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).
Figure 1. The “ viral shunt” . Energy, in the form of fixed carbon, is provided to the marine environment via photosynthesis by the primary producers. The fixed carbon, or photosynthate, supports new biomass and respiration of the primary producers. In turn, the primary producers are consumed by grazers (copepods, fish, etc.), who are eaten by bigger predators. A significant amount of photosynthate is also released as particulate and dissolved organic matter (P-D-OM), which supports heterotrophic microbial growth (both bacteria and archaea). The viruses and protists kill similar proportions of the microbes, and the lysed cells then join the P-D-OM pool, which feeds more heterotrophic microbes. The result is more carbon respired, thereby increasing the trophic transfer efficiency of nutrients and energy through the marine foodweb. Adapted from Suttle (2005).

Viral influence in phytoplankton community composition

It is evident, from their effect on algal blooms and cyanobacteria, that viruses are also in a unique position to influence community species composition. Even if viruses were to cause only a small proportion of the mortality of a group of organisms, they could still have a profound effect on the relative proportions of different species or strains in the community (Hennes, Suttle, and Chan, 1995; Waterbury and Valois, 1993). Considering that viral infection is density dependent and that the majority of marine viruses appear to have narrow host specificity, then a particular species or strain becomes more susceptible to infection as its density increases. This may help explaining Hutchinson’s “ paradox of plankton” on the coexistence of unexpected phytoplankton diversity (Hutchinson, 1961). Competition theory would predict just one or a few competitive winners, however viral activity probably assists because the competitive dominants become particularly susceptible to infection, whereas rare species are relatively protected (Fuhrman and Suttle, 1993). With this “ killing the winner” strategy (Thingstad, 2000) viruses become a driving force for community composition and succession, both at the interspecific (Brussaard et al., 2005; Castberg et al., 2001; Larsen et al., 2001) and intraspecific (Martinez-Martinez et al., 2006; Muhling et al., 2005; Tarutani, Nagasaki, and Yamaguchi, 2000) levels.

