Infection by a Giant Virus (AaV) Induces Widespread Physiological Reprogramming in Aureococcus anophagefferens CCMP1984 – A Harmful Bloom Algae

While viruses with distinct phylogenetic origins and different nucleic acid types can infect and lyse eukaryotic phytoplankton, “giant” dsDNA viruses have been found to be associated with important ecological processes, including the collapse of algal blooms. However, the molecular aspects of giant virus–host interactions remain largely unknown. Aureococcus anophagefferens virus (AaV), a giant virus in the Mimiviridae clade, is known to play a critical role in regulating the fate of brown tide blooms caused by the pelagophyte Aureococcus anophagefferens. To understand the physiological response of A. anophagefferens CCMP1984 upon AaV infection, we studied the transcriptomic landscape of this host–virus pair over an entire infection cycle using a RNA-sequencing approach. A massive transcriptional response of the host was evident as early as 5 min post-infection, with modulation of specific processes likely related to both host defense mechanism(s) and viral takeover of the cell. Infected Aureococcus showed a relative suppression of host-cell transcripts associated with photosynthesis, cytoskeleton formation, fatty acid, and carbohydrate biosynthesis. In contrast, host cell processes related to protein synthesis, polyamine biosynthesis, cellular respiration, transcription, and RNA processing were overrepresented compared to the healthy cultures at different stages of the infection cycle. A large number of redox active host-selenoproteins were overexpressed, which suggested that viral replication and assembly progresses in a highly oxidative environment. The majority (99.2%) of annotated AaV genes were expressed at some point during the infection cycle and demonstrated a clear temporal–expression pattern and an increasing relative expression for the majority of the genes through the time course. We detected a putative early promoter motif for AaV, which was highly similar to the early promoter elements of two other Mimiviridaemembers, indicating some degree of evolutionary conservation of gene regulation within this clade. This large-scale transcriptome study provides insights into the Aureococcus cells infected by a giant virus and establishes a foundation to test hypotheses regarding metabolic and regulatory processes critical for AaV and other Mimiviridae members

Influence of light on the infection of Aureococcus anophagefferens CCMP 1984 by a “giant virus”

The pelagophyte Aureococcus anophagefferens has caused recurrent brown tide blooms along the northeast coast of the United States since the mid-1980\u27s, and more recently spread to other regions of the globe. These blooms, due to the high cell densities, are associated with severe light attenuation that destroys the sea grass beds which provide the basis for many fisheries. Data collected by transmission electron microscopy, PCR, and metatranscriptomic studies of the blooms, support the hypothesis that large dsDNA viruses play a role in bloom dynamics. While a large (~140 nm) icosahedral virus, with a 371 kbp genome, was first isolated more than a decade ago, the constraints imposed by environmental parameters on bloom infection dynamics by Aureococcus anophagefferens Virus, (AaV) remain unknown. To investigate the role light plays in infection by this virus, we acclimated A. anophagefferens to light intensities of 30 (low), 60 (medium) or 90 μmol photons m-2 s-1 (high) and infected cultures at these irradiance levels. Moreover, we completed light shift experiments where acclimated cultures were exposed to even lower light intensities (0, 5, and 15 μmol photons m-2 s-1) consistent with irradiance found during the peak of the bloom when cell concentrations are highest. The abundance of viruses produced per lytic event (burst size) was lower in the low irradiance acclimated cultures compared to the medium and high acclimated cultures. Transferring infected cultures to more-limiting light availabilities further decreased burst size and increased the length of time it took for cultures to lyse, regardless of acclimation irradiance level. A hypothetical mechanism for the reduced efficiency of the infection cycle in low light due to ribosome biogenesis was predicted from pre-existing transcriptomes. Overall, these studies provide a framework for understanding light effects on infection dynamics over the course of the summer months when A. anophagefferens blooms occur

The Complicated and Confusing Ecology of Microcystis Blooms

Blooms of the toxin-producing cyanobacterium are increasing globally, leading to the loss of ecosystem services, threats to human health, as well as the deaths of pets and husbandry animals. While nutrient availability is a well-known driver of algal biomass, the factors controlling who is present in fresh waters are more complicated. possesses multiple strategies to adapt to temperature, light, changes in nutrient chemistry, herbivory, and parasitism that provide a selective advantage over its competitors. Moreover, its ability to alter ecosystem pH provides it a further advantage that helps exclude many of its planktonic competitors. While decades of nutrient monitoring have provided us with the tools to predict the accumulation of phytoplankton biomass, here, we point to factors on the horizon that may inform us why is presently the dominant bloom former in freshwaters around the world

