Benjamin J. Tully

Microbial communities associated with ferromanganese nodules and the surrounding sediments

The formation and maintenance of deep-sea ferromanganese/polymetallic nodules still remains a mystery 140 years after their discovery. The wealth of rare metals concentrated in these nodules has spurred global interest in exploring the mining potential of these resources. The prevailing theory of abiotic formation has been called into question and the role of microbial metabolisms in nodule development is now an area of active research. To understand the community structure of microbes associated with nodules and their surrounding sediment, we performed targeted sequencing of the V4 hypervariable region of the 16S rRNA gene from three nodules collected from the central South Pacific. Results have shown that the microbial communities of the nodules are significantly distinct from the communities in the surrounding sediments, and that the interiors of the nodules harbor communities different from the exterior. This suggests not only differences in potential metabolisms between the nodule and sediment communities, but also differences in the dominant metabolisms of interior and exterior communities. We identified several operational taxonomic units (OTUs) unique to both the nodule and sediment environments. The identified OTUs were assigned putative taxonomic identifications, including two OTUs only found associated with the nodules, which were assigned to the α-Proteobacteria. Finally, we explored the diversity of the most assigned taxonomic group, the Thaumarchaea MG-1, which revealed novel OTUs compared to previous research from the region and suggests a potential role as a source of fixed carbon for ammonia oxidizing archaea in the environment.

The reconstruction of 2,631 draft metagenome-assembled genomes from the global oceans

Microorganisms play a crucial role in mediating global biogeochemical cycles in the marine environment. By reconstructing the genomes of environmental organisms through metagenomics, researchers are able to study the metabolic potential of Bacteria and Archaea that are resistant to isolation in the laboratory. Utilizing the large metagenomic dataset generated from 234 samples collected during the Tara Oceans circumnavigation expedition, we were able to assemble 102 billion paired-end reads into 562 million contigs, which in turn were co-assembled and consolidated in to 7.2 million contigs ≥2 kb in length. Approximately 1 million of these contigs were binned to reconstruct draft genomes. In total, 2,631 draft genomes with an estimated completion of ≥50% were generated (1,491 draft genomes >70% complete; 603 genomes >90% complete). A majority of the draft genomes were manually assigned phylogeny based on sets of concatenated phylogenetic marker genes and/or 16S rRNA gene sequences. The draft genomes are now publically available for the research community at-large.

Prospects for the Study of Evolution in the Deep Biosphere

Since the days of Darwin, scientists have used the framework of the theory of evolution to explore the interconnectedness of life on Earth and adaptation of organisms to the ever-changing environment. The advent of molecular biology has advanced and accelerated the study of evolution by allowing direct examination of the genetic material that ultimately determines the phenotypes upon which selection acts. The study of evolution has been furthered through examination of microbial evolution, with large population numbers, short generation times, and easily extractable DNA. Such work has spawned the study of microbial biogeography, with the realization that concepts developed in population genetics may be applicable to microbial genomes (Martiny et al., 2006; Manhes and Velicer, 2011). Microbial biogeography and adaptation has been examined in many different environments. Here we argue that the deep biosphere is a unique environment for the study of evolution and list specific factors that can be considered and where the studies may be performed. This publication is the result of the NSF-funded Center for Dark Energy Biosphere Investigations (C-DEBI) theme team on Evolution (www.darkenergybiosphere.org).

BinSanity: unsupervised clustering of environmental microbial assemblies using coverage and affinity propagation

Metagenomics has become an integral part of defining microbial diversity in various environments. Many ecosystems have characteristically low biomass and few cultured representatives. Linking potential metabolisms to phylogeny in environmental microorganisms is important for interpreting microbial community functions and the impacts these communities have on geochemical cycles. However, with metagenomic studies there is the computational hurdle of ‘binning’ contigs into phylogenetically related units or putative genomes. Binning methods have been implemented with varying approaches such as k-means clustering, Gaussian mixture models, hierarchical clustering, neural networks, and two-way clustering; however, many of these suffer from biases against low coverage/abundance organisms and closely related taxa/strains. We are introducing a new binning method, BinSanity, that utilizes the clustering algorithm affinity propagation (AP), to cluster assemblies using coverage with compositional based refinement (tetranucleotide frequency and percent GC content) to optimize bins containing multiple source organisms. This separation of composition and coverage based clustering reduces bias for closely related taxa. BinSanity was developed and tested on artificial metagenomes varying in size and complexity. Results indicate that BinSanity has a higher precision, recall, and Adjusted Rand Index compared to five commonly implemented methods. When tested on a previously published environmental metagenome, BinSanity generated high completion and low redundancy bins corresponding with the published metagenome-assembled genomes.

