Cyanorak v2.1: a scalable information system dedicated to the visualization and expert curation of marine and brackish picocyanobacteria genomes.

Cyanorak v2.1 (http://www.sb-roscoff.fr/cyanorak) is an information system dedicated to visualizing, comparing and curating the genomes of Prochlorococcus, Synechococcus and Cyanobium, the most abundant photosynthetic microorganisms on Earth. The database encompasses sequences from 97 genomes, covering most of the wide genetic diversity known so far within these groups, and which were split into 25,834 clusters of likely orthologous groups (CLOGs). The user interface gives access to genomic characteristics, accession numbers as well as an interactive map showing strain isolation sites. The main entry to the database is through search for a term (gene name, product, etc.), resulting in a list of CLOGs and individual genes. Each CLOG benefits from a rich functional annotation including EggNOG, EC/K numbers, GO terms, TIGR Roles, custom-designed Cyanorak Roles as well as several protein motif predictions. Cyanorak also displays a phyletic profile, indicating the genotype and pigment type for each CLOG, and a genome viewer (Jbrowse) to visualize additional data on each genome such as predicted operons, genomic islands or transcriptomic data, when available. This information system also includes a BLAST search tool, comparative genomic context as well as various data export options. Altogether, Cyanorak v2.1 constitutes an invaluable, scalable tool for comparative genomics of ecologically relevant marine microorganisms.

Light color acclimation is a key process in the global ocean distribution of Synechococcus cyanobacteria.

Marine Synechococcus cyanobacteria are major contributors to global oceanic primary production and exhibit a unique diversity of photosynthetic pigments, allowing them to exploit a wide range of light niches. However, the relationship between pigment content and niche partitioning has remained largely undetermined due to the lack of a single-genetic marker resolving all pigment types (PTs). Here, we developed and employed a robust method based on three distinct marker genes (cpcBA, mpeBA, and mpeW) to estimate the relative abundance of all known Synechococcus PTs from metagenomes. Analysis of the Tara Oceans dataset allowed us to reveal the global distribution of Synechococcus PTs and to define their environmental niches. Green-light specialists (PT 3a) dominated in warm, green equatorial waters, whereas blue-light specialists (PT 3c) were particularly abundant in oligotrophic areas. Type IV chromatic acclimaters (CA4-A/B), which are able to dynamically modify their light absorption properties to maximally absorb green or blue light, were unexpectedly the most abundant PT in our dataset and predominated at depth and high latitudes. We also identified populations in which CA4 might be nonfunctional due to the lack of specific CA4 genes, notably in warm high-nutrient low-chlorophyll areas. Major ecotypes within clades I-IV and CRD1 were preferentially associated with a particular PT, while others exhibited a wide range of PTs. Altogether, this study provides important insights into the ecology of Synechococcus and highlights the complex interactions between vertical phylogeny, pigmentation, and environmental parameters that shape Synechococcus community structure and evolution.

Delineating ecologically significant taxonomic units from global patterns of marine picocyanobacteria.

Prochlorococcus and Synechococcus are the two most abundant and widespread phytoplankton in the global ocean. To better understand the factors controlling their biogeography, a reference database of the high-resolution taxonomic marker petB, encoding cytochrome b6, was used to recruit reads out of 109 metagenomes from the Tara Oceans expedition. An unsuspected novel genetic diversity was unveiled within both genera, even for the most abundant and well-characterized clades, and 136 divergent petB sequences were successfully assembled from metagenomic reads, significantly enriching the reference database. We then defined Ecologically Significant Taxonomic Units (ESTUs)-that is, organisms belonging to the same clade and occupying a common oceanic niche. Three major ESTU assemblages were identified along the cruise transect for Prochlorococcus and eight for Synechococcus Although Prochlorococcus HLIIIA and HLIVA ESTUs codominated in iron-depleted areas of the Pacific Ocean, CRD1 and the yet-to-be cultured EnvB were the prevalent Synechococcus clades in this area, with three different CRD1 and EnvB ESTUs occupying distinct ecological niches with regard to iron availability and temperature. Sharp community shifts were also observed over short geographic distances-for example, around the Marquesas Islands or between southern Indian and Atlantic Oceans-pointing to a tight correlation between ESTU assemblages and specific physico-chemical parameters. Together, this study demonstrates that there is a previously overlooked, ecologically meaningful, fine-scale diversity within some currently defined picocyanobacterial ecotypes, bringing novel insights into the ecology, diversity, and biology of the two most abundant phototrophs on Earth.

WiseScaffolder: an algorithm for the semi-automatic scaffolding of Next Generation Sequencing data.

