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
C 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
C-labeled carbon from diatom extracellular polymeric substances (
C-dEPS) by fungi correlated with the expression of fungal genes encoding carbohydrate-active enzymes. Phylogenetic analysis of genes from
C-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
C-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
C-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.
High‐throughput sequencing of the 16S rRNA gene on the Illumina platform is commonly used to assess microbial diversity in environmental samples. The MiniSeq, Illumina's latest benchtop sequencer, ...enables more cost‐efficient DNA sequencing relative to larger Illumina sequencing platforms (e.g., MiSeq). Here we used a modified custom primer sequencing approach to test the fidelity of the MiniSeq for high‐throughput sequencing of the V4 hypervariable region of 16S rRNA genes from complex communities in environmental samples. To this end, we designed additional sequencing primers that enabled application of a dual‐index barcoding method on the MiniSeq. A mock community was sequenced alongside the environmental samples in four different sequencing runs as a quality control benchmark. We were able to recapture a realistic richness of the mock community in all sequencing runs, and identify meaningful differences in alpha and beta diversity in the environmental samples. Furthermore, rarefaction analysis indicated diversity in many environmental samples was close to saturation. These results show that the MiniSeq can produce similar quantities of high‐quality V4 reads compared to the MiSeq, yet is a cost‐effective option for any laboratory interested in performing high‐throughput 16S rRNA gene sequencing.
Innovation in next‐generation DNA sequencing technology continues to contribute to the democratization of 16S rRNA gene sequencing, which is now often carried out on the Illumina MiSeq and HiSeq platforms. Here, we describe a 16S rRNA gene sequencing protocol for the latest benchtop high‐throughput sequencer, the Illumina MiniSeq, which provides comparable quantity and quality of data, but at significantly reduced cost compared to larger and more expensive sequencers. This opens the opportunity for smaller labs to perform their own 16S sequencing independently.
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
C 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.
C-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
C from diatom exopolysaccharides (mostly
,
,
, and
) also assimilated
C-bicarbonate under anoxic conditions in sediment incubations. Moreover, many transcriptionally active taxa from the seafloor community (mostly sulfate-reducing Deltaproteobacteria and sulfide-oxidizing
) that assimilated
C-bicarbonate under sediment anoxic conditions also assimilated
C 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.
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
C 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.
Benthic environments harbor highly diverse and complex microbial communities that control carbon fluxes, but the role of specific uncultivated microbial groups in organic matter turnover is poorly ...understood. In this study, quantitative DNA stable isotope probing (DNA-qSIP) was used for the first time to link uncultivated populations of bacteria and archaea to carbon turnover in lacustrine surface sediments. After 1-week incubations in the dark with
Cbicarbonate, DNA-qSIP showed that ammonia-oxidizing archaea (AOA) were the dominant active chemolithoautotrophs involved in the production of new organic matter. Natural
C-labeled organic matter was then obtained by incubating sediments in the dark for 2.5 months with
Cbicarbonate, followed by extraction and concentration of high-molecular-weight (HMW) (>50-kDa) organic matter. qSIP showed that the labeled organic matter was turned over within 1 week by 823 microbial populations (operational taxonomic units OTUs) affiliated primarily with heterotrophic
,
,
, and
However, several OTUs affiliated with the candidate microbial taxa
,
,
,
,
,
, and
, groups known only from genomic signatures, also contributed to biomass turnover. Of these 823 labeled OTUs, 52% (primarily affiliated with
) also became labeled in 1-week incubations with
Cbicarbonate, indicating that they turned over carbon faster than OTUs that were labeled only in incubations with
C-labeled HMW organic matter. These taxa consisted primarily of uncultivated populations within the
,
,
, and
, highlighting their ecological importance. Our study helps define the role of several poorly understood, uncultivated microbial groups in the turnover of benthic carbon derived from "dark" primary production.
Little is known about the ecological role of uncultivated microbial populations in carbon turnover in benthic environments. To better understand this, we used quantitative stable isotope probing (qSIP) to quantify the abundance of diverse, specific groups of uncultivated bacteria and archaea involved in autotrophy and heterotrophy in a benthic lacustrine habitat. Our results provide quantitative evidence for active heterotrophic and autotrophic metabolism of several poorly understood microbial groups, thus demonstrating their relevance for carbon turnover in benthic settings. Archaeal ammonia oxidizers were significant drivers of
"dark" primary production supporting the growth of heterotrophic bacteria. These findings expand our understanding of the microbial populations within benthic food webs and the role of uncultivated microbes in benthic carbon turnover.
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.
ABSTRACT
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.
Terrestrial hydrothermal springs and aquifers are excellent sites to study microbial biogeography because of their high physicochemical heterogeneity across relatively limited geographic regions. In ...this study, we performed 16S rRNA gene sequencing and metagenomic analyses of the microbial diversity of 11 different geothermal aquifers and springs across the tectonically active Biga Peninsula (Turkey). Across geothermal settings ranging in temperature from 43 to 79°C, one of the most highly represented groups in both 16S rRNA gene and metagenomic datasets was affiliated with the uncultivated phylum "
Bipolaricaulota" (former "
Acetothermia" and OP1 division). The highest relative abundance of "
Bipolaricaulota" was observed in a 68°C geothermal brine sediment, where it dominated the microbial community, representing 91% of all detectable 16S rRNA genes. Correlation analysis of "
Bipolaricaulota" operational taxonomic units (OTUs) with physicochemical parameters indicated that salinity was the strongest environmental factor measured associated with the distribution of this novel group in geothermal fluids. Correspondingly, analysis of 23 metagenome-assembled genomes (MAGs) revealed two distinct groups of "
Bipolaricaulota" MAGs based on the differences in carbon metabolism: one group encoding the bacterial Wood-Ljungdahl pathway (WLP) for H
dependent CO
fixation is selected for at lower salinities, and a second heterotrophic clade that lacks the WLP that was selected for under hypersaline conditions in the geothermal brine sediment. In conclusion, our results highlight that the biogeography of "
Bipolaricaulota" taxa is strongly correlated with salinity in hydrothermal ecosystems, which coincides with key differences in carbon acquisition strategies. The exceptionally high relative abundance of apparently heterotrophic representatives of this novel candidate Phylum in geothermal brine sediment observed here may help to guide future enrichment experiments to obtain representatives in pure culture.
