A two-step process for epigenetic inheritance in Arabidopsis.

In Arabidopsis, multisubunit RNA polymerases IV and V orchestrate RNA-directed DNA methylation (RdDM) and transcriptional silencing, but what identifies the loci to be silenced is unclear. We show that heritable silent locus identity at a specific subset of RdDM targets requires HISTONE DEACETYLASE 6 (HDA6) acting upstream of Pol IV recruitment and siRNA biogenesis. At these loci, epigenetic memory conferring silent locus identity is erased in hda6 mutants such that restoration of HDA6 activity cannot restore siRNA biogenesis or silencing. Silent locus identity is similarly lost in mutants for the cytosine maintenance methyltransferase, MET1. By contrast, pol IV or pol V mutants disrupt silencing without erasing silent locus identity, allowing restoration of Pol IV or Pol V function to restore silencing. Collectively, these observations indicate that silent locus specification and silencing are separable steps that together account for epigenetic inheritance of the silenced state.

Bacterial community assembly based on functional genes rather than species.

The principles underlying the assembly and structure of complex microbial communities are an issue of long-standing concern to the field of microbial ecology. We previously analyzed the community membership of bacterial communities associated with the green macroalga Ulva australis, and proposed a competitive lottery model for colonization of the algal surface in an attempt to explain the surprising lack of similarity in species composition across different algal samples. Here we extend the previous study by investigating the link between community structure and function in these communities, using metagenomic sequence analysis. Despite the high phylogenetic variability in microbial species composition on different U. australis (only 15% similarity between samples), similarity in functional composition was high (70%), and a core of functional genes present across all algal-associated communities was identified that were consistent with the ecology of surface- and host-associated bacteria. These functions were distributed widely across a variety of taxa or phylogenetic groups. This observation of similarity in habitat (niche) use with respect to functional genes, but not species, together with the relative ease with which bacteria share genetic material, suggests that the key level at which to address the assembly and structure of bacterial communities may not be "species" (by means of rRNA taxonomy), but rather the more functional level of genes.

Catalytic promiscuity in the biosynthesis of cyclic peptide secondary metabolites in planktonic marine cyanobacteria.

Our understanding of secondary metabolite production in bacteria has been shaped primarily by studies of attached varieties such as symbionts, pathogens, and soil bacteria. Here we show that a strain of the single-celled, planktonic marine cyanobacterium Prochlorococcus-which conducts a sizable fraction of photosynthesis in the oceans-produces many cyclic, lanthionine-containing peptides (lantipeptides). Remarkably, in Prochlorococcus MIT9313 a single promiscuous enzyme transforms up to 29 different linear ribosomally synthesized peptides into a library of polycyclic, conformationally constrained products with highly diverse ring topologies. Genes encoding this system are found in variable abundances across the oceans-with a hot spot in a Galapagos hypersaline lagoon-suggesting they play a habitat- and/or community-specific role. The extraordinarily efficient pathway for generating structural diversity enables these cyanobacteria to produce as many secondary metabolites as model antibiotic-producing bacteria, but with much smaller genomes.

Epigenetic Targeting of Adipocytes Inhibits High-Grade Serous Ovarian Cancer Cell Migration and Invasion.

Ovarian cancer (OC) cells frequently metastasize to the omentum, and adipocytes play a significant role in ovarian tumor progression. Therapeutic interventions targeting aberrant DNA methylation in ovarian tumors have shown promise in the clinic, but the effects of epigenetic therapy on the tumor microenvironment are understudied. Here, we examined the effect of adipocytes on OC cell behavior in culture and impact of targeting DNA methylation in adipocytes on OC metastasis. The presence of adipocytes increased OC cell migration and invasion, and proximal and direct coculture of adipocytes increased OC proliferation alone or after treatment with carboplatin. Treatment of adipocytes with hypomethylating agent guadecitabine decreased migration and invasion of OC cells toward adipocytes. Subcellular protein fractionation of adipocytes treated with guadecitabine revealed decreased DNA methyltransferase 1 (DNMT1) levels even in the presence of DNA synthesis inhibitor, aphidicolin. Methyl-Capture- and RNA-sequencing analysis of guadecitabine-treated adipocytes revealed derepression of tumor-suppressor genes and epithelial-mesenchymal transition inhibitors. SUSD2, a secreted tumor suppressor downregulated by promoter CpG island methylation in adipocytes, was upregulated after guadecitabine treatment, and recombinant SUSD2 decreased OC cell migration and invasion. Integrated analysis of the methylomic and transcriptomic data identified pathways associated with inhibition of matrix metalloproteases and fatty acid α-oxidation, suggesting a possible mechanism of how epigenetic therapy of adipocytes decreases metastasis. In conclusion, the effect of DNMT inhibitor on fully differentiated adipocytes suggests that hypomethylating agents may affect the tumor microenvironment to decrease cancer cell metastasis.Implications: Epigenetic targeting of tumor microenvironment can affect metastatic behavior of ovarian cancer cells. Mol Cancer Res; 16(8); 1226-40. ©2018 AACR.

