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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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At least 127 records · Page 7
All sourcesNASA NTRSDOE OSTI
DOE OSTI2022

High-quality genome of the basidiomycete yeast Dioszegia hungarica PDD-24b-2 isolated from cloud water

The genome of the basidiomycete yeast Dioszegia hungarica strain PDD-24b-2 isolated from cloud water at the summit of puy de $D\hat{o}me$ (France) was sequenced using a hybrid PacBio and Illumina sequencing strategy. The obtained assembled genome of 20.98 Mb and a GC content of 57% is structured in 16 large-scale contigs ranging from 90 kb to 5.56Mb, and another 27.2 kb contig representing the complete circular mitochondrial genome. In total, 8,234 proteins were predicted from the genome sequence. The mitochondrial genome shows 16.2% cgu codon usage for arginine but has no canonical cognate tRNA to translate this codon. Detected transposable element (TE)-related sequences account for about 0.63% of the assembled genome. A dataset of 2,068 hand-picked public environmental metagenomes, representing over 20 Tbp of raw reads, was probed for D. hungarica related ITS sequences, and revealed worldwide distribution of this species, particularly in aerial habitats. Growth experiments suggested a psychrophilic phenotype and the ability to disperse by producing ballistospores. The high-quality assembled genome obtained for this D. hungarica strain will help investigate the behavior and ecological functions of this species in the environment.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

OrthoPhyl—streamlining large-scale, orthology-based phylogenomic studies of bacteria at broad evolutionary scales

Abstract There are a staggering number of publicly available bacterial genome sequences (at writing, 2.0 million assemblies in NCBI's GenBank alone), and the deposition rate continues to increase. This wealth of data begs for phylogenetic analyses to place these sequences within an evolutionary context. A phylogenetic placement not only aids in taxonomic classification but informs the evolution of novel phenotypes, targets of selection, and horizontal gene transfer. Building trees from multi-gene codon alignments is a laborious task that requires bioinformatic expertise, rigorous curation of orthologs, and heavy computation. Compounding the problem is the lack of tools that can streamline these processes for building trees from large-scale genomic data. Here we present OrthoPhyl, which takes bacterial genome assemblies and reconstructs trees from whole genome codon alignments. The analysis pipeline can analyze an arbitrarily large number of input genomes (>1200 tested here) by identifying a diversity-spanning subset of assemblies and using these genomes to build gene models to infer orthologs in the full dataset. To illustrate the versatility of OrthoPhyl, we show three use cases: E. coli/Shigella, Brucella/Ochrobactrum and the order Rickettsiales. We compare trees generated with OrthoPhyl to trees generated with kSNP3 and GToTree along with published trees using alternative methods. We show that OrthoPhyl trees are consistent with other methods while incorporating more data, allowing for greater numbers of input genomes, and more flexibility of analysis.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Thermophilic site-specific recombination system for rapid insertion of heterologous DNA into the Clostridium thermocellum chromosome

Clostridium thermocellum is an anaerobic thermophile capable of producing ethanol and other commodity chemicals from lignocellulosic biomass. The insertion of heterologous DNA into the C. thermocellum chromosome is currently achieved via a time-consuming homologous recombination process, where a single stable insertion can take 2–4 weeks or more to construct. In this work, we developed a thermostable version of the Serine recombinase Assisted Genome Engineering (tSAGE) approach for gene insertion in C. thermocellum utilizing a site-specific recombinase from Geobacillus sp. Y412MC61, enabling quick and easy insertion of DNA into the chromosome for accelerated genetic tool screening and heterologous gene expression. Using tSAGE, chromosomal insertion of plasmid DNA occurred at a maximum transformation efficiency of 5 × 10 3 CFU/µg, which is comparable to the transformation efficiency of a replicating control plasmid in C. thermocellum. Using tSAGE, we chromosomally integrated and characterized 17 reporter genes, 15 homologous and 31 heterologous constitutive promoters of varying strengths, 4 inducible promoters, and 5 riboswitches in C. thermocellum. We also determined that a 6–7 nucleotide gap between the ribosome binding site (RBS) and the start codon is optimal for high expression by employing a library of superfolder green fluorescent protein expression constructs driven by our strongest tested promoter (P clo1313_1194 ) with different distances between the RBS and start codon. The tools developed here will aid in accelerating C. thermocellum strain engineering for producing sustainable fuels and chemicals directly from plant biomass.

