Environmental constraints on cyanomyophage abundance in the subtropical Pacific Ocean

Viruses are abundant in the world’s oceans and are thought to be important participants in marine biogeochemical cycling. Of these viruses, cyanophages are considered especially important because they infect and lyse cyanobacteria, which are some of the main primary producers in marine environments. Cyanophages are thought to influence the abundance and diversity of cyanobacterial populations and impart significant mortality, thereby affecting primary productivity and microbial community structure. Despite their ecological relevance, little is known about how environmental factors shape cyanophage abundance and diversity over large temporal and spatial scales. To address this gap in knowledge, seawater samples were collected during a research cruise transect from Honolulu, HI to San Diego, CA. The Myoviridae family of cyanophage was targeted for this study because of its perceived ecological dominance and the availability of molecular probes which can be used to measure their diversity and quantify abundance. The g20 gene (which codes for portal vertex protein in myoviruses) was targeted by an established primer set and used as a proxy for cyanomyophage abundance in qPCR assays. Initial analysis of quantification data has revealed significant correlations between cyanomyophage abundance and depth, dissolved inorganic carbon concentration, and total viral abundance. Total viral abundance was also significantly correlated with depth. The lack of trends between viral abundance and other environmental variables may have been due to the temporal offset in the phage-host relationship, which needs to be taken into consideration in future studies

Quantification and Ecological Perspectives on Cyanophage and Aquatic Viruses

The field of viral ecology is still relatively new and many processes by which viruses influence hosts are still widely unknown. One problem is that there are few standardized techniques in virus ecology, making comparisons of data very difficult. To better understand the methodology, we first set out to make a video showing the process for the viral production assay using the “dilution and reoccurrence” method, which has become the standard to analyze production rates in aquatic ecosystems. Using this method, we also determined the production rates of viruses during a seasonal pelagic phytoplankton bloom during a cruise off the coast of the north island of New Zealand in the subtropical Pacific Ocean. Other biotic and abiotic parameters were also compared throughout the bloom. Production rates were within normal ranges, but showed that viruses were very important for the remobilization of nutrients in the nitrogen-limited system. It is well known that the cyanobacterial genera Synechococcus and Prochlorococcus thrive in the world’s oceans with Synechococcus and other cyanobacterial species also succeeding in freshwater ecosystems. Cyanophages are viruses which infect cyanobacteria and many studies have investigated their diversity using the portal vertex g20 gene in the Cyanomyoviridae family. Although we know that there is significant genetic richness in these phage in marine and freshwater environments, information on their numerical distributions is rare. Using quantitative PCR with the g20 gene, we determined that cyanomyoviruses are ubiquitous and abundant in the Atlantic and Pacific Oceans as well as within Lake Erie. Using statistical analyses we were able to find correlations between cyanomyoviruses and other biotic and abiotic parameters: in the Sargasso Sea, cyanomyovirus abundance correlated well to biology, but in the other systems there was no significant correlation to biological abundances. This suggests that the constraints of this group of viruses may be different in different aquatic realms

First description of a cyanophage infecting the cyanobacterium Arthrospira platensis (Spirulina)

International audienceCyanobacteria constitute a versatile group of photosynthetic bacteria of immense commercial and ecological importance. Some species of this group are cultivated and sold as food because of their high nutritional value. This is typically the case for Arthrospira platensis. We describe for the first time a virus infecting this economically important filamentous cyanobacterium isolated from culture pools located in the South of France. This virus could be observed and discriminated easily from other particles with flow cytometry. Based on morphology and molecular investigation, it was proposed that the virus belongs to the cyanopodovirus group with a capsid and short tail of about 120 and 20 nm, respectively. Finally, the virus appeared to be highly specific (very narrow host range) to A. platensis

Population Dynamics and Diversity of Viruses, Bacteria and Phytoplankton in a Shallow Eutrophic Lake

