ALBERT

Description: Beneath the seafloor, microbial life subsists in isolation from the surface world under persistent energy limitation. The nature and extent of genomic evolution in subseafloor microbes have been unknown. Here, we show that the genomes of Thalassospira bacterial populations cultured from million-year-old subseafloor sediments evolve in clonal populations by point mutation, with a relatively low rate of homologous recombination and elevated numbers of pseudogenes. Ratios of nonsynonymous to synonymous substitutions correlate with the accumulation of pseudogenes, consistent with a role for genetic drift in the subseafloor strains but not in type strains of Thalassospira isolated from the surface world. Consistent with this, pangenome analysis reveals that the subseafloor bacterial genomes have a significantly lower number of singleton genes than the type strains, indicating a reduction in recent gene acquisitions. Numerous insertion-deletion events and pseudogenes were present in a flagellar operon of the subseafloor bacteria, indicating that motility is nonessential in these million-year-old subseafloor sediments. This genomic evolution in subseafloor clonal populations coincided with a phenotypic difference: all subseafloor isolates have a lower rate of growth under laboratory conditions than the Thalassospira xiamenensis type strain. Our findings demonstrate that the long-term physical isolation of Thalassospira, in the absence of recombination, has resulted in clonal populations whereby reduced access to novel genetic material from neighbors has resulted in the fixation of new mutations that accumulate in genomes over millions of years.

Description: Thermodynamic models predict that H2 is energetically favorable for seafloor microbial life, but how H2 affects anabolic processes in seafloor-associated communities is poorly understood. Here, we used quantitative 13C DNA stable isotope probing (qSIP) to quantify the effect of H2 on carbon assimilation by microbial taxa synthesizing 13C-labeled DNA that are associated with partially serpentinized peridotite rocks from the equatorial Mid-Atlantic Ridge. The rock-hosted seafloor community was an order of magnitude more diverse compared to the seawater community directly above the rocks. With added H2, peridotite-associated taxa increased assimilation of 13C-bicarbonate and 13C-acetate into 16S rRNA genes of operational taxonomic units by 146% (±29%) and 55% (±34%), respectively, which correlated with enrichment of H2-oxidizing NiFe-hydrogenases encoded in peridotite-associated metagenomes. The effect of H2 on anabolism was phylogenetically organized, with taxa affiliated with Atribacteria, Nitrospira, and Thaumarchaeota exhibiting the most significant increases in 13C-substrate assimilation in the presence of H2. In SIP incubations with added H2, an order of magnitude higher number of peridotite rock-associated taxa assimilated 13C-bicarbonate, 13C-acetate, and 13C-formate compared to taxa that were not associated with peridotites. Collectively, these findings indicate that the unique geochemical nature of the peridotite-hosted ecosystem has selected for H2-metabolizing, rock-associated taxa that can increase anabolism under high H2 concentrations. Because ultramafic rocks are widespread in slow-, and ultraslow-spreading oceanic lithosphere, continental margins, and subduction zones where H2 is formed in copious amounts, the link between H2 and carbon assimilation demonstrated here may be widespread within these geological settings.

Description: The genomes of the Asgard superphylum of Archaea hold clues pertaining to the nature of the host cell that acquired the mitochondrion at the origin of eukaryotes1,2,3,4. Representatives of the Asgard candidate phylum Candidatus Lokiarchaeota (Lokiarchaeon) have the capacity for acetogenesis and fermentation5,6,7, but how their metabolic activity responds to environmental conditions is poorly understood. Here, we show that in anoxic Namibian shelf sediments, Lokiarchaeon gene expression levels are higher than those of bacterial phyla and increase with depth below the seafloor. Lokiarchaeon gene expression was significantly different across a hypoxic–sulfidic redox gradient, whereby genes involved in growth, fermentation and H2-dependent carbon fixation had the highest expression under the most reducing (sulfidic) conditions. Quantitative stable isotope probing revealed that anaerobic utilization of CO2 and diatomaceous extracellular polymeric substances by Lokiarchaeon was higher than the bacterial average, consistent with higher expression of Lokiarchaeon genes, including those involved in transport and fermentation of sugars and amino acids. The quantitative stable isotope probing and gene expression data demonstrate homoacetogenic activity of Candidatus Lokiarchaeota, whereby fermentative H2 production from organic substrates is coupled with the Wood–Ljungdahl carbon fixation pathway8. The high energetic efficiency provided by homoacetogenesis8 helps to explain the elevated metabolic activity of Lokiarchaeon in this anoxic, energy-limited setting.

