A rapid method for measuring ATP + ADP + AMP in marine sediment

In this report, I describe a method for rapid measurement of total adenylate (ATP + ADP + AMP) in marine sediment samples for estimating microbial biomass. A simple ‘boil and dilute’ method is described here, whereby adding boiled MilliQ water to sediments increases the detection limit for ATP + ADP + AMP up to 100-fold. The lowered detection limit of this method enabled the detection ATP + ADP + AMP in relatively low-biomass sub-seafloor sediment cores with 104 16S rRNA gene copies per gram. Concentrations of ATP + ADP + AMP correlated with 16S rRNA gene concentrations from bacteria and archaea across six different sites that range in water depth from 1 to 6000 m indicating that the ATP + ADP + AMP method can be used as an additional biomass proxy. In deep sea microbial communities, the ratio of ATP + ADP + AMP concentrations to 16S rRNA genes >1 m below seafloor was significantly lower compared to communities in the upper 30 cm of sediment, which may be due to reduced cell sizes and or lower ATP + ADP + AMP concentrations per cell in the deep sea sub-seafloor biosphere. The boil and dilute method for ATP + ADP + AMP is demonstrated here to have a detection limit sufficient for measuring low biomass communities from deep sea sub-seafloor cores. The method can be applied to frozen samples, enabling measurements of ATP + ADP + AMP from frozen sediment cores stored in core repositories from past and future international drilling campaigns

Exploring the Abundance, Metabolic Potential, and Gene Expression of Subseafloor Chloroflexi in Million-year-old Oxic and Anoxic Abyssal Clay

Chloroflexi are widespread in subsurface environments, and recent studies indicate that they represent a major fraction of the communities in subseafloor sediment. Here, we compare the abundance, diversity, metabolic potential, and gene expression of Chloroflexi from three abyssal sediment cores from the western North Atlantic Gyre (water depth \u3e5400 m) covering up to 15 million years of sediment deposition, where Chloroflexi were found to represent major components of the community at all sites. Chloroflexi communities die off in oxic red clay over 10 to 15 million years, and gene expression was below detection. In contrast, Chloroflexi abundance and gene expression at the anoxic abyssal clay site increase below the seafloor and peak in 2 to 3 million-year-old sediment, indicating a comparably higher activity. Metatranscriptomes from the anoxic site reveal increased expression of Chloroflexi genes involved in cell wall biogenesis, protein turnover, inorganic ion transport, defense mechanisms and prophages. Phylogenetic analysis shows that these Chloroflexi are closely related to homoacetogenic subseafloor clades and actively transcribe genes involved in sugar fermentations, gluconeogenesis and Wood-Ljungdahl pathway in the subseafloor. Concomitant expression of cell division genes indicates that these putative homoacetogenic Chloroflexi are actively growing in these million-year-old anoxic abyssal sediments

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

© The Author(s), 2019. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in ISME Journal (2019), doi:10.1038/s41396-019-0373-4.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.This work was supported by a grant OR 417/1-1 from the Deutsche Forschungsgemeinschaft, and a Junior Researcher Fund grant from LMU Munich to WDO. This work was performed in part, through the Master’s Program in Geobiology and Paleontology (MGAP) at LMU Munich

Genealogical analyses of multiple loci of litostomatean ciliates (Protista, Ciliophora, Litostomatea)

© The Author(s), 2012. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Molecular Phylogenetics and Evolution 66 (2012): 397-411, doi:10.1016/j.ympev.2012.06.024.The class Litostomatea is a highly diverse ciliate taxon comprising hundreds of free-living and endocommensal species. However, their traditional morphology-based classification conflicts with 18S rRNA gene phylogenies indicating (1) a deep bifurcation of the Litostomatea into Rhynchostomatia and Haptoria + Trichostomatia, and (2) body polarization and simplification of the oral apparatus as main evolutionary trends in the Litostomatea. To test whether 18S rRNA molecules provide a suitable proxy for litostomatean evolutionary history, we used eighteen new ITS1-5.8S rRNA-ITS2 region sequences from various free-living litostomatean orders. These single- and multiple-locus analyses are in agreement with previous 18S rRNA gene phylogenies, supporting that both 18S rRNA gene and ITS region sequences are effective tools for resolving phylogenetic relationships among the litostomateans. Despite insertions, deletions and mutational saturations in the ITS region, the present study shows that ITS1 and ITS2 molecules can be used to infer phylogenetic relationships not only at species level but also at higher taxonomic ranks when their secondary structure information is utilized to aid alignment.Financial support was provided by the Austrian Science Foundation (FWF Projects P-19699-B17 and P-20360-B17 to Wilhelm Foissner), the Slovak Scientific Grant Agency (VEGA Project 1/0600/11 to Peter Vd’ačný), and US NSF Grants (Projects MCB-0348341 and DEB-0816840 to Slava S. Epstein)

