Microbial Dark Matter

Metagenomes encode an enormous diversity of proteins, reflecting a multiplicity of functions and activities1,2 . Exploration of this vast sequence space has been limited to a comparative analysis against reference microbial genomes and protein families derived from those genomes. Here, to examine the scale of yet untapped functional diversity beyond what is currently possible through the lens of reference genomes, we develop a computational approach to generate reference-free protein families from the sequence space in metagenomes...

To date, the vast majority of prokaryotic organisms escapes detailed characterization because they cannot be isolated in axenic cultures. These organisms are referred to as microbial dark matter (MDM). Targeted labelling and sorting of these microorganisms paves the way for single-cell, enrichment or cultivation approaches. In this review, we describe an array of different methods ranging from labeling-free to specific labelling techniques. In addition, different cell sorting methods and their combinations with targeting strategies are summarized and downstream applications like sequencing and cultivation are reviewed...

Owing to the ineffectiveness of traditional culture techniques for the vast majority of microbial species, culture-independent analyses utilizing next-generation sequencing and bioinformatics have become essential for gaining insight into microbial ecology and function. This mini-review focuses on two essential methods for obtaining genetic information from uncultured prokaryotes, metagenomics and single-cell genomics. We analyzed the registration status of uncultured prokaryotic genome data from major public databases and assessed the advantages and limitations of both the methods...

No abstract text is available yet for this article.

Deserts occupy one-third of the Earth's terrestrial surface and represent a potentially significant reservoir of microbial biodiversity, yet the majority of desert microorganisms remain uncharacterized and are seen as "microbial dark matter". Here, we introduce a multi-omics strategy, culturomics-based metagenomics (CBM) that integrates large-scale cultivation, full-length 16S rRNA gene amplicon, and shotgun metagenomic sequencing. The results showed that CBM captured a significant amount of taxonomic and functional diversity missed in direct sequencing by increasing the recovery of amplicon sequence variants (ASVs) and high/medium-quality metagenome-assembled genomes (MAGs)...

Enormous viral populations have been identified in activated sludge systems, but their ecological and biochemical roles in landfill leachate treatment plants remain poorly understood. To address this knowledge gap, we conducted an in-depth analysis using 36 metagenomic datasets that we collected and sequenced during a half-year time-series sampling campaign at six sites in a full-scale landfill leachate treatment plant (LLTP), elucidating viral distribution, virus‒host dynamics, virus-encoded auxiliary metabolic genes (AMGs), and viral contributions to the spread of virulence and antibiotic resistance genes...

Patescibacteria, also known as the candidate phyla radiation (CPR), are a diverse group of bacteria that constitute a disproportionately large fraction of microbial dark matter. Its few cultivated members, belonging mostly to Saccharibacteria, grow as epibionts on host Actinobacteria. Due to a lack of suitable tools, the genetic basis of this lifestyle and other unique features of Patescibacteira remain unexplored. Here, we show that Saccharibacteria exhibit natural competence, and we exploit this property for their genetic manipulation...

Archaea constitute a substantial fraction of marine microbial biomass and play critical roles in the biogeochemistry of oceans. However, studies on their distribution and ecology in the Arctic Ocean are relatively scarce. Here, we studied the distributions of archaea and archaeal ammonia monooxygenase (amoA) gene in the western Arctic Ocean, using the amplicon sequencing approach from the sea surface to deep waters up to 3040 m depth. A total of five archaeal phyla, Nitrososphaerota, "Euryarchaeota", "Halobacteriota," "Nanoarchaeota", and Candidatus Thermoplasmatota, were detected...

Microbes continually shape Earth's biochemical and physical landscapes by inhabiting diverse metabolic niches. Despite the important role microbes play in ecosystem functioning, most microbial species remain unknown highlighting a gap in our understanding of structured complex ecosystems. To elucidate the relevance of these unknown taxa, often referred to as "microbial dark matter," the integration of multiple high throughput sequencing technologies was used to evaluate the co-occurrence and connectivity of all microbes within the community...

Bacteria affiliated with the Candidate Phyla Radiation (CPR) are a hyper-diverse group of ultra-small bacteria with versatile yet sparse metabolisms. However, most insights into this group come from a surprisingly small number of environments, and recovery of CPR bacteria from soils has been hindered due to their extremely low abundance within complex microbial assemblages. In this study we enriched soil samples from 14 different soil fertility treatments for ultra-small (<0.45 μm) bacteria in order to study rare soil CPR...

Cryoconite, the dark sediment on the surface of glaciers, often aggregates into oval or irregular granules serving as biogeochemical factories. They reduce a glacier's albedo, act as biodiversity hotspots by supporting aerobic and anaerobic microbial communities, constitute one of the organic matter (OM) sources on glaciers, and are a feeder for micrometazoans. Although cryoconite granules have multiple roles on glaciers, their formation is poorly understood. Cyanobacteria are ubiquitous and abundant engineers of cryoconite hole ecosystems...

