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We sequenced two metagenomes of sediments from the East Fork Poplar Creek in the Oak Ridge Reservation (Oak Ridge, TN), a natural stream that has been contaminated with Hg from upstream sources, and we reconstructed 28 metagenome-assembled genomes of novel prokaryotic species.

The conversion of mercury (Hg) to monomethylmercury (MMHg) is a critical area of concern in global Hg cycling. Periphyton biofilms may harbor significant amounts of MMHg but little is known about the Hg-methylating potential of the periphyton microbiome. Therefore, we used high-throughput amplicon sequencing of the 16S rRNA gene, ITS2 region, and Hg methylation gene pair (hgcAB) to characterize the archaea/bacteria, fungi, and Hg-methylating microorganisms in periphyton communities grown in a contaminated watershed in East Tennessee (United States). Furthermore, we examined how nutrient amendments (nitrate and/or phosphate) altered periphyton community structure and function. We found that bacterial/archaeal richness in experimental conditions decreased in summer and increased in autumn relative to control treatments, while fungal diversity generally increased in summer and decreased in autumn relative to control treatments. Interestingly, the Hg-methylating communities were dominated by Proteobacteria followed by Candidatus Atribacteria across both seasons. Surprisingly, Hg methylation potential correlated with numerous bacterial families that do not contain hgcAB, suggesting that the overall microbiome structure of periphyton communities influences rates of Hg transformation within these microbial mats. To further explore these complex community interactions, we performed a microbial network analysis and found that the nitrate-amended treatment resulted in the highest number of hub taxa that also corresponded with enhanced Hg methylation potential. This work provides insight into community interactions within the periphyton microbiome that may contribute to Hg cycling and will inform future research that will focus on establishing mixed microbial consortia to uncover mechanisms driving shifts in Hg cycling within periphyton habitats.

The gene pairhgcABis essential for microbial mercury methylation. Our understanding of its abundance and diversity in nature is rapidly evolving. In this study we developed a new broad-range primer set forhgcAB, plus an expandedhgcABreference library, and used these to characterize Hg-methylating communities from diverse environments. We applied this new Hg-methylator database to assign taxonomy tohgcAsequences from clone, amplicon, and metagenomic datasets. We evaluated potential biases introduced in primer design, sequence length, and classification, and suggest best practices for studying Hg-methylator diversity. Our study confirms the emerging picture of an expanded diversity of HgcAB-encoding microbes in many types of ecosystems, with abundant putative mercury methylatorsNitrospiraeandChloroflexiin several new environments including salt marsh and peat soils. Other common microbes encoding HgcAB includedPhycisphaerae, Aminicenantes, Spirochaetes, andElusimicrobia. Gene abundance data also corroborate the important role of two “classic” groups of methylators (DeltaproteobacteriaandMethanomicrobia) in many environments, but generally show a scarcity ofhgcAB+Firmicutes. The new primer set was developed to specifically targethgcABsequences found in nature, reducing degeneracy and providing increased sensitivity while maintaining broad diversity capture. We evaluated mock communities to confirm primer improvements, including culture spikes to environmental samples with variable DNA extraction and PCR amplification efficiencies. For select sites, this new workflow was combined with direct high-throughputhgcABsequencing. ThehgcABdiversity generated by direct amplicon sequencing confirmed the potential for novel Hg-methylators previously identified using metagenomic screens. A new phylogenetic analysis using sequences from freshwater, saline, and terrestrial environments showedDeltaproteobacteriaHgcA sequences generally clustered among themselves, while metagenome-resolved HgcA sequences in other phyla tended to cluster by environment, suggesting horizontal gene transfer into many clades. HgcA from marine metagenomes often formed distinct subtrees from those sequenced from freshwater ecosystems. Overall the majority of HgcA sequences branch from a cluster of HgcAB fused proteins related toThermococci, Atribacteria(candidate division OP9),Aminicenantes(OP8), andChloroflexi. The improved primer set and library, combined with direct amplicon sequencing, provide a significantly improved assessment of the abundance and diversity ofhgcAB+ microbes in nature.Contribution to the Field StatementThe gene pairhgcABis essential for microbial production of the neurotoxin methylmercury. In recent years these genes have been used as biomarkers to determine the potential of a microbiome to generate methylmercury via PCR amplification using degenerate primers from several research groups. However, improved techniques for capturinghgcABdiversity are necessary for identifying the major environmental producers of the neurotoxin as well as the expanding diversity of novel putative methylators, and the genes’ evolutionary history. The work described herein advanceshgcABdetection in environmental samples through an updated primer set coupled with a direct high-throughput sequencing method that enables broader diversity capture. We provide an expandedhgcABsequence reference library that allows for more sensitive and robust estimations of Hg-methylator diversity and potential for MeHg generation in the environment. ThehgcABdiversity generated by high-throughput sequencing confirms the potential for novel Hg-methylators previously only identified using metagenomic screens. This study provides a significantly improved assessment of the abundance and diversity ofhgcAB+ microbes in nature. By expanding our understanding of the microbial metabolic clades associated with mercury methylation, this work improves our ability to predict environmental conditions that drive production and accumulation of the neurotoxin in aquatic ecosystems.

