Evolutionary genomics of the cold-adapted diatom Fragilariopsis cylindrus.

The Southern Ocean houses a diverse and productive community of organisms. Unicellular eukaryotic diatoms are the main primary producers in this environment, where photosynthesis is limited by low concentrations of dissolved iron and large seasonal fluctuations in light, temperature and the extent of sea ice. How diatoms have adapted to this extreme environment is largely unknown. Here we present insights into the genome evolution of a cold-adapted diatom from the Southern Ocean, Fragilariopsis cylindrus, based on a comparison with temperate diatoms. We find that approximately 24.7 per cent of the diploid F. cylindrus genome consists of genetic loci with alleles that are highly divergent (15.1 megabases of the total genome size of 61.1 megabases). These divergent alleles were differentially expressed across environmental conditions, including darkness, low iron, freezing, elevated temperature and increased CO2. Alleles with the largest ratio of non-synonymous to synonymous nucleotide substitutions also show the most pronounced condition-dependent expression, suggesting a correlation between diversifying selection and allelic differentiation. Divergent alleles may be involved in adaptation to environmental fluctuations in the Southern Ocean.

Designer diatom episomes delivered by bacterial conjugation.

Eukaryotic microalgae hold great promise for the bioproduction of fuels and higher value chemicals. However, compared with model genetic organisms such as Escherichia coli and Saccharomyces cerevisiae, characterization of the complex biology and biochemistry of algae and strain improvement has been hampered by the inefficient genetic tools. To date, many algal species are transformable only via particle bombardment, and the introduced DNA is integrated randomly into the nuclear genome. Here we describe the first nuclear episomal vector for diatoms and a plasmid delivery method via conjugation from Escherichia coli to the diatoms Phaeodactylum tricornutum and Thalassiosira pseudonana. We identify a yeast-derived sequence that enables stable episome replication in these diatoms even in the absence of antibiotic selection and show that episomes are maintained as closed circles at copy number equivalent to native chromosomes. This highly efficient genetic system facilitates high-throughput functional characterization of algal genes and accelerates molecular phytoplankton research.

Genomes and gene expression across light and productivity gradients in eastern subtropical Pacific microbial communities.

Transitions in community genomic features and biogeochemical processes were examined in surface and subsurface chlorophyll maximum (SCM) microbial communities across a trophic gradient from mesotrophic waters near San Diego, California to the oligotrophic Pacific. Transect end points contrasted in thermocline depth, rates of nitrogen and CO2 uptake, new production and SCM light intensity. Relative to surface waters, bacterial SCM communities displayed greater genetic diversity and enrichment in putative sulfur oxidizers, multiple actinomycetes, low-light-adapted Prochlorococcus and cell-associated viruses. Metagenomic coverage was not correlated with transcriptional activity for several key taxa within Bacteria. Low-light-adapted Prochlorococcus, Synechococcus, and low abundance gamma-proteobacteria enriched in the>3.0-µm size fraction contributed disproportionally to global transcription. The abundance of these groups also correlated with community functions, such as primary production or nitrate uptake. In contrast, many of the most abundant bacterioplankton, including SAR11, SAR86, SAR112 and high-light-adapted Prochlorococcus, exhibited low levels of transcriptional activity and were uncorrelated with rate processes. Eukaryotes such as Haptophytes and non-photosynthetic Aveolates were prevalent in surface samples while Mamielles and Pelagophytes dominated the SCM. Metatranscriptomes generated with ribosomal RNA-depleted mRNA (total mRNA) coupled to in vitro polyadenylation compared with polyA-enriched mRNA revealed a trade-off in detection eukaryotic organelle and eukaryotic nuclear origin transcripts, respectively. Gene expression profiles of SCM eukaryote populations, highly similar in sequence identity to the model pelagophyte Pelagomonas sp. CCMP1756, suggest that pelagophytes are responsible for a majority of nitrate assimilation within the SCM.

Transcriptomic analysis of metabolic function in the giant kelp, Macrocystis pyrifera, across depth and season.