Identification of Pol IV and RDR2-dependent precursors of 24 nt siRNAs guiding de novo DNA methylation in Arabidopsis.

In Arabidopsis thaliana, abundant 24 nucleotide small interfering RNAs (24 nt siRNA) guide the cytosine methylation and silencing of transposons and a subset of genes. 24 nt siRNA biogenesis requires nuclear RNA polymerase IV (Pol IV), RNA-dependent RNA polymerase 2 (RDR2) and DICER-like 3 (DCL3). However, siRNA precursors are mostly undefined. We identified Pol IV and RDR2-dependent RNAs (P4R2 RNAs) that accumulate in dcl3 mutants and are diced into 24 nt RNAs by DCL3 in vitro. P4R2 RNAs are mostly 26-45 nt and initiate with a purine adjacent to a pyrimidine, characteristics shared by Pol IV transcripts generated in vitro. RDR2 terminal transferase activity, also demonstrated in vitro, may account for occasional non-templated nucleotides at P4R2 RNA 3' termini. The 24 nt siRNAs primarily correspond to the 5' or 3' ends of P4R2 RNAs, suggesting a model whereby siRNAs are generated from either end of P4R2 duplexes by single dicing events.

Mapping RNA Sequences that Contact Viral Capsid Proteins in Virions.

We have adapted the methodology of CLIP-seq (Crosslinking-Immunoprecipitation and DNA Sequencing) to map the segments of encapsidated RNAs that contact the protein shells of virions. Results from the protocol report on the RNA sequences that contact the viral capsid.

Geoarchaeota: a new candidate phylum in the Archaea from high-temperature acidic iron mats in Yellowstone National Park.

Geothermal systems in Yellowstone National Park (YNP) provide an outstanding opportunity to understand the origin and evolution of metabolic processes necessary for life in extreme environments including low pH, high temperature, low oxygen and elevated concentrations of reduced iron. Previous phylogenetic studies of acidic ferric iron mats from YNP have revealed considerable diversity of uncultivated and undescribed archaea. The goal of this study was to obtain replicate de novo genome assemblies for a dominant archaeal population inhabiting acidic iron-oxide mats in YNP. Detailed analysis of conserved ribosomal and informational processing genes indicates that the replicate assemblies represent a new candidate phylum within the domain Archaea referred to here as 'Geoarchaeota' or 'novel archaeal group 1 (NAG1)'. The NAG1 organisms contain pathways necessary for the catabolism of peptides and complex carbohydrates as well as a bacterial-like Form I carbon monoxide dehydrogenase complex likely used for energy conservation. Moreover, this novel population contains genes involved in the metabolism of oxygen including a Type A heme copper oxidase, a bd-type terminal oxidase and a putative oxygen-sensing protoglobin. NAG1 has a variety of unique bacterial-like cofactor biosynthesis and transport genes and a Type3-like CRISPR system. Discovery of NAG1 is critical to our understanding of microbial community structure and function in extant thermophilic iron-oxide mats of YNP, and will provide insight regarding the evolution of Archaea in early Earth environments that may have important analogs active in YNP today.

Prokaryotic Super Program Advisory Committee DOE Joint Genome Institute, Walnut Creek, CA, March 27, 2013.

The Prokaryotic Super Program Advisory Committee met on March 27, 2013 for their annual review the Prokaryotic Super Program at the DOE Joint Genome Institute. As is the case with any site visit or program review, the objective is to evaluate progress in meeting organizational objectives, provide feedback to from the user-community and to assist the JGI in formulating plans for the coming year. The advisors want to commend the JGI for its central role in developing new technologies and capabilities, and for catalyzing the formation of new collaborative user communities. Highlights of the post-meeting exchanges among the advisors focused on the importance of programmatic initiatives including: • GEBA, which serves as a phylogenetic "base-map" on which our knowledge of functional diversity can be layered. • FEBA, which promises to provide new insights into the physiological capabilities of prokaryotes under highly standardized conditions. • Single-cell genomics technology, which is seen to significantly enhance our ability to interpret genomic and metagenomic data and broaden the scope of the GEBA program to encompass at least a part of the microbial "dark-matter". • IMG, which is seen to play a central role in JGI programs and is viewed as a strategically important asset in the JGI portfolio. On this latter point, the committee encourages the formation of a strategic relationship between IMG and the Kbase to ensure that the intelligence, deep knowledge and experience captured in the former is not lost. The committee strongly urges the DOE to continue its support for maintaining this critical resource.

Community structure and functional gene profile of bacteria on healthy and diseased thalli of the red seaweed Delisea pulchra.