Paddy soils from mercury (Hg)-contaminated rice fields in Guizhou, China were studied with respect to total mercury (THg) and methylmercury (MeHg) concentrations as well as Bacterial and Archaeal community composition. Total Hg (0.25-990 μg g-1) and MeHg (1.3-30.5 ng g-1) varied between samples. Pyrosequencing (454 FLX) of the hypervariable v1-v3 regions of the 16S rRNA genes showed that Proteobacteria, Actinobacteria, Chloroflexi, Acidobacteria, Euryarchaeota, and Crenarchaeota were dominant in all samples. The Bacterial α-diversity was higher in samples with relatively Low THg and MeHg and decreased with increasing THg and MeHg concentrations. In contrast, Archaeal α-diversity increased with increasing of MeHg concentrations but did not correlate with changes in THg concentrations. Overall, the methylation gene hgcAB copy number increased with both increasing THg and MeHg concentrations. The microbial communities at High THg and High MeHg appear to be adapted by species that are both Hg resistant and carry hgcAB genes for MeHg production. The relatively high abundance of both sulfate-reducing δ-Proteobacteria and methanogenic Archaea, as well as their positive correlations with increasing THg and MeHg concentrations, suggests that these microorganisms are the primary Hg-methylators in the rice paddy soils in Guizhou, China.

Two genes, hgcA and hgcB , are essential for microbial mercury (Hg) methylation. Detection and estimation of their abundance, in conjunction with Hg concentration, bioavailability, and biogeochemistry, are critical in determining potential hot spots of methylmercury (MeHg) generation in at-risk environments. We developed broad-range degenerate PCR primers spanning known hgcAB genes to determine the presence of both genes in diverse environments. These primers were tested against an extensive set of pure cultures with published genomes, including 13 Deltaproteobacteria , nine Firmicutes , and nine methanogenic Archaea genomes. A distinct PCR product at the expected size was confirmed for all hgcAB + strains tested via Sanger sequencing. Additionally, we developed clade-specific degenerate quantitative PCR (qPCR) primers that targeted hgcA for each of the three dominant Hg-methylating clades. The clade-specific qPCR primers amplified hgcA from 64%, 88%, and 86% of tested pure cultures of Deltaproteobacteria , Firmicutes , and Archaea , respectively, and were highly specific for each clade. Amplification efficiencies and detection limits were quantified for each organism. Primer sensitivity varied among species based on sequence conservation. Finally, to begin to evaluate the utility of our primer sets in nature, we tested hgcA and hgcAB recovery from pure cultures spiked into sand and soil. These novel quantitative molecular tools designed in this study will allow for more accurate identification and quantification of the individual Hg-methylating groups of microorganisms in the environment. The resulting data will be essential in developing accurate and robust predictive models of Hg methylation potential, ideally integrating the geochemistry of Hg methylation to the microbiology and genetics of hgcAB . IMPORTANCE The neurotoxin methylmercury (MeHg) poses a serious risk to human health. MeHg production in nature is associated with anaerobic microorganisms. The recent discovery of the Hg-methylating gene pair, hgcA and hgcB , has allowed us to design and optimize molecular probes against these genes within the genomic DNA for microorganisms known to methylate Hg. The protocols designed in this study allow for both qualitative and quantitative assessments of pure-culture or environmental samples. With these protocols in hand, we can begin to study the distribution of Hg-methylating organisms in nature via a cultivation-independent strategy.

