Exploring novel alkane-degradation pathways in uncultured bacteria from the North Atlantic Ocean.

IMPORTANCE: Petroleum pollution in the ocean has increased because of rapid population growth and modernization, requiring urgent remediation. Our understanding of the metabolic response of native microbial communities to oil spills is not well understood. Here, we explored the baseline hydrocarbon-degrading communities of a subarctic Atlantic region to uncover the metabolic potential of the bacteria that inhabit the surface and subsurface water. We conducted enrichments with a 13C-labeled hydrocarbon to capture the fraction of the community actively using the hydrocarbon. We then combined this approach with metagenomics to identify the metabolic potential of this hydrocarbon-degrading community. This revealed previously undescribed uncultured bacteria with unique metabolic mechanisms involved in aerobic hydrocarbon degradation, indicating that temperature may be pivotal in structuring hydrocarbon-degrading baseline communities. Our findings highlight gaps in our understanding of the metabolic complexity of hydrocarbon degradation by native marine microbial communities.

Well-hidden methanogenesis in deep, organic-rich sediments of Guaymas Basin.

Deep marine sediments (>1mbsf) harbor ~26% of microbial biomass and are the largest reservoir of methane on Earth. Yet, the deep subsurface biosphere and controls on its contribution to methane production remain underexplored. Here, we use a multidisciplinary approach to examine methanogenesis in sediments (down to 295 mbsf) from sites with varying degrees of thermal alteration (none, past, current) at Guaymas Basin (Gulf of California) for the first time. Traditional (13C/12C and D/H) and multiply substituted (13CH3D and 12CH2D2) methane isotope measurements reveal significant proportions of microbial methane at all sites, with the largest signal at the site with past alteration. With depth, relative microbial methane decreases at differing rates between sites. Gibbs energy calculations confirm methanogenesis is exergonic in Guaymas sediments, with methylotrophic pathways consistently yielding more energy than the canonical hydrogenotrophic and acetoclastic pathways. Yet, metagenomic sequencing and cultivation attempts indicate that methanogens are present in low abundance. We find only one methyl-coenzyme M (mcrA) sequence within the entire sequencing dataset. Also, we identify a wide diversity of methyltransferases (mtaB, mttB), but only a few sequences phylogenetically cluster with methylotrophic methanogens. Our results suggest that the microbial methane in the Guaymas subsurface was produced over geologic time by relatively small methanogen populations, which have been variably influenced by thermal sediment alteration. Higher resolution metagenomic sampling may clarify the modern methanogen community. This study highlights the importance of using a multidisciplinary approach to capture microbial influences in dynamic, deep subsurface settings like Guaymas Basin.

Inference and reconstruction of the heimdallarchaeial ancestry of eukaryotes.

In the ongoing debates about eukaryogenesis-the series of evolutionary events leading to the emergence of the eukaryotic cell from prokaryotic ancestors-members of the Asgard archaea play a key part as the closest archaeal relatives of eukaryotes1. However, the nature and phylogenetic identity of the last common ancestor of Asgard archaea and eukaryotes remain unresolved2-4. Here we analyse distinct phylogenetic marker datasets of an expanded genomic sampling of Asgard archaea and evaluate competing evolutionary scenarios using state-of-the-art phylogenomic approaches. We find that eukaryotes are placed, with high confidence, as a well-nested clade within Asgard archaea and as a sister lineage to Hodarchaeales, a newly proposed order within Heimdallarchaeia. Using sophisticated gene tree and species tree reconciliation approaches, we show that analogous to the evolution of eukaryotic genomes, genome evolution in Asgard archaea involved significantly more gene duplication and fewer gene loss events compared with other archaea. Finally, we infer that the last common ancestor of Asgard archaea was probably a thermophilic chemolithotroph and that the lineage from which eukaryotes evolved adapted to mesophilic conditions and acquired the genetic potential to support a heterotrophic lifestyle. Our work provides key insights into the prokaryote-to-eukaryote transition and a platform for better understanding the emergence of cellular complexity in eukaryotic cells.

Expansion of Armatimonadota through marine sediment sequencing describes two classes with unique ecological roles.

