Rohwer Wegley-Kelly Lab

Forest Rohwer, PhD

Education

Forest Rohwer, PhD

  • University of California San Diego/San Diego State University
    • Joint Doctoral Program in Molecular Biology, 09/92-07/97 advisor: Kathleen McGuire 
    • Doctoral dissertation: Interleukin-2 activation of intracellular signaling pathways: Effects on gene expression, proliferation, and apoptosis, pp. 1-174. 1997.
  • University of Idaho, Letters and Science, 09/91-05/92
  • College of Idaho, Gipson Scholar in Biology, Chemistry, and History, 09/87-05/91

Awards and Honors

  • List in The World's Most Influential Scientific Minds by Thomson Reuters. 2014.
  • Outstanding Faculty. College of Science. SDSU. 2014.
  • Teacher Scholar. SDSU Biology. 2014.
  • SDSU Phi Beta Kappa. 2013.
  • American Academy of Microbiology Fellow. 2013.
  • SDSU Alumni Association Award for Outstanding Faculty Contributions - "Monty". 2011.
  • Achievement Award. Sigma Xi. 2009.
  • International Society of Microbial Ecology Young Investigators Award. 2008.
  • Canadian Institute for Advanced Research (CIFAR) Fellow in Integrated Microbial Biodiversity Program. 2007.
  • American Academy for Advancement of Science (AAAS) Fellow. 2006.
  • SDSU Foundation Technology Transfer Office Honorable Mention of Inventorship. 2002.
  • ARCS. 1995 and 1996. Achievement award for young scientists. SDSU.       
  • Sigma Xi. 1993. Grant-in-aid for graduate students. SDSU.
  • NSF Research Experience for Undergraduates (REU). 1991. University of Nevada, Reno.
  • Graduated summa cum laude. 1991. College of Idaho.
  • Gipson Scholar. 1988-1991. For academic scholarship. College of Idaho.

Professional Experience

  • San Diego State University. Professor. 2009-present
  • San Diego State University. Associate Professor. 2007-2008
  • San Diego State University. Assistant Professor. 2003-2007
  • San Diego State University. Adjunct Assistant Professor. 2001-2003
  • Scripps Institution of Oceanography. Postdoctoral researcher with Dr. Farooq Azam. 1997-2001

Curriculum vitae

Download Dr. Rohwer's complete CV (download PDF).

Books

  • Life in Our Phage World. Forest Rohwer, Merry Youle, Heather Maughan, Nao Hisakawa. Illustrations by Leah Pantéa and Ben Darby. 2015. Wholon.

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    Life in Our Phage World! Cover ArtWe share the Earth with more than 10,000,000,000,000,000,000,000,000,000,000 phages. Everywhere they thrive, from well-fed guts to near-boiling acidic springs, from cryoconite holes to endolithic fissures. They travel from one microbial host to the next as virions, their genetic weapons packaged inside a protective protein shell. If you could lay all of these nanoscopic phage virions side-by-side, the line-up would stretch over 42 million light years. Through their daily shenanigans they kill or collaborate with their microbial hosts to spur microbial evolution and maintain ecosystem functioning. We have learned much about them since their discovery by Frederick Twort a century ago. They also taught us that DNA, not protein, is the hereditary material, unraveled the triplet genetic code, and offered their enzymes as indispensible tools for the molecular biology revolution. More contributions will be forthcoming since the vast majority of phages await discovery. Phage genomes harbor the world's largest cache of unexplored genetic diversity, and we now have the equipment needed to go prospecting.

    Although there are field guides to birds, insects, wild flowers, even Bacteria, there was no such handbook to guide the phage explorer. Forest Rohwer decided to correct this oversight, for novice and expert alike, and thus was born Life in Our Phage World. A diverse collection of 30 phages are featured. Each phage is characterized by its distinctive traits, including details about its genome, habitat, lifestyle, global range, and close relatives. The beauty of its intricate virion is captured in a pen-and-ink portrait by artist Benjamin Darby. Each phage also stars in a carefully researched action story relating how that phage encounters, exploits, kills, or otherwise manipulates its host. These behaviors are imaginatively illustrated by fine artist Leah L. Pantéa. Eight researchers that work closely with phages also relate their experiences as inhabitants of the phage world.

    Rohwer has years of first-hand experience with the phage multitudes in ecosystems ranging from coral reefs to the human lung to arctic waters. He pioneered the key metagenomic methods now widely used to catalog and characterize Earth's microbial and viral life. Despite research advances, most people, many scientists included, remain unaware of the ongoing drama in our phage world. In anticipation of 2015, the centennial of phage discovery, Forest assembled a cadre of writers, artists, scientists, and a cartographer and set them to work. The result? This alluring field guide-a feast for the imagination and a celebration of phage diversity.

  • Coral Reefs in the Microbial Seas. Forest Rohwer with Merry Youle. Illustrations by Derek Vosten. 2010. Plaid Productions.

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    Coral Reefs in the Microbial Seas Cover ArtFor millennia, coral reefs have flourished as one of the planet's most magnificent natural wonders. As Earth's most biodiverse ecosystem-surpassing even the rainforests-they are home to a cooperative network ranging from immense fish to sunlight-capturing algae to invisible microbes. Just how critical the microbes in particular are for coral reef health is finally understood thanks to recent discoveries. Coral Reefs in the Microbial Seas is the first book to unveil the complete story of how these relationships uphold coral reef health and what impact human activity has on this delicate balance.

Publications

  • Rohwer F and AM Segall (2015) A century of phage lessons. Nature 528(7580):46-48 26632584 10.1038/528046a View Abstract · DOI Record
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    No abstract available.

  • Andreas Haas, Marine Guibert, Anja Foerschner, Tim Co, Sandi Calhoun, Emma George, Mark Hatay, Elizabeth Dinsdale, Stuart A. Sandin, Jennifer E. Smith, Mark J.A. Vermeij, Ben Felts, Philip Dustan, Peter Salamon, Forest Rohwer (2015) Can we measure beauty? Computational evaluation of coral reef aesthetics PeerJ 3(e1390): https://dx.doi.org/10.7717/peerj.1390 View Abstract · DOI Record
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    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 for N ≥ 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.

  • Lim YW, Haynes M, Furlan M, Robertson CE, Harris JK, Rohwer F (2014) Purifying the impure: sequencing metagenomes and metatranscriptomes from complex animal-associated samples. J Vis Exp (94): View Abstract · Pubmed Record
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    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.

  • Hevroni G, Enav H, Rohwer F, Béjà O (2014) Diversity of viral photosystem-I psaA genes. ISME J : View Abstract · Pubmed Record
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    Marine photosynthesis is one of the major contributors to the global carbon cycle and the world&#039;s oxygen supply. This process is largely driven by cyanobacteria, namely Synechococcus and Prochlorococcus. Genes encoding photosystem-II (PSII) reaction center proteins are found in many cyanophage genomes, and are expressed during the infection of their hosts. On the basis of metagenomics, cyanophage photosystem-I (PSI) gene cassettes were recently discovered with two gene arrangements psaJF&rarr;C&rarr;A&rarr;B&rarr;K&rarr;E&rarr;D and psaD&rarr;C&rarr;A&rarr;B. It was suggested that the horizontal transfer of PSII and PSI genes is increasing phage fitness. To better understand their diversity, we designed degenerate primers to cover a wide diversity of organisms, and using PCR we targeted the psaC&rarr;A arrangement, which is unique to cyanophages cassettes. We examined viral concentrates from four islands in the Pacific Ocean and found samples containing the psaC&rarr;A arrangement. Analyses of the amplified viral psaA gene revealed six subgroups varying in their level of similarity and %G+C content, suggesting that the diversity of cyanophage PSI genes is greater than originally thought.The ISME Journal advance online publication, 23 December 2014; doi:10.1038/ismej.2014.244.

  • Quinn RA, Whiteson K, Lim YW, Salamon P, Bailey B, Mienardi S, Sanchez SE, Blake D, Conrad D, Rohwer F (2014) A Winogradsky-based culture system shows an association between microbial fermentation and cystic fibrosis exacerbation. ISME J : View Abstract · Pubmed Record
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    There is a poor understanding of how the physiology of polymicrobial communities in cystic fibrosis (CF) lungs contributes to pulmonary exacerbations and lung function decline. In this study, a microbial culture system based on the principles of the Winogradsky column (WinCF system) was developed to study the physiology of CF microbes. The system used glass capillary tubes filled with artificial sputum medium to mimic a clogged airway bronchiole. Chemical indicators were added to observe microbial physiology within the tubes. Characterization of sputum samples from seven patients showed variation in pH, respiration, biofilm formation and gas production, indicating that the physiology of CF microbial communities varied among patients. Incubation of homogenized tissues from an explant CF lung mirrored responses of a Pseudomonas aeruginosa pure culture, supporting evidence that end-stage lungs are dominated by this pathogen. Longitudinal sputum samples taken through two exacerbation events in a single patient showed that a two-unit drop in pH and a 30% increase in gas production occurred in the tubes prior to exacerbation, which was reversed with antibiotic treatment. Microbial community profiles obtained through amplification and sequencing of the 16S rRNA gene showed that fermentative anaerobes became more abundant during exacerbation and were then reduced during treatment where P. aeruginosa became the dominant bacterium. Results from the WinCF experiments support the model where two functionally different CF microbial communities exist, the persistent Climax Community and the acute Attack Community. Fermentative anaerobes are hypothesized to be the core members of the Attack Community and production of acidic and gaseous products from fermentation may drive developing exacerbations. Treatment targeting the Attack Community may better resolve exacerbations and resulting lung damage.The ISME Journal advance online publication, 16 December 2014; doi:10.1038/ismej.2014.234.

  • Haas AF, Knowles B, Lim YW, McDole Somera T, Kelly LW, Hatay M, Rohwer F (2014) Unraveling the unseen players in the ocean - a field guide to water chemistry and marine microbiology. J Vis Exp (93):e52131 View Abstract · Pubmed Record
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    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.

  • Zarei S, Mirtar A, Rohwer F, Salamon P (2014) Stochastic tracking of infection in a CF lung. PLoS ONE 9(10):e111245 (PMC4216002) View Abstract · Pubmed Record
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    Magnetic Resonance Imaging (MRI) and Computed Tomography (CT) scan are the two ubiquitous imaging sources that physicians use to diagnose patients with Cystic Fibrosis (CF) or any other Chronic Obstructive Pulmonary Disease (COPD). Unfortunately the cost constraints limit the frequent usage of these medical imaging procedures. In addition, even though both CT scan and MRI provide mesoscopic details of a lung, in order to obtain microscopic information a very high resolution is required. Neither MRI nor CT scans provide micro level information about the location of infection in a binary tree structure the binary tree structure of the human lung. In this paper we present an algorithm that enhances the current imaging results by providing estimated micro level information concerning the location of the infection. The estimate is based on a calculation of the distribution of possible mucus blockages consistent with available information using an offline Metropolis-Hastings algorithm in combination with a real-time interpolation scheme. When supplemented with growth rates for the pockets of mucus, the algorithm can also be used to estimate how lung functionality as manifested in spirometric tests will change in patients with CF or COPD.

  • Grasis JA, Lachnit T, Anton-Erxleben F, Lim YW, Schmieder R, Fraune S, Franzenburg S, Insua S, Machado G, Haynes M, Little M, Kimble R, Rosenstiel P, Rohwer FL, Bosch TC (2014) Species-specific viromes in the ancestral holobiont Hydra. PLoS ONE 9(10):e109952 (PMC4208763) View Abstract · Pubmed Record
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    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.

  • Lim YW, Cuevas DA, Silva GG, Aguinaldo K, Dinsdale EA, Haas AF, Hatay M, Sanchez SE, Wegley-Kelly L, Dutilh BE, Harkins TT, Lee CC, Tom W, Sandin SA, Smith JE, Zgliczynski B, Vermeij MJ, Rohwer F, Edwards RA (2014) Sequencing at sea: challenges and experiences in Ion Torrent PGM sequencing during the 2013 Southern Line Islands Research Expedition. PeerJ 2:e520 (PMC4145072) View Abstract · Pubmed Record
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    Genomics and metagenomics have revolutionized our understanding of marine microbial ecology and the importance of microbes in global geochemical cycles. However, the process of DNA sequencing has always been an abstract extension of the research expedition, completed once the samples were returned to the laboratory. During the 2013 Southern Line Islands Research Expedition, we started the first effort to bring next generation sequencing to some of the most remote locations on our planet. We successfully sequenced twenty six marine microbial genomes, and two marine microbial metagenomes using the Ion Torrent PGM platform on the Merchant Yacht Hanse Explorer. Onboard sequence assembly, annotation, and analysis enabled us to investigate the role of the microbes in the coral reef ecology of these islands and atolls. This analysis identified phosphonate as an important phosphorous source for microbes growing in the Line Islands and reinforced the importance of L-serine in marine microbial ecosystems. Sequencing in the field allowed us to propose hypotheses and conduct experiments and further sampling based on the sequences generated. By eliminating the delay between sampling and sequencing, we enhanced the productivity of the research expedition. By overcoming the hurdles associated with sequencing on a boat in the middle of the Pacific Ocean we proved the flexibility of the sequencing, annotation, and analysis pipelines.

  • Kelly LW, Williams GJ, Barott KL, Carlson CA, Dinsdale EA, Edwards RA, Haas AF, Haynes M, Lim YW, McDole T, Nelson CE, Sala E, Sandin SA, Smith JE, Vermeij MJ, Youle M, Rohwer F (2014) Local genomic adaptation of coral reef-associated microbiomes to gradients of natural variability and anthropogenic stressors. Proc. Natl. Acad. Sci. U.S.A. 111(28):10227-32 (PMC4104888) View Abstract · Pubmed Record
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    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.

  • Quistad SD, Stotland A, Barott KL, Smurthwaite CA, Hilton BJ, Grasis JA, Wolkowicz R, Rohwer FL (2014) Evolution of TNF-induced apoptosis reveals 550 My of functional conservation. Proc. Natl. Acad. Sci. U.S.A. 111(26):9567-72 (PMC4084427) View Abstract · Pubmed Record
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    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 (HuTNF&alpha;). HuTNF&alpha; 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 &lt; 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.

  • Whiteson KL, Meinardi S, Lim YW, Schmieder R, Maughan H, Quinn R, Blake DR, Conrad D, Rohwer F (2014) Breath gas metabolites and bacterial metagenomes from cystic fibrosis airways indicate active pH neutral 2,3-butanedione fermentation. ISME J 8(6):1247-58 (PMC4030226) View Abstract · Pubmed Record
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    The airways of cystic fibrosis (CF) patients are chronically colonized by patient-specific polymicrobial communities. The conditions and nutrients available in CF lungs affect the physiology and composition of the colonizing microbes. Recent work in bioreactors has shown that the fermentation product 2,3-butanediol mediates cross-feeding between some fermenting bacteria and Pseudomonas aeruginosa, and that this mechanism increases bacterial current production. To examine bacterial fermentation in the respiratory tract, breath gas metabolites were measured and several metagenomes were sequenced from CF and non-CF volunteers. 2,3-butanedione was produced in nearly all respiratory tracts. Elevated levels in one patient decreased during antibiotic treatment, and breath concentrations varied between CF patients at the same time point. Some patients had high enough levels of 2,3-butanedione to irreversibly damage lung tissue. Antibiotic therapy likely dictates the activities of 2,3-butanedione-producing microbes, which suggests a need for further study with larger sample size. Sputum microbiomes were dominated by P. aeruginosa, Streptococcus spp. and Rothia mucilaginosa, and revealed the potential for 2,3-butanedione biosynthesis. Genes encoding 2,3-butanedione biosynthesis were disproportionately abundant in Streptococcus spp, whereas genes for consumption of butanedione pathway products were encoded by P. aeruginosa and R. mucilaginosa. We propose a model where low oxygen conditions in CF lung lead to fermentation and a decrease in pH, triggering 2,3-butanedione fermentation to avoid lethal acidification. We hypothesize that this may also increase phenazine production by P. aeruginosa, increasing reactive oxygen species and providing additional electron acceptors to CF microbes.

  • Freeman SE, Rohwer FL, D'Spain GL, Friedlander AM, Gregg AK, Sandin SA, Buckingham MJ (2014) The origins of ambient biological sound from coral reef ecosystems in the Line Islands archipelago. J. Acoust. Soc. Am. 135(4):1775-88 View Abstract · Pubmed Record
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    Although ambient biological underwater sound was first characterized more than 60 years ago, attributing specific components of ambient sound to their creators remains a challenge. Noise produced by snapping shrimp typically dominates the ambient spectra near tropical coasts, but significant unexplained spectral variation exists. Here, evidence is presented indicating that a discernible contribution to the ambient sound field over coral reef ecosystems in the Line Islands archipelago originates from the interaction of hard-shelled benthic macro-organisms with the coral substrate. Recordings show a broad spectral peak centered between 14.30 and 14.63&thinsp;kHz, incoherently added to a noise floor typically associated with relatively &quot;white&quot; snapping shrimp sounds. A 4.6 to 6.2&thinsp;dB increase of pressure spectral density level in the 11 to 17&thinsp;kHz band occurs simultaneously with an increase in benthic invertebrate activity at night, quantified through time-lapse underwater photography. Spectral-level-filtered recordings of hermit crabs Clibanarius diugeti in quiet aquarium conditions reveal that transient sounds produced by the interaction between the crustaceans&#039; carapace, shell, and coral substrate are spectrally consistent with Line Islands recordings. Coral reef ecosystems are highly interconnected and subtle yet important ecological changes may be detected quantitatively through passive monitoring that utilizes the acoustic byproducts of biological activity.