To increase knowledge of transcript diversity for the giant kelp, Macrocystis pyrifera, and assess gene expression across naturally occurring depth gradients in light, temperature and nutrients, we sequenced four cDNA libraries created from blades collected at the sea surface and at 18 m depth during the winter and summer. Comparative genomics cluster analyses revealed novel gene families (clusters) in existing brown alga expressed sequence tag data compared with other related algal groups, a pattern also seen with the addition of M. pyrifera sequences. Assembly of 228 Mbp of sequence generated c. 9000 isotigs and c. 12,000 open reading frames. Annotations were assigned using families of hidden Markov models for c. 11% of open reading frames; M. pyrifera had highest similarity to other members of the Phaeophyceae, namely Ectocarpus siliculosus and Laminaria digitata. Quantitative polymerase chain reaction of transcript targets verified depth-related differences in gene expression; stress response and light-harvesting transcripts, especially members of the LI818 (also known as LHCSR) family, showed high expression in the surface compared with 18 m depth, while some nitrogen acquisition transcripts (e.g. nitrite reductase) were upregulated at depth compared with the surface, supporting a conceptual biological model of depth-dependent physiology.

Metagenomes of Mediterranean coastal lagoons.

Coastal lagoons, both hypersaline and freshwater, are common, but still understudied ecosystems. We describe, for the first time, using high throughput sequencing, the extant microbiota of two large and representative Mediterranean coastal lagoons, the hypersaline Mar Menor, and the freshwater Albufera de Valencia, both located on the south eastern coast of Spain. We show there are considerable differences in the microbiota of both lagoons, in comparison to other marine and freshwater habitats. Importantly, a novel uncultured sulfur oxidizing Alphaproteobacteria was found to dominate bacterioplankton in the hypersaline Mar Menor. Also, in the latter prokaryotic cyanobacteria were almost exclusively comprised by Synechococcus and no Prochlorococcus was found. Remarkably, the microbial community in the freshwaters of the hypertrophic Albufera was completely in contrast to known freshwater systems, in that there was a near absence of well known and cosmopolitan groups of ultramicrobacteria namely Low GC Actinobacteria and the LD12 lineage of Alphaproteobacteria.

Influence of cobalamin scarcity on diatom molecular physiology and identification of a cobalamin acquisition protein.

Diatoms are responsible for ~40% of marine primary production and are key players in global carbon cycling. There is mounting evidence that diatom growth is influenced by cobalamin (vitamin B(12)) availability. This cobalt-containing micronutrient is only produced by some bacteria and archaea but is required by many diatoms and other eukaryotic phytoplankton. Despite its potential importance, little is known about mechanisms of cobalamin acquisition in diatoms or the impact of cobalamin scarcity on diatom molecular physiology. Proteomic profiling and RNA-sequencing transcriptomic analysis of the cultured diatoms Phaeodactylum tricornutum and Thalassiosira pseudonana revealed three distinct strategies used by diatoms to cope with low cobalamin: increased cobalamin acquisition machinery, decreased cobalamin demand, and management of reduced methionine synthase activity through changes in folate and S-adenosyl methionine metabolism. One previously uncharacterized protein, cobalamin acquisition protein 1 (CBA1), was up to 160-fold more abundant under low cobalamin availability in both diatoms. Autologous overexpression of CBA1 revealed association with the outside of the cell and likely endoplasmic reticulum localization. Cobalamin uptake rates were elevated in strains overexpressing CBA1, directly linking this protein to cobalamin acquisition. CBA1 is unlike characterized cobalamin acquisition proteins and is the only currently identified algal protein known to be implicated in cobalamin uptake. The abundance and widespread distribution of transcripts encoding CBA1 in environmental samples suggests that cobalamin is an important nutritional factor for phytoplankton. Future study of CBA1 and other molecular signatures of cobalamin scarcity identified here will yield insight into the evolution of cobalamin utilization and facilitate monitoring of cobalamin starvation in oceanic diatom communities.

Genomic insights to SAR86, an abundant and uncultivated marine bacterial lineage.