et al., The draft genome of the moss model, Physcomitrella patens, comprised approximately 2000 unordered scaffolds. In order to enable analyses of genome structure and evolution we generated a chromosome-scale genome assembly using genetic linkage as well as (end) sequencing of long DNA fragments. We find that 57% of the genome comprises transposable elements (TEs), some of which may be actively transposing during the life cycle. Unlike in flowering plant genomes, gene- and TE-rich regions show an overall even distribution along the chromosomes. However, the chromosomes are mono-centric with peaks of a class of Copia elements potentially coinciding with centromeres. Gene body methylation is evident in 5.7% of the protein-coding genes, typically coinciding with low GC and low expression. Some giant virus insertions are transcriptionally active and might protect gametes from viral infection via siRNA mediated silencing. Structure-based detection methods show that the genome evolved via two rounds of whole genome duplications (WGDs), apparently common in mosses but not in liverworts and hornworts. Several hundred genes are present in colinear regions conserved since the last common ancestor of plants. These syntenic regions are enriched for functions related to plant-specific cell growth and tissue organization. The P. patens genome lacks the TE-rich pericentromeric and gene-rich distal regions typical for most flowering plant genomes. More non-seed plant genomes are needed to unravel how plant genomes evolve, and to understand whether the P. patens genome structure is typical for mosses or bryophytes., The work conducted by the US Department of Energy Joint Genome Institute is supported by the Office of Science of the US Department of Energy under Contract No. DE-AC02-05CH11231. Support to RR and SAR by the German Research Foundation (DFG RE 837/10-2), the Excellence Initiative of the German Federal and State Governments (EXC 294), and by the German Federal Ministry of Education and Research (BMBF FRISYS), is highly appreciated. CoGe is supported by the US National Science Foundation under Award Numbers IOS-339156 and IOS-1444490, CyVerse is supported by the U.S. National Science Foundation under Award Numbers DBI-0735191 and DBI-1265383. YK and ACC are grateful for support from the UK Biological Sciences and Biotechnology Research Council (Grant BB/F001797/1). KV acknowledges the Multidisciplinary Research Partnership ‘Bioinformatics: from nucleotides to networks’ Project (no 01MR0410W) of Ghent University. JC is grateful for support from the Spanish Ministerio de Economía y Competitividad (Grant AGL2013-43244-R). RSQ is grateful to Monsanto (St. Louis, MO, USA) for sequencing genomic DNA of P. patens accession Kaskaskia. The 1000 Plants (1 KP) initiative, led by GKSW, is funded by the Alberta Ministry of Innovation and Advanced Education, Alberta Innovates Technology Futures (AITF), Innovates Centres of Research Excellence (iCORE), Musea Ventures, BGI-Shenzhen and China National Genebank (CNGB). TW was supported by EMBO Long-Term Fellowships (ALTF 1166-2011) and by Marie Curie Actions (European Commission EMBOCOFUND2010, GA-2010-267146). The work conducted at PGSB was supported by the German Research Foundation (SFB924) and German Ministry of Education and Research (BMBF, 031A536/de.NBI).

© This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication. Using in-field bioreactors, we investigated the influence of exogenous microorganisms in groundwater planktonic and biofilm microbial communities as part of the Integrated Field Research Challenge (IFRC). After an acclimation period with source groundwater, bioreactors received either filtered (0.22 μM filter) or unfiltered well groundwater in triplicate and communities were tracked routinely for 23 days after filtration was initiated. To address geochemical influences, the planktonic phase was assayed periodically for protein, organic acids, physico-/geochemical measurements and bacterial community (via 16S rRNA gene sequencing), while biofilms (i.e. microbial growth on sediment coupons) were targeted for bacterial community composition at the completion of the experiment (23 d). Based on Bray-Curtis distance, planktonic bacterial community composition varied temporally and between treatments (filtered, unfiltered bioreactors). Notably, filtration led to an increase in the dominant genus, Zoogloea relative abundance over time within the planktonic community, while remaining relatively constant when unfiltered. At day 23, biofilm communities were more taxonomically and phylogenetically diverse and substantially different from planktonic bacterial communities; however, the biofilm bacterial communities were similar regardless of filtration. These results suggest that although planktonic communities were sensitive to groundwater filtration, bacterial biofilm communities were stable and resistant to filtration. Bioreactors are useful tools in addressing questions pertaining to microbial community assembly and succession. These data provide a first step in understanding how an extrinsic factor, such as a groundwater inoculation and flux of microbial colonizers, impact how microbial communities assemble in environmental systems.