Functional redundancy revealed by the deletion of the mimivirus GMC-oxidoreductase genes.

The mimivirus 1.2 Mb genome was shown to be organized into a nucleocapsid-like genomic fiber encased in the nucleoid compartment inside the icosahedral capsid. The genomic fiber protein shell is composed of a mixture of two GMC-oxidoreductase paralogs, one of them being the main component of the glycosylated layer of fibrils at the surface of the virion. In this study, we determined the effect of the deletion of each of the corresponding genes on the genomic fiber and the layer of surface fibrils. First, we deleted the GMC-oxidoreductase, the most abundant in the genomic fiber, and determined its structure and composition in the mutant. As expected, it was composed of the second GMC-oxidoreductase and contained 5- and 6-start helices similar to the wild-type fiber. This result led us to propose a model explaining their coexistence. Then we deleted the GMC-oxidoreductase, the most abundant in the layer of fibrils, to analyze its protein composition in the mutant. Second, we showed that the fitness of single mutants and the double mutant were not decreased compared with the wild-type viruses under laboratory conditions. Third, we determined that deleting the GMC-oxidoreductase genes did not impact the glycosylation or the glycan composition of the layer of surface fibrils, despite modifying their protein composition. Because the glycosylation machinery and glycan composition of members of different clades are different, we expanded the analysis of the protein composition of the layer of fibrils to members of the B and C clades and showed that it was different among the three clades and even among isolates within the same clade. Taken together, the results obtained on two distinct central processes (genome packaging and virion coating) illustrate an unexpected functional redundancy in members of the family Mimiviridae, suggesting this may be the major evolutionary force behind their giant genomes.

The compact genome of the sponge Oopsacas minuta (Hexactinellida) is lacking key metazoan core genes.

BACKGROUND: Explaining the emergence of the hallmarks of bilaterians is a central focus of evolutionary developmental biology-evodevo-and evolutionary genomics. For this purpose, we must both expand and also refine our knowledge of non-bilaterian genomes, especially by studying early branching animals, in particular those in the metazoan phylum Porifera. RESULTS: We present a comprehensive analysis of the first whole genome of a glass sponge, Oopsacas minuta, a member of the Hexactinellida. Studying this class of sponge is evolutionary relevant because it differs from the three other Porifera classes in terms of development, tissue organization, ecology, and physiology. Although O. minuta does not exhibit drastic body simplifications, its genome is among the smallest of animal genomes sequenced so far, and surprisingly lacks several metazoan core genes (including Wnt and several key transcription factors). Our study also provides the complete genome of a symbiotic Archaea dominating the associated microbial community: a new Thaumarchaeota species. CONCLUSIONS: The genome of the glass sponge O. minuta differs from all other available sponge genomes by its compactness and smaller number of encoded proteins. The unexpected loss of numerous genes previously considered ancestral and pivotal for metazoan morphogenetic processes most likely reflects the peculiar syncytial tissue organization in this group. Our work further documents the importance of convergence during animal evolution, with multiple convergent evolution of septate-like junctions, electrical-signaling and multiciliated cells in metazoans.

An Update on Eukaryotic Viruses Revived from Ancient Permafrost.

One quarter of the Northern hemisphere is underlain by permanently frozen ground, referred to as permafrost. Due to climate warming, irreversibly thawing permafrost is releasing organic matter frozen for up to a million years, most of which decomposes into carbon dioxide and methane, further enhancing the greenhouse effect. Part of this organic matter also consists of revived cellular microbes (prokaryotes, unicellular eukaryotes) as well as viruses that have remained dormant since prehistorical times. While the literature abounds on descriptions of the rich and diverse prokaryotic microbiomes found in permafrost, no additional report about "live" viruses have been published since the two original studies describing pithovirus (in 2014) and mollivirus (in 2015). This wrongly suggests that such occurrences are rare and that "zombie viruses" are not a public health threat. To restore an appreciation closer to reality, we report the preliminary characterizations of 13 new viruses isolated from seven different ancient Siberian permafrost samples, one from the Lena river and one from Kamchatka cryosol. As expected from the host specificity imposed by our protocol, these viruses belong to five different clades infecting Acanthamoeba spp. but not previously revived from permafrost: Pandoravirus, Cedratvirus, Megavirus, and Pacmanvirus, in addition to a new Pithovirus strain.