Biofuels↗
DOE OSTI2021

Assessment of Clonal Expansion Using CarcSeq Measurement of Lung Cancer Driver Mutations and Correlation With Mouse Strain- and Sex-Related Incidence of Spontaneous Lung Neoplasia

Quantification of variation in levels of spontaneously occurring cancer driver mutations (CDMs) was developed to assess clonal expansion and predict future risk of neoplasm development. Specifically, an error-corrected next-generation sequencing method, CarcSeq, and a mouse CarcSeq panel (analogous to human and rat panels) were developed and used to quantify low-frequency mutations in a panel of amplicons enriched in hotspot CDMs. Mutations in a subset of panel amplicons, Braf, Egfr, Kras, Stk11, and Tp53, were related to incidence of lung neoplasms at 2 years. This was achieved by correlating median absolute deviation (MAD) from the overall median mutant fraction (MF) measured in the lung DNA of 16-week-old male and female, B6C3F1 and CD-1 mice (10 mice/sex/strain) with percentages of spontaneous alveolar/bronchioloalveolar adenomas and carcinomas reported in bioassay control groups. A total of 1586 mouse lung mutants with MFs > x 10-4 were recovered. The ratio of nonsynonymous to synonymous mutations was used to assess the proportion of recovered mutations conferring a positive selective advantage. The greatest ratio was observed in what is considered the most lung tumor-sensitive model examined, male B6C3F1 mice. Of the recurrent, nonsynonymous mouse mutations recovered, 55.5% have been reported in human tumors, with many located in or around the mouse equivalent of human cancer hotspot codons. MAD for the same subset of amplicons measured in normal human lung DNA samples showed a correlation of moderate strength and borderline significance with age (a cancer risk factor), as well as age-related cumulative lung cancer risk, suggesting MAD may inform species extrapolation.

Toxicology↗
DOE OSTI2026

Miscanthus giganteus Biolistic Transformation Using the Visible RUBY Red Marker Gene to Monitor Transformation Efficiency

Miscanthus × giganteus ( M × g ) is a high-yielding perennial C4 bioenergy crop, but genetic improvement by breeding is constrained by triploid sterility and clonal propagation. Improving genetic transformation methods for M × g would provide opportunities for advantageous trait introgression. Use of an easy to phenotype reporter gene is a promising strategy to improve transformation processes and efficiency. This study presents an efficient novel method for biolistic transformation of inflorescence-derived callus in M × g and demonstrates its efficacy using RUBY, a betalain-based noninvasive reporter that is visible throughout the transformation process. RUBY expression ( Zea mays codon optimized) was visible from callus stage through plantlet development into maturity. RUBY expressing independently transformed plants were confirmed by hygromycin phosphotransferase ELISA and by genomic PCR demonstrating that the RUBY phenotype is sufficient for screening transformants. The Zea mays codon optimized hygromycin selection marker was driven by previously established promoters for Miscanthus, ZmUBI and 2×35S, while the RUBY gene expression was controlled by a known Zea mays C4 promoter, Brachypodium UBI10, newly employed in Miscanthus. The construct containing the 2×35S promoter for hygromycin had a 15.1% transformation efficiency while the ZmUBI promoter had a 20.5% transformation efficiency. This study provides a novel, highly efficient protocol for successful biolistic transformation of M × g for stable expression. This study also demonstrates that RUBY expression can be used as a convenient and powerful monitor of transformation in ongoing and future work to engineer M × g into an improved bioproduct feedstock.