Gene expression in the deep biosphere

Author Posting. © The Author(s), 2013. This is the author's version of the work. It is posted here by permission of Nature Publishing Group for personal use, not for redistribution. The definitive version was published in Nature 499 (2013): 205-208, doi:10.1038/nature12230.Scientific ocean drilling has revealed a deep biosphere of widespread microbial life in sub-seafloor sediment. Microbial metabolism in the marine subsurface likely plays an important role in global biogeochemical cycles1-3 but deep biosphere activities are not well understood1. Here, we describe and analyze the first subseafloor metatranscriptomes from anaerobic Peru Margin sediment up to 159 meters below seafloor (mbsf) represented by over 1 billion cDNA sequence reads. Anaerobic metabolism of amino acids, carbohydrates, and lipids appear to be dominant metabolic processes, and profiles of dissimilatory sulfite reductase (Dsr) transcripts are consistent with porewater sulfate concentration profiles1. Moreover, transcripts involved in cell division increase as a function of microbial cell concentration, indicating that increases in subseafloor microbial abundance are a function of cell division across all three domains of life. These data support calculations1 and models4 of subseafloor microbial metabolism and represent the first holistic picture of deep biosphere activities.This work was fostered by a Center for Dark Energy Biosphere Investigations (CDEBI) grant OCE-0939564 to WO and a NSF IOS grant 1238801 to JFB.2013-12-1

White and green rust chimneys accumulate RNA in a ferruginous chemical garden

Mechanisms of nucleic acid accumulation were likely critical to life's emergence in the ferruginous oceans of the early Earth. How exactly prebiotic geological settings accumulated nucleic acids from dilute aqueous solutions, is poorly understood. As a possible solution to this concentration problem, we simulated the conditions of prebiotic low-temperature alkaline hydrothermal vents in co-precipitation experiments to investigate the potential of ferruginous chemical gardens to accumulate nucleic acids via sorption. The injection of an alkaline solution into an artificial ferruginous solution under anoxic conditions (O2 < 0.01% of present atmospheric levels) and at ambient temperatures, caused the precipitation of amakinite (“white rust”), which quickly converted to chloride-containing fougerite (“green rust”). RNA was only extractable from the ferruginous solution in the presence of a phosphate buffer, suggesting RNA in solution was bound to Fe2+ ions. During chimney formation, this iron-bound RNA rapidly accumulated in the white and green rust chimney structure from the surrounding ferruginous solution at the fastest rates in the initial white rust phase and correspondingly slower rates in the following green rust phase. This represents a new mechanism for nucleic acid accumulation in the ferruginous oceans of the early Earth, in addition to wet-dry cycles and may have helped to concentrate RNA in a dilute prebiotic ocean

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 [C-13]bicarbonate, DNA-qSIP showed that ammoniaoxidizing archaea (AOA) were the dominant active chemolithoautotrophs involved in the production of new organic matter. Natural C-13-labeled organic matter was then obtained by incubating sediments in the dark for 2.5 months with [C-13] 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 [C-13] bicarbonate, indicating that they turned over carbon faster than OTUs that were labeled only in incubations with C-13-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

