Forest Rohwer

In this chapter we discuss changing approaches to viral discovery and human health, summarize the current understanding of the human-associated viral community, and review contemporary methods in viral metagenomics. The virome is the community of viruses that populate an organism or ecosystem at any given time. This includes the “core” set of commensal viruses that do not give rise to clinical symptoms or viremia, combined with any acute or persistent infections that may be present. Recent technological advances enable us to sequence viral genomes without culturing or cloning. These methods permit not only the discovery of a wider range of viral pathogens, but also a broader assessment of the human virome in the absence of clinically recognized disease. A new focus in contemporary virology is the natural viral community of the human body. This will provide a background for recognition of emerging and previously unrecognized viruses. It should be possible to detect viral infection before the emergence of symptoms, which will have significant implications for health-care delivery.

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.

Background: Saturated brines are extreme environments of low diversity.Salinibacter ruberis 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.Results: Three regions of the sequenced isolate were scarcely represented in the metagenome thus appearing to vary among co-occurringS. rubercells. 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.Conclusion: The genomic organisation ofS. rubervariable 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.

Summary: 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 ∼50 km).

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'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 “marine-ness” 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.

Background: 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. Results: 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. Conclusion: 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.

Background: 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. Results: 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. Conclusion: 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.

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.