We have studied the temporal variation in viral abundances and community assemblage in the eutrophic Lake Loosdrecht through epifluorescence microscopy and pulsed field gel electrophoresis (PFGE). The virioplankton community was a dynamic component of the aquatic community, with abundances ranging between 5.5 × 107 and 1.3 × 108 virus-like particles ml−1 and viral genome sizes ranging between 30 and 200 kb. Both viral abundances and community composition followed a distinct seasonal cycle, with high viral abundances observed during spring and summer. Due to the selective and parasitic nature of viral infection, it was expected that viral and host community dynamics would covary both in abundances and community composition. The temporal dynamics of the bacterial and cyanobacterial communities, as potential viral hosts, were studied in addition to a range of environmental parameters to relate these to viral community dynamics. Cyanobacterial and bacterial communities were studied applying epifluorescence microscopy, flow cytometry, and denaturing gradient gel electrophoresis (DGGE). Both bacterial and cyanobacterial communities followed a clear seasonal cycle. Contrary to expectations, viral abundances were neither correlated to abundances of the most dominant plankton groups in Lake Loosdrecht, the bacteria and the filamentous cyanobacteria, nor could we detect a correlation between the assemblage of viral and bacterial or cyanobacterial communities during the overall period. Only during short periods of strong fluctuations in microbial communities could we detect viral community assemblages to covary with cyanobacterial and bacterial communities. Methods with a higher specificity and resolution are probably needed to detect the more subtle virus–host interactions. Viral abundances did however relate to cyanobacterial community assemblage and showed a significant positive correlation to Chl-a as well as prochlorophytes, suggesting that a significant proportion of the viruses in Lake Loosdrecht may be phytoplankton and more specific cyanobacterial viruses. Temporal changes in bacterial abundances were significantly related to viral community assemblage, and vice versa, suggesting an interaction between viral and bacterial communities in Lake Loosdrecht

The P-SSP7 Cyanophage Has a Linear Genome with Direct Terminal Repeats

P-SSP7 is a T7-like phage that infects the cyanobacterium Prochlorococcus MED4. MED4 is a member of the high-light-adapted Prochlorococcus ecotypes that are abundant in the surface oceans and contribute significantly to primary production. P-SSP7 has become a model system for the investigation of T7-like phages that infect Prochlorococcus. It was classified as T7-like based on genome content and organization. However, because its genome assembled as a circular molecule, it was thought to be circularly permuted and to lack the direct terminal repeats found in other T7-like phages. Here we sequenced the ends of the P-SSP7 genome and found that the genome map is linear and contains a 206 bp repeat at both genome ends. Furthermore, we found that a 728 bp region of the genome originally placed downstream of the last ORF is actually located upstream of the first ORF on the genome map. These findings suggest that P-SSP7 is likely to use the direct terminal repeats for genome replication and packaging in a similar manner to other T7-like phages. Moreover, these results highlight the importance of experimentally verifying the ends of phage genomes, and will facilitate the use of P-SSP7 as a model for the correct assembly and end determination of the many T7-like phages isolated from the marine environment that are currently being sequenced

Detection of virus mRNA within infected host cells using an isothermal nucleic acid amplification assay: marine cyanophage gene expression within Synechococcus sp

Abstract Background Signal-Mediated Amplification of RNA Technology (SMART) is an isothermal nucleic acid amplification technology, developed for the detection of specific target sequences, either RNA (for expression) or DNA. Cyanophages are viruses that infect cyanobacteria. Marine cyanophages are ubiquitous in the surface layers of the ocean where they infect members of the globally important genus Synechococcus. Results Here we report that the SMART assay allowed us to differentiate between infected and non-infected host cultures. Expression of the cyanophage strain S-PM2 portal vertex gene (g20) was detected from infected host Synechococcus sp. WH7803 cells. Using the SMART assay, we demonstrated that g20 mRNA peaked 240 – 360 minutes post-infection, allowing us to characterise this as a mid to late transcript. g20 DNA was also detected, peaking 10 hours post-infection, coinciding with the onset of host lysis. Conclusion The SMART assay is based on isothermal nucleic acid amplification, allowing the detection of specific sequences of DNA or RNA. It was shown to be suitable for differentiating between virus-infected and non-infected host cultures and for the detection of virus gene expression: the first reported use of this technology for such applications.</p