RUBY↗
DOE OSTI2013

Small Genomes and Sparse Metabolisms of Sediment-Associated Bacteria from Four Candidate Phyla

Cultivation-independent surveys of microbial diversity have revealed many bacterial phyla that lack cultured representatives. These lineages, referred to as candidate phyla, have been detected across many environments. Here, we deeply sequenced microbial communities from acetate-stimulated aquifer sediment to recover the complete and essentially complete genomes of single representatives of the candidate phyla SR1, WWE3, TM7, and OD1. All four of these genomes are very small, 0.7 to 1.2 Mbp, and have large inventories of novel proteins. Additionally, all lack identifiable biosynthetic pathways for several key metabolites. The SR1 genome uses the UGA codon to encode glycine, and the same codon is very rare in the OD1 genome, suggesting that the OD1 organism could also transition to alternate coding. Interestingly, the relative abundance of the members of SR1 increased with the appearance of sulfide in groundwater, a pattern mirrored by a member of the phylum Tenericutes . All four genomes encode type IV pili, which may be involved in interorganism interaction. On the basis of these results and other recently published research, metabolic dependence on other organisms may be widely distributed across multiple bacterial candidate phyla. Few or no genomic sequences exist for members of the numerous bacterial phyla lacking cultivated representatives, making it difficult to assess their roles in the environment. This paper presents three complete and one essentially complete genomes of members of four candidate phyla, documents consistently small genome size, and predicts metabolic capabilities on the basis of gene content. These metagenomic analyses expand our view of a lifestyle apparently common across these candidate phyla.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2017

Refactoring the Genetic Code for Increased Evolvability

ABSTRACT The standard genetic code is robust to mutations during transcription and translation. Point mutations are likely to be synonymous or to preserve the chemical properties of the original amino acid. Saturation mutagenesis experiments suggest that in some cases the best-performing mutant requires replacement of more than a single nucleotide within a codon. These replacements are essentially inaccessible to common error-based laboratory engineering techniques that alter a single nucleotide per mutation event, due to the extreme rarity of adjacent mutations. In this theoretical study, we suggest a radical reordering of the genetic code that maximizes the mutagenic potential of single nucleotide replacements. We explore several possible genetic codes that allow a greater degree of accessibility to the mutational landscape and may result in a hyperevolvable organism that could serve as an ideal platform for directed evolution experiments. We then conclude by evaluating the challenges of constructing such recoded organisms and their potential applications within the field of synthetic biology. IMPORTANCE The conservative nature of the genetic code prevents bioengineers from efficiently accessing the full mutational landscape of a gene via common error-prone methods. Here, we present two computational approaches to generate alternative genetic codes with increased accessibility. These new codes allow mutational transitions to a larger pool of amino acids and with a greater extent of chemical differences, based on a single nucleotide replacement within the codon, thus increasing evolvability both at the single-gene and at the genome levels. Given the widespread use of these techniques for strain and protein improvement, along with more fundamental evolutionary biology questions, the use of recoded organisms that maximize evolvability should significantly improve the efficiency of directed evolution, library generation, and fitness maximization.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Pangenomes suggest ecological-evolutionary responses to experimental soil warming

