns microbial communities, in particular from extreme habitats, into a valuable resource for the discovery of novel enzymes with potential applications in biotechnology. Different strategies for their uncovering such as bioprospecting, which relies mainly on metagenomic approaches in combination with sequence-based bioinformatic analyses, have emerged; yet accurate function prediction of their proteomes and deciphering the in vivo activity of an enzyme remains challenging.ResultsHere, we present environmental activity-based protein profiling (eABPP), a multi-omics approach that extends genome-resolved metagenomics with mass spectrometry-based ABPP. This combination allows direct profiling of environmental community samples in their native habitat and the identification of active enzymes based on their function, even without sequence or structural homologies to annotated enzyme families. eABPP thus bridges the gap between environmental genomics, correct function annotation, and in vivo enzyme activity. As a showcase, we report the successful identification of active thermostable serine hydrolases from eABPP of natural microbial communities from two independent hot springs in Kamchatka, Russia.ConclusionsBy reporting enzyme activities within an ecosystem in their native state, we anticipate that eABPP will not only advance current methodological approaches to sequence homology-guided enzyme discovery from environmental ecosystems for subsequent biocatalyst development but also contributes to the ecological investigation of microbial community interactions by dissecting their underlying molecular mechanisms.

Genome-resolved metagenomics reveals diverse taxa and metabolic complexity in Antarctic lake microbial structures.

Lake Untersee, a lake in Antarctica that is perennially covered with ice, is home to unique microbial structures that are not lithified. We have evaluated the structure of the community and its metabolic potential across the pigmented upper layers and the sediment-enriched deeper layers in these pinnacle and cone-shaped microbial structures using metagenomics. These microbial structures are inhabited by distinct communities. The upper layers of the cone-shaped structures have a higher abundance of the cyanobacterial MAG Microcoleus, while the pinnacle-shaped structures have a higher abundance of Elainellacea MAG. This suggests that cyanobacteria influence the morphologies of the mats. We identified stark contrasts in the composition of the community and its metabolic potential between the upper and lower layers of the mat. The upper layers of the mat, which receive light, have an increased abundance of photosynthetic pathways. In contrast, the lower layer has an increased abundance of heterotrophic pathways. Our results also showed that Lake Untersee is the first Antarctic lake with a substantial presence of ammonia-oxidizing Nitrospiracea and amoA genes. The genomic capacity for recycling biological molecules was prevalent across metagenome-assembled genomes (MAGs) that cover 19 phyla. This highlights the importance of nutrient scavenging in ultra-oligotrophic environments. Overall, our study provides new insights into the formation of microbial structures and the potential metabolic complexity of Antarctic laminated microbial mats. These mats are important environments for biodiversity that drives biogeochemical cycling in polar deserts.

Genome-resolved metagenomics reveals the effect of nutrient availability on bacterial genomic properties across 44 European freshwater lakes.

Understanding intricate microbial interactions in the environment is crucial. This is especially true for the relationships between nutrients and bacteria, as phosphorus, nitrogen and organic carbon availability are known to influence bacterial population dynamics. It has been suggested that low nutrient conditions prompt the evolutionary process of genome streamlining. This process helps conserve scarce nutrients and allows for proliferation. Genome streamlining is associated with genomic properties such as %GC content, genes encoding sigma factors, percent coding regions, gene redundancy, and functional shifts in processes like cell motility and ATP binding cassette transporters, among others. The current study aims to unveil the impact of nutrition on the genome size, %GC content, and functional properties of pelagic freshwater bacteria. We do this at finer taxonomic resolutions for many metagenomically characterized communities. Our study confirms the interplay of trophic level and genomic properties. It also highlights that different nutrient types, particularly phosphorus and nitrogen, impact these properties differently. We observed a covariation of functional traits with genome size. Larger genomes exhibit enriched pathways for motility, environmental interaction, and regulatory genes. ABC transporter genes reflect the availability of nutrients in the environment, with small genomes presumably relying more on metabolites from other organisms. We also discuss the distinct strategies different phyla adopt to adapt to oligotrophic environments. The findings contribute to our understanding of genomic adaptations within complex microbial communities.

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.

Microbial Sources and Sinks of Nitrous Oxide during Organic Waste Composting.

Composting is widely used for organic waste management and is also a major source of nitrous oxide (N2O) emission. New insight into microbial sources and sinks is essential for process regulation to reduce N2O emission from composting. This study used genome-resolved metagenomics to decipher the genomic structures and physiological behaviors of individual bacteria for N2O sources and sinks during composting. Results showed that several nosZ-lacking denitrifiers in feedstocks drove N2O emission at the beginning of the composting. Such emission became negligible at the thermophilic stage, as high temperatures inhibited all denitrifiers for N2O production except for those containing nirK. The nosZ-lacking denitrifiers were notably enriched to increase N2O production at the cooling stage. Nevertheless, organic biodegradation limited energy availability for chemotaxis and flagellar assembly to restrain nirKS-containing denitrifiers for nitrate reduction toward N2O sources but insignificantly interrupt norBC- and nosZ-containing bacteria (particularly nosZ-containing nondenitrifiers) for N2O sinks by capturing N2O and nitric oxide (NO) for energy production, thereby reducing N2O emission at the mature stage. Furthermore, nosZII-type bacteria included all nosZ-containing nondenitrifiers and dominated N2O sinks. Thus, targeted strategies can be developed to restrict the physiological behaviors of nirKS-containing denitrifiers and expand the taxonomic distribution of nosZ for effective N2O mitigation in composting.