An investigation into the adsorption of cyanophages to their cyanobacterial hosts

Cyanophages, viruses that infect cyanobacteria, are known to be abundant throughout the world’s oceans. They are important because of the ecological significance of their hosts which are prominent primary producers. In the natural environment cyanobacteria undergo light-dark cycles, which might be expected to exert significant effects on the way in which cyanophages reproduce. The results in this study show how light plays an important role in cyanophage adsorption to the host cell using a model system consisting of cyanophage S-PM2 and Synechococcus sp. WH7803. An initial investigation of the role of light on phage adsorption revealed a striking light-dependence. In the dark, the phage S-PM2 was virtually not capable of adsorbing to WH7803, but adsorption resumed as soon as the light was switched on. This light-dependent phage adsorption was not just limited to the phage S-PM2, four out of nine other cyanophages showed the same effect. The host photosynthetic activity and light/dark cycles were demonstrated not to influence phage adsorption. The presence of the photosynthetic reaction centre gene psbA in cyanophage genomes was not associated with the light-dependent phage adsorption. No photoreceptor was detected from the phage S-PM2 particle. A phage-resistant mutant that S-PM2 can’t adsorb to WH7803 was isolated. A putative multicopper oxidase was found to be absent from the outer membrane fraction of the mutant. This outer membrane fraction in the wild type showed a moderate phage neutralisation activity (up to ~ 30%). To test whether the putative multicopper oxidase was the S-PM2 receptor, a recombinant WH7803 strain was constructed by inactivating the putative multicopper oxidase gene. As S-PM2 can still adsorb to the knockout mutant as efficiently as to the wild type, it suggests that the multicopper oxidase is not the phage receptor and that loss of the putative multicopper oxidase is probably a pleiotropic consequence of the loss of the S-PM2 receptor or other components, such as lipopolysaccharide, that is needed for a successful S-PM2 adsorption

CHARACTERISATION OF LATENT INFECTIONS lN AQUATIC CYANOBACTERIA AND MICROALGAE

Aquatic photosynthetic microorganisms were surveyed (algae and cyanobacteria) for novel lysogenic/latent viruses and new methodology was established, using a variety of techniques such as AFC, electron microscopy, and molecular tools. The first study assessed Symbiodinium sp. cultures as a model system to investigate the induction of potential latent vi ruses. The study of Symbiodinium sp. showed that ca. 37% of the strains tested had a group of filamentous VLPs that is inducible by UV-C treatment. Extrapolation of this virus-host interaction and its effects on zooxanthellae viability provides a novel link to the impact of latent infection on symbiotic dinoflagellates of cnidarians and the subsequent disruption of the reef ecosystems. The second study examined the interaction of a freshwater cyanobacterium and its inducible VLPs. The work carried out on Pseudanabaena, strain PPt10905 suggests that this freshwater cyanobacterium harbours a prophage. An unusual interaction was observed in this freshwater cyanobacterium, where the abundance of carboxysome-like particles increased 10 times in heat-treated cultures. The cyanobacterium PPt10905, its inducible VLPs and the co-occurring increase in carboxysomes could be a new mechanism in which lysogeny benefits freshwater cyanobacteria, possibly increasing the host's photosynthetic efficiency. Finally, the third study investigated the presence of latent viruses in the Plymouth culture collection of marine algae. The characterisation and isolation of inducible viruses from this algal culture collection has revealed much novel information on the prevalence of latent viruses in algae. From the 30 algal species examined in this study, over 35% appear to contain an inducible infectious agent. AFC and TEM images have confirmed the presence of VLPs, and thin sections of UV-induced cultures further supported the presence of VLPs in the UV-induced cultures. This work's contribution increases the knowledge of latent and temperate viruses of aquatic microbes, which are underrepresented in previous studies. Additionally, this research established novel techniques for the study of unique biological interactions between aquatic viruses and their hosts that will facilitate and improve subsequent investigation of similar systems

The effects of nutrient limitation and cyanophage on heterotrophic microbial diversity

