Seasonal dynamics of natural Ostreococcus viral infection at the single cell level using VirusFISH.

Ostreococcus is a cosmopolitan marine genus of phytoplankton found in mesotrophic and oligotrophic waters, and the smallest free-living eukaryotes known to date, with a cell diameter close to 1 µm. Ostreococcus has been extensively studied as a model system to investigate viral-host dynamics in culture, yet the impact of viruses in naturally occurring populations is largely unknown. Here, we used Virus Fluorescence in situ Hybridization (VirusFISH) to visualize and quantify viral-host dynamics in natural populations of Ostreococcus during a seasonal cycle in the central Cantabrian Sea (Southern Bay of Biscay). Ostreococcus were predominantly found during summer and autumn at surface and 50 m depth, in coastal, mid-shelf and shelf waters, representing up to 21% of the picoeukaryotic communities. Viral infection was only detected in surface waters, and its impact was variable but highest from May to July and November to December, when up to half of the population was infected. Metatranscriptomic data available from the mid-shelf station unveiled that the Ostreococcus population was dominated by the species O. lucimarinus. This work represents a proof of concept that the VirusFISH technique can be used to quantify the impact of viruses on targeted populations of key microbes from complex natural communities.

Combining Nanopore and Illumina Sequencing Permits Detailed Analysis of Insertion Mutations and Structural Variations Produced by PEG-Mediated Transformation in Ostreococcus tauri.

Ostreococcus tauri is a simple unicellular green alga representing an ecologically important group of phytoplankton in oceans worldwide. Modern molecular techniques must be developed in order to understand the mechanisms that permit adaptation of microalgae to their environment. We present for the first time in O. tauri a detailed characterization of individual genomic integration events of foreign DNA of plasmid origin after PEG-mediated transformation. Vector integration occurred randomly at a single locus in the genome and mainly as a single copy. Thus, we confirmed the utility of this technique for insertional mutagenesis. While the mechanism of double-stranded DNA repair in the O. tauri model remains to be elucidated, we clearly demonstrate by genome resequencing that the integration of the vector leads to frequent structural variations (deletions/insertions and duplications) and some chromosomal rearrangements in the genome at the insertion loci. Furthermore, we often observed variations in the vector sequence itself. From these observations, we speculate that a nonhomologous end-joining-like mechanism is employed during random insertion events, as described in plants and other freshwater algal models. PEG-mediated transformation is therefore a promising molecular biology tool, not only for functional genomic studies, but also for biotechnological research in this ecologically important marine alga.

Visualization of Viral Infection Dynamics in a Unicellular Eukaryote and Quantification of Viral Production Using Virus Fluorescence in situ Hybridization.

One of the major challenges in viral ecology is to assess the impact of viruses in controlling the abundance of specific hosts in the environment. To this end, techniques that enable the detection and quantification of virus-host interactions at the single-cell level are essential. With this goal in mind, we implemented virus fluorescence in situ hybridization (VirusFISH) using as a model the marine picoeukaryote Ostreococcus tauri and its virus Ostreococcus tauri virus 5 (OtV5). VirusFISH allowed the visualization and quantification of the proportion of infected cells during an infection cycle in experimental conditions. We were also able to quantify the abundance of free viruses released during cell lysis, discriminating OtV5 from other mid-level fluorescence phages in our non-axenic infected culture that were not easily distinguishable with flow cytometry. Our results showed that although the major lysis of the culture occurred between 24 and 48 h after OtV5 inoculation, some new viruses were already produced between 8 and 24 h. With this work, we demonstrate that VirusFISH is a promising technique to study specific virus-host interactions in non-axenic cultures and establish a framework for its application in complex natural communities.

Virus-host coexistence in phytoplankton through the genomic lens.

