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Schonknecht, G, Chen WH, Ternes CM, Barbier GG, Shrestha RP, Stanke M, Brautigam A, Baker BJ, Banfield JF, Garavito RM, Carr K, Wilkerson C, Rensing SA, Gagneul D, Dickenson NE, Oesterhelt C, Lercher MJ, Weber APM.  2013.  Gene transfer from bacteria and archaea facilitated evolution of an extremophilic eukaryote. Science. 339:1207-10.   10.1126/science.1231707   AbstractWebsite

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.

Trentacoste, EM, Shrestha RP, Smith SR, Gle C, Hartmann AC, Hildebrand M, Gerwick WH.  2013.  Metabolic engineering of lipid catabolism increases microalgal lipid accumulation without compromising growth. Proceedings of the National Academy of Sciences of the United States of America. 110:19748-19753.   10.1073/pnas.1309299110   AbstractWebsite

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.

Shrestha, RP, Haerizadeh F, Hildebrand M.  2013.  Molecular Genetic Manipulation of Microalgae: Principles and Applications. Handbook of Microalgal Culture. Second Edition pp.146-167. ( Richmond A, Hu Q, Eds.).: Blackwell Publishing Ltd
Shrestha, RP, Tesson B, Norden-Krichmar T, Federowicz S, Hildebrand M, Allen AE.  2012.  Whole transcriptome analysis of the silicon response of the diatom Thalassiosira pseudonana. BMC Genomics. 13 Abstract

Background: Silicon 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. Results: Three 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. Conclusions: Analysis 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.

Franssen, SU, Shrestha RP, Brautigam A, Bornberg-Bauer E, Weber APM.  2011.  Comprehensive transcriptome analysis of the highly complex Pisum sativum genome using next generation sequencing. BMC Genomics. 12   10.1186/1471-2164-12-227   AbstractWebsite

Background: The 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. Results: We 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. Conclusions: We 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.

Lapidot, M, Shrestha RP, Weinstein Y, Arad S.  2010.  Red microalgae: from basic know-how to biotechnology. Life as We Know It. Cellular Origin, Life in Extreme Habitats and Astrobiology. ( Seckbach J, Ed.).: Springer Abstract
Oesterhelt, C, Vogelbein S, Shrestha RP, Stanke M, Weber APM.  2008.  The genome of the thermoacidophilic red microalga Galdieria sulphuraria encodes a small family of secreted class III peroxidases that might be involved in cell wall modification. Planta. 227:353-362.   10.1007/s00425-007-0622-z   AbstractWebsite

We report the identification of a small family of secreted class III plant peroxidases (Prx) from the genome of the unicellular thermoacidophilic red alga Galdieria sulphuraria (Cyanidiaceae). Apart from two class I ascorbate peroxidases and one cytochrome c peroxidase, the red algal genome encodes four class III plant peroxidases, thus complementing the short list of algal cell wall peroxidases (Passardi et al. in Genomics 89:567-579, 2007). We have characterized the family gene structure, analyzed the extracellular space and cell wall fraction of G. sulphuraria for the presence of peroxidase activity and used shotgun proteomics to identify candidate extracellular peroxidases. For a detailed enzymatic characterization, we have purified a secreted peroxidase (GsPrx04) from the cell-free medium using hydrophobic interaction chromatography. The enzyme proved heat and acid-stable and exhibited an apparent molecular mass of 40 kDa. Comparative genomics between endolithically growing G. sulphuraria and a close relative, the obligatory aquatic, cell wall-less Cyanidioschyzon merolae, revealed that class III peroxidases only occur in the terrestrial microalga, thus supporting the key function of these enzymes in the process of land colonization.

Brautigam, A, Shrestha RP, Whitten D, Wilkerson CG, Carr KM, Froehlich JE, Weber APM.  2008.  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. Journal of Biotechnology. 136:44-53.   10.1016/j.jbiotec.2008.02.007   AbstractWebsite

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. (C) 2008 Elsevier B.V. All rights reserved.

Weber, AP, Barbier G, Shrestha RP, Horst RJ, Minoda A, Oesterhelt C.  2007.  A genomics approach to understanding the biology of thermo-acidophilic red algae. Algae and cyanobacteria in extreme environments. ( Seckbach J, Ed.).:505-518., Dordrecht: Springer Abstract

Algae and Cyanobacteria in Extreme Environments is a unique collection of essays, contributed by leading scientists from around the world, devoted to algae??? and some related microbes??? observed in unexpected harsh habitats, which it seems are an oasis or Garden of Eden for these organisms. This timely book on Extremophilic algae, including its especially impressive micrographs, may provide clues about the edges of life on Earth and possibly elsewhere in the universe. Defining locations from the anthropomorphic point of view, the environments explored ranges from severe and distant to???n.

Hoef-Emden, K, Shrestha RP, Lapidot M, Weinstein Y, Melkonian M, Arad S.  2005.  Actin phylogeny and intron distribution in bangiophyte red algae (rhodoplantae). Journal of Molecular Evolution. 61:360-371.   10.1007/s00239-004-0366-3   AbstractWebsite

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.

Shrestha, RP, Weinstein Y, Bar-Zvi D, Arad S.  2004.  A glycoprotein noncovalently associated with cell-wall polysaccharide of the red microalga Porphyridium sp. (Rhodophyta). Journal of Phycology. 40:568-580.   10.1111/j.1529-8817.2004.02177.x   AbstractWebsite

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.

Ucko, M, Shrestha RP, Mesika P, Bar-Zvi D, Arad S.  1999.  Glycoprotein moiety in the cell wall of the red microalga Porphyridium sp. (Rhodophyta) as the biorecognition site for the Crypthecodinium cohnii-like dinoflagellate. Journal of Phycology. 35:1276-1281.   10.1046/j.1529-8817.1999.3561276.x   AbstractWebsite

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 alpha-D-mannosyl and alpha-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 is the recognition site and prey-prevention results from the blocking of this site on the cell wall.

Shrestha, RP, Joshi KK.  1993.  Effects of Phospamidon and Monocrotophos on seed germination, external morphology and meiotic behavior in Vicia faba. Proceedings of Eighth General Meeting of Nepal Botanical Society, Kathmandu, 1993. :143-152. Abstract