Roshan P. Shrestha

Gene Transfer from Bacteria and Archaea Facilitated Evolution of an Extremophilic Eukaryote

Hot, Toxic Eukaryote Unusually, the single-celled eukaryote red alga, Galdieria sulphuraria, can thrive in hot, acidic springs. This organism is endowed with extraordinary metabolic talents and can consume a variety of strange carbohydrates, as well as turn on photosynthesis when the food runs out. Schönknecht et al. (p. 1207; see the Perspective by Rocha) discerned from phylogenetic analysis of its genome that during its evolution, G. sulphuraria appears to have commandeered at least 75 bacterial and archaeal genes by horizontal gene transfer and then applied gene expansion to boost its metabolic repertoire. A mosaic of genes acquired from various phyla enable a red alga to grow abundantly in hot, acidic, and toxic niches. [Also see Perspective by Rocha] Some microbial eukaryotes, such as the extremophilic red alga Galdieria sulphuraria, live in hot, toxic metal-rich, acidic environments. To elucidate the underlying molecular mechanisms of adaptation, we sequenced the 13.7-megabase genome of G. sulphuraria. This alga shows an enormous metabolic flexibility, growing either photoautotrophically or heterotrophically on more than 50 carbon sources. Environmental adaptation seems to have been facilitated by horizontal gene transfer from various bacteria and archaea, often followed by gene family expansion. At least 5% of protein-coding genes of G. sulphuraria were probably acquired horizontally. These proteins are involved in ecologically important processes ranging from heavy-metal detoxification to glycerol uptake and metabolism. Thus, our findings show that a pan-domain gene pool has facilitated environmental adaptation in this unicellular eukaryote.

Metabolic engineering of lipid catabolism increases microalgal lipid accumulation without compromising growth

Significance As global CO2 levels rise and fossil fuel abundance decreases, the development of alternative fuels becomes increasingly imperative. Biologically derived fuels, and specifically those from microalgae, are promising sources, but improvements throughout the production process are required to reduce cost. Increasing lipid yields in microalgae without compromising growth has great potential to improve economic feasibility. We report that disrupting lipid catabolism is a practical approach to increase lipid yields in microalgae without affecting growth or biomass. We developed transgenic strains through targeted metabolic engineering that show increased lipid accumulation, biomass, and lipid yields. The target enzyme’s ubiquity suggests that this approach can be applied broadly to improve the economic feasibility of algal biofuels in other groups of microalgae. Biologically derived fuels are viable alternatives to traditional fossil fuels, and microalgae are a particularly promising source, but improvements are required throughout the production process to increase productivity and reduce cost. Metabolic engineering to increase yields of biofuel-relevant lipids in these organisms without compromising growth is an important aspect of advancing economic feasibility. We report that the targeted knockdown of a multifunctional lipase/phospholipase/acyltransferase increased lipid yields without affecting growth in the diatom Thalassiosira pseudonana. Antisense-expressing knockdown strains 1A6 and 1B1 exhibited wild-type–like growth and increased lipid content under both continuous light and alternating light/dark conditions. Strains 1A6 and 1B1, respectively, contained 2.4- and 3.3-fold higher lipid content than wild-type during exponential growth, and 4.1- and 3.2-fold higher lipid content than wild-type after 40 h of silicon starvation. Analyses of fatty acids, lipid classes, and membrane stability in the transgenic strains suggest a role for this enzyme in membrane lipid turnover and lipid homeostasis. These results demonstrate that targeted metabolic manipulations can be used to increase lipid accumulation in eukaryotic microalgae without compromising growth.