  • Quinn RA, Lim YW, Maughan H, Conrad D, Rohwer F, Whiteson KL (2014) Biogeochemical forces shape the composition and physiology of polymicrobial communities in the cystic fibrosis lung. MBio 5(2):e00956-13 (PMC3967525) View Abstract · Pubmed Record
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    The cystic fibrosis (CF) lung contains thick mucus colonized by opportunistic pathogens which adapt to the CF lung environment over decades. The difficulty associated with sampling airways has impeded a thorough examination of the biochemical microhabitats these pathogens are exposed to. An indirect approach is to study the responses of microbial communities to these microhabitats, facilitated by high-throughput sequencing of microbial DNA and RNA from sputum samples. Microbial metagenomes and metatranscriptomes were sequenced from multiple CF patients, and the reads were assigned taxonomy and function through sequence homology to NCBI and the Kyoto Encyclopedia of Genes and Genomes (KEGG) database hierarchies. For a comparison, saliva microbial metagenomes from the Human Microbiome Project (HMP) were also analyzed. These analyses identified that functions encoded and expressed by CF microbes were significantly enriched for amino acid catabolism, folate biosynthesis, and lipoic acid biosynthesis. The data indicate that the community uses oxidative phosphorylation as a major energy source but that terminal electron acceptors were diverse. Nitrate reduction was the most abundant anaerobic respiratory pathway, and genes for nitrate reductase were largely assigned to Pseudomonas and Rothia. Although many reductive pathways of the nitrogen cycle were present, the cycle was incomplete, because the oxidative pathways were absent. Due to the abundant amino acid catabolism and incomplete nitrogen cycle, the CF microbial community appears to accumulate ammonia. This finding was verified experimentally using a CF bronchiole culture model system. The data also revealed abundant sensing and transport of iron, ammonium, zinc, and other metals along with a low-oxygen environment. This study reveals the core biochemistry and physiology of the CF microbiome. IMPORTANCE The cystic fibrosis (CF) microbial community is complex and adapts to the environmental conditions of the lung over the lifetime of a CF patient. This analysis illustrates the core functions of the CF microbial community in the context of CF lung biochemistry. There are many studies of the metabolism and physiology of individual microbes within the CF lung, but none that collectively analyze data from the whole microbiome. Understanding the core metabolism of microbes that inhabit the CF lung can provide new targets for novel therapies. The fundamental processes that CF pathogens rely on for survival may represent an Achilles heel for this pathogenic community. Novel therapies that are designed to disrupt understudied survival strategies of the CF microbial community may succeed against otherwise untreatable or antibiotic-resistant microbes.

  • Lim YW, Evangelista JS, Schmieder R, Bailey B, Haynes M, Furlan M, Maughan H, Edwards R, Rohwer F, Conrad D (2014) Clinical insights from metagenomic analysis of sputum samples from patients with cystic fibrosis. J. Clin. Microbiol. 52(2):425-37 (PMC3911355) View Abstract · Pubmed Record
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    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.

  • Kryshtafovych A, Moult J, Bales P, Bazan JF, Biasini M, Burgin A, Chen C, Cochran FV, Craig TK, Das R, Fass D, Garcia-Doval C, Herzberg O, Lorimer D, Luecke H, Ma X, Nelson DC, van Raaij MJ, Rohwer F, Segall A, Seguritan V, Zeth K, Schwede T (2014) Challenging the state of the art in protein structure prediction: Highlights of experimental target structures for the 10th Critical Assessment of Techniques for Protein Structure Prediction Experiment CASP10. Proteins 82 Suppl 2:26-42 (PMC4072496) View Abstract · Pubmed Record
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    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.

  • Reyes A, Wu M, McNulty NP, Rohwer FL, Gordon JI (2013) Gnotobiotic mouse model of phage-bacterial host dynamics in the human gut. Proc. Natl. Acad. Sci. U.S.A. 110(50):20236-41 (PMC3864308) View Abstract · Pubmed Record
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    Bacterial viruses (phages) are the most abundant biological group on Earth and are more genetically diverse than their bacterial prey/hosts. To characterize their role as agents shaping gut microbial community structure, adult germ-free mice were colonized with a consortium of 15 sequenced human bacterial symbionts, 13 of which harbored one or more predicted prophages. One member, Bacteroides cellulosilyticus WH2, was represented by a library of isogenic transposon mutants that covered 90% of its genes. Once assembled, the community was subjected to a staged phage attack with a pool of live or heat-killed virus-like particles (VLPs) purified from the fecal microbiota of five healthy humans. Shotgun sequencing of DNA from the input pooled VLP preparation plus shotgun sequencing of gut microbiota samples and purified fecal VLPs from the gnotobiotic mice revealed a reproducible nonsimultaneous pattern of attack extending over a 25-d period that involved five phages, none described previously. This system allowed us to (i) correlate increases in specific phages present in the pooled VLPs with reductions in the representation of particular bacterial taxa, (ii) provide evidence that phage resistance occurred because of ecological or epigenetic factors, (iii) track the origin of each of the five phages among the five human donors plus the extent of their genome variation between and within recipient mice, and (iv) establish the dramatic in vivo fitness advantage that a locus within a B. cellulosilyticus prophage confers upon its host. Together, these results provide a defined community-wide view of phage-bacterial host dynamics in the gut.

  • Barr JJ, Youle M, Rohwer F (2013) Innate and acquired bacteriophage-mediated immunity. Bacteriophage 3(3):e25857 (PMC3821666) View Abstract · Pubmed Record
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    We recently described a novel, non-host-derived, phage-mediated immunity active at mucosal surfaces, the main site of pathogen entry in metazoans. In that work, we showed that phage T4 adheres to mucus glycoproteins via immunoglobulin-like domains displayed on its capsid. This adherence positions the phage in mucus surfaces where they are more likely to encounter and kill bacteria, thereby benefiting both the phage and its metazoan host. We presented this phage-metazoan symbiosis based on an exclusively lytic model of phage infection. Here we extend our bacteriophage adherence to mucus (BAM) model to consider the undoubtedly more complex dynamics in vivo. We hypothesize how mucus-adherent phages, both lytic and temperate, might impact the commensal microbiota as well as protect the metazoan epithelium from bacterial invasion. We suggest that BAM may provide both an innate and an acquired antimicrobial immunity.

  • Haas AF, Nelson CE, Rohwer F, Wegley-Kelly L, Quistad SD, Carlson CA, Leichter JJ, Hatay M, Smith JE (2013) Influence of coral and algal exudates on microbially mediated reef metabolism. PeerJ 1:e108 (PMC3719129) View Abstract · Pubmed Record
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    Benthic primary producers in tropical reef ecosystems can alter biogeochemical cycling and microbial processes in the surrounding seawater. In order to quantify these influences, we measured rates of photosynthesis, respiration, and dissolved organic carbon (DOC) exudate release by the dominant benthic primary producers (calcifying and non-calcifying macroalgae, turf-algae and corals) on reefs of Mo&#039;orea French Polynesia. Subsequently, we examined planktonic and benthic microbial community response to these dissolved exudates by measuring bacterial growth rates and oxygen and DOC fluxes in dark and daylight incubation experiments. All benthic primary producers exuded significant quantities of DOC (roughly 10% of their daily fixed carbon) into the surrounding water over a diurnal cycle. The microbial community responses were dependent upon the source of the exudates and whether the inoculum of microbes included planktonic or planktonic plus benthic communities. The planktonic and benthic microbial communities in the unamended control treatments exhibited opposing influences on DO concentration where respiration dominated in treatments comprised solely of plankton and autotrophy dominated in treatments with benthic plus plankon microbial communities. Coral exudates (and associated inorganic nutrients) caused a shift towards a net autotrophic microbial metabolism by increasing the net production of oxygen by the benthic and decreasing the net consumption of oxygen by the planktonic microbial community. In contrast, the addition of algal exudates decreased the net primary production by the benthic communities and increased the net consumption of oxygen by the planktonic microbial community thereby resulting in a shift towards net heterotrophic community metabolism. When scaled up to the reef habitat, exudate-induced effects on microbial respiration did not outweigh the high oxygen production rates of benthic algae, such that reef areas dominated with benthic primary producers were always estimated to be net autotrophic. However, estimates of microbial consumption of DOC at the reef scale surpassed the DOC exudation rates suggesting net consumption of DOC at the reef-scale. In situ mesocosm experiments using custom-made benthic chambers placed over different types of benthic communities exhibited identical trends to those found in incubation experiments. Here we provide the first comprehensive dataset examining direct primary producer-induced, and indirect microbially mediated alterations of elemental cycling in both benthic and planktonic reef environments over diurnal cycles. Our results highlight the variability of the influence of different benthic primary producers on microbial metabolism in reef ecosystems and the potential implications for energy transfer to higher trophic levels during shifts from coral to algal dominance on reefs.

  • Haas AF, Gregg AK, Smith JE, Abieri ML, Hatay M, Rohwer F (2013) Visualization of oxygen distribution patterns caused by coral and algae. PeerJ 1:e106 (PMC3719126) View Abstract · Pubmed Record
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    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 &plusmn; 7.7 &micro;mol O2 L(- 1)) and daylight (97.9 &plusmn; 27.5 &micro;mol 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.

  • Lim YW, Schmieder R, Haynes M, Furlan M, Matthews TD, Whiteson K, Poole SJ, Hayes CS, Low DA, Maughan H, Edwards R, Conrad D, Rohwer F (2013) Mechanistic model of Rothia mucilaginosa adaptation toward persistence in the CF lung, based on a genome reconstructed from metagenomic data. PLoS ONE 8(5):e64285 (PMC3667864) View Abstract · Pubmed Record
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    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.

  • Barr JJ, Auro R, Furlan M, Whiteson KL, Erb ML, Pogliano J, Stotland A, Wolkowicz R, Cutting AS, Doran KS, Salamon P, Youle M, Rohwer F (2013) Bacteriophage adhering to mucus provide a non-host-derived immunity. Proc. Natl. Acad. Sci. U.S.A. 110(26):10771-6 (PMC3696810) View Abstract · Pubmed Record
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    Mucosal surfaces are a main entry point for pathogens and the principal sites of defense against infection. Both bacteria and phage are associated with this mucus. Here we show that phage-to-bacteria ratios were increased, relative to the adjacent environment, on all mucosal surfaces sampled, ranging from cnidarians to humans. In vitro studies of tissue culture cells with and without surface mucus demonstrated that this increase in phage abundance is mucus dependent and protects the underlying epithelium from bacterial infection. Enrichment of phage in mucus occurs via binding interactions between mucin glycoproteins and Ig-like protein domains exposed on phage capsids. In particular, phage Ig-like domains bind variable glycan residues that coat the mucin glycoprotein component of mucus. Metagenomic analysis found these Ig-like proteins present in the phages sampled from many environments, particularly from locations adjacent to mucosal surfaces. Based on these observations, we present the bacteriophage adherence to mucus model that provides a ubiquitous, but non-host-derived, immunity applicable to mucosal surfaces. The model suggests that metazoan mucosal surfaces and phage coevolve to maintain phage adherence. This benefits the metazoan host by limiting mucosal bacteria, and benefits the phage through more frequent interactions with bacterial hosts. The relationships shown here suggest a symbiotic relationship between phage and metazoan hosts that provides a previously unrecognized antimicrobial defense that actively protects mucosal surfaces.

  • Frank JA, Lorimer D, Youle M, Witte P, Craig T, Abendroth J, Rohwer F, Edwards RA, Segall AM, Burgin AB (2013) Structure and function of a cyanophage-encoded peptide deformylase. ISME J 7(6):1150-60 (PMC3660681) View Abstract · Pubmed Record
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    Bacteriophages encode auxiliary metabolic genes that support more efficient phage replication. For example, cyanophages carry several genes to maintain host photosynthesis throughout infection, shuttling the energy and reducing power generated away from carbon fixation and into anabolic pathways. Photodamage to the D1/D2 proteins at the core of photosystem II necessitates their continual replacement. Synthesis of functional proteins in bacteria requires co-translational removal of the N-terminal formyl group by a peptide deformylase (PDF). Analysis of marine metagenomes to identify phage-encoded homologs of known metabolic genes found that marine phages carry PDF genes, suggesting that their expression during infection might benefit phage replication. We identified a PDF homolog in the genome of Synechococcus cyanophage S-SSM7. Sequence analysis confirmed that it possesses the three absolutely conserved motifs that form the active site in PDF metalloproteases. Phylogenetic analysis placed it within the Type 1B subclass, most closely related to the Arabidopsis chloroplast PDF, but lacking the C-terminal &alpha;-helix characteristic of that group. PDF proteins from this phage and from Synechococcus elongatus were expressed and characterized. The phage PDF is the more active enzyme and deformylates the N-terminal tetrapeptides from D1 proteins more efficiently than those from ribosomal proteins. Solution of the X-ray/crystal structures of those two PDFs to 1.95 &Aring; resolution revealed active sites identical to that of the Type 1B Arabidopsis chloroplast PDF. Taken together, these findings show that many cyanophages encode a PDF with a D1 substrate preference that adds to the repertoire of genes used by phages to maintain photosynthetic activities.

  • Nelson CE, Goldberg SJ, Wegley Kelly L, Haas AF, Smith JE, Rohwer F, Carlson CA (2013) Coral and macroalgal exudates vary in neutral sugar composition and differentially enrich reef bacterioplankton lineages. ISME J 7(5):962-79 (PMC3635233) View Abstract · Pubmed Record
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    Increasing algal cover on tropical reefs worldwide may be maintained through feedbacks whereby algae outcompete coral by altering microbial activity. We hypothesized that algae and coral release compositionally distinct exudates that differentially alter bacterioplankton growth and community structure. We collected exudates from the dominant hermatypic coral holobiont Porites spp. and three dominant macroalgae (one each Ochrophyta, Rhodophyta and Chlorophyta) from reefs of Mo&#039;orea, French Polynesia. We characterized exudates by measuring dissolved organic carbon (DOC) and fractional dissolved combined neutral sugars (DCNSs) and subsequently tracked bacterioplankton responses to each exudate over 48&thinsp;h, assessing cellular growth, DOC/DCNS utilization and changes in taxonomic composition (via 16S rRNA amplicon pyrosequencing). Fleshy macroalgal exudates were enriched in the DCNS components fucose (Ochrophyta) and galactose (Rhodophyta); coral and calcareous algal exudates were enriched in total DCNS but in the same component proportions as ambient seawater. Rates of bacterioplankton growth and DOC utilization were significantly higher in algal exudate treatments than in coral exudate and control incubations with each community selectively removing different DCNS components. Coral exudates engendered the smallest shift in overall bacterioplankton community structure, maintained high diversity and enriched taxa from Alphaproteobacteria lineages containing cultured representatives with relatively few virulence factors (VFs) (Hyphomonadaceae and Erythrobacteraceae). In contrast, macroalgal exudates selected for less diverse communities heavily enriched in copiotrophic Gammaproteobacteria lineages containing cultured pathogens with increased VFs (Vibrionaceae and Pseudoalteromonadaceae). Our results demonstrate that algal exudates are enriched in DCNS components, foster rapid growth of bacterioplankton and select for bacterial populations with more potential VFs than coral exudates.

  • Mokili JL, Dutilh BE, Lim YW, Schneider BS, Taylor T, Haynes MR, Metzgar D, Myers CA, Blair PJ, Nosrat B, Wolfe ND, Rohwer F (2013) Identification of a novel human papillomavirus by metagenomic analysis of samples from patients with febrile respiratory illness. PLoS ONE 8(3):e58404 (PMC3600855) View Abstract · Pubmed Record
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    As part of a virus discovery investigation using a metagenomic approach, a highly divergent novel Human papillomavirus type was identified in pooled convenience nasal/oropharyngeal swab samples collected from patients with febrile respiratory illness. Phylogenetic analysis of the whole genome and the L1 gene reveals that the new HPV identified in this study clusters with previously described gamma papillomaviruses, sharing only 61.1% (whole genome) and 63.1% (L1) sequence identity with its closest relative in the Papillomavirus episteme (PAVE) database. This new virus was named HPV_SD2 pending official classification. The complete genome of HPV-SD2 is 7,299 bp long (36.3% G/C) and contains 7 open reading frames (L2, L1, E6, E7, E1, E2 and E4) and a non-coding long control region (LCR) between L1 and E6. The metagenomic procedures, coupled with the bioinformatic methods described herein are well suited to detect small circular genomes such as those of human papillomaviruses.

  • Rohwer F, Barott K (2013) Viral information. Biol Philos 28(2):283-297 (PMC3585991) View Abstract · Pubmed Record
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    Viruses are major drivers of global biogeochemistry and the etiological agents of many diseases. They are also the winners in the game of life: there are more viruses on the planet than cellular organisms and they encode most of the genetic diversity on the planet. In fact, it is reasonable to view life as a viral incubator. Nevertheless, most ecological and evolutionary theories were developed, and continue to be developed, without considering the virosphere. This means these theories need to be to reinterpreted in light of viral knowledge or we need to develop new theory from the viral point-of-view. Here we briefly introduce our viral planet and then address a major outstanding question in biology: why is most of life viral? A key insight is that during an infection cycle the original virus is completely broken down and only the associated information is passed on to the next generation. This is different for cellular organisms, which must pass on some physical part of themselves from generation to generation. Based on this premise, it is proposed that the thermodynamic consequences of physical information (e.g., Landauer&#039;s principle) are observed in natural viral populations. This link between physical and genetic information is then used to develop the Viral Information Hypothesis, which states that genetic information replicates itself to the detriment of system energy efficiency (i.e., is viral in nature). Finally, we show how viral information can be tested, and illustrate how this novel view can explain existing ecological and evolutionary theories from more fundamental principles.

