Diuron tolerance and potential degradation by pelagic microbiomes in the Great Barrier Reef lagoon.

Diuron is a herbicide commonly used in agricultural areas where excess application causes it to leach into rivers, reach sensitive marine environments like the Great Barrier Reef (GBR) lagoon and pose risks to marine life. To investigate the impact of diuron on whole prokaryotic communities that underpin the marine food web and are integral to coral reef health, GBR lagoon water was incubated with diuron at environmentally-relevant concentration (8 µg/L), and sequenced at specific time points over the following year. 16S rRNA gene amplicon profiling revealed no significant short- or long-term effect of diuron on microbiome structure. The relative abundance of prokaryotic phototrophs was not significantly altered by diuron, which suggests that they were largely tolerant at this concentration. Assembly of a metagenome derived from waters sampled at a similar location in the GBR lagoon did not reveal the presence of mutations in the cyanobacterial photosystem that could explain diuron tolerance. However, resident phages displayed several variants of this gene and could potentially play a role in tolerance acquisition. Slow biodegradation of diuron was reported in the incubation flasks, but no correlation with the relative abundance of heterotrophs was evident. Analysis of metagenomic reads supports the hypothesis that previously uncharacterized hydrolases carried by low-abundance species may mediate herbicide degradation in the GBR lagoon. Overall, this study offers evidence that pelagic phototrophs of the GBR lagoon may be more tolerant of diuron than other tropical organisms, and that heterotrophs in the microbial seed bank may have the potential to degrade diuron and alleviate local anthropogenic stresses to inshore GBR ecosystems.

Marine microbial communities of the Great Barrier Reef lagoon are influenced by riverine floodwaters and seasonal weather events.

The role of microorganisms in maintaining coral reef health is increasingly recognized. Riverine floodwater containing herbicides and excess nutrients from fertilizers compromises water quality in the inshore Great Barrier Reef (GBR), with unknown consequences for planktonic marine microbial communities and thus coral reefs. In this baseline study, inshore GBR microbial communities were monitored along a 124 km long transect between 2011 and 2013 using 16S rRNA gene amplicon sequencing. Members of the bacterial orders Rickettsiales (e.g., Pelagibacteraceae) and Synechococcales (e.g., Prochlorococcus), and of the archaeal class Marine Group II were prevalent in all samples, exhibiting a clear seasonal dynamics. Microbial communities near the Tully river mouth included a mixture of taxa from offshore marine sites and from the river system. The environmental parameters collected could be summarized into four groups, represented by salinity, rainfall, temperature and water quality, that drove the composition of microbial communities. During the wet season, lower salinity and a lower water quality index resulting from higher river discharge corresponded to increases in riverine taxa at sites near the river mouth. Particularly large, transient changes in microbial community structure were seen during the extreme wet season 2010-11, and may be partially attributed to the effects of wind and waves, which resuspend sediments and homogenize the water column in shallow near-shore regions. This work shows that anthropogenic floodwaters and other environmental parameters work in conjunction to drive the spatial distribution of microorganisms in the GBR lagoon, as well as their seasonal and daily dynamics.

Evaluation of viral genome assembly and diversity estimation in deep metagenomes.

BACKGROUND: Viruses have unique properties, small genome and regions of high similarity, whose effects on metagenomic assemblies have not been characterized so far. This study uses diverse in silico simulated viromes to evaluate how extensively genomes can be assembled using different sequencing platforms and assemblers. Further, it investigates the suitability of different methods to estimate viral diversity in metagenomes. RESULTS: We created in silico metagenomes mimicking various platforms at different sequencing depths. The CLC assembler revealed subpar compared to IDBA_UD and CAMERA , which are metagenomic-specific. Up to a saturation point, Illumina platforms proved more capable of reconstructing large portions of viral genomes compared to 454. Read length was an important factor for limiting chimericity, while scaffolding marginally improved contig length and accuracy. The genome length of the various viruses in the metagenomes did not significantly affect genome reconstruction, but the co-existence of highly similar genomes was detrimental. When evaluating diversity estimation tools, we found that PHACCS results were more accurate than those from CatchAll and clustering, which were both orders of magnitude above expected. CONCLUSIONS: Assemblers designed specifically for the analysis of metagenomes should be used to facilitate the creation of high-quality long contigs. Despite the high coverage possible, scientists should not expect to always obtain complete genomes, because their reconstruction may be hindered by co-existing species bearing highly similar genomic regions. Further development of metagenomics-oriented assemblers may help bypass these limitations in future studies. Meanwhile, the lack of fully reconstructed communities keeps methods to estimate viral diversity relevant. While none of the three methods tested had absolute precision, only PHACCS was deemed suitable for comparative studies.

