Coccolithovirus Ehv-84

Standards in Genomic Sciences (2011) 5:1-11 DOI:10.4056/sigs.1884581 Draft genome sequence of the coccolithovirus EhV-84 Jozef I. Nissimov1, Charlotte A. Worthy1,2, Paul Rooks1, Johnathan A. Napier2, Susan A. Kimmance1, Matthew R Henn3, Hiroyuki Ogata4, Michael J. Allen1* 1Plymouth Marine Laboratory, Prospect Place, The Hoe, Plymouth, PL1 3DH, UK 2Department of Biological Chemistry, Rothamsted Research, Harpenden, Herts AL5 2JQ 3The Broad Institute of MIT and Harvard, Cambridge, Massachusetts, United States of America 4Structural and Genomic Information Laboratory, CNRS-UPR2589, Mediterranean Institute of Microbiology (IFR-88), Aix-Marseille University, 163 avenue de Luminy Case 934, FR- 13288 Marseille, France *Corresponding author: Michael J. Allen ([email protected]) Keywords: coccolithovirus, marine, phycodnavirus, algae, virus The Coccolithoviridae is a recently discovered group of viruses that infect the marine coccoli- thophorid Emiliania huxleyi. Emiliania huxleyi virus 84 (EhV-84) has a 160 -180 nm diameter icosahedral structure and a genome of approximately 400 kbp. Here we describe the structural and genomic features of this virus, together with a near complete draft genome sequence (~99%) and its annotation. This is the fourth genome sequence of a member of the coccolithovirus family. Introduction Coccolithoviruses infect the cosmopolitan marine 2005 to reveal a genome of 407,339 bp. Two fur- microalgae, Emiliania huxleyi [1]. These algae are ther strains, EhV-163 and EhV-99B1 were isolated capable of forming vast blooms which can be seen in 2000 and 1999 respectively from a Norwegian from space and can cover up to 100, 000 km2 oc- fjord and have had their partial genomes also se- curring in the top 50-100 m of the water column, quenced [7,8]. All coccolithoviruses known to date with a cellular density of more than a million cells have been isolated from the English Channel and a per liter of seawater [2]. E. huxleyi has become a Norwegian fjord. Here we present a summary species crucial to the study of global biogeochemi- classification and a set of features for coccolithovi- cal cycling [3-5]. The elegant calcium carbonate rus strain EhV-84, the second English Channel scales (known as coccoliths) which it produces coccolithovirus sequenced, together with the de- intracellularly and the scale of its blooms have scription of the sequencing and annotation of its made E. huxleyi an essential model organism for genome. marine primary productivity and global carbon cycling [6]. Coccolithoviruses have been shown to Classification and features be a major cause of coccolithophore bloom termi- All coccolithoviruses to date have been isolated nation and their pivotal role in global biogeochem- from E. huxleyi algal blooms in temperate and sub ical cycling has gained increasing attention. Cocco- temperate oceanic waters. Maximum likelihood lithovirus abundances typically reach 107 per ml phylogenetic analysis of available DNA polyme- in natural seawater under bloom conditions and rase gene sequences (DNA pol), one of the viral 108 -109 per ml under laboratory culture. The kingdom’s phylogenetic markers (equivalent to model coccolithovirus strain EhV-86 (AJ890364), 16S rDNA sequences in bacteria) indicates that the and two other similar but genetically distinct closest related viral strain to EhV-84 is EhV-86 strains, EhV-84 and EhV-88 were isolated in 1999 and EhV-88 (Figure 1). Both of these strains were from a coccolithophore bloom in the English isolated from the English Channel in the same year Channel. EhV-86 was sequenced in its entirety in The Genomic Standards Consortium Coccolithovirus EhV-84 as EhV-84 [13]. The English Channel EhVs that (PBCV-1), Micromonas pusilla virus SP1 (MpV- were isolated in 1999 (EhV-84, EhV-86 and EhV- SP1), Chrysochromulina brevifilum virus PW1 88) are more similar to other strains from the (CbV-PW1), Ectocarpus siliculosus virus 1 (EsV-1), English Channel such as EhV-201, EhV-203, EhV- Heterosigma akashiwo virus 01 (HaV-01) are in- 207 and EhV-208 isolated two years later in 2001, cluded here as an additional reference and they than strains such as EhV-163 and EhV-99B1 that cluster outside the EhVs genus. The EhV-84 virion are from a different geographical location; i.e. a structure has icosahedral morphology, a diameter Norwegian fjord. Interestingly EhV-202 seems to of 160 -180 nm (Figure 2), and is similar to other be the most different of all strains sequenced to coccolithoviruses (and phycodnaviruses in gener- date and this is also evident from full genome se- al) [14]. Isolation and general phylogenetic cha- quencing (data not published). Other algal viruses racteristics are outlined in Table 1. such as Paramecium bursaria Chlorella virus Figure 1. Multiple Sequence Alignment of the DNA pol (DNA polymerase) gene of ten coccolithoviruses (EhVs) and five other algal viruses. The evolutionary history was inferred using the Neighbor-Joining method [9]. The bootstrap consensus tree inferred from 1000 replicates is taken to represent the evolutionary history of the taxa analyzed [10]. The per- centage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) are shown next to the branches when greater than 50% [10]. The evolutionary distances were computed using the Maximum Composite Likelihood method [11] and are in the units of the number of base substitutions per site. All positions contain- ing gaps and missing data were eliminated from the dataset (Complete deletion option). Phylogenetic analyses were conducted in MEGA4 [12]. 