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Search indexed NASA NTRS and DOE OSTI research on propulsion, heat transfer, battery materials and energy systems. Follow report and document links to the original sources.

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All sourcesNASA NTRSDOE OSTI
DOE OSTI2012

Exploring the Switchgrass Transcriptome Using Second-Generation Sequencing Technology

Switchgrass (Panicum virgatum L.) is a C4 perennial grass and widely popular as an important bioenergy crop. To accelerate the pace of developing high yielding switchgrass cultivars adapted to diverse environmental niches, the generation of genomic resources for this plant is necessary. The large genome size and polyploid nature of switchgrass makes whole genome sequencing a daunting task even with current technologies. Exploring the transcriptional landscape using next generation sequencing technologies provides a viable alternative to whole genome sequencing in switchgrass. Switchgrass cDNA libraries from germinating seedlings, emerging tillers, flowers, and dormant seeds were sequenced using Roche 454 GS-FLX Titanium technology, generating 980,000 reads with an average read length of 367 bp. De novo assembly generated 243,600 contigs with an average length of 535 bp. Using the foxtail millet genome as a reference greatly improved the assembly and annotation of switchgrass ESTs. Comparative analysis of the 454-derived switchgrass EST reads with other sequenced monocots including Brachypodium, sorghum, rice and maize indicated a 70– 80% overlap. RPKM analysis demonstrated unique transcriptional signatures of the four tissues analyzed in this study. More than 24,000 ESTs were identified in the dormant seed library. In silico analysis indicated that there are more than 2000 ESTSSRs in this collection. Expression of several orphan ESTs was confirmed by RT-PCR. We estimate that about 90% of the switchgrass gene space has been covered in this analysis. This study nearly doubles the amount of EST information for switchgrass currently in the public domain. The celerity and economical nature of second-generation sequencing technologies provide an in-depth view of the gene space of complex genomes like switchgrass. Sequence analysis of closely related members of the NAD + -malic enzyme type C4 grasses such as the model system Setaria viridis can serve as a viable proxy for the switchgrass genome.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2012

Sequencing of Seven Haloarchaeal Genomes Reveals Patterns of Genomic Flux

We report the sequencing of seven genomes from two haloarchaeal genera, Haloferax and Haloarcula. Ease of cultivation and the existence of well-developed genetic and biochemical tools for several diverse haloarchaeal species make haloarchaea a model group for the study of archaeal biology. The unique physiological properties of these organisms also make them good candidates for novel enzyme discovery for biotechnological applications. Seven genomes were sequenced to ,206coverage and assembled to an average of 50 contigs (range 5 scaffolds - 168 contigs). Comparisons of protein-coding gene compliments revealed large-scale differences in COG functional group enrichment between these genera. Analysis of genes encoding machinery for DNA metabolism reveals genera-specific expansions of the general transcription factor TATA binding protein as well as a history of extensive duplication and horizontal transfer of the proliferating cell nuclear antigen. Insights gained from this study emphasize the importance of haloarchaea for investigation of archaeal biology.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2012

Manipulating Fatty Acid Biosynthesis in Microalgae for Biofuel through Protein-Protein Interactions

Microalgae are a promising feedstock for renewable fuels, and algal metabolic engineering can lead to crop improvement, thus accelerating the development of commercially viable biodiesel production from algae biomass. We demonstrate that protein-protein interactions between the fatty acid acyl carrier protein (ACP) and thioesterase (TE) govern fatty acid hydrolysis within the algal chloroplast. Using green microalga Chlamydomonas reinhardtii (Cr) as a model, a structural simulation of docking CrACP to CrTE identifies a protein-protein recognition surface between the two domains. A virtual screen reveals plant TEs with similar in silico binding to CrACP. Employing an activity-based crosslinking probe designed to selectively trap transient protein-protein interactions between the TE and ACP, we demonstrate in vitro that CrTE must functionally interact with CrACP to release fatty acids, while TEs of vascular plants show no mechanistic crosslinking to CrACP. This is recapitulated in vivo, where overproduction of the endogenous CrTE increased levels of short-chain fatty acids and engineering plant TEs into the C. reinhardtii chloroplast did not alter the fatty acid profile. These findings highlight the critical role of protein-protein interactions in manipulating fatty acid biosynthesis for algae biofuel engineering as illuminated by activity-based probes.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2012