Comprehensive transcriptome analysis of the highly complex Pisum sativum genome using next generation sequencing

BackgroundThe garden pea, Pisum sativum, is among the best-investigated legume plants and of significant agro-commercial relevance. Pisum sativum has a large and complex genome and accordingly few comprehensive genomic resources exist.ResultsWe analyzed the pea transcriptome at the highest possible amount of accuracy by current technology. We used next generation sequencing with the Roche/454 platform and evaluated and compared a variety of approaches, including diverse tissue libraries, normalization, alternative sequencing technologies, saturation estimation and diverse assembly strategies. We generated libraries from flowers, leaves, cotyledons, epi- and hypocotyl, and etiolated and light treated etiolated seedlings, comprising a total of 450 megabases. Libraries were assembled into 324,428 unigenes in a first pass assembly.A second pass assembly reduced the amount to 81,449 unigenes but caused a significant number of chimeras. Analyses of the assemblies identified the assembly step as a major possibility for improvement. By recording frequencies of Arabidopsis orthologs hit by randomly drawn reads and fitting parameters of the saturation curve we concluded that sequencing was exhaustive. For leaf libraries we found normalization allows partial recovery of expression strength aside the desired effect of increased coverage. Based on theoretical and biological considerations we concluded that the sequence reads in the database tagged the vast majority of transcripts in the aerial tissues. A pathway representation analysis showed the merits of sampling multiple aerial tissues to increase the number of tagged genes. All results have been made available as a fully annotated database in fasta format.ConclusionsWe conclude that the approach taken resulted in a high quality - dataset which serves well as a first comprehensive reference set for the model legume pea. We suggest future deep sequencing transcriptome projects of species lacking a genomics backbone will need to concentrate mainly on resolving the issues of redundancy and paralogy during transcriptome assembly.

Low-coverage massively parallel pyrosequencing of cDNAs enables proteomics in non-model species: Comparison of a species-specific database generated by pyrosequencing with databases from related species for proteome analysis of pea chloroplast envelopes

Proteomics is a valuable tool for establishing and comparing the protein content of defined tissues, cell types, or subcellular structures. Its use in non-model species is currently limited because the identification of peptides critically depends on sequence databases. In this study, we explored the potential of a preliminary cDNA database for the non-model species Pisum sativum created by a small number of massively parallel pyrosequencing (MPSS) runs for its use in proteomics and compared it to comprehensive cDNA databases from Medicago truncatula and Arabidopsis thaliana created by Sanger sequencing. Each database was used to identify proteins from a pea leaf chloroplast envelope preparation. It is shown that the pea database identified more proteins with higher accuracy, although the sequence quality was low and the sequence contigs were short compared to databases from model species. Although the number of identified proteins in non-species-specific databases could potentially be increased by lowering the threshold for successful protein identifications, this strategy markedly increases the number of wrongly identified proteins. The identification rate with non-species-specific databases correlated with spectral abundance but not with the predicted membrane helix content, and strong conservation is necessary but not sufficient for protein identification with a non-species-specific database. It is concluded that massively parallel sequencing of cDNAs substantially increases the power of proteomics in non-model species.

Whole transcriptome analysis of the silicon response of the diatom Thalassiosira pseudonana

BackgroundSilicon plays important biological roles, but the mechanisms of cellular responses to silicon are poorly understood. We report the first analysis of cell cycle arrest and recovery from silicon starvation in the diatom Thalassiosira pseudonana using whole genome microarrays.ResultsThree known responses to silicon were examined, 1) silicified cell wall synthesis, 2) recovery from silicon starvation, and 3) co-regulation with silicon transporter (SIT) genes. In terms of diatom cell wall formation, thus far only cell surface proteins and proteins tightly associated with silica have been characterized. Our analysis has identified new genes potentially involved in silica formation, and other genes potentially involved in signaling, trafficking, protein degradation, glycosylation and transport, which provides a larger-scale picture of the processes involved. During silicon starvation, an overrepresentation of transcription and translation related genes were up-regulated, indicating that T. pseudonana is poised to rapidly recover from silicon starvation and resume cell cycle progression upon silicon replenishment. This is in contrast to other types of limitation, and provides the first molecular data explaining the well-established environmental response of diatoms to grow as blooms and to out-compete other classes of microalgae for growth. Comparison of our data with a previous diatom cell cycle analysis indicates that assignment of the cell cycle specific stage of particular cyclins and cyclin dependent kinases should be re-evaluated. Finally, genes co-varying in expression with the SITs enabled identification of a new class of diatom-specific proteins containing a unique domain, and a putative silicon efflux protein.ConclusionsAnalysis of the T. pseudonana microarray data has provided a wealth of new genes to investigate previously uncharacterized cellular phenomenon related to silicon metabolism, silicon’s interaction with cellular components, and environmental responses to silicon.

