Phages are viruses that specifically infect bacteria and were first discovered early in the 20th century. Initial interest was focused on the potential for using phages as therapeutic tools in the fight against bacterial infectious diseases (Bordet, 1925). Because of the specificity of host-virus interactions, they were an important component to the development of modern molecular biology (Safferman et al., 1965; Mann, 2005). In recent years, attention has been diverted to studies in aquatic environments, with phages being recognized as major players in the oceanic biogeochemical cycles (Wilhem and Suttle, 2002), representing the greatest potential genetic resource in the biosphere.
Microcystis spp. (Kützing ex Lemmermann 1907) has been one of the most well-studied members of the Cyanobacteria group. The fact that they are ubiquitous in most aquatic environments where bloom episodes with potential toxin (microcystin) production occur (often in freshwater lakes and reservoirs, Carmichael, 1992; Chorus and Bartram 1999), clearly justifies the high amount of research on the ecology of this Genus (Long et al,. 2001; Maria et al., 2003; Stone and Bress, 2007; Liu et al., 2008). Surface water sources are major contributors for drinking water production worldwide, and Microcystis spp. bloom episodes have been reported as source of adverse effects in water quality, impacting both ecological quality and diverse human activities (Chorus and Bartram 1999; Burns, 2008). Consumption of water contaminated with microcystin (i.e. hepatoxin) release/produced by Microcystis spp. has been proven to cause human death and liver chronic disease (Falconer and Baresford 1983; Teixeira and Costa, 1993; Azevedo and Carmichael, 2002), and other harmful effects have been reported such as livestock, wildlife and fish kills (Olson, 1960; McBarron and May, 1966; Pearl 1988). Whereas most studies have been focused on environmental factors contributing to Microcystis spp. ecology and bloom dynamics, some attention has been given to studying biological parameters ((Paerl, 1988; Orr and Jones, 1998; Bertasi et al., 2003). Predation and competition have been studied (Yang et al., 2004), and there are recent works reporting lytic cyanophage activity controlling Microcystis spp. populations (Pathmalal et al., 2001; Tucker and Pollard, 2005; Yoshida et al., 2006).
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Aquatic research has shown that viruses represent a significant and dynamic component of microbial communities: their number generally exceeding the number of bacteria by 10 to 100 times or more (Proctor and Furhman, 1990; Furhman and Noble, 1995; Cochran and Paul, 1998). Around 1989, transmission electron microscopy (TEM) was used to show that viruses were more abundant than previously expected in pelagic aquatic systems (Proctor and Furhman, 1990). Fuhrman et al. (1999) provides an excellent review of the decade of aquatic viral discovery, attending to concentrations of viruses found in the 1990's and the roles of viruses assessed through experimental manipulation (e.g. Fuhrman and Noble, 1995). Since then, other review articles have emerged, some discussing the roles of aquatic viruses in climate change and oceanic mixing and transport.
Viruses are ubiquitous, abundant and temporally dynamic members of aquatic communities. Since they depend upon their hosts for replication, the relative abundance of specific virus types roughly parallels that of the organisms they infect (Hambly and Suttle, 2005).There is more biological diversity within viruses than in all the rest of the bacterial, plant, and animal kingdoms put together (principles of virology chapter 1, 4th edition). This is the result of the success of viruses in parasitizing all known groups of living organisms, and understanding this diversity is the key to comprehending the interactions of viruses with their hosts.
The host DNA is digested
New phage DNA forms using nucleotide from former host DNA
The host cell transcribes the phage DNA and translates viral RNA produging proteins
Assembly of new phage is complete. A phage-encoded enzyme causes the host to lyse
New phages are released to start a new infection cycle
The chromossome and the integrated prophage replicates. This can go on through many cell divisions
Triggers induce prophage excision: the host enters the lytic stage
The phage DNA integrates into host' chromossome becoming a nonreactive prophage
The bacteriophage binds to the bacteria (host)
The phage DNA enters the host cell
Always on Time
Marked to Standard
Figure 1.1 - Bacteriophage life cycle strategies (modified from Sadava et al., 2009).
