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Current investigations into phage-host interactions are dependent on extrapolating knowledge from (meta)genomes. Interestingly, 60 - 95% of all phage sequences share no homology to current annotated proteins. As a result, a large proportion of phage genes are annotated as hypothetical. This reality heavily affects the annotation of both structural and auxiliary metabolic genes. Here we present phenomic methods designed to capture the physiological response(s) of a selected host during expression of one of these unknown phage genes. Multi-phenotype Assay Plates (MAPs) are used to monitor the diversity of host substrate utilization and subsequent biomass formation, while metabolomics provides bi-product analysis by monitoring metabolite abundance and diversity. Both tools are used simultaneously to provide a phenotypic profile associated with expression of a single putative phage open reading frame (ORF). Representative results for both methods are compared, highlighting the phenotypic profile differences of a host carrying either putative structural or metabolic phage genes. In addition, the visualization techniques and high throughput computational pipelines that facilitated experimental analysis are presented.

It is 3:00 AM and a thousand-pound rosette is swinging off the back of the ship. The rosette cradles dozens of Niskin bottles that contain water samples retrieved from various depths. As the crew laboriously secures all moving parts, you get soaked with 4 °C seawater setting up filters to capture the viruses and microbes in the water samples, the efforts hindered by the ship plowing through 25+-foot swells. Conical tubes roll around the bench and the microscope has been slammed so often that it is almost impossible to check that the filters are working. Two hours later the ship has arrived at the next station and the rosette is dropped back into the deep. This is your chance to catch some hours of sleep before doing it all over again. Only 3 weeks more to go. Drifting off in the bunk, you dream of sending undergraduate “volunteers” on the next research cruise.

Many human diseases are caused by pathogens that produce exotoxins. The genes that encode these exotoxins are frequently encoded by mobile DNA elements such as plasmids or phage. Mobile DNA elements can move exotoxin genes among microbial hosts, converting avirulent bacteria into pathogens. Phage and bacteria from water, soil, and sediment environments represent a potential reservoir of phage- and plasmid-encoded exotoxin genes. The genes encoding exotoxins that are the causes of cholera, diphtheria, enterohemorrhagic diarrhea, and Staphylococcus aureus food poisoning were found in soil, sediment, and water samples by standard PCR assays from locations where the human diseases are uncommon or nonexistent. On average, at least one of the target exotoxin genes was detected in approximately 15% of the more than 300 environmental samples tested. The results of standard PCR assays were confirmed by quantitative PCR (QPCR) and Southern dot blot analyses. Agreement between the results of the standard PCR and QPCR ranged from 63% to 84%; and the agreement between standard PCR and Southern dot blots ranged from 50% to 66%. Both the cholera and shiga exotoxin genes were also found in the free phage DNA fraction. The results indicate that phage-encoded exotoxin genes are widespread and mobile in terrestrial and aquatic environments.

The maintenance and dosing of aquatic organisms, such as corals and mollusks, are essential for ecotoxicology studies, yet it is difficult to maintain many of these sensitive organisms for an extended period. Consequently, many previous aquatic ecotoxicology experiments have been limited in their number of replicates and maintained in one or a few experimental aquaria, with only a limited number of stressors tested in each experiment. Here we describe a modular system that overcomes many of the difficulties of maintaining large numbers of sensitive aquatic organisms in separate containers, and allows testing of a large suite of stressors in each experiment. The AADAMS (aquatic automated dosing and maintenance system) allows testing of 40 independent stressors with 10 independent replicates per stressor (400 individuals total). The AADAMS provides surge and regular water changes simultaneously with accurate dosing via Venturi valves. In a series of experiments over a 1-year period, the AADAMS was used to test the effects of various factors affecting water quality on Caribbean coral reefs. Roofing tar and road asphalt were two of the most damaging pollutants tested, with LD50 values (lethal dose that killed 50% of the corals) of 0.013 g L–1 and 0.079 g L–1, respectively, thus suggesting that runoff from roads and near-shore construction could be contributing to reef decline. The AADAMS is an accurate, reliable system for highly replicated ecotoxicological studies of sensitive aquatic organisms, which are important indicators of ecosystem health.

Coral reefs are suffering a long-term global decline, yet the causes remain contentious. The role of poor water quality in this decline is particularly unclear, with most previous studies providing only weak correlations between elevated nutrient levels and coral mortality. Here we experimentally show that routinely measured components of water quality (nitrate, phosphate, ammonia) do not cause substantial coral mortality. In contrast, dissolved organic carbon (DOC), which is rarely measured on reefs, does. Elevated DOC levels also accelerate the growth rate of microbes living in the corals’ surface mucopolysaccharide layer by an order of magnitude, suggesting that mortality occurs due to a disruption of the balance between the coral and its associated microbiota. We propose a model by which elevated DOC levels cause Caribbean reefs to shift further from coral to macroalgal dominance. Increasing DOC levels on coral reefs should be recognized as a threat and routinely monitored.

There are an estimated 10(31) viruses on Earth, most of which are phages that infect bacteria. Metagenomic analyses have shown that environmental viral communities are incredibly diverse. There are an estimated 5000 viral genotypes in 200 liters of seawater and possibly a million different viral genotypes in one kilogram of marine sediment. By contrast, some culturing and molecular studies have found that viruses move between different biomes. Together, these findings suggest that viral diversity could be high on a local scale but relatively limited globally. Also, by moving between environments, viruses can facilitate horizontal gene transfer.

Summary In August, more than 350 conferees from 24 countries attended the ASM Conference on the New Phage Biology, in Key Biscayne, Florida. This meeting, also called the Phage Summit, was the first major international gathering in decades devoted exclusively to phage biology. What emerged from the 5 days of the Summit was a clear perspective on the explosive resurgence of interest in all aspects of bacteriophage biology. The classic phage systems like λ and T4, reinvigorated by structural biology, bioinformatics and new molecular and cell biology tools, remain model systems of unequalled power and facility for studying fundamental biological issues. In addition, the New Phage Biology is also populated by basic and applied scientists focused on ecology, evolution, nanotechnology, bacterial pathogenesis and phage-based immunologics, therapeutics and diagnostics, resulting in a heightened interest in bacteriophages per se, rather than as a model system. Besides constituting another landmark in the long history of a field begun by d’Herelle and Twort during the early 20th century, the Summit provided a unique venue for establishment of new interactive networks for collaborative efforts between scientists of many different backgrounds, interests and expertise.