Disease is increasingly viewed as a major factor in the ecology of marine communities and its impact appears to be increasing with environmental change, such as global warming. The temperate macroalga Delisea pulchra bleaches in Southeast Australia during warm summer periods, a phenomenon which previous studies have indicated is caused by a temperature induced bacterial disease. In order to better understand the ecology of this disease, the bacterial communities associated with threes type of samples was investigated using 16S rRNA gene and environmental shotgun sequencing: 1) unbleached (healthy) D. pulchra 2) bleached parts of D. pulchra and 3) apparently healthy tissue adjacent to bleached regions. Phylogenetic differences between healthy and bleached communities mainly reflected relative changes in the taxa Colwelliaceae, Rhodobacteraceae, Thalassomonas and Parvularcula. Comparative metagenomics showed clear difference in the communities of healthy and diseased D. pulchra as reflected by changes in functions associated with transcriptional regulation, cation/multidrug efflux and non-ribosomal peptide synthesis. Importantly, the phylogenetic and functional composition of apparently healthy tissue adjacent to bleached sections of the thalli indicated that changes in the microbial communities already occur in the absence of visible tissue damage. This shift in unbleached sections might be due to the decrease in furanones, algal metabolites which are antagonists of bacterial quorum sensing. This study reveals the complex shift in the community composition associated with bleaching of Delisea pulchra and together with previous studies is consistent with a model in which elevated temperatures reduce levels of chemical defenses in stressed thalli, leading to colonization or proliferation by opportunistic pathogens or scavengers.

Influence of nutrients and currents on the genomic composition of microbes across an upwelling mosaic.

Metagenomic data sets were generated from samples collected along a coastal to open ocean transect between Southern California Bight and California Current waters during a seasonal upwelling event, providing an opportunity to examine the impact of episodic pulses of cold nutrient-rich water into surface ocean microbial communities. The data set consists of ~5.8 million predicted proteins across seven sites, from three different size classes: 0.1-0.8, 0.8-3.0 and 3.0-200.0 µm. Taxonomic and metabolic analyses suggest that sequences from the 0.1-0.8 µm size class correlated with their position along the upwelling mosaic. However, taxonomic profiles of bacteria from the larger size classes (0.8-200 µm) were less constrained by habitat and characterized by an increase in Cyanobacteria, Bacteroidetes, Flavobacteria and double-stranded DNA viral sequences. Functional annotation of transmembrane proteins indicate that sites comprised of organisms with small genomes have an enrichment of transporters with substrate specificities for amino acids, iron and cadmium, whereas organisms with larger genomes have a higher percentage of transporters for ammonium and potassium. Eukaryotic-type glutamine synthetase (GS) II proteins were identified and taxonomically classified as viral, most closely related to the GSII in Mimivirus, suggesting that marine Mimivirus-like particles may have played a role in the transfer of GSII gene functions. Additionally, a Planctomycete bloom was sampled from one upwelling site providing a rare opportunity to assess the genomic composition of a marine Planctomycete population. The significant correlations observed between genomic properties, community structure and nutrient availability provide insights into habitat-driven dynamics among oligotrophic versus upwelled marine waters adjoining each other spatially.

Functional genomic signatures of sponge bacteria reveal unique and shared features of symbiosis.

Sponges form close relationships with bacteria, and a remarkable phylogenetic diversity of yet-uncultured bacteria has been identified from sponges using molecular methods. In this study, we use a comparative metagenomic analysis of the bacterial community in the model sponge Cymbastela concentrica and in the surrounding seawater to identify previously unrecognized genomic signatures and functions for sponge bacteria. We observed a surprisingly large number of transposable insertion elements, a feature also observed in other symbiotic bacteria, as well as a set of predicted mechanisms that may defend the sponge community against the introduction of foreign DNA and hence contribute to its genetic resilience. Moreover, several shared metabolic interactions between bacteria and host include vitamin production, nutrient transport and utilization, and redox sensing and response. Finally, an abundance of protein-protein interactions mediated through ankyrin and tetratricopeptide repeat proteins could represent a mechanism for the sponge to discriminate between food and resident bacteria. These data provide new insight into the evolution of symbiotic diversity, microbial metabolism and host-microbe interactions in sponges.

Environmental genome shotgun sequencing of the Sargasso Sea.

We have applied "whole-genome shotgun sequencing" to microbial populations collected en masse on tangential flow and impact filters from seawater samples collected from the Sargasso Sea near Bermuda. A total of 1.045 billion base pairs of nonredundant sequence was generated, annotated, and analyzed to elucidate the gene content, diversity, and relative abundance of the organisms within these environmental samples. These data are estimated to derive from at least 1800 genomic species based on sequence relatedness, including 148 previously unknown bacterial phylotypes. We have identified over 1.2 million previously unknown genes represented in these samples, including more than 782 new rhodopsin-like photoreceptors. Variation in species present and stoichiometry suggests substantial oceanic microbial diversity.