ABSTRACT Below-ground carbon transformations that contribute to healthy soils represent a natural climate change mitigation, but newly acquired traits adaptive to climate stress may alter microbial feedback mechanisms. To better define microbial evolutionary responses to long-term climate warming, we study microorganisms from an ongoing in situ soil warming experiment where, for over three decades, temperate forest soils are continuously heated at 5°C above ambient. We hypothesize that across generations of chronic warming, genomic signatures within diverse bacterial lineages reflect adaptations related to growth and carbon utilization. From our bacterial culture collection isolated from experimental heated and control plots, we sequenced genomes representing dominant taxa sensitive to warming, including lineages of Actinobacteria, Alphaproteobacteria, and Betaproteobacteria. We investigated genomic attributes and functional gene content to identify signatures of adaptation. Comparative pangenomics revealed accessory gene clusters related to central metabolism, competition, and carbon substrate degradation, with few functional annotations explicitly associated with long-term warming. Trends in functional gene patterns suggest genomes from heated plots were relatively enriched in central carbohydrate and nitrogen metabolism pathways, while genomes from control plots were relatively enriched in amino acid and fatty acid metabolism pathways. We observed that genomes from heated plots had less codon bias, suggesting potential adaptive traits related to growth or growth efficiency. Codon usage bias varied for organisms with similar 16S rrn operon copy number, suggesting that these organisms experience different selective pressures on growth efficiency. Our work suggests the emergence of lineage-specific trends as well as common ecological-evolutionary microbial responses to climate change. IMPORTANCE Anthropogenic climate change threatens soil ecosystem health in part by altering below-ground carbon cycling carried out by microbes. Microbial evolutionary responses are often overshadowed by community-level ecological responses, but adaptive responses represent potential changes in traits and functional potential that may alter ecosystem function. We predict that microbes are adapting to climate change stressors like soil warming. To test this, we analyzed the genomes of bacteria from a soil warming experiment where soil plots have been experimentally heated 5°C above ambient for over 30 years. While genomic attributes were unchanged by long-term warming, we observed trends in functional gene content related to carbon and nitrogen usage and genomic indicators of growth efficiency. These responses may represent new parameters in how soil ecosystems feedback to the climate system.

Choudoir, Mallory J. (ORCID:0000000291175150)↗
DOE OSTI2014

Quality scores for 32,000 genomes

More than 80% of the microbial genomes in GenBank are of ‘draft’ quality (12,553 draft vs. 2,679 finished, as of October, 2013). In this study, we have examined all the microbial DNA sequences available for complete, draft, and Sequence Read Archive genomes in GenBank as well as three other major public databases, and assigned quality scores for more than 30,000 prokaryotic genome sequences. Scores were assigned using four categories: the completeness of the assembly, the presence of full-length rRNA genes, tRNA composition and the presence of a set of 102 conserved genes in prokaryotes. Most (~88%) of the genomes had quality scores of 0.8 or better and can be safely used for standard comparative genomics analysis. We compared genomes across factors that may influence the score. We found that although sequencing depth coverage of over 100x did not ensure a better score, sequencing read length was a better indicator of sequencing quality. With few exceptions, most of the 30,000 genomes have nearly all the 102 essential genes. The score can be used to set thresholds for screening data when analyzing “all published genomes” and reference data is either not available or not applicable. The scores highlighted organisms for which commonly used tools do not perform well. This information can be used to improve tools and to serve a broad group of users as more diverse organisms are sequenced. Finally and unexpectedly, the comparison of predicted tRNAs across 15,000 high quality genomes showed that anticodons beginning with an ‘A’ (codons ending with a ‘U’) are almost non-existent, with the exception of one arginine codon (CGU); this has been noted previously in the literature for a few genomes, but not with the depth found here.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2019

Quantitative principles of cis-translational control by general mRNA sequence features in eukaryotes

General translational cis-elements are present in the mRNAs of all genes and affect the recruitment, assembly, and progress of preinitiation complexes and the ribosome under many physiological states. These elements include mRNA folding, upstream open reading frames, specific nucleotides flanking the initiating AUG codon, protein coding sequence length, and codon usage. The quantitative contributions of these sequence features and how and why they coordinate to control translation rates are not well understood.