BACKGROUND: The sequencing depth provided by high-throughput sequencing technologies has allowed a rise in the number of de novo sequenced genomes that could potentially be closed without further sequencing. However, genome scaffolding and closure require costly human supervision that often results in genomes being published as drafts. A number of automatic scaffolders were recently released, which improved the global quality of genomes published in the last few years. Yet, none of them reach the efficiency of manual scaffolding. RESULTS: Here, we present an innovative semi-automatic scaffolder that additionally helps with chimerae resolution and generates valuable contig maps and outputs for manual improvement of the automatic scaffolding. This software was tested on the newly sequenced marine cyanobacterium Synechococcus sp. WH8103 as well as two reference datasets used in previous studies, Rhodobacter sphaeroides and Homo sapiens chromosome 14 (http://gage.cbcb.umd.edu/). The quality of resulting scaffolds was compared to that of three other stand-alone scaffolders: SSPACE, SOPRA and SCARPA. For all three model organisms, WiseScaffolder produced better results than other scaffolders in terms of contiguity statistics (number of genome fragments, N50, LG50, etc.) and, in the case of WH8103, the reliability of the scaffolds was confirmed by whole genome alignment against a closely related reference genome. We also propose an efficient computer-assisted strategy for manual improvement of the scaffolding, using outputs generated by WiseScaffolder, as well as for genome finishing that in our hands led to the circularization of the WH8103 genome. CONCLUSION: Altogether, WiseScaffolder proved more efficient than three other scaffolders for both prokaryotic and eukaryotic genomes and is thus likely applicable to most genome projects. The scaffolding pipeline described here should be of particular interest to biologists wishing to take advantage of the high added value of complete genomes.

Metagenomic analyses of a microbial assemblage in a subglacial lake beneath the Vatnajökull ice cap, Iceland.

Skaftárkatlar are two subglacial lakes located beneath the Vatnajökull ice cap in Iceland associated with geothermal and volcanic activity. Previous studies of these lakes with ribosomal gene (16S rDNA) tag sequencing revealed a limited diversity of bacteria adapted to cold, dark, and nutrient-poor waters. In this study, we present analyses of metagenomes from the lake which give new insights into its microbial ecology. Analyses of the 16S rDNA genes in the metagenomes confirmed the existence of a low-diversity core microbial assemblage in the lake and insights into the potential metabolisms of the dominant members. Seven taxonomic genera, Sulfuricurvum, Sulfurospirillum, Acetobacterium, Pelobacter/Geobacter, Saccharibacteria, Caldisericum, and an unclassified member of Prolixibacteraceae, comprised more than 98% of the rDNA reads in the library. Functional characterisation of the lake metagenomes revealed complete metabolic pathways for sulphur cycling, nitrogen metabolism, carbon fixation via the reverse Krebs cycle, and acetogenesis. These results show that chemolithoautotrophy constitutes the main metabolism in this subglacial ecosystem. This assemblage and its metabolisms are not reflected in enrichment cultures, demonstrating the importance of in situ investigations of this environment.

Differential global distribution of marine picocyanobacteria gene clusters reveals distinct niche-related adaptive strategies.

The ever-increasing number of available microbial genomes and metagenomes provides new opportunities to investigate the links between niche partitioning and genome evolution in the ocean, especially for the abundant and ubiquitous marine picocyanobacteria Prochlorococcus and Synechococcus. Here, by combining metagenome analyses of the Tara Oceans dataset with comparative genomics, including phyletic patterns and genomic context of individual genes from 256 reference genomes, we show that picocyanobacterial communities thriving in different niches possess distinct gene repertoires. We also identify clusters of adjacent genes that display specific distribution patterns in the field (eCAGs) and are thus potentially involved in the same metabolic pathway and may have a key role in niche adaptation. Several eCAGs are likely involved in the uptake or incorporation of complex organic forms of nutrients, such as guanidine, cyanate, cyanide, pyrimidine, or phosphonates, which might be either directly used by cells, for example for the biosynthesis of proteins or DNA, or degraded to inorganic nitrogen and/or phosphorus forms. We also highlight the enrichment of eCAGs involved in polysaccharide capsule biosynthesis in Synechococcus populations thriving in both nitrogen- and phosphorus-depleted areas vs. low-iron (Fe) regions, suggesting that the complexes they encode may be too energy-consuming for picocyanobacteria thriving in the latter areas. In contrast, Prochlorococcus populations thriving in Fe-depleted areas specifically possess an alternative respiratory terminal oxidase, potentially involved in the reduction of Fe(III) to Fe(II). Altogether, this study provides insights into how phytoplankton communities populate oceanic ecosystems, which is relevant to understanding their capacity to respond to ongoing climate change.

Global Phylogeography of Marine Synechococcus in Coastal Areas Reveals Strong Community Shifts.