Virus-microbe interactions in the ocean are commonly described by "boom and bust" dynamics, whereby a numerically dominant microorganism is lysed and replaced by a virus-resistant one. Here, we isolated a microalga strain and its infective dsDNA virus whose dynamics are characterized instead by parallel growth of both the microalga and the virus. Experimental evolution of clonal lines revealed that this viral production originates from the lysis of a minority of virus-susceptible cells, which are regenerated from resistant cells. Whole-genome sequencing demonstrated that this resistant-susceptible switch involved a large deletion on one chromosome. Mathematical modeling explained how the switch maintains stable microalga-virus population dynamics consistent with their observed growth pattern. Comparative genomics confirmed an ancient origin of this "accordion" chromosome despite a lack of sequence conservation. Together, our results show how dynamic genomic rearrangements may account for a previously overlooked coexistence mechanism in microalgae-virus interactions.

Mantoniella beaufortii and Mantoniella baffinensis sp. nov. (Mamiellales, Mamiellophyceae), two new green algal species from the high arctic1.

Members of the class Mamiellophyceae comprise species that can dominate picophytoplankton diversity in polar waters. Yet, polar species are often morphologically indistinguishable from temperate species, although clearly separated by molecular features. Here we examine four Mamiellophyceae strains from the Canadian Arctic. The 18S rRNA and Internal Transcribed Spacer 2 (ITS2) gene phylogeny place these strains within the family Mamiellaceae (Mamiellales, Mamiellophyceae) in two separate clades of the genus Mantoniella. ITS2 synapomorphies support their placement as two new species, Mantoniella beaufortii and Mantoniella baffinensis. Both species have round green cells with diameter between 3 and 5 µm, one long flagellum and a short flagellum (~1 µm) and are covered by spiderweb-like scales, making both species similar to other Mantoniella species. Morphologically, M. beaufortii and M. baffinensis are most similar to the cosmopolitan M. squamata with only minor differences in scale structure distinguishing them. Screening of global marine metabarcoding data sets indicates M. beaufortii has only been recorded in seawater and sea ice samples from the Arctic, while no environmental barcode matches M. baffinensis. Like other Mamiellophyceae genera that have distinct polar and temperate species, the polar distribution of these new species suggests they are cold or ice-adapted Mantoniella species.

Seasonal Dynamics of Algae-Infecting Viruses and Their Inferred Interactions with Protists.

Viruses are a highly abundant, dynamic, and diverse component of planktonic communities that have key roles in marine ecosystems. We aimed to reveal the diversity and dynamics of marine large dsDNA viruses infecting algae in the Northern Skagerrak, South Norway through the year by metabarcoding, targeting the major capsid protein (MCP) and its correlation to protist diversity and dynamics. Metabarcoding results demonstrated a high diversity of algal viruses compared to previous metabarcoding surveys in Norwegian coastal waters. We obtained 313 putative algal virus operational taxonomic units (vOTUs), all classified by phylogenetic analyses to either the Phycodnaviridae or Mimiviridae families, most of them in clades without any cultured or environmental reference sequences. The viral community showed a clear temporal variation, with some vOTUs persisting for several months. The results indicate co-occurrences between abundant viruses and potential hosts during long periods. This study gives new insights into the virus-algal host dynamics and provides a baseline for future studies of algal virus diversity and temporal dynamics.

Simplified Transformation of Ostreococcus tauri Using Polyethylene Glycol.

Ostreococcustauri is an easily cultured representative of unicellular algae (class Mamiellophyceae) that abound in oceans worldwide. Eight complete 13-22 Mb genomes of phylogenetically divergent species within this class are available, and their DNA sequences are nearly always present in metagenomic data produced from marine samples. Here we describe a simplified and robust transformation protocol for the smallest of these algae (O. tauri). Polyethylene glycol (PEG) treatment was much more efficient than the previously described electroporation protocol. Short (2 min or less) incubation times in PEG gave >104 transformants per microgram DNA. The time of cell recovery after transformation could be reduced to a few hours, permitting the experiment to be done in a day rather than overnight as used in previous protocols. DNA was randomly inserted in the O. tauri genome. In our hands PEG was 20-40-fold more efficient than electroporation for the transformation of O. tauri, and this improvement will facilitate mutagenesis of all of the dispensable genes present in the tiny O. tauri genome.

Viruses of Polar Aquatic Environments.