Effect of oxygen minimum zone formation on communities of marine protists

Author Posting. © The Author(s), 2012. This is the author's version of the work. It is posted here by permission of Nature Publishing Group for personal use, not for redistribution. The definitive version was published in The ISME Journal 6 (2012): 1586–1601, doi:10.1038/ismej.2012.7.Changes in ocean temperature and circulation patterns compounded by human activities are leading to oxygen minimum zone expansion with concomitant alteration in nutrient and climate active trace gas cycling. Here, we report the response of microbial eukaryote populations to seasonal changes in water column oxygen-deficiency using Saanich Inlet, a seasonally anoxic fjord on the coast of Vancouver Island British Columbia, as a model ecosystem. We combine small subunit ribosomal RNA gene sequencing approaches with multivariate statistical methods to reveal shifts in operational taxonomic units during successive stages of seasonal stratification and renewal. A meta-analysis is used to identify common and unique patterns of community composition between Saanich Inlet and the anoxic/sulfidic Cariaco Basin (Venezuela) and Framvaren Fjord (Norway) to show shared and unique responses of microbial eukaryotes to oxygen and sulfide in these three environments. Our analyses also reveal temporal fluctuations in rare populations of microbial eukaryotes, particularly anaerobic ciliates, that may be of significant importance to the biogeochemical cycling of methane in oxygen minimum zones.This work was performed under the auspices of the US Department of Energy's Office of Science, Biological and Environmental Research Program, and by the University of California, Lawrence Berkeley National Laboratory, Lawrence Livermore National Laboratory under Contract No., and Los Alamos National Laboratory (Contract No. DE-AC02-05CH11231, DE-AC52-07NA27344, DE-AC02-06NA25396), the Natural Sciences and Engineering Research Council (NSERC) of Canada 328256-07 and STPSC 356988, Canada Foundation for Innovation (CFI) 17444; Canadian Institute for Advanced Research (CIFAR), NSF MCB-0348407 to VE, NSF Center for Deep Energy Biosphere Investigations, and the Center for Bioinorganic Chemistry (CEBIC).2012-09-0

Prevalence of partnerships between bacteria and ciliates in oxygen-depleted marine water columns

© The Author(s), 2012. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Frontiers in Microbiology 3 (2012): 341, doi:10.3389/fmicb.2012.00341.Symbioses between Bacteria, Archaea, and Eukarya in deep-sea marine environments represent a means for eukaryotes to exploit otherwise inhospitable habitats. Such symbioses are abundant in many low-oxygen benthic marine environments, where the majority of microbial eukaryotes contain prokaryotic symbionts. Here, we present evidence suggesting that in certain oxygen-depleted marine water-column habitats, the majority of microbial eukaryotes are also associated with prokaryotic cells. Ciliates (protists) associated with bacteria were found to be the dominant eukaryotic morphotype in the haloclines of two different deep-sea hypersaline anoxic basins (DHABs) in the Eastern Mediterranean Sea. These findings are compared to associations between ciliates and bacteria documented from the permanently anoxic waters of the Cariaco Basin (Caribbean Sea). The dominance of ciliates exhibiting epibiotic bacteria across three different oxygen-depleted marine water column habitats suggests that such partnerships confer a fitness advantage for ciliates in these environments.This work was funded by NSF grant OCE-0849578 and to Virginia P. Edgcomb and Joan M. Bernhard, and OCE-1061774 to Virginia P. Edgcomb and Craig Taylor (WHOI)

Biodegradation of textile waste by marine bacterial communities enhanced by light

Knowledge of biofilm formation on pollutants in the marine realm is expanding, but how communities respond to substrates during colonization remains poorly understood. Here, we assess community assembly and respiration in response to two different micropollutants, virgin high‐density polyethylene (HDPE) microbeads and textile fibres under different light settings. Raman characterization, high‐throughput DNA sequencing data, quantitative PCR, and respiration measurements reveal how a stimulation of aerobic respiration by micropollutants is translated into selection for significantly different communities colonizing the substrates. Despite the lack of evidence for biodegradation of HDPE, an increased abundance and respiration of bacterial taxa closely related to hydrocarbonoclastic Kordiimonas spp. and Alteromonas spp. in the presence of textile waste highlights their biodegradation potential. Incubations with textile fibres exhibited significantly higher respiration rates in the presence of light, which could be partially explained by photochemical dissolution of the textile waste into smaller bioavailable compounds. Our results suggest that the development and increased respiration of these unique microbial communities may potentially play a role in the bioremediation of the relatively long‐lived textile pollutants in marine habitats, and that the respiration of heterotrophic hydrocarbon‐degrading bacteria colonizing marine pollutants can be stimulated by light