Description: Ammonia-oxidizing archaea (AOA) dominate microbial communities throughout oxic subseafloor sediment deposited over millions of years in the North Atlantic Ocean. Rates of nitrification correlated with the abundance of these dominant AOA populations, whose metabolism is characterized by ammonia oxidation, mixotrophic utilization of organic nitrogen, deamination, and the energetically efficient chemolithoautotrophic hydroxypropionate/hydroxybutyrate carbon fixation cycle. These AOA thus have the potential to couple mixotrophic and chemolithoautotrophic metabolism via mixotrophic deamination of organic nitrogen, followed by oxidation of the regenerated ammonia for additional energy to fuel carbon fixation. This metabolic feature likely reduces energy loss and improves AOA fitness under energy-starved, oxic conditions, thereby allowing them to outcompete other taxa for millions of years.

Description: Oxygen minimum zones (OMZs) are hot spots for redox-sensitive nitrogen transformations fueled by sinking organic matter. In comparison, the regulating role of sulfur-cycling microbes in marine OMZs, their impact on carbon cycling in pelagic and benthic habitats, and activities below the seafloor remain poorly understood. Using 13C DNA stable isotope probing (SIP) and metatranscriptomics, we explored microbial guilds involved in sulfur and carbon cycling from the ocean surface to the subseafloor on the Namibian shelf. There was a clear separation in microbial community structure across the seawater-seafloor boundary, which coincided with a 100-fold-increased concentration of microbial biomass and unique gene expression profiles of the benthic communities. 13C-labeled 16S rRNA genes in SIP experiments revealed carbon-assimilating taxa and their distribution across the sediment-water interface. Most of the transcriptionally active taxa among water column communities that assimilated 13C from diatom exopolysaccharides (mostly Bacteroidetes, Actinobacteria, Alphaproteobacteria, and Planctomycetes) also assimilated 13C-bicarbonate under anoxic conditions in sediment incubations. Moreover, many transcriptionally active taxa from the seafloor community (mostly sulfate-reducing Deltaproteobacteria and sulfide-oxidizing Gammaproteobacteria) that assimilated 13C-bicarbonate under sediment anoxic conditions also assimilated 13C from diatom exopolysaccharides in the surface ocean and OMZ waters. Despite strong selection at the sediment-water interface, many taxa related to either planktonic or benthic communities were found to be present at low abundance and actively assimilating carbon under both sediment and water column conditions. In austral winter, mixing of shelf waters reduces stratification and suspends sediments from the seafloor into the water column, potentially spreading metabolically versatile microbes across niches. IMPORTANCE Microbial activities in oxygen minimum zones (OMZs) transform inorganic fixed nitrogen into greenhouse gases, impacting the Earth's climate and nutrient equilibrium. Coastal OMZs are predicted to expand with global change and increase carbon sedimentation to the seafloor. However, the role of sulfur-cycling microbes in assimilating carbon in marine OMZs and related seabed habitats remain poorly understood. Using 13C DNA stable isotope probing and metatranscriptomics, we explore microbial guilds involved in sulfur and carbon cycling from ocean surface to subseafloor on the Namibian shelf. Despite strong selection and differential activities across the sediment-water interface, many active taxa were identified in both planktonic and benthic communities, either fixing inorganic carbon or assimilating organic carbon from algal biomass. Our data show that many planktonic and benthic microbes linked to the sulfur cycle can cross redox boundaries when mixing of the shelf waters reduces stratification and suspends seafloor sediment particles into the water column.

