Noriko Cassman CMGT Rio de Janeiro, Brasil Nov. 17, 2012 Viral metagenomics in marine environments or, Why Perl is Actually Useful Noriko Cassman CMGT Rio de Janeiro, Brasil Nov. 17, 2012

Ignoring the viruses of your environment? Think again! 10^6 bacteria and 10^7 viruses in each ml of seawater Metagenomics: linking community genotypes and functional potential to ecology Bacterial vs. viral metagenomic analysis Viruses don't do actual metabolism

Viral Ecology Plus <1% culturable so we use genomics Viral marker genes (phoH, psb) Metagenomes: do we get random samples of all viruses? Targeted metagenomes: let's look at dsDNA phages <10% similarity of OMZ viruses to databases so we turn to other biology-inspired methods

Enter Evolution: DNA phages affect all trophic levels of an ecosystem Phages are predators and pool of genes for bacteria Host cells are required for bacteriophage replication Most abundant replicating entities in the world Viruses infect all DNA-based organisms. There are even viruses that infect other viruses. So viruses are important in all levels of an ecosystem. Breitbart et. al. 2011

Two Major Phage Lifecycles within the Host Cell LYTIC CYCLE Induction LYSOGENIC CYCLE

Phage lysis releases nutrients and structures bacterial communities LYTIC CYCLE Viral infection and lysis of cells releases as much as 40% of dissolved organic material back into the ocean by rough estimates. By infecting according to which cells are available in an environment, phages naturally target the most abundant member of a bacterial community. This is called Kill the Winner dynamics. However, the big picture of what viruses and especially phage are doing in the ocean is far from complete. For instance, Kill the Winner only takes into account one-to-one phage/host infectivity.

Phage lysogeny provides a “horizontal gene pool” for hosts Transduction in the environment results in the evolution of bacterial genes residing in phage and prophage genomes The host cell then expresses the successful prophage genes During the process of transduction, phages cut and package some of the bacterial genome when they resume a lytic nature. Some phage packaging proteins are notoriously sloppy and we have long since harnessed them for use in the laboratory to transfer bacterial genes via viruses. Prophage refers to the phage before it resumes a lytic nature. This close genetic relationship between phages and hosts results in pervasive horizontal gene transfer in the ocean. LYSOGENIC CYCLE

Prophage presence may change a host's phenotype Lysogenic conversion: when prophage gene expression causes drastic phenotypic change Vibrio cholerae phage CTXφ 60% of bacterial genomes contain at least one prophage Within bacterial species, the presence of one or more permanent prophage differentiates between strains Horizontal gene transfer of metabolically active genes within phages sometimes results in lysogenic conversion. For example, Vibrio bacteria with the phage encoding the cholera toxin becomes virulent. Many studies have pinpointed prophage genomes within bacterial genomes. In studies of E. coli, Salmonella, and Vibrio species, the difference between strains within a species is sometimes due to the presence or absence of a permanent prophage. It remains to be seen if this is an actual cause of strain and maybe also species differentiation, apart from ordinary vertical hereditary transfer of genetic material.

Hunting for Prophages in Bacterial Genomes Well-studied recombination reactions in phages when inserting into bacterial genome Catalyzed by phage integrases or recombinases attB attP attL attR 3 - 20 nucleotide repeats 12/25/2017

What can we do with a viral metagenomic dataset? “Classic” metagenomics tells us about viral ecology: taxonomic potential and functional role But phages coevolve with their bacterial hosts Lytic phages Lysogenic prophages Permanent prophages So we can use metagenomes to investigate both ecology and evolution of viruses Random samples are good!

Enter OMZs: Phages tend toward lysogeny in extreme environments Oxygen Minimum Zones are unique, extreme environments Normal seawater contains 5 ml/L dissolved O OMZs contain a permanent anoxic layer with <1ml/L dissolved O We sampled the Chilean OMZ which is a part of the Eastern Tropical Southern Pacific OMZ. This figure shows oxygen concentrations at 200 m worldwide in 2009.

