In metagenome analysis, computational methods for assembly, taxonomic profiling and binning are key components facilitating downstream biological data interpretation. However, a lack of consensus about benchmarking datasets and evaluation metrics complicates proper performance assessment. The Critical Assessment of Metagenome Interpretation (CAMI) challenge has engaged the global developer community to benchmark their programs on datasets of unprecedented complexity and realism. Benchmark metagenomes were generated from ~700 newly sequenced microorganisms and ~600 novel viruses and plasmids, including genomes with varying degrees of relatedness to each other and to publicly available ones and representing common experimental setups. Across all datasets, assembly and genome binning programs performed well for species represented by individual genomes, while performance was substantially affected by the presence of related strains. Taxonomic profiling and binning programs were proficient at high taxonomic ranks, with a notable performance decrease below the family level. Parameter settings substantially impacted performances, underscoring the importance of program reproducibility. While highlighting current challenges in computational metagenomics, the CAMI results provide a roadmap for software selection to answer specific research questions.

Many marine bacteria have evolved to grow optimally at either high (copiotrophic) or low (oligotrophic) nutrient concentrations, enabling different species to colonize distinct trophic habitats in the oceans. Here, we compare the genome sequences of two bacteria, Photobacterium angustum S14 and Sphingopyxis alaskensis RB2256, that serve as useful model organisms for copiotrophic and oligotrophic modes of life and specifically relate the genomic features to trophic strategy for these organisms and define their molecular mechanisms of adaptation. We developed a model for predicting trophic lifestyle from genome sequence data and tested >400,000 proteins representing >500 million nucleotides of sequence data from 126 genome sequences with metagenome data of whole environmental samples. When applied to available oceanic metagenome data (e.g., the Global Ocean Survey data) the model demonstrated that oligotrophs, and not the more readily isolatable copiotrophs, dominate the ocean's free-living microbial populations. Using our model, it is now possible to define the types of bacteria that specific ocean niches are capable of sustaining.microbial adaptation and ecology ͉ microbial genomics and metagenomics ͉ monitoring environmental health ͉ trophic adaptation T he marine environment is the largest habitat on Earth, accounting for Ͼ90% of the biosphere by volume and harboring microorganisms responsible for Ϸ50% of total global primary production. Within this environment, marine bacteria (and archaea) play a pivotal role in biogeochemical cycles while constantly assimilating, storing, transforming, exporting, and remineralizing the largest pool of organic carbon on the planet (1).Nutrient levels in pelagic waters are not uniform. Large expanses of water are relatively nutrient depleted (e.g., oligotrophic open ocean water), whereas other zones are relatively nutrient rich (e.g., copiotrophic coastal and estuarine waters). Local variations in nutrient content can occur because of physical processes, including upwelling of nutrient rich deep waters or aeolian and riverine deposition, or biological processes such as phytoplankton blooms or aggregation of particulate organic matter. In addition, heterogeneity in ocean waters is not limited to gross differences in nutrient concentrations, but extends to microscale patchiness that occurs throughout the continuum of ocean nutrient concentrations (2).In ecological terms, bacteria are generally defined as rstrategists, having a small body, short generation time, and highly dispersible offspring. Although this strategy is broadly true compared with macroorganisms, bacteria have evolved a wide range of growth and survival strategies to maximize reproductive success. In particular, nutrient type and availability have provided strong selective pressure for defining lifestyle strategies among marine bacteria. However, although a large number of copiotrophic marine organisms (and fewer oligotrophs) have been cultured, the study of trophic strategy has been impaired by a lack of unders...