Marine sediments comprise one of the largest environments on the planet, and their microbial inhabitants are significant players in global carbon and nutrient cycles. Recent studies using metagenomic techniques have shown the complexity of these communities and identified novel microorganisms from the ocean floor. Here, we obtained 77 metagenome-assembled genomes (MAGs) from the bacterial phylum Armatimonadota in the Guaymas Basin, Gulf of California, and the Bohai Sea, China. These MAGs comprise two previously undescribed classes within Armatimonadota, which we propose naming Hebobacteria and Zipacnadia. They are globally distributed in hypoxic and anoxic environments and are dominant members of deep-sea sediments (up to 1.95% of metagenomic raw reads). The classes described here also have unique metabolic capabilities, possessing pathways to reduce carbon dioxide to acetate via the Wood-Ljungdahl pathway (WLP) and generating energy through the oxidative branch of glycolysis using carbon dioxide as an electron sink, maintaining the redox balance using the WLP. Hebobacteria may also be autotrophic, not previously identified in Armatimonadota. Furthermore, these Armatimonadota may play a role in sulfur and nitrogen cycling, using the intermediate compounds hydroxylamine and sulfite. Description of these MAGs enhances our understanding of diversity and metabolic potential within anoxic habitats worldwide.

Functional divergence of teleost carbonic anhydrase 4.

The functional role of membrane-bound carbonic anhydrases (CAs) has been of keen interest in the past decade, and in particular, studies have linked CA in red muscle, heart, and eye to enhanced tissue oxygen extraction in bony fishes (teleosts). However, the number of purported membrane-bound CA isoforms in teleosts, combined with the imperfect system of CA isoform nomenclature, present roadblocks for ascribing physiological functions to particular CA isoforms across different teleost lineages. Here we developed an organizational framework for membrane-bound CAs in teleosts, providing the latest phylogenetic analysis of extant CA4 and CA4-like isoforms. Our data confirm that there are three distinct isoforms of CA4 (a, b, and c) that are conserved across major teleost lineages, with the exception of CA4c gene being lost in salmonids. Tissue distribution analyses suggest CA4a functions in oxygen delivery across teleost lineages, while CA4b may be specialized for renal acid-base balance and ion regulation. This work provides an important foundation for researchers to elucidate the functional significance of CA4 isoforms in fishes.

New globally distributed bacterial phyla within the FCB superphylum.

Microbes in marine sediments play crucial roles in global carbon and nutrient cycling. However, our understanding of microbial diversity and physiology on the ocean floor is limited. Here, we use phylogenomic analyses of thousands of metagenome-assembled genomes (MAGs) from coastal and deep-sea sediments to identify 55 MAGs that are phylogenetically distinct from previously described bacterial phyla. We propose that these MAGs belong to 4 novel bacterial phyla (Blakebacterota, Orphanbacterota, Arandabacterota, and Joyebacterota) and a previously proposed phylum (AABM5-125-24), all of them within the FCB superphylum. Comparison of their rRNA genes with public databases reveals that these phyla are globally distributed in different habitats, including marine, freshwater, and terrestrial environments. Genomic analyses suggest these organisms are capable of mediating key steps in sedimentary biogeochemistry, including anaerobic degradation of polysaccharides and proteins, and respiration of sulfur and nitrogen. Interestingly, these genomes code for an unusually high proportion (~9% on average, up to 20% per genome) of protein families lacking representatives in public databases. Genes encoding hundreds of these protein families colocalize with genes predicted to be involved in sulfur reduction, nitrogen cycling, energy conservation, and degradation of organic compounds. Our findings advance our understanding of bacterial diversity, the ecological roles of these bacteria, and potential links between novel gene families and metabolic processes in the oceans.

Genomes of six viruses that infect Asgard archaea from deep-sea sediments.

Asgard archaea are globally distributed prokaryotic microorganisms related to eukaryotes; however, viruses that infect these organisms have not been described. Here, using metagenome sequences recovered from deep-sea hydrothermal sediments, we characterize six relatively large (up to 117 kb) double-stranded DNA (dsDNA) viral genomes that infected two Asgard archaeal phyla, Lokiarchaeota and Helarchaeota. These viruses encode Caudovirales-like structural proteins, as well as proteins distinct from those described in known archaeal viruses. Their genomes contain around 1-5% of genes associated with eukaryotic nucleocytoplasmic large DNA viruses (NCLDVs) and appear to be capable of semi-autonomous genome replication, repair, epigenetic modifications and transcriptional regulation. Moreover, Helarchaeota viruses may hijack host ubiquitin systems similar to eukaryotic viruses. Genomic analysis of these Asgard viruses reveals that they contain features of both prokaryotic and eukaryotic viruses, and provides insights into their potential infection and host interaction mechanisms.

Large-scale protein level comparison of Deltaproteobacteria reveals cohesive metabolic groups.