To determine how marine organisms respond to anthropogenic and natural stressors, it is important to treat these organisms with suspected ecotoxins in situ. It is also desirable to dose marine organisms with solutions for physiological studies (e.g., nutrients labeled with stable isotopes). The most appropriate tool for these types of studies is a syringe pump. However, all current syringe pumps use electrical motors and require a continuous electrical supply, which makes them unsuitable for most field deployments, particularly underwater. Here we describe a syringe pump that is powered by compressed air from standard SCUBA tanks. SPIDERS (Syringe Pumps Independently Deployable for Emission and Release of Solutions) allow the precise delivery of 20 independent treatment solutions. The rate that solutions are injected from SPIDERS can be adjusted from a couple of hours to 1 wk. Both the SCUBA tanks and treatment syringes can be changed underwater, permitting continuous and accurate dosing for long-term deployments. We also tested a variety of emitter designs that allow mixing of solutions with the surrounding water. SPIDERS are an adaptable platform for in situ treatment of benthic marine organisms that will allow ecotoxicology and other treatment studies to be moved from aquaria to the field.

Anthropogenic inputs, including organic carbon and nutrient loading, are increasingly changing the water quality on coral reefs. Herein we show that treating Montastraea annularis, Agaricia tenuifolia and Porites furcata with various organic carbon sources (starch, lactose, arabinose and mannose) results in different species-specific and carbon-specific pathologies and rates of mortality. The variation in the pathological characteristics caused by stressors showed that visual cues for determining coral health and disease may be misleading. The probability of mortality increased significantly over time with continual exposure to several of the stressors, suggesting that chronic stressors may be more harmful than acute stressors. In contrast to the organic carbon sources, high concentrations of nutrients (phosphate, ammonium and nitrate) did not directly kill corals. The variation in coral responses to anthropogenic stressors means that changes on disturbed coral reefs will depend on the type of and duration of exposure to the stressor, as well as on the species of coral.

White band disease type I (WBD I) has been a major cause of the dramatic decline of Acroporid coral populations throughout the Caribbean during the last two decades, yet the aetiological agent of this disease is unknown. In this study, the bacterial communities associated with both healthy and diseased Acropora species were compared by 16S rDNA analyses. The bacterial communities of both healthy and diseased Acropora spp. were dominated by a single ribotype with 90% identity to a bacterium in the order Rickettsiales. Screening by nested PCR specific to the coral-associated Rickettsiales 1 (CAR1) bacterium showed that this microbe was widespread in both healthy and diseased A. cervicornis and A. palmata corals from 'healthy' (i.e. low WBD I incidence) and 'stressed' reefs (i.e. high WBD I incidence). These results indicate that there were no dramatic changes in the composition of the microbial community associated with WBD I. CAR1 was also associated with non-Acroporid corals of the Caribbean, as well as with two Acroporid corals native to the Pacific. CAR1 was not present in the water column. This bacterium was also absent from preserved Caribbean Acroporid samples collected between 1937 and 1980 before the outbreak of WBD I. These results suggest CAR1 is a relatively new bacterial associate of Acroporids and that a non-bacterial pathogen might be the cause of WBD I.

Viruses are abundant in all known ecosystems. In the present study, we tested the possibility that viruses from one biome can successfully propagate in another. Viral concentrates were prepared from different near-shore marine sites, lake water, marine sediments, and soil. The concentrates were added to microcosms containing dissolved organic matter as a food source (after filtration to allow 100-kDa particles to pass through) and a 3% (vol/vol) microbial inoculum from a marine water sample (after filtration through a 0.45-μm-pore-size filter). Virus-like particle abundances were then monitored using direct counting. Viral populations from lake water, marine sediments, and soil were able to replicate when they were incubated with the marine microbes, showing that viruses can move between different ecosystems and propagate. These results imply that viruses can laterally transfer DNA between microbes in different biomes.

The coral holobiont includes the coral, zooxanthellae, fungi, endolithic algae, and >30 species of Bacteria. Using culture-independent techniques, we now show that Archaea are also abundant and widespread on corals. Sequence analyses of Archaea on 3 species of Caribbean corals revealed that coral-associated Archaea are novel, diverse, and include representatives from both the Crenarchaeota and Euryarchaeota. Unlike zooxanthellae and Bacteria, the Archaea do not appear to form species-specific associations with reef-building corals. Fluorescent in situ hybridizations with peptide nucleic acid (PNA) probes showed that Archaea were present at >107 cells cm-2 on Porites astreoides, comprising nearly half of the prokaryotic community. This study and one by Kellogg (Mar Ecol Prog Ser 273:81-88) show that Archaea are abundant, diverse, and potentially important components of the coral holobiont.

Two bacteriophages of an environmental isolate of Vibrio parahaemolyticus were isolated and sequenced. The VP16T and VP16C phages were separated from a mixed lysate based on plaque morphology and exhibit 73 to 88% sequence identity over about 80% of their genomes. Only about 25% of their predicted open reading frames are similar to genes with known functions in the GenBank database. Both phages have cos sites and open reading frames encoding proteins closely related to coliphage lambda's terminase protein (the large subunit). Like in coliphage lambda and other siphophages, a large operon in each phage appears to encode proteins involved in DNA packaging and capsid assembly and presumably in host lysis; we refer to this as the structural operon. In addition, both phages have open reading frames closely related to genes encoding DNA polymerase and helicase proteins. Both phages also encode several putative transcription regulators, an apparent polypeptide deformylase, and a protein related to a virulence-associated protein, VapE, of Dichelobacter nodosus . Despite the similarity of the proteins and genome organization, each of the phages also encodes a few proteins not encoded by the other. We did not identify genes closely related to genes encoding integrase proteins belonging to either the tyrosine or serine recombinase family, and we have no evidence so far that these phages can lysogenize the V. parahaemolyticus strain 16 host. Surprisingly for active lytic viruses, the two phages have a codon usage that is very different than that of the host, suggesting the possibility that they may be relative newcomers to growth in V. parahaemolyticus . The DNA sequences should allow us to characterize the lifestyles of VP16T and VP16C and the interactions between these phages and their host at the molecular level, as well as their relationships to other marine and nonmarine phages.