  • Lim YW, Schmieder R, Haynes M, Willner D, Furlan M, Youle M, Abbott K, Edwards R, Evangelista J, Conrad D, Rohwer F (2013) Metagenomics and metatranscriptomics: windows on CF-associated viral and microbial communities. J. Cyst. Fibros. 12(2):154-64 (PMC3534838) View Abstract · Pubmed Record
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    Samples collected from CF patient airways often contain large amounts of host-derived nucleic acids that interfere with recovery and purification of microbial and viral nucleic acids. This study describes metagenomic and metatranscriptomic methods that address these issues. Microbial and viral metagenomes, and microbial metatranscriptomes, were successfully prepared from sputum samples from five adult CF patients. Contaminating host DNA was dramatically reduced in the metagenomes. Each CF patient presented a unique microbiome; in some Pseudomonas aeruginosa was replaced by other opportunistic bacteria. Even though the taxonomic composition of the microbiomes is very different, the metabolic potentials encoded by the community are very similar. The viral communities were dominated by phages that infect major CF pathogens. The metatranscriptomes reveal differential expression of encoded metabolic potential with changing health status. Microbial and viral metagenomics combined with microbial transcriptomics characterize the dynamic polymicrobial communities found in CF airways, revealing both the taxa present and their current metabolic activities. These approaches can facilitate the development of individualized treatment plans and novel therapeutic approaches.

  • Janouškovec J, Horák A, Barott KL, Rohwer FL, Keeling PJ (2013) Environmental distribution of coral-associated relatives of apicomplexan parasites. ISME J 7(2):444-7 (PMC3554414) View Abstract · Pubmed Record
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    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 &#039;contamination&#039; 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.

  • Conrad D, Haynes M, Salamon P, Rainey PB, Youle M, Rohwer F (2013) Cystic fibrosis therapy: a community ecology perspective. Am. J. Respir. Cell Mol. Biol. 48(2):150-6 (PMC3604065) View Abstract · Pubmed Record
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    Current therapy for cystic fibrosis (CF) focuses on minimizing the microbial community and the host&#039;s immune response through the aggressive use of airway clearance techniques, broad-spectrum antibiotics, and treatments that break down the pervasive endobronchial biofilm. Antibiotic selection is typically based on the susceptibility of individual microbial strains to specific antibiotics in vitro. Often this approach cannot accurately predict medical outcomes because of factors both technical and biological. Recent culture-independent assessments of the airway microbial and viral communities demonstrated that the CF airway infection is considerably more complex and dynamic than previously appreciated. Understanding the ecological and evolutionary pressures that shape these communities is critically important for the optimal use of current therapies (in both the choice of therapy and timing of administration) and the development of newer strategies. The climax-attack model (CAM) presented here, grounded in basic ecological principles, postulates the existence of two major functional communities. The attack community consists of transient viral and microbial populations that induce strong innate immune responses. The resultant intense immune response creates microenvironments that facilitate the establishment of a climax community that is slower-growing and inherently resistant to antibiotic therapy. Newer methodologies, including sequence-based metagenomic analysis, can track not only the taxonomic composition but also the metabolic capabilities of these changing viral and microbial communities over time. Collecting this information for CF airways will enable the mathematical modeling of microbial community dynamics during disease progression. The resultant understanding of airway communities and their effects on lung physiology will facilitate the optimization of CF therapies.

  • Barott KL, Rohwer FL (2012) Unseen players shape benthic competition on coral reefs. Trends Microbiol. 20(12):621-8 View Abstract · Pubmed Record
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    Recent work has shown that hydrophilic and hydrophobic organic matter (OM) from algae disrupts the function of the coral holobiont and promotes the invasion of opportunistic pathogens, leading to coral morbidity and mortality. Here we refer to these dynamics as the (3)DAM [dissolved organic matter (DOM), direct contact, disease, algae and microbes] model. There is considerable complexity in coral-algae interactions; turf algae and macroalgae promote heterotrophic microbial overgrowth of coral, macroalgae also directly harm the corals via hydrophobic OM, whereas crustose coralline algae generally encourage benign microbial communities. In addition, complex flow patterns transport OM and pathogens from algae to downstream corals, and direct algal contact enhances their delivery. These invisible players (microbes, viruses, and OM) are important drivers of coral reefs because they have non-linear responses to disturbances and are the first to change in response to perturbations, providing near real-time trajectories for a coral reef, a vital metric for conservation and restoration.

  • Zarei S, Mirtar A, Rohwer F, Conrad DJ, Theilmann RJ, Salamon P (2012) Mucus distribution model in a lung with cystic fibrosis. Comput Math Methods Med 2012:970809 (PMC3483681) View Abstract · Pubmed Record
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    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&#039; 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.

  • Cassman N, Prieto-Davó A, Walsh K, Silva GG, Angly F, Akhter S, Barott K, Busch J, McDole T, Haggerty JM, Willner D, Alarcón G, Ulloa O, DeLong EF, Dutilh BE, Rohwer F, Dinsdale EA (2012) Oxygen minimum zones harbour novel viral communities with low diversity. Environ. Microbiol. 14(11):3043-65 View Abstract · Pubmed Record
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    Oxygen minimum zones (OMZs) are oceanographic features that affect ocean productivity and biodiversity, and contribute to ocean nitrogen loss and greenhouse gas emissions. Here we describe the viral communities associated with the Eastern Tropical South Pacific (ETSP) OMZ off Iquique, Chile for the first time through abundance estimates and viral metagenomic analysis. The viral-to-microbial ratio (VMR) in the ETSP OMZ fluctuated in the oxycline and declined in the anoxic core to below one on several occasions. The number of viral genotypes (unique genomes as defined by sequence assembly) ranged from 2040 at the surface to 98 in the oxycline, which is the lowest viral diversity recorded to date in the ocean. Within the ETSP OMZ viromes, only 4.95% of genotypes were shared between surface and anoxic core viromes using reciprocal BLASTn sequence comparison. ETSP virome comparison with surface marine viromes (Sargasso Sea, Gulf of Mexico, Kingman Reef, Chesapeake Bay) revealed a dissimilarity of ETSP OMZ viruses to those from other oceanic regions. From the 1.4 million non-redundant DNA sequences sampled within the altered oxygen conditions of the ETSP OMZ, more than 97.8% were novel. Of the average 3.2% of sequences that showed similarity to the SEED non-redundant database, phage sequences dominated the surface viromes, eukaryotic virus sequences dominated the oxycline viromes, and phage sequences dominated the anoxic core viromes. The viral community of the ETSP OMZ was characterized by fluctuations in abundance, taxa and diversity across the oxygen gradient. The ecological significance of these changes was difficult to predict; however, it appears that the reduction in oxygen coincides with an increased shedding of eukaryotic viruses in the oxycline, and a shift to unique viral genotypes in the anoxic core.

  • McDole T, Nulton J, Barott KL, Felts B, Hand C, Hatay M, Lee H, Nadon MO, Nosrat B, Salamon P, Bailey B, Sandin SA, Vargas-Angel B, Youle M, Zgliczynski BJ, Brainard RE, Rohwer F (2012) Assessing coral reefs on a Pacific-wide scale using the microbialization score. PLoS ONE 7(9):e43233 (PMC3436891) View Abstract · Pubmed Record
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    The majority of the world&#039;s coral reefs are in various stages of decline. While a suite of disturbances (overfishing, eutrophication, and global climate change) have been identified, the mechanism(s) of reef system decline remain elusive. Increased microbial and viral loading with higher percentages of opportunistic and specific microbial pathogens have been identified as potentially unifying features of coral reefs in decline. Due to their relative size and high per cell activity, a small change in microbial biomass may signal a large reallocation of available energy in an ecosystem; that is the microbialization of the coral reef. Our hypothesis was that human activities alter the energy budget of the reef system, specifically by altering the allocation of metabolic energy between microbes and macrobes. To determine if this is occurring on a regional scale, we calculated the basal metabolic rates for the fish and microbial communities at 99 sites on twenty-nine coral islands throughout the Pacific Ocean using previously established scaling relationships. From these metabolic rate predictions, we derived a new metric for assessing and comparing reef health called the microbialization score. The microbialization score represents the percentage of the combined fish and microbial predicted metabolic rate that is microbial. Our results demonstrate a strong positive correlation between reef microbialization scores and human impact. In contrast, microbialization scores did not significantly correlate with ocean net primary production, local chla concentrations, or the combined metabolic rate of the fish and microbial communities. These findings support the hypothesis that human activities are shifting energy to the microbes, at the expense of the macrobes. Regardless of oceanographic context, the microbialization score is a powerful metric for assessing the level of human impact a reef system is experiencing.

  • Reyes A, Semenkovich NP, Whiteson K, Rohwer F, Gordon JI (2012) Going viral: next-generation sequencing applied to phage populations in the human gut. Nat. Rev. Microbiol. 10(9):607-17 (PMC3596094) View Abstract · Pubmed Record
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    Over the past decade, researchers have begun to characterize viral diversity using metagenomic methods. These studies have shown that viruses, the majority of which infect bacteria, are probably the most genetically diverse components of the biosphere. Here, we briefly review the incipient rise of a phage biology renaissance, which has been catalysed by advances in next-generation sequencing. We explore how work characterizing phage diversity and lifestyles in the human gut is changing our view of ourselves as supra-organisms. Finally, we discuss how a renewed appreciation of phage dynamics may yield new applications for phage therapies designed to manipulate the structure and functions of our gut microbiomes.

  • Youle M, Rohwer F, Stacy A, Whiteley M, Steel BC, Delalez NJ, Nord AL, Berry RM, Armitage JP, Kamoun S, Hogenhout S, Diggle SP, Gurney J, Pollitt EJ, Boetius A, Cary SC (2012) The Microbial Olympics. Nat. Rev. Microbiol. 10(8):583-8 View Abstract · Pubmed Record
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    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 &#039;rules of the game&#039; 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.

  • Janouškovec J, Horák A, Barott KL, Rohwer FL, Keeling PJ (2012) Global analysis of plastid diversity reveals apicomplexan-related lineages in coral reefs. Curr. Biol. 22(13):R518-9 View Abstract · Pubmed Record
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    No abstract available.

  • Angly FE, Willner D, Rohwer F, Hugenholtz P, Tyson GW (2012) Grinder: a versatile amplicon and shotgun sequence simulator. Nucleic Acids Res. 40(12):e94 (PMC3384353) View Abstract · Pubmed Record
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    We introduce Grinder (http://sourceforge.net/projects/biogrinder/), an open-source bioinformatic tool to simulate amplicon and shotgun (genomic, metagenomic, transcriptomic and metatranscriptomic) datasets from reference sequences. This is the first tool to simulate amplicon datasets (e.g. 16S rRNA) widely used by microbial ecologists. Grinder can create sequence libraries with a specific community structure, &alpha; and &beta; diversities and experimental biases (e.g. chimeras, gene copy number variation) for commonly used sequencing platforms. This versatility allows the creation of simple to complex read datasets necessary for hypothesis testing when developing bioinformatic software, benchmarking existing tools or designing sequence-based experiments. Grinder is particularly useful for simulating clinical or environmental microbial communities and complements the use of in vitro mock communities.

  • Pride DT, Salzman J, Haynes M, Rohwer F, Davis-Long C, White RA, Loomer P, Armitage GC, Relman DA (2012) Evidence of a robust resident bacteriophage population revealed through analysis of the human salivary virome. ISME J 6(5):915-26 (PMC3329113) View Abstract · Pubmed Record
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    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&thinsp;267&thinsp;695 virome reads from viral particles and compared them with 263&thinsp;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&thinsp;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.

  • Barott KL, Rodriguez-Mueller B, Youle M, Marhaver KL, Vermeij MJ, Smith JE, Rohwer FL (2012) Microbial to reef scale interactions between the reef-building coral Montastraea annularis and benthic algae. Proc. Biol. Sci. 279(1733):1655-64 (PMC3282354) View Abstract · Pubmed Record
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    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.

  • Kelly LW, Barott KL, Dinsdale E, Friedlander AM, Nosrat B, Obura D, Sala E, Sandin SA, Smith JE, Vermeij MJ, Williams GJ, Willner D, Rohwer F (2012) Black reefs: iron-induced phase shifts on coral reefs. ISME J 6(3):638-49 (PMC3280131) View Abstract · Pubmed Record
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    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, cyanobacterial mats 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 and maintain black reefs. Comparative surveys show that the live coral cover was reduced from 40 to 60% to &lt;10% on black reefs on Millennium, Tabuaeran and Kingman. These three sites are relatively large (&gt;0.75 km(2)). 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 times higher 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.

  • Mokili JL, Rohwer F, Dutilh BE (2012) Metagenomics and future perspectives in virus discovery. Curr Opin Virol 2(1):63-77 View Abstract · Pubmed Record
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    Monitoring the emergence and re-emergence of viral diseases with the goal of containing the spread of viral agents requires both adequate preparedness and quick response. Identifying the causative agent of a new epidemic is one of the most important steps for effective response to disease outbreaks. Traditionally, virus discovery required propagation of the virus in cell culture, a proven technique responsible for the identification of the vast majority of viruses known to date. However, many viruses cannot be easily propagated in cell culture, thus limiting our knowledge of viruses. Viral metagenomic analyses of environmental samples suggest that the field of virology has explored less than 1% of the extant viral diversity. In the last decade, the culture-independent and sequence-independent metagenomic approach has permitted the discovery of many viruses in a wide range of samples. Phylogenetically, some of these viruses are distantly related to previously discovered viruses. In addition, 60-99% of the sequences generated in different viral metagenomic studies are not homologous to known viruses. In this review, we discuss the advances in the area of viral metagenomics during the last decade and their relevance to virus discovery, clinical microbiology and public health. We discuss the potential of metagenomics for characterization of the normal viral population in a healthy community and identification of viruses that could pose a threat to humans through zoonosis. In addition, we propose a new model of the Koch&#039;s postulates named the &#039;Metagenomic Koch&#039;s Postulates&#039;. Unlike the original Koch&#039;s postulates and the Molecular Koch&#039;s postulates as formulated by Falkow, the metagenomic Koch&#039;s postulates focus on the identification of metagenomic traits in disease cases. The metagenomic traits that can be traced after healthy individuals have been exposed to the source of the suspected pathogen.

  • Ghai R, Pašić L, Fernández AB, Martin-Cuadrado AB, Mizuno CM, McMahon KD, Papke RT, Stepanauskas R, Rodriguez-Brito B, Rohwer F, Sánchez-Porro C, Ventosa A, Rodríguez-Valera F (2012) New abundant microbial groups in aquatic hypersaline environments. Sci Rep 1:135 (PMC3216616) View Abstract · Pubmed Record
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    We describe the microbiota of two hypersaline saltern ponds, one of intermediate salinity (19%) and a NaCl saturated crystallizer pond (37%) using pyrosequencing. The analyses of these metagenomes (nearly 784 Mb) reaffirmed the vast dominance of Haloquadratum walsbyi but also revealed novel, abundant and previously unsuspected microbial groups. We describe for the first time, a group of low GC Actinobacteria, related to freshwater Actinobacteria, abundant in low and intermediate salinities. Metagenomic assembly revealed three new abundant microbes: a low-GC euryarchaeon with the lowest GC content described for any euryarchaeon, a high-GC euryarchaeon and a gammaproteobacterium related to Alkalilimnicola and Nitrococcus. Multiple displacement amplification and sequencing of the genome from a single archaeal cell of the new low GC euryarchaeon suggest a photoheterotrophic and polysaccharide-degrading lifestyle and its relatedness to the recently described lineage of Nanohaloarchaea. These discoveries reveal the combined power of an unbiased metagenomic and single cell genomic approach.

  • Willner D, Haynes MR, Furlan M, Hanson N, Kirby B, Lim YW, Rainey PB, Schmieder R, Youle M, Conrad D, Rohwer F (2012) Case studies of the spatial heterogeneity of DNA viruses in the cystic fibrosis lung. Am. J. Respir. Cell Mol. Biol. 46(2):127-31 (PMC3361360) View Abstract · Pubmed Record
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    Microbial communities in the lungs of patients with cystic fibrosis (CF) and chronic obstructive pulmonary disease (COPD) have been shown to be spatially heterogeneous. Viral communities may also vary spatially, leading to localized viral populations and infections. Here, we characterized viral communities from multiple areas of the lungs of two patients with late-stage CF using metagenomics, that is, the explanted lungs from a transplant patient and lungs acquired postmortem. All regions harbored eukaryotic viruses that may infect the human host, notably herpesviruses, anelloviruses, and papillomaviruses. In the highly diseased apical lobes of explant lungs, viral diversity was extremely low, and only eukaryotic viruses were present. The absence of phage suggests that CF-associated microbial biofilms may escape top-down controls by phage predation. The phages present in other lobes of explant lungs and in all lobes of postmortem lungs comprised distinct communities, and encoded genes for clinically important microbial phenotypes, including small colony variants and antibiotic resistance. Based on the these observations, we postulate that viral communities in CF lungs are spatially distinct and contribute to CF pathology by augmenting the metabolic potential of resident microbes, as well as by directly damaging lung tissue via carcinomas and herpesviral outbreaks.

  • Willner D, Haynes MR, Furlan M, Schmieder R, Lim YW, Rainey PB, Rohwer F, Conrad D (2012) Spatial distribution of microbial communities in the cystic fibrosis lung. ISME J 6(2):471-4 (PMC3260497) View Abstract · Pubmed Record
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    Cystic fibrosis (CF) is a common fatal genetic disorder with mortality most often resulting from microbial infections of the lungs. Culture-independent studies of CF-associated microbial communities have indicated that microbial diversity in the CF airways is much higher than suggested by culturing alone. However, these studies have relied on indirect methods to sample the CF lung such as expectorated sputum and bronchoalveolar lavage (BAL). Here, we characterize the diversity of microbial communities in tissue sections from anatomically distinct regions of the CF lung using barcoded 16S amplicon pyrosequencing. Microbial communities differed significantly between different areas of the lungs, and few taxa were common to microbial communities in all anatomical regions surveyed. Our results indicate that CF lung infections are not only polymicrobial, but also spatially heterogeneous suggesting that treatment regimes tailored to dominant populations in sputum or BAL samples may be ineffective against infections in some areas of the lung.