Most proteins adopt a well defined three-dimensional structure; however, it is increasingly recognized that some proteins can exist with at least two stable conformations. Recently, a class of intracellular chloride ion channel proteins (CLICs) has been shown to exist in both soluble and integral membrane forms. The structure of the soluble form of CLIC1 is typical of a soluble glutathione S-transferase superfamily protein but contains a glutaredoxin-like active site. In this study we show that on oxidation CLIC1 undergoes a reversible transition from a monomeric to a non-covalent dimeric state due to the formation of an intramolecular disulfide bond (Cys-24 -Cys-59). We have determined the crystal structure of this oxidized state and show that a major structural transition has occurred, exposing a large hydrophobic surface, which forms the dimer interface. The oxidized CLIC1 dimer maintains its ability to form chloride ion channels in artificial bilayers and vesicles, whereas a reducing environment prevents the formation of ion channels by CLIC1. Mutational studies show that both Cys-24 and Cys-59 are required for channel activity.Chloride ion channels control a variety of cellular processes that are central to normal function and disease states (1). The CLIC 1 family is a recently identified class of Cl Ϫ channel proteins that consists of seven members (p64, parchorin, CLIC1-5) (2, 3). A conserved C-terminal CLIC module of ϳ240 amino acids is present in each member of the family with several members containing additional, unrelated Nterminal domains. Most CLICs are localized to intracellular membranes and have been linked to functions including apoptosis, pH, and cell cycle regulation (4 -6). The CLIC ion channels are unusual in that they possess both soluble and integral membrane forms (2). In this regard they are similar to some bacterial toxins and several classes of intracellular proteins including Bcl-x L and the annexins (7). Our understanding of how such dual natured proteins enter the membrane is limited by the dearth of high resolution structures for key states in this process.We have recently determined the crystal structure of a soluble monomeric form of CLIC1 (8) and found that it is a structural homologue of the GST superfamily of proteins (9). This soluble form of CLIC1 consists of two domains, the N-domain possessing a thioredoxin fold closely resembling glutaredoxin and an all ␣-helical C-domain, which is typical of the GST superfamily. CLIC1 contains an intact glutathione-binding site that was shown to covalently bind glutathione via a conserved CLIC cysteine residue, Cys-24. This led to the suggestion that CLIC1 function may be under redox control, possibly via reactive oxygen or nitrogen species.The structure and stoichiometry of the integral membrane form of the CLIC proteins is still unclear. Electrophysiology of purified, soluble (Escherichia coli-expressed) recombinant CLIC1 in reconstituted artificial bilayers shows that CLIC1 alone is sufficient for chloride ion channel formation (8, 1...

CLIC1 (NCC27) is a member of the highly conserved class of chloride ion channels that exists in both soluble and integral membrane forms. Purified CLIC1 can integrate into synthetic lipid bilayers forming a chloride channel with similar properties to those observed in vivo. The structure of the soluble form of CLIC1 has been determined at 1.4-Å resolution. The protein is monomeric and structurally homologous to the glutathione S-transferase superfamily, and it has a redox-active site resembling glutaredoxin. The structure of the complex of CLIC1 with glutathione shows that glutathione occupies the redox-active site, which is adjacent to an open, elongated slot lined by basic residues. Integration of CLIC1 into the membrane is likely to require a major structural rearrangement, probably of the N-domain (residues 1-90), with the putative transmembrane helix arising from residues in the vicinity of the redox-active site. The structure indicates that CLIC1 is likely to be controlled by redox-dependent processes.Chloride ion channels, located both within the plasma membrane and other internal cell membranes (1, 2), are involved in diverse physiological processes. They are known to participate in the control of secretion and absorption of salt, regulation of membrane potentials, organellar acidification, and cell volume homeostasis (3). Malfunction in these channels can lead to severe disease states (4).Chloride channels fall into several classes based on their sequence relationships. The three best characterized classes are the ligand-gated receptor channels (␥-aminobutyric acid and glycine receptors), the cystic fibrosis transmembrane conductance regulator family, and the ClC chloride ion channels (1, 2). A new class of chloride ion channel, the "chloride intracellular channels" (CLICs), 1 has recently been characterized at a molecular level. To date, there are seven members of the CLIC family: CLIC1 (NCC27) (5), CLIC2 (6), CLIC3 (7), CLIC4 (8), CLIC5 (9), p64 (10), and parchorin (11). All of these proteins exist as soluble globular proteins that can form ion channels in organellar and plasma membranes (5,7,8,(12)(13)(14)(15). Five of the CLIC proteins are each composed of ϳ240 residues, while the longer p64 and parchorin consist of distinct amino-terminal domains followed by the 240-residue CLIC module. This module has recently been shown to share weak sequence homology with the glutathione S-transferase (GST) superfamily (16).The CLIC proteins are expressed in a wide variety of tissues and appear to have diverse physiological functions. p64 is associated with kidney function (17), while CLIC1 and CLIC4 appear to have a broad tissue distribution (5,8,18,19). Several CLICs interact with protein kinases (7,11,20). CLICs are associated with a variety of intracellular membranes including the nuclear membrane (5), the endoplasmic reticular membrane (8), large dense-core vesicles (19), mitochondria (21), trans-Golgi vesicles (22), and secretory vesicles (23). Parchorin forms the chloride channel in water-secreting cells,...