Larger eukaryotes adapt poorly to hypoxic waters Bacteria may help establish or maintain the low oxygen conditions found in OMZs Hypothesis: lysogenic phages increase bacterial fitness in the OMZ Ulloa et al 2011 http://www.cksinfo.com/clipart/animals/wateranimals/fishes/-happy-fish_001.png

OMZ viral metagenome collection Dec 2009 Collected 80 L water at anoxic 200 m Niskin rosette Concentrated samples to 500 ml using tangential flow filtration

Prophage and viral metagenome extraction Incubation with Mitomycin C overnight 0.22 μm filtered “Prophage metagenome” extracted Incubated without Mitomycin C overnight “Viral metagenome” extracted Thurber et al. 2009 + Mitomycin C

OMZ Datasets 2008 2009 Depth St. 3 St. 5 10m or 55m 90m 200m Brazil data to come (6 more metagenomes) OMZ Datasets 2008 2009 Depth St. 3 St. 5 10m or 55m Viral 90m 200m Viral+bact Viral+prophage+bact Viral+prophage White et. al. 2012

Homology-based Analysis: viral and prophage comparisons Homology based approaches Best bidir BLAST results against Phantome database, ACLAME proteins, Integrase database Metabolism information Carbohydrate metabolism (link to anaerobic gut phages) Homology-based Analysis: viral and prophage comparisons Metagenomic reads 90% identity contigs (NEWBLER) 95% NR clusters (UCLUST) Best tBLASTx, BLASTx and psiBLAST match percentages ACLAME db, Phantome proteins, Phantome contigs, custom Integrase db Best BLASTx match percentages SEED nr, RefSeq db (MGRAST, RTMG, STAMP)

Significantly different level 2 subsystems (from BLASTx against subsystems on MGRAST at evalue<10-5). Fisher’s exact test was used to find the overrepresented functions in each metagenome (DP Asymptotic continuity correction). The viral metagenome (BLUE) and the prophage metagenome (ORANGE) are different. NOTE: excluded phage capsid and phage capsid protein functions. Viral and prophage metagenomic analyses with homology-based comparisons are a good start

Non-homology based Methods: Viral and Prophage Comparisons Non-homology based approaches Nucleotide ratio frequencies (from MetaVir) Cross assembly info (percentage assembled with bact, assembly of singletons) k-mer frequencies* Alpha/beta diversity* Non-homology based Methods: Viral and Prophage Comparisons Ecology Dinucleotide ratio frequencies (METAVIR) Comparisons with public viromes (METAVIR) Alpha/beta diversity estimates (PHACCS) Prophage metagenome is good to collect! K-mer frequencies (custom Perl scripts) Contig/cluster frequencies (custom Perl scripts) OMZ virome overlap (bidirectional BLASTn) Contig mapping from cross-assemblies (NEWBLER, CRASS)

BLAST-based Comparison of Viral and Prophage MG with other viromes (Cluster Tree)

BLAST-based Comparison of Viral and Prophage MG with other viromes (MDS plot)

A tBLASTx-based comparison between OMZ viromes from the MetaVir pipeline. A tBLASTx is a translated protein query against a translated protein subject. A similarity score was computed for each virome pair by summing the best BLAST hit scores. The scoring matrix was clustered using R and the pvclust package and an MDS plot drawn (all through the MetaVir pipeline).

Contig Coverage over depths in the OMZ – conserved gene regions

Non-homology based approaches Nucleotide ratio frequencies (from MetaVir) Cross assembly info (percentage assembled with bact, assembly of singletons) k-mer frequencies* Alpha/beta diversity* Non-homology based methods: Viral and Prophage and Bacterial Metagenomes Cross-assembly with bacterial metagenomes (NEWBLER, CRASS) Prophage insertion site identification (PhiSpy) Virome and microbiome overlap (bidirectional BLASTn) CRISPR identification (CRISPR recognition tool)

Viral genomes contain genes that may become important for bacterial survival Reyes et al, Nature 2010

Full picture: linking contribution of phage to microbial community metabolisms

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