Biotechnology & Applied Microbiology↗
DOE OSTI2022

MetaPop: a pipeline for macro- and microdiversity analyses and visualization of microbial and viral metagenome-derived populations

Abstract Background Microbes and their viruses are hidden engines driving Earth’s ecosystems from the oceans and soils to humans and bioreactors. Though gene marker approaches can now be complemented by genome-resolved studies of inter-(macrodiversity) and intra-(microdiversity) population variation, analytical tools to do so remain scattered or under-developed. Results Here, we introduce MetaPop, an open-source bioinformatic pipeline that provides a single interface to analyze and visualize microbial and viral community metagenomes at both the macro - and microdiversity levels. Macrodiversity estimates include population abundances and α- and β-diversity. Microdiversity calculations include identification of single nucleotide polymorphisms, novel codon-constrained linkage of SNPs, nucleotide diversity ( π and θ ), and selective pressures (pN/pS and Tajima’s D ) within and fixation indices ( F ST ) between populations. MetaPop will also identify genes with distinct codon usage. Following rigorous validation, we applied MetaPop to the gut viromes of autistic children that underwent fecal microbiota transfers and their neurotypical peers. The macrodiversity results confirmed our prior findings for viral populations (microbial shotgun metagenomes were not available) that diversity did not significantly differ between autistic and neurotypical children. However, by also quantifying microdiversity, MetaPop revealed lower average viral nucleotide diversity ( π ) in autistic children. Analysis of the percentage of genomes detected under positive selection was also lower among autistic children, suggesting that higher viral π in neurotypical children may be beneficial because it allows populations to better “bet hedge” in changing environments. Further, comparisons of microdiversity pre- and post-FMT in autistic children revealed that the delivery FMT method (oral versus rectal) may influence viral activity and engraftment of microdiverse viral populations, with children who received their FMT rectally having higher microdiversity post-FMT. Overall, these results show that analyses at the macro level alone can miss important biological differences. Conclusions These findings suggest that standardized population and genetic variation analyses will be invaluable for maximizing biological inference, and MetaPop provides a convenient tool package to explore the dual impact of macro - and microdiversity across microbial communities.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2026

Data for Miscanthus giganteus Biolistic Transformation Using the Visible RUBY Red Marker Gene to Monitor Transformation Efficiency

Miscanthus × giganteus ( M × g ) is a high- yielding perennial C4 bioenergy crop, but genetic improvement by breeding is constrained by triploid sterility and clonal propagation. Improving genetic transformation methods for M × g would provide opportunities for advantageous trait introgression. Use of an easy to phenotype reporter gene is a promising strategy to improve transformation processes and efficiency. This study presents an efficient novel method for biolistic transformation of inflorescence- derived callus in M × g and demonstrates its efficacy using RUBY, a betalain-based noninvasive reporter that is visible throughout the transformation process. RUBY expression ( Zea mays codon optimized) was visible from callus stage through plantlet development into maturity. RUBY expressing independently transformed plants were confirmed by hygromycin phosphotransferase ELISA and by genomic PCR demonstrating that the RUBY phenotype is sufficient for screening transformants. The Zea mays codon optimized hygromycin selection marker was driven by previously established promoters for Miscanthus, ZmUBI and 2×35S, while the RUBY gene expression was controlled by a known Zea mays C4 promoter, Brachypodium UBI10, newly employed in Miscanthus. The construct containing the 2×35S promoter for hygromycin had a 15.1% transformation efficiency while the ZmUBI promoter had a 20.5% transformation efficiency. This study provides a novel, highly efficient protocol for successful biolistic transformation of M × g for stable expression. This study also demonstrates that RUBY expression can be used as a convenient and powerful monitor of transformation in ongoing and future work to engineer M × g into an improved bioproduct feedstock. **NOTE: in "TableS2_ProtocolComparison.csv", the data from row 665 to 971 should be removed.