The poles constitute 14% of the Earth's biosphere: The aquatic Arctic surrounded by land in the north, and the frozen Antarctic continent surrounded by the Southern Ocean. In spite of an extremely cold climate in addition to varied topographies, the polar aquatic regions are teeming with microbial life. Even in sub-glacial regions, cellular life has adapted to these extreme environments where perhaps there are traces of early microbes on Earth. As grazing by macrofauna is limited in most of these polar regions, viruses are being recognized for their role as important agents of mortality, thereby influencing the biogeochemical cycling of nutrients that, in turn, impact community dynamics at seasonal and spatial scales. Here, we review the viral diversity in aquatic polar regions that has been discovered in the last decade, most of which has been revealed by advances in genomics-enabled technologies, and we reflect on the vast extent of the still-to-be explored polar microbial diversity and its "enigmatic virosphere".

Genome Analyses of the Microalga Picochlorum Provide Insights into the Evolution of Thermotolerance in the Green Lineage.

While the molecular events involved in cell responses to heat stress have been extensively studied, our understanding of the genetic basis of basal thermotolerance, and particularly its evolution within the green lineage, remains limited. Here, we present the 13.3-Mb haploid genome and transcriptomes of a halotolerant and thermotolerant unicellular green alga, Picochlorum costavermella (Trebouxiophyceae) to investigate the evolution of the genomic basis of thermotolerance. Differential gene expression at high and standard temperatures revealed that more of the gene families containing up-regulated genes at high temperature were recently evolved, and less originated at the ancestor of green plants. Inversely, there was an excess of ancient gene families containing transcriptionally repressed genes. Interestingly, there is a striking overlap between the thermotolerance and halotolerance transcriptional rewiring, as more than one-third of the gene families up-regulated at 35 °C were also up-regulated under variable salt concentrations in Picochlorum SE3. Moreover, phylogenetic analysis of the 9,304 protein coding genes revealed 26 genes of horizontally transferred origin in P. costavermella, of which five were differentially expressed at higher temperature. Altogether, these results provide new insights about how the genomic basis of adaptation to halo- and thermotolerance evolved in the green lineage.

Rapidity of Genomic Adaptations to Prasinovirus Infection in a Marine Microalga.

Prasinoviruses are large dsDNA viruses commonly found in aquatic systems worldwide, where they can infect and lyse unicellular prasinophyte algae such as Ostreococcus. Host susceptibility is virus strain-specific, but resistance of susceptible Ostreococcus tauri strains to a virulent virus arises frequently. In clonal resistant lines that re-grow, viruses are usually present for many generations, and genes clustered on chromosome 19 show physical rearrangements and differential expression. Here, we investigated changes occurring during the first two weeks after inoculation of the prasinovirus OtV5. By serial dilutions of cultures at the time of inoculation, we estimated the frequency of resistant cells arising in virus-challenged O. tauri cultures to be 10-3⁻10-4 of the inoculated population. Re-growing resistant cells were detectable by flow cytometry 3 days post-inoculation (dpi), visible re-greening of cultures occurred by 6 dpi, and karyotypic changes were visually detectable at 8 dpi. Resistant cell lines showed a modified spectrum of host-virus specificities and much lower levels of OtV5 adsorption.

Prasinovirus Attack of Ostreococcus Is Furtive by Day but Savage by Night.

