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Single-cell genomics and transcriptomics can provide reliable context for assembled genome fragments and gene expression activity on the level of individual prokaryotic genomes. These methods are rapidly emerging as an essential complement to cultivation-based, metagenomics, metatranscriptomics, and microbial community-focused research approaches by allowing direct access to information from individual microorganisms, even from deep-branching phylogenetic groups that currently lack cultured representatives. Their integration and binning with environmental ‘omics data already provides unprecedented insights into microbial diversity and metabolic potential, enabling us to provide information on individual organisms and the structure and dynamics of natural microbial populations in complex environments. This review highlights the pitfalls and recent advances in the field of single-cell omics and its importance in microbiological and biotechnological studies.Key points• Single-cell omics expands the tree of life through the discovery of novel organisms, genes, and metabolic pathways.• Disadvantages of metagenome-assembled genomes are overcome by single-cell omics.• Functional analysis of single cells explores the heterogeneity of gene expression.• Technical challenges still limit this field, thus prompting new method developments.

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 Marine deep subsurface sediments were once thought to be devoid of eukaryotic life, but advances in molecular technology have unlocked the presence and activity of well-known closely related terrestrial and marine fungi. Commonly detected fungi in deep marine sediment environments includes Penicillium , Aspergillus , Cladosporium , Fusarium , and Schizophyllum , which could have important implications in carbon and nitrogen cycling in this isolated environment. In order to determine the diversity and unknown metabolic capabilities of fungi in deep-sea sediments, their genomes need to be fully analyzed. In this study, two Penicillium species were isolated from South Pacific Gyre sediment enrichments during Integrated Ocean Drilling Program Expedition 329. The inner gyre has very limited productivity, organic carbon, and nutrients. Results Here, we present high-quality genomes of two proposed novel Penicillium species using Illumina HiSeq and PacBio sequencing technologies. Single-copy homologues within the genomes were compared to other closely related genomes using OrthoMCL and maximum-likelihood estimation, which showed that these genomes were novel species within the genus Penicillium . We propose to name isolate SPG-F1 as Penicillium pacificasedimenti sp. nov. and SPG-F15 as Penicillium pacificagyrus sp. nov. The resulting genome sizes were 32.6 Mbp and 36.4 Mbp, respectively, and both genomes were greater than 98% complete as determined by the presence of complete single-copy orthologs. The transposable elements for each genome were 4.87% for P . pacificasedimenti and 10.68% for P . pacificagyrus . A total of 12,271 genes were predicted in the P . pacificasedimenti genome and 12,568 genes in P . pacificagyrus . Both isolates contained genes known to be involved in the degradation of recalcitrant carbon, amino acids, and lignin-derived carbon. Conclusions Our results provide the first constructed genomes of novel Penicillium isolates from deep marine sediments, which will be useful for future studies of marine subsurface fungal diversity and function. Furthermore, these genomes shed light on the potential impact fungi in marine sediments and the subseafloor could have on global carbon and nitrogen biogeochemical cycles and how they may be persisting in the most energy-limited sedimentary biosphere.

The plethora of stress factors that can damage microbial cells has evolved sophisticated stress response mechanisms. While existing bioreporters can monitor individual responses, sensors for detecting multimodal stress responses in living microorganisms are still lacking. Orthogonally detectable red, green, and blue fluorescent proteins combined in a single plasmid, dubbed RGB-S reporter, enable simultaneous, independent, and real-time analysis of the transcriptional response of Escherichia coli using three promoters which report physiological stress (PosmY for RpoS), genotoxicity (PsulA for SOS), and cytotoxicity (PgrpE for RpoH). The bioreporter is compatible with standard analysis and Fluorescent Activated Cell Sorting (FACS) combined with subsequent transcriptome analysis. Various stressors, including the biotechnologically relevant 2-propanol, activate one, two, or all three stress responses, which can significantly impact non-stress-related metabolic pathways. Implemented in microfluidic cultivation with confocal fluorescence microscopy imaging, the RGB-S reporter enabled spatiotemporal analysis of live biofilms revealing stratified subpopulations of bacteria with heterogeneous stress responses.