Past and present giant viruses diversity explored through permafrost metagenomics.

Giant viruses are abundant in aquatic environments and ecologically important through the metabolic reprogramming of their hosts. Less is known about giant viruses from soil even though two of them, belonging to two different viral families, were reactivated from 30,000-y-old permafrost samples. This suggests an untapped diversity of Nucleocytoviricota in this environment. Through permafrost metagenomics we reveal a unique diversity pattern and a high heterogeneity in the abundance of giant viruses, representing up to 12% of the sum of sequence coverage in one sample. Pithoviridae and Orpheoviridae-like viruses were the most important contributors. A complete 1.6 Mb Pithoviridae-like circular genome was also assembled from a 42,000-y-old sample. The annotation of the permafrost viral sequences revealed a patchwork of predicted functions amidst a larger reservoir of genes of unknown functions. Finally, the phylogenetic reconstructions not only revealed gene transfers between cells and viruses, but also between viruses from different families.

Metagenomic survey of the microbiome of ancient Siberian permafrost and modern Kamchatkan cryosols.

In the context of global warming, the melting of Arctic permafrost raises the threat of a reemergence of microorganisms some of which were shown to remain viable in ancient frozen soils for up to half a million years. In order to evaluate this risk, it is of interest to acquire a better knowledge of the composition of the microbial communities found in this understudied environment. Here, we present a metagenomic analysis of 12 soil samples from Russian Arctic and subarctic pristine areas: Chukotka, Yakutia and Kamchatka, including nine permafrost samples collected at various depths. These large datasets (9.2 × 1011 total bp) were assembled (525 313 contigs > 5 kb), their encoded protein contents predicted, and then used to perform taxonomical assignments of bacterial, archaeal and eukaryotic organisms, as well as DNA viruses. The various samples exhibited variable DNA contents and highly diverse taxonomic profiles showing no obvious relationship with their locations, depths or deposit ages. Bacteria represented the largely dominant DNA fraction (95%) in all samples, followed by archaea (3.2%), surprisingly little eukaryotes (0.5%), and viruses (0.4%). Although no common taxonomic pattern was identified, the samples shared unexpected high frequencies of ß-lactamase genes, almost 0.9 copy/bacterial genome. In addition to known environmental threats, the particularly intense warming of the Arctic might thus enhance the spread of bacterial antibiotic resistances, today's major challenge in public health. ß-Lactamases were also observed at high frequency in other types of soils, suggesting their general role in the regulation of bacterial populations.

Validation of predicted anonymous proteins simply using Fisher's exact test.

Motivation: Genomes sequencing has become the primary (and often the sole) experimental method to characterize newly discovered organisms, in particular from the microbial world (bacteria, archaea, viruses). This generates an ever increasing number of predicted proteins the existence of which is unwarranted, in particular among those without homolog in model organisms. As a last resort, the computation of the selection pressure from pairwise alignments of the corresponding 'Open Reading Frames' (ORFs) can be used to validate their existences. However, this approach is error-prone, as not usually associated with a significance test. Results: We introduce the use of the straightforward Fisher's exact test as a postprocessing of the results provided by the popular CODEML sequence comparison software. The respective rates of nucleotide changes at the nonsynonymous versus synonymous position (as determined by CODEML) are turned into entries into a 2 × 2 contingency table, the probability of which is computed under the Null hypothesis that they should not behave differently if the ORFs do not encode actual proteins. Using the genome sequences of two recently isolated giant viruses, we show that strong negative selection pressures do not always provide a solid argument in favor of the existence of proteins.

Characterization of Mollivirus kamchatka, the First Modern Representative of the Proposed Molliviridae Family of Giant Viruses.