Reef-building corals associate with a diverse array of eukaryotic and noneukaryotic microbes. Best known are dinoflagellates in the genus Symbiodinium ("zooxanthellae"), which are photosynthetic symbionts found in all reef-building corals. Once considered a single species, they are now recognized as several large, genetically diverse groups that often co-occur within a single host species or colony. Variation among Symbiodinium in host identities, tolerance to stress, and ability to colonize hosts has been documented, but there is little information on the ecology of zooxanthellar free-living stages and how different zooxanthellae perform as partners. Other microbial associates of reef corals are much less well known, but studies indicate that individual coral colonies host diverse assemblages of bacteria, some of which seem to have species-specific associations. This diversity of microbial associates has important evolutionary and ecological implications. Most mutualisms evolve as balanced reciprocations that allow partners to detect cheaters, particularly when partners are potentially diverse and can be transmitted horizontally. Thus, environmental stresses that incapacitate the ability of partners to reciprocate can destabilize associations by eliciting rejection by their hosts. Coral bleaching (the loss of zooxanthellae) and coral diseases, both increasing over the last several decades, may be examples of stress-related mutualistic instability.

Phages are viruses whose hosts are bacterial cells. They identify their hosts by specific receptor molecules on the outside of the host cell. Once the phages find their specific receptors, they bind to the bacterial cell and inject their nucleic acid inside the cell. The binding between phage and host can be so specific that only certain strains of a single species can be infected. In this communication, the specificity of phage P22 for Salmonella typhimurium LT2 is exploited to allow the detection of Salmonella in the presence of other bacterial species. In particular, the dsDNA of P22 is bound to SYBR gold, a highly sensitive, fluorescent nucleic acid stain. When multiple phages infect the same cell, the fluorescence emissions of the phage DNA inside the cell allow it to be imaged using an epifluorescence microscope. The advantages of using phages as the bacterial recognition element in a sensor over antibodies are discussed.

Interleukin-2 (IL-2) is the major growth factor of activated T lymphocytes. By inducing cell cycle progression and protection from apoptosis in these cells, IL-2 is involved in the successful execution of an immune response. Upon binding its receptor, IL-2 activates a variety of signal transduction pathways, including the Ras/mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) and Janus kinase (JAK)/STAT cascades. In addition, activation of phosphatidylinositol 3-kinase (PI3K) and several of its downstream targets has also been shown. However, the coupling of STAT3 serine phosphorylation to PI3K in response to IL-2 has yet to be shown in either T cell lines or primary human T cells. This report shows that the PI3K inhibitors LY294002 and wortmannin block activation of MEK and ERK by IL-2 in primary human T cells. Moreover, these inhibitors significantly reduce IL-2-triggered STAT3 serine phosphorylation without affecting STAT5 serine phosphorylation. Analysis of the effects of these inhibitors on cell cycle progression and apoptosis strongly suggests that PI3K-mediated events, which includes STAT3 activation, are involved in IL-2-mediated cell proliferation but not cell survival. Finally, results presented illustrate that in primary human T cells, activation of Akt is insufficient for IL-2-induced anti-apoptosis. Thus, these results demonstrate that IL-2 stimulates PI3K-dependent events that correlate with cell cycle progression, but not anti-apoptosis, in activated primary human T cells.

The natural beauty of coral reefs attracts millions of tourists worldwide resulting in substantial revenues for the adjoining economies. Although their visual appearance is a pivotal factor attracting humans to coral reefs current monitoring protocols exclusively target biogeochemical parameters, neglecting changes in their aesthetic appearance. Here we introduce a standardized computational approach to assess coral reef environments based on 109 visual features designed to evaluate the aesthetic appearance of art. The main feature groups include color intensity and diversity of the image, relative size, color, and distribution of discernable objects within the image, and texture. Specific coral reef aesthetic values combining all 109 features were calibrated against an established biogeochemical assessment (NCEAS) using machine learning algorithms. These values were generated for ∼2,100 random photographic images collected from 9 coral reef locations exposed to varying levels of anthropogenic influence across 2 ocean systems. Aesthetic values proved accurate predictors of the NCEAS scores (root mean square error < 5 forN≥ 3) and significantly correlated to microbial abundance at each site. This shows that mathematical approaches designed to assess the aesthetic appearance of photographic images can be used as an inexpensive monitoring tool for coral reef ecosystems. It further suggests that human perception of aesthetics is not purely subjective but influenced by inherent reactions towards measurable visual cues. By quantifying aesthetic features of coral reef systems this method provides a cost efficient monitoring tool that targets one of the most important socioeconomic values of coral reefs directly tied to revenue for its local population.

Viruses are the most common biological entities in the oceans by an order of magnitude. However, very little is known about their diversity. Here we report a genomic analysis of two uncultured marine viral communities. Over 65% of the sequences were not significantly similar to previously reported sequences, suggesting that much of the diversity is previously uncharacterized. The most common significant hits among the known sequences were to viruses. The viral hits included sequences from all of the major families of dsDNA tailed phages, as well as some algal viruses. Several independent mathematical models based on the observed number of contigs predicted that the most abundant viral genome comprised 2–3% of the total population in both communities, which was estimated to contain between 374 and 7,114 viral types. Overall, diversity of the viral communities was extremely high. The results also showed that it would be possible to sequence the entire genome of an uncultured marine viral community.