  • Haas AF, Nelson CE, Wegley Kelly L, Carlson CA, Rohwer F, Leichter JJ, Wyatt A, Smith JE (2011) Effects of coral reef benthic primary producers on dissolved organic carbon and microbial activity. PLoS ONE 6(11):e27973 (PMC3220721) View Abstract · Pubmed Record
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    Benthic primary producers in marine ecosystems may significantly alter biogeochemical cycling and microbial processes in their surrounding environment. To examine these interactions, we studied dissolved organic matter release by dominant benthic taxa and subsequent microbial remineralization in the lagoonal reefs of Moorea, French Polynesia. Rates of photosynthesis, respiration, and dissolved organic carbon (DOC) release were assessed for several common benthic reef organisms from the backreef habitat. We assessed microbial community response to dissolved exudates of each benthic producer by measuring bacterioplankton growth, respiration, and DOC drawdown in two-day dark dilution culture incubations. Experiments were conducted for six benthic producers: three species of macroalgae (each representing a different algal phylum: Turbinaria ornata--Ochrophyta; Amansia rhodantha--Rhodophyta; Halimeda opuntia--Chlorophyta), a mixed assemblage of turf algae, a species of crustose coralline algae (Hydrolithon reinboldii) and a dominant hermatypic coral (Porites lobata). Our results show that all five types of algae, but not the coral, exuded significant amounts of labile DOC into their surrounding environment. In general, primary producers with the highest rates of photosynthesis released the most DOC and yielded the greatest bacterioplankton growth; turf algae produced nearly twice as much DOC per unit surface area than the other benthic producers (14.0&plusmn;2.8 &micro;mol hâ

  • Sharon I, Battchikova N, Aro EM, Giglione C, Meinnel T, Glaser F, Pinter RY, Breitbart M, Rohwer F, Béjà O (2011) Comparative metagenomics of microbial traits within oceanic viral communities. ISME J 5(7):1178-90 (PMC3146289) View Abstract · Pubmed Record
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    Viral genomes often contain genes recently acquired from microbes. In some cases (for example, psbA) the proteins encoded by these genes have been shown to be important for viral replication. In this study, using a unique search strategy on the Global Ocean Survey (GOS) metagenomes in combination with marine virome and microbiome pyrosequencing-based datasets, we characterize previously undetected microbial metabolic capabilities concealed within the genomes of uncultured marine viral communities. A total of 34 microbial gene families were detected on 452 viral GOS scaffolds. The majority of auxiliary metabolic genes found on these scaffolds have never been reported in phages. Host genes detected in viruses were mainly divided between genes encoding for different energy metabolism pathways, such as electron transport and newly identified photosystem genes, or translation and post-translation mechanism related. Our findings suggest previously undetected ways, in which marine phages adapt to their hosts and improve their fitness, including translation and post-translation level control over the host rather than the already known transcription level control.

  • Ng TF, Willner DL, Lim YW, Schmieder R, Chau B, Nilsson C, Anthony S, Ruan Y, Rohwer F, Breitbart M (2011) Broad surveys of DNA viral diversity obtained through viral metagenomics of mosquitoes. PLoS ONE 6(6):e20579 (PMC3108952) View Abstract · Pubmed Record
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    Viruses are the most abundant and diverse genetic entities on Earth; however, broad surveys of viral diversity are hindered by the lack of a universal assay for viruses and the inability to sample a sufficient number of individual hosts. This study utilized vector-enabled metagenomics (VEM) to provide a snapshot of the diversity of DNA viruses present in three mosquito samples from San Diego, California. The majority of the sequences were novel, suggesting that the viral community in mosquitoes, as well as the animal and plant hosts they feed on, is highly diverse and largely uncharacterized. Each mosquito sample contained a distinct viral community. The mosquito viromes contained sequences related to a broad range of animal, plant, insect and bacterial viruses. Animal viruses identified included anelloviruses, circoviruses, herpesviruses, poxviruses, and papillomaviruses, which mosquitoes may have obtained from vertebrate hosts during blood feeding. Notably, sequences related to human papillomaviruses were identified in one of the mosquito samples. Sequences similar to plant viruses were identified in all mosquito viromes, which were potentially acquired through feeding on plant nectar. Numerous bacteriophages and insect viruses were also detected, including a novel densovirus likely infecting Culex erythrothorax. Through sampling insect vectors, VEM enables broad survey of viral diversity and has significantly increased our knowledge of the DNA viruses present in mosquitoes.

  • Barott KL, Rodriguez-Brito B, Janouškovec J, Marhaver KL, Smith JE, Keeling P, Rohwer FL (2011) Microbial diversity associated with four functional groups of benthic reef algae and the reef-building coral Montastraea annularis. Environ. Microbiol. 13(5):1192-204 View Abstract · Pubmed Record
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    The coral reef benthos is primarily colonized by corals and algae, which are often in direct competition with one another for space. Numerous studies have shown that coral-associated Bacteria are different from the surrounding seawater and are at least partially species specific (i.e. the same bacterial species on the same coral species). Here we extend these microbial studies to four of the major ecological functional groups of algae found on coral reefs: upright and encrusting calcifying algae, fleshy algae, and turf algae, and compare the results to the communities found on the reef-building coral Montastraea annularis. It was found using 16S rDNA tag pyrosequencing that the different algal genera harbour characteristic bacterial communities, and these communities were generally more diverse than those found on corals. While the majority of coral-associated Bacteria were related to known heterotrophs, primarily consuming carbon-rich coral mucus, algal-associated communities harboured a high percentage of autotrophs. The majority of algal-associated autotrophic Bacteria were Cyanobacteria and may be important for nitrogen cycling on the algae. There was also a rich diversity of photosynthetic eukaryotes associated with the algae, including protists, diatoms, and other groups of microalgae. Together, these observations support the hypothesis that coral reefs are a vast landscape of distinctive microbial communities and extend the holobiont concept to benthic algae.

  • Willner D, Furlan M, Schmieder R, Grasis JA, Pride DT, Relman DA, Angly FE, McDole T, Mariella RP, Rohwer F, Haynes M (2011) Metagenomic detection of phage-encoded platelet-binding factors in the human oral cavity. Proc. Natl. Acad. Sci. U.S.A. 108 Suppl :4547-53 (PMC3063595) View Abstract · Pubmed Record
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    The human oropharynx is a reservoir for many potential pathogens, including streptococcal species that cause endocarditis. Although oropharyngeal microbes have been well described, viral communities are essentially uncharacterized. We conducted a metagenomic study to determine the composition of oropharyngeal DNA viral communities (both phage and eukaryotic viruses) in healthy individuals and to evaluate oropharyngeal swabs as a rapid method for viral detection. Viral DNA was extracted from 19 pooled oropharyngeal swabs and sequenced. Viral communities consisted almost exclusively of phage, and complete genomes of several phage were recovered, including Escherichia coli phage T3, Propionibacterium acnes phage PA6, and Streptococcus mitis phage SM1. Phage relative abundances changed dramatically depending on whether samples were chloroform treated or filtered to remove microbial contamination. pblA and pblB genes of phage SM1 were detected in the metagenomes. pblA and pblB mediate the attachment of S. mitis to platelets and play a significant role in S. mitis virulence in the endocardium, but have never previously been detected in the oral cavity. These genes were also identified in salivary metagenomes from three individuals at three time points and in individual saliva samples by PCR. Additionally, we demonstrate that phage SM1 can be induced by commonly ingested substances. Our results indicate that the oral cavity is a reservoir for pblA and pblB genes and for phage SM1 itself. Further studies will determine the association between pblA and pblB genes in the oral cavity and the risk of endocarditis.

  • Alperovitch-Lavy A, Sharon I, Rohwer F, Aro EM, Glaser F, Milo R, Nelson N, Béjà O (2011) Reconstructing a puzzle: existence of cyanophages containing both photosystem-I and photosystem-II gene suites inferred from oceanic metagenomic datasets. Environ. Microbiol. 13(1):24-32 View Abstract · Pubmed Record
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    Cyanobacteria play a key role in marine photosynthesis, which contributes to the global carbon cycle and to the world oxygen supply. Genes encoding the photosystem-II (PSII) reaction centre are found in many cyanophage genomes, and it was suggested that the horizontal transfer of these genes might be involved in increasing phage fitness. Recently, evidence for the existence of phages carrying Photosystem-I (PSI) genes was also reported. Here, using a combination of different marine metagenomic datasets and a unique crossing of the datasets, we now describe the finding of phages that, as in plants and cyanobacteria, contain both PSII and PSI genes. In addition, these phages also contain NADH dehydrogenase genes. The presence of modified PSII and PSI genes in the same viral entities in combination with electron transfer proteins like NAD(P)H dehydrogenase (NDH-1) strongly points to a role in perturbation of the cyanobacterial host photosynthetic electron flow. We therefore suggest that, depending on the physiological condition of the infected cyanobacterial host, the viruses may use different options to maximize survival. The modified PSI may alternate between functioning with PSII in linear electron transfer and contributing to the production of both NADPH and ATP or functioning independently of PSII in cyclic mode via the NDH-1 complex and thus producing only ATP.

  • Reyes A, Haynes M, Hanson N, Angly FE, Heath AC, Rohwer F, Gordon JI (2010) Viruses in the faecal microbiota of monozygotic twins and their mothers. Nature 466(7304):334-8 (PMC2919852) View Abstract · Pubmed Record
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    Viral diversity and life cycles are poorly understood in the human gut and other body habitats. Phages and their encoded functions may provide informative signatures of a human microbiota and of microbial community responses to various disturbances, and may indicate whether community health or dysfunction is manifest after apparent recovery from a disease or therapeutic intervention. Here we report sequencing of the viromes (metagenomes) of virus-like particles isolated from faecal samples collected from healthy adult female monozygotic twins and their mothers at three time points over a one-year period. We compared these data sets with data sets of sequenced bacterial 16S ribosomal RNA genes and total-faecal-community DNA. Co-twins and their mothers share a significantly greater degree of similarity in their faecal bacterial communities than do unrelated individuals. In contrast, viromes are unique to individuals regardless of their degree of genetic relatedness. Despite remarkable interpersonal variations in viromes and their encoded functions, intrapersonal diversity is very low, with &gt;95% of virotypes retained over the period surveyed, and with viromes dominated by a few temperate phages that exhibit remarkable genetic stability. These results indicate that a predatory viral-microbial dynamic, manifest in a number of other characterized environmental ecosystems, is notably absent in the very distal intestine.

  • Schmieder R, Lim YW, Rohwer F, Edwards R (2010) TagCleaner: Identification and removal of tag sequences from genomic and metagenomic datasets. BMC Bioinformatics 11:341 (PMC2910026) View Abstract · Pubmed Record
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    Sequencing metagenomes that were pre-amplified with primer-based methods requires the removal of the additional tag sequences from the datasets. The sequenced reads can contain deletions or insertions due to sequencing limitations, and the primer sequence may contain ambiguous bases. Furthermore, the tag sequence may be unavailable or incorrectly reported. Because of the potential for downstream inaccuracies introduced by unwanted sequence contaminations, it is important to use reliable tools for pre-processing sequence data. TagCleaner is a web application developed to automatically identify and remove known or unknown tag sequences allowing insertions and deletions in the dataset. TagCleaner is designed to filter the trimmed reads for duplicates, short reads, and reads with high rates of ambiguous sequences. An additional screening for and splitting of fragment-to-fragment concatenations that gave rise to artificial concatenated sequences can increase the quality of the dataset. Users may modify the different filter parameters according to their own preferences. TagCleaner is a publicly available web application that is able to automatically detect and efficiently remove tag sequences from metagenomic datasets. It is easily configurable and provides a user-friendly interface. The interactive web interface facilitates export functionality for subsequent data processing, and is available at http://edwards.sdsu.edu/tagcleaner.

  • Barott KL, Caselle JE, Dinsdale EA, Friedlander AM, Maragos JE, Obura D, Rohwer FL, Sandin SA, Smith JE, Zgliczynski B (2010) The lagoon at Caroline/Millennium atoll, Republic of Kiribati: natural history of a nearly pristine ecosystem. PLoS ONE 5(6):e10950 (PMC2880600) View Abstract · Pubmed Record
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    A series of surveys were carried out to characterize the physical and biological parameters of the Millennium Atoll lagoon during a research expedition in April of 2009. Millennium is a remote coral atoll in the Central Pacific belonging to the Republic of Kiribati, and a member of the Southern Line Islands chain. The atoll is among the few remaining coral reef ecosystems that are relatively pristine. The lagoon is highly enclosed, and was characterized by reticulate patch and line reefs throughout the center of the lagoon as well as perimeter reefs around the rim of the atoll. The depth reached a maximum of 33.3 m in the central region of the lagoon, and averaged between 8.8 and 13.7 m in most of the pools. The deepest areas were found to harbor large platforms of Favia matthaii, which presumably provided a base upon which the dominant corals (Acropora spp.) grew to form the reticulate reef structure. The benthic algal communities consisted mainly of crustose coralline algae (CCA), microfilamentous turf algae and isolated patches of Halimeda spp. and Caulerpa spp. Fish species richness in the lagoon was half of that observed on the adjacent fore reef. The lagoon is likely an important nursery habitat for a number of important fisheries species including the blacktip reef shark and Napoleon wrasse, which are heavily exploited elsewhere around the world but were common in the lagoon at Millennium. The lagoon also supports an abundance of giant clams (Tridacna maxima). Millennium lagoon provides an excellent reference of a relatively undisturbed coral atoll. As with most coral reefs around the world, the lagoon communities of Millennium may be threatened by climate change and associated warming, acidification and sea level rise, as well as sporadic local resource exploitation which is difficult to monitor and enforce because of the atoll&#039;s remote location. While the remote nature of Millennium has allowed it to remain one of the few nearly pristine coral reef ecosystems in the world, it is imperative that this ecosystem receives protection so that it may survive for future generations.

  • Rodriguez-Brito B, Li L, Wegley L, Furlan M, Angly F, Breitbart M, Buchanan J, Desnues C, Dinsdale E, Edwards R, Felts B, Haynes M, Liu H, Lipson D, Mahaffy J, Martin-Cuadrado AB, Mira A, Nulton J, Pasić L, Rayhawk S, Rodriguez-Mueller J, Rodriguez-Valera F, Salamon P, Srinagesh S, Thingstad TF, Tran T, Thurber RV, Willner D, Youle M, Rohwer F (2010) Viral and microbial community dynamics in four aquatic environments. ISME J 4(6):739-51 View Abstract · Pubmed Record
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    The species composition and metabolic potential of microbial and viral communities are predictable and stable for most ecosystems. This apparent stability contradicts theoretical models as well as the viral-microbial dynamics observed in simple ecosystems, both of which show Kill-the-Winner behavior causing cycling of the dominant taxa. Microbial and viral metagenomes were obtained from four human-controlled aquatic environments at various time points separated by one day to &gt;1 year. These environments were maintained within narrow geochemical bounds and had characteristic species composition and metabolic potentials at all time points. However, underlying this stability were rapid changes at the fine-grained level of viral genotypes and microbial strains. These results suggest a model wherein functionally redundant microbial and viral taxa are cycling at the level of viral genotypes and virus-sensitive microbial strains. Microbial taxa, viral taxa, and metabolic function persist over time in stable ecosystems and both communities fluctuate in a Kill-the-Winner manner at the level of viral genotypes and microbial strains.

  • He S, Kunin V, Haynes M, Martin HG, Ivanova N, Rohwer F, Hugenholtz P, McMahon KD (2010) Metatranscriptomic array analysis of 'Candidatus Accumulibacter phosphatis'-enriched enhanced biological phosphorus removal sludge. Environ. Microbiol. 12(5):1205-17 View Abstract · Pubmed Record
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    Here we report the first metatranscriptomic analysis of gene expression and regulation of &#039;Candidatus Accumulibacter&#039;-enriched lab-scale sludge during enhanced biological phosphorus removal (EBPR). Medium density oligonucleotide microarrays were generated with probes targeting most predicted genes hypothesized to be important for the EBPR phenotype. RNA samples were collected at the early stage of anaerobic and aerobic phases (15 min after acetate addition and switching to aeration respectively). We detected the expression of a number of genes involved in the carbon and phosphate metabolisms, as proposed by EBPR models (e.g. polyhydroxyalkanoate synthesis, a split TCA cycle through methylmalonyl-CoA pathway, and polyphosphate formation), as well as novel genes discovered through metagenomic analysis. The comparison between the early stage anaerobic and aerobic gene expression profiles showed that expression levels of most genes were not significantly different between the two stages. The majority of upregulated genes in the aerobic sample are predicted to encode functions such as transcription, translation and protein translocation, reflecting the rapid growth phase of Accumulibacter shortly after being switched to aerobic conditions. Components of the TCA cycle and machinery involved in ATP synthesis were also upregulated during the early aerobic phase. These findings support the predictions of EBPR metabolic models that the oxidation of intracellularly stored carbon polymers through the TCA cycle provides ATP for cell growth when oxygen becomes available. Nitrous oxide reductase was among the very few Accumulibacter genes upregulated in the anaerobic sample, suggesting that its expression is likely induced by the deprivation of oxygen.

