Quantifying genome-specific carbon fixation in a 750-meter deep subsurface hydrothermal microbial community.

Dissolved inorganic carbon has been hypothesized to stimulate microbial chemoautotrophic activity as a biological sink in the carbon cycle of deep subsurface environments. Here, we tested this hypothesis using quantitative DNA stable isotope probing of metagenome-assembled genomes (MAGs) at multiple 13C-labeled bicarbonate concentrations in hydrothermal fluids from a 750-m deep subsurface aquifer in the Biga Peninsula (Turkey). The diversity of microbial populations assimilating 13C-labeled bicarbonate was significantly different at higher bicarbonate concentrations, and could be linked to four separate carbon-fixation pathways encoded within 13C-labeled MAGs. Microbial populations encoding the Calvin-Benson-Bassham cycle had the highest contribution to carbon fixation across all bicarbonate concentrations tested, spanning 1-10 mM. However, out of all the active carbon-fixation pathways detected, MAGs affiliated with the phylum Aquificae encoding the reverse tricarboxylic acid (rTCA) pathway were the only microbial populations that exhibited an increased 13C-bicarbonate assimilation under increasing bicarbonate concentrations. Our study provides the first experimental data supporting predictions that increased bicarbonate concentrations may promote chemoautotrophy via the rTCA cycle and its biological sink for deep subsurface inorganic carbon.

Does subtotal resection ameliorate hypothalamic morbidity in pediatric craniopharyngioma? A 30-year retrospective cohort study.

OBJECTIVE: The optimal extent of resection of craniopharyngiomas to minimize the long-term risks of hypothalamic and endocrine dysfunction (obesity and panhypopituitarism) in children remains uncertain. The purpose of this study was to report long-term outcomes of pediatric patients with craniopharyngioma undergoing surgical treatment and to study rates of endocrinological and hypothalamic dysfunction in association with extent of resection. METHODS: This retrospective study was performed in a cohort of children who underwent resection for craniopharyngioma at Children's of Alabama between 1990 and 2020. The primary outcome was hypothalamic dysfunction defined as a 0.5 increase in body mass index (BMI) Z-score and as a BMI > 2 SDs with or without psychiatric disturbances. Univariable analysis was performed using ANOVA, Wilcoxon rank-sum test, Pearson's chi-square test, and Fisher's exact test as appropriate. Missing data on the primary outcome were handled via multiple imputations. Relative risks were estimated using a multivariable generalized linear model with a priori variables selected using a modified Poisson regression approach with robust error variance to estimate risk ratios. RESULTS: The cohort includes 39 patients (24 girls and 15 boys; age range 1 month-16 years) who underwent resection of craniopharyngioma at the authors' center between 1990 and 2020. The preoperative goal of treatment was cyst decompression (CD) in 5, subtotal resection (STR) in 13, and gross-total resection (GTR) in 21 patients. The median long-term follow-up after surgery was 8.11 years (average 8.21, range 0.4-24.33 years). Univariate analysis demonstrated a statistically significant increase in hypothalamic dysfunction in patients undergoing GTR when compared to those undergoing STR or CD at 1 month postoperatively (p = 0.006) and 6-11 months postoperatively (p = 0.010), but with this difference not persisting beyond 1 year. Multivariable analysis showed patients older than 10 years at time of surgery to be the most affected and at highest risk of developing significant hypothalamic dysfunction. There was no significant difference in pituitary or neurological function between the STR/CD and GTR groups at 12-24 months or at most recent follow-up. There was no significant difference in BMI Z-scores between the STR/CD and GTR groups at 6-12 months or at most recent follow-up. CONCLUSIONS: Both STR and GTR of craniopharyngioma were associated with significant endocrinological sequelae after 1 year. These potential complications should be discussed with patients and their families, and postoperative protocols should include early nutritional and endocrinological interventions with endocrinologist consultation.

Carbon metabolism and biogeography of candidate phylum "Candidatus Bipolaricaulota" in geothermal environments of Biga Peninsula, Turkey.

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 "Candidatus Bipolaricaulota" (former "Ca. Acetothermia" and OP1 division). The highest relative abundance of "Ca. 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 "Ca. 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 "Ca. Bipolaricaulota" MAGs based on the differences in carbon metabolism: one group encoding the bacterial Wood-Ljungdahl pathway (WLP) for H2 dependent CO2 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 "Ca. 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.

Synthesis and Biological Evaluation of Trehalose-based Bi-aryl Derivatives as C-type Lectin Ligands.