The origin of eukaryotic cells represents a key transition in cellular evolution and is closely tied to outstanding questions about mitochondrial endosymbiosis [1, 2]. For example, gene-rich mitochondrial genomes are thought to be indicative of an ancient divergence, but this relies on unexamined assumptions about endosymbiont-to-host gene transfer [3-5]. Here, we characterize Ancoracysta twista, a new predatory flagellate that is not closely related to any known lineage in 201-protein phylogenomic trees and has a unique morphology, including a novel type of extrusome (ancoracyst). The Ancoracysta mitochondrion has a gene-rich genome with a coding capacity exceeding that of all other eukaryotes except the distantly related jakobids and Diphylleia, and it uniquely possesses heterologous, nucleus-, and mitochondrion-encoded cytochrome c maturase systems. To comprehensively examine mitochondrial genome reduction, we also assembled mitochondrial genomes from picozoans and colponemids and re-annotated existing mitochondrial genomes using hidden Markov model gene profiles. This revealed over a dozen previously overlooked mitochondrial genes at the level of eukaryotic supergroups. Analysis of trends over evolutionary time demonstrates that gene transfer to the nucleus was non-linear, that it occurred in waves of exponential decrease, and that much of it took place comparatively early, massively independently, and with lineage-specific rates. This process has led to differential gene retention, suggesting that gene-rich mitochondrial genomes are not a product of their early divergence. Parallel transfer of mitochondrial genes and their functional replacement by new nuclear factors are important in models for the origin of eukaryotes, especially as major gaps in our knowledge of eukaryotic diversity at the deepest level remain unfilled.

Coral reefs are the most biodiverse of all marine ecosystems; however, very little is known about prokaryotic diversity in these systems. To address this issue, we sequenced over 1000 bacterial 16S rDNAs from 3 massive coral species (Montastraea franksi, Diploria strigosa, and Porites astreoides) in Panama and Bermuda. Analysis of only 14 coral samples yielded 430 distinct bacterial ribotypes. Statistical analyses suggest that additional sequencing would have resulted in a total of 6000 bacterial ribotypes. Half of the sequences shared

The accessibility of high-throughput sequencing has revolutionized many fields of biology. In order to better understand host-associated viral and microbial communities, a comprehensive workflow for DNA and RNA extraction was developed. The workflow concurrently generates viral and microbial metagenomes, as well as metatranscriptomes, from a single sample for next-generation sequencing. The coupling of these approaches provides an overview of both the taxonomical characteristics and the community encoded functions. The presented methods use Cystic Fibrosis (CF) sputum, a problematic sample type, because it is exceptionally viscous and contains high amount of mucins, free neutrophil DNA, and other unknown contaminants. The protocols described here target these problems and successfully recover viral and microbial DNA with minimal human DNA contamination. To complement the metagenomics studies, a metatranscriptomics protocol was optimized to recover both microbial and host mRNA that contains relatively few ribosomal RNA (rRNA) sequences. An overview of the data characteristics is presented to serve as a reference for assessing the success of the methods. Additional CF sputum samples were also collected to (i) evaluate the consistency of the microbiome profiles across seven consecutive days within a single patient, and (ii) compare the consistency of metagenomic approach to a 16S ribosomal RNA gene-based sequencing. The results showed that daily fluctuation of microbial profiles without antibiotic perturbation was minimal and the taxonomy profiles of the common CF-associated bacteria were highly similar between the 16S rDNA libraries and metagenomes generated from the hypotonic lysis (HL)-derived DNA. However, the differences between 16S rDNA taxonomical profiles generated from total DNA and HL-derived DNA suggest that hypotonic lysis and the washing steps benefit in not only removing the human-derived DNA, but also microbial-derived extracellular DNA that may misrepresent the actual microbial profiles.

It has been stated that certain chains of biological reactions can go to near completion in both directions as needed without any exterior driving force. This claim represents a thermodynamic impossibility. Yet, this impossibility is of a type not usually covered by the traditional list of impossibility devices known as perpetual motion machines. Rather, it represents a perpetual motion machine of the third kind (PM3) that becomes impossible only in finite time. At non-vanishing rate the chain of biological reactions above would constitute a finite-time perpetual motion machine. The resolution of this quandary leads directly to a notion of staging free energy: the free energy invested in choreographing all the actors of a biochemical reaction directing them to the needed places at the needed times. We conclude by noting that biological systems make ample use of invertible degrees of freedom that often operate near PM3 limits.

Here we introduce a series of thoroughly tested and well standardized research protocols adapted for use in remote marine environments. The sampling protocols include the assessment of resources available to the microbial community (dissolved organic carbon, particulate organic matter, inorganic nutrients), and a comprehensive description of the viral and bacterial communities (via direct viral and microbial counts, enumeration of autofluorescent microbes, and construction of viral and microbial metagenomes). We use a combination of methods, which represent a dispersed field of scientific disciplines comprising already established protocols and some of the most recent techniques developed. Especially metagenomic sequencing techniques used for viral and bacterial community characterization, have been established only in recent years, and are thus still subjected to constant improvement. This has led to a variety of sampling and sample processing procedures currently in use. The set of methods presented here provides an up to date approach to collect and process environmental samples. Parameters addressed with these protocols yield the minimum on information essential to characterize and understand the underlying mechanisms of viral and microbial community dynamics. It gives easy to follow guidelines to conduct comprehensive surveys and discusses critical steps and potential caveats pertinent to each technique.