Prasinoviruses are large DNA viruses that infect diverse genera of green microalgae worldwide in aquatic ecosystems, but molecular knowledge of their life cycles is lacking. Several complete genomes of both these viruses and their marine algal hosts are now available and have been used to show the pervasive presence of these species in microbial metagenomes. We have analyzed the life cycle of Ostreococcus tauri virus 5 (OtV5), a lytic virus, using transcriptome sequencing (RNA-Seq) from 12 time points of healthy or infected Ostreococcus tauri cells over a day/night cycle in culture. In the day, viral gene transcription remained low while host nitrogen metabolism gene transcription was initially strongly repressed for two successive time points before being induced for 8 h, but during the night, viral transcription increased steeply while host nitrogen metabolism genes were repressed and many host functions that are normally reduced in the dark appeared to be compensated either by genes expressed from the virus or by increased expression of a subset of 4.4% of the host's genes. Some host cells underwent lysis progressively during the night, but a larger proportion were lysed the following morning. Our data suggest that the life cycles of algal viruses mirror the diurnal rhythms of their hosts.IMPORTANCE Prasinoviruses are common in marine environments, and although several complete genomes of these viruses and their hosts have been characterized, little is known about their life cycles. Here we analyze in detail the transcriptional changes occurring over a 27-h-long experiment in a natural diurnal rhythm, in which the growth of host cells is to some extent synchronized, so that host DNA replication occurs late in the day or early in the night and cell division occurs during the night. Surprisingly, viral transcription remains quiescent over the daytime, when the most energy (from light) is available, but during the night viral transcription activates, accompanied by expression of a few host genes that are probably required by the virus. Although our experiment was accomplished in the lab, cyclical changes have been documented in host transcription in the ocean. Our observations may thus be relevant for eukaryotic phytoplankton in natural environments.

Population genomics of picophytoplankton unveils novel chromosome hypervariability.

Tiny photosynthetic microorganisms that form the picoplankton (between 0.3 and 3 µm in diameter) are at the base of the food web in many marine ecosystems, and their adaptability to environmental change hinges on standing genetic variation. Although the genomic and phenotypic diversity of the bacterial component of the oceans has been intensively studied, little is known about the genomic and phenotypic diversity within each of the diverse eukaryotic species present. We report the level of genomic diversity in a natural population of Ostreococcus tauri (Chlorophyta, Mamiellophyceae), the smallest photosynthetic eukaryote. Contrary to the expectations of clonal evolution or cryptic species, the spectrum of genomic polymorphism observed suggests a large panmictic population (an effective population size of 1.2 × 107) with pervasive evidence of sexual reproduction. De novo assemblies of low-coverage chromosomes reveal two large candidate mating-type loci with suppressed recombination, whose origin may pre-date the speciation events in the class Mamiellophyceae. This high genetic diversity is associated with large phenotypic differences between strains. Strikingly, resistance of isolates to large double-stranded DNA viruses, which abound in their natural environment, is positively correlated with the size of a single hypervariable chromosome, which contains 44 to 156 kb of strain-specific sequences. Our findings highlight the role of viruses in shaping genome diversity in marine picoeukaryotes.

A Viral Immunity Chromosome in the Marine Picoeukaryote, Ostreococcus tauri.

Micro-algae of the genus Ostreococcus and related species of the order Mamiellales are globally distributed in the photic zone of world's oceans where they contribute to fixation of atmospheric carbon and production of oxygen, besides providing a primary source of nutrition in the food web. Their tiny size, simple cells, ease of culture, compact genomes and susceptibility to the most abundant large DNA viruses in the sea render them attractive as models for integrative marine biology. In culture, spontaneous resistance to viruses occurs frequently. Here, we show that virus-producing resistant cell lines arise in many independent cell lines during lytic infections, but over two years, more and more of these lines stop producing viruses. We observed sweeping over-expression of all genes in more than half of chromosome 19 in resistant lines, and karyotypic analyses showed physical rearrangements of this chromosome. Chromosome 19 has an unusual genetic structure whose equivalent is found in all of the sequenced genomes in this ecologically important group of green algae.

Metagenomic insights into strategies of carbon conservation and unusual sulfur biogeochemistry in a hypersaline Antarctic lake.

