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Despite >130 years of microbial cultivation studies, many microorganisms remain resistant to traditional cultivation approaches, including numerous candidate phyla of bacteria and archaea. Unraveling the mysteries of these candidate phyla is a grand challenge in microbiology and is especially important in habitats where they are abundant, including some extreme environments and low-energy ecosystems. Over the past decade, parallel advances in DNA amplification, DNA sequencing and computing have enabled rapid progress on this problem, particularly through metagenomics and single-cell genomics. Although each approach suffers limitations, metagenomics and single-cell genomics are particularly powerful when combined synergistically. Studies focused on extreme environments have revealed the first substantial genomic information for several candidate phyla, encompassing putative acidophiles (Parvarchaeota), halophiles (Nanohaloarchaeota), thermophiles (Acetothermia, Aigarchaeota, Atribacteria, Calescamantes, Korarchaeota, and Fervidibacteria), and piezophiles (Gracilibacteria). These data have enabled insights into the biology of these organisms, including catabolic and anabolic potential, molecular adaptations to life in extreme environments, unique genomic features such as stop codon reassignments, and predictions about cell ultrastructure. In addition, the rapid expansion of genomic coverage enabled by these studies continues to yield insights into the early diversification of microbial lineages and the relationships within and between the phyla of Bacteria and Archaea. In the next 5 years, the genomic foliage within the tree of life will continue to grow and the study of yet-uncultivated candidate phyla will firmly transition into the post-genomic era.

Microbial communities drive biogeochemical cycles through networks of metabolite exchange that are structured along energetic gradients. As energy yields become limiting, these networks favor co-metabolic interactions to maximize energy disequilibria. Here we apply single-cell genomics, metagenomics, and metatranscriptomics to study bacterial populations of the abundant “microbial dark matter” phylum Marinimicrobia along defined energy gradients. We show that evolutionary diversification of major Marinimicrobia clades appears to be closely related to energy yields, with increased co-metabolic interactions in more deeply branching clades. Several of these clades appear to participate in the biogeochemical cycling of sulfur and nitrogen, filling previously unassigned niches in the ocean. Notably, two Marinimicrobia clades, occupying different energetic niches, express nitrous oxide reductase, potentially acting as a global sink for the greenhouse gas nitrous oxide. Little is known about Marinimicrobia, a group of bacteria that are prevalent in the oceans. Here, the authors study global populations of Marinimicrobia using single-cell genomics, metagenomics and metatranscriptomics, showing potential co-metabolic interactions and participation in the sulfur and nitrogen cycles.

Rare microbial lineages, such as members of the candidate phyla radiation (CPR) bacteria and Diapherotrites, Parvarchaeota, Aenigmarchaeota, Nanoarchaeota, and Nanohaloarchaeota (DPANN) archaea, are increasingly recognized as key components of microbial communities in natural systems. Yet, their global distribution, biogeographic patterns, and broader role in shaping microbial community structure across diverse ecosystems remain poorly characterized. Here, we analyzed 2860 metagenomes spanning nine ecosystems using a curated reference database and a bias-aware taxonomic filtering approach to quantify the richness, relative abundance, and structural influence of low-abundance microbial taxa on community structure across a wide range of ecosystems. Our findings reveal that rare taxa, primarily CPR and DPANN, disproportionately shape microbial community dissimilarities across global ecosystems. We observed that the richness of these two groups, that drives community structure variation, increases with latitude, peaking in temperate regions, thereby contrasting classical latitudinal diversity patterns and suggesting unique biogeographic drivers. CPR and DPANN were predominantly enriched in free-living environments, particularly groundwater and soil, then in host-associated habitats, consistent with niche specialization shaped by environmental filtering and dispersal constraints. These findings challenge abundance-centric assumptions in microbial ecology and highlight the need to integrate low-abundance taxa into macroecological frameworks. Fully resolving their ecological functions, however, will require targeted experimental and multi-omics investigations.

Neonatal sepsis (NS) kills 750,000 infants every year. Effectively treating NS requires timely diagnosis and antimicrobial therapy matched to the causative pathogens, but most blood cultures for suspected NS do not recover a causative pathogen. We refer to these suspected but unidentified pathogens as microbial dark matter. Given these low culture recovery rates, many non-culture-based technologies are being explored to diagnose NS, including PCR, 16S amplicon sequencing, and whole metagenomic sequencing. However, few of these newer technologies are scalable or sustainable globally. To reduce worldwide deaths from NS, one possibility may be performing population-wide pathogen discovery. Because pathogen transmission patterns can vary across space and time, computational models can be built to predict the pathogens responsible for NS by region and season. This approach could help to optimally treat patients, decreasing deaths from NS and increasing antimicrobial stewardship until effective diagnostics that are scalable become available globally.