Deltaproteobacteria, now proposed to be the phyla Desulfobacterota, Myxococcota, and SAR324, are ubiquitous in marine environments and play essential roles in global carbon, sulfur, and nutrient cycling. Despite their importance, our understanding of these bacteria is biased towards cultured organisms. Here we address this gap by compiling a genomic catalog of 1 792 genomes, including 402 newly reconstructed and characterized metagenome-assembled genomes (MAGs) from coastal and deep-sea sediments. Phylogenomic analyses reveal that many of these novel MAGs are uncultured representatives of Myxococcota and Desulfobacterota that are understudied. To better characterize Deltaproteobacteria diversity, metabolism, and ecology, we clustered ~1 500 genomes based on the presence/absence patterns of their protein families. Protein content analysis coupled with large-scale metabolic reconstructions separates eight genomic clusters of Deltaproteobacteria with unique metabolic profiles. While these eight clusters largely correspond to phylogeny, there are exceptions where more distantly related organisms appear to have similar ecological roles and closely related organisms have distinct protein content. Our analyses have identified previously unrecognized roles in the cycling of methylamines and denitrification among uncultured Deltaproteobacteria. This new view of Deltaproteobacteria diversity expands our understanding of these dominant bacteria and highlights metabolic abilities across diverse taxa.

Brockarchaeota, a novel archaeal phylum with unique and versatile carbon cycling pathways.

Geothermal environments, such as hot springs and hydrothermal vents, are hotspots for carbon cycling and contain many poorly described microbial taxa. Here, we reconstructed 15 archaeal metagenome-assembled genomes (MAGs) from terrestrial hot spring sediments in China and deep-sea hydrothermal vent sediments in Guaymas Basin, Gulf of California. Phylogenetic analyses of these MAGs indicate that they form a distinct group within the TACK superphylum, and thus we propose their classification as a new phylum, 'Brockarchaeota', named after Thomas Brock for his seminal research in hot springs. Based on the MAG sequence information, we infer that some Brockarchaeota are uniquely capable of mediating non-methanogenic anaerobic methylotrophy, via the tetrahydrofolate methyl branch of the Wood-Ljungdahl pathway and reductive glycine pathway. The hydrothermal vent genotypes appear to be obligate fermenters of plant-derived polysaccharides that rely mostly on substrate-level phosphorylation, as they seem to lack most respiratory complexes. In contrast, hot spring lineages have alternate pathways to increase their ATP yield, including anaerobic methylotrophy of methanol and trimethylamine, and potentially use geothermally derived mercury, arsenic, or hydrogen. Their broad distribution and their apparent anaerobic metabolic versatility indicate that Brockarchaeota may occupy previously overlooked roles in anaerobic carbon cycling.

Evolutionary history of dimethylsulfoniopropionate (DMSP) demethylation enzyme DmdA in marine bacteria.

Dimethylsulfoniopropionate (DMSP), an osmolyte produced by oceanic phytoplankton and bacteria, is primarily degraded by bacteria belonging to the Roseobacter lineage and other marine Alphaproteobacteria via DMSP-dependent demethylase A protein (DmdA). To date, the evolutionary history of DmdA gene family is unclear. Some studies indicate a common ancestry between DmdA and GcvT gene families and a co-evolution between Roseobacter and the DMSP-producing-phytoplankton around 250 million years ago (Mya). In this work, we analyzed the evolution of DmdA under three possible evolutionary scenarios: (1) a recent common ancestor of DmdA and GcvT, (2) a coevolution between Roseobacter and the DMSP-producing-phytoplankton, and (3) an enzymatic adaptation for utilizing DMSP in marine bacteria prior to Roseobacter origin. Our analyses indicate that DmdA is a new gene family originated from GcvT genes by duplication and functional divergence driven by positive selection before a coevolution between Roseobacter and phytoplankton. Our data suggest that Roseobacter acquired dmdA by horizontal gene transfer prior to an environment with higher DMSP. Here, we propose that the ancestor that carried the DMSP demethylation pathway genes evolved in the Archean, and was exposed to a higher concentration of DMSP in a sulfur-rich atmosphere and anoxic ocean, compared to recent Roseobacter eco-orthologs (orthologs performing the same function under different conditions), which should be adapted to lower concentrations of DMSP.

Diversity, ecology and evolution of Archaea.