Bacteria in the 16S rRNA clade SAR86 are among the most abundant uncultivated constituents of microbial assemblages in the surface ocean for which little genomic information is currently available. Bioinformatic techniques were used to assemble two nearly complete genomes from marine metagenomes and single-cell sequencing provided two more partial genomes. Recruitment of metagenomic data shows that these SAR86 genomes substantially increase our knowledge of non-photosynthetic bacteria in the surface ocean. Phylogenomic analyses establish SAR86 as a basal and divergent lineage of γ-proteobacteria, and the individual genomes display a temperature-dependent distribution. Modestly sized at 1.25-1.7 Mbp, the SAR86 genomes lack several pathways for amino-acid and vitamin synthesis as well as sulfate reduction, trends commonly observed in other abundant marine microbes. SAR86 appears to be an aerobic chemoheterotroph with the potential for proteorhodopsin-based ATP generation, though the apparent lack of a retinal biosynthesis pathway may require it to scavenge exogenously-derived pigments to utilize proteorhodopsin. The genomes contain an expanded capacity for the degradation of lipids and carbohydrates acquired using a wealth of tonB-dependent outer membrane receptors. Like the abundant planktonic marine bacterial clade SAR11, SAR86 exhibits metabolic streamlining, but also a distinct carbon compound specialization, possibly avoiding competition.

The origin of a derived superkingdom: how a gram-positive bacterium crossed the desert to become an archaeon.

BACKGROUND: The tree of life is usually rooted between archaea and bacteria. We have previously presented three arguments that support placing the root of the tree of life in bacteria. The data have been dismissed because those who support the canonical rooting between the prokaryotic superkingdoms cannot imagine how the vast divide between the prokaryotic superkingdoms could be crossed. RESULTS: We review the evidence that archaea are derived, as well as their biggest differences with bacteria. We argue that using novel data the gap between the superkingdoms is not insurmountable. We consider whether archaea are holophyletic or paraphyletic; essential to understanding their origin. Finally, we review several hypotheses on the origins of archaea and, where possible, evaluate each hypothesis using bioinformatics tools. As a result we argue for a firmicute ancestry for archaea over proposals for an actinobacterial ancestry. CONCLUSION: We believe a synthesis of the hypotheses of Lake, Gupta, and Cavalier-Smith is possible where a combination of antibiotic warfare and viral endosymbiosis in the bacilli led to dramatic changes in a bacterium that resulted in the birth of archaea and eukaryotes. REVIEWERS: This article was reviewed by Patrick Forterre, Eugene Koonin, and Gáspár Jékely.

Save the tree of life or get lost in the woods.

BACKGROUND: The wealth of prokaryotic genomic data available has revealed that the histories of many genes are inconsistent, leading some to question the value of the tree of life hypothesis. It has been argued that a tree-like representation requires suppressing too much information, and that a more pluralistic approach is necessary for understanding prokaryotic evolution. We argue that trees may still be a useful representation for evolutionary histories in light of new data. RESULTS: Genomic data alone can be highly misleading when trying to resolve the tree of life. We present evidence from protein abundance data sets that genomic conservation greatly underestimates functional conservation. Function follows more of a tree-like structure than genetic material, even in the presence of horizontal transfer. We argue that the tree of cells must be incorporated into any new synthesis in order to place horizontal transfers into their proper selective context. We also discuss the role data sources other than primary sequence can play in resolving the tree of cells. CONCLUSIONS: The tree of life is alive, but not well. Construction of the tree of cells has been viewed as the end goal of the study of evolution, where in reality we need to consider it more of a starting point. We propose a duality where we must consider variation of genetic material in terms of networks and selection of cellular function in terms of trees. Otherwise one gets lost in the woods of neutral evolution.

History of biological metal utilization inferred through phylogenomic analysis of protein structures.

The fundamental chemistry of trace elements dictates the molecular speciation and reactivity both within cells and the environment at large. Using protein structure and comparative genomics, we elucidate several major influences this chemistry has had upon biology. All of life exhibits the same proteome size-dependent scaling for the number of metal-binding proteins within a proteome. This fundamental evolutionary constant shows that the selection of one element occurs at the exclusion of another, with the eschewal of Fe for Zn and Ca being a defining feature of eukaryotic proteomes. Early life lacked both the structures required to control intracellular metal concentrations and the metal-binding proteins that catalyze electron transport and redox transformations. The development of protein structures for metal homeostasis coincided with the emergence of metal-specific structures, which predominantly bound metals abundant in the Archean ocean. Potentially, this promoted the diversification of emerging lineages of Archaea and Bacteria through the establishment of biogeochemical cycles. In contrast, structures binding Cu and Zn evolved much later, providing further evidence that environmental availability influenced the selection of the elements. The late evolving Zn-binding proteins are fundamental to eukaryotic cellular biology, and Zn bioavailability may have been a limiting factor in eukaryotic evolution. The results presented here provide an evolutionary timeline based on genomic characteristics, and key hypotheses can be tested by alternative geochemical methods.