The phylum Chloroflexota (formerly Chloroflexi) encompasses metabolically diverse bacteria that often have high prevalence in terrestrial and aquatic habitats, some even with biotechnological application. However, there is substantial disagreement in public databases which lineage should be considered a member of the phylum and at what taxonomic level. Here, we addressed these issues through extensive phylogenomic analyses. The analyses were based on a collection of >5000 Chloroflexota genomes and metagenome-assembled genomes (MAGs) from public databases, novel environmental sites, as well as newly generated MAGs from publicly available sequence reads via an improved binning approach incorporating covariance information. Based on calculated relative evolutionary divergence, we propose that Candidatus Dormibacterota should be listed as a class (i.e., Ca. Dormibacteria) within Chloroflexota together with the classes Anaerolineae, Chloroflexia, Dehalococcoidia, Ktedonobacteria, Ca. Limnocylindria, Thermomicrobia, and two other classes containing only uncultured members. All other Chloroflexota lineages previously listed at the class rank appear to be rather orders or families in the Anaerolineae and Dehalococcoidia, which contain the vast majority of genomes and exhibited the strongest phylogenetic radiation within the phylum. Furthermore, the study suggests that a common ecophysiological capability of members of the phylum is to successfully cope with low energy fluxes.

Single cell genomics (SCG) is a is a complementary method to metagenomics for exploring the genomes of uncultivated microorganisms. However, due to the minute amounts of DNA in the individual microbial cell, an amplification step is required before sequencing. Unfortunately, this reaction is notoriously costly and does not amplify all genomic regions equally well, preventing high-throughput applications and leading to incomplete and biased genomes. Here, we show a simple volume reduction approach to make SCG more feasible.

Single cell genomics (SCG) is a is a complementary method to metagenomics for exploring the genomes of uncultivated microorganisms. However, due to the minute amounts of DNA in the individual microbial cell, an amplification step is required before sequencing. Unfortunately, this reaction is notoriously costly and does not amplify all genomic regions equally well, preventing high-throughput applications and leading to incomplete and biased genomes. Here, we show a simple volume reduction approach to make SCG more feasible.

Single cell genomics (SCG) can provide reliable context for assembled genome fragments on the level of individual prokaryotic genomes and has rapidly emerged as an essential complement to cultivation-based and metagenomics research approaches. Targeted cell sorting approaches, which enable the selection of specific taxa by fluorescent labeling, compatible with subsequent single cell genomics offers an opportunity to access genetic information from rare biosphere members which would have otherwise stayed hidden as microbial dark matter.

Undoubtedly, Earth s first redox revolution, which culminated 2.4 billion years ago in the Great Oxidation Event (GOE), fundamentally altered the resources available to microbial communities, leading to novel ecological competitions and evolutionary innovations. These eco-evolutionary dynamics are largely unexplored, particularly at the molecular level. Here, we hypothesize that such dynamics in the wake of the GOE explain the otherwise paradoxical evolutionary history of metal use in nitrogen fixation by nitrogenase. This ancient metalloenzyme exists in three isozymes, with distinct metal cofactors. Recent research demonstrates that the most ancient isozyme, emerging a billion years or more before the GOE, required a molybdenum (Mo) based cofactor. Alternative nitrogenases using iron (Fe) or vanadium (V) cofactors evolved after the GOE. This history is puzzling because Mo availability in the environment increased after the GOE, while Fe availability decreased, due to the contrasting environmental redox behaviors of these elements. Why, then, did the alternatives emerge only after the GOE? Using a simple model constrained by known microbial Mo quotas, we demonstrate that a strong selection pressure for use of metals in nitrogenase other than Mo is a likely consequence of competition between nitrogen fixing prokaryotes and nitrate reducing microbes, which require Mo for nitrate reduction and assimilation. This competition would have intensified after the GOE due to increasing environmental availability of nitrate, explaining the evolutionary timing of the nitrogenase isozymes. Ecological resource competition therefore emerges as a third driver of metallome evolution in deep-time, alongside the relative environmental availabilities and adaptive advantages of particular metals.Competing Interest StatementThe authors have declared no competing interest.