Marine Synechococcus comprise a numerically and ecologically prominent phytoplankton group, playing a major role in both carbon cycling and trophic networks in all oceanic regions except in the polar oceans. Despite their high abundance in coastal areas, our knowledge of Synechococcus communities in these environments is based on only a few local studies. Here, we use the global metagenome data set of the Ocean Sampling Day (June 21st, 2014) to get a snapshot of the taxonomic composition of coastal Synechococcus communities worldwide, by recruitment on a reference database of 141 picocyanobacterial genomes, representative of the whole Prochlorococcus, Synechococcus, and Cyanobium diversity. This allowed us to unravel drastic community shifts over small to medium scale gradients of environmental factors, in particular along European coasts. The combined analysis of the phylogeography of natural populations and the thermophysiological characterization of eight strains, representative of the four major Synechococcus lineages (clades I to IV), also brought novel insights about the differential niche partitioning of clades I and IV, which most often co-dominate the Synechococcus community in cold and temperate coastal areas. Altogether, this study reveals several important characteristics and specificities of the coastal communities of Synechococcus worldwide. IMPORTANCE Synechococcus is the second most abundant phytoplanktonic organism on Earth, and its wide genetic diversity allowed it to colonize all the oceans except for polar waters, with different clades colonizing distinct oceanic niches. In recent years, the use of global metagenomics data sets has greatly improved our knowledge of "who is where" by describing the distribution of Synechococcus clades or ecotypes in the open ocean. However, little is known about the global distribution of Synechococcus ecotypes in coastal areas, where Synechococcus is often the dominant phytoplanktonic organism. Here, we leverage the global Ocean Sampling Day metagenomics data set to describe Synechococcus community composition in coastal areas worldwide, revealing striking community shifts, in particular along the coasts of Europe. As temperature appears as an important driver of the community composition, we also characterize the thermal preferenda of 8 Synechococcus strains, bringing new insights into the adaptation to temperature of the dominant Synechococcus clades.

Diversity and Evolution of Pigment Types in Marine Synechococcus Cyanobacteria.

Synechococcus cyanobacteria are ubiquitous and abundant in the marine environment and contribute to an estimated 16% of the ocean net primary productivity. Their light-harvesting complexes, called phycobilisomes (PBS), are composed of a conserved allophycocyanin core, from which radiates six to eight rods with variable phycobiliprotein and chromophore content. This variability allows Synechococcus cells to optimally exploit the wide variety of spectral niches existing in marine ecosystems. Seven distinct pigment types or subtypes have been identified so far in this taxon based on the phycobiliprotein composition and/or the proportion of the different chromophores in PBS rods. Most genes involved in their biosynthesis and regulation are located in a dedicated genomic region called the PBS rod region. Here, we examine the variability of gene content and organization of this genomic region in a large set of sequenced isolates and natural populations of Synechococcus representative of all known pigment types. All regions start with a tRNA-PheGAA and some possess mobile elements for DNA integration and site-specific recombination, suggesting that their genomic variability relies in part on a "tycheposon"-like mechanism. Comparison of the phylogenies obtained for PBS and core genes revealed that the evolutionary history of PBS rod genes differs from the core genome and is characterized by the co-existence of different alleles and frequent allelic exchange. We propose a scenario for the evolution of the different pigment types and highlight the importance of incomplete lineage sorting in maintaining a wide diversity of pigment types in different Synechococcus lineages despite multiple speciation events.

Unusual organization, complexity and redundancy at the Escherichia coli hcp-hcr operon promoter.

Expression from the Escherichia coli hcp-hcr operon promoter is optimally induced during anaerobic conditions in the presence of nitrite. This expression depends on transcription activation by FNR (fumarate and nitrate reduction regulator), which binds to a target centred at position -72.5 upstream of the transcript start site. Mutational analysis was exploited to identify the corresponding -10 and -35 hexamer elements. A DNA site for NarL and NarP, located at position -104.5, plays only a minor role, whereas NsrR binding to a DNA target centred at position +6 plays a major role in induction of the hcp-hcr operon promoter. Electrophoretic mobility-shift assays show that NsrR binds to this target. The consequences of this for the kinetics of induction of the hcp-hcr operon are discussed.

Evolutionary Mechanisms of Long-Term Genome Diversification Associated With Niche Partitioning in Marine Picocyanobacteria.

