Dongying Wu

The Sorcerer II Global Ocean Sampling Expedition: Northwest Atlantic through Eastern Tropical Pacific

The worlds oceans contain a complex mixture of micro-organisms that are for the most part, uncharacterized both genetically and biochemically. We report here a metagenomic study of the marine planktonic microbiota in which surface (mostly marine) water samples were analyzed as part of the Sorcerer II Global Ocean Sampling expedition. These samples, collected across a several-thousand km transect from the North Atlantic through the Panama Canal and ending in the South Pacific yielded an extensive dataset consisting of 7.7 million sequencing reads (6.3 billion bp). Though a few major microbial clades dominate the planktonic marine niche, the dataset contains great diversity with 85% of the assembled sequence and 57% of the unassembled data being unique at a 98% sequence identity cutoff. Using the metadata associated with each sample and sequencing library, we developed new comparative genomic and assembly methods. One comparative genomic method, termed “fragment recruitment,” addressed questions of genome structure, evolution, and taxonomic or phylogenetic diversity, as well as the biochemical diversity of genes and gene families. A second method, termed “extreme assembly,” made possible the assembly and reconstruction of large segments of abundant but clearly nonclonal organisms. Within all abundant populations analyzed, we found extensive intra-ribotype diversity in several forms: (1) extensive sequence variation within orthologous regions throughout a given genome; despite coverage of individual ribotypes approaching 500-fold, most individual sequencing reads are unique; (2) numerous changes in gene content some with direct adaptive implications; and (3) hypervariable genomic islands that are too variable to assemble. The intra-ribotype diversity is organized into genetically isolated populations that have overlapping but independent distributions, implying distinct environmental preference. We present novel methods for measuring the genomic similarity between metagenomic samples and show how they may be grouped into several community types. Specific functional adaptations can be identified both within individual ribotypes and across the entire community, including proteorhodopsin spectral tuning and the presence or absence of the phosphate-binding gene PstS.

A phylogeny-driven genomic encyclopaedia of Bacteria and Archaea.

Sequencing of bacterial and archaeal genomes has revolutionized our understanding of the many roles played by microorganisms. There are now nearly 1,000 completed bacterial and archaeal genomes available, most of which were chosen for sequencing on the basis of their physiology. As a result, the perspective provided by the currently available genomes is limited by a highly biased phylogenetic distribution. To explore the value added by choosing microbial genomes for sequencing on the basis of their evolutionary relationships, we have sequenced and analysed the genomes of 56 culturable species of Bacteria and Archaea selected to maximize phylogenetic coverage. Analysis of these genomes demonstrated pronounced benefits (compared to an equivalent set of genomes randomly selected from the existing database) in diverse areas including the reconstruction of phylogenetic history, the discovery of new protein families and biological properties, and the prediction of functions for known genes from other organisms. Our results strongly support the need for systematic ‘phylogenomic’ efforts to compile a phylogeny-driven ‘Genomic Encyclopedia of Bacteria and Archaea’ in order to derive maximum knowledge from existing microbial genome data as well as from genome sequences to come.

Improving the coverage of the cyanobacterial phylum using diversity-driven genome sequencing

The cyanobacterial phylum encompasses oxygenic photosynthetic prokaryotes of a great breadth of morphologies and ecologies; they play key roles in global carbon and nitrogen cycles. The chloroplasts of all photosynthetic eukaryotes can trace their ancestry to cyanobacteria. Cyanobacteria also attract considerable interest as platforms for “green” biotechnology and biofuels. To explore the molecular basis of their different phenotypes and biochemical capabilities, we sequenced the genomes of 54 phylogenetically and phenotypically diverse cyanobacterial strains. Comparison of cyanobacterial genomes reveals the molecular basis for many aspects of cyanobacterial ecophysiological diversity, as well as the convergence of complex morphologies without the acquisition of novel proteins. This phylum-wide study highlights the benefits of diversity-driven genome sequencing, identifying more than 21,000 cyanobacterial proteins with no detectable similarity to known proteins, and foregrounds the diversity of light-harvesting proteins and gene clusters for secondary metabolite biosynthesis. Additionally, our results provide insight into the distribution of genes of cyanobacterial origin in eukaryotic nuclear genomes. Moreover, this study doubles both the amount and the phylogenetic diversity of cyanobacterial genome sequence data. Given the exponentially growing number of sequenced genomes, this diversity-driven study demonstrates the perspective gained by comparing disparate yet related genomes in a phylum-wide context and the insights that are gained from it.

Metabolic complementarity and genomics of the dual bacterial symbiosis of sharpshooters.