Microbes trapped in permanently frozen paleosoils (permafrost) are the focus of increasing research in the context of global warming. Our previous investigations led to the discovery and reactivation of two Acanthamoeba-infecting giant viruses, Mollivirus sibericum and Pithovirus sibericum, from a 30,000-year old permafrost layer. While several modern pithovirus strains have since been isolated, no contemporary mollivirus relative was found. We now describe Mollivirus kamchatka, a close relative to M. sibericum, isolated from surface soil sampled on the bank of the Kronotsky River in Kamchatka, Russian Federation. This discovery confirms that molliviruses have not gone extinct and are at least present in a distant subarctic continental location. This modern isolate exhibits a nucleocytoplasmic replication cycle identical to that of M. sibericum Its spherical particle (0.6 µm in diameter) encloses a 648-kb GC-rich double-stranded DNA genome coding for 480 proteins, of which 61% are unique to these two molliviruses. The 461 homologous proteins are highly conserved (92% identical residues, on average), despite the presumed stasis of M. sibericum for the last 30,000 years. Selection pressure analyses show that most of these proteins contribute to virus fitness. The comparison of these first two molliviruses clarify their evolutionary relationship with the pandoraviruses, supporting their provisional classification in a distinct family, the Molliviridae, pending the eventual discovery of intermediary missing links better demonstrating their common ancestry.IMPORTANCE Virology has long been viewed through the prism of human, cattle, or plant diseases, leading to a largely incomplete picture of the viral world. The serendipitous discovery of the first giant virus visible under a light microscope (i.e., >0.3 µm in diameter), mimivirus, opened a new era of environmental virology, now incorporating protozoan-infecting viruses. Planet-wide isolation studies and metagenome analyses have shown the presence of giant viruses in most terrestrial and aquatic environments, including upper Pleistocene frozen soils. Those systematic surveys have led authors to propose several new distinct families, including the Mimiviridae, Marseilleviridae, Faustoviridae, Pandoraviridae, and Pithoviridae We now propose to introduce one additional family, the Molliviridae, following the description of M. kamchatka, the first modern relative of M. sibericum, previously isolated from 30,000-year-old arctic permafrost.

Exploration of the propagation of transpovirons within Mimiviridae reveals a unique example of commensalism in the viral world.

Acanthamoeba-infecting Mimiviridae are giant viruses with dsDNA genome up to 1.5 Mb. They build viral factories in the host cytoplasm in which the nuclear-like virus-encoded functions take place. They are themselves the target of infections by 20-kb-dsDNA virophages, replicating in the giant virus factories and can also be found associated with 7-kb-DNA episomes, dubbed transpovirons. Here we isolated a virophage (Zamilon vitis) and two transpovirons respectively associated to B- and C-clade mimiviruses. We found that the virophage could transfer each transpoviron provided the host viruses were devoid of a resident transpoviron (permissive effect). If not, only the resident transpoviron originally isolated from the corresponding virus was replicated and propagated within the virophage progeny (dominance effect). Although B- and C-clade viruses devoid of transpoviron could replicate each transpoviron, they did it with a lower efficiency across clades, suggesting an ongoing process of adaptive co-evolution. We analysed the proteomes of host viruses and virophage particles in search of proteins involved in this adaptation process. This study also highlights a unique example of intricate commensalism in the viral world, where the transpoviron uses the virophage to propagate and where the Zamilon virophage and the transpoviron depend on the giant virus to replicate, without affecting its infectious cycle.

Unexpected invasion of miniature inverted-repeat transposable elements in viral genomes.

BACKGROUND: Transposable elements (TEs) are common and often present with high copy numbers in cellular genomes. Unlike in cellular organisms, TEs were previously thought to be either rare or absent in viruses. Almost all reported TEs display only one or two copies per viral genome. In addition, the discovery of pandoraviruses with genomes up to 2.5-Mb emphasizes the need for biologists to rethink the fundamental nature of the relationship between viruses and cellular life. RESULTS: Herein, we performed the first comprehensive analysis of miniature inverted-repeat transposable elements (MITEs) in the 5170 viral genomes for which sequences are currently available. Four hundred and fifty one copies of ten miniature inverted-repeat transposable elements (MITEs) were found and each MITE had reached relatively large copy numbers (some up to 90) in viruses. Eight MITEs belonging to two DNA superfamilies (hobo/Activator/Tam3 and Chapaev-Mirage-CACTA) were for the first time identified in viruses, further expanding the organismal range of these two superfamilies. TEs may play important roles in shaping the evolution of pandoravirus genomes, which were here found to be very rich in MITEs. We also show that putative autonomous partners of seven MITEs are present in the genomes of viral hosts, suggesting that viruses may borrow the transpositional machinery of their cellular hosts' autonomous elements to spread MITEs and colonize their own genomes. The presence of seven similar MITEs in viral hosts, suggesting horizontal transfers (HTs) as the major mechanism for MITEs propagation. CONCLUSIONS: Our discovery highlights that TEs contribute to shape genome evolution of pandoraviruses. We concluded that as for cellular organisms, TEs are part of the pandoraviruses' diverse mobilome.