It is a well-known fact that viruses have complex life cycles (Figure 1) and that the relation between host and the phage is diverse, having different degrees of complexity.
Bacteriophages are diverse and distinct types of phage virions may carry single- or double-stranded dsDNA (double stranded DNA) or RNA, and the details of their replication cycles reflects this diversity (Casjens, 2003). The dsDNA phages can be divided into lytic and temperate virus groups, each of which is extremely diverse by itself.
Lytic dsDNA phages infect bacterial cells and at different degrees of complexity. Bacteriophages are widely diverse and distinct types of phage virions may carry single- or double-stranded dsDNA (double stranded DNA) or RNA, and the details of their replication cycles reflect this diversity (Casjens, 2003). The dsDNA phages can be divided into lytic and temperate virus groups, each of which is extremely diverse by itself. Lytic dsDNA phages infect bacterial cells and always program the synthesis of progeny virions, which are then released from the infected (dead) host cell. In contrast, temperate dsDNA phages are able to establish a stable relationship with their host bacteria in which the phage DNA is replicated. This process is done in concert with the host's chromosome, and virus genes that are disadvantageous to the host are not expressed. Such long-term association of bacteriophages with bacterial cells was first described in the 1920s (Bordet, 1925), but its acceptance and the understanding of the real nature of this association was only achieved latter on (Lwoff, 1953; 1966). Succeeding work has shown that, during this process phage DNA is integrated into one of the native replicons of the host (Campbell, 1962; Freifelder and Meselson, 1970). Lysogeny is defined as the process whereby many phages develop a symbiotic relationship with their hosts (Ackerman and Dubow, 1987) and phages capable of such process are named temperate phages reflecting their capability of establishing such silent infections by integration of their genomes (as prophages) into one of the host's replicons (Paul, 2008). It should be noted however that most research within the aquatic viral ecology field has been devoted to the role and diversity of lytic viruses in aquatic environments.
Cyanophages are viruses that infect cyanobacteria, which are prokaryotic micro-algae that can form noxious blooms in lakes and reservoirs. Cyanobacteria are potentially toxigenic, producing potent hepatotoxins, called microcystins (MCs) (Carmichael, 1996), that have caused many cases of animal and human poisoning (Pouria et al., 1998). Previously, most studies have focused on relationships among the cyanobacteria bloom dynamics and the changes in physicochemical factors promoting cyanobacteria growth in the aquatic environment (Reis, 2005; Galvão et al., 2008).The major bloom-forming cyanobacterial species Microcystis aeruginosa forms noxious blooms in many eutrophic freshwater lakes, ponds, and reservoirs. Microcystis aeruginosa strains have the potential for the production of potent hepatotoxins called MCs within the environment (Carmichael, 1996). These potent toxins have caused many cases of animal and human poisoning (Pouria et al., 1998). Previously, most studies have focused on relationships among the cyanobacterial bloom dynamics and the changes in physicochemical factors (nutrients, light, and temperature) that influence cyanobacterial growth in the aquatic environment (Whitton and Pots, 2000; Reis, 2005; Galvão et al., 2008).
Since the discovery that viruses are widespread in marine ecosystems (Bergh et al., 1989), cyanophages that can infect cyanobacteria have been thought to be an alternative factor that may control the succession of cyanobacterial blooms (Suttle, 2000; Mann, 2003; Okunishi et al., 2003). In addition, cyanophages can also influence the clonal composition of the host Synechococcus communities (Waterbury and Valois, 1993) and could account for some of the cyanobacterial diversity observed in natural communities (Wommack and Colwell, 2000). Nevertheless, little is known about how freshwater cyanophages can affect the abundance and clonal composition of cyanobacterial blooms in lakes over time (Yoshida et al., 2008). The mechanisms of virus-host interactions are missing from our present understanding of lakes ecosystems processes (Yoshida et al., 2007, 2008c).