Evidence for a regulatory role of diatom silicon transporters in cellular silicon responses.

ABSTRACT The utilization of silicon by diatoms has both global and small-scale implications, from oceanic primary productivity to nanotechnological applications of their silica cell walls. The sensing and transport of silicic acid are key aspects of understanding diatom silicon utilization. At low silicic acid concentrations (<30 μM), transport mainly occurs through silicic acid transport proteins (SITs), and at higher concentrations it occurs through diffusion. Previous analyses of the SITs were done either in heterologous systems or without a distinction between individual SITs. In the present study, we examined individual SITs in Thalassiosira pseudonana in terms of transcript and protein abundance in response to different silicic acid regimes and examined knockdown lines to evaluate the role of the SITs in transport, silica incorporation, and lipid accumulation resulting from silicon starvation. SIT1 and SIT2 were localized in the plasma membrane, and protein levels were generally inversely correlated with cellular silicon needs, with a distinct response being found when the two SITs were compared. We developed highly effective approaches for RNA interference and antisense knockdowns, the first such approaches developed for a centric diatom. SIT knockdown differentially affected the uptake of silicon and the incorporation of silicic acid and resulted in the induction of lipid accumulation under silicon starvation conditions far earlier than in the wild-type cells, suggesting that the cells were artificially sensing silicon limitation. The data suggest that the transport role of the SITs is relatively minor under conditions with sufficient silicic acid. Their primary role is to sense silicic acid levels to evaluate whether the cell can proceed with its cell wall formation and division processes.

A GLYCOPROTEIN NONCOVALENTLY ASSOCIATED WITH CELL-WALL POLYSACCHARIDE OF THE RED MICROALGA PORPHYRIDIUM SP. (RHODOPHYTA)1

The cells of the red microalga Porphyridium sp. (UTEX 637) are encapsulated in a cell wall of a negatively charged mucilaginous polysaccharide complex composed of 10 different sugars, sulfate, and proteins. In this work, we studied the proteins associated with the cell‐wall polysaccharide. A number of noncovalently associated proteins were resolved by SDS‐PAGE, but no covalently bound proteins were detected. The most prominent protein detected was a 66‐kDa glycoprotein consisting of a polypeptide of approximately 58 kDa and a glycan moiety of approximately 8 kDa containing N‐linked terminal mannose. In size‐exclusion chromatography, the 66‐kDa protein was coeluted with the polysaccharide and could be separated from the polysaccharide only after denaturation of the protein, indicating that the 66‐kDa protein was tightly bound to the polysaccharide. Western blot analysis revealed that the 66‐kDa protein was specific to Porphyridium sp. and P. cruentum, because it was not detected in the other species of red microalgae examined. Indirect immunofluorescence assay confirmed the location of the protein in the algal cell wall. The sequence of cDNA clone encoding the 66‐kDa glycoprotein, detected in our in‐house expressed sequence tag database of Porphyridium sp., revealed that this is a novel protein with no similarity to any protein in the public domain databases and our in‐house expressed sequence tag database of the red microalga Rhodella reticulata. The 66‐kDa protein bound polysaccharides from red algae but not from those of other origins tested. Possible roles of the 66‐kDa protein in the biosynthesis of the polysaccharide are discussed.

