Isolation and characterisation of novel viruses infecting marine phytoplankton

Viruses are the most abundant biological agents in the global marine environment. Through cellular lysis viruses influence many biogeochemical and ecological processes, including energy and nutrient cycling, host distribution and abundance, algal bloom control and genetic transfer. Nano- and picophytoplankton are ubiquitous in the world’s oceans and are responsible for a high proportion of the annual global carbon fixation. However, relatively few viruses have been isolated and described that infect these key primary producers and little is known of their diversity, dynamics or propagation strategies. The aims of this study were to detect, isolate and characterise novel marine viruses that infect these important members of the phytoplankton assemblage. Screening of seawater samples for viruses that infect a broad representation of nano and picophytoplankton species was undertaken here. To enable this, a large culture collection of 106 phytoplankton species was established and used to screen seawater samples for viruses on a weekly basis over a two year period. A total of 12 novel viruses infecting the prasinophyte species’ Ostreococcus tauri and Micromonas pusilla were isolated from seawater sampled in coastal waters of the Western English Channel. Viruses were purified by plaque purification or liquid serial dilution techniques. Characterisation of novel virus isolates included growth kinetics, visualisation using transmission electron microscopy, host range analysis and estimates of viral genome sizes using pulsed field gel electrophoresis. Phylogenetic analysis of these viruses was conducted based on the sequence of the conserved DNA polymerase gene. Genome sequencing of two of the viruses infecting O. tauri was completed and revealed many exciting features, including a suite of genes hitherto unreported, or with rare occurrence, in viruses. Evidence is presented for horizontal gene transfer between viruses isolated in this study and their hosts, as well as between other eukaryotic and bacterial sources. Functional characterisation of the viral genomes sequenced and described in this study will provide clearer insights into viral dynamics and evolutionary history

Marine prasinoviruses and their tiny plankton hosts: A review

This is the final version. Available on open access from MDPI via the DOI in this recordViruses play a crucial role in the marine environment, promoting nutrient recycling and biogeochemical cycling and driving evolutionary processes. Tiny marine phytoplankton called prasinophytes are ubiquitous and significant contributors to global primary production and biomass. A number of viruses (known as prasinoviruses) that infect these important primary producers have been isolated and characterised over the past decade. Here we review the current body of knowledge about prasinoviruses and their interactions with their algal hosts. Several genes, including those encoding for glycosyltransferases, methyltransferases and amino acid synthesis enzymes, which have never been identified in viruses of eukaryotes previously, have been detected in prasinovirus genomes. The host organisms are also intriguing; most recently, an immunity chromosome used by a prasinophyte in response to viral infection was discovered. In light of such recent, novel discoveries, we discuss why the cellular simplicity of prasinophytes makes for appealing model host organism–virus systems to facilitate focused and detailed investigations into the dynamics of marine viruses and their intimate associations with host species. We encourage the adoption of the prasinophyte Ostreococcus and its associated viruses as a model host–virus system for examination of cellular and molecular processes in the marine environment

Isolation and characterisation of novel viruses infecting marine phytoplankton

Viruses are the most abundant biological agents in the global marine environment. Through cellular lysis viruses influence many biogeochemical and ecological processes, including energy and nutrient cycling, host distribution and abundance, algal bloom control and genetic transfer. Nano- and picophytoplankton are ubiquitous in the world’s oceans and are responsible for a high proportion of the annual global carbon fixation. However, relatively few viruses have been isolated and described that infect these key primary producers and little is known of their diversity, dynamics or propagation strategies. The aims of this study were to detect, isolate and characterise novel marine viruses that infect these important members of the phytoplankton assemblage. Screening of seawater samples for viruses that infect a broad representation of nano and picophytoplankton species was undertaken here. To enable this, a large culture collection of 106 phytoplankton species was established and used to screen seawater samples for viruses on a weekly basis over a two year period. A total of 12 novel viruses infecting the prasinophyte species’ Ostreococcus tauri and Micromonas pusilla were isolated from seawater sampled in coastal waters of the Western English Channel. Viruses were purified by plaque purification or liquid serial dilution techniques. Characterisation of novel virus isolates included growth kinetics, visualisation using transmission electron microscopy, host range analysis and estimates of viral genome sizes using pulsed field gel electrophoresis. Phylogenetic analysis of these viruses was conducted based on the sequence of the conserved DNA polymerase gene. Genome sequencing of two of the viruses infecting O. tauri was completed and revealed many exciting features, including a suite of genes hitherto unreported, or with rare occurrence, in viruses. Evidence is presented for horizontal gene transfer between viruses isolated in this study and their hosts, as well as between other eukaryotic and bacterial sources. Functional characterisation of the viral genomes sequenced and described in this study will provide clearer insights into viral dynamics and evolutionary history.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Genetic, morphological and growth characterisation of a new Roseofilum strain (Oscillatoriales, Cyanobacteria) associated with coral black band disease

