Jennifer H. Wisecaver

Dinoflagellate Genome Evolution

The dinoflagellates are an ecologically important group of microbial eukaryotes that have evolved many novel genomic characteristics. They possess some of the largest nuclear genomes among eukaryotes arranged on permanently condensed liquid-crystalline chromosomes. Recent advances have revealed the presence of genes arranged in tandem arrays, trans-splicing of messenger RNAs, and a reduced role for transcriptional regulation compared to other eukaryotes. In contrast, the mitochondrial and plastid genomes have the smallest gene content among functional eukaryotic organelles. Dinoflagellate biology and genome evolution have been dramatically influenced by lateral transfer of individual genes and large-scale transfer of genes through endosymbiosis. Next-generation sequencing technologies have only recently made genome-scale analyses of these organisms possible, and these new methods are helping researchers better understand the biology and evolution of this enigmatic group of eukaryotes.

Evolution of Saxitoxin Synthesis in Cyanobacteria and Dinoflagellates

Dinoflagellates produce a variety of toxic secondary metabolites that have a significant impact on marine ecosystems and fisheries. Saxitoxin (STX), the cause of paralytic shellfish poisoning, is produced by three marine dinoflagellate genera and is also made by some freshwater cyanobacteria. Genes involved in STX synthesis have been identified in cyanobacteria but are yet to be reported in the massive genomes of dinoflagellates. We have assembled comprehensive transcriptome data sets for several STX-producing dinoflagellates and a related non-toxic species and have identified 265 putative homologs of 13 cyanobacterial STX synthesis genes, including all of the genes directly involved in toxin synthesis. Putative homologs of four proteins group closely in phylogenies with cyanobacteria and are likely the functional homologs of sxtA, sxtG, and sxtB in dinoflagellates. However, the phylogenies do not support the transfer of these genes directly between toxic cyanobacteria and dinoflagellates. SxtA is split into two proteins in the dinoflagellates corresponding to the N-terminal portion containing the methyltransferase and acyl carrier protein domains and a C-terminal portion with the aminotransferase domain. Homologs of sxtB and N-terminal sxtA are present in non-toxic strains, suggesting their functions may not be limited to saxitoxin production. Only homologs of the C-terminus of sxtA and sxtG were found exclusively in toxic strains. A more thorough survey of STX+ dinoflagellates will be needed to determine if these two genes may be specific to SXT production in dinoflagellates. The A. tamarense transcriptome does not contain homologs for the remaining STX genes. Nevertheless, we identified candidate genes with similar predicted biochemical activities that account for the missing functions. These results suggest that the STX synthesis pathway was likely assembled independently in the distantly related cyanobacteria and dinoflagellates, although using some evolutionarily related proteins. The biological role of STX is not well understood in either cyanobacteria or dinoflagellates. However, STX production in these two ecologically distinct groups of organisms suggests that this toxin confers a benefit to producers that we do not yet fully understand.

Transcriptome analysis reveals nuclear-encoded proteins for the maintenance of temporary plastids in the dinoflagellate Dinophysis acuminata.

BackgroundDinophysis is exceptional among dinoflagellates, possessing plastids derived from cryptophyte algae. Although Dinophysis can be maintained in pure culture for several months, the genus is mixotrophic and needs to feed either to acquire plastids (a process known as kleptoplastidy) or obtain growth factors necessary for plastid maintenance. Dinophysis does not feed directly on cryptophyte algae, but rather on a ciliate (Myrionecta rubra) that has consumed the cryptophytes and retained their plastids. Despite the apparent absence of cryptophyte nuclear genes required for plastid function, Dinophysis can retain cryptophyte plastids for months without feeding.ResultsTo determine if this dinoflagellate has nuclear-encoded genes for plastid function, we sequenced cDNA from Dinophysis acuminata, its ciliate prey M. rubra, and the cryptophyte source of the plastid Geminigera cryophila. We identified five proteins complete with plastid-targeting peptides encoded in the nuclear genome of D. acuminata that function in photosystem stabilization and metabolite transport. Phylogenetic analyses show that the genes are derived from multiple algal sources indicating some were acquired through horizontal gene transfer.ConclusionsThese findings suggest that D. acuminata has some functional control of its plastid, and may be able to extend the useful life of the plastid by replacing damaged transporters and protecting components of the photosystem from stress. However, the dearth of plastid-related genes compared to other fully phototrophic algae suggests that D. acuminata does not have the nuclear repertoire necessary to maintain the plastid permanently.