Prior to 2005, only a very few phage strains, including SM-1 and SM-2, were reported to be lytic for M. aeruginosa (Safferman et al., 1969; Fox et al., 1976). In spite of such promising reports, the M. aeruginosa NRC-1 strain reported to be sensitive to SM-1 and SM-2 was later found to be a Synechococcus strain, so SM-1 and SM-2 are phages that infect Synechococcus sp.Phillips et al., (1990) described the isolation of a lytic agent that formed plaques on lawns of an M. aeruginosa strain but this agent was never identified.
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Thus, the cyanophages that infect M. aeruginosa have not been characterized or cultured previously to the work of Tucker and Pollard (2005). These authors identified two types of Podovirus-like particles that inhibited growth of M. aeruginosa in natural lake samples collected during a M. aeruginosa bloom. Later on, Yoshida et al., (2006) reported the first isolation and characterization of a cyanophage Ma-LMM01 (M. aeruginosa Lake Mikata Myoviridae 01, according to the nomenclature for cyanophages proposed by Suttle, 2000). The Ma-LMM01 phage particles contain a linear dsDNA of about 165 kb and four major polypeptides of 84, 47, 38, and 26 kDa by using SDS-PAGE (Yoshida et al., 2006). Results from whole genome sequencing further revealed the presence of a homologue of a site specific recombinase used by temperate phages to integrate the phage genome into the bacterial chromosome (Groth and Calos, 2004) and also prophage antirepressor genes. Yoshida et al., 2008a argue that this cyanophage could display a prophage state in some hosts, but also that transfer of those genes by transposable elements could be possible. The same authors have been developing research on this single cyanophage-M. aeruginosa pair (Yoshida et al., 2008b), but the fact that the isolated phage only infects a single host strain, somehow refrains extrapolations of this singular relationship to unknown ecological relationship's amongst cyanophages and respective Microcystis spp. hosts. Ma-LMM01 is a dsDNA virus with a contractile tail therefore placed onto the Myoviridae family (Yoshida et al., 2008a). This viral family processes at least six subgroups (T4-, P1-, P2-, Mu-, SPO1-, and H-like phages) (Fauquet et al., 2005) that exhibit high genomic diversity.
Given that only one lytic cyanophage has been sequenced, even less is known about triggers potentially inducing temperate phages in Microcystis spp. Extensive research has been done concerning induction of temperate phages in Synechococcus spp. from marine and estuarine systems. However no references were found concerning testing commonly used triggers for temperate phage induction in Microcystis spp.. There have been a few reportson triggers of temperate phage in filamentous cyanobacteria (e.g. Rimon and Oppenheim, 1975), and more recently work done in marine unicellular cyanobacteria (Synechococcus spp.) (Ortmann et al., 2002). Given the fact that induction of temperate phages by physical and/or chemical triggers could potentially represent an important contributor for the regulation of Microcystis spp. in natural environments, it is remarkable the lack of studies devoted to these issues.
Overall, the ecological impact of cyanophages on Microcystis spp. is not clear nevertheless, reports have suggested that phage may play an important role in regulating bloom dynamics. For instance, Manage et al., (1999) observed that an increase in cyanophage titers was accompanied by a large decrease in the abundance of M. aeruginosa in a natural freshwater environment.
Unraveling the biology of phages and their relationship with their hosts is mandatory to understanding microbial systems and their exploitation (Clokie et al., 2011). Analysis of host-phage systems and response of Microcystis strains to temperate phage induction triggers is expected to increase our understanding of the ecology and physiology of toxic cyanobacterial blooms.
1.1 - Objectives
The main goal of this dissertation was to develop an understanding of the role of viruses in Microcystis bloom regulation, specifically the previously poorly investigated temperate phages. A major objective was to isolate and characterize the relationship between cyanophages and host: Microcystis spp. This main goal results from previous work (Reis, 2005; Galvão, et al., 2008) demonstrating that Microcystis blooms in Algarve, Portugal freshwater reservoirs are not bottom-up regulated.
To accomplish this objective, in a globally meaningful way, the following specific goals were achieved:
Isolate and characterize a small uni-algal Microcystis collection;
Compare three methodological approaches for the concentration and successful isolation of cyanophages from real world water samples from around the world
To develop a standardized approach and useful protocol for the detection and isolation of Microcystis's cyanophages
4) To study the cyanophage:host relationship.