Marine viruses are critically important in the regulation of biogeochemical cycles and host microbial communities. In this study, we tested whether the indirect effects of virus predation on a phototroph (i.e., Synechococcus) affected the composition of co-occurring heterotrophic bacteria under nitrogen and phosphorus limitation in long-term chemostat experiments. Using 454 Titanium barcoded pyrosequencing of the 16S rRNA gene, microbial diversity and technical (i.e., sequencing) reproducibility were assessed for nine individual chemostats across five different time points. A total of 325,142 reads were obtained; 194,778 high-quality, non-cyanobacterial sequences were assigned to 110 OTUs. Our results show high reproducibility with most communities clustering closest with their technical replicate, and a similar distribution of taxonomic assignments across replicates. The most abundant phylum was Proteobacteria, with Cyanobacteria representing only 20% of the sequences. OTU-based analyses revealed similar trends across chemostats; Sulfitobacter was the dominant genus while Pseudomonas was unique to the phosphorus-limited chemostats. A statistical examination of biological replicates revealed significant differences between the nitrogen- and phosphorus-limited treatments (p = 0.0001) and time (p = 0.0001), as well as a significant interaction between nutrient limitation and time (p = 0.0091). These results demonstrate the relative importance of nutrient-limitation as a potential primary driver of non-target heterotrophic community change as opposed to the indirect effects of viruses on a marine food web

Molecular approaches to the study of marine cyanophages

Cyanophages are thought to play an important role in the mortality and clonal composition of marine Synechococcus spp., and have been shown to be widespread throughout the world's oceans. However, relatively little research has been made into the molecular analysis of marine cyanophages. This study continued previous research to develop molecular probes (PCR primers) which would specifically detect cyanophages which infect marine Synechococcus spp., and be used to interrogate natural marine cyanophage populations. An attempt was made to develop a rapid technique for quantifying marine cyanophages, using competitive PCR (cPCR). For the development of cyanophage-specific PCR primers, several cyanophages which infected Synechococcus sp. strains WH7803 and WH80 18 were isolated from coastal Bermuda and the Sargasso Sea. A region of DNA had previously been found which showed homology amongst several marine cyanophages, and to T4 gene 20, which encodes a minor capsid protein. Homologues from three cyanophages were completely sequenced, and two, potentially cyanophage-specific, PCR primers were designed. The primers detected only marine cyanophages which belonged to the family Myoviridae, regardless of the geographical location of their isolation. They also detected cyanophages which infected different marine Synechococcus spp. strains, and therefore provide a more comprehensive tool than infective methods. The primers were able to detect as few as 190 cyanophages Ilr1, which would correspond to an in situ concentration of 103 PFU mH. The PCR should therefore detect most natural concentrations of marine cyanophages in surface waters, especially with prior concentration from seawater. Preliminary experiments showed that PCR products could be obtained from as little as I III of un concentrated seawater. PCR therefore provides a sensitive method for the detection of marine cyanophages, which is far more rapid than traditional infection techniques. Quantification by cPCR was attempted. An internal competitor was constructed, and a calibration curve was drawn for three cyanophages, with a loglinear relationship over ca. three orders of magnitude of cyanophage numbers. This demonstrates that rapid quantification of a known marine cyanophage is possible. However, cPCR of the three different cyanophages resulted in three different calibration curves. Hence, quantification of a marine sample containing a mixture of cyanophages was not yet possible. The cyanophage-specific primers were then applied to marine samples which were collected whilst on the AMT-2 cruise, from Port Stanley (Falkland Islands) to Plymouth (UK). Cyanophages were concentrated by tangential flow filtration, and PCR products were obtained from most of the surface samples throughout the Atlantic Ocean. Products from some of the stations were sequenced, providing novel genetic information of natural marine cyanophage populations. The results showed that cyanophage populations were highly diverse, with at least twelve genetically different cyanomyoviruses in one sample. Some sequences obtained from the same sample were clearly very similar to each other, whilst others within a sample could be as diverse as those isolated from different oceans. However, very similar sequences were obtained from some samples separated by thousands of miles, in different hemispheres, or even in different oceans