A distinct and active bacterial community in cold oxygenated fluids circulating beneath the western flank of the Mid-Atlantic ridge.

The rock-hosted, oceanic crustal aquifer is one of the largest ecosystems on Earth, yet little is known about its indigenous microorganisms. Here we provide the first phylogenetic and functional description of an active microbial community residing in the cold oxic crustal aquifer. Using subseafloor observatories, we recovered crustal fluids and found that the geochemical composition is similar to bottom seawater, as are cell abundances. However, based on relative abundances and functional potential of key bacterial groups, the crustal fluid microbial community is heterogeneous and markedly distinct from seawater. Potential rates of autotrophy and heterotrophy in the crust exceeded those of seawater, especially at elevated temperatures (25 °C) and deeper in the crust. Together, these results reveal an active, distinct, and diverse bacterial community engaged in both heterotrophy and autotrophy in the oxygenated crustal aquifer, providing key insight into the role of microbial communities in the ubiquitous cold dark subseafloor biosphere.

Potential Mechanisms for Microbial Energy Acquisition in Oxic Deep-Sea Sediments

ABSTRACT The South Pacific Gyre (SPG) possesses the lowest rates of sedimentation, surface chlorophyll concentration, and primary productivity in the global oceans. As a direct result, deep-sea sediments are thin and contain small amounts of labile organic carbon. It was recently shown that the entire SPG sediment column is oxygenated and may be representative of up to a third of the global marine environment. To understand the microbial processes that contribute to the removal of the labile organic matter at the water-sediment interface, a sediment sample was collected and subjected to metagenomic sequencing and analyses. Analysis of nine partially reconstructed environmental genomes, which represent approximately one-third of the microbial community, revealed that the members of the SPG surface sediment microbial community are phylogenetically distinct from surface/upper-ocean organisms. These genomes represent a wide distribution of novel organisms, including deep-branching Alphaproteobacteria, two novel organisms within the Proteobacteria, and new members of the Nitrospirae, Nitrospinae, and candidate phylum NC10. These genomes contain evidence for microbially mediated metal (iron/manganese) oxidation and carbon fixation linked to nitrification. Additionally, despite hypothesized energy limitation, members of the SPG microbial community had motility and chemotaxis genes and possessed mechanisms for the degradation of high-molecular-weight organic matter. This study contributes to our understanding of the metabolic potential of microorganisms in deep-sea oligotrophic sediments and their impact on local carbon geochemistry. IMPORTANCE This research provides insight into the microbial metabolic potential of organisms inhabiting oxygenated deep-sea marine sediments. Current estimates suggest that these environments account for up to a third of the global marine sediment habitat. Nine novel deep-sea microbial genomes were reconstructed from a metagenomic data set and expand the limited number of environmental genomes from deep-sea sediment environments. This research provides phylogeny-linked insight into critical metabolisms, including carbon fixation associated with nitrification, which is assignable to members of the marine group 1 Thaumarchaeota, Nitrospinae, and Nitrospirae and neutrophilic metal (iron/manganese) oxidation assignable to a novel proteobacterium.

A dynamic microbial community with high functional redundancy inhabits the cold, oxic subseafloor aquifer