Black band disease (BBD) is a common disease of reef-building corals with a worldwide distribution that causes tissue loss at a rate of up to 3 cm/day. Critical fora mechanistic understanding of the disease's aetiology is the cultivation of its proposed pathogen, filamentous cyanobacteria (genus Roseofilum). Here, we optimise existing protocols for the isolation and cultivation of Roseofilum cyanobacteria using a new strain from the central Great Barrier Reef. We demonstrate that the isolation of this bacterium via inoculation onto agar plates was highly effective with a low percentage agar of 0.6% and that growth monitoring was most sensitive with fluorescence measurements of chlorophyll-a (440/685 nm). Cell growth curves in liquid and solid media were generated for the first time for this cyanobacterium and showed best growth rates for the previously untested L1-medium (growth rate k = 0.214 biomass/day; doubling time t(gen) = 4.67 days). Our results suggest that the trace metals contained in L1-medium maximise biomass increase over time for this cyanobacteriurn. Since the newly isolated Roseofilum strain is genetically closest to Pseudoscillatoria coralii, but in terms of pigmentation and cell size closer to Roseofilum reptotaenium, we formally merge the two species into a single taxon by providing an emended species description, Roseofilum reptotaenium (Rasoulouniriana) Casamatta emend. Following this optimized protocol is recommended for fast isolation and cultivation of Roseofilum cyanobacteria, for growth curve generation in strain comparisons and for maximisation of biomass in genetic studies

From cholera to corals: viruses as drivers of virulence in a major coral bacterial pathogen

Disease is an increasing threat to reef-building corals. One of the few identified pathogens of coral disease is the bacterium Vibrio coralliilyticus. In Vibrio cholerae, infection by a bacterial virus (bacteriophage) results in the conversion of non-pathogenic strains to pathogenic strains and this can lead to cholera pandemics. Pathogenicity islands encoded in the V. cholerae genome play an important role in pathogenesis. Here we analyse five whole genome sequences of V. coralliilyticus to examine whether virulence is similarly driven by horizontally acquired elements. We demonstrate that bacteriophage genomes encoding toxin genes with homology to those found in pathogenic V. cholerae are integrated in V. coralliilyticus genomes. Virulence factors located on chromosomal pathogenicity islands also exist in some strains of V. coralliilyticus. The presence of these genetic signatures indicates virulence in V. coralliilyticus is driven by prophages and other horizontally acquired elements. Screening for pathogens of coral disease should target conserved regions in these elements

CRISPR-Cas defense system and potential prophages in cyanobacteria associated with the coral black band disease

Understanding how pathogens maintain their virulence is critical to developing tools to mitigate disease in animal populations. We sequenced and assembled the first draft genome of Roseofilum reptotaenium AO1, the dominant cyanobacterium underlying pathogenicity of the virulent coral black band disease (BBD), and analyzed parts of the BBD-associated Geitlerinema sp. BBD_1991 genome in silico. Both cyanobacteria are equipped with an adaptive, heritable clustered regularly interspaced short palindromic repeats (CRISPR)-Cas defense system type I-D and have potential virulence genes located within several prophage regions. The defense system helps to prevent infection by viruses and mobile genetic elements via identification of short fingerprints of the intruding DNA, which are stored as templates in the bacterial genome, in so-called CRISPRs. Analysis of CRISPR target sequences (protospacers) revealed an unusually high number of self-targeting spacers in R. reptotaenium AO1 and extraordinary long CRIPSR arrays of up to 260 spacers in Geitlerinema sp. BBD_1991. The self-targeting spacers are unlikely to be a form of autoimmunity; instead these target an incomplete lysogenic bacteriophage. Lysogenic virus induction experiments with mitomycin C and UV light did not reveal an actively replicating virus population in R. reptotaenium AO1 cultures, suggesting that phage functionality is compromised or excision could be blocked by the CRISPR-Cas system. Potential prophages were identified in three regions of R. reptotaenium AO1 and five regions of Geitlerinema sp. BBD_1991, containing putative BBD relevant virulence genes, such as an NAD-dependent epimerase/dehydratase (a homolog in terms of functionality to the third and fourth most expressed gene in BBD), lysozyme/metalloendopeptidases and other lipopolysaccharide modification genes. To date, viruses have not been considered to be a component of the BBD consortium or a contributor to the virulence of R. reptotaenium AO1 and Geitlerinema sp. BBD_(1)991. We suggest that the presence of virulence genes in potential prophage regions, and the CRISPR-Cas defense systems are evidence of an arms race between the respective cyanobacteria and their bacteriophage predators. The presence of such a defense system likely reduces the number of successful bacteriophage infections and mortality in the cyanobacteria, facilitating the progress of BBD

Genome Sequence of Stenotrophomonas maltophilia PML168, Which Displays Baeyer-Villiger Monooxygenase Activity

Stenotrophomonas maltophilia PML168 was isolated from Wembury Beach on the English Coast from a rock pool following growth and selection on agar plates. Here we present the permanent draft genome sequence, which has allowed prediction of function for several genes encoding enzymes relevant to industrial biotechnology, including a novel flavoprotein monooxygenase