Structural analysis of polarizing indels: an emerging consensus on the root of the tree of life.

BACKGROUND: The root of the tree of life has been a holy grail ever since Darwin first used the tree as a metaphor for evolution. New methods seek to narrow down the location of the root by excluding it from branches of the tree of life. This is done by finding traits that must be derived, and excluding the root from the taxa those traits cover. However the two most comprehensive attempts at this strategy, performed by Cavalier-Smith and Lake et al., have excluded each other's rootings. RESULTS: The indel polarizations of Lake et al. rely on high quality alignments between paralogs that diverged before the last universal common ancestor (LUCA). Therefore, sequence alignment artifacts may skew their conclusions. We have reviewed their data using protein structure information where available. Several of the conclusions are quite different when viewed in the light of structure which is conserved over longer evolutionary time scales than sequence. We argue there is no polarization that excludes the root from all Gram-negatives, and that polarizations robustly exclude the root from the Archaea. CONCLUSION: We conclude that there is no contradiction between the polarization datasets. The combination of these datasets excludes the root from every possible position except near the Chloroflexi.

Nothing about protein structure classification makes sense except in the light of evolution.

In this, the 200th anniversary of Charles Darwin's birth and the 150th anniversary of the publication of the Origin of Species, it is fitting to revisit the classification of protein structures from an evolutionary perspective. Existing classifications use homologous sequence relationships, but knowing that structure is much more conserved that sequence creates an iterative loop from which structures can be further classified beyond that of the domain, thereby teasing out distant evolutionary relationships. The desired classification scheme is then one in which a fold is merely semantics and structure can be classified as either ancestral or derived.

Sm/Lsm genes provide a glimpse into the early evolution of the spliceosome.

The spliceosome, a sophisticated molecular machine involved in the removal of intervening sequences from the coding sections of eukaryotic genes, appeared and subsequently evolved rapidly during the early stages of eukaryotic evolution. The last eukaryotic common ancestor (LECA) had both complex spliceosomal machinery and some spliceosomal introns, yet little is known about the early stages of evolution of the spliceosomal apparatus. The Sm/Lsm family of proteins has been suggested as one of the earliest components of the emerging spliceosome and hence provides a first in-depth glimpse into the evolving spliceosomal apparatus. An analysis of 335 Sm and Sm-like genes from 80 species across all three kingdoms of life reveals two significant observations. First, the eukaryotic Sm/Lsm family underwent two rapid waves of duplication with subsequent divergence resulting in 14 distinct genes. Each wave resulted in a more sophisticated spliceosome, reflecting a possible jump in the complexity of the evolving eukaryotic cell. Second, an unusually high degree of conservation in intron positions is observed within individual orthologous Sm/Lsm genes and between some of the Sm/Lsm paralogs. This suggests that functional spliceosomal introns existed before the emergence of the complete Sm/Lsm family of proteins; hence, spliceosomal machinery with considerably fewer components than today's spliceosome was already functional.

Rethinking proteasome evolution: two novel bacterial proteasomes.

The proteasome is a multisubunit structure that degrades proteins. Protein degradation is an essential component of regulation because proteins can become misfolded, damaged, or unnecessary. Proteasomes and their homologues vary greatly in complexity: from HslV (heat shock locus v), which is encoded by 1 gene in bacteria, to the eukaryotic 20S proteasome, which is encoded by more than 14 genes. Despite this variation in complexity, all the proteasomes are composed of homologous subunits. We searched 238 complete bacterial genomes for structures related to the proteasome and found evidence of two novel groups of bacterial proteasomes. The first, which we name Anbu, is sparsely distributed among cyanobacteria and proteobacteria. We hypothesize that Anbu must be very ancient because of its distribution within the cyanobacteria, and that it has been lost in many more recent species. We also present evidence for a fourth type of bacterial proteasome found in a few beta-proteobacteria, which we call beta-proteobacteria proteasome homologue (BPH). Sequence and structural analyses show that Anbu and BPH are both distinct from known bacterial proteasomes but have homologous structures. Anbu is encoded by one gene, so we postulate a duplication of Anbu created the 20S proteasome. Anbu's function appears to be related to transglutaminase activity, not the general stress response associated with HslV. We have found different combinations of Anbu, BPH, and HslV within these bacterial genomes, which raises questions about specialized protein degradation systems.