Groundtruthing Next-Gen Sequencing for Microbial Ecology–Biases and Errors in Community Structure Estimates from PCR Amplicon Pyrosequencing

Analysis of microbial communities by high-throughput pyrosequencing of SSU rRNA gene PCR amplicons has transformed microbial ecology research and led to the observation that many communities contain a diverse assortment of rare taxa–a phenomenon termed the Rare Biosphere. Multiple studies have investigated the effect of pyrosequencing read quality on operational taxonomic unit (OTU) richness for contrived communities, yet there is limited information on the fidelity of community structure estimates obtained through this approach. Given that PCR biases are widely recognized, and further unknown biases may arise from the sequencing process itself, a priori assumptions about the neutrality of the data generation process are at best unvalidated. Furthermore, post-sequencing quality control algorithms have not been explicitly evaluated for the accuracy of recovered representative sequences and its impact on downstream analyses, reducing useful discussion on pyrosequencing reads to their diversity and abundances. Here we report on community structures and sequences recovered for in vitro-simulated communities consisting of twenty 16S rRNA gene clones tiered at known proportions. PCR amplicon libraries of the V3–V4 and V6 hypervariable regions from the in vitro-simulated communities were sequenced using the Roche 454 GS FLX Titanium platform. Commonly used quality control protocols resulted in the formation of OTUs with >1% abundance composed entirely of erroneous sequences, while over-aggressive clustering approaches obfuscated real, expected OTUs. The pyrosequencing process itself did not appear to impose significant biases on overall community structure estimates, although the detection limit for rare taxa may be affected by PCR amplicon size and quality control approach employed. Meanwhile, PCR biases associated with the initial amplicon generation may impose greater distortions in the observed community structure.

54 ENVIRONMENTAL SCIENCES↗
DOE OSTI2012

Genomic Comparison of Escherichia coli O104:H4 Isolates from 2009 and 2011 Reveals Plasmid, and Prophage Heterogeneity, Including Shiga Toxin Encoding Phage stx2

In May of 2011, an enteroaggregative Escherichia coli O104:H4 strain that had acquired a Shiga toxin 2-converting phage caused a large outbreak of bloody diarrhea in Europe which was notable for its high prevalence of hemolytic uremic syndrome cases. Several studies have described the genomic inventory and phylogenies of strains associated with the outbreak and a collection of historical E. coli O104:H4 isolates using draft genome assemblies. We present the complete, closed genome sequences of an isolate from the 2011 outbreak (2011C–3493) and two isolates from cases of bloody diarrhea that occurred in the Republic of Georgia in 2009 (2009EL–2050 and 2009EL–2071). Comparative genome analysis indicates that, while the Georgian strains are the nearest neighbors to the 2011 outbreak isolates sequenced to date, structural and nucleotide-level differences are evident in the Stx2 phage genomes, the mer/tet antibiotic resistance island, and in the prophage and plasmid profiles of the strains, including a previously undescribed plasmid with homology to the pMT virulence plasmid of Yersinia pestis. In addition, multiphenotype analysis showed that 2009EL–2071 possessed higher resistance to polymyxin and membrane-disrupting agents. Finally, we show evidence by electron microscopy of the presence of a common phage morphotype among the European and Georgian strains and a second phage morphotype among the Georgian strains. The presence of at least two stx2 phage genotypes in host genetic backgrounds that may derive from a recent common ancestor of the 2011 outbreak isolates indicates that the emergence of stx2 phage-containing E. coli O104:H4 strains probably occurred more than once, or that the current outbreak isolates may be the result of a recent transfer of a new stx2 phage element into a pre-existing stx2-positive genetic background.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2013

PCR-Induced Transitions Are the Major Source of Error in Cleaned Ultra-Deep Pyrosequencing Data