  • Pasić L, Rodriguez-Mueller B, Martin-Cuadrado AB, Mira A, Rohwer F, Rodriguez-Valera F. (2010) Metagenomic islands of hyperhalophiles: the case of Salinibacter ruber. BMC Genomics 10:570 (PMC2800850) View Abstract · Pubmed Record
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    Saturated brines are extreme environments of low diversity. Salinibacter ruber is the only bacterium that inhabits this environment in significant numbers. In order to establish the extent of genetic diversity in natural populations of this microbe, the genomic sequence of reference strain DSM 13855 was compared to metagenomic fragments recovered from climax saltern crystallizers and obtained with 454 sequencing technology. This kind of analysis reveals the presence of metagenomic islands, i.e. highly variable regions among the different lineages in the population. Three regions of the sequenced isolate were scarcely represented in the metagenome thus appearing to vary among co-occurring S. ruber cells. These metagenomic islands showed evidence of extensive genomic corruption with atypically low GC content, low coding density, high numbers of pseudogenes and short hypothetical proteins. A detailed analysis of island gene content showed that the genes in metagenomic island 1 code for cell surface polysaccharides. The strain-specific genes of metagenomic island 2 were found to be involved in biosynthesis of cell wall polysaccharide components. Finally, metagenomic island 3 was rich in DNA related enzymes. The genomic organisation of S. ruber variable genomic regions showed a number of convergences with genomic islands of marine microbes studied, being largely involved in variable cell surface traits. This variation at the level of cell envelopes in an environment devoid of grazing pressure probably reflects a global strategy of bacteria to escape phage predation.

  • Angly FE, Willner D, Prieto-Davó A, Edwards RA, Schmieder R, Vega-Thurber R, Antonopoulos DA, Barott K, Cottrell MT, Desnues C, Dinsdale EA, Furlan M, Haynes M, Henn MR, Hu Y, Kirchman DL, McDole T, McPherson JD, Meyer F, Miller RM, Mundt E, Naviaux RK, Rodriguez-Mueller B, Stevens R, Wegley L, Zhang L, Zhu B, Rohwer F (2009) The GAAS metagenomic tool and its estimations of viral and microbial average genome size in four major biomes. PLoS Comput. Biol. 5(12):e1000593 (PMC2781106) View Abstract · Pubmed Record
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    Metagenomic studies characterize both the composition and diversity of uncultured viral and microbial communities. BLAST-based comparisons have typically been used for such analyses; however, sampling biases, high percentages of unknown sequences, and the use of arbitrary thresholds to find significant similarities can decrease the accuracy and validity of estimates. Here, we present Genome relative Abundance and Average Size (GAAS), a complete software package that provides improved estimates of community composition and average genome length for metagenomes in both textual and graphical formats. GAAS implements a novel methodology to control for sampling bias via length normalization, to adjust for multiple BLAST similarities by similarity weighting, and to select significant similarities using relative alignment lengths. In benchmark tests, the GAAS method was robust to both high percentages of unknown sequences and to variations in metagenomic sequence read lengths. Re-analysis of the Sargasso Sea virome using GAAS indicated that standard methodologies for metagenomic analysis may dramatically underestimate the abundance and importance of organisms with small genomes in environmental systems. Using GAAS, we conducted a meta-analysis of microbial and viral average genome lengths in over 150 metagenomes from four biomes to determine whether genome lengths vary consistently between and within biomes, and between microbial and viral communities from the same environment. Significant differences between biomes and within aquatic sub-biomes (oceans, hypersaline systems, freshwater, and microbialites) suggested that average genome length is a fundamental property of environments driven by factors at the sub-biome level. The behavior of paired viral and microbial metagenomes from the same environment indicated that microbial and viral average genome sizes are independent of each other, but indicative of community responses to stressors and environmental conditions.

  • Barott K, Smith J, Dinsdale E, Hatay M, Sandin S, Rohwer F (2009) Hyperspectral and physiological analyses of coral-algal interactions. PLoS ONE 4(11):e8043 (PMC2778555) View Abstract · Pubmed Record
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    Space limitation leads to competition between benthic, sessile organisms on coral reefs. As a primary example, reef-building corals are in direct contact with each other and many different species and functional groups of algae. Here we characterize interactions between three coral genera and three algal functional groups using a combination of hyperspectral imaging and oxygen microprofiling. We also performed in situ interaction transects to quantify the relative occurrence of these interaction on coral reefs. These studies were conducted in the Southern Line Islands, home to some of the most remote and near-pristine reefs in the world. Our goal was to determine if different types of coral-coral and coral-algal interactions were characterized by unique fine-scale physiological signatures. This is the first report using hyperspectral imaging for characterization of marine benthic organisms at the micron scale and proved to be a valuable tool for discriminating among different photosynthetic organisms. Consistent patterns emerged in physiology across different types of competitive interactions. In cases where corals were in direct contact with turf or macroalgae, there was a zone of hypoxia and altered pigmentation on the coral. In contrast, interaction zones between corals and crustose coralline algae (CCA) were not hypoxic and the coral tissue was consistent across the colony. Our results suggest that at least two main characteristic coral interaction phenotypes exist: 1) hypoxia and coral tissue disruption, seen with interactions between corals and fleshy turf and/or some species of macroalgae, and 2) no hypoxia or tissue disruption, seen with interactions between corals and some species of CCA. Hyperspectral imaging in combination with oxygen profiling provided useful information on competitive interactions between benthic reef organisms, and demonstrated that some turf and fleshy macroalgae can be a constant source of stress for corals, while CCA are not.

  • Rohwer F, Prangishvili D, Lindell D (2009) Roles of viruses in the environment. Environ. Microbiol. 11(11):2771-4 View Abstract · Pubmed Record
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    No abstract available.

  • Rodriguez-Valera F, Martin-Cuadrado AB, Rodriguez-Brito B, Pasić L, Thingstad TF, Rohwer F, Mira A (2009) Explaining microbial population genomics through phage predation. Nat. Rev. Microbiol. 7(11):828-36 View Abstract · Pubmed Record
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    The remarkable differences that have been detected by metagenomics in the genomes of strains of the same bacterial species are difficult to reconcile with the widely accepted paradigm that periodic selection within bacterial populations will regularly purge genomic diversity by clonal replacement. We have found that many of the genes that differ between strains affect regions that are potential phage recognition targets. We therefore propose the constant-diversity dynamics model, in which the diversity of prokaryotic populations is preserved by phage predation. We provide supporting evidence for this model from metagenomics, mathematical analysis and computer simulations. Periodic selection and phage predation dynamics are not mutually exclusive; we compare their predictions to shed light on the ecological circumstances under which each type of dynamics could predominate.

  • Angly F, Youle M, Nosrat B, Srinagesh S, Rodriguez-Brito B, McNairnie P, Deyanat-Yazdi G, Breitbart M, Rohwer F (2009) Genomic analysis of multiple Roseophage SIO1 strains. Environ. Microbiol. 11(11):2863-73 View Abstract · Pubmed Record
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    Roseophage SIO1 is a lytic marine phage that infects Roseobacter SIO67, a member of the Roseobacter clade of near-shore alphaproteobacteria. Roseophage SIO1 was first isolated in 1989 and sequenced in 2000. We have re-sequenced and re-annotated the original isolate. Our current annotation could only assign functions to seven additional open reading frames, indicating that, despite the advances in bioinformatics tools and increased genomic resources, we are still far from being able to translate phage genomic sequences into biological functions. In 2001, we isolated four new strains of Roseophage SIO1 from California near-shore locations. The genomes of all four were sequenced and compared against the original Roseophage SIO1 isolated in 1989. A high degree of conservation was evident across all five genomes; comparisons at the nucleotide level yielded an average 97% identity. The observed differences were clustered in protein-encoding regions and were mostly synonymous. The one strain that was found to possess an expanded host range also showed notable changes in putative tail protein-coding regions. Despite the possibly rapid evolution of phage and the mostly uncharacterized diversity found in viral metagenomic data sets, these findings indicate that viral genomes such as the genome of SIO1-like Roseophages can be stably maintained over ecologically significant time and distance (i.e. over a decade and approximately 50 km).

  • Willner D, Furlan M, Haynes M, Schmieder R, Angly FE, Silva J, Tammadoni S, Nosrat B, Conrad D, Rohwer F (2009) Metagenomic analysis of respiratory tract DNA viral communities in cystic fibrosis and non-cystic fibrosis individuals. PLoS ONE 4(10):e7370 (PMC2756586) View Abstract · Pubmed Record
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    The human respiratory tract is constantly exposed to a wide variety of viruses, microbes and inorganic particulates from environmental air, water and food. Physical characteristics of inhaled particles and airway mucosal immunity determine which viruses and microbes will persist in the airways. Here we present the first metagenomic study of DNA viral communities in the airways of diseased and non-diseased individuals. We obtained sequences from sputum DNA viral communities in 5 individuals with cystic fibrosis (CF) and 5 individuals without the disease. Overall, diversity of viruses in the airways was low, with an average richness of 175 distinct viral genotypes. The majority of viral diversity was uncharacterized. CF phage communities were highly similar to each other, whereas Non-CF individuals had more distinct phage communities, which may reflect organisms in inhaled air. CF eukaryotic viral communities were dominated by a few viruses, including human herpesviruses and retroviruses. Functional metagenomics showed that all Non-CF viromes were similar, and that CF viromes were enriched in aromatic amino acid metabolism. The CF metagenomes occupied two different metabolic states, probably reflecting different disease states. There was one outlying CF virome which was characterized by an over-representation of Guanosine-5&#039;-triphosphate,3&#039;-diphosphate pyrophosphatase, an enzyme involved in the bacterial stringent response. Unique environments like the CF airway can drive functional adaptations, leading to shifts in metabolic profiles. These results have important clinical implications for CF, indicating that therapeutic measures may be more effective if used to change the respiratory environment, as opposed to shifting the taxonomic composition of resident microbiota.

  • Sharon I, Alperovitch A, Rohwer F, Haynes M, Glaser F, Atamna-Ismaeel N, Pinter RY, Partensky F, Koonin EV, Wolf YI, Nelson N, Béjà O (2009) Photosystem I gene cassettes are present in marine virus genomes. Nature 461(7261):258-62 View Abstract · Pubmed Record
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    Cyanobacteria of the Synechococcus and Prochlorococcus genera are important contributors to photosynthetic productivity in the open oceans. Recently, core photosystem II (PSII) genes were identified in cyanophages and proposed to function in photosynthesis and in increasing viral fitness by supplementing the host production of these proteins. Here we show evidence for the presence of photosystem I (PSI) genes in the genomes of viruses that infect these marine cyanobacteria, using pre-existing metagenomic data from the global ocean sampling expedition as well as from viral biomes. The seven cyanobacterial core PSI genes identified in this study, psaA, B, C, D, E, K and a unique J and F fusion, form a cluster in cyanophage genomes, suggestive of selection for a distinct function in the virus life cycle. The existence of this PSI cluster was confirmed with overlapping and long polymerase chain reaction on environmental DNA from the Northern Line Islands. Potentially, the seven proteins encoded by the viral genes are sufficient to form an intact monomeric PSI complex. Projection of viral predicted peptides on the cyanobacterial PSI crystal structure suggested that the viral-PSI components might provide a unique way of funnelling reducing power from respiratory and other electron transfer chains to the PSI.

  • Vega Thurber R, Willner-Hall D, Rodriguez-Mueller B, Desnues C, Edwards RA, Angly F, Dinsdale E, Kelly L, Rohwer F (2009) Metagenomic analysis of stressed coral holobionts. Environ. Microbiol. 11(8):2148-63 View Abstract · Pubmed Record
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    The coral holobiont is the community of metazoans, protists and microbes associated with scleractinian corals. Disruptions in these associations have been correlated with coral disease, but little is known about the series of events involved in the shift from mutualism to pathogenesis. To evaluate structural and functional changes in coral microbial communities, Porites compressa was exposed to four stressors: increased temperature, elevated nutrients, dissolved organic carbon loading and reduced pH. Microbial metagenomic samples were collected and pyrosequenced. Functional gene analysis demonstrated that stressors increased the abundance of microbial genes involved in virulence, stress resistance, sulfur and nitrogen metabolism, motility and chemotaxis, fatty acid and lipid utilization, and secondary metabolism. Relative changes in taxonomy also demonstrated that coral-associated microbiota (Archaea, Bacteria, protists) shifted from a healthy-associated coral community (e.g. Cyanobacteria, Proteobacteria and the zooxanthellae Symbiodinium) to a community (e.g. Bacteriodetes, Fusobacteria and Fungi) of microbes often found on diseased corals. Additionally, low-abundance Vibrio spp. were found to significantly alter microbiome metabolism, suggesting that the contribution of a just a few members of a community can profoundly shift the health status of the coral holobiont.

  • Willner D, Thurber RV, Rohwer F (2009) Metagenomic signatures of 86 microbial and viral metagenomes. Environ. Microbiol. 11(7):1752-66 View Abstract · Pubmed Record
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    Previous studies have shown that dinucleotide abundances capture the majority of variation in genome signatures and are useful for quantifying lateral gene transfer and building molecular phylogenies. Metagenomes contain a mixture of individual genomes, and might be expected to lack compositional signatures. In many metagenomic data sets the majority of sequences have no significant similarities to known sequences and are effectively excluded from subsequent analyses. To circumvent this limitation, di-, tri- and tetranucleotide abundances of 86 microbial and viral metagenomes consisting of short pyrosequencing reads were analysed to provide a method which includes all sequences that can be used in combination with other analysis to increase our knowledge about microbial and viral communities. Both principal component analysis and hierarchical clustering showed definitive groupings of metagenomes drawn from similar environments. Together these analyses showed that dinucleotide composition, as opposed to tri- and tetranucleotides, defines a metagenomic signature which can explain up to 80% of the variance between biomes, which is comparable to that obtained by functional genomics. Metagenomes with anomalous content were also identified using dinucleotide abundances. Subsequent analyses determined that these metagenomes were contaminated with exogenous DNA, suggesting that this approach is a useful metric for quality control. The predictive strength of the dinucleotide composition also opens the possibility of assigning ecological classifications to unknown fragments. Environmental selection may be responsible for this dinucleotide signature through direct selection of specific compositional signals; however, simulations suggest that the environment may select indirectly by promoting the increased abundance of a few dominant taxa.

  • Rohwer F, Thurber RV (2009) Viruses manipulate the marine environment. Nature 459(7244):207-12 View Abstract · Pubmed Record
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    Marine viruses affect Bacteria, Archaea and eukaryotic organisms and are major components of the marine food web. Most studies have focused on their role as predators and parasites, but many of the interactions between marine viruses and their hosts are much more complicated. A series of recent studies has shown that viruses have the ability to manipulate the life histories and evolution of their hosts in remarkable ways, challenging our understanding of this almost invisible world.

  • Thurber RV, Haynes M, Breitbart M, Wegley L, Rohwer F (2009) Laboratory procedures to generate viral metagenomes. Nat Protoc 4(4):470-83 View Abstract · Pubmed Record
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    This collection of laboratory protocols describes the steps to collect viruses from various samples with the specific aim of generating viral metagenome sequence libraries (viromes). Viral metagenomics, the study of uncultured viral nucleic acid sequences from different biomes, relies on several concentration, purification, extraction, sequencing and heuristic bioinformatic methods. No single technique can provide an all-inclusive approach, and therefore the protocols presented here will be discussed in terms of hypothetical projects. However, care must be taken to individualize each step depending on the source and type of viral-particles. This protocol is a description of the processes we have successfully used to: (i) concentrate viral particles from various types of samples, (ii) eliminate contaminating cells and free nucleic acids and (iii) extract, amplify and purify viral nucleic acids. Overall, a sample can be processed to isolate viral nucleic acids suitable for high-throughput sequencing in approximately 1 week.

  • Breitbart M, Hoare A, Nitti A, Siefert J, Haynes M, Dinsdale E, Edwards R, Souza V, Rohwer F, Hollander D (2009) Metagenomic and stable isotopic analyses of modern freshwater microbialites in Cuatro Ciénegas, Mexico. Environ. Microbiol. 11(1):16-34 View Abstract · Pubmed Record
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    Ancient biologically mediated sedimentary carbonate deposits, including stromatolites and other microbialites, provide insight into environmental conditions on early Earth. The primary limitation to interpreting these records is our lack of understanding regarding microbial processes and the preservation of geochemical signatures in contemporary microbialite systems. Using a combination of metagenomic sequencing and isotopic analyses, this study describes the identity, metabolic potential and chemical processes of microbial communities from living microbialites from Cuatro Ci&eacute;negas, Mexico. Metagenomic sequencing revealed a diverse, redox-dependent microbial community associated with the microbialites. The microbialite community is distinct from other marine and freshwater microbial communities, and demonstrates extensive environmental adaptation. The microbialite metagenomes contain a large number of genes involved in the production of exopolymeric substances and the formation of biofilms, creating a complex, spatially structured environment. In addition to the spatial complexity of the biofilm, microbial activity is tightly controlled by sensory and regulatory systems, which allow for coordination of autotrophic and heterotrophic processes. Isotopic measurements of the intracrystalline organic matter demonstrate the importance of heterotrophic respiration of photoautotrophic biomass in the precipitation of calcium carbonate. The genomic and stable isotopic data presented here significantly enhance our evolving knowledge of contemporary biomineralization processes, and are directly applicable to studies of ancient microbialites.

  • Vega Thurber RL, Barott KL, Hall D, Liu H, Rodriguez-Mueller B, Desnues C, Edwards RA, Haynes M, Angly FE, Wegley L, Rohwer FL (2008) Metagenomic analysis indicates that stressors induce production of herpes-like viruses in the coral Porites compressa. Proc. Natl. Acad. Sci. U.S.A. 105(47):18413-8 (PMC2584576) View Abstract · Pubmed Record
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    During the last several decades corals have been in decline and at least one-third of all coral species are now threatened with extinction. Coral disease has been a major contributor to this threat, but little is known about the responsible pathogens. To date most research has focused on bacterial and fungal diseases; however, viruses may also be important for coral health. Using a combination of empirical viral metagenomics and real-time PCR, we show that Porites compressa corals contain a suite of eukaryotic viruses, many related to the Herpesviridae. This coral-associated viral consortium was found to shift in response to abiotic stressors. In particular, when exposed to reduced pH, elevated nutrients, and thermal stress, the abundance of herpes-like viral sequences rapidly increased in 2 separate experiments. Herpes-like viral sequences were rarely detected in apparently healthy corals, but were abundant in a majority of stressed samples. In addition, surveys of the Nematostella and Hydra genomic projects demonstrate that even distantly related Cnidarians contain numerous herpes-like viral genes, likely as a result of latent or endogenous viral infection. These data support the hypotheses that corals experience viral infections, which are exacerbated by stress, and that herpes-like viruses are common in Cnidarians.