Mutualistic intracellular symbiosis between bacteria and insects is a widespread phenomenon that has contributed to the global success of insects. The symbionts, by provisioning nutrients lacking from diets, allow various insects to occupy or dominate ecological niches that might otherwise be unavailable. One such insect is the glassy-winged sharpshooter (Homalodisca coagulata), which feeds on xylem fluid, a diet exceptionally poor in organic nutrients. Phylogenetic studies based on rRNA have shown two types of bacterial symbionts to be coevolving with sharpshooters: the gamma-proteobacterium Baumannia cicadellinicola and the Bacteroidetes species Sulcia muelleri. We report here the sequencing and analysis of the 686,192–base pair genome of B. cicadellinicola and approximately 150 kilobase pairs of the small genome of S. muelleri, both isolated from H. coagulata. Our study, which to our knowledge is the first genomic analysis of an obligate symbiosis involving multiple partners, suggests striking complementarity in the biosynthetic capabilities of the two symbionts: B. cicadellinicola devotes a substantial portion of its genome to the biosynthesis of vitamins and cofactors required by animals and lacks most amino acid biosynthetic pathways, whereas S. muelleri apparently produces most or all of the essential amino acids needed by its host. This finding, along with other results of our genome analysis, suggests the existence of metabolic codependency among the two unrelated endosymbionts and their insect host. This dual symbiosis provides a model case for studying correlated genome evolution and genome reduction involving multiple organisms in an intimate, obligate mutualistic relationship. In addition, our analysis provides insight for the first time into the differences in symbionts between insects (e.g., aphids) that feed on phloem versus those like H. coagulata that feed on xylem. Finally, the genomes of these two symbionts provide potential targets for controlling plant pathogens such as Xylella fastidiosa, a major agroeconomic problem, for which H. coagulata and other sharpshooters serve as vectors of transmission.

One bacterial cell, one complete genome.

While the bulk of the finished microbial genomes sequenced to date are derived from cultured bacterial and archaeal representatives, the vast majority of microorganisms elude current culturing attempts, severely limiting the ability to recover complete or even partial genomes from these environmental species. Single cell genomics is a novel culture-independent approach, which enables access to the genetic material of an individual cell. No single cell genome has to our knowledge been closed and finished to date. Here we report the completed genome from an uncultured single cell of Candidatus Sulcia muelleri DMIN. Digital PCR on single symbiont cells isolated from the bacteriome of the green sharpshooter Draeculacephala minerva bacteriome allowed us to assess that this bacteria is polyploid with genome copies ranging from approximately 200–900 per cell, making it a most suitable target for single cell finishing efforts. For single cell shotgun sequencing, an individual Sulcia cell was isolated and whole genome amplified by multiple displacement amplification (MDA). Sanger-based finishing methods allowed us to close the genome. To verify the correctness of our single cell genome and exclude MDA-derived artifacts, we independently shotgun sequenced and assembled the Sulcia genome from pooled bacteriomes using a metagenomic approach, yielding a nearly identical genome. Four variations we detected appear to be genuine biological differences between the two samples. Comparison of the single cell genome with bacteriome metagenomic sequence data detected two single nucleotide polymorphisms (SNPs), indicating extremely low genetic diversity within a Sulcia population. This study demonstrates the power of single cell genomics to generate a complete, high quality, non-composite reference genome within an environmental sample, which can be used for population genetic analyzes.

Complete reannotation of the Arabidopsis genome: methods, tools, protocols and the final release

BackgroundSince the initial publication of its complete genome sequence, Arabidopsis thaliana has become more important than ever as a model for plant research. However, the initial genome annotation was submitted by multiple centers using inconsistent methods, making the data difficult to use for many applications.ResultsOver the course of three years, TIGR has completed its effort to standardize the structural and functional annotation of the Arabidopsis genome. Using both manual and automated methods, Arabidopsis gene structures were refined and gene products were renamed and assigned to Gene Ontology categories. We present an overview of the methods employed, tools developed, and protocols followed, summarizing the contents of each data release with special emphasis on our final annotation release (version 5).ConclusionOver the entire period, several thousand new genes and pseudogenes were added to the annotation. Approximately one third of the originally annotated gene models were significantly refined yielding improved gene structure annotations, and every protein-coding gene was manually inspected and classified using Gene Ontology terms.

