Horizontal gene transfer of an entire metabolic pathway between a eukaryotic alga and its DNA virus

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Plankton Viruses – abundance and host mortality

“ The concentration of bacteriophages in natural unpolluted waters is in general believed to be low, and they have therefore been considered ecologically unimportant. Using a new method for quantitative enumeration, we have found up to 2.5×10 8 virus particles per millilitre in natural waters. These concentrations indicate that virus infection may be an important factor in the ecological control of planktonic micro-organisms, and that viruses might mediate genetic exchange among bacteria in natural aquatic environments.” ( from Bergh et al., 1989).
When, some twenty years ago, Bergh and his colleagues resumed their newest discovery in the paragraph transcribed above, few would have predicted that high viral abundances in seawater would gain such a profound influence on our understanding of biological oceanographic processes, evolution and geochemical cycling. A recent extraordinary extrapolation of those numbers, which takes into account the average amount of viruses (3×10 9 per l) and the total volume of the oceans (1.3×10 21 per l), predicts that the ocean waters can contain around 1030 viruses (Suttle, 2005b). This implies that, after bacteria, viruses represent the second largest carbon reservoir in the planet. Numerous studies have demonstrated that in the oceans the composition and abundance of the viral community is directly related to the dynamics of the microbial plankton (comprising hetero and auto trophic bacteria and protists) (for extensive reviews check Breitbart et al., 2007; Fuhrman, 1999; Suttle, 2005a; Suttle, 2005b; Wommack and Colwell, 2000). In general virioplankton abundance varies with depth (Hara et al., 1996), along trophic gradients (Noble and Fuhrman, 2000), and during the course of phytoplankton blooming events (Brussaard et al., 2004b; Castberg et al., 2002).
The majority of the virioplankton consists of bacteriophages, and their abundance (on average around 1010 l-1) follows the same general pattern as bacteria (Maranger, Bird, and Juniper, 1994; Wommack et al., 1992). This claim is supported by observations such as the ability of changes in bacterial abundance to predict changes in viral abundance (Hara et al., 1996), the greater abundance of bacteria over all of other planktonic hosts (Boehme et al., 1993), and the predominance of viruses within the virioplankton with bacteriophage-sized genomes (Wommack et al., 1999). Moreover, phages are estimated to be responsible for about 10-50% of the total bacterial mortality in surface waters (Fuhrman and Noble, 1995; Steward, Smith, and Azam, 1996; Suttle, 1994; Weinbauer et al., 1995).
The data relating to the abundance and impact of eukaryotic phytoplankton viruses (herein referred as algal viruses) is not as extensive as for marine bacteriophages. Nevertheless, evidence is also accumulating that viruses assume a clear role in the control of eukaryotic phytoplankton dynamics. Algal viruses have now been isolated from many geographic locations, including both freshwater and marine environments, and ranging from oligotrophic to eutrophic ecosystems, and even sediments (Brussaard et al., 2004b; Castberg et al., 2002; Cottrell and Suttle, 1991; Jacobsen, Bratbak, and Heldal, 1996; Lawrence, Chan, and Suttle, 2001; Nagasaki and Yamaguchi, 1997; Sandaa et al., 2001; Suttle and Chan, 1995). Most of the algal-virus systems in culture today correspond to large double stranded DNA viruses, which belong to the Phycodnaviridae (for an extensive review check Brussaard, 2004a). Although not as numerous yet as their DNA counterparts, RNA algal viruses have also been isolated and described (Tai et al., 2003; Tomaru et al., 2004).
The Phycodnaviridae are a diverse group of viruses, but their common ancestry is clear at the molecular level. Since the discovery that the DNA pol gene is highly conserved within this group, it became possible to design PCR primers that theoretically cover the majority of the phycodnaviruses (Short and Suttle, 1999). Using these tools several studies have demonstrated the wide distribution of the Phycodnaviridae in all studied aquatic environments (Clasen and Suttle, 2009; Short and Suttle, 2002; Short and Suttle, 2003). More recently, new metagenomic data have corroborated those results (Monier, Claverie, and Ogata, 2008; Monier et al., 2008).
Algal viruses have often been associated with the termination of phytoplankton blooms (Bratbak, Egge, and Heldal, 1993; Brussaard et al., 1996b; Castberg et al., 2001; Jacquet et al., 2002; Nagasaki et al., 1994), however there is growing evidence that, by limiting host population size, these viruses can also play a significant role in preventing the development of bloom events (Larsen et al., 2001; Suttle and Chan, 1994; Tomaru et al., 2007). A considerable decrease in photosynthetic rate was demonstrated by researchers adding natural virus concentrates to algal populations, suggesting the potential for a reciprocal viral control of global primary productivity (Suttle, 1992; Suttle, Chan, and Cottrell, 1990). Reports of viral lysis rates of phytoplankton in the field are still limited. There is evidence though that viral lysis is responsible for massive cell mortality (rates up to 0.3 d-1), particularly during the decline of algal blooms (Brussaard et al., 1996a; Brussaard et al., 1995), but also in oligotrophic ecosystems (Agusti and Duarte, 2000; Agusti and Sanchez, 2002).