Viruses and genetic exchange

Virus-host interaction is often promiscuous at the genetic level, a situation that creates a different opportunity for marine viruses to affect genetic exchange in the oceanic realm. This can happen between virus and cellular organisms (direct hosts or not), and among different viruses (especially in situations of co-infection). Recognizing the magnitude and characteristics of horizontal gene transfers (HGT) in the oceans is important from an ecological point of view, and in our case especially important when trying to incorporate viral impact factors in models that try predict phytoplankton dynamics.
HGT can happen during the course of both lysogenic and lytic viral infections. A persistent virus has its genome incorporated in the genome of its host “waiting” for a stimulus that will trigger a lytic infection. At that moment new virions are formed and passed onto new host cells. To present date, and to the author’s knowledge, plankton viruses with lysogenic strategies have only been documented in marine phages. The occurrence of lysogeny in freshwater filamentous cyanobacteria has been known for more than 35 years (Padan, Shilo, and Oppenhei.Ab, 1972), but only now are we starting to understand the real magnitude of this phenomenon. The generalized occurrence of lysogeny involving marine phages has been extensively documented (Jiang and Paul, 1996; Jiang and Paul, 1998a; Jiang and Paul, 1998b; McDaniel, delaRosa, and Paul, 2006; Weinbauer and Suttle, 1996; Weinbauer and Suttle, 1999). Recent estimates point to roughly half of marine bacterial isolates containing prophages (Paul, 2008).
HGT can also occur between virus and host in the course of lytic infections. Such situations are usually denounced by close phylogenetic identity between host and virus homologous genes, confirming that either the viruses “stole” the genes from its host, or vice-versa. Evidence for this type of “direct” HGT is be coming more and more abundant with the progressive sequencing of genomes belonging to marine organisms and their respective viruses. As for lysogeny, it was with phages and their prokaryote hosts that the first evidence started to appear. One of the clearest and most interesting examples regards the cyanophages and their photosynthesis genes. Cyanophages infect the abundant cyanobacterial genera, Synechococcus and Prochlorococcus. Sequencing of these viral genomes showed that they commonly carry genes involved in photosynthesis (Lindell et al., 2004; Mann et al., 2005; Millard et al., 2004). These genes include the highlight-inducible (hli) gene, as well as psbA and psbD, which encode the photosystem II (PSII) core reaction-centre proteins D1 and D2, respectively (Sullivan et al., 2005; Sullivan et al., 2006). The D1 protein is of particular interest because it is the most labile protein in PSII and the most likely to be rate limiting. During the lytic cycle, most of the host’s transcription and translation is shut down by phage, which replaces like for like function with its own virally encoded proteins. Because phage must maintain the proton motive force if they are to lyse the host, they need to prolong photosynthesis during the infection cycle. Thus, the cyanophage-encoded D1 proteins are expressed during the infection cycle, countering the virally induced decline in host gene expression (Clokie et al., 2006; Lindell et al., 2005). It is thought that by encoding psbA and other genes involved in photosynthesis, phages manipulate their host systems to generate the energy necessary for viral production. Still concerning cyanophages Sullivan and co-workers (2005) have also demonstrated the presence of an aldolase family gene (talC), that could facilitate alternative routes of carbon metabolism during infection; and phosphate-inducible genes (phoH and pstS), that are likely to be important for phage and host responses to phosphate stress, a commonly limiting nutrient in marine systems.
Regarding eukaryotic phytoplankton, examples of direct HGT are also starting to appear. Sequencing of the nucleo-cytoplasmic large DNA virus (NCLDV) Emiliania huxleyi Virus (EhV) revealed the presence of some unexpected genes. The most striking example is a unique sphingolipid biosynthesis pathway (SBP) (Wilson et al., 2005b), which was later concluded to be imported from its host Emiliania huxleyi (Monier et al., 2009). Sphingolipids are membrane lipids present in all eukaryotes and some prokaryotes. The SBP can ultimately lead to the production of ceramide, a central molecule often involved in signal transduction and control of cell death, namely apoptosis mechanisms (Hannun, 1996; Hannun and Obeid, 1995; Hannun and Obeid, 2002; Pettus, Chalfant, and Hannun, 2002). Other examples of viral control of host apoptosis have already been documented (McLean et al., 2008; Roulston, Marcellus, and Branton, 1999). When the new viral SBP was discovered hypotheses were immediately drawn on the possibility of EhV using its own virally encoded SBP to control the death of its host. An important part of this thesis works focused precisely on trying to explain the origin and function of this EhV metabolic pathway.