Adaptations to Submarine Hydrothermal Environments Exemplified by the Genome of Nautilia profundicola

Submarine hydrothermal vents are model systems for the Archaean Earth environment, and some sites maintain conditions that may have favored the formation and evolution of cellular life. Vents are typified by rapid fluctuations in temperature and redox potential that impose a strong selective pressure on resident microbial communities. Nautilia profundicola strain Am-H is a moderately thermophilic, deeply-branching Epsilonproteobacterium found free-living at hydrothermal vents and is a member of the microbial mass on the dorsal surface of vent polychaete, Alvinella pompejana. Analysis of the 1.7-Mbp genome of N. profundicola uncovered adaptations to the vent environment—some unique and some shared with other Epsilonproteobacterial genomes. The major findings included: (1) a diverse suite of hydrogenases coupled to a relatively simple electron transport chain, (2) numerous stress response systems, (3) a novel predicted nitrate assimilation pathway with hydroxylamine as a key intermediate, and (4) a gene (rgy) encoding the hallmark protein for hyperthermophilic growth, reverse gyrase. Additional experiments indicated that expression of rgy in strain Am-H was induced over 100-fold with a 20°C increase above the optimal growth temperature of this bacterium and that closely related rgy genes are present and expressed in bacterial communities residing in geographically distinct thermophilic environments. N. profundicola, therefore, is a model Epsilonproteobacterium that contains all the genes necessary for life in the extreme conditions widely believed to reflect those in the Archaean biosphere—anaerobic, sulfur, H2- and CO2-rich, with fluctuating redox potentials and temperatures. In addition, reverse gyrase appears to be an important and common adaptation for mesophiles and moderate thermophiles that inhabit ecological niches characterized by rapid and frequent temperature fluctuations and, as such, can no longer be considered a unique feature of hyperthermophiles.

Systematic identification of gene families for use as "markers" for phylogenetic and phylogeny-driven ecological studies of bacteria and archaea and their major subgroups.

With the astonishing rate that genomic and metagenomic sequence data sets are accumulating, there are many reasons to constrain the data analyses. One approach to such constrained analyses is to focus on select subsets of gene families that are particularly well suited for the tasks at hand. Such gene families have generally been referred to as “marker” genes. We are particularly interested in identifying and using such marker genes for phylogenetic and phylogeny-driven ecological studies of microbes and their communities (e.g., construction of species trees, phylogenetic based assignment of metagenomic sequence reads to taxonomic groups, phylogeny-based assessment of alpha- and beta-diversity of microbial communities from metagenomic data). We therefore refer to these as PhyEco (for phylogenetic and phylogenetic ecology) markers. The dual use of these PhyEco markers means that we needed to develop and apply a set of somewhat novel criteria for identification of the best candidates for such markers. The criteria we focused on included universality across the taxa of interest, ability to be used to produce robust phylogenetic trees that reflect as much as possible the evolution of the species from which the genes come, and low variation in copy number across taxa. We describe here an automated protocol for identifying potential PhyEco markers from a set of complete genome sequences. The protocol combines rapid searching, clustering and phylogenetic tree building algorithms to generate protein families that meet the criteria listed above. We report here the identification of PhyEco markers for different taxonomic levels including 40 for “all bacteria and archaea”, 114 for “all bacteria (greatly expanding on the ∼30 commonly used), and 100 s to 1000 s for some of the individual phyla of bacteria. This new list of PhyEco markers should allow much more detailed automated phylogenetic and phylogenetic ecology analyses of these groups than possible previously.

Extraordinary phylogenetic diversity and metabolic versatility in aquifer sediment

Microorganisms in the subsurface represent a substantial but poorly understood component of the Earth’s biosphere. Subsurface environments are complex and difficult to characterize; thus, their microbiota have remained as a ‘dark matter’ of the carbon and other biogeochemical cycles. Here we deeply sequence two sediment-hosted microbial communities from an aquifer adjacent to the Colorado River, CO, USA. No single organism represents more than ~1% of either community. Remarkably, many bacteria and archaea in these communities are novel at the phylum level or belong to phyla lacking a sequenced representative. The dominant organism in deeper sediment, RBG-1, is a member of a new phylum. On the basis of its reconstructed complete genome, RBG-1 is metabolically versatile. Its wide respiration-based repertoire may enable it to respond to the fluctuating redox environment close to the water table. We document extraordinary microbial novelty and the importance of previously unknown lineages in sediment biogeochemical transformations.

Characterization of bacterial communities associated with deep-sea corals on Gulf of Alaska seamounts.

ABSTRACT Although microbes associated with shallow-water corals have been reported, deepwater coral microbes are poorly characterized. A cultivation-independent analysis of Alaskan seamount octocoral microflora showed that Proteobacteria (classes Alphaproteobacteria and Gammaproteobacteria), Firmicutes, Bacteroidetes, and Acidobacteria dominate and vary in abundance. More sampling is needed to understand the basis and significance of this variation.