In nature, the complexity and structure of microbial communities varies widely, ranging from a few species to thousands of species, and from highly structured to highly unstructured communities. Here, we describe the identity and functional capacity of microbial populations within distinct layers of a pristine, marine-derived, meromictic (stratified) lake (Ace Lake) in Antarctica. Nine million open reading frames were analyzed, representing microbial samples taken from six depths of the lake size fractionated on sequential 3.0, 0.8 and 0.1 lm filters, and including metaproteome data from matching 0.1 lm filters. We determine how the interactions of members of this highly structured and moderately complex community define the biogeochemical fluxes throughout the entire lake. Our view is that the health of this delicate ecosystem is dictated by the effects of the polar light cycle on the dominant role of green sulfur bacteria in primary production and nutrient cycling, and the influence of viruses/phage and phage resistance on the cooperation between members of the microbial community right throughout the lake. To test our assertions, and develop a framework applicable to other microbially driven ecosystems, we developed a mathematical model that describes how cooperation within a microbial system is impacted by periodic fluctuations in environmental parameters on key populations of microorganisms. Our study reveals a mutualistic structure within the microbial community throughout the lake that has arisen as the result of mechanistic interactions between the physico-chemical parameters and the selection of individual members of the community. By exhaustively describing and modelling interactions in Ace Lake, we have developed an approach that may be applicable to learning how environmental perturbations affect the microbial dynamics in more complex aquatic systems.

Viruses are abundant ubiquitous members of microbial communities and in the marine environment affect population structure and nutrient cycling by infecting and lysing primary producers. Antarctic lakes are microbially dominated ecosystems supporting truncated food webs in which viruses exert a major influence on the microbial loop. Here we report the discovery of a virophage (relative of the recently described Sputnik virophage) that preys on phycodnaviruses that infect prasinophytes (phototrophic algae). By performing metaproteogenomic analysis on samples from Organic Lake, a hypersaline meromictic lake in Antarctica, complete virophage and near-complete phycodnavirus genomes were obtained. By introducing the virophage as an additional predator of a predator-prey dynamic model we determined that the virophage stimulates secondary production through the microbial loop by reducing overall mortality of the host and increasing the frequency of blooms during polar summer light periods. Virophages remained abundant in the lake 2 y later and were represented by populations with a high level of major capsid protein sequence variation (25-100% identity). Virophage signatures were also found in neighboring Ace Lake (in abundance) and in two tropical lakes (hypersaline and fresh), an estuary, and an ocean upwelling site. These findings indicate that virophages regulate host-virus interactions, influence overall carbon flux in Organic Lake, and play previously unrecognized roles in diverse aquatic ecosystems.metagenomics | metaproteomics | East Antarctica | Vestfold Hills | microbial ecology

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.

Metagenomic samples from oceans around the globe were used to examine the biogeography of the dominant marine heterotrophic bacterial clade, SAR11. Analysis uncovers evidence of adaptive radiation in response to environmental parameters, particularly temperature.