Compared to bacteria, our knowledge of archaeal biology is limited. Historically, microbiologists have mostly relied on culturing and single-gene diversity surveys to understand Archaea in nature. However, only six of the 27 currently proposed archaeal phyla have cultured representatives. Advances in genomic sequencing and computational approaches are revolutionizing our understanding of Archaea. The recovery of genomes belonging to uncultured groups from the environment has resulted in the description of several new phyla, many of which are globally distributed and are among the predominant organisms on the planet. In this Review, we discuss how these genomes, together with long-term enrichment studies and elegant in situ measurements, are providing insights into the metabolic capabilities of the Archaea. We also debate how such studies reveal how important Archaea are in mediating an array of ecological processes, including global carbon and nutrient cycles, and how this increase in archaeal diversity has expanded our view of the tree of life and early archaeal evolution, and has provided new insights into the origin of eukaryotes.

Author Correction: Diversity, ecology and evolution of Archaea.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Understanding the Mechanisms Behind the Response to Environmental Perturbation in Microbial Mats: A Metagenomic-Network Based Approach.

To date, it remains unclear how anthropogenic perturbations influence the dynamics of microbial communities, what general patterns arise in response to disturbance, and whether it is possible to predict them. Here, we suggest the use of microbial mats as a model of study to reveal patterns that can illuminate the ecological processes underlying microbial dynamics in response to stress. We traced the responses to anthropogenic perturbation caused by water depletion in microbial mats from Cuatro Cienegas Basin (CCB), Mexico, by using a time-series spatially resolved analysis in a novel combination of three computational approaches. First, we implemented MEBS (Multi-genomic Entropy-Based Score) to evaluate the dynamics of major biogeochemical cycles across spatio-temporal scales with a single informative value. Second, we used robust Time Series-Ecological Networks (TS-ENs) to evaluate the total percentage of interactions at different taxonomic levels. Lastly, we utilized network motifs to characterize specific interaction patterns. Our results indicate that microbial mats from CCB contain an enormous taxonomic diversity with at least 100 phyla, mainly represented by members of the rare biosphere (RB). Statistical ecological analyses point out a clear involvement of anaerobic guilds related to sulfur and methane cycles during wet versus dry conditions, where we find an increase in fungi, photosynthetic, and halotolerant taxa. TS-ENs indicate that in wet conditions, there was an equilibrium between cooperation and competition (positive and negative relationships, respectively), while under dry conditions there is an over-representation of negative relationships. Furthermore, most of the keystone taxa of the TS-ENs at family level are members of the RB and the microbial mat core highlighting their crucial role within the community. Our results indicate that microbial mats are more robust to perturbation due to redundant functions that are likely shared among community members in the highly connected TS-ENs with density values close to one (≈0.9). Finally, we provide evidence that suggests that a large taxonomic diversity where all community members interact with each other (low modularity), the presence of permanent of low-abundant taxa, and an increase in competition can be potential buffers against environmental disturbance in microbial mats.

MEBS, a software platform to evaluate large (meta)genomic collections according to their metabolic machinery: unraveling the sulfur cycle.

The increasing number of metagenomic and genomic sequences has dramatically improved our understanding of microbial diversity, yet our ability to infer metabolic capabilities in such datasets remains challenging. We describe the Multigenomic Entropy Based Score pipeline (MEBS), a software platform designed to evaluate, compare, and infer complex metabolic pathways in large "omic" datasets, including entire biogeochemical cycles. MEBS is open source and available through https://github.com/eead-csic-compbio/metagenome_Pfam_score. To demonstrate its use, we modeled the sulfur cycle by exhaustively curating the molecular and ecological elements involved (compounds, genes, metabolic pathways, and microbial taxa). This information was reduced to a collection of 112 characteristic Pfam protein domains and a list of complete-sequenced sulfur genomes. Using the mathematical framework of relative entropy (H΄), we quantitatively measured the enrichment of these domains among sulfur genomes. The entropy of each domain was used both to build up a final score that indicates whether a (meta)genomic sample contains the metabolic machinery of interest and to propose marker domains in metagenomic sequences such as DsrC (PF04358). MEBS was benchmarked with a dataset of 2107 non-redundant microbial genomes from RefSeq and 935 metagenomes from MG-RAST. Its performance, reproducibility, and robustness were evaluated using several approaches, including random sampling, linear regression models, receiver operator characteristic plots, and the area under the curve metric (AUC). Our results support the broad applicability of this algorithm to accurately classify (AUC = 0.985) hard-to-culture genomes (e.g., Candidatus Desulforudis audaxviator), previously characterized ones, and metagenomic environments such as hydrothermal vents, or deep-sea sediment. Our benchmark indicates that an entropy-based score can capture the metabolic machinery of interest and can be used to efficiently classify large genomic and metagenomic datasets, including uncultivated/unexplored taxa.