Growing evidence of HGT events involving viruses and cellular organisms other than their direct hosts, so called indirect transfer, is also accumulating. The most notable examples regard bacterial-like genes present in protist and metazoan viruses (Dunigan, Fitzgerald, and Van Etten, 2006; Iyer et al., 2006; Suzan-Monti, La Scola, and Raoult, 2006). Possible explanations for the mechanisms involving this type of genetic transfer are still rudimentary. A recent study from Fillée et al. (2008) has provided some clues. Partial results suggest that indirect HGT seems to be more frequent in viruses whose eukaryotic hosts graze on bacteria. Chlorella and Mimivirus (whose hosts feed on bacteria), and EhV and EsV (which infect free leaving microalgae that do not graze on bacteria) show marked variation in bacterial-like genes. While there is a general increase in bacterial gene number with genome size, the strongest dichotomy appears between the Chlorella Phycodnaviruses and Mimivirus, which are considerably enriched for bacterial genes, in contrast to Phycodnaviruses EhV86 and EsV-1 which are not. Moreover, very few mobile genetic elements (MGE) of bacterial origin could be found in these latter two algal viruses (Filee, Pouget, and Chandler, 2008).
The development of new metagenomic sequencing techniques has brought the study of HGT to a new level. A considerable portion of the genes present in the viromes analysed so far share very close homology with genes found in both eukaryotic and prokaryotic databases. A metagenomic study of 9 biomes, in which 42 distinct viromes were characterized, found that all the functional diversity present in the microbial metagenomes was also present in the viromes (Dinsdale et al., 2008). A striking example was the totally unexpected discovery of motility related genes present in the viromes. It also became clear that the acquisition of these proteins by the viral community was not random. For example, in the viromes, flagellar biosynthesis protein FlhA, the chemotaxis response regulator proteins CheA and CheB and deacylases were overrepresented when compared to their presence in the microbial genomes. In another study Sharon et al. (2007) reported that up to 60% of the psbA genes in surface water are of phage origin. Moreover, phage genes were shown to be undergoing an independent selection for distinct D1 proteins, and also different viral psbA genes are being expressed in the environment. Recently, it was demonstrated that photosystem 1 gene cassettes are also present in cyanophage genomes (Sharon et al., 2009). Regarding eukaryotic hosts Monier et al. (2007) analysed a large dataset of Large Eukaryotic DNA Virus genomes and reported the presence of many genes putatively associated with the control of host defence systems, such as innate/adaptive immune systems or apoptosis pathways.
All this evidence adds further credence to the idea that viral communities represent reservoirs of genetic diversity, with viruses themselves serving as potential vectors of genetic information among host communities and ecosystems. HGT are rather rare events on an individual scale, but analysed on a global planetary scale this phenomenon assumes a totally different magnitude. Fuhrman (1999) proposed an exercise to infer global oceanic HGT frequency involving marine bacteria. Considering the great abundance of potential cellular hosts (typical bacterial abundances, for example, are around 109 l-1 in the euphotic zone) and huge volume of the sea (~3.6×10 7 km3 in the top 100 m), coupled with generation times on the order of a day, implies that an event with a probability has low as 10-20 per generation would be occurring about a million times per day.
On the other hand the relevance of HGT between virus and their hosts is also under scrutiny from an evolutionary perspective. As mentioned previously, the origin of viruses and cells has been under intense debate, especially after the discovery of large DNA viruses such as the EhV or the Mimivirus. One hypothesis proposes that these viruses represent ancient cellular forms that gained viral form by progressive loss of genes (Claverie, 2006; Suzan-Monti, La Scola, and Raoult, 2006). Along similar lines of thought hypotheses have been drawn that viruses may have appeared before the separation of the current cellular domains, and consequently influenced the entire evolution of life as we know it (Forterre, 2006a; Forterre, 2006b; Forterre and Gadelle, 2009; Hendrix, 1999). Other authors propose that large DNA viruses are the result of a tendency to indiscriminately acquire genes from all different “horizontal” sources (direct hosts or not) (Koonin, 2005; Moreira and Brochier-Armanet, 2008; Moreira and Lopez-Garcia, 2005). On the contrary according to Monier et al (2007) despite the fact that HGT events play a significant role in the dynamics of gene transfer between the different reservoirs of genetic diversity in the oceans, such events still account for only a minority of the gene composition found in most viruses. This observation suggests that the extremely large sizes of the genomes of some large viruses (for example the Mimivirus) are not due to recent accretion of foreign genes. By extrapolation, the capacity to capture foreign genes is unlikely to be the major factor that determines the tremendous variation in genome size for DNA viruses (Claverie et al., 2006).
Clearly viral HGT, its magnitude and impacts, remain a very hot topic in today’s virology. The recognition of HGT events is highly dependent on the capacity of recognizing homologies between potentially phylogeneticaly close DNA sequences. To that extent we must not forget that the great majority of the genes present in NCLDV genomes, or in the viral metagenomic databases, remain of unknown function given their dissimilarity with the actual characterized genetic diversity (for example see Raoult et al., 2004; Wilson et al., 2005b). This situation can be the result of a very old origin and/or rapid parallel evolution of viral genes. Hence, even if for a few genes the probability of correctly identifying HGT events is high, the reality is that on the whole it remains difficult to determine the extent of HGT events in these large viral genomes.