Holobionts are species-specific associations between macro- and microorganisms. On coral reefs, the benthic coverage of coral and algal holobionts varies due to natural and anthropogenic forcings. Different benthic macroorganisms are predicted to have specific microbiomes. In contrast, local environmental factors are predicted to select for specific metabolic pathways in microbes. To reconcile these two predictions, we hypothesized that adaptation of microbiomes to local conditions is facilitated by the horizontal transfer of genes responsible for specific metabolic capabilities. To test this hypothesis, microbial metagenomes were sequenced from 22 coral reefs at 11 Line Islands in the central Pacific that together span a wide range of biogeochemical and anthropogenic influences. Consistent with our hypothesis, the percent cover of major benthic functional groups significantly correlated with particular microbial taxa. Reefs with higher coral cover had a coral microbiome with higher abundances of Alphaproteobacteria (such as Rhodobacterales and Sphingomonadales), whereas microbiomes of algae-dominated reefs had higher abundances of Gammaproteobacteria (such as Alteromonadales, Pseudomonadales, and Vibrionales), Betaproteobacteria, and Bacteriodetes. In contrast to taxa, geography was the strongest predictor of microbial community metabolism. Microbial communities on reefs with higher nutrient availability (e.g., equatorial upwelling zones) were enriched in genes involved in nutrient-related metabolisms (e.g., nitrate and nitrite ammonification, Ton/Tol transport, etc.). On reefs further from the equator, microbes had more genes encoding chlorophyll biosynthesis and photosystems I/II. These results support the hypothesis that core microbiomes are determined by holobiont macroorganisms, and that those core taxa adapt to local conditions by selecting for advantageous metabolic genes.

The Precambrian explosion led to the rapid appearance of most major animal phyla alive today. It has been argued that the complexity of life has steadily increased since that event. Here we challenge this hypothesis through the characterization of apoptosis in reef-building corals, representatives of some of the earliest animals. Bioinformatic analysis reveals that all of the major components of the death receptor pathway are present in coral with high-predicted structural conservation with Homo sapiens. The TNF receptor-ligand superfamilies (TNFRSF/TNFSF) are central mediators of the death receptor pathway, and the predicted proteome of Acropora digitifera contains more putative coral TNFRSF members than any organism described thus far, including humans. This high abundance of TNFRSF members, as well as the predicted structural conservation of other death receptor signaling proteins, led us to wonder what would happen if corals were exposed to a member of the human TNFSF (HuTNFα). HuTNFα was found to bind directly to coral cells, increase caspase activity, cause apoptotic blebbing and cell death, and finally induce coral bleaching. Next, immortalized human T cells (Jurkats) expressing a functional death receptor pathway (WT) and a corresponding Fas-associated death domain protein (FADD) KO cell line were exposed to a coral TNFSF member (AdTNF1) identified and purified here. AdTNF1 treatment resulted in significantly higher cell death (P < 0.0001) in WT Jurkats compared with the corresponding FADD KO, demonstrating that coral AdTNF1 activates the H. sapiens death receptor pathway. Taken together, these data show remarkable conservation of the TNF-induced apoptotic response representing 550 My of functional conservation.

While in nucleotide sequencing, the analysis of DNA from complex mixtures of organisms is common, this is not yet true for mass spectrometric data analysis of complex mixtures. The comparative analyses of mass spectrometry data of microbial communities at the molecular level is difficult to perform, especially in the context of a host. The challenge does not lie in generating the mass spectrometry data, rather much of the difficulty falls in the realm of how to derive relevant information from this data. The informatics based techniques to visualize and organize datasets are well established for metagenome sequencing; however, due to the scarcity of informatics strategies in mass spectrometry, it is currently difficult to cross correlate two very different mass spectrometry data sets from microbial communities and their hosts. We highlight that molecular networking can be used as an organizational tool of tandem mass spectrometry data, automated database search for rapid identification of metabolites, and as a workflow to manage and compare mass spectrometry data from complex mixtures of organisms. To demonstrate this platform, we show data analysis from hard corals and a human lung associated with cystic fibrosis.

Bacteriophages (phages) are the viruses of bacteria. The impact of phages on bacterial pathogenesis may be divided into two major themes, transduction and predation. In this chapter, the authors take a phage-centered view of the ecology of the phage-bacterium relationship, looking in particular for unappreciated subtleties that might impact pathogen formation, disease progression, or the phage-induced destruction of bacterial populations. The impact of phages on bacterial pathogenesis occurs primarily over the course of phage infection. Transduction is described as the phage-mediated movement of genetic material from one bacterium to another. Generalized transduction consequently does not result in DNA serial transfer except via subsequent accidental means. Researchers need to be aware not just of the traditional laboratory characterization of phage biology but also of how to interpret that biology from an ecological perspective. Phages are also often compared by restriction fragment length polymorphism analysis and Southern blotting. The major advantage of working with cultured phages is the potential for further investigations into phage biology. Metagenomic analyses are another way to identify uncultured phages within environmental communities. Population modeling also suggests that uncultured phage genomes from the environment may be sequenced to completion by the shotgun approach. Obtaining and propagating more virulence factor (VF)-encoding phages in culture, including currently unidentified toxin-carrying phages sequenced from the environment, will provide valuable information concerning host range and other aspects of phage biology that are relevant to bacterial pathogenesis.

In this study we characterized the prokaryotic microbiota associated with the reef-building coral Montastraea franksi in a culture-independent manner by sequencing 16S rDNAs. The majority of the bacteria identified by this method were novel species and belonged to a wide variety of microbial groups, with cyanobacteria and α-proteobacteria being the most abundant. In contrast, the bacteria cultured from the same M. franksi samples were closely related to previously described bacteria and consisted mostly of γ-proteobacteria. These results show that the microbial diversity associated with corals can be greatly underestimated when relying exclusively on culture-based methods. Our analyses also showed that one α-proteobacteria species was present in all M. franksi samples isolated from five reefs separated by up to 10 km. This finding is suggestive of a specific microbe–coral association.

In the following report, thermal cycling coupled with random 10-mers as primers was used to construct randomly amplified shotgun libraries (RASLs). This approach allowed shotgun libraries to be constructed from nanogram quantities of input DNA. RASLs contained inserts from throughout a target genome in an unbiased fashion and did not appear to contain chimeric sequences. This protocol should be useful for shotgun sequencing the genomes of unculturable organisms and rapidly producing shotgun libraries from cosmids, fosmids, yeast artificial chromosomes (YACs), and bacterial artificial chromosomes (BACs).