The identification of Mincle as the C-type lectin receptor on innate immune cells responsible for binding TDM and the realization that this receptor could be key to productive vaccines for mycobacterial infection has raised interest in the development of synthetic Mincle ligands as novel adjuvants. We recently reported on the synthesis and evaluation of Brartemicin analog UM-1024 that demonstrated Mincle agonist activity, exhibiting potent Th1/Th17 adjuvant activity that was greater than that of trehalose dibehenate (TDB). Our pursuit to understand Mincle/ligand relationships and improve the pharmacologic properties of the ligands has expanded and continues to reveal new and exciting structure activity relationships. Herein we report the synthesis of novel bi-aryl trehalose derivatives in good to excellent yields. These compounds were evaluated for their ability to engage the human Mincle receptor and tested for the induction of cytokines from human peripheral blood mononuclear cells. A preliminary structure-activity relationship (SAR) of these novel bi-aryl derivatives revealed that bi-aryl trehalose ligand 3D showed relatively high potency in cytokine production in comparison to trehalose glycolipid adjuvant TDB and the natural ligand TDM and induced dose-dependent, Mincle selective stimulation in hMincle HEK reporter cells. Also, through computational studies, we provide an insight into the potential mode of binding of 6,6'-Biaryl trehalose compounds on human Mincle receptor.

Predicting of the current and future geographical distribution of Laurus nobilis L. under the effects of climate change.

Today, climate change affects all living things on earth. It also leads to serious losses in terms of biodiversity, ecosystem services, and human welfare. In this context, Laurus nobilis L. is a very important species for Turkey, and the Mediterranean countries. This research aimed to simulate the current distribution of the suitable habitat for L. nobilis in Turkey and to predict its possible range shifts in future climate scenarios. To predict the geographical distribution of L. nobilis, the study used the maximum-entropy algorithm-based MaxEnt 3.4.1 with seven bioclimatic variables created using the Community Climate System Model 4.0 (CCSM4) and the prediction models RCP4.5-8.5 for the years 2050-2070. The results indicated that the most important bioclimatic variables that shape the distribution of L. nobilis are BIO11-mean temperature of coldest quarter, and BIO7-annual temperature range. Two climate change scenarios predicted that the geographical distribution of L. nobilis would increase slightly and then decrease in the future. However, the spatial change analysis showed that the general geographical distribution area of L. nobilis did not change significantly, but the "moderate," "high," and "very high" suitable habitats changed towards "low" suitable habitats. These changes were particularly effective in Turkey's Mediterranean region, which shows that climate change is instrumental in determining the future of the Mediterranean ecosystem. Therefore, suitability mapping and change analysis of potential future bioclimatic habitats can help in planning for land use, conservation, and ecological restoration of L. nobilis.

Microbial Activities and Selection from Surface Ocean to Subseafloor on the Namibian Continental Shelf.

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.

Quantifying the effects of hydrogen on carbon assimilation in a seafloor microbial community associated with ultramafic rocks.

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.

Carbon assimilating fungi from surface ocean to subseafloor revealed by coupled phylogenetic and stable isotope analysis.

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.

Genome Evolution in Bacteria Isolated from Million-Year-Old Subseafloor Sediment.

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. IMPORTANCE 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 Thalassospira 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 Thalassospira that have accumulated mutations in their genomes over millions of years.

Design of Trehalose-Based Amide/Sulfonamide C-type Lectin Receptor Signaling Compounds.

Mincle agonists have been shown to induce inflammatory cytokine production, such as tumor necrosis factor-alpha (TNF) and promote the development of a Th1/Th17 immune response that might be crucial to development of effective vaccination against pathogens such as Mycobacterium tuberculosis. As an expansion of our previous work, a library of 6,6'-amide and sulfonamide α,α-d-trehalose compounds with various substituents on the aromatic ring was synthesized efficiently in good to excellent yields. These compounds were evaluated for their ability to activate the human C-type lectin receptor Mincle by the induction of cytokines from human peripheral blood mononuclear cells. A preliminary structure-activity relationship (SAR) of these novel trehalose diamides and sulfonamides revealed that aryl amide-linked trehalose compounds demonstrated improved activity and relatively high potency cytokine production compared to the Mincle ligand trehalose dibehenate adjuvant (TDB) and the natural ligand trehalose dimycolate (TDM) inducing dose-dependent and human-Mincle-specific stimulation in a HEK reporter cell line.

Atribacteria Reproducing over Millions of Years in the Atlantic Abyssal Subseafloor.

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.

6,6'-Aryl trehalose analogs as potential Mincle ligands.

6,6'-Aryl trehalose derivatives have been synthesized with a view towards identifying novel Th-17-inducing vaccine adjuvants based on the high affinity Mincle ligand Brartemicin. The initial structure-activity relationships of these novel trehalose-based compounds were investigated. All compounds have been evaluated for their ability to engage the Mincle receptor and induce a potential pro-Th17 cytokine profile from human peripheral blood mononuclear cells based on IL-6 production in human peripheral blood mononuclear cells. The preliminary biological characterization of the designed analogs presented in this paper should aid in the future design and testing of more affine ligands that may foster the discovery of novel adjuvants with improved pharmacological properties.