Ultra-deep pyrosequencing (UDPS) is used to identify rare sequence variants. The sequence depth is influenced by several factors including the error frequency of PCR and UDPS. This study investigated the characteristics and source of errors in raw and cleaned UDPS data. UDPS of a 167-nucleotide fragment of the HIV-1 SG3Δenv plasmid was performed on the Roche/454 platform. The plasmid was diluted to one copy, PCR amplified and subjected to bidirectional UDPS on three occasions. The dataset consisted of 47,693 UDPS reads. Raw UDPS data had an average error frequency of 0.30% per nucleotide site. Most errors were insertions and deletions in homopolymeric regions. We used a cleaning strategy that removed almost all indel errors, but had little effect on substitution errors, which reduced the error frequency to 0.056% per nucleotide. In cleaned data the error frequency was similar in homopolymeric and non-homopolymeric regions, but varied considerably across sites. These site-specific error frequencies were moderately, but still significantly, correlated between runs (r = 0.15–0.65) and between forward and reverse sequencing directions within runs (r = 0.33–0.65). Furthermore, transition errors were 48-times more common than transversion errors (0.052% vs. 0.001%; p<0.0001). Collectively the results indicate that a considerable proportion of the sequencing errors that remained after data cleaning were generated during the PCR that preceded UDPS. A majority of the sequencing errors that remained after data cleaning were introduced by PCR prior to sequencing, which means that they will be independent of platform used for next-generation sequencing. The transition vs. transversion error bias in cleaned UDPS data will influence the detection limits of rare mutations and sequence variants.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2013

Detection of Bacillus anthracis DNA in Complex Soil and Air Samples Using Next-Generation Sequencing

Bacillus anthracis is the potentially lethal etiologic agent of anthrax disease, and is a significant concern in the realm of biodefense. One of the cornerstones of an effective biodefense strategy is the ability to detect infectious agents with a high degree of sensitivity and specificity in the context of a complex sample background. The nature of the B. anthracis genome, however, renders specific detection difficult, due to close homology with B. cereus and B. thuringiensis. We therefore elected to determine the efficacy of next-generation sequencing analysis and microarrays for detection of B. anthracis in an environmental background. We applied next-generation sequencing to titrated genome copy numbers of B. anthracis in the presence of background nucleic acid extracted from aerosol and soil samples. We found next-generation sequencing to be capable of detecting as few as 10 genomic equivalents of B. anthracis DNA per nanogram of background nucleic acid. Detection was accomplished by mapping reads to either a defined subset of reference genomes or to the full GenBank database. Moreover, sequence data obtained from B. anthracis could be reliably distinguished from sequence data mapping to either B. cereus or B. thuringiensis. We also demonstrated the efficacy of a microbial census microarray in detecting B. anthracis in the same samples, representing a cost-effective and high-throughput approach, complementary to next-generation sequencing. Our results, in combination with the capacity of sequencing for providing insights into the genomic characteristics of complex and novel organisms, suggest that these platforms should be considered important components of a biosurveillance strategy.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2014

Bayesian Analysis of Congruence of Core Genes in Prochlorococcus and Synechococcus and Implications on Horizontal Gene Transfer

It is often suggested that horizontal gene transfer is so ubiquitous in microbes that the concept of a phylogenetic tree representing the pattern of vertical inheritance is oversimplified or even positively misleading. “Universal proteins” have been used to infer the organismal phylogeny, but have been criticized as being only the “tree of one percent.” Currently, few options exist for those wishing to rigorously assess how well a universal protein phylogeny, based on a relative handful of well-conserved genes, represents the phylogenetic histories of hundreds of genes. Here, we address this problem by proposing a visualization method and a statistical test within a Bayesian framework. We use the genomes of marine cyanobacteria, a group thought to exhibit substantial amounts of HGT, as a test case. We take 379 orthologous gene families from 28 cyanobacteria genomes and estimate the Bayesian posterior distributions of trees – a “treecloud” – for each, as well as for a concatenated dataset based on putative “universal proteins.” We then calculate the average distance between trees within and between all treeclouds on various metrics and visualize this high-dimensional space with non-metric multidimensional scaling (NMMDS). We show that the tree space is strongly clustered and that the universal protein treecloud is statistically significantly closer to the center of this tree space than any individual gene treecloud. We apply several commonly-used tests for incongruence/HGT and show that they agree HGT is rare in this dataset, but make different choices about which genes were subject to HGT. Our results show that the question of the representativeness of the “tree of one percent” is a quantitative empirical question, and that the phylogenetic central tendency is a meaningful observation even if many individual genes disagree due to the various sources of incongruence.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2014