Complete Genome Sequence of Cyanobacterial Siphovirus KBS2A

Abstract We present the genome of a cyanosiphovirus (KBS2A) that infects a marine Synechococcus sp. (strain WH7803). Unique to this genome, relative to other sequenced cyanosiphoviruses, is the absence of elements associated with integration into the host chromosome, suggesting this virus may not be able to establish a lysogenic relationship. Go to: GENOME ANNOUNCEMENT As obligate parasites, viruses can regulate their host population dynamics but also influence the structure and productivity of microbial communities (1, 2). Synechococcus species are an abundant and ecologically important group of Cyanobacteria found in freshwater and marine ecosystems worldwide. Virus-cyanobacterium interactions may have important implications for global biogeochemical cycles. The most commonly isolated cyanophages are myoviruses and podoviruses (3, 4). Siphoviruses are a third group of viruses that infect cyanobacteria, but they have received less attention (5). The genomes of 5 cyanosiphoviruses have recently become available: that of P-SS2, a siphovirus infecting Prochlorococcus (MIT9313) (6), followed by the cyanosiphoviruses S-CBS1, S-CBS2, S-CBS3, and S-CBS4, isolated from the Chesapeake Bay Estuary, all infecting Synechococcus populations (5). Here, we present the complete genome of cyanosiphophage (KBS2A, originally named KBS-S-2A), a virus that infects Synechococcus sp. strain WH7803. The virus was isolated by plaque assay from the Chesapeake Bay by plating on Synechococcus sp. WH7803. Purified virus DNA was submitted to the Broad Institute as part of the Marine Phage Sequencing Project, where it was sequenced to ~30-fold coverage using 454 pyrosequencing. Translated open reading frames (ORFs) were compared with known protein sequences using the BLASTp program. ORF annotation was aided by the use of PSI-BLAST, HHpred, gene size, and domain conservation. The genome size of KBS2A is 40,658 bp. In total, 64 ORFs have been predicted in this genome; of these, 43 have homologues in databases, and among them, 33 have been assigned to a putative function. For most (88%) predicted ORFs with homologues, homology has been found with the other cyanosiphovirus genomes. We compared the genomic arrangements of the 6 sequenced cyanosiphoviruses using dot plot and global gene homology and found no common genomic organization, suggesting strong mosaicism in the cyanosiphoviruses. In cyanophages, cyanobacterium-related proteins can be found and are often associated with photosynthesis and transcriptional regulation (6). In previously sequenced cyanosiphovirus genomes (5, 6), numerous viral genes (6 to 40 per genome) possess homology with host genes. In the case of the KBS2A genome, only 3 ORFs (coding for RNA polymerase sigma factor RpoD, HNH endonuclease, and a putative DNA polymerase) show such homology, implying less exchange (and potentially interaction) with the host genome. The first annotated cyanosiphovirus genome (that of P-SS2) showed the presence of genes identified as encoding an integrase and excisionase, which are enzymes that allow for phage integration into the host’s genome (6). Moreover, the annotation of cyanosiphoviruses S-CBS1 and S-CBS3 led to the discovery of a prophage-like structure in two sequenced Synechococcus elongatus strains (5). In phage genomes, tRNA genes serve as indicators of potential phage integration by site-specific recombination (7, 8), although recent models have offered alternative suggestions for the role of these genes (9). Sequences of this nature can, however, be found in the P-SS2 and S-CBS4 genomes. No such features (tRNAs, integrases, etc.) were found in the genome of KBS2A, suggesting that this siphovirus might be an exclusively lytic phage rather than a temperate phage. Nucleotide sequence accession number. The complete sequence of the Synechococcus phage KBS2A genome can be accessed under the GenBank accession no. HQ634187. doi: 10.1128/genomeA.00472-1

Cryopreservation of Paramecium bursaria Chlorella Virus-1 during an active infection cycle of its host

Best practices in laboratory culture management often include cryopreservation of microbiota, but this can be challenging with some virus particles. By preserving viral isolates researchers can mitigate genetic drift and laboratory-induced selection, thereby maintaining genetically consistent strains between experiments. To this end, we developed a method to cryopreserve the model, green-alga infecting virus, Paramecium bursaria Chlorella virus 1 (PBCV-1). We explored cryotolerance of the infectivity of this virus particle, whereby freezing without cryoprotectants was found to maintain the highest infectivity (~2.5%). We then assessed the cryopreservation potential of PBCV-1 during an active infection cycle in its Chlorella variabilisNC64A host, and found that virus survivorship was highest (69.5 ± 16.5%) when the infected host is cryopreserved during mid-late stages of infection (i.e., coinciding with virion assembly). The most optimal condition for cryopreservation was observed at 240 minutes post-infection. Overall, utilizing the cell as a vehicle for viral cryopreservation resulted in 24.9–30.1 fold increases in PBCV-1 survival based on 95% confidence intervals of frozen virus particles and virus cryopreserved at 240 minutes post-infection. Given that cryoprotectants are often naturally produced by psychrophilic organisms, we suspect that cryopreservation of infected hosts may be a reliable mechanism for virus persistence in non-growth permitting circumstances in the environment, such as ancient permafrosts