Author Correction: Metabolic activity analyses demonstrate that Lokiarchaeon exhibits homoacetogenesis in sulfidic marine sediments.

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

Aryl Trehalose Derivatives as Vaccine Adjuvants for Mycobacterium tuberculosis.

Mycobacterium tuberculosis (Mtb) continues to be a major health threat worldwide, and the development of Mtb vaccines could play a pivotal role in the prevention and control of this devastating epidemic. Th17-mediated immunity has been implicated in disease protection correlates of immune protection against Mtb. Currently, there are no approved adjuvants capable of driving a Th17 response in a vaccine setting. Recent clinical trial results using trehalose dibehenate have demonstrated a formulation-dependant proof of concept adjuvant system CAF01 capable of inducing long-lived protection. We have discovered a new class of Th17-inducing vaccine adjuvants based on the natural product Brartemicin. We synthesized and evaluated the capacity of a library of aryl trehalose derivatives to drive immunostimulatory reresponses and evaluated the structure-activity relationships in terms of the ability to engage the Mincle receptor and induce production of innate cytokines from human and murine cells. We elaborated on the structure-activity relationship of the new scaffold and demonstrated the ability of the lead entity to induce a pro-Th17 cytokine profile from primary human peripheral blood mononuclear cells and demonstrated efficacy in generating antibodies in combination with tuberculosis antigen M72 in a mouse model.

Metabolic activity analyses demonstrate that Lokiarchaeon exhibits homoacetogenesis in sulfidic marine sediments.

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 eukaryotes1-4. Representatives of the Asgard candidate phylum Candidatus Lokiarchaeota (Lokiarchaeon) have the capacity for acetogenesis and fermentation5-7, 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.

Archaea dominate oxic subseafloor communities over multimillion-year time scales.

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.

Quantifying population-specific growth in benthic bacterial communities under low oxygen using H218O.

The benthos in estuarine environments often experiences periods of regularly occurring hypoxic and anoxic conditions, dramatically impacting biogeochemical cycles. How oxygen depletion affects the growth of specific uncultivated microbial populations within these diverse benthic communities, however, remains poorly understood. Here, we applied H218O quantitative stable isotope probing (qSIP) in order to quantify the growth of diverse, uncultured bacterial populations in response to low oxygen concentrations in estuarine sediments. Over the course of 7- and 28-day incubations with redox conditions spanning from hypoxia to euxinia (sulfidic), 18O labeling of bacterial populations exhibited different patterns consistent with micro-aerophilic, anaerobic, facultative anaerobic, and aerotolerant anaerobic growth. 18O-labeled populations displaying anaerobic growth had a significantly non-random phylogenetic distribution, exhibited by numerous clades currently lacking cultured representatives within the Planctomycetes, Actinobacteria, Latescibacteria, Verrucomicrobia, and Acidobacteria. Genes encoding the beta-subunit of the dissimilatory sulfate reductase (dsrB) became 18O labeled only during euxinic conditions. Sequencing of these 18O-labeled dsrB genes showed that Acidobacteria were the dominant group of growing sulfate-reducing bacteria, highlighting their importance for sulfur cycling in estuarine sediments. Our findings provide the first experimental constraints on the redox conditions underlying increased growth in several groups of "microbial dark matter", validating hypotheses put forth by earlier metagenomic studies.

Linking Uncultivated Microbial Populations and Benthic Carbon Turnover by Using Quantitative Stable Isotope Probing.

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 [13C]bicarbonate, DNA-qSIP showed that ammonia-oxidizing archaea (AOA) were the dominant active chemolithoautotrophs involved in the production of new organic matter. Natural 13C-labeled organic matter was then obtained by incubating sediments in the dark for 2.5 months with [13C]bicarbonate, 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 Proteobacteria, Chloroflexi, Verrucomicrobia, and Bacteroidetes However, several OTUs affiliated with the candidate microbial taxa Latescibacteria, Omnitrophica, Aminicentantes, Cloacimonates, AC1, Bathyarchaeota, and Woesearchaeota, groups known only from genomic signatures, also contributed to biomass turnover. Of these 823 labeled OTUs, 52% (primarily affiliated with Proteobacteria) also became labeled in 1-week incubations with [13C]bicarbonate, indicating that they turned over carbon faster than OTUs that were labeled only in incubations with 13C-labeled HMW organic matter. These taxa consisted primarily of uncultivated populations within the Firmicutes, Bacteroidetes, Verrucomicrobia, and Chloroflexi, 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.IMPORTANCE 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 in situ "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.