New genomic data and analyses challenge the traditional vision of animal epithelium evolution.

BACKGROUND: The emergence of epithelia was the foundation of metazoan expansion. Epithelial tissues are a hallmark of metazoans deeply rooted in the evolution of their complex developmental morphogenesis processes. However, studies on the epithelial features of non-bilaterians are still sparse and it remains unclear whether the last common metazoan ancestor possessed a fully functional epithelial toolkit or if it was acquired later during metazoan evolution. RESULTS: To investigate the early evolution of animal epithelia, we sequenced the genome and transcriptomes of two new sponge species to characterize epithelial markers such as the E-cadherin complex and the polarity complexes for all classes (Calcarea, Demospongiae, Hexactinellida, Homoscleromorpha) of sponges (phylum Porifera) and compare them with their homologues in Placozoa and in Ctenophora. We found that Placozoa and most sponges possess orthologues of all essential genes encoding proteins characteristic of bilaterian epithelial cells, as well as their conserved interaction domains. In stark contrast, we found that ctenophores lack several major polarity complex components such as the Crumbs complex and Scribble. Furthermore, the E-cadherin ctenophore orthologue exhibits a divergent cytoplasmic domain making it unlikely to interact with its canonical cytoplasmic partners. CONCLUSIONS: These unexpected findings challenge the current evolutionary paradigm on the emergence of epithelia. Altogether, our results raise doubt on the homology of protein complexes and structures involved in cell polarity and adhesive-type junctions between Ctenophora and Bilateria epithelia.

Noumeavirus replication relies on a transient remote control of the host nucleus.

Acanthamoeba are infected by a remarkable diversity of large dsDNA viruses, the infectious cycles of which have been characterized using genomics, transcriptomics and electron microscopy. Given their gene content and the persistence of the host nucleus throughout their infectious cycle, the Marseilleviridae were initially assumed to fully replicate in the cytoplasm. Unexpectedly, we find that their virions do not incorporate the virus-encoded transcription machinery, making their replication nucleus-dependent. However, instead of delivering their DNA to the nucleus, the Marseilleviridae initiate their replication by transiently recruiting the nuclear transcription machinery to their cytoplasmic viral factory. The nucleus recovers its integrity after becoming leaky at an early stage. This work highlights the importance of virion proteomic analyses to complement genome sequencing in the elucidation of the replication scheme and evolution of large dsDNA viruses.

Benthic protists: the under-charted majority.

Marine protist diversity inventories have largely focused on planktonic environments, while benthic protists have received relatively little attention. We therefore hypothesize that current diversity surveys have only skimmed the surface of protist diversity in marine sediments, which may harbor greater diversity than planktonic environments. We tested this by analyzing sequences of the hypervariable V4 18S rRNA from benthic and planktonic protist communities sampled in European coastal regions. Despite a similar number of OTUs in both realms, richness estimations indicated that we recovered at least 70% of the diversity in planktonic protist communities, but only 33% in benthic communities. There was also little overlap of OTUs between planktonic and benthic communities, as well as between separate benthic communities. We argue that these patterns reflect the heterogeneity and diversity of benthic habitats. A comparison of all OTUs against the Protist Ribosomal Reference database showed that a higher proportion of benthic than planktonic protist diversity is missing from public databases; similar results were obtained by comparing all OTUs against environmental references from NCBI's Short Read Archive. We suggest that the benthic realm may therefore be the world's largest reservoir of marine protist diversity, with most taxa at present undescribed.