The IL-2 growth hormone is the major growth factor of activated T lymphocytes during a developing immune response. IL-2 is required not only for cell cycle progression but also to protect Ag-activated T cells from programmed cell death. In several cell types, activation of NF-κB and/or activating protein-1 (AP-1) has been demonstrated to be extremely important in blocking apoptosis. To determine whether either or both of these transcription factors are involved in cell survival or cell cycle progression in response to IL-2, primary human T cells responsive to the growth factor were analyzed for NF-κB and AP-1 activation. The current study clearly demonstrates that IL-2 does not induce IκBα degradation or NF-κB activation in primary human T cells that respond to IL-2 by entering the cell cycle and avoiding apoptosis. Similarly, IL-2 neither activates JNK nor increases AP-1 binding activity to a consensus o-tetradecanoylphorbol 13-acetate (TPA) response element. On the other hand, the growth factor does induce the activation of STAT3 and STAT5 in these cells, as has been previously demonstrated. These data show that neither NF-κB nor AP-1 activation is required for IL-2-mediated survival or cell cycle progression in activated primary human T cells.

One hundred years after the first description of viruses that infect bacterial cells, the contribution of these bacteriophages to fundamental biology, biotechnology and human health continues unabated and deserves celebration.

As DNA sequencing becomes faster and cheaper, genomics-based approaches are being explored for their use in personalized diagnoses and treatments. Here, we provide a proof of principle for disease monitoring using personal metagenomic sequencing and traditional clinical microbiology by focusing on three adults with cystic fibrosis (CF). The CF lung is a dynamic environment that hosts a complex ecosystem composed of bacteria, viruses, and fungi that can vary in space and time. Not surprisingly, the microbiome data from the induced sputum samples we collected revealed a significant amount of species diversity not seen in routine clinical laboratory cultures. The relative abundances of several species changed as clinical treatment was altered, enabling the identification of the climax and attack communities that were proposed in an earlier work. All patient microbiomes encoded a diversity of mechanisms to resist antibiotics, consistent with the characteristics of multidrug-resistant microbial communities that are commonly observed in CF patients. The metabolic potentials of these communities differed by the health status and recovery route of each patient. Thus, this pilot study provides an example of how metagenomic data might be used with clinical assessments for the development of treatments tailored to individual patients.

Recent evidence showing host specificity of colonizing bacteria supports the view that multicellular organisms are holobionts comprised of the macroscopic host in synergistic interdependence with a heterogeneous and host-specific microbial community. Whereas host-bacteria interactions have been extensively investigated, comparatively little is known about host-virus interactions and viral contribution to the holobiont. We sought to determine the viral communities associating with different Hydra species, whether these viral communities were altered with environmental stress, and whether these viruses affect the Hydra-associated holobiont. Here we show that each species of Hydra harbors a diverse host-associated virome. Primary viral families associated with Hydra are Myoviridae, Siphoviridae, Inoviridae, and Herpesviridae. Most Hydra-associated viruses are bacteriophages, a reflection of their involvement in the holobiont. Changes in environmental conditions alter the associated virome, increase viral diversity, and affect the metabolism of the holobiont. The specificity and dynamics of the virome point to potential viral involvement in regulating microbial associations in the Hydra holobiont. While viruses are generally regarded as pathogenic agents, our study suggests an evolutionary conserved ability of viruses to function as holobiont regulators and, therefore, constitutes an emerging paradigm shift in host-microbe interactions.

Algae-derived dissolved organic matter has been hypothesized to induce mortality of reef building corals. One proposed killing mechanism is a zone of hypoxia created by rapidly growing microbes. To investigate this hypothesis, biological oxygen demand (BOD) optodes were used to quantify the change in oxygen concentrations of microbial communities following exposure to exudates generated by turf algae and crustose coralline algae (CCA). BOD optodes were embedded with microbial communities cultured from Montastraea annularis and Mussismilia hispida, and respiration was measured during exposure to turf and CCA exudates. The oxygen concentrations along the optodes were visualized with a low-cost Submersible Oxygen Optode Recorder (SOOpR) system. With this system we observed that exposure to exudates derived from turf algae stimulated higher oxygen drawdown by the coral-associated bacteria than CCA exudates or seawater controls. Furthermore, in both turf and CCA exudate treatments, all microbial communities (coral-, algae-associated and pelagic) contributed significantly to the observed oxygen drawdown. This suggests that the driving factor for elevated oxygen consumption rates is the source of exudates rather than the initially introduced microbial community. Our results demonstrate that exudates from turf algae may contribute to hypoxia-induced coral stress in two different coral genera as a result of increased biological oxygen demand of the local microbial community. Additionally, the SOOpR system developed here can be applied to measure the BOD of any culturable microbe or microbial community.

For the last two decades, CASP has assessed the state of the art in techniques for protein structure prediction and identified areas which required further development. CASP would not have been possible without the prediction targets provided by the experimental structural biology community. In the latest experiment, CASP10, more than 100 structures were suggested as prediction targets, some of which appeared to be extraordinarily difficult for modeling. In this article, authors of some of the most challenging targets discuss which specific scientific question motivated the experimental structure determination of the target protein, which structural features were especially interesting from a structural or functional perspective, and to what extent these features were correctly reproduced in the predictions submitted to CASP10. Specifically, the following targets will be presented: the acid-gated urea channel, a difficult to predict transmembrane protein from the important human pathogen Helicobacter pylori; the structure of human interleukin (IL)-34, a recently discovered helical cytokine; the structure of a functionally uncharacterized enzyme OrfY from Thermoproteus tenax formed by a gene duplication and a novel fold; an ORFan domain of mimivirus sulfhydryl oxidase R596; the fiber protein gene product 17 from bacteriophage T7; the bacteriophage CBA-120 tailspike protein; a virus coat protein from metagenomic samples of the marine environment; and finally, an unprecedented class of structure prediction targets based on engineered disulfide-rich small proteins.