Microbial single-cell genomics (SCG) provides access to the genomes of rare and uncultured microorganisms and is a complementary method to metagenomics. Due to the femtogram-levels of DNA in a single microbial cell, sequencing the genome requires whole genome amplification (WGA) as a preliminary step. However, the most common WGA method, multiple displacement amplification (MDA), is known to be costly and biased against specific genomic regions, preventing high-throughput applications and resulting in uneven genome coverage. Thus, obtaining high-quality genomes from many taxa, especially minority members of microbial communities, becomes difficult. Here, we present a volume reduction approach that significantly reduces costs while improving genome coverage and uniformity of DNA amplification products in standard 384-well plates. Our results demonstrate that further volume reduction in specialized and complex setups (e.g., microfluidic chips) is likely unnecessary to obtain higher-quality microbial genomes. This volume reduction method makes SCG more feasible for future studies, thus helping to broaden our knowledge on the diversity and function of understudied and uncharacterized microorganisms in the environment.

Background Increasing our knowledge of soil biodiversity is fundamental to forecast changes in ecosystem functions under global change scenarios. All multicellular organisms are now known to be holobionts, containing large assemblages of microbial species. Soil fauna is now known to have thousands of species living within them. However, we know very little about the identity and function of host microbiome in contrasting soil faunal groups, across different terrestrial biomes, or at a large spatial scale. Here, we examined the microbiomes of multiple functionally important soil fauna in contrasting terrestrial ecosystems across China. Results Different soil fauna had diverse and unique microbiomes, which were also distinct from those in surrounding soils. These unique microbiomes were maintained within taxa across diverse sampling sites and in contrasting terrestrial ecosystems. The microbiomes of nematodes, potworms, and earthworms were more difficult to predict using environmental data, compared to those of collembolans, oribatid mites, and predatory mites. Although stochastic processes were important, deterministic processes, such as host selection, also contributed to the assembly of unique microbiota in each taxon of soil fauna. Microbial biodiversity, unique microbial taxa, and microbial dark matter (defined as unidentified microbial taxa) all increased with trophic levels within the soil food web. Conclusions Our findings demonstrate that soil animals are important as repositories of microbial biodiversity, and those at the top of the food web harbor more diverse and unique microbiomes. This hidden source of biodiversity is rarely considered in biodiversity and conservation debates and stresses the importance of preserving key soil invertebrates. 6kheVvGhGfd4H1y_K4HwoR Video abstract

The data reported in this paper are among the first relating to the microbiology of hyper-arid, very high altitude deserts and they provide base line information on the structure of actinobacterial communities. The high mountain Cerro Chajnantor landscape of the Central Andes in northern Chile is exposed to the world’s most intense levels of solar radiation and its impoverished soils are severely desiccated. The purpose of this research was to define the actinobacterial community structures in soils at altitudes ranging from 3000 to 5000 m above sea level. Pyrosequencing surveys have revealed an extraordinary degree of microbial dark matter at these elevations that includes novel candidate actinobacterial classes, orders and families. Ultraviolet-B irradiance and a range of edaphic factors were found to be highly significant in determining community compositions at family and genus levels of diversity.

The Gaia hypothesis, proposed 50 years ago, posits that the Earth's biosphere, atmosphere, hydrosphere and lithosphere interact as a cybernetic system, maintaining the long-term habitability of the planet. The resulting chemical composition of the atmosphere, oceans and crust is unique as compared to the other planets of our solar system, and due to the presence of life. Together these components comprise the biosphere, the life support system of the planet, with most of the essential processes carried out by microbes. Over a half of the elements in the periodic table are now known to have some biological role with many having complex biogeochemical cycles. The global microbiome inhabits a wide range of environments including deep into the Earth's crust, with a population of ∼10 cells and more than a trillion species. Deep sequencing projects have revealed hitherto unknown phyla and 'microbial dark matter'. The discoveries of conductive pili and cable bacteria have demonstrated that microbes transfer electrons to and from external sources, sometimes over significant distances, while research on quorum sensing and the plethora of microbial volatile organic substances have provided new insights into how microbes communicate. These advances in microbiology have expanded our understanding how Gaia could actually work.

The bacterial tree of life has recently undergone significant expansion, chiefly from candidate phyla retrieved through genome-resolved metagenomics. Bypassing the need for genome availability, we present a snapshot of bacterial phylogenetic diversity based on the recovery of high-quality SSU rRNA gene sequences extracted from nearly 7000 metagenomes and all available reference genomes. We illuminate taxonomic richness within established bacterial phyla together with environmental distribution patterns, providing a revised framework for future phylogeny-driven sequencing efforts.

Background The 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. Results The mineralogy and geochemistry of the terrace profile revealed large water level fluctuations in the lake, scarcity of organic carbon, and high concentration of SO 4 2- -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 CO 2 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. Conclusions The 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. EPcqnK6xUD66cr6MUL2q3U Video Abstract