The 474-Kilobase-Pair Complete Genome Sequence of CeV-01B, a Virus Infecting Haptolina (Chrysochromulina) ericina (Prymnesiophyceae).

We report the complete genome sequence of CeV-01B, a large double-stranded DNA virus infecting the unicellular marine phytoplankton Haptolina (formerly Chrysochromulina) ericina. CeV-01B and its closest relative Phaeocystis globosa virus define an emerging subclade of the Megaviridae family with smaller genomes and particles than the originally described giant Mimiviridae infecting Acanthamoeba.

Draft Genome Sequence of an Alphaproteobacterium Associated with the Mediterranean Sponge Oscarella lobularis.

While sequencing DNA purified from the homoscleromorph sponge Oscarella lobularis, we detected a large number of reads with strong similarity to available alphaproteobacteria gene sequences of family Rhodobacteraceae. Here, we present the genome sequence of this putative sponge symbiont that we propose to designate as "Candidatus Rhodobacter lobularis."

In Vivo Evolution of Bacterial Resistance in Two Cases of Enterobacter aerogenes Infections during Treatment with Imipenem.

Infections caused by multidrug resistant (MDR) bacteria are a major concern worldwide. Changes in membrane permeability, including decreased influx and/or increased efflux of antibiotics, are known as key contributors of bacterial MDR. Therefore, it is of critical importance to understand molecular mechanisms that link membrane permeability to MDR in order to design new antimicrobial strategies. In this work, we describe genotype-phenotype correlations in Enterobacter aerogenes, a clinically problematic and antibiotic resistant bacterium. To do this, series of clinical isolates have been periodically collected from two patients during chemotherapy with imipenem. The isolates exhibited different levels of resistance towards multiple classes of antibiotics, consistently with the presence or the absence of porins and efflux pumps. Transport assays were used to characterize membrane permeability defects. Simultaneous genome-wide analysis allowed the identification of putative mutations responsible for MDR. The genome of the imipenem-susceptible isolate G7 was sequenced to closure and used as a reference for comparative genomics. This approach uncovered several loci that were specifically mutated in MDR isolates and whose products are known to control membrane permeability. These were omp35 and omp36, encoding the two major porins; rob, encoding a global AraC-type transcriptional activator; cpxA, phoQ and pmrB, encoding sensor kinases of the CpxRA, PhoPQ and PmrAB two-component regulatory systems, respectively. This report provides a comprehensive analysis of membrane alterations relative to mutational steps in the evolution of MDR of a recognized nosocomial pathogen.

Mitochondrial Genome Sequence of the Glass Sponge Oopsacas minuta.

We report the complete mitochondrial genome sequence of the Mediterranean glass sponge Oopsacas minuta. This 19-kb mitochondrial genome has 24 noncoding genes (22 tRNAs and 2 rRNAs) and 14 protein-encoding genes coding for 11 subunits of respiratory chain complexes and 3 ATP synthase subunits.

Marine protist diversity in European coastal waters and sediments as revealed by high-throughput sequencing.

Although protists are critical components of marine ecosystems, they are still poorly characterized. Here we analysed the taxonomic diversity of planktonic and benthic protist communities collected in six distant European coastal sites. Environmental deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) from three size fractions (pico-, nano- and micro/mesoplankton), as well as from dissolved DNA and surface sediments were used as templates for tag pyrosequencing of the V4 region of the 18S ribosomal DNA. Beta-diversity analyses split the protist community structure into three main clusters: picoplankton-nanoplankton-dissolved DNA, micro/mesoplankton and sediments. Within each cluster, protist communities from the same site and time clustered together, while communities from the same site but different seasons were unrelated. Both DNA and RNA-based surveys provided similar relative abundances for most class-level taxonomic groups. Yet, particular groups were overrepresented in one of the two templates, such as marine alveolates (MALV)-I and MALV-II that were much more abundant in DNA surveys. Overall, the groups displaying the highest relative contribution were Dinophyceae, Diatomea, Ciliophora and Acantharia. Also, well represented were Mamiellophyceae, Cryptomonadales, marine alveolates and marine stramenopiles in the picoplankton, and Monadofilosa and basal Fungi in sediments. Our extensive and systematic sequencing of geographically separated sites provides the most comprehensive molecular description of coastal marine protist diversity to date.