Fig. S1: Overview of bioinformatics pipeline

Current research posits that all multicellular organisms live in symbioses with associated microorganisms and form so-called metaorganisms or holobionts. Cnidarian metaorganisms are of specific interest given that stony corals provide the foundation of the globally threatened coral reef ecosystems. To gain first insight into viruses associated with the coral model system Aiptasia (sensu Exaiptasia pallida), we analyzed an existing RNA-Seq dataset of aposymbiotic, partially populated, and fully symbiotic Aiptasia CC7 anemones with Symbiodinium. Our approach included the selective removal of anemone host and algal endosymbiont sequences and subsequent microbial sequence annotation. Of a total of 297 million raw sequence reads, 8.6 million (∼3%) remained after host and endosymbiont sequence removal. Of these, 3,293 sequences could be assigned as of viral origin. Taxonomic annotation of these sequences suggests that Aiptasia is associated with a diverse viral community, comprising 116 viral taxa covering 40 families. The viral assemblage was dominated by viruses from the families Herpesviridae (12.00%), Partitiviridae (9.93%), and Picornaviridae (9.87%). Despite an overall stable viral assemblage, we found that some viral taxa exhibited significant changes in their relative abundance when Aiptasia engaged in a symbiotic relationship with Symbiodinium. Elucidation of viral taxa consistently present across all conditions revealed a core virome of 15 viral taxa from 11 viral families, encompassing many viruses previously reported as members of coral viromes. Despite the non-random selection of viral genetic material due to the nature of the sequencing data analyzed, our study provides a first insight into the viral community associated with Aiptasia. Similarities of the Aiptasia viral community with those of corals corroborate the application of Aiptasia as a model system to study coral holobionts. Further, the change in abundance of certain viral taxa across different symbiotic states suggests a role of viruses in the algal endosymbiosis, but the functional significance of this remains to be determined

Host-Associated Bacteriophage Isolation and Preparation for Viral Metagenomics.

Prokaryotic viruses, or bacteriophages, are viruses that infect bacteria and archaea. These viruses have been known to associate with host systems for decades, yet only recently have their influence on the regulation of host-associated bacteria been appreciated. These studies have been conducted in many host systems, from the base of animal life in the Cnidarian phylum to mammals. These prokaryotic viruses are useful for regulating the number of bacteria in a host ecosystem and for regulating the strains of bacteria useful for the microbiome. These viruses are likely selected by the host to maintain bacterial populations. Viral metagenomics allows researchers to profile the communities of viruses associating with animal hosts, and importantly helps to determine the functional role these viruses play. Further, viral metagenomics show the sphere of viral involvement in gene flow and gene shuffling in an ever-changing host environment. The influence of prokaryotic viruses could, therefore, have a clear impact on host health

Evolutionary consequences of viral resistance in the marine picoeukaryote Ostreococcus tauri

In marine environments, eukaryotic marine microalgae coexist with the viruses that infect them. Marine microalgae are the main primary producers in the oceans and are at the base of the marine food web. Viruses play important roles in top-down control of algae populations, cycling of organic matter, and as evolutionary drivers of their hosts. Algae must adapt in response to the strong selection pressure that viruses impose for resistance to infection. In addition to biotic selection pressures such as viral infections, algae must also adapt to their abiotic environment. Global climate change is affecting temperature, salinity, pH, light and nutrient concentrations in the oceans, particularly in surface waters, where microalgae live. Currently, little is known about how consistent the effects of viruses on their hosts are, whether the cost of host resistance varies across environments, and whether there is a trade-off between maintaining resistance to viruses and adapting to other environmental changes. The marine picoeukaryote Ostreococcus tauri is abundant in Mediterranean lagoons, where it experiences large fluctuations in environmental conditions and co-occurs with lytic viruses (Ostreococcus tauri viruses – OtVs). Viral infection causes lysis of susceptible (S) cells, however a small proportion of cells are resistant (R) and avoid lysis. Some resistant O. tauri populations can coexist with infectious viruses, and it has been proposed that these viruses are produced by a minority of susceptible cells within a mainly resistant population. These populations are referred to as resistant producers (RP). Virus production in RP populations is unstable and eventually they shift to R populations. I used O. tauri and one of its viruses, OtV5, as a model system to investigate whether cells that are susceptible or resistant to virus infection adapt to environmental change differently and whether there is a cost of being resistant. For the first time, I evolved susceptible and resistant hosts of a marine alga separately under a range of environments and directly compared their plastic and evolved responses. I showed that resistant populations of O. tauri maintained their resistance for more than 200 generations in the absence of viruses across all environments, indicating that the resistance mechanism is difficult to reverse. Furthermore, I did not detect a cost of being resistant, as measured by population growth rate and competitive ability. Virus production in RP populations stopped in all environments and all populations became R. In addition, I found that virus production in RP O. tauri populations can fluctuate before completely ceasing, and that phosphate affected the length of time it took for virus production to stop. These results, combined with mathematical modelling of O. tauri infection dynamics, provide support for the prediction that RP populations consist of a mixed population of susceptible and resistant cells. By examining multiple environments and resistance types, we can better understand first, how microalgae populations adapt to environmental change and second, the ecological and evolutionary consequences of maintaining resistance to viruses in common marine picoeukaryotes