Virioplankton as catalysts of global nutrient cycles

Viruses are constantly and actively influencing the marine microbial loop (Azam et al., 1994). Lytic infection of the primary producers converts cells into viruses plus cellular debris. This debris is made up of dissolved molecules (monomers, oligomers and polymers) plus colloids and cell fragments (Shibata et al., 1997), most of which is operationally defined as dissolved and particulate organic matter (P-D-OM). Most or all of the lysis products, which contain substantial amounts of major nutrients (C, N, P) and trace nutrients (e.g. Fe), will eventually become available to bacteria (Bratbak et al., 1990; Gobler et al., 1997; Middelboe et al., 2003; Poorvin et al., 2004; Proctor and Fuhrman, 1990). This will provoke an increase in bacterial production and respiration, and reduce protist and animal production, an effect called the “ viral shunt” (Fig. 1). This sequestration of materials in viru ses, bacteria and dissolved matter may lead to better retention of nutrients in the euphotic zone in virus-infected systems, because more material remains in small non-sinking forms (Shibata et al., 1997). On the other hand reduced viral activity may result in more material in larger organisms, which either sink themselves or as detritus, transporting carbon and inorganic nutrients from the euphotic zone to the deep sea (Fuhrman, 1999; Suttle, 2005b).

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Biogeochemical and ecological roles

As photosynthesizers and calcifying organisms, coccolithophores assume a rather complex and extremely important role on the regulation of the Earth’s system, mainly in what regards carbon flux between atmosphere/ocean/lithosphere (Fig. 7). Coccolithophores are unicellular photosynthetic organisms, and hence integral part of the oceanic phytoplankton. Phytoplankton uses light energy to sequester dissolved carbon dioxide (CO2) and produce particulate organic carbon (POC), and oxygen (O2). This so-called photosynthetic process [CO2+H2O CH2O (POC) + O2] participates to maintain the atmospheric CO2 concentration 150 to 220 ppmv below what it would be if phytoplankton did not exist (Falkowski et al., 2000). It is estimated that 25% of the carbon fixed by phytoplankton is exported to the deep oceans, in a total of 11 to 16 Gt of carbon per year (Falkowski, Barber, and Smetacek, 1998; Laws et al., 2000). The complex system of oceanic biological and physico-chemical processes that transport carbon from the epipelagic zone to the abyssal ocean floor is designated the “biological pump” (Vo lk and Hoffert, 1985).

Phylogeny and evolution

The phylogenetic position of the coccolithoviruses is still in debate with high uncertainty regarding its evolutionary history. Several independent phylogenetic studies (Allen et al., 2006c; Larsen et al., 2008; Schroeder et al., 2002; Wilson et al., 2006) have always placed the EhV within the family Phycodnaviridae. However, the coccolithovirus do not cluster with the other Prymnesiovirus identified to date (whose hosts are phylogeneticaly close to E. huxleyi), but instead occupy a very deep position in the phycodnavirus clade (Fig. 3). This differentiation from the other members of the Phycodnaviridae led to the creation of the new genus Coccolithovirus.
The 6 RNA polymerase subunits present in the EhV genome (unique among the known phycodnaviruses) add to the singularity of the coccolithoviruses among other algal viruses. Since ancestral NCLDV contained the RNA polymerase function, it is likely that of all the phycodnaviruses sequenced so far, EhV-86 represents the virus with the lifestyle most similar to the ancestral virus (Allen et al., 2006d). The change in lifestyle represented by this loss of RNA polymerase function (i.e. from nuclear independence to nuclear dependent transcription) probably contributes to the high diversity among present day genera in the Phycodnaviridae.