A Quantitative Comparison of Single-Cell Whole Genome Amplification Methods

Single-cell sequencing is emerging as an important tool for studies of genomic heterogeneity. Whole genome amplification (WGA) is a key step in single-cell sequencing workflows and a multitude of methods have been introduced. Here, we compare three state-of-the-art methods on both bulk and single-cell samples of E. coli DNA: Multiple Displacement Amplification (MDA), Multiple Annealing and Looping Based Amplification Cycles (MALBAC), and the PicoPLEX single-cell WGA kit (NEB-WGA). We considered the effects of reaction gain on coverage uniformity, error rates and the level of background contamination. We compared the suitability of the different WGA methods for the detection of copy-number variations, for the detection of single-nucleotide polymorphisms and for de-novo genome assembly. No single method performed best across all criteria and significant differences in characteristics were observed; the choice of which amplifier to use will depend strongly on the details of the type of question being asked in any given experiment.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2016

Surveillance for Western equine encephalitis St. Louis encephalitis and West Nile viruses using reverse transcription loop-mediated isothermal amplification

In this study, collection of mosquitoes and testing for vector-borne viruses is a key surveillance activity that directly influences the vector control efforts of public health agencies, including determining when and where to apply insecticides. Vector control districts in California routinely monitor for three human pathogenic viruses including West Nile virus (WNV), Western equine encephalitis virus (WEEV), and St. Louis encephalitis virus (SLEV). Reverse transcription quantitative polymerase chain reaction (RT-qPCR) offers highly sensitive and specific detection of these three viruses in a single multiplex reaction, but this technique requires costly, specialized equipment that is generally only available in centralized public health laboratories. We report the use of reverse transcription loop-mediated isothermal amplification (RT-LAMP) to detect WNV, WEEV, and SLEV RNA extracted from pooled mosquito samples collected in California, including novel primer sets for specific detection of WEEV and SLEV, targeting the nonstructural protein 4 (nsP4) gene of WEEV and the 3’ untranslated region (3’-UTR) of SLEV. Our WEEV and SLEV RT-LAMP primers allowed detection of <0.1 PFU/reaction of their respective targets in <30 minutes, and exhibited high specificity without cross reactivity when tested against a panel of alphaviruses and flaviviruses. Furthermore, the SLEV primers do not cross-react with WNV, despite both viruses being closely related members of the Japanese encephalitis virus complex. The SLEV and WEEV primers can also be combined in a single RT-LAMP reaction, with discrimination between amplicons by melt curve analysis. Although RT-qPCR is approximately one order of magnitude more sensitive than RT-LAMP for all three targets, the RT-LAMP technique is less instrumentally intensive than RT-qPCR and provides a more cost-effective method of vector-borne virus surveillance.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2016

Brucella melitensis Methionyl-tRNA-Synthetase (MetRS), a Potential Drug Target for Brucellosis