Comparative genomics of biotechnologically important yeasts

Significance The highly diverse Ascomycete yeasts have enormous biotechnological potential. Collectively, these yeasts convert a broad range of substrates into useful compounds, such as ethanol, lipids, and vitamins, and can grow in extremes of temperature, salinity, and pH. We compared 29 yeast genomes with the goal of correlating genetics to useful traits. In one rare species, we discovered a genetic code that translates CUG codons to alanine rather than canonical leucine. Genome comparison enabled correlation of genes to useful metabolic properties and showed the synteny of the mating-type locus to be conserved over a billion years of evolution. Our study provides a roadmap for future biotechnological exploitations. Ascomycete yeasts are metabolically diverse, with great potential for biotechnology. Here, we report the comparative genome analysis of 29 taxonomically and biotechnologically important yeasts, including 16 newly sequenced. We identify a genetic code change, CUG-Ala, in Pachysolen tannophilus in the clade sister to the known CUG-Ser clade. Our well-resolved yeast phylogeny shows that some traits, such as methylotrophy, are restricted to single clades, whereas others, such as l-rhamnose utilization, have patchy phylogenetic distributions. Gene clusters, with variable organization and distribution, encode many pathways of interest. Genomics can predict some biochemical traits precisely, but the genomic basis of others, such as xylose utilization, remains unresolved. Our data also provide insight into early evolution of ascomycetes. We document the loss of H3K9me2/3 heterochromatin, the origin of ascomycete mating-type switching, and panascomycete synteny at the MAT locus. These data and analyses will facilitate the engineering of efficient biosynthetic and degradative pathways and gateways for genomic manipulation.

The evolution of fungal metabolic pathways.

Fungi contain a remarkable range of metabolic pathways, sometimes encoded by gene clusters, enabling them to digest most organic matter and synthesize an array of potent small molecules. Although metabolism is fundamental to the fungal lifestyle, we still know little about how major evolutionary processes, such as gene duplication (GD) and horizontal gene transfer (HGT), have interacted with clustered and non-clustered fungal metabolic pathways to give rise to this metabolic versatility. We examined the synteny and evolutionary history of 247,202 fungal genes encoding enzymes that catalyze 875 distinct metabolic reactions from 130 pathways in 208 diverse genomes. We found that gene clustering varied greatly with respect to metabolic category and lineage; for example, clustered genes in Saccharomycotina yeasts were overrepresented in nucleotide metabolism, whereas clustered genes in Pezizomycotina were more common in lipid and amino acid metabolism. The effects of both GD and HGT were more pronounced in clustered genes than in their non-clustered counterparts and were differentially distributed across fungal lineages; specifically, GD, which was an order of magnitude more abundant than HGT, was most frequently observed in Agaricomycetes, whereas HGT was much more prevalent in Pezizomycotina. The effect of HGT in some Pezizomycotina was particularly strong; for example, we identified 111 HGT events associated with the 15 Aspergillus genomes, which sharply contrasts with the 60 HGT events detected for the 48 genomes from the entire Saccharomycotina subphylum. Finally, the impact of GD within a metabolic category was typically consistent across all fungal lineages, whereas the impact of HGT was variable. These results indicate that GD is the dominant process underlying fungal metabolic diversity, whereas HGT is episodic and acts in a category- or lineage-specific manner. Both processes have a greater impact on clustered genes, suggesting that metabolic gene clusters represent hotspots for the generation of fungal metabolic diversity.