CopyRighter: a rapid tool for improving the accuracy of microbial community profiles through lineage-specific gene copy number correction.

BACKGROUND: Culture-independent molecular surveys targeting conserved marker genes, most notably 16S rRNA, to assess microbial diversity remain semi-quantitative due to variations in the number of gene copies between species. RESULTS: Based on 2,900 sequenced reference genomes, we show that 16S rRNA gene copy number (GCN) is strongly linked to microbial phylogenetic taxonomy, potentially under-representing Archaea in amplicon microbial profiles. Using this relationship, we inferred the GCN of all bacterial and archaeal lineages in the Greengenes database within a phylogenetic framework. We created CopyRighter, new software which uses these estimates to correct 16S rRNA amplicon microbial profiles and associated quantitative (q)PCR total abundance. CopyRighter parses microbial profiles and, because GCN estimates are pre-computed for all taxa in the reference taxonomy, rapidly corrects GCN bias. Software validation with in silico and in vitro mock communities indicated that GCN correction results in more accurate estimates of microbial relative abundance and improves the agreement between metagenomic and amplicon profiles. Analyses of human-associated and anaerobic digester microbiomes illustrate that correction makes tangible changes to estimates of qPCR total abundance, α and ß diversity, and can significantly change biological interpretation. For example, human gut microbiomes from twins were reclassified into three rather than two enterotypes after GCN correction. CONCLUSIONS: The CopyRighter bioinformatic tools permits rapid correction of GCN in microbial surveys, resulting in improved estimates of microbial abundance, α and ß diversity.

The Bio-Community Perl toolkit for microbial ecology.

The development of bioinformatic solutions for microbial ecology in Perl is limited by the lack of modules to represent and manipulate microbial community profiles from amplicon and meta-omics studies. Here we introduce Bio-Community, an open-source, collaborative toolkit that extends BioPerl. Bio-Community interfaces with commonly used programs using various file formats, including BIOM, and provides operations such as rarefaction and taxonomic summaries. Bio-Community will help bioinformaticians to quickly piece together custom analysis pipelines and develop novel software. Availability an implementation: Bio-Community is cross-platform Perl code available from http://search.cpan.org/dist/Bio-Community under the Perl license. A readme file describes software installation and how to contribute.

Future reef decalcification under a business-as-usual CO2 emission scenario.

Increasing atmospheric partial pressure of CO2 (pCO2) is a major threat to coral reefs, but some argue that the threat is mitigated by factors such as the variability in the response of coral calcification to acidification, differences in bleaching susceptibility, and the potential for rapid adaptation to anthropogenic warming. However the evidence for these mitigating factors tends to involve experimental studies on corals, as opposed to coral reefs, and rarely includes the influence of multiple variables (e.g., temperature and acidification) within regimes that include diurnal and seasonal variability. Here, we demonstrate that the inclusion of all these factors results in the decalcification of patch-reefs under business-as-usual scenarios and reduced, although positive, calcification under reduced-emission scenarios. Primary productivity was found to remain constant across all scenarios, despite significant bleaching and coral mortality under both future scenarios. Daylight calcification decreased and nocturnal decalcification increased sharply from the preindustrial and control conditions to the future scenarios of low (reduced emissions) and high (business-as-usual) increases in pCO2. These changes coincided with deeply negative carbonate budgets, a shift toward smaller carbonate sediments, and an increase in the abundance of sediment microbes under the business-as-usual emission scenario. Experimental coral reefs demonstrated highest net calcification rates and lowest rates of coral mortality under preindustrial conditions, suggesting that reef processes may not have been able to keep pace with the relatively minor environmental changes that have occurred during the last century. Taken together, our results have serious implications for the future of coral reefs under business-as-usual environmental changes projected for the coming decades and century.

Oxygen minimum zones harbour novel viral communities with low diversity.

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.

Grinder: a versatile amplicon and shotgun sequence simulator.

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, α and ß 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.

Phage-bacteria relationships and CRISPR elements revealed by a metagenomic survey of the rumen microbiome.