The rock-hosted subseafloor crustal aquifer harbors a reservoir of microbial life that may influence global marine biogeochemical cycles. Here we utilized metagenomic libraries of crustal fluid samples from North Pond, located on the flanks of the Mid-Atlantic Ridge, a site with cold, oxic subseafloor fluid circulation within the upper basement to query microbial diversity. Twenty-one samples were collected during a 2-year period to examine potential microbial metabolism and community dynamics. We observed minor changes in the geochemical signatures over the 2 years, yet the microbial community present in the crustal fluids underwent large shifts in the dominant taxonomic groups. An analysis of 195 metagenome-assembled genomes (MAGs) were generated from the data set and revealed a connection between litho- and autotrophic processes, linking carbon fixation to the oxidation of sulfide, sulfur, thiosulfate, hydrogen, and ferrous iron in members of the Proteobacteria, specifically the Alpha-, Gamma- and Zetaproteobacteria, the Epsilonbacteraeota and the Planctomycetes. Despite oxic conditions, analysis of the MAGs indicated that members of the microbial community were poised to exploit hypoxic or anoxic conditions through the use of microaerobic cytochromes, such as cbb3- and bd-type cytochromes, and alternative electron acceptors, like nitrate and sulfate. Temporal and spatial trends from the MAGs revealed a high degree of functional redundancy that did not correlate with the shifting microbial community membership, suggesting functional stability in mediating subseafloor biogeochemical cycles. Collectively, the repeated sampling at multiple sites, together with the successful binning of hundreds of genomes, provides an unprecedented data set for investigation of microbial communities in the cold, oxic crustal aquifer.

290 metagenome-assembled genomes from the Mediterranean Sea: a resource for marine microbiology

The Tara Oceans Expedition has provided large, publicly-accessible microbial metagenomic datasets from a circumnavigation of the globe. Utilizing several size fractions from the samples originating in the Mediterranean Sea, we have used current assembly and binning techniques to reconstruct 290 putative draft metagenome-assembled bacterial and archaeal genomes, with an estimated completion of ≥50%, and an additional 2,786 bins, with estimated completion of 0–50%. We have submitted our results, including initial taxonomic and phylogenetic assignments, for the putative draft genomes to open-access repositories for the scientific community to use in ongoing research.

Comparative genomics of planktonic Flavobacteriaceae from the Gulf of Maine using metagenomic data

BackgroundThe Gulf of Maine is an important biological province of the Northwest Atlantic with high productivity year round. From an environmental Sanger-based metagenome, sampled in summer and winter, we were able to assemble and explore the partial environmental genomes of uncultured members of the class Flavobacteria. Each of the environmental genomes represents organisms that compose less than 1% of the total microbial metagenome.ResultsFour partial environmental genomes were assembled with varying degrees of estimated completeness (37%–84% complete) and were analyzed from a perspective of gathering information regarding niche partitioning between co-occurring organisms. Comparative genomics revealed potentially important niche partitioning genomic variations, including iron transporters and genes associated with cell attachment and polymer degradation. Analysis of large syntenic regions helped reveal potentially ecologically relevant variations for Flavobacteriaceae in the Gulf of Maine, such as arginine biosynthesis, and identify a putative genomic island incorporating novel exogenous genes from the environment.ConclusionsBiogeographic analysis revealed flavobacteria species with distinct abundance patterns suggesting the presence of local blooms relative to the other species, as well as seasonally selected organisms. The analysis of genomic content for the Gulf of Maine Flavobacteria supports the hypothesis of a particle-associated lifestyle and specifically highlights a number of putative coding sequences that may play a role in the remineralization of particulate organic matter. And lastly, analysis of the underlying sequences for each assembled genome revealed seasonal and nonseasonal variants of specific genes implicating a dynamic interaction between individuals within the species.

De Novo Sequences of Haloquadratum walsbyi from Lake Tyrrell, Australia, Reveal a Variable Genomic Landscape

Hypersaline systems near salt saturation levels represent an extreme environment, in which organisms grow and survive near the limits of life. One of the abundant members of the microbial communities in hypersaline systems is the square archaeon, Haloquadratum walsbyi. Utilizing a short-read metagenome from Lake Tyrrell, a hypersaline ecosystem in Victoria, Australia, we performed a comparative genomic analysis of H. walsbyi to better understand the extent of variation between strains/subspecies. Results revealed that previously isolated strains/subspecies do not fully describe the complete repertoire of the genomic landscape present in H. walsbyi. Rearrangements, insertions, and deletions were observed for the Lake Tyrrell derived Haloquadratum genomes and were supported by environmental de novo sequences, including shifts in the dominant genomic landscape of the two most abundant strains. Analysis pertaining to halomucins indicated that homologs for this large protein are not a feature common for all species of Haloquadratum. Further, we analyzed ATP-binding cassette transporters (ABC-type transporters) for evidence of niche partitioning between different strains/subspecies. We were able to identify unique and variable transporter subunits from all five genomes analyzed and the de novo environmental sequences, suggesting that differences in nutrient and carbon source acquisition may play a role in maintaining distinct strains/subspecies.