Organic Lake is a shallow, marine-derived hypersaline lake in the Vestfold Hills, Antarctica that has the highest reported concentration of dimethylsulfide (DMS) in a natural body of water. To determine the composition and functional potential of the microbial community and learn about the unusual sulfur chemistry in Organic Lake, shotgun metagenomics was performed on size-fractionated samples collected along a depth profile. Eucaryal phytoflagellates were the main photosynthetic organisms. Bacteria were dominated by the globally distributed heterotrophic taxa Marinobacter, Roseovarius and Psychroflexus. The dominance of heterotrophic degradation, coupled with low fixation potential, indicates possible net carbon loss. However, abundant marker genes for aerobic anoxygenic phototrophy, sulfur oxidation, rhodopsins and CO oxidation were also linked to the dominant heterotrophic bacteria, and indicate the use of photo- and lithoheterotrophy as mechanisms for conserving organic carbon. Similarly, a high genetic potential for the recycling of nitrogen compounds likely functions to retain fixed nitrogen in the lake. Dimethylsulfoniopropionate (DMSP) lyase genes were abundant, indicating that DMSP is a significant carbon and energy source. Unlike marine environments, DMSP demethylases were less abundant, indicating that DMSP cleavage is the likely source of high DMS concentration. DMSP cleavage, carbon mixotrophy (photoheterotrophy and lithoheterotrophy) and nitrogen remineralization by dominant Organic Lake bacteria are potentially important adaptations to nutrient constraints. In particular, carbon mixotrophy relieves the extent of carbon oxidation for energy production, allowing more carbon to be used for biosynthetic processes. The study sheds light on how the microbial community has adapted to this unique Antarctic lake environment.

Key microbial drivers in Antarctic aquatic environments.

Antarctica is arguably the world's most important continent for influencing the Earth's climate and ocean ecosystem function. The unique physico-chemical properties of the Southern Ocean enable high levels of microbial primary production to occur. This not only forms the base of a significant fraction of the global oceanic food web, but leads to the sequestration of anthropogenic CO2 and its transport to marine sediments, thereby removing it from the atmosphere; the Southern Ocean accounts for ~ 30% of global ocean uptake of CO2 despite representing ~ 10% of the total surface area of the global ocean. The Antarctic continent itself harbors some liquid water, including a remarkably diverse range of surface and subglacial lakes. Being one of the remaining natural frontiers, Antarctica delivers the paradox of needing to be protected from disturbance while requiring scientific endeavor to discover what is indigenous and learn how best to protect it. Moreover, like many natural environments on Earth, in Antarctica, microorganisms dominate the genetic pool and biomass of the colonizable niches and play the key roles in maintaining proper ecosystem function. This review puts into perspective insight that has been and can be gained about Antarctica's aquatic microbiota using molecular biology, and in particular, metagenomic approaches.

Simple high-throughput annotation pipeline (SHAP).

SUMMARY: SHAP (simple high-throughput annotation pipeline) is a lightweight and scalable sequence annotation pipeline capable of supporting research efforts that generate or utilize large volumes of DNA sequence data. The software provides Grid capable analysis, relational storage and Web-based full-text searching of annotation results. Implemented in Java, SHAP recognizes the limited resources of many smaller research groups. AVAILABILITY: Source code is freely available under GPLv3 at https://sourceforge.net/projects/shap. CONTACT: matt.demaere@unsw.edu.au; r.cavicchioli@unsw.edu.au.

Virophage control of antarctic algal host-virus dynamics.

Viruses are abundant ubiquitous members of microbial communities and in the marine environment affect population structure and nutrient cycling by infecting and lysing primary producers. Antarctic lakes are microbially dominated ecosystems supporting truncated food webs in which viruses exert a major influence on the microbial loop. Here we report the discovery of a virophage (relative of the recently described Sputnik virophage) that preys on phycodnaviruses that infect prasinophytes (phototrophic algae). By performing metaproteogenomic analysis on samples from Organic Lake, a hypersaline meromictic lake in Antarctica, complete virophage and near-complete phycodnavirus genomes were obtained. By introducing the virophage as an additional predator of a predator-prey dynamic model we determined that the virophage stimulates secondary production through the microbial loop by reducing overall mortality of the host and increasing the frequency of blooms during polar summer light periods. Virophages remained abundant in the lake 2 y later and were represented by populations with a high level of major capsid protein sequence variation (25-100% identity). Virophage signatures were also found in neighboring Ace Lake (in abundance) and in two tropical lakes (hypersaline and fresh), an estuary, and an ocean upwelling site. These findings indicate that virophages regulate host-virus interactions, influence overall carbon flux in Organic Lake, and play previously unrecognized roles in diverse aquatic ecosystems.