GLYCOPROTEIN MOIETY IN THE CELL WALL OF THE RED MICROALGA PORPHYRIDIUM SP. (RHODOPHYTA) AS THE BIORECOGNITION SITE FOR THE CRYPTHECODINIUM COHNII‐LIKE DINOFLAGELLATE

The Crypthecodinium cohnii‐like heterotrophic dinoflagellate preys on the cells of the red microalga Porphyridium sp. UTEX 637, and not on other microalgae. The dinoflagellate contains enzymes that degrade the cell wall complex of this species of alga and not that of other red microalgae. The cells of the red microalgae are encapsulated within a cell wall complex composed of about 10 sugars, sulfate, and proteins. We previously hypothesized that the dinoflagellate recognizes the cell wall of this alga. In this study, we have shown that the biorecognition site is the 66‐kDa glycoprotein in the algal cell wall complex. The methodology used in this study was based on changing the algal cell wall composition and examining the prey and chemosensory response of the dinoflagellate. The dinoflagellate was not attracted to the cell wall of other red microalgae, which are similar to that of Porphyridium sp., or to sugars composing its cell wall. However, the dinoflagellate preyed on and was attracted to Porphyridium sp. mutants (DCB resistant) having modified cell wall polysaccharide composition, probably because the 66‐kDa cell wall glycoprotein was not changed. The dinoflagellate did not respond chemotactically to enzymatically degraded cell wall complex. Treatment of the cell wall complex with antiserum to the 66‐kDa glycoprotein or with the lectin concanavalin A (con A), which binds specifically to α‐d‐mannosyl and α‐d‐glucosyl residues, did not affect the chemotactic attraction. However, prey by the dinoflagellate was prevented when the algal cells were blocked with antiserum specific to the 66‐kDa glycoprotein or with con A. These latter results provide direct proof that the 66‐kDa cell wall glycoprotein isthe recognition site and prey‐prevention results from the blocking of this site on the cell wall.

Actin Phylogeny and Intron Distribution in Bangiophyte Red Algae(Rhodoplantae)

The molecular phylogeny of red algal actin genes, with emphasis on the paraphyletic “Bangiophyceae,” was examined and compared to the rhodophyte SSU rDNA phylogeny. Nineteen new genomic actin sequences and seven SSU rDNA sequences were obtained and subjected to diverse phylogenetic analyses (maximum likelihood, distance/neighbor-joining, maximum parsimony, Bayesian analyses, and, with respect to protein sequences, also quartet puzzling). The actin trees confirmed most of the major clades found in the SSU rDNA phylogenies, although with a lower resolution. An actin gene duplication in the florideophycean lineage is reported, presumably related to an increased complexity of sexual reproduction. In addition, the distribution and characteristics of spliceosomal introns found in some of the actin sequences were examined. Introns were found in almost all florideophycean actin genes, whereas only two bangiophyte sequences contained introns. One intron in the florideophycean actin genes was also found in metazoan, and, shifted by one or two nucleotides, in a glaucocystophyte, a cryptophyte, and two fungal actin genes, and thus may be an ancient intron.

Applications of Imaging Flow Cytometry for Microalgae

The ability to image large numbers of cells at high resolution enhances flow cytometric analysis of cells and cell populations. In particular, the ability to image intracellular features adds a unique aspect to analyses, and can enable correlation between molecular phenomena resulting in alterations in cellular phenotype. Unicellular microalgae are amenable to high-throughput analysis to capture the diversity of cell types in natural samples, or diverse cellular responses in clonal populations, especially using imaging cytometry. Using examples from our laboratory, we review applications of imaging cytometry, specifically using an Amnis(®) ImageStream(®)X instrument, to characterize photosynthetic microalgae. Some of these examples highlight advantages of imaging flow cytometry for certain research objectives, but we also include examples that would not necessarily require imaging and could be performed on a conventional cytometer to demonstrate other concepts in cytometric evaluation of microalgae. We demonstrate the value of these approaches for (1) analysis of populations, (2) documentation of cellular features, and (3) analysis of gene expression.