  • McDaniel L, Breitbart M, Mobberley J, Long A, Haynes M, Rohwer F, Paul JH (2008) Metagenomic analysis of lysogeny in Tampa Bay: implications for prophage gene expression. PLoS ONE 3(9):e3263 (PMC2533394) View Abstract · Pubmed Record
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    Phage integrase genes often play a role in the establishment of lysogeny in temperate phage by catalyzing the integration of the phage into one of the host&#039;s replicons. To investigate temperate phage gene expression, an induced viral metagenome from Tampa Bay was sequenced by 454/Pyrosequencing. The sequencing yielded 294,068 reads with 6.6% identifiable. One hundred-three sequences had significant similarity to integrases by BLASTX analysis (e &lt; or =0.001). Four sequences with strongest amino-acid level similarity to integrases were selected and real-time PCR primers and probes were designed. Initial testing with microbial fraction DNA from Tampa Bay revealed 1.9 x 10(7), and 1300 gene copies of Vibrio-like integrase and Oceanicola-like integrase L(-1) respectively. The other two integrases were not detected. The integrase assay was then tested on microbial fraction RNA extracted from 200 ml of Tampa Bay water sampled biweekly over a 12 month time series. Vibrio-like integrase gene expression was detected in three samples, with estimated copy numbers of 2.4-1280 L(-1). Clostridium-like integrase gene expression was detected in 6 samples, with estimated copy numbers of 37 to 265 L(-1). In all cases, detection of integrase gene expression corresponded to the occurrence of lysogeny as detected by prophage induction. Investigation of the environmental distribution of the two expressed integrases in the Global Ocean Survey Database found the Vibrio-like integrase was present in genome equivalents of 3.14% of microbial libraries and all four viral metagenomes. There were two similar genes in the library from British Columbia and one similar gene was detected in both the Gulf of Mexico and Sargasso Sea libraries. In contrast, in the Arctic library eleven similar genes were observed. The Clostridium-like integrase was less prevalent, being found in 0.58% of the microbial and none of the viral libraries. These results underscore the value of metagenomic data in discovering signature genes that play important roles in the environment through their expression, as demonstrated by integrases in lysogeny.

  • Dinsdale EA, Rohwer F (2008) Dissecting microbial employment. Nat. Biotechnol. 26(9):997-8 View Abstract · Pubmed Record
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    No abstract available.

  • Marhaver KL, Edwards RA, Rohwer F (2008) Viral communities associated with healthy and bleaching corals. Environ. Microbiol. 10(9):2277-86 (PMC2702503) View Abstract · Pubmed Record
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    The coral holobiont is the integrated assemblage of the coral animal, its symbiotic algae, protists, fungi and a diverse consortium of Bacteria and Archaea. Corals are a model system for the study of symbiosis, the breakdown of which can result in disease and mortality. Little is known, however, about viruses that infect corals and their symbionts. Here we present metagenomic analyses of the viral communities associated with healthy and partially bleached specimens of the Caribbean reef-building coral Diploria strigosa. Surprisingly, herpes-like sequences accounted for 4-8% of the total sequences in each metagenome; this abundance of herpes-like sequences is unprecedented in other marine viral metagenomes. Viruses similar to those that infect algae and plants were also present in the coral viral assemblage. Among the phage identified, cyanophages were abundant in both healthy and bleaching corals and vibriophages were also present. Therefore, coral-associated viruses could potentially infect all components of the holobiont--coral, algal and microbial. Thus, we expect viruses to figure prominently in the preservation and breakdown of coral health.

  • Breitbart M, Haynes M, Kelley S, Angly F, Edwards RA, Felts B, Mahaffy JM, Mueller J, Nulton J, Rayhawk S, Rodriguez-Brito B, Salamon P, Rohwer F (2008) Viral diversity and dynamics in an infant gut. Res. Microbiol. 159(5):367-73 View Abstract · Pubmed Record
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    Metagenomic sequencing of DNA viruses from the feces of a healthy week-old infant revealed a viral community with extremely low diversity. The identifiable sequences were dominated by phages, which likely influence the diversity and abundance of co-occurring microbes. The most abundant fecal viral sequences did not originate from breast milk or formula, suggesting a non-dietary initial source of viruses. Certain sequences were stable in the infant&#039;s gut over the first 3 months of life, but microarray experiments demonstrated that the overall viral community composition changed dramatically between 1 and 2 weeks of age.

  • Tringe SG, Zhang T, Liu X, Yu Y, Lee WH, Yap J, Yao F, Suan ST, Ing SK, Haynes M, Rohwer F, Wei CL, Tan P, Bristow J, Rubin EM, Ruan Y (2008) The airborne metagenome in an indoor urban environment. PLoS ONE 3(4):e1862 (PMC2270337) View Abstract · Pubmed Record
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    The indoor atmosphere is an ecological unit that impacts on public health. To investigate the composition of organisms in this space, we applied culture-independent approaches to microbes harvested from the air of two densely populated urban buildings, from which we analyzed 80 megabases genomic DNA sequence and 6000 16S rDNA clones. The air microbiota is primarily bacteria, including potential opportunistic pathogens commonly isolated from human-inhabited environments such as hospitals, but none of the data contain matches to virulent pathogens or bioterror agents. Comparison of air samples with each other and nearby environments suggested that the indoor air microbes are not random transients from surrounding outdoor environments, but rather originate from indoor niches. Sequence annotation by gene function revealed specific adaptive capabilities enriched in the air environment, including genes potentially involved in resistance to desiccation and oxidative damage. This baseline index of air microbiota will be valuable for improving designs of surveillance for natural or man-made release of virulent pathogens.

  • Dinsdale EA, Edwards RA, Hall D, Angly F, Breitbart M, Brulc JM, Furlan M, Desnues C, Haynes M, Li L, McDaniel L, Moran MA, Nelson KE, Nilsson C, Olson R, Paul J, Brito BR, Ruan Y, Swan BK, Stevens R, Valentine DL, Thurber RV, Wegley L, White BA, Rohwer F (2008) Functional metagenomic profiling of nine biomes. Nature 452(7187):629-32 View Abstract · Pubmed Record
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    Microbial activities shape the biogeochemistry of the planet and macroorganism health. Determining the metabolic processes performed by microbes is important both for understanding and for manipulating ecosystems (for example, disruption of key processes that lead to disease, conservation of environmental services, and so on). Describing microbial function is hampered by the inability to culture most microbes and by high levels of genomic plasticity. Metagenomic approaches analyse microbial communities to determine the metabolic processes that are important for growth and survival in any given environment. Here we conduct a metagenomic comparison of almost 15 million sequences from 45 distinct microbiomes and, for the first time, 42 distinct viromes and show that there are strongly discriminatory metabolic profiles across environments. Most of the functional diversity was maintained in all of the communities, but the relative occurrence of metabolisms varied, and the differences between metagenomes predicted the biogeochemical conditions of each environment. The magnitude of the microbial metabolic capabilities encoded by the viromes was extensive, suggesting that they serve as a repository for storing and sharing genes among their microbial hosts and influence global evolutionary and metabolic processes.

  • Krause L, Diaz NN, Goesmann A, Kelley S, Nattkemper TW, Rohwer F, Edwards RA, Stoye J (2008) Phylogenetic classification of short environmental DNA fragments. Nucleic Acids Res. 36(7):2230-9 (PMC2367736) View Abstract · Pubmed Record
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    Metagenomics is providing striking insights into the ecology of microbial communities. The recently developed massively parallel 454 pyrosequencing technique gives the opportunity to rapidly obtain metagenomic sequences at a low cost and without cloning bias. However, the phylogenetic analysis of the short reads produced represents a significant computational challenge. The phylogenetic algorithm CARMA for predicting the source organisms of environmental 454 reads is described. The algorithm searches for conserved Pfam domain and protein families in the unassembled reads of a sample. These gene fragments (environmental gene tags, EGTs), are classified into a higher-order taxonomy based on the reconstruction of a phylogenetic tree of each matching Pfam family. The method exhibits high accuracy for a wide range of taxonomic groups, and EGTs as short as 27 amino acids can be phylogenetically classified up to the rank of genus. The algorithm was applied in a comparative study of three aquatic microbial samples obtained by 454 pyrosequencing. Profound differences in the taxonomic composition of these samples could be clearly revealed.

  • Desnues C, Rodriguez-Brito B, Rayhawk S, Kelley S, Tran T, Haynes M, Liu H, Furlan M, Wegley L, Chau B, Ruan Y, Hall D, Angly FE, Edwards RA, Li L, Thurber RV, Reid RP, Siefert J, Souza V, Valentine DL, Swan BK, Breitbart M, Rohwer F (2008) Biodiversity and biogeography of phages in modern stromatolites and thrombolites. Nature 452(7185):340-3 View Abstract · Pubmed Record
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    Viruses, and more particularly phages (viruses that infect bacteria), represent one of the most abundant living entities in aquatic and terrestrial environments. The biogeography of phages has only recently been investigated and so far reveals a cosmopolitan distribution of phage genetic material (or genotypes). Here we address this cosmopolitan distribution through the analysis of phage communities in modern microbialites, the living representatives of one of the most ancient life forms on Earth. On the basis of a comparative metagenomic analysis of viral communities associated with marine (Highborne Cay, Bahamas) and freshwater (Pozas Azules II and Rio Mesquites, Mexico) microbialites, we show that some phage genotypes are geographically restricted. The high percentage of unknown sequences recovered from the three metagenomes (&gt;97%), the low percentage similarities with sequences from other environmental viral (n = 42) and microbial (n = 36) metagenomes, and the absence of viral genotypes shared among microbialites indicate that viruses are genetically unique in these environments. Identifiable sequences in the Highborne Cay metagenome were dominated by single-stranded DNA microphages that were not detected in any other samples examined, including sea water, fresh water, sediment, terrestrial, extreme, metazoan-associated and marine microbial mats. Finally, a marine signature was present in the phage community of the Pozas Azules II microbialites, even though this environment has not been in contact with the ocean for tens of millions of years. Taken together, these results prove that viruses in modern microbialites display biogeographical variability and suggest that they may be derived from an ancient community.

  • Dinsdale EA, Pantos O, Smriga S, Edwards RA, Angly F, Wegley L, Hatay M, Hall D, Brown E, Haynes M, Krause L, Sala E, Sandin SA, Thurber RV, Willis BL, Azam F, Knowlton N, Rohwer F (2008) Microbial ecology of four coral atolls in the Northern Line Islands. PLoS ONE 3(2):e1584 (PMC2253183) View Abstract · Pubmed Record
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    Microbes are key players in both healthy and degraded coral reefs. A combination of metagenomics, microscopy, culturing, and water chemistry were used to characterize microbial communities on four coral atolls in the Northern Line Islands, central Pacific. Kingman, a small uninhabited atoll which lies most northerly in the chain, had microbial and water chemistry characteristic of an open ocean ecosystem. On this atoll the microbial community was equally divided between autotrophs (mostly Prochlorococcus spp.) and heterotrophs. In contrast, Kiritimati, a large and populated ( approximately 5500 people) atoll, which is most southerly in the chain, had microbial and water chemistry characteristic of a near-shore environment. On Kiritimati, there were 10 times more microbial cells and virus-like particles in the water column and these microbes were dominated by heterotrophs, including a large percentage of potential pathogens. Culturable Vibrios were common only on Kiritimati. The benthic community on Kiritimati had the highest prevalence of coral disease and lowest coral cover. The middle atolls, Palmyra and Tabuaeran, had intermediate densities of microbes and viruses and higher percentages of autotrophic microbes than either Kingman or Kiritimati. The differences in microbial communities across atolls could reflect variation in 1) oceaonographic and/or hydrographic conditions or 2) human impacts associated with land-use and fishing. The fact that historically Kingman and Kiritimati did not differ strongly in their fish or benthic communities (both had large numbers of sharks and high coral cover) suggest an anthropogenic component in the differences in the microbial communities. Kingman is one of the world&#039;s most pristine coral reefs, and this dataset should serve as a baseline for future studies of coral reef microbes. Obtaining the microbial data set, from atolls is particularly important given the association of microbes in the ongoing degradation of coral reef ecosystems worldwide.

  • Sandin SA, Smith JE, Demartini EE, Dinsdale EA, Donner SD, Friedlander AM, Konotchick T, Malay M, Maragos JE, Obura D, Pantos O, Paulay G, Richie M, Rohwer F, Schroeder RE, Walsh S, Jackson JB, Knowlton N, Sala E (2008) Baselines and degradation of coral reefs in the Northern Line Islands. PLoS ONE 3(2):e1548 (PMC2244711) View Abstract · Pubmed Record
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    Effective conservation requires rigorous baselines of pristine conditions to assess the impacts of human activities and to evaluate the efficacy of management. Most coral reefs are moderately to severely degraded by local human activities such as fishing and pollution as well as global change, hence it is difficult to separate local from global effects. To this end, we surveyed coral reefs on uninhabited atolls in the northern Line Islands to provide a baseline of reef community structure, and on increasingly populated atolls to document changes associated with human activities. We found that top predators and reef-building organisms dominated unpopulated Kingman and Palmyra, while small planktivorous fishes and fleshy algae dominated the populated atolls of Tabuaeran and Kiritimati. Sharks and other top predators overwhelmed the fish assemblages on Kingman and Palmyra so that the biomass pyramid was inverted (top-heavy). In contrast, the biomass pyramid at Tabuaeran and Kiritimati exhibited the typical bottom-heavy pattern. Reefs without people exhibited less coral disease and greater coral recruitment relative to more inhabited reefs. Thus, protection from overfishing and pollution appears to increase the resilience of reef ecosystems to the effects of global warming.

  • Kunin V, He S, Warnecke F, Peterson SB, Garcia Martin H, Haynes M, Ivanova N, Blackall LL, Breitbart M, Rohwer F, McMahon KD, Hugenholtz P (2008) A bacterial metapopulation adapts locally to phage predation despite global dispersal. Genome Res. 18(2):293-7 (PMC2203627) View Abstract · Pubmed Record
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    Using a combination of bacterial and phage-targeted metagenomics, we analyzed two geographically remote sludge bioreactors enriched in a single bacterial species Candidatus Accumulibacter phosphatis (CAP). We inferred unrestricted global movement of this species and identified aquatic ecosystems as the primary environmental reservoirs facilitating dispersal. Highly related and geographically remote CAP strains differed principally in genomic regions encoding phage defense mechanisms. We found that CAP populations were high density, clonal, and nonrecombining, providing natural targets for &quot;kill-the-winner&quot; phage predation. Community expression analysis demonstrated that phages were consistently active in the bioreactor community. Genomic signatures linking CAP to past phage exposures were observed mostly between local phage and host. We conclude that CAP strains disperse globally but must adapt to phage predation pressure locally.

  • Wegley L, Edwards R, Rodriguez-Brito B, Liu H, Rohwer F (2007) Metagenomic analysis of the microbial community associated with the coral Porites astreoides. Environ. Microbiol. 9(11):2707-19 View Abstract · Pubmed Record
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    The coral holobiont is a dynamic assemblage of the coral animal, zooxanthellae, endolithic algae and fungi, Bacteria,Archaea and viruses. Zooxanthellae and some Bacteria form relatively stable and species-specific associations with corals. Other associations are less specific; coral-associated Archaea differ from those in the water column, but the same archaeal species may be found on different coral species. It has been hypothesized that the coral animal can adapt to differing ecological niches by &#039;switching&#039; its microbial associates. In the case of corals and zooxanthellae, this has been termed adaptive bleaching and it has important implications for carbon cycling within the coral holobiont and ultimately the survival of coral reefs. However, the roles of other components of the coral holobiont are essentially unknown. To better understand these other coral associates, a fractionation procedure was used to separate the microbes, mitochondria and viruses from the coral animal cells and zooxanthellae. The resulting metagenomic DNA was sequenced using pyrosequencing. Fungi, Bacteria and phage were the most commonly identified organisms in the metagenome. Three of the four fungal phyla were represented, including a wide diversity of fungal genes involved in carbon and nitrogen metabolism, suggesting that the endolithic community is more important than previously appreciated. In particular, the data suggested that endolithic fungi could be converting nitrate and nitrite to ammonia, which would enable fixed nitrogen to cycle within the coral holobiont. The most prominent bacterial groups were Proteobacteria (68%), Firmicutes (10%), Cyanobacteria (7%) and Actinobacteria (6%). Functionally, the bacterial community was primarily heterotrophic and included a number of pathways for the degradation of aromatic compounds, the most abundant being the homogentisate pathway. The most abundant phage family was the ssDNA Microphage and most of the eukaryotic viruses were most closely related to those known to infect aquatic organisms. This study provides a metabolic and taxonomic snapshot of microbes associated with the reef-building coral Porites astreoides and presents a basis for understanding how coral-microbial interactions structure the holobiont and coral reefs.