Genome-resolved metagenomics identifies novel active microbes in biogeochemical cycling within methanol-enriched soil.

Metagenome assembled genomes (MAGs), generated from sequenced 13C-labelled DNA from 13C-methanol enriched soils, were binned using an ensemble approach. This method produced a significantly larger number of higher-quality MAGs compared to direct binning approaches. These MAGs represent both the primary methanol utilizers and the secondary utilizers labelled via cross-feeding and predation on the labelled methylotrophs, including numerous uncultivated taxa. Analysis of these MAGs enabled the identification of multiple metabolic pathways within these active taxa that have climatic relevance relating to nitrogen, sulfur and trace gas metabolism. This includes denitrification, dissimilatory nitrate reduction to ammonium, ammonia oxidation and metabolism of organic sulfur species. The binning of viral sequence data also yielded extensive viral MAGs, identifying active viral replication by both lytic and lysogenic phages within the methanol-enriched soils. These MAGs represent a valuable resource for characterizing biogeochemical cycling within terrestrial environments.

A comparison of short-read, HiFi long-read, and hybrid strategies for genome-resolved metagenomics.

Shotgun metagenomics enables the reconstruction of complex microbial communities at a high level of detail. Such an approach can be conducted using both short-read and long-read sequencing data, as well as a combination of both. To assess the pros and cons of these different approaches, we used 22 fecal DNA extracts collected weekly for 11 weeks from two respective lab mice to study seven performance metrics over four combinations of sequencing depth and technology: (i) 20 Gbp of Illumina short-read data, (ii) 40 Gbp of short-read data, (iii) 20 Gbp of PacBio HiFi long-read data, and (iv) 40 Gbp of hybrid (20 Gbp of short-read +20 Gbp of long-read) data. No strategy was best for all metrics; instead, each one excelled across different metrics. The long-read approach yielded the best assembly statistics, with the highest N50 and lowest number of contigs. The 40 Gbp short-read approach yielded the highest number of refined bins. Finally, the hybrid approach yielded the longest assemblies and the highest mapping rate to the bacterial genomes. Our results suggest that while long-read sequencing significantly improves the quality of reconstructed bacterial genomes, it is more expensive and requires deeper sequencing than short-read approaches to recover a comparable amount of reconstructed genomes. The most optimal strategy is study-specific and depends on how researchers assess the trade-off between the quantity and quality of recovered genomes.IMPORTANCEMice are an important model organism for understanding the gut microbiome. When studying these gut microbiomes using DNA techniques, researchers can choose from technologies that use short or long DNA reads. In this study, we perform an extensive benchmark between short- and long-read DNA sequencing for studying mice gut microbiomes. We find that no one approach was best for all metrics and provide information that can help guide researchers in planning their experiments.

Refinement of the "Candidatus Accumulibacter" genus based on metagenomic analysis of biological nutrient removal (BNR) pilot-scale plants operated with reduced aeration.

Members of the "Candidatus Accumulibacter" genus are widely studied as key polyphosphate-accumulating organisms (PAOs) in biological nutrient removal (BNR) facilities performing enhanced biological phosphorus removal (EBPR). This diverse lineage includes 18 "Ca. Accumulibacter" species, which have been proposed based on the phylogenetic divergence of the polyphosphate kinase 1 (ppk1) gene and genome-scale comparisons of metagenome-assembled genomes (MAGs). Phylogenetic classification based on the 16S rRNA genetic marker has been difficult to attain because most "Ca. Accumulibacter" MAGs are incomplete and often do not include the rRNA operon. Here, we investigate the "Ca. Accumulibacter" diversity in pilot-scale treatment trains performing BNR under low dissolved oxygen (DO) conditions using genome-resolved metagenomics. Using long-read sequencing, we recovered medium- and high-quality MAGs for 5 of the 18 "Ca. Accumulibacter" species, all with rRNA operons assembled, which allowed a reassessment of the 16S rRNA-based phylogeny of this genus and an analysis of phylogeny based on the 23S rRNA gene. In addition, we recovered a cluster of MAGs that based on 16S rRNA, 23S rRNA, ppk1, and genome-scale phylogenetic analyses do not belong to any of the currently recognized "Ca. Accumulibacter" species for which we propose the new species designation "Ca. Accumulibacter jenkinsii" sp. nov. Relative abundance evaluations of the genus across all pilot plant operations revealed that regardless of the operational mode, "Ca. A. necessarius" and "Ca. A. propinquus" accounted for more than 40% of the "Ca. Accumulibacter" community, whereas the newly proposed "Ca. A. jenkinsii" accounted for about 5% of the "Ca. Accumulibacter" community.IMPORTANCEOne of the main drivers of energy use and operational costs in activated sludge processes is the amount of oxygen provided to enable biological phosphorus and nitrogen removal. Wastewater treatment facilities are increasingly considering reduced aeration to decrease energy consumption, and whereas successful BNR has been demonstrated in systems with minimal aeration, an adequate understanding of the microbial communities that facilitate nutrient removal under these conditions is still lacking. In this study, we used genome-resolved metagenomics to evaluate the diversity of the "Candidatus Accumulibacter" genus in pilot-scale plants operating with minimal aeration. We identified the "Ca. Accumulibacter" species enriched under these conditions, including one novel species for which we propose "Ca. Accumulibacter jenkinsii" sp. nov. as its designation. Furthermore, the MAGs obtained for five additional "Ca. Accumulibacter" species further refine the phylogeny of the "Ca. Accumulibacter" genus and provide new insight into its diversity within unconventional biological nutrient removal systems.