We investigated Brucella melitensis methionyl-tRNA-synthetase (BmMetRS) with molecular, structural and phenotypic methods to learn if BmMetRS is a promising target for brucellosis drug development. Recombinant BmMetRS was expressed, purified from wild type Brucella melitensis biovar Abortus 2308 strain ATCC/CRP #DD-156 and screened by a thermal melt assay against a focused library of one hundred previously classified methionyl-tRNA-synthetase inhibitors of the blood stage form of Trypanosoma brucei. Three compounds showed appreciable shift of denaturation temperature and were selected for further studies on inhibition of the recombinant enzyme activity and cell viability against wild type B. melitensis strain 16M. BmMetRS protein complexed with these three inhibitors resolved into three-dimensional crystal structures and was analyzed. All three selected methionyl-tRNA-synthetase compounds inhibit recombinant BmMetRS enzymatic functions in an aminoacylation assay at varying concentrations. Furthermore, growth inhibition of B. melitensis strain 16M by the compounds was shown. Inhibitor-BmMetRS crystal structure models were used to illustrate the molecular basis of the enzyme inhibition. Our current data suggests that BmMetRS is a promising target for brucellosis drug development. However, further studies are needed to optimize lead compound potency, efficacy and safety as well as determine the pharmacokinetics, optimal dosage, and duration for effective treatment.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2022

Tracking ebolavirus genomic drift with a resequencing microarray

Filoviruses are emerging pathogens that cause acute fever with high fatality rate and present a global public health threat. During the 2013–2016 Ebola virus outbreak, genome sequencing allowed the study of virus evolution, mutations affecting pathogenicity and infectivity, and tracing the viral spread. In 2018, early sequence identification of the Ebolavirus as EBOV in the Democratic Republic of the Congo supported the use of an Ebola virus vaccine. However, field-deployable sequencing methods are needed to enable a rapid public health response. Resequencing microarrays (RMA) are a targeted method to obtain genomic sequence on clinical specimens rapidly, and sensitively, overcoming the need for extensive bioinformatic analysis. This study presents the design and initial evaluation of an ebolavirus resequencing microarray (Ebolavirus-RMA) system for sequencing the major genomic regions of four Ebolaviruses that cause disease in humans. The design of the Ebolavirus-RMA system is described and evaluated by sequencing repository samples of three Ebolaviruses and two EBOV variants. The ability of the system to identify genetic drift in a replicating virus was achieved by sequencing the ebolavirus glycoprotein gene in a recombinant virus cultured under pressure from a neutralizing antibody. Comparison of the Ebolavirus-RMA results to the Genbank database sequence file with the accession number given for the source RNA and Ebolavirus-RMA results compared to Next Generation Sequence results of the same RNA samples showed up to 99% agreement.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Structure of the T. brucei kinetoplastid RNA editing substrate-binding complex core component, RESC5

Kinetoplastid protists such as Trypanosoma brucei undergo an unusual process of mitochondrial uridine (U) insertion and deletion editing termed kinetoplastid RNA editing (kRNA editing). This extensive form of editing, which is mediated by guide RNAs (gRNAs), can involve the insertion of hundreds of Us and deletion of tens of Us to form a functional mitochondrial mRNA transcript. kRNA editing is catalyzed by the 20 S editosome/RECC. However, gRNA directed, processive editing requires the RNA editing substrate binding complex (RESC), which is comprised of 6 core proteins, RESC1-RESC6. To date there are no structures of RESC proteins or complexes and because RESC proteins show no homology to proteins of known structure, their molecular architecture remains unknown. RESC5 is a key core component in forming the foundation of the RESC complex. To gain insight into the RESC5 protein we performed biochemical and structural studies. We show that RESC5 is monomeric and we report the T . brucei RESC5 crystal structure to 1.95 Å. RESC5 harbors a dimethylarginine dimethylaminohydrolase-like (DDAH) fold. DDAH enzymes hydrolyze methylated arginine residues produced during protein degradation. However, RESC5 is missing two key catalytic DDAH residues and does bind DDAH substrate or product. Implications of the fold for RESC5 function are discussed. This structure provides the first structural view of an RESC protein.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Insights from a workplace SARS-CoV-2 specimen collection program, with genomes placed into global sequence phylogeny