Description: Fungi are ubiquitous in the ocean and hypothesized to be important members of marine ecosystems, but their roles in the marine carbon cycle are poorly understood. Here, we use 13C DNA stable isotope probing coupled with phylogenetic analyses to investigate carbon assimilation within diverse communities of planktonic and benthic fungi in the Benguela Upwelling System (Namibia). Across the redox stratified water column and in the underlying sediments, assimilation of 13C-labeled carbon from diatom extracellular polymeric substances (13C-dEPS) by fungi correlated with the expression of fungal genes encoding carbohydrate-active enzymes. Phylogenetic analysis of genes from 13C-labeled metagenomes revealed saprotrophic lineages related to the facultative yeast Malassezia were the main fungal foragers of pelagic dEPS. In contrast, fungi living in the underlying sulfidic sediments assimilated more 13C-labeled carbon from chemosynthetic bacteria compared to dEPS. This coincided with a unique seafloor fungal community and dissolved organic matter composition compared to the water column, and a 100-fold increased fungal abundance within the subseafloor sulfide-nitrate transition zone. The subseafloor fungi feeding on 13C-labeled chemolithoautotrophs under anoxic conditions were affiliated with Chytridiomycota and Mucoromycota that encode cellulolytic and proteolytic enzymes, revealing polysaccharide and protein-degrading fungi that can anaerobically decompose chemosynthetic necromass. These subseafloor fungi, therefore, appear to be specialized in organic matter that is produced in the sediments. Our findings reveal that the phylogenetic diversity of fungi across redox stratified marine ecosystems translates into functionally relevant mechanisms helping to structure carbon flow from primary producers in marine microbiomes from the surface ocean to the subseafloor.

Description: How microbial metabolism is translated into cellular reproduction under energy-limited settings below the seafloor over long timescales is poorly understood. Here, we show that microbial abundance increases an order of magnitude over a 5 million-year-long sequence in anoxic subseafloor clay of the abyssal North Atlantic Ocean. This increase in biomass correlated with an increased number of transcribed protein-encoding genes that included those involved in cytokinesis, demonstrating that active microbial reproduction outpaces cell death in these ancient sediments. Metagenomes, metatranscriptomes, and 16S rRNA gene sequencing all show that the actively reproducing community was dominated by the candidate phylum “Candidatus Atribacteria,” which exhibited patterns of gene expression consistent with fermentative, and potentially acetogenic, metabolism. “Ca. Atribacteria” dominated throughout the 8 million-year-old cored sequence, despite the detection limit for gene expression being reached in 5 million-year-old sediments. The subseafloor reproducing “Ca. Atribacteria” also expressed genes encoding a bacterial microcompartment that has potential to assist in secondary fermentation by recycling aldehydes and, thereby, harness additional power to reduce ferredoxin and NAD+. Expression of genes encoding the Rnf complex for generation of chemiosmotic ATP synthesis were also detected from the subseafloor “Ca. Atribacteria,” as well as the Wood-Ljungdahl pathway that could potentially have an anabolic or catabolic function. The correlation of this metabolism with cytokinesis gene expression and a net increase in biomass over the million-year-old sampled interval indicates that the “Ca. Atribacteria” can perform the necessary catabolic and anabolic functions necessary for cellular reproduction, even under energy limitation in millions-of-years-old anoxic sediments. IMPORTANCE The deep subseafloor sedimentary biosphere is one of the largest ecosystems on Earth, where microbes subsist under energy-limited conditions over long timescales. It remains poorly understood how mechanisms of microbial metabolism promote increased fitness in these settings. We discovered that the candidate bacterial phylum “Candidatus Atribacteria” dominated a deep-sea subseafloor ecosystem, where it exhibited increased transcription of genes associated with acetogenic fermentation and reproduction in million-year-old sediment. We attribute its improved fitness after burial in the seabed to its capabilities to derive energy from increasingly oxidized metabolites via a bacterial microcompartment and utilize a potentially reversible Wood-Ljungdahl pathway to help meet anabolic and catabolic requirements for growth. Our findings show that “Ca. Atribacteria” can perform all the necessary catabolic and anabolic functions necessary for cellular reproduction, even under energy limitation in anoxic sediments that are millions of years old.