Sphingolipid biosynthesis gene sequences from E. huxleyi CCMP1516

The genome sequence data of E. huxleyi CCMP1516 strain were produced by the International E. huxleyi Genome Sequencing Consortium in collaboration with the US Department of Energy Joint Genome Institute (http://www.jgi.doe.gov/). The genome sequence data are being analyzed by the consortium members and will be published elsewhere. The amino acid sequences corresponding to the seven EhV-86 sphingolipid biosynthesis genes (Table 1) were used to identify their homologs in the E. huxleyi genome sequences, using BLASTP searches (Altschul et al., 1997) against the host’s ORFeome (the JGI reduced protein set as of April 4, 2008; E-value<10-20). For the detection of E. huxleyi 3-KSR homolog, 3-KSR homologs from green plants (Arabidopsis thaliana and Ostreococcus tauri) were used as TBLASTN queries.

PCR-amplification and sequencing of sphingolipid biosynthesis genes from host and virus strains

Six E. huxleyi and eleven EhV strains were chosen by taking into account their distant geographical origins (Table S1) and distinct behavior regarding susceptibility to EhV infection (data not shown). To extract E. huxleyi DNA, 250 ml of late exponential growing cultures were harvested by centrifugation (14000 rpm for 2 mins). A 0.5ml pellet was recovered and initially treated with proteinase K (5 mg/ml) in a lysis buffer containing 20 mM EDTA, pH 8 and 0.5% SDS (w/v) at 65 ºC for 1 h. Major cell debris was removed by adding 600 µl of phenol to each sample and centrifuging at maximum speed for 10 min. The top layer was recovered and the DNA was extracted using an equal volume of chloroform:isoamyl alcohol (24:1). The DNA was precipitated with the addition of 0.5 × volume 7.5 M ammonium acetate, pH 7.5, and 2.5 × vo lume absolute ethanol. The pellet was washed 3 times in 300 µl of ice-cold 70% ethanol, a fter which it was dried and re-suspended in 30 µl of DNase free water. The virus isolates we re directly used as DNA template for PCR without prior DNA purification. The on-line application Primer3 (Rozen and Skaletsky, 2000) was used to design primers that target homologous regions in both host and viral genes (Table S2). The PCR reaction was set up as follows: 1 µl of DNA template (extracted DNA in case of the hosts, viral isolate in the case of the virus) was added to a 25 µl reaction mixture which contained: 1 U Taq DNA polymerase (Promega), 1 × PCR reaction buffer ( Promega), BSA, 0.25 mM dNTPs, 2.5 mM MgCl2, 10 pmol of each primer. The PCR was conducted in a PTC-100™ cycler (MJ Research) with an initial denaturin g step of 95 ºC (5 min), followed by 35 cycles of denaturing at 95 ºC (60 s), annealing at 56 ºC (60 s), and extension at 74 ºC (60 s). A SequiTherm EXCEL II DNA Sequencing Kit-LC (EpicentreTechnologies) with a LI-COR Automated DNA Sequencer was used to sequence the PCR products. Obtained sequence data were deposited in GenBank (accession numbers from FJ531546 to FJ531633).

Table of contents :

Résumé (in French)
Table of contents
Avant propos
Chapter 1. Introduction 
1. Virus – life’s lubricant
2. Phycodnaviridae
3. Coccolithophores
4. Emiliania huxleyi
5. Thesis Objectives
Chapter 2. Coccolithovirus – a review
Chapter 3. Horizontal gene transfer of an entire metabolic pathway between a eukaryotic alga and its DNA virus
Supplementary data
Chapter 4. Host-virus shift of the sphingolipid pathway along an Emiliania huxleyi bloom: survival of the fattest 
Supplementary data
Chapter 5. Novel transcription features unveiled during natural coccolithovirus infection
Supplementary data
Chapter 6. Short report on attempts to isolate new coccolithophore viruses 
Chapter 7. Final discussion and perspectives 
Perspectives for future research
Annexe A. Horizontal gene transfer between Emiliania huxleyi and viruses 
Annexe B. Uncoupling of Emiliania huxleyi photosynthesis: virus infection versus nutrient stress 


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