Untangling the multiple monooxygenases of Mycobacterium chubuense strain NBB4, a versatile hydrocarbon degrader.

Mycobacterium strain NBB4 was isolated on ethene as part of a bioprospecting study searching for novel monooxygenase (MO) enzymes of interest to biocatalysis and bioremediation. Previous work indicated that strain NBB4 contained an unprecedented diversity of MO genes, and we hypothesized that each MO type would support growth on a distinct hydrocarbon substrate. Here, we attempted to untangle the relationships between MO types and hydrocarbon substrates. Strain NBB4 was shown to grow on C2 -C4 alkenes and C2 -C16 alkanes. Complete gene clusters encoding six different monooxygenases were recovered from a fosmid library, including homologues of ethene MO (etnABCD), propene MO (pmoABCD), propane MO (smoABCD), butane MO (smoXYB1C1Z), cytochrome P450 (CYP153; fdx-cyp-fdr) and alkB (alkB-rubA1-rubA2). Catabolic enzymes involved in ethene assimilation (EtnA, EtnC, EtnD, EtnE) and alkane assimilation (alcohol and aldehyde dehydrogenases) were identified by proteomics, and we showed for the first time that stress response proteins (catalase/peroxidase, chaperonins) were induced by growth on C2 -C5 alkanes and ethene. Surprisingly, none of the identified MO genes could be specifically associated with oxidation of small alkanes, and thus the nature of the gaseous alkane MO in NBB4 remains mysterious.

An integrative study of a meromictic lake ecosystem in Antarctica.

In nature, the complexity and structure of microbial communities varies widely, ranging from a few species to thousands of species, and from highly structured to highly unstructured communities. Here, we describe the identity and functional capacity of microbial populations within distinct layers of a pristine, marine-derived, meromictic (stratified) lake (Ace Lake) in Antarctica. Nine million open reading frames were analyzed, representing microbial samples taken from six depths of the lake size fractionated on sequential 3.0, 0.8 and 0.1 µm filters, and including metaproteome data from matching 0.1 µm filters. We determine how the interactions of members of this highly structured and moderately complex community define the biogeochemical fluxes throughout the entire lake. Our view is that the health of this delicate ecosystem is dictated by the effects of the polar light cycle on the dominant role of green sulfur bacteria in primary production and nutrient cycling, and the influence of viruses/phage and phage resistance on the cooperation between members of the microbial community right throughout the lake. To test our assertions, and develop a framework applicable to other microbially driven ecosystems, we developed a mathematical model that describes how cooperation within a microbial system is impacted by periodic fluctuations in environmental parameters on key populations of microorganisms. Our study reveals a mutualistic structure within the microbial community throughout the lake that has arisen as the result of mechanistic interactions between the physico-chemical parameters and the selection of individual members of the community. By exhaustively describing and modelling interactions in Ace Lake, we have developed an approach that may be applicable to learning how environmental perturbations affect the microbial dynamics in more complex aquatic systems.

RSF1010-like plasmids in Australian Salmonella enterica serovar Typhimurium and origin of their sul2-strA-strB antibiotic resistance gene cluster.

Salmonella enterica serovar Typhimurium phage type 9 isolates resistant to streptomycin and sulfonamide have been recovered from both bovine and human sources in Australia. This study aimed to identify the resistance genes and their location. Polymerase chain reaction was used to screen for resistance genes and sul2 (sulphonamide resistance) and strA and strB (streptomycin resistance) were detected. A small streptomycin and sulfonamide resistance plasmid carrying the three resistance genes was recovered from these isolates by transformation and was shown to be essentially identical to the small IncQ plasmid RSF1010. The sequences of one plasmid, pSRC15, and RSF1010 differed at only a few positions that may be errors in the older sequence. RSF1010 has been recovered from many species and in many countries since its first isolation in the early 1970s. We conclude that this plasmid has persisted unchanged in the environment for over 30 years. The antibiotic resistance gene cluster containing strA, strB, and sul2 genes has clearly arisen from other known entities by a combination of transposition and homologous recombination using a short segment of homology. This resistance gene cluster is now widely distributed in plasmids and genomic islands in a number of contexts.