  • Fierer N, Breitbart M, Nulton J, Salamon P, Lozupone C, Jones R, Robeson M, Edwards RA, Felts B, Rayhawk S, Knight R, Rohwer F, Jackson RB (2007) Metagenomic and small-subunit rRNA analyses reveal the genetic diversity of bacteria, archaea, fungi, and viruses in soil. Appl. Environ. Microbiol. 73(21):7059-66 (PMC2074941) View Abstract · Pubmed Record
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    Recent studies have highlighted the surprising richness of soil bacterial communities; however, bacteria are not the only microorganisms found in soil. To our knowledge, no study has compared the diversities of the four major microbial taxa, i.e., bacteria, archaea, fungi, and viruses, from an individual soil sample. We used metagenomic and small-subunit RNA-based sequence analysis techniques to compare the estimated richness and evenness of these groups in prairie, desert, and rainforest soils. By grouping sequences at the 97% sequence similarity level (an operational taxonomic unit [OTU]), we found that the archaeal and fungal communities were consistently less even than the bacterial communities. Although total richness levels are difficult to estimate with a high degree of certainty, the estimated number of unique archaeal or fungal OTUs appears to rival or exceed the number of unique bacterial OTUs in each of the collected soils. In this first study to comprehensively survey viral communities using a metagenomic approach, we found that soil viruses are taxonomically diverse and distinct from the communities of viruses found in other environments that have been surveyed using a similar approach. Within each of the four microbial groups, we observed minimal taxonomic overlap between sites, suggesting that soil archaea, bacteria, fungi, and viruses are globally as well as locally diverse.

  • Beman JM, Roberts KJ, Wegley L, Rohwer F, Francis CA (2007) Distribution and diversity of archaeal ammonia monooxygenase genes associated with corals. Appl. Environ. Microbiol. 73(17):5642-7 (PMC2042080) View Abstract · Pubmed Record
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    Corals are known to harbor diverse microbial communities of Bacteria and Archaea, yet the ecological role of these microorganisms remains largely unknown. Here we report putative ammonia monooxygenase subunit A (amoA) genes of archaeal origin associated with corals. Multiple DNA samples drawn from nine coral species and four different reef locations were PCR screened for archaeal and bacterial amoA genes, and archaeal amoA gene sequences were obtained from five different species of coral collected in Bocas del Toro, Panama. The 210 coral-associated archaeal amoA sequences recovered in this study were broadly distributed phylogenetically, with most only distantly related to previously reported sequences from coastal/estuarine sediments and oceanic water columns. In contrast, the bacterial amoA gene could not be amplified from any of these samples. These results offer further evidence for the widespread presence of the archaeal amoA gene in marine ecosystems, including coral reefs.

  • Hoffmann KH, Rodriguez-Brito B, Breitbart M, Bangor D, Angly F, Felts B, Nulton J, Rohwer F, Salamon P (2007) Power law rank-abundance models for marine phage communities. FEMS Microbiol. Lett. 273(2):224-8 View Abstract · Pubmed Record
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    Metagenomic analyses suggest that the rank-abundance curve for marine phage communities follows a power law distribution. A new type of power law dependence based on a simple model in which a modified version of Lotka-Volterra predator-prey dynamics is sampled uniformly in time is presented. Biologically, the model embodies a kill the winner hypothesis and a neutral evolution hypothesis. The model can match observed power law distributions and uses very few parameters that are readily identifiable and characterize phage ecosystems. The model makes new untested predictions: (1) it is unlikely that the most abundant phage genotype will be the same at different time points and (2) the long-term decay of isolated phage populations follows a power law.

  • Casas V, Rohwer F (2007) Phage metagenomics. Meth. Enzymol. 421:259-68 View Abstract · Pubmed Record
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    The vast majority of novel DNA sequences deposited in the databases now comes from environmental phage DNA sequences. Methods are presented for the cloning and sequencing of phage DNA that might otherwise be lethal to bacterial host vectors or contain modified DNA bases that prevent standard cloning of such sequences. In addition, methods are presented for the isolation of viral particles directly from soil and sediment environmental samples or from large volumes of environmental water samples. The viral particles are then purified by cesium-chloride density centrifugation followed by DNA extraction. This purified viral metagenomic DNA is then used for cloning and sequencing.

  • Rohwer F (2007) Real-time microbial ecology. Environ. Microbiol. 9(1):10 View Abstract · Pubmed Record
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    No abstract available.

  • Angly FE, Felts B, Breitbart M, Salamon P, Edwards RA, Carlson C, Chan AM, Haynes M, Kelley S, Liu H, Mahaffy JM, Mueller JE, Nulton J, Olson R, Parsons R, Rayhawk S, Suttle CA, Rohwer F (2006) The marine viromes of four oceanic regions. PLoS Biol. 4(11):e368 (PMC1634881) View Abstract · Pubmed Record
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    Viruses are the most common biological entities in the marine environment. There has not been a global survey of these viruses, and consequently, it is not known what types of viruses are in Earth&#039;s oceans or how they are distributed. Metagenomic analyses of 184 viral assemblages collected over a decade and representing 68 sites in four major oceanic regions showed that most of the viral sequences were not similar to those in the current databases. There was a distinct &quot;marine-ness&quot; quality to the viral assemblages. Global diversity was very high, presumably several hundred thousand of species, and regional richness varied on a North-South latitudinal gradient. The marine regions had different assemblages of viruses. Cyanophages and a newly discovered clade of single-stranded DNA phages dominated the Sargasso Sea sample, whereas prophage-like sequences were most common in the Arctic. However most viral species were found to be widespread. With a majority of shared species between oceanic regions, most of the differences between viral assemblages seemed to be explained by variation in the occurrence of the most common viral species and not by exclusion of different viral genomes. These results support the idea that viruses are widely dispersed and that local environmental conditions enrich for certain viral types through selective pressure.

  • Wegley L, Mosier-Boss P, Lieberman S, Andrews J, Graff-Baker A, Rohwer F (2006) Rapid estimation of microbial numbers in water using bulk fluorescence. Environ. Microbiol. 8(10):1775-82 View Abstract · Pubmed Record
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    Enumeration of microbial cells without culturing is an essential technique for microbial ecology and water quality evaluation. Here we show that bulk fluorescence using the SYBR Gold DNA stain can be used to rapidly quantify microbial cells per millilitre in fresh, marine and estuarine waters. The bulk fluorescence method is comparable to estimating cell concentrations in cultures using optical density; however, this enhanced method enables the user to estimate microbial numbers at lower concentration (&gt; 10(5) cells ml(-1)) found in environmental samples. The technique worked in both single-cell and 96-well plate fluorescent spectrophotometers. Differences of approximately 10(5) cells per millilitre were discernible and the precision of the bulk fluorescence was higher than direct counting by epifluorescent microscopy. Treatment with DNase I increased sensitivity by lowering background noise attributed to free DNA. This technique is simple, rapid, inexpensive and adaptable for automatically estimating microbial numbers in water samples.

  • Casas V, Miyake J, Balsley H, Roark J, Telles S, Leeds S, Zurita I, Breitbart M, Bartlett D, Azam F, Rohwer F (2006) Widespread occurrence of phage-encoded exotoxin genes in terrestrial and aquatic environments in Southern California. FEMS Microbiol. Lett. 261(1):141-9 View Abstract · Pubmed Record
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    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.

  • Krause L, Diaz NN, Bartels D, Edwards RA, Pühler A, Rohwer F, Meyer F, Stoye J (2006) Finding novel genes in bacterial communities isolated from the environment. Bioinformatics 22(14):e281-9 View Abstract · Pubmed Record
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    Novel sequencing techniques can give access to organisms that are difficult to cultivate using conventional methods. When applied to environmental samples, the data generated has some drawbacks, e.g. short length of assembled contigs, in-frame stop codons and frame shifts. Unfortunately, current gene finders cannot circumvent these difficulties. At the same time, the automated prediction of genes is a prerequisite for the increasing amount of genomic sequences to ensure progress in metagenomics. We introduce a novel gene finding algorithm that incorporates features overcoming the short length of the assembled contigs from environmental data, in-frame stop codons as well as frame shifts contained in bacterial sequences. The results show that by searching for sequence similarities in an environmental sample our algorithm is capable of detecting a high fraction of its gene content, depending on the species composition and the overall size of the sample. The method is valuable for hunting novel unknown genes that may be specific for the habitat where the sample is taken. Finally, we show that our algorithm can even exploit the limited information contained in the short reads generated by 454 technology for the prediction of protein coding genes. The program is freely available upon request.

  • Smith JE, Shaw M, Edwards RA, Obura D, Pantos O, Sala E, Sandin SA, Smriga S, Hatay M, Rohwer FL (2006) Indirect effects of algae on coral: algae-mediated, microbe-induced coral mortality. Ecol. Lett. 9(7):835-45 View Abstract · Pubmed Record
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    Declines in coral cover are generally associated with increases in the abundance of fleshy algae. In many cases, it remains unclear whether algae are responsible, directly or indirectly, for coral death or whether they simply settle on dead coral surfaces. Here, we show that algae can indirectly cause coral mortality by enhancing microbial activity via the release of dissolved compounds. When coral and algae were placed in chambers together but separated by a 0.02 mum filter, corals suffered 100% mortality. With the addition of the broad-spectrum antibiotic ampicillin, mortality was completely prevented. Physiological measurements showed complementary patterns of increasing coral stress with proximity to algae. Our results suggest that as human impacts increase and algae become more abundant on reefs a positive feedback loop may be created whereby compounds released by algae enhance microbial activity on live coral surfaces causing mortality of corals and further algal growth.

  • Edwards RA, Rodriguez-Brito B, Wegley L, Haynes M, Breitbart M, Peterson DM, Saar MO, Alexander S, Alexander EC, Rohwer F (2006) Using pyrosequencing to shed light on deep mine microbial ecology. BMC Genomics 7:57 (PMC1483832) View Abstract · Pubmed Record
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    Contrasting biological, chemical and hydrogeological analyses highlights the fundamental processes that shape different environments. Generating and interpreting the biological sequence data was a costly and time-consuming process in defining an environment. Here we have used pyrosequencing, a rapid and relatively inexpensive sequencing technology, to generate environmental genome sequences from two sites in the Soudan Mine, Minnesota, USA. These sites were adjacent to each other, but differed significantly in chemistry and hydrogeology. Comparisons of the microbes and the subsystems identified in the two samples highlighted important differences in metabolic potential in each environment. The microbes were performing distinct biochemistry on the available substrates, and subsystems such as carbon utilization, iron acquisition mechanisms, nitrogen assimilation, and respiratory pathways separated the two communities. Although the correlation between much of the microbial metabolism occurring and the geochemical conditions from which the samples were isolated could be explained, the reason for the presence of many pathways in these environments remains to be determined. Despite being physically close, these two communities were markedly different from each other. In addition, the communities were also completely different from other microbial communities sequenced to date. We anticipate that pyrosequencing will be widely used to sequence environmental samples because of the speed, cost, and technical advantages. Furthermore, subsystem comparisons rapidly identify the important metabolisms employed by the microbes in different environments.

  • Rodriguez-Brito B, Rohwer F, Edwards RA (2006) An application of statistics to comparative metagenomics. BMC Bioinformatics 7:162 (PMC1473205) View Abstract · Pubmed Record
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    Metagenomics, sequence analyses of genomic DNA isolated directly from the environments, can be used to identify organisms and model community dynamics of a particular ecosystem. Metagenomics also has the potential to identify significantly different metabolic potential in different environments. Here we use a statistical method to compare curated subsystems, to predict the physiology, metabolism, and ecology from metagenomes. This approach can be used to identify those subsystems that are significantly different between metagenome sequences. Subsystems that were overrepresented in the Sargasso Sea and Acid Mine Drainage metagenome when compared to non-redundant databases were identified. The methodology described herein applies statistics to the comparisons of metabolic potential in metagenomes. This analysis reveals those subsystems that are more, or less, represented in the different environments that are compared. These differences in metabolic potential lead to several testable hypotheses about physiology and metabolism of microbes from these ecosystems.

  • Yu Y, Breitbart M, McNairnie P, Rohwer F (2006) FastGroupII: a web-based bioinformatics platform for analyses of large 16S rDNA libraries. BMC Bioinformatics 7:57 (PMC1386709) View Abstract · Pubmed Record
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    High-throughput sequencing makes it possible to rapidly obtain thousands of 16S rDNA sequences from environmental samples. Bioinformatic tools for the analyses of large 16S rDNA sequence databases are needed to comprehensively describe and compare these datasets. FastGroupII is a web-based bioinformatics platform to dereplicate large 16S rDNA libraries. FastGroupII provides users with the option of four different dereplication methods, performs rarefaction analysis, and automatically calculates the Shannon-Wiener Index and Chao1. FastGroupII was tested on a set of 16S rDNA sequences from coral-associated Bacteria. The different grouping algorithms produced similar, but not identical, results. This suggests that 16S rDNA datasets need to be analyzed in multiple ways when being used for community ecology studies. FastGroupII is an effective bioinformatics tool for the trimming and dereplication of 16S rDNA sequences. Several standard diversity indices are calculated, and the raw sequences are prepared for downstream analyses.

  • Zhang T, Breitbart M, Lee WH, Run JQ, Wei CL, Soh SW, Hibberd ML, Liu ET, Rohwer F, Ruan Y (2006) RNA viral community in human feces: prevalence of plant pathogenic viruses. PLoS Biol. 4(1):e3 (PMC1310650) View Abstract · Pubmed Record
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    The human gut is known to be a reservoir of a wide variety of microbes, including viruses. Many RNA viruses are known to be associated with gastroenteritis; however, the enteric RNA viral community present in healthy humans has not been described. Here, we present a comparative metagenomic analysis of the RNA viruses found in three fecal samples from two healthy human individuals. For this study, uncultured viruses were concentrated by tangential flow filtration, and viral RNA was extracted and cloned into shotgun viral cDNA libraries for sequencing analysis. The vast majority of the 36,769 viral sequences obtained were similar to plant pathogenic RNA viruses. The most abundant fecal virus in this study was pepper mild mottle virus (PMMV), which was found in high concentrations--up to 10(9) virions per gram of dry weight fecal matter. PMMV was also detected in 12 (66.7%) of 18 fecal samples collected from healthy individuals on two continents, indicating that this plant virus is prevalent in the human population. A number of pepper-based foods tested positive for PMMV, suggesting dietary origins for this virus. Intriguingly, the fecal PMMV was infectious to host plants, suggesting that humans might act as a vehicle for the dissemination of certain plant viruses.

  • Breitbart M, Rohwer F (2005) Method for discovering novel DNA viruses in blood using viral particle selection and shotgun sequencing. BioTechniques 39(5):729-36 View Abstract · Pubmed Record
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    Rapid identification of viruses is needed to monitor the blood supply for emerging threats. Here we present a method that meets these criteria and allows for the shotgun sequencing of novel, uncultured DNA viruses directly from human blood. This method employs selection based on the physical properties of viruses combined with sequence-independent amplification and cloning. We show that both single- and double-stranded DNA viruses can be recovered from blood samples using this approach. In addition, we report the discovery of novel anellovirus sequences in the blood of healthy donors. PCR primers designed to amplify these novel anellovirus sequences were then used to verify the presence of these viruses in the general donor population.

  • Breitbart M, Rohwer F (2005) Here a virus, there a virus, everywhere the same virus? Trends Microbiol. 13(6):278-84 View Abstract · Pubmed Record
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    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.

  • Paul JH, Williamson SJ, Long A, Authement RN, John D, Segall AM, Rohwer FL, Androlewicz M, Patterson S (2005) Complete genome sequence of phiHSIC, a pseudotemperate marine phage of Listonella pelagia. Appl. Environ. Microbiol. 71(6):3311-20 (PMC1151857) View Abstract · Pubmed Record
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    The genome for the marine pseudotemperate member of the Siphoviridae phiHSIC has been sequenced using a combination of linker amplification library construction, restriction digest library construction, and primer walking. phiHSIC enters into a pseudolysogenic relationship with its host, Listonella pelagia, characterized by sigmoidal growth curves producing &gt;10(9) cells/ml and &gt;10(11) phage/ml. The genome (37,966 bp; G+C content, 44%) contained 47 putative open reading frames (ORFs), 17 of which had significant BLASTP hits in GenBank, including a beta subunit of DNA polymerase III, a helicase, a helicase-like subunit of a resolvasome complex, a terminase, a tail tape measure protein, several phage-like structural proteins, and 1 ORF that may assist in host pathogenicity (an ADP ribosyltransferase). The genome was circularly permuted, with no physical ends detected by sequencing or restriction enzyme digestion analysis, and lacked a cos site. This evidence is consistent with a headful packaging mechanism similar to that of Salmonella phage P22 and Shigella phage Sf6. Because none of the phage-like ORFs were closely related to any existing phage sequences in GenBank (i.e., none more than 62% identical and most &lt;25% identical at the amino acid level), phiHSIC is unique among phages that have been sequenced to date. These results further emphasize the need to sequence phages from the marine environment, perhaps the largest reservoir of untapped genetic information.

  • Edwards RA, Rohwer F (2005) Viral metagenomics. Nat. Rev. Microbiol. 3(6):504-10 View Abstract · Pubmed Record
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    Viruses, most of which infect microorganisms, are the most abundant biological entities on the planet. Identifying and measuring the community dynamics of viruses in the environment is complicated because less than one percent of microbial hosts have been cultivated. Also, there is no single gene that is common to all viral genomes, so total uncultured viral diversity cannot be monitored using approaches analogous to ribosomal DNA profiling. Metagenomic analyses of uncultured viral communities circumvent these limitations and can provide insights into the composition and structure of environmental viral communities.