Ocean plankton. Environmental characteristics of Agulhas rings affect interocean plankton transport.

Agulhas rings provide the principal route for ocean waters to circulate from the Indo-Pacific to the Atlantic basin. Their influence on global ocean circulation is well known, but their role in plankton transport is largely unexplored. We show that, although the coarse taxonomic structure of plankton communities is continuous across the Agulhas choke point, South Atlantic plankton diversity is altered compared with Indian Ocean source populations. Modeling and in situ sampling of a young Agulhas ring indicate that strong vertical mixing drives complex nitrogen cycling, shaping community metabolism and biogeochemical signatures as the ring and associated plankton transit westward. The peculiar local environment inside Agulhas rings may provide a selective mechanism contributing to the limited dispersal of Indian Ocean plankton populations into the Atlantic.

Patterns of rare and abundant marine microbial eukaryotes.

BACKGROUND: Biological communities are normally composed of a few abundant and many rare species. This pattern is particularly prominent in microbial communities, in which most constituent taxa are usually extremely rare. Although abundant and rare subcommunities may present intrinsic characteristics that could be crucial for understanding community dynamics and ecosystem functioning, microbiologists normally do not differentiate between them. Here, we investigate abundant and rare subcommunities of marine microbial eukaryotes, a crucial group of organisms that remains among the least-explored biodiversity components of the biosphere. We surveyed surface waters of six separate coastal locations in Europe, independently considering the picoplankton, nanoplankton, and microplankton/mesoplankton organismal size fractions. RESULTS: Deep Illumina sequencing of the 18S rRNA indicated that the abundant regional community was mostly structured by organismal size fraction, whereas the rare regional community was mainly structured by geographic origin. However, some abundant and rare taxa presented similar biogeography, pointing to spatiotemporal structure in the rare microeukaryote biosphere. Abundant and rare subcommunities presented regular proportions across samples, indicating similar species-abundance distributions despite taxonomic compositional variation. Several taxa were abundant in one location and rare in other locations, suggesting large oscillations in abundance. The substantial amount of metabolically active lineages found in the rare biosphere suggests that this subcommunity constitutes a diversity reservoir that can respond rapidly to environmental change. CONCLUSIONS: We propose that marine planktonic microeukaryote assemblages incorporate dynamic and metabolically active abundant and rare subcommunities, with contrasting structuring patterns but fairly regular proportions, across space and time.

Vampires in the oceans: predatory cercozoan amoebae in marine habitats.

Vampire amoebae (vampyrellids) are predators of algae, fungi, protozoa and small metazoans known primarily from soils and in freshwater habitats. They are among the very few heterotrophic naked, filose and reticulose protists that have received some attention from a morphological and ecological point of view over the last few decades, because of the peculiar mode of feeding of known species. Yet, the true extent of their biodiversity remains largely unknown. Here we use a complementary approach of culturing and sequence database mining to address this issue, focusing our efforts on marine environments, where vampyrellids are very poorly known. We present 10 new vampyrellid isolates, 8 from marine or brackish sediments, and 2 from soil or freshwater sediment. Two of the former correspond to the genera Thalassomyxa Grell and Penardia Cash for which sequence data were previously unavailable. Small-subunit ribosomal DNA analysis confirms they are all related to previously sequenced vampyrellids. An exhaustive screening of the NCBI GenBank database and of 454 sequence data generated by the European BioMarKs consortium revealed hundreds of distinct environmental vampyrellid sequences. We show that vampyrellids are much more diverse than previously thought, especially in marine habitats. Our new isolates, which cover almost the full phylogenetic range of vampyrellid sequences revealed in this study, offer a rare opportunity to integrate data from environmental DNA surveys with phenotypic information. However, the very large genetic diversity we highlight within vampyrellids (especially in marine sediments and soils) contrasts with the paradoxically low morphological distinctiveness we observed across our isolates.