2 Standards in Genomic Sciences Nissimov et al. Table 1. Classification and general features of Emiliania huxleyi virus 84 according to the MIGS recommendations [15]. MIGS ID Property Term Evidence code Domain: Viruses, dsDNA viruses, no RNA stage TAS [16] Class: NCLDNA (Nucleo-Cytoplasmic Large DNA) TAS [16] Current classification Family: Phycodnaviridae TAS [16] Genus: Coccolithovirus TAS [16] Species: Emiliania huxleyi virus 84 TAS [16] Virion shape Icosahedral IDA MIGS-6 Habitat Oceanic, Coastal TAS [16] MIGS-15 Biotic relationship Obligate intracellular parasite of Emiliania huxleyi TAS [16] MIGS-14 Pathogenicity Lytic virus of Emiliania huxleyi TAS [16] MIGS-4 Geographic location English Channel, UK TAS [16] MIGS-5 Sample collection time July 26, 1999 TAS [16] MIGS-4.1 Latitude 50.15 N TAS [16] MIGS-4.2 Longitude 4.13 E TAS [16] MIGS-4.3 Depth 15 m TAS [16] Evidence codes - IDA: Inferred from Direct Assay; TAS: Traceable Author Statement (i.e., a direct report exists in the literature); NAS: Non-traceable Author Statement (i.e., not directly observed for the living, isolated sample, but based on a generally accepted property for the species, or anecdotal evidence). These evidence codes are from the Gene Ontology project [17]. Figure 2. Transmission electron micrograph of an EhV-84 virion. http://standardsingenomics.org 3 Coccolithovirus EhV-84 Genome sequencing and annotation Genome project history The Marine Microbiology Initiative (MMI) of the for sequencing on the basis of its global impor- Gordon & Betty Moore Foundation aims to gener- tance in the demise of E. huxleyi blooms [13], the ate new knowledge about the composition, func- horizontal gene transfer events observed in other tion, and ecological role of the microbial commun- coccolithovirus genomes [18], the metabolic po- ities that serve as the basis of the food webs of the tential displayed by its large genome size and its oceans and that facilitate the flow of nitrogen, car- possible manipulation of signaling pathways such bon, and energy in the ocean. In an effort to un- as programmed cell death in its host organism derstand the ecology and evolution of marine [8,19]. phage and viruses and to explore the diversity and The genome project is deposited in the The Inte- ecological roles of entire phage/viral communities grated Microbial Genomes (IMG) system and the through metagenomics, the Broad Institute colla- complete genome sequence and annotation are borated with MMI and researchers whose se- available in GenBank (JF974290). Genome sequencing nominations were chosen by the Marine quencing, finishing and annotation were per- Phage, Virus, and Virome Selection Committee to formed by the Broad Institute. A summary of the generate genomic sequence and annotation of eco- project information is shown in Table 2. logically important phage. EhV-84 was nominated Table 2. Genome sequencing project information MIGS ID Property Term MIGS-31 Finishing quality Finished (>99%) Number of contigs 9 Average contig size 43,980 Largest contig size 97,445 Assembly size (using large contigs) 395,820 Assembly coverage ("peak Depth") 36.16 Total number of reads used 28,526 MIGS-29 Sequencing platforms 454 MIGS-30 Assemblers Newbler Version 2.3 PostRelease-11.19.2009 MIGS-32 Gene calling method Broad Institute Automated Phage Annotation Protocol [20] GenBank ID JF974290 GOLD ID N/A Gordon & Betty Moore Foundation's Marine Microbiology Project relevance Initiative. Emiliania huxleyi virus 84- G3248. Growth conditions and DNA isolation Genome sequencing and assembly Emiliania huxleyi strain CCMP 2090 was grown in The genome of strain EhV-84 was sequenced us- 1 liter cultures (f/2 nutrient media) in the labora- ing the 454 FLX pyrosequencing platform tory under a light/dark cycle of 16/8 respectively, (Roche/454, Branford, CT, USA). Library construc- at a temperature of 16°C. Once the cultures were tion, and sequencing were performed as previous- at mid exponential growth (i.e. 4 × 106 ml-1), they ly described [20]. General protocols for library were infected with an EhV-84 lysate at an MOI ra- construction can be found at [22]. De novo genome tio of 1:1. Infection, host death and viral produc- assembly of resulting reads was performed using tion were confirmed by flow cytometry. Fresh vi- the Newbler v2.3 assembly software package as rus lysate was filtered through a 0.2 µm pore 47 previously described [20].

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