  • Purdy A, Rohwer F, Edwards R, Azam F, Bartlett DH (2005) A glimpse into the expanded genome content of Vibrio cholerae through identification of genes present in environmental strains. J. Bacteriol. 187(9):2992-3001 (PMC1082809) View Abstract · Pubmed Record
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    Vibrio cholerae has multiple survival strategies which are reflected both in its broad distribution in many aquatic environments and its high genotypic diversity. To obtain additional information regarding the content of the V. cholerae genome, suppression subtractive hybridization (SSH) was used to prepare libraries of DNA sequences from two southern California coastal isolates which are divergent or absent in the clinical strain V. cholerae O1 El Tor N16961. More than 1,400 subtracted clones were sequenced. This revealed the presence of novel sequences encoding functions related to cell surface structures, transport, metabolism, signal transduction, luminescence, mobile elements, stress resistance, and virulence. Flanking sequence information was determined for loci of interest, and the distribution of these sequences was assessed for a collection of V. cholerae strains obtained from southern California and Mexican environments. This led to the surprising observation that sequences related to the toxin genes toxA, cnf1, and exoY are widespread and more common in these strains than those of the cholera toxin genes which are a hallmark of the pandemic strains of V. cholerae. Gene transfer among these strains could be facilitated by a 4.9-kbp plasmid discovered in one isolate, which possesses similarity to plasmids from other environmental vibrios. By investigating some of the nucleotide sequence basis for V. cholerae genotypic diversity, DNA fragments have been uncovered which could promote survival in coastal environments. Furthermore, a set of genes has been described which could be involved in as yet undiscovered interactions between V. cholerae and eukaryotic organisms.

  • Sullivan MB, Coleman ML, Weigele P, Rohwer F, Chisholm SW (2005) Three Prochlorococcus cyanophage genomes: signature features and ecological interpretations. PLoS Biol. 3(5):e144 (PMC1079782) View Abstract · Pubmed Record
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    The oceanic cyanobacteria Prochlorococcus are globally important, ecologically diverse primary producers. It is thought that their viruses (phages) mediate population sizes and affect the evolutionary trajectories of their hosts. Here we present an analysis of genomes from three Prochlorococcus phages: a podovirus and two myoviruses. The morphology, overall genome features, and gene content of these phages suggest that they are quite similar to T7-like (P-SSP7) and T4-like (P-SSM2 and P-SSM4) phages. Using the existing phage taxonomic framework as a guideline, we examined genome sequences to establish &quot;core&quot; genes for each phage group. We found the podovirus contained 15 of 26 core T7-like genes and the two myoviruses contained 43 and 42 of 75 core T4-like genes. In addition to these core genes, each genome contains a significant number of &quot;cyanobacterial&quot; genes, i.e., genes with significant best BLAST hits to genes found in cyanobacteria. Some of these, we speculate, represent &quot;signature&quot; cyanophage genes. For example, all three phage genomes contain photosynthetic genes (psbA, hliP) that are thought to help maintain host photosynthetic activity during infection, as well as an aldolase family gene (talC) that could facilitate alternative routes of carbon metabolism during infection. The podovirus genome also contains an integrase gene (int) and other features that suggest it is capable of integrating into its host. If indeed it is, this would be unprecedented among cultured T7-like phages or marine cyanophages and would have significant evolutionary and ecological implications for phage and host. Further, both myoviruses contain phosphate-inducible genes (phoH and pstS) that are likely to be important for phage and host responses to phosphate stress, a commonly limiting nutrient in marine systems. Thus, these marine cyanophages appear to be variations of two well-known phages-T7 and T4-but contain genes that, if functional, reflect adaptations for infection of photosynthetic hosts in low-nutrient oceanic environments.

  • Angly F, Rodriguez-Brito B, Bangor D, McNairnie P, Breitbart M, Salamon P, Felts B, Nulton J, Mahaffy J, Rohwer F (2005) PHACCS, an online tool for estimating the structure and diversity of uncultured viral communities using metagenomic information. BMC Bioinformatics 6:41 (PMC555943) View Abstract · Pubmed Record
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    Phages, viruses that infect prokaryotes, are the most abundant microbes in the world. A major limitation to studying these viruses is the difficulty of cultivating the appropriate prokaryotic hosts. One way around this limitation is to directly clone and sequence shotgun libraries of uncultured viral communities (i.e., metagenomic analyses). PHACCS http://phage.sdsu.edu/phaccs, Phage Communities from Contig Spectrum, is an online bioinformatic tool to assess the biodiversity of uncultured viral communities. PHACCS uses the contig spectrum from shotgun DNA sequence assemblies to mathematically model the structure of viral communities and make predictions about diversity. PHACCS builds models of possible community structure using a modified Lander-Waterman algorithm to predict the underlying contig spectrum. PHACCS finds the most appropriate structure model by optimizing the model parameters until the predicted contig spectrum is as close as possible to the experimental one. This model is the basis for making estimates of uncultured viral community richness, evenness, diversity index and abundance of the most abundant genotype. PHACCS analysis of four different environmental phage communities suggests that the power law is an important rank-abundance form to describe uncultured viral community structure. The estimates support the fact that the four phage communities were extremely diverse and that phage community biodiversity and structure may be correlated with that of their hosts.

  • Adhya S, Black L, Friedman D, Hatfull G, Kreuzer K, Merril C, Oppenheim A, Rohwer F, Young R (2005) 2004 ASM Conference on the New Phage Biology: the 'Phage Summit'. Mol. Microbiol. 55(5):1300-14 View Abstract · Pubmed Record
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    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 lambda 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&#039;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.

  • Breitbart M, Bhagooli R, Griffin S, Johnston I, Rohwer F (2005) Microbial communities associated with skeletal tumors on Porites compressa. FEMS Microbiol. Lett. 243(2):431-6 View Abstract · Pubmed Record
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    Coral tumors are atypical skeletal forms found on coral reefs worldwide. Here we present an analysis of the microbial communities associated with skeletal tumors on the coral Porites compressa. Microbial growth rates on both healthy and tumorous P. compressa were decoupled from the surrounding water column. Microbial communities associated with tumorous colonies had a significantly faster growth rate than those associated with healthy P. compressa. The microbial community associated with the tumors contained more culturable Vibrio spp. and could utilize more carbon sources than the microbes associated with healthy colonies. Presence of tumors affected the composition and dynamics of the microbial population associated with the entire colony.

  • Casas V, Kline DI, Wegley L, Yu Y, Breitbart M, Rohwer F (2004) Widespread association of a Rickettsiales-like bacterium with reef-building corals. Environ. Microbiol. 6(11):1137-48 View Abstract · Pubmed Record
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    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 &#039;healthy&#039; (i.e. low WBD I incidence) and &#039;stressed&#039; 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.

  • Sano E, Carlson S, Wegley L, Rohwer F (2004) Movement of viruses between biomes. Appl. Environ. Microbiol. 70(10):5842-6 (PMC522096) View Abstract · Pubmed Record
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    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-microm-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.

  • Lindell D, Sullivan MB, Johnson ZI, Tolonen AC, Rohwer F, Chisholm SW (2004) Transfer of photosynthesis genes to and from Prochlorococcus viruses. Proc. Natl. Acad. Sci. U.S.A. 101(30):11013-8 (PMC503735) View Abstract · Pubmed Record
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    Comparative genomics gives us a new window into phage-host interactions and their evolutionary implications. Here we report the presence of genes central to oxygenic photosynthesis in the genomes of three phages from two viral families (Myoviridae and Podoviridae) that infect the marine cyanobacterium Prochlorococcus. The genes that encode the photosystem II core reaction center protein D1 (psbA), and a high-light-inducible protein (HLIP) (hli) are present in all three genomes. Both myoviruses contain additional hli gene types, and one of them encodes the second photosystem II core reaction center protein D2 (psbD), whereas the other encodes the photosynthetic electron transport proteins plastocyanin (petE) and ferredoxin (petF). These uninterrupted, full-length genes are conserved in their amino acid sequence, suggesting that they encode functional proteins that may help maintain photosynthetic activity during infection. Phylogenetic analyses show that phage D1, D2, and HLIP proteins cluster with those from Prochlorococcus, indicating that they are of cyanobacterial origin. Their distribution among several Prochlorococcus clades further suggests that the genes encoding these proteins were transferred from host to phage multiple times. Phage HLIPs cluster with multicopy types found exclusively in Prochlorocococus, suggesting that phage may be mediating the expansion of the hli gene family by transferring these genes back to their hosts after a period of evolution in the phage. These gene transfers are likely to play a role in the fitness landscape of hosts and phages in the surface oceans.

  • Breitbart M, Miyake JH, Rohwer F (2004) Global distribution of nearly identical phage-encoded DNA sequences. FEMS Microbiol. Lett. 236(2):249-56 View Abstract · Pubmed Record
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    Phages, the most abundant biological entities on the planet, play important roles in biogeochemical cycling, horizontal gene transfer, and defining microbial community composition. However, very little is known about phage diversity or biogeography, and there has not yet been a systematic effort to compare the phages found in different ecosystems. Here, we report that T7-like Podophage DNA polymerase sequences occur in every major biome investigated, including marine, freshwater, sediment, terrestrial, extreme, and metazoan-associated. The majority of these sequences belong to a unique clade that is only distantly related to cultured isolates. Some identical T7-like phage-encoded DNA polymerase genes from this clade were &gt;99% conserved at the nucleotide level in multiple different environments, suggesting that these phages are moving between biomes in recent evolutionary time and that the global genomic pool for T7-like phages may be smaller than previously hypothesized.

  • Breitbart M, Felts B, Kelley S, Mahaffy JM, Nulton J, Salamon P, Rohwer F (2004) Diversity and population structure of a near-shore marine-sediment viral community. Proc. Biol. Sci. 271(1539):565-74 (PMC1691639) View Abstract · Pubmed Record
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    Viruses, most of which are phage, are extremely abundant in marine sediments, yet almost nothing is known about their identity or diversity. We present the metagenomic analysis of an uncultured near-shore marine-sediment viral community. Three-quarters of the sequences in the sample were not related to anything previously reported. Among the sequences that could be identified, the majority belonged to double-stranded DNA phage. Temperate phage were more common than lytic phage, suggesting that lysogeny may be an important lifestyle for sediment viruses. Comparisons between the sediment sample and previously sequenced seawater viral communities showed that certain phage phylogenetic groups were abundant in all marine viral communities, while other phage groups were under-represented or absent. This &#039;marineness&#039; suggests that marine phage are derived from a common set of ancestors. Several independent mathematical models, based on the distribution of overlapping shotgun sequence fragments from the library, were used to show that the diversity of the viral community was extremely high, with at least 10(4) viral genotypes per kilogram of sediment and a Shannon index greater than 9 nats. Based on these observations we propose that marine-sediment viral communities are one of the largest unexplored reservoirs of sequence space on the planet.

  • Breitbart M, Wegley L, Leeds S, Schoenfeld T, Rohwer F (2004) Phage community dynamics in hot springs. Appl. Environ. Microbiol. 70(3):1633-40 (PMC368299) View Abstract · Pubmed Record
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    In extreme thermal environments such as hot springs, phages are the only known microbial predators. Here we present the first study of prokaryotic and phage community dynamics in these environments. Phages were abundant in hot springs, reaching concentrations of a million viruses per milliliter. Hot spring phage particles were resistant to shifts to lower temperatures, possibly facilitating DNA transfer out of these extreme environments. The phages were actively produced, with a population turnover time of 1 to 2 days. Phage-mediated microbial mortality was significant, making phage lysis an important component of hot spring microbial food webs. Together, these results show that phages exert an important influence on microbial community structure and energy flow in extreme thermal environments.

  • Seguritan V, Feng IW, Rohwer F, Swift M, Segall AM (2003) Genome sequences of two closely related Vibrio parahaemolyticus phages, VP16T and VP16C. J. Bacteriol. 185(21):6434-47 (PMC219397) View Abstract · Pubmed Record
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    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&#039;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.

  • Knowlton N, Rohwer F (2003) Multispecies microbial mutualisms on coral reefs: the host as a habitat. Am. Nat. 162(4 Suppl):S51-62 View Abstract · Pubmed Record
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    Reef-building corals associate with a diverse array of eukaryotic and noneukaryotic microbes. Best known are dinoflagellates in the genus Symbiodinium (&quot;zooxanthellae&quot;), 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.

  • Breitbart M, Hewson I, Felts B, Mahaffy JM, Nulton J, Salamon P, Rohwer F (2003) Metagenomic analyses of an uncultured viral community from human feces. J. Bacteriol. 185(20):6220-3 (PMC225035) View Abstract · Pubmed Record
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    Here we present the first metagenomic analyses of an uncultured viral community from human feces, using partial shotgun sequencing. Most of the sequences were unrelated to anything previously reported. The recognizable viruses were mostly siphophages, and the community contained an estimated 1,200 viral genotypes.

  • Fung MM, Rohwer F, McGuire KL (2003) IL-2 activation of a PI3K-dependent STAT3 serine phosphorylation pathway in primary human T cells. Cell. Signal. 15(6):625-36 View Abstract · Pubmed Record
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    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.

  • Rohwer F (2003) Global phage diversity. Cell 113(2):141 View Abstract · Pubmed Record
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    Ten new mycobacteriophage genomes presented by show that most phage diversity remains uncharacterized. Extrapolation suggests that less than 0.0002% of the global phage metagenome has been sampled. The new genomes also contain a number of potential virulence factors that may be important in pathogenesis.

  • Paul JH, Sullivan MB, Segall AM, Rohwer F (2002) Marine phage genomics. Comp. Biochem. Physiol. B, Biochem. Mol. Biol. 133(4):463-76 View Abstract · Pubmed Record
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    Marine phages are the most abundant biological entities in the oceans. They play important roles in carbon cycling through marine food webs, gene transfer by transduction and conversion of hosts by lysogeny. The handful of marine phage genomes that have been sequenced to date, along with prophages in marine bacterial genomes, and partial sequencing of uncultivated phages are yielding glimpses of the tremendous diversity and physiological potential of the marine phage community. Common gene modules in diverse phages are providing the information necessary to make evolutionary comparisons. Finally, deciphering phage genomes is providing clues about the adaptive response of phages and their hosts to environmental cues.

  • Breitbart M, Salamon P, Andresen B, Mahaffy JM, Segall AM, Mead D, Azam F, Rohwer F (2002) Genomic analysis of uncultured marine viral communities. Proc. Natl. Acad. Sci. U.S.A. 99(22):14250-5 (PMC137870) View Abstract · Pubmed Record
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    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.

  • Rohwer F, Edwards R (2002) The Phage Proteomic Tree: a genome-based taxonomy for phage. J. Bacteriol. 184(16):4529-35 (PMC135240) View Abstract · Pubmed Record
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    There are approximately 10(31) phage in the biosphere, making them the most abundant biological entities on the planet. Despite their great numbers and ubiquitous presence, very little is known about phage biodiversity, biogeography, or phylogeny. Information is limited, in part, because the current ICTV taxonomical system is based on culturing phage and measuring physical parameters of the free virion. No sequence-based taxonomic systems have previously been established for phage. We present here the &quot;Phage Proteomic Tree,&quot; which is based on the overall similarity of 105 completely sequenced phage genomes. The Phage Proteomic Tree places phage relative to both their near neighbors and all other phage included in the analysis. This method groups phage into taxa that predicts several aspects of phage biology and highlights genetic markers that can be used for monitoring phage biodiversity. We propose that the Phage Proteomic Tree be used as the basis of a genome-based taxonomical system for phage.

  • Hagström A, Pommier T, Rohwer F, Simu K, Stolte W, Svensson D, Zweifel UL (2002) Use of 16S ribosomal DNA for delineation of marine bacterioplankton species. Appl. Environ. Microbiol. 68(7):3628-33 (PMC126765) View Abstract · Pubmed Record
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    All of the marine bacterioplankton-derived 16S ribosomal DNA sequences previously deposited in GenBank were reanalyzed to determine the number of bacterial species in the oceanic surface waters. These sequences have been entered into the database since 1990. The rate of new additions reached a peak in 1999 and subsequently leveled off, suggesting that much of the marine microbial species richness has been sampled. When the GenBank sequences were dereplicated by using 97% similarity as a cutoff, 1,117 unique ribotypes were found. Of the unique sequences, 609 came from uncultured environmental clones and 508 came from cultured bacteria. We conclude that the apparent bacterioplankton species richness is relatively low.

  • Quistad SD, Grasis JA, Barr JJ, Rohwer FL (2017) Viruses and the origin of microbiome selection and immunity. ISME 11:835-840 10.1038/ismej.2016.182 View Abstract · DOI Record
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    The last common metazoan ancestor (LCMA) emerged over half a billion years ago. These complex metazoans provided newly available niche space for viruses and microbes. Modern day contemporaries, such as cnidarians, suggest that the LCMA consisted of two cell layers: a basal endoderm and a mucus-secreting ectoderm, which formed a surface mucus layer (SML). Here we propose a model for the origin of metazoan immunity based on external and internal microbial selection mechanisms. In this model, the SML concentrated bacteria and their associated viruses (phage) through physical dynamics (that is, the slower flow fields near a diffusive boundary layer), which selected for mucin-binding capabilities. The concentration of phage within the SML provided the LCMA with an external microbial selective described by the bacteriophage adherence to mucus (BAM) model. In the BAM model, phage adhere to mucus protecting the metazoan host against invading, potentially pathogenic bacteria. The same fluid dynamics that concentrated phage and bacteria in the SML also concentrated eukaryotic viruses. As eukaryotic viruses competed for host intracellular niche space, those viruses that provided the LCMA with immune protection were maintained. If a resident virus became pathogenic or if a non-beneficial infection occurred, we propose that tumor necrosis factor (TNF)-mediated programmed cell death, as well as other apoptosis mechanisms, were utilized to remove virally infected cells. The ubiquity of the mucosal environment across metazoan phyla suggest that both BAM and TNF-induced apoptosis emerged during the Precambrian era and continue to drive the evolution of metazoan immunity.

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Rohwer Laboratory, San Diego State University.

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Forest Rohwer, PhD
Professor of Biology
SDSU Dept. of Biology

frohwer@gmail.com