A protocol for enumeration of aquatic viruses by epifluorescence microscopy using Anodisc™ 13 membranes

<p>Abstract</p> <p>Background</p> <p>Epifluorescence microscopy is a common method used to enumerate virus-like particles (VLP) from environmental samples and relies on the use of filter membranes with pore sizes < 0.02 μm; the most commonly used protocols employ 25 mm Anodisc™ membranes with a built-in support ring. Other filters with small pore sizes exist, including the 13 mm Anodisc™ membranes without a support ring. However, the use of these membranes for viral enumeration has not been previously reported.</p> <p>Results</p> <p>Here we describe a modified protocol for 13 mm Anodisc membranes that uses a custom filter holder that can be readily constructed in individual investigators' laboratories from commercially available Swinnex^®filter holders. We compared VLP concentrations obtained from phage lysates and seawater samples using both Anodisc membranes, as well as Nuclepore™ small pore-size membranes (0.015 or 0.030 μm). The 13 mm Anodisc membranes gave comparable estimates of VLP abundance to those obtained with the 25 mm Anodisc membranes when similar staining methods were employed. Both Nuclepore membranes typically gave an order of magnitude lower VLP abundance values for environmental samples.</p> <p>Conclusions</p> <p>The 13 mm Anodisc membranes are less costly and require smaller sample volumes than their 25 mm counterpart making them ideal for large-scale studies and sample replication. This method increases the options of reliable approaches available for quantifying VLP from environmental samples.</p

Genome Sequence of the Sulfitobacter sp. Strain 2047-Infecting Lytic Phage ΦCB2047-B

We announce the complete genome sequence of a lytic podovirus, ΦCB2047-B, which infects the bacterium Sulfitobacter sp. strain 2047, a member of theRoseobacter clade. Genome analysis revealed ΦCB2047-B to be an N4-like phage, with its genome having high nucleotide similarity to other N4-like roseophage genomes

Genome Sequences of Two Temperate Phages, ΦCB2047-A and ΦCB2047-C, Infecting Sulfitobacter sp. Strain 2047

We announce the complete genome sequences of two temperate Podoviridae,Sulfitobacter phages ΦCB2047-A and ΦCB2047-C, which infect Sulfitobacter sp. strain 2047, a member of the Roseobacter clade. This is the first report of temperate podophage infecting members of the Sulfitobacter genus of theRoseobacter clade

Plasticity of Total and Intracellular Phosphorus Quotas in Microcystis aeruginosa Cultures and Lake Erie Algal Assemblages

Blooms of the potentially toxic cyanobacterium Microcystis are common events globally, and as a result significant resources continue to be dedicated to monitoring and controlling these events. Recent studies have shown that a significant proportion of total cell-associated phosphorus (P) in marine phytoplankton can be surface adsorbed; as a result studies completed to date do not accurately report the P demands of these organisms. In this study we measure the total cell-associated and intracellular P as well as growth rates of two toxic strains of Microcystis aeruginosa Kütz grown under a range of P concentrations. The results show that the intracellular P pool in Microcystis represents a percentage of total cell-associated P (50–90%) similar to what has been reported for actively growing algae in marine systems. Intracellular P concentrations (39–147 fg cell−1) generally increased with increasing P concentrations in the growth medium, but growth rate and the ratio of total cell-associated to intracellular P remained generally stable. Intracellular P quotas and growth rates in cells grown under the different P treatments illustrate the ability of this organism to successfully respond to changes in ambient P loads, and thus have implications for ecosystem scale productivity models employing P concentrations to predict algal bloom events

Estimating Virus Production Rates in Aquatic Systems

Viruses are pervasive components of marine and freshwater systems, and are known to be significant agents of microbial mortality. Developing quantitative estimates of this process is critical as we can then develop better models of microbial community structure and function as well as advance our understanding of how viruses work to alter aquatic biogeochemical cycles. The virus reduction technique allows researchers to estimate the rate at which virus particles are released from the endemic microbial community. In brief, the abundance of free (extracellular) viruses is reduced in a sample while the microbial community is maintained at near ambient concentration. The microbial community is then incubated in the absence of free viruses and the rate at which viruses reoccur in the sample (through the lysis of already infected members of the community) can be quantified by epifluorescence microscopy or, in the case of specific viruses, quantitative PCR. These rates can then be used to estimate the rate of microbial mortality due to virus-mediated cell lysis