Viruses are the most abundant biological entities on the planet and play an important role in balancing microbes within an ecosystem and facilitating horizontal gene transfer. Although bacteriophages are abundant in rumen environments, little is known about the types of viruses present or their interaction with the rumen microbiome. We undertook random pyrosequencing of virus-enriched metagenomes (viromes) isolated from bovine rumen fluid and analysed the resulting data using comparative metagenomics. A high level of diversity was observed with up to 28,000 different viral genotypes obtained from each environment. The majority (~78%) of sequences did not match any previously described virus. Prophages outnumbered lytic phages approximately 2:1 with the most abundant bacteriophage and prophage types being associated with members of the dominant rumen phyla (Firmicutes and Proteobacteria). Metabolic profiling based on SEED subsystems revealed an enrichment of sequences with putative functional roles in DNA and protein metabolism, but a surprisingly low proportion of sequences assigned to carbohydrate and amino acid metabolism. We expanded our analysis to include previously described metagenomic data and 14 reference genomes. Clustered regularly interspaced short palindromic repeats (CRISPR) were detected in most of the microbial genomes, suggesting previous interactions between viral and microbial communities.

Metagenomic analysis of DNA viruses in a wastewater treatment plant in tropical climate.

Viruses have been detected in the different stages of wastewater treatment plants (WWTPs) at concentrations of 10(8) -10(10) ml(-1) of virus-like particles (VLPs), 10-1000 times higher than in natural aquatic environments, suggesting that WWTPs can be considered as an important reservoir and source of viruses. This study revealed novel diversity and function with the DNA viral communities in the influent, activated sludge, anaerobic digester, and effluent of a domestic WWTP using metagenomics. WWTP was a very specific environment, with less than 5% of the > 936 000 metagenomic sequences obtained (∼70-119 Mbp per sample) similar to sequences present in other environmental viromes. Many viruses found in the WWTP were novel, resulting in only < 5-20% of the reads being phylogenetically or functionally assigned. DNA metabolism was observed as the most abundant function with DNA methylase detected at levels 4.2-fold higher than other published viromes, while carbohydrate and amino acids metabolisms were 3.7- and 4.2-fold less abundant respectively. These specific aspects of the WWTP community functions are likely due to high biomass concentration, turnover rate and microbial activity in WWTPs, and likely include mechanisms that help viruses increase their infectivity. Among ∼500 genotypes estimated in individual WWTP viromes, > 82% were shared. These data suggested that VLPs of most viral types could be present between 1 and 30 days in the process before they were discharged. Viruses in WWTP and the discharged ones can have potential impacts on the functioning of the wastewater treatment system and on the dynamics of microbial community in the surrounding aquatic environments respectively.

Phage encoded H-NS: a potential achilles heel in the bacterial defence system.

The relationship between phage and their microbial hosts is difficult to elucidate in complex natural ecosystems. Engineered systems performing enhanced biological phosphorus removal (EBPR), offer stable, lower complexity communities for studying phage-host interactions. Here, metagenomic data from an EBPR reactor dominated by Candidatus Accumulibacter phosphatis (CAP), led to the recovery of three complete and six partial phage genomes. Heat-stable nucleoid structuring (H-NS) protein, a global transcriptional repressor in bacteria, was identified in one of the complete phage genomes (EPV1), and was most similar to a homolog in CAP. We infer that EPV1 is a CAP-specific phage and has the potential to repress up to 6% of host genes based on the presence of putative H-NS binding sites in the CAP genome. These genes include CRISPR associated proteins and a Type III restriction-modification system, which are key host defense mechanisms against phage infection. Further, EPV1 was the only member of the phage community found in an EBPR microbial metagenome collected seven months prior. We propose that EPV1 laterally acquired H-NS from CAP providing it with a means to reduce bacterial defenses, a selective advantage over other phage in the EBPR system. Phage encoded H-NS could constitute a previously unrecognized weapon in the phage-host arms race.

Next generation sequence assembly with AMOS.

A Modular Open-Source Assembler (AMOS) was designed to offer a modular approach to genome assembly. AMOS includes a wide range of tools for assembly, including the lightweight de novo assemblers Minimus and Minimo, and Bambus 2, a robust scaffolder able to handle metagenomic and polymorphic data. This protocol describes how to configure and use AMOS for the assembly of Next Generation sequence data. Additionally, we provide three tutorial examples that include bacterial, viral, and metagenomic datasets with specific tips for improving assembly quality.

Metagenomic detection of phage-encoded platelet-binding factors in the human oral cavity.

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.

Viruses in the faecal microbiota of monozygotic twins and their mothers.

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 >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.

Viral and microbial community dynamics in four aquatic environments.

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 >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.

The GAAS metagenomic tool and its estimations of viral and microbial average genome size in four major biomes.

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.

Metagenomic analysis of respiratory tract DNA viral communities in cystic fibrosis and non-cystic fibrosis individuals.

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'-triphosphate,3'-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.

Genomic analysis of multiple Roseophage SIO1 strains.

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).

Metagenomic analysis of stressed coral holobionts.

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.