The impaired mucociliary clearance in individuals with Cystic Fibrosis (CF) enables opportunistic pathogens to colonize CF lungs. Here we show that Rothia mucilaginosa is a common CF opportunist that was present in 83% of our patient cohort, almost as prevalent as Pseudomonas aeruginosa (89%). Sequencing of lung microbial metagenomes identified unique R. mucilaginosa strains in each patient, presumably due to evolution within the lung. The de novo assembly of a near-complete R. mucilaginosa (CF1E) genome illuminated a number of potential physiological adaptations to the CF lung, including antibiotic resistance, utilization of extracellular lactate, and modification of the type I restriction-modification system. Metabolic characteristics predicted from the metagenomes suggested R. mucilaginosa have adapted to live within the microaerophilic surface of the mucus layer in CF lungs. The results also highlight the remarkable evolutionary and ecological similarities of many CF pathogens; further examination of these similarities has the potential to guide patient care and treatment.

Planar optodes were used to visualize oxygen distribution patterns associated with a coral reef associated green algae (Chaetomorpha sp.) and a hermatypic coral (Favia sp.) separately, as standalone organisms, and placed in close proximity mimicking coral-algal interactions. Oxygen patterns were assessed in light and dark conditions and under varying flow regimes. The images show discrete high oxygen concentration regions above the organisms during lighted periods and low oxygen in the dark. Size and orientation of these areas were dependent on flow regime. For corals and algae in close proximity the 2D optodes show areas of extremely low oxygen concentration at the interaction interfaces under both dark (18.4 ± 7.7 µmol O2 L(- 1)) and daylight (97.9 ± 27.5 µmol O2 L(- 1)) conditions. These images present the first two-dimensional visualization of oxygen gradients generated by benthic reef algae and corals under varying flow conditions and provide a 2D depiction of previously observed hypoxic zones at coral algae interfaces. This approach allows for visualization of locally confined, distinctive alterations of oxygen concentrations facilitated by benthic organisms and provides compelling evidence for hypoxic conditions at coral-algae interaction zones.

Holobionts are assemblages of microbial symbionts and their macrobial host. As extant representatives of some of the oldest macro-organisms, corals and algae are important for understanding how holobionts develop and interact with one another. Using untargeted metabolomics, we show that non-self interactions altered the coral metabolome more than self-interactions (i.e. different or same genus, respectively). Platelet activating factor (PAF) and Lyso-PAF, central inflammatory modulators in mammals, were major lipid components of the coral holobionts. When corals were damaged during competitive interactions with algae, PAF increased along with expression of the gene encoding Lyso-PAF acetyltransferase; the protein responsible for converting Lyso-PAF to PAF. This shows that self and non-self recognition among some of the oldest extant holobionts involve bioactive lipids identical to those in highly derived taxa like humans. This further strengthens the hypothesis that major players of the immune response evolved during the pre-Cambrian.

Nature 531, 466–470 (2016); doi:10.1038/nature17193 In this Article, the ‘Predator–prey modelling’ section of the Methods shows Lotka–Volterra equations. Although these equations are meant to present a basic Lotka–Volterra model, the term ‘N/K’ in the second equation was inadvertently introduced during proofing, which makes the equations reflect the Piggyback-the-Winner model rather than basic Lotka–Volterra.

Phages can exploit their bacterial hosts by lytic infection, when many viral particles are released at cell lysis, or by lysogeny, when phages integrate into the host’s genome. We recently proposed a new dynamic model of bacteria–phage interactions in which lysogeny predominates at high microbial abundance and growth rates. This model, named Piggyback-the-Winner (PtW), contrasts to current accepted models on the frequency of lysis and lysogeny and predicts that phages integrate into their hosts’ genomes as prophages when microbial abundances and growth rates are high. According to PtW, switching to the temperate life cycle reduces phage predation control on bacterial abundance and confers superinfection exclusion, preventing that a closely-related phage infects the same bacterial cell. Here we examine how PtW is important for metazoans. Specifically, we postulate that PtW and the recently described bacteriophage adherence to mucus (BAM) model are strongly interrelated and have an important role in the development of the microbiome. In BAM, phage produced by the microbiome attach to mucins and protect underlying epithelial cells from invading bacteria. Spatial structuring of the mucus creates a gradient of phage replication strategies consistent with PtW. We predict that lysogeny is favored at the top mucosal layer and lytic predation predominates in the bacteria-sparse intermediary layers. The lysogeny confers competitive advantage to commensals against niche invasion and the lytic infection eliminates potential pathogens from deeper mucus layers.

A lineage of plastid-bearing eukaryotic microbes that is closely related to apicomplexan parasites was recently found in a specific association with coral reefs (apicomplexan-related lineage-V, or ARL-V). Here, we address the possible nature of this association using plastid ‘contamination’ in fine-scale bacterial sequence surveys. In a transect between corals and associated macroalgae, ARL-V is specifically associated with the coral, in contrast to all microalgal types (including diatoms, haptophytes, pelagophytes and photosynthetic apicomplexan relatives, Chromera and Vitrella), which are associated with macroalgae. ARL-V is associated with at least 20 species of symbiotic corals through extended time periods and large geographic distances. It is significantly enriched in healthy coral tissue and shallow reef depths. Altogether, the evidence points to a specific relationship between ARL-V and corals, and is suggestive of symbiosis, perhaps based on photosynthesis.

Cystic fibrosis (CF) is the most common autosomal recessive disease in Caucasians with a reported incidence of 1 in every 3200 live births. Most strikingly, CF is associated with early mortality. Host in flammatory responses result in airway mucus plugging, airway wall edema, and eventual destruction of airway wall support structure. Despite aggressive treatment, the median age of survival is approximately 38 years. This work is the first attempt to parameterize the distributions of mucus in a CF lung as a function of time. By default, the model makes arbitrary choices at each stage of the construction process, whereby the simplest choice is made. The model is sophisticated enough to fit the average CF patients' spirometric data over time and to identify several interesting parameters: probability of colonization, mucus volume growth rate, and scarring rate. Extensions of the model appropriate for describing the dynamics of single patient MRI data are also discussed.