Comparative genome sequencing reveals genomic signature of extreme desiccation tolerance in the anhydrobiotic midge

Anhydrobiosis represents an extreme example of tolerance adaptation to water loss, where an organism can survive in an ametabolic state until water returns. Here we report the first comparative analysis examining the genomic background of extreme desiccation tolerance, which is exclusively found in larvae of the only anhydrobiotic insect, Polypedilum vanderplanki. We compare the genomes of P. vanderplanki and a congeneric desiccation-sensitive midge P. nubifer. We determine that the genome of the anhydrobiotic species specifically contains clusters of multi-copy genes with products that act as molecular shields. In addition, the genome possesses several groups of genes with high similarity to known protective proteins. However, these genes are located in distinct paralogous clusters in the genome apart from the classical orthologues of the corresponding genes shared by both chironomids and other insects. The transcripts of these clustered paralogues contribute to a large majority of the mRNA pool in the desiccating larvae and most likely define successful anhydrobiosis. Comparison of expression patterns of orthologues between two chironomid species provides evidence for the existence of desiccation-specific gene expression systems in P. vanderplanki.

Fungal metabolic gene clusters-caravans traveling across genomes and environments.

Metabolic gene clusters (MGCs), physically co-localized genes participating in the same metabolic pathway, are signature features of fungal genomes. MGCs are most often observed in specialized metabolism, having evolved in individual fungal lineages in response to specific ecological needs, such as the utilization of uncommon nutrients (e.g., galactose and allantoin) or the production of secondary metabolic antimicrobial compounds and virulence factors (e.g., aflatoxin and melanin). A flurry of recent studies has shown that several MGCs, whose functions are often associated with fungal virulence as well as with the evolutionary arms race between fungi and their competitors, have experienced horizontal gene transfer (HGT). In this review, after briefly introducing HGT as a source of gene innovation, we examine the evidence for HGTs involvement on the evolution of MGCs and, more generally of fungal metabolism, enumerate the molecular mechanisms that mediate such transfers and the ecological circumstances that favor them, as well as discuss the types of evidence required for inferring the presence of HGT in MGCs. The currently available examples indicate that transfers of entire MGCs have taken place between closely related fungal species as well as distant ones and that they sometimes involve large chromosomal segments. These results suggest that the HGT-mediated acquisition of novel metabolism is an ongoing and successful ecological strategy for many fungal species.

Bio-crude transcriptomics: Gene discovery and metabolic network reconstruction for the biosynthesis of the terpenome of the hydrocarbon oil-producing green alga, Botryococcus braunii race B (Showa)*

BackgroundMicroalgae hold promise for yielding a biofuel feedstock that is sustainable, carbon-neutral, distributed, and only minimally disruptive for the production of food and feed by traditional agriculture. Amongst oleaginous eukaryotic algae, the B race of Botryococcus braunii is unique in that it produces large amounts of liquid hydrocarbons of terpenoid origin. These are comparable to fossil crude oil, and are sequestered outside the cells in a communal extracellular polymeric matrix material. Biosynthetic engineering of terpenoid bio-crude production requires identification of genes and reconstruction of metabolic pathways responsible for production of both hydrocarbons and other metabolites of the alga that compete for photosynthetic carbon and energy.ResultsA de novo assembly of 1,334,609 next-generation pyrosequencing reads form the Showa strain of the B race of B. braunii yielded a transcriptomic database of 46,422 contigs with an average length of 756 bp. Contigs were annotated with pathway, ontology, and protein domain identifiers. Manual curation allowed the reconstruction of pathways that produce terpenoid liquid hydrocarbons from primary metabolites, and pathways that divert photosynthetic carbon into tetraterpenoid carotenoids, diterpenoids, and the prenyl chains of meroterpenoid quinones and chlorophyll. Inventories of machine-assembled contigs are also presented for reconstructed pathways for the biosynthesis of competing storage compounds including triacylglycerol and starch. Regeneration of S-adenosylmethionine, and the extracellular localization of the hydrocarbon oils by active transport and possibly autophagy are also investigated.ConclusionsThe construction of an annotated transcriptomic database, publicly available in a web-based data depository and annotation tool, provides a foundation for metabolic pathway and network reconstruction, and facilitates further omics studies in the absence of a genome sequence for the Showa strain of B. braunii, race B. Further, the transcriptome database empowers future biosynthetic engineering approaches for strain improvement and the transfer of desirable traits to heterologous hosts.