Detailed molecular composition of solid phase extracted dissolved organic matter (SPEDOM ) collected from fractured-rock groundwater was compared to connected surface river water at two different watersheds in the unconfined chalk aquifer of Champagne in France using full scan ultrahigh resolution electrospray and photoionization Fourier transform ion cyclotron mass spectrometry (FT-ICR MS), Orbitrap tandem MS (MS/MS) and 1 H magnetic resonance spectroscopy (NMR). 1 H NMR spectroscopy indicated that groundwater SPEDOM carried a higher contribution of aliphatic compounds while surface river waters SPEDOM were enriched in carboxyl-rich alicyclic molecules (CRAM), acetate derivatives and oxygenated units...

The land-use of organic fertilizers is considered as an important sustainable method for resource utilization, which may have an impact on the microplastic behaviors in the soil. Here, a 240-d dark culture experiment was conducted to reveal the degradation and biofilm characteristics of degradable and refractory granule microplastics in soil and soil-fertilizer systems. The results indicated that microplastics generally exhibited a weak weight loss as well as a specific etiolation on the surface after the culture, especially polyvinyl-chloride and polyhydroxyalkanoates (PHA)...

Excessive nitrogen (N) fertilization in agroecological systems increases nitrous oxide (N2 O) emissions. 3,4-dimethylpyrazole phosphate (DMPP) is used to mitigate N2 O losses. The influence of DMPP efficiency on N2 O mitigation was clearly affected by spatiotemporal heterogeneity. Using field and incubation experiments combined with metagenomic sequencing, we aimed to investigate DMPP efficiency and the underlying microbial mechanisms in dark-brown (Siping, SP), fluvo-aquic (Cangzhou, CZ; Xinxiang, XX), and red soil (Wenzhou, WZ) from diverse climatic zones...

The order Saprospirales , a group of bacteria involved in complex degradation pathways, comprises three officially described families: Saprospiraceae , Lewinellaceae , and Haliscomenobacteraceae . These collectively contain 17 genera and 31 species. The current knowledge on Saprospirales diversity is the product of traditional isolation methods, with the inherited limitations of culture-based approaches. This study utilized the extensive information available in public sequence repositories combined with recent analytical tools to evaluate the global evidence-based diversity of the Saprospirales order...

BACKGROUND: Archaea of the order Thermoplasmatales are widely distributed in natural acidic areas and are amongst the most acidophilic prokaryotic organisms known so far. These organisms are difficult to culture, with currently only six genera validly published since the discovery of Thermoplasma acidophilum in 1970. Moreover, known great diversity of uncultured Thermoplasmatales represents microbial dark matter and underlines the necessity of efforts in cultivation and study of these archaea...

Dissolved organic matter (DOM)-mediated divalent mercury (Hg(II)) aging kinetics play a crucial role in controlling Hg(II) transformation and bioavailability in natural aquatic environments. However, the differential environmental behaviors of new and aged Hg(II) in a same reaction system remains unknown. In this study, multi-isotope tracing was used to investigate the impacts of binding site and sulfidation during DOM-mediated Hg(II) aging processes on Hg(II) reduction and microbial methylation in the same reaction system...

Transcriptomics, or more specifically mRNA sequencing, is a powerful tool to study gene expression at the single-cell level (scRNA-seq) which enables new insights into a plethora of biological processes. While methods for single-cell RNA-seq in eukaryotes are well established, application to prokaryotes is still challenging. Reasons for that are rigid and diverse cell wall structures hampering lysis, the lack of polyadenylated transcripts impeding mRNA enrichment, and minute amounts of RNA requiring amplification steps before sequencing...

A large number of microbes are not able to form colonies using agar-plating methods, which is one of the reasons that cultivation based on solid media leaves the majority of microbial diversity in the environment inaccessible. We developed a new Non-Colony-Forming Liquid Cultivation method (NCFLC) that can selectively isolate non-colony-forming microbes that exclusively grow in liquid culture. The NCFLC method involves physically separating cells using dilution-to-extinction (DTE) cultivation and then selecting those that could not grow on a solid medium...

More than 20,000 species of prokaryotes (less than 1% of the estimated number of Earth's microbial species) have been described thus far. However, the vast majority of microbes that inhabit extreme environments remain uncultured and this group is termed "microbial dark matter." Little is known regarding the ecological functions and biotechnological potential of these underexplored extremophiles, thus representing a vast untapped and uncharacterized biological resource. Advances in microbial cultivation approaches are key for a detailed and comprehensive characterization of the roles of these microbes in shaping the environment and, ultimately, for their biotechnological exploitation, such as for extremophile-derived bioproducts (extremozymes, secondary metabolites, CRISPR Cas systems, and pigments, among others), astrobiology, and space exploration...