Every four years, the Olympic Games plays host to competitors who have built on their natural talent by training for many years to become the best in their chosen discipline. Similar spirit and endeavour can be found throughout the microbial world, in which every day is a competition to survive and thrive. Microorganisms are trained through evolution to become the fittest and the best adapted to a particular environmental niche or lifestyle, and to innovate when the 'rules of the game' are changed by alterations to their natural habitats. In this Essay, we honour the best competitors in the microbial world by inviting them to take part in the inaugural Microbial Olympics.

The Marine Microbial Food Web (MMFW) includes heterotrophic microbes and their protist and viral predators. These microbes consume dissolved organic matter thereby making the MMFW a major component of global biogeochemical and energy cycles. However, quantification of the MMFW contribution to these cycles is dependent on a handful of techniques, all of which require laboratory-derived conversion factors. Here we describe a differential calorimeter capable of measuring the small amounts of heat produced by marine microbes and viruses at natural populations. Using this ultra-sensitive calorimeter, we show that heat production in the presence of viruses is significantly larger than in their absence. This increased heat output occurs despite a net decrease in the number of microbes. This provides direct evidence for top-down control of microbial populations by viruses and shows that there is increased re-mineralization. A comparative statics model was developed to interpret the calorimeter measurements. The spirit of the model is thermodynamic ‐ it restricts its view to net changes in the populations and net heat produced. The model predicts that approximately 25% of the total heat production during the growth phase of a pelagic microbial community is due directly to viral activities. This result has implications for the energy budget of our planet and for climate prediction.

The Line Islands are calcium carbonate coral reef platforms located in iron-poor regions of the central Pacific. Natural terrestrial run-off of iron is non-existent and aerial deposition is extremely low. However, a number of ship groundings have occurred on these atolls. The reefs surrounding the shipwreck debris are characterized by high benthic cover of turf algae, macroalgae, cyanobacterialmats and corallimorphs, as well as particulate-laden, cloudy water. These sites also have very low coral and crustose coralline algal cover and are call black reefs because of the dark-colored benthic community and reduced clarity of the overlying water column. Here we use a combination of benthic surveys, chemistry, metagenomics and microcosms to investigate if and how shipwrecks initiate andmaintain black reefs. Comparative surveys show that the live coral cover was reduced from 40 to 60% too10% on black reefs on Millennium, Tabuaeran and Kingman. These three sites are relatively large (40.75 km2). The phase shift occurs rapidly; the Kingman black reef formed within 3 years of the ship grounding. Iron concentrations in algae tissue from the Millennium black reef site were six timeshigher than in algae collected from reference sites. Metagenomic sequencing of the Millennium Atoll black reef-associated microbial community was enriched in iron-associated virulence genes and known pathogens. Microcosm experiments showed that corals were killed by black reef rubble through microbial activity. Together these results demonstrate that shipwrecks and their associated iron pose significant threats to coral reefs in iron-limited regions.

Viruses are regarded as peripheral oddities in most ecological and evolutionary theory, as well as in the supporting field and laboratory work. This is a major mistake. After all, there are more of them, they reproduce more quickly, they evolve more rapidly, and they are part of every biome. Viruses, the most diverse biological entities on the planet, are also the least characterized in terms of their genetic, taxonomic, and functional diversity. They are the dark matter of the biological universe. In this chapter, we begin by counting viruses, then we estimate their diversity. With their vast numbers, great diversity, and rapid rates of mutation and recombination, viruses are exploring sequence space at a phenomenal rate. They exchange genes among themselves and with their hosts; they move genes globally from biome to biome. Everything viral is in rapid evolutionary and ecological movement, and this movement reverberates throughout the biosphere.

Viruses are the most abundant known infectious agents on the planet and are significant drivers of diversity in a variety of ecosystems. Although there have been numerous studies of viral communities, few have focused on viruses within the indigenous human microbiota. We analyzed 2 267 695 virome reads from viral particles and compared them with 263 516 bacterial 16S rRNA gene sequences from the saliva of five healthy human subjects over a 2- to 3-month period, in order to improve our understanding of the role viruses have in the complex oral ecosystem. Our data reveal viral communities in human saliva dominated by bacteriophages whose constituents are temporally distinct. The preponderance of shared homologs between the salivary viral communities in two unrelated subjects in the same household suggests that environmental factors are determinants of community membership. When comparing salivary viromes to those from human stool and the respiratory tract, each group was distinct, further indicating that habitat is of substantial importance in shaping human viromes. Compared with coexisting bacteria, there was concordance among certain predicted host–virus pairings such as Veillonella and Streptococcus, whereas there was discordance among others such as Actinomyces. We identified 122 728 virulence factor homologs, suggesting that salivary viruses may serve as reservoirs for pathogenic gene function in the oral environment. That the vast majority of human oral viruses are bacteriophages whose putative gene function signifies some have a prominent role in lysogeny, suggests these viruses may have an important role in helping shape the microbial diversity in the human oral cavity.

Competition between reef-building corals and benthic algae is of key importance for reef dynamics. These interactions occur on many spatial scales, ranging from chemical to regional. Using microprobes, 16S rDNA pyrosequencing and underwater surveys, we examined the interactions between the reef-building coral Montastraea annularis and four types of benthic algae. The macroalgae Dictyota bartayresiana and Halimeda opuntia , as well as a mixed consortium of turf algae, caused hypoxia on the adjacent coral tissue. Turf algae were also associated with major shifts in the bacterial communities at the interaction zones, including more pathogens and virulence genes. In contrast to turf algae, interactions with crustose coralline algae (CCA) and M. annularis did not appear to be antagonistic at any scale. These zones were not hypoxic, the microbes were not pathogen-like and the abundance of coral–CCA interactions was positively correlated with per cent coral cover. We propose a model in which fleshy algae (i.e. some species of turf and fleshy macroalgae) alter benthic competition dynamics by stimulating bacterial respiration and promoting invasion of virulent bacteria on corals. This gives fleshy algae a competitive advantage over corals when human activities, such as overfishing and eutrophication, remove controls on algal abundance. Together, these results demonstrate the intricate connections and mechanisms that structure coral reefs.