In 2020, the Department of Energy established the National Virtual Biotechnology Laboratory (NVBL) to address key challenges associated with COVID-19. As part of that effort, Pacific Northwest National Laboratory (PNNL) established a capability to collect and analyze specimens from employees who self-reported symptoms consistent with the disease. During the spring and fall of 2021, 688 specimens were screened for SARS-CoV-2, with 64 (9.3%) testing positive using reverse-transcriptase quantitative PCR (RT-qPCR). Of these, 36 samples were released for research. All 36 positive samples released for research were sequenced and genotyped. Here, the relationship between patient age and viral load as measured by Ct values was measured and determined to be only weakly significant. Consensus sequences for each sample were placed into a global phylogeny and transmission dynamics were investigated, revealing that the closest relative for many samples was from outside of Washington state, indicating mixing of viral pools within geographic regions.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Crystal structure of the Arabidopsis SPIRAL2 C-terminal domain reveals a p80-Katanin-like domain

Epidermal cells of dark-grown plant seedlings reorient their cortical microtubule arrays in response to blue light from a net lateral orientation to a net longitudinal orientation with respect to the long axis of cells. The molecular mechanism underlying this microtubule array reorientation involves katanin, a microtubule severing enzyme, and a plant-specific microtubule associated protein called SPIRAL2. Katanin preferentially severs longitudinal microtubules, generating seeds that amplify the longitudinal array. Upon severing, SPIRAL2 binds nascent microtubule minus ends and limits their dynamics, thereby stabilizing the longitudinal array while the lateral array undergoes net depolymerization. To date, no experimental structural information is available for SPIRAL2 to help inform its mechanism. To gain insight into SPIRAL2 structure and function, we determined a 1.8 Å resolution crystal structure of the Arabidopsis thaliana SPIRAL2 C-terminal domain. The domain is composed of seven core α-helices, arranged in an α-solenoid. Amino-acid sequence conservation maps primarily to one face of the domain involving helices α1, α3, α5, and an extended loop, the α6-α7 loop. The domain fold is similar to, yet structurally distinct from the C-terminal domain of Ge-1 (an mRNA decapping complex factor involved in P-body localization) and, surprisingly, the C-terminal domain of the katanin p80 regulatory subunit. The katanin p80 C-terminal domain heterodimerizes with the MIT domain of the katanin p60 catalytic subunit, and in metazoans, binds the microtubule minus-end factors CAMSAP3 and ASPM. Structural analysis predicts that SPIRAL2 does not engage katanin p60 in a mode homologous to katanin p80. The SPIRAL2 structure highlights an interesting evolutionary convergence of domain architecture and microtubule minus-end localization between SPIRAL2 and katanin complexes, and establishes a foundation upon which structure-function analysis can be conducted to elucidate the role of this domain in the regulation of plant microtubule arrays.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2014

Human APOBEC3 Induced Mutation of Human Immunodeficiency Virus Type-1 Contributes to Adaptation and Evolution in Natural Infection

Human APOBEC3 proteins are cytidine deaminases that contribute broadly to innate immunity through the control of exogenous retrovirus replication and endogenous retroelement retrotransposition. As an intrinsic antiretroviral defense mechanism, APOBEC3 proteins induce extensive guanosine-to-adenosine (G-to-A) mutagenesis and inhibit synthesis of nascent human immunodeficiency virus-type 1 (HIV-1) cDNA. Human APOBEC3 proteins have additionally been proposed to induce infrequent, potentially non-lethal G-to-A mutations that make subtle contributions to sequence diversification of the viral genome and adaptation though acquisition of beneficial mutations. Using single-cycle HIV-1 infections in culture and highly parallel DNA sequencing, we defined trinucleotide contexts of the edited sites for APOBEC3D, APOBEC3F, APOBEC3G, and APOBEC3H. We then compared these APOBEC3 editing contexts with the patterns of G-to-A mutations in HIV-1 DNA in cells obtained sequentially from ten patients with primary HIV-1 infection. Viral substitutions were highest in the preferred trinucleotide contexts of the edited sites for the APOBEC3 deaminases. Consistent with the effects of immune selection, amino acid changes accumulated at the APOBEC3 editing contexts located within human leukocyte antigen (HLA)- appropriate epitopes that are known or predicted to enable peptide binding. Thus, APOBEC3 activity may induce mutations that influence the genetic diversity and adaptation of the HIV-1 population in natural infection.

59 BASIC BIOLOGICAL SCIENCES↗