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The phycodnaviruses are a family of large dsDNA viruses that infect a very diverse group of aquatic eukaryotic organisms. The phycodnaviruses isolated and characterized so far infect different protist lineages comprising green algae, haptophytes, and stramenopiles, as well as multicellular organisms belonging to the brown algae group. They are generally very large viruses that contain also some of the largest viral genomes ever found. Among the phycodnaviruses we find Emiliania huxleyi Virus, which has been the central object of study throughout this thesis.

Taxonomy and distribution

The phycodnavirus group comprise a genetically diverse (Dunigan, Fitzgerald, and Van Etten, 2006; Iyer et al., 2006), yet morphologically similar, family of large icosahedral viruses that infect marine or freshwater eukaryotic organisms. Their big dsDNA genomes can range from 180 kb to 560 kb (Van Etten et al., 2002). To present date members of the Phycodnaviridae are grouped into six genera (named after the hosts groups they infect): Chlorovirus, Coccolithovirus, Prasinovirus, Prymnesiovirus, Phaeovirus, and Raphidovirus (Table 1). We should also mention here the mimivirus group. These are huge dsDNA viruses (genome reaching up to 1.2 Mb) that, so far, have been found to infect amoeba (Raoult et al., 2004). Even if their potential hosts are not algae, cumulating evidence indicates that they occupy a phylogenetic position within the phycodnaviridae (Larsen et al., 2008; Monier et al., 2008; Wu et al., 2009). From herein in this text, all mentions to phycodnaviruses should be regarded as that wider group that includes also the mimivirus.
Phycodnaviruses are widely distributed in nature. Viral isolates have been obtained from eutrophic and oligotrophic water masses, and even sediments (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). These probably represent only a tiny fraction of the enormous diversity of the existing phycodnaviruses. Other culture independent techniques have allowed a glimpse into the magnitude of their variability and dispersion. Given their large size phycodnaviruses can be identified and quantified using flow cytometric techniques (Brussaard, 2004b). Marie and colleagues (Marie et al., 1999) recurred to such techniques to show that a clearly distinct group of phycodnaviruses was always present in sea water samples from mesotrophic through oligotrophic environments. Moreover, genetic fingerprints based on polymerase chain reaction (PCR), denaturing gradient gel electrophoresis (DGGE), and metagenomic sequencing reveal that phycodnaviruses are very diverse and a regular component of all aquatic environments (Chen and Suttle, 1995; Chen, Suttle, and Short, 1996; Larsen et al., 2008; Monier, Claverie, and Ogata, 2008; Short and Suttle, 2002; Short and Suttle, 2003).
To date several phycodnavirus genomes have been completely sequenced. They correspond to representatives of the genera chlorovirus (Zhang et al., 1994), coccolithovirus (Wilson et al., 2005b), phaeovirus (Delaroque et al., 2001; Schroeder et al., 2009), prasinovirus (Derelle et al., 2008; Weynberg et al., 2009), and mimivirus (Raoult et al., 2004). Evolutionary analysis of their genomes places them within a major, monophyletic assemblage of large eukaryotic dsDNA viruses termed the Nucleo-Cytoplasmic Large DNA Viruses (NCLDVs) (Fig. 2) (Allen et al., 2006c; Derelle et al., 2008; Iyer, Aravind, and Koonin, 2001; Iyer et al., 2006; Raoult et al., 2004). Five families are currently attributed to the NCLDVs clade, including Poxviridae, Iridoviridae, Asfarviridae, Phycodnaviridae. The inclusion of the phycodnaviruses within the NCLDVs is significant for, as the name suggests, it implies a likely propagation mechanism where replication would initiate in the nucleus, and be completed in the cytoplasm (Iyer et al., 2006; Raoult et al., 2004; Villarreal and DeFilippis, 2000). A total of nine gene products are present in all NCLDVs identified to date, and 33 more gene products are present in at least two of these five viral families (Iyer, Aravind, and Koonin, 2001; Raoult et al., 2004). Phylogeny of the NCLDVs constructed by cladistic analysis indicates that the major families may have diverged prior to the divergence of the major eukaryotic lineages 1-2 billion years ago (Iyer et al., 2006; Raoult et al., 2004). Regarding the Phycodnaviridae, the finding that only 14 genes (from a pool of approximately 1000 genes) are shared between three genomes from different genera (chlorovirus, coccolithovirus and phaeovirus) supports the idea that these groups also diverged a long time ago (Allen et al., 2006c).

Table of contents :

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
Chapter 4. Host-virus shift of the sphingolipid pathway along an Emiliania huxleyi bloom: survival of the fattest 94
Chapter 5. Novel transcription features unveiled during natural coccolithovirus infection
Chapter 6. Short report on attempts to isolate new coccolithophore viruses 
Chapter 7. Final discussion and perspectives 
Perspectives for future research


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