Thermodynamic models predict that H
is energetically favorable for seafloor microbial life, but how H
affects anabolic processes in seafloor-associated communities is poorly understood. Here, we used ...quantitative
C DNA stable isotope probing (qSIP) to quantify the effect of H
on carbon assimilation by microbial taxa synthesizing
C-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 H
, peridotite-associated taxa increased assimilation of
C-bicarbonate and
C-acetate into 16S rRNA genes of operational taxonomic units by 146% (±29%) and 55% (±34%), respectively, which correlated with enrichment of H
-oxidizing NiFe-hydrogenases encoded in peridotite-associated metagenomes. The effect of H
on anabolism was phylogenetically organized, with taxa affiliated with Atribacteria, Nitrospira, and Thaumarchaeota exhibiting the most significant increases in
C-substrate assimilation in the presence of H
. In SIP incubations with added H
, an order of magnitude higher number of peridotite rock-associated taxa assimilated
C-bicarbonate,
C-acetate, and
C-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 H
-metabolizing, rock-associated taxa that can increase anabolism under high H
concentrations. Because ultramafic rocks are widespread in slow-, and ultraslow-spreading oceanic lithosphere, continental margins, and subduction zones where H
is formed in copious amounts, the link between H
and carbon assimilation demonstrated here may be widespread within these geological settings.
Nitrate pollution of freshwaters and methane emissions into the
atmosphere are crucial factors in deteriorating the quality of
drinking water and in contributing to global climate change. The n-damo ...(nitrite-dependent anaerobic methane oxidation), nitrate-dependent anaerobic methane
oxidation and the anaerobic oxidation of ammonium (anammox) represent
two microbially mediated processes that can reduce nitrogen loading of
aquatic ecosystems and associated methane emissions to the
atmosphere. Here, we report vertical concentration and stable-isotope
profiles of CH4, NO3-, NO2-, and
NH4+ in the water column of Fohnsee (lake in southern
Bavaria, Germany) that may indicate linkages between denitrification, anaerobic oxidation of methane (AOM), and anammox. At
a water depth from 12 to 20 m, a methane–nitrate transition
zone (NMTZ) was observed, where δ13C values of methane
and δ15N and δ18O of dissolved nitrate
markedly increased in concert with decreasing concentrations of
methane and nitrate. These data patterns, together with the results of
a simple 1-D diffusion model linked with a degradation term, show that
the nonlinear methane concentration profile cannot be explained by
diffusion and that microbial oxidation of methane coupled with
denitrification under anaerobic conditions is the most parsimonious
explanation for these data trends. In the methane zone at the bottom
of the NMTZ (20 to 22 m) δ15N of ammonium increased
by 4 ‰, while ammonium concentrations decreased. In
addition, a strong 15N enrichment of dissolved nitrate was
observed at a water depth of 20 m, suggesting that anammox
is occurring together with denitrification. The conversion of
nitrite to N2 and nitrate during anammox is associated with
an inverse N isotope fractionation and may explain the observed
increasing offset (Δδ15N) of 26 ‰
between δ15N values of dissolved nitrate and nitrite at a
water depth of 20 m compared to the Δδ15Nnitrate-nitrite of 11 ‰ obtained in
the NMTZ at a water depth between 16 and 18 m. The associated
methane concentration and stable-isotope profiles indicate that some
of the denitrification may be coupled to AOM, an observation supported
by an increased concentration of bacteria known to be involved in
n-damo/denitrification with AOM (NC10 and
Crenothrix) and anammox (“Candidatus
Anammoximicrobium”) whose concentrations were highest in the methane
and ammonium oxidation zones, respectively. This study shows the
potential for a coupling of microbially mediated nitrate-dependent
methane oxidation with anammox in stratified freshwater ecosystems,
which may be important for affecting both methane emissions and
nitrogen concentrations in lakes.
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
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
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
, 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.
The nature and extent of genomic evolution in subseafloor microbial populations subsisting for millions of years below the seafloor are unknown. Subseafloor populations have ultralow metabolic rates that are hypothesized to restrict reproduction and, consequently, the spread of new traits. Our findings demonstrate that genomes of cultivated bacterial strains from the genus
isolated from million-year-old abyssal sediment exhibit greatly reduced levels of homologous recombination, elevated numbers of pseudogenes, and genome-wide evidence of relaxed purifying selection. These substitutions and pseudogenes are fixed into the population, suggesting that the genome evolution of these bacteria has been dominated by genetic drift. Thus, reduced recombination, stemming from long-term physical isolation, resulted in small clonal populations of
that have accumulated mutations in their genomes over millions of years.
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