Marine picocyanobacteria of the genera Prochlorococcus and Synechococcus are the most abundant photosynthetic organisms on Earth, an ecological success thought to be linked to the differential partitioning of distinct ecotypes into specific ecological niches. However, the underlying processes that governed the diversification of these microorganisms and the appearance of niche-related phenotypic traits are just starting to be elucidated. Here, by comparing 81 genomes, including 34 new Synechococcus, we explored the evolutionary processes that shaped the genomic diversity of picocyanobacteria. Time-calibration of a core-protein tree showed that gene gain/loss occurred at an unexpectedly low rate between the different lineages, with for instance 5.6 genes gained per million years (My) for the major Synechococcus lineage (sub-cluster 5.1), among which only 0.71/My have been fixed in the long term. Gene content comparisons revealed a number of candidates involved in nutrient adaptation, a large proportion of which are located in genomic islands shared between either closely or more distantly related strains, as identified using an original network construction approach. Interestingly, strains representative of the different ecotypes co-occurring in phosphorus-depleted waters (Synechococcus clades III, WPC1, and sub-cluster 5.3) were shown to display different adaptation strategies to this limitation. In contrast, we found few genes potentially involved in adaptation to temperature when comparing cold and warm thermotypes. Indeed, comparison of core protein sequences highlighted variants specific to cold thermotypes, notably involved in carotenoid biosynthesis and the oxidative stress response, revealing that long-term adaptation to thermal niches relies on amino acid substitutions rather than on gene content variation. Altogether, this study not only deciphers the respective roles of gene gains/losses and sequence variation but also uncovers numerous gene candidates likely involved in niche partitioning of two key members of the marine phytoplankton.

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.

Development of a targeted metagenomic approach to study a genomic region involved in light harvesting in marine Synechococcus.

Synechococcus, one of the most abundant cyanobacteria in marine ecosystems, displays a broad pigment diversity. However, the in situ distribution of pigment types remains largely unknown. In this study, we combined flow cytometry cell sorting, whole-genome amplification, and fosmid library construction to target a genomic region involved in light-harvesting complex (phycobilisome) biosynthesis and regulation. Synechococcus community composition and relative contamination by heterotrophic bacteria were assessed at each step of the pipeline using terminal restriction fragment length polymorphism targeting the petB and 16S rRNA genes, respectively. This approach allowed us to control biases inherent to each method and select reliable WGA products to construct a fosmid library from a natural sample collected off Roscoff (France). Sequencing of 25 fosmids containing the targeted region led to the assembly of whole or partial phycobilisome regions. Most contigs were assigned to clades I and IV consistent with the known dominance of these clades in temperate coastal waters. However, one of the fosmids contained genes distantly related to their orthologs in reference genomes, suggesting that it belonged to a novel phylogenetic clade. Altogether, this study provides novel insights into Synechococcus community structure and pigment type diversity at a representative coastal station of the English Channel.

A gene island with two possible configurations is involved in chromatic acclimation in marine Synechococcus.

Synechococcus, the second most abundant oxygenic phototroph in the marine environment, harbors the largest pigment diversity known within a single genus of cyanobacteria, allowing it to exploit a wide range of light niches. Some strains are capable of Type IV chromatic acclimation (CA4), a process by which cells can match the phycobilin content of their phycobilisomes to the ambient light quality. Here, we performed extensive genomic comparisons to explore the diversity of this process within the marine Synechococcus radiation. A specific gene island was identified in all CA4-performing strains, containing two genes (fciA/b) coding for possible transcriptional regulators and one gene coding for a phycobilin lyase. However, two distinct configurations of this cluster were observed, depending on the lineage. CA4-A islands contain the mpeZ gene, encoding a recently characterized phycoerythrobilin lyase-isomerase, and a third, small, possible regulator called fciC. In CA4-B islands, the lyase gene encodes an uncharacterized relative of MpeZ, called MpeW. While mpeZ is expressed more in blue light than green light, this is the reverse for mpeW, although only small phenotypic differences were found among chromatic acclimaters possessing either CA4 island type. This study provides novel insights into understanding both diversity and evolution of the CA4 process.

Insights into the role of DNA methylation in diatoms by genome-wide profiling in Phaeodactylum tricornutum.

DNA cytosine methylation is a widely conserved epigenetic mark in eukaryotes that appears to have critical roles in the regulation of genome structure and transcription. Genome-wide methylation maps have so far only been established from the supergroups Archaeplastida and Unikont. Here we report the first whole-genome methylome from a stramenopile, the marine model diatom Phaeodactylum tricornutum. Around 6% of the genome is intermittently methylated in a mosaic pattern. We find extensive methylation in transposable elements. We also detect methylation in over 320 genes. Extensive gene methylation correlates strongly with transcriptional silencing and differential expression under specific conditions. By contrast, we find that genes with partial methylation tend to be constitutively expressed. These patterns contrast with those found previously in other eukaryotes. By going beyond plants, animals and fungi, this stramenopile methylome adds significantly to our understanding of the evolution of DNA methylation in eukaryotes.