Examining the Evolution of the Regulatory Circuit Controlling Secondary Metabolism and Development in the Fungal Genus Aspergillus

Filamentous fungi produce diverse secondary metabolites (SMs) essential to their ecology and adaptation. Although each SM is typically produced by only a handful of species, global SM production is governed by widely conserved transcriptional regulators in conjunction with other cellular processes, such as development. We examined the interplay between the taxonomic narrowness of SM distribution and the broad conservation of global regulation of SM and development in Aspergillus, a diverse fungal genus whose members produce well-known SMs such as penicillin and gliotoxin. Evolutionary analysis of the 2,124 genes comprising the 262 SM pathways in four Aspergillus species showed that most SM pathways were species-specific, that the number of SM gene orthologs was significantly lower than that of orthologs in primary metabolism, and that the few conserved SM orthologs typically belonged to non-homologous SM pathways. RNA sequencing of two master transcriptional regulators of SM and development, veA and mtfA, showed that the effects of deletion of each gene, especially veA, on SM pathway regulation were similar in A. fumigatus and A. nidulans, even though the underlying genes and pathways regulated in each species differed. In contrast, examination of the role of these two regulators in development, where 94% of the underlying genes are conserved in both species showed that whereas the role of veA is conserved, mtfA regulates development in the homothallic A. nidulans but not in the heterothallic A. fumigatus. Thus, the regulation of these highly conserved developmental genes is divergent, whereas–despite minimal conservation of target genes and pathways–the global regulation of SM production is largely conserved. We suggest that the evolution of the transcriptional regulation of secondary metabolism in Aspergillus represents a novel type of regulatory circuit rewiring and hypothesize that it has been largely driven by the dramatic turnover of the target genes involved in the process.

A Global Coexpression Network Approach for Connecting Genes to Specialized Metabolic Pathways in Plants

Global gene coexpression network analysis is a powerful approach for identifying known and novel plant-specialized metabolic pathways, irrespective of the genomic location of the constitutive genes. Plants produce diverse specialized metabolites (SMs), but the genes responsible for their production and regulation remain largely unknown, hindering efforts to tap plant pharmacopeia. Given that genes comprising SM pathways exhibit environmentally dependent coregulation, we hypothesized that genes within a SM pathway would form tight associations (modules) with each other in coexpression networks, facilitating their identification. To evaluate this hypothesis, we used 10 global coexpression data sets, each a meta-analysis of hundreds to thousands of experiments, across eight plant species to identify hundreds of coexpressed gene modules per data set. In support of our hypothesis, 15.3 to 52.6% of modules contained two or more known SM biosynthetic genes, and module genes were enriched in SM functions. Moreover, modules recovered many experimentally validated SM pathways, including all six known to form biosynthetic gene clusters (BGCs). In contrast, bioinformatically predicted BGCs (i.e., those lacking an associated metabolite) were no more coexpressed than the null distribution for neighboring genes. These results suggest that most predicted plant BGCs are not genuine SM pathways and argue that BGCs are not a hallmark of plant specialized metabolism. We submit that global gene coexpression is a rich, largely untapped resource for discovering the genetic basis and architecture of plant natural products.