Gene Editing↗
DOE OSTI2025

Sodium azide mutagenesis induces a unique pattern of mutations

The nature and effect of mutations are of fundamental importance to the evolutionary process. The generation of mutations with mutagens has also played important roles in genetics. Applications of mutagens include dissecting the genetic basis of trait variation, inducing desirable traits in crops, and understanding the nature of genetic load. Previous studies of sodium azide-induced mutations have reported single nucleotide variants (SNVs) found in individual genes. To characterize the nature of mutations induced by sodium azide, we analyze whole-genome sequencing (WGS) of 11 barley lines derived from sodium azide mutagenesis, where all lines were selected for diminution of plant fitness owing to induced mutations. We contrast observed mutagen-induced variants with those found in standing variation in WGS of 13 barley landraces. Here, we report indels that are two orders of magnitude more abundant than expected based on nominal mutation rates. We found induced SNVs are very specific, with C → T changes occurring in a context followed by another C on the same strand (or the reverse complement). The codons most affected by the mutagen include the sodium azide-specific CC motif (or the reverse complement), resulting in a handful of amino acid changes and few stop codons. The specific nature of induced mutations suggests that mutagens could be chosen based on experimental goals. Sodium azide would not be ideal for gene knockouts but will create many missense mutations with more subtle effects on protein function.

Genetics & Heredity↗
DOE OSTI2016

Genetic Variability of West Nile Virus in U.S. Blood Donors from the 2012 Epidemic Season

West Nile virus (WNV) is an arbovirus maintained in nature in a bird-mosquito enzootic cycle which can also infect other vertebrates including humans. WNV is now endemic in the United States (U.S.), causing yearly outbreaks that have resulted in an estimated total of 4–5 million human infections. Over 41,700 cases of West Nile disease, including 18,810 neuroinvasive cases and 1,765 deaths, were reported to the CDC between 1999 and 2014. In 2012, the second largest West Nile outbreak in the U.S. was reported, which caused 5,674 cases and 286 deaths. WNV continues to evolve, and three major WNV lineage I genotypes (NY99, WN02, and SW/WN03) have been described in the U.S. since introduction of the virus in 1999. We report here the WNV sequences obtained from 19 human samples acquired during the 2012 U.S. outbreak and our examination of the evolutionary dynamics in WNV isolates sequenced from 1999–2012. Maximum-likelihood and Bayesian methods were used to perform the phylogenetic analyses. Selection pressure analyses were performed with the HyPhy package using the Datamonkey web-server. Using different codon-based and branch-site selection models, we detected a number of codons subjected to positive pressure in WNV genes. Thirteen of the 19 completely sequenced isolates from 10 U.S. states were genetically similar, sharing up to 55 nucleotide mutations and 4 amino acid substitutions when compared with the prototype isolate WN-NY99. Altogether, these analyses showed that following a brief contraction in 2008–2009, WNV genetic divergence in the U.S. continued to increase in 2012, and that closely related variants were found across a broad geographic range of the U.S., coincident with the second-largest WNV outbreak in U.S. history.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2016

Reconstructing the backbone of the Saccharomycotina yeast phylogeny using genome-scale data

Understanding the phylogenetic relationships among the yeasts of the subphylum Saccharomycotina is a prerequisite for understanding the evolution of their metabolisms and ecological lifestyles. In the last two decades, the use of rDNA and multilocus data sets has greatly advanced our understanding of the yeast phylogeny, but many deep relationships remain unsupported. In contrast, phylogenomic analyses have involved relatively few taxa and lineages that were often selected with limited considerations for covering the breadth of yeast biodiversity. Here we used genome sequence data from 86 publicly available yeast genomes representing nine of the 11 known major lineages and 10 nonyeast fungal outgroups to generate a 1233-gene, 96-taxon data matrix. Species phylogenies reconstructed using two different methods (concatenation and coalescence) and two data matrices (amino acids or the first two codon positions) yielded identical and highly supported relationships between the nine major lineages. Aside from the lineage comprised by the family Pichiaceae, all other lineages were monophyletic. Most interrelationships among yeast species were robust across the two methods and data matrices. Furthermore, eight of the 93 internodes conflicted between analyses or data sets, including the placements of: the clade defined by species that have reassigned the CUG codon to encode serine, instead of leucine; the clade defined by a whole genome duplication; and the species Ascoidea rubescens . These phylogenomic analyses provide a robust roadmap for future comparative work across the yeast subphylum in the disciplines of taxonomy, molecular genetics, evolutionary biology, ecology, and biotechnology. To further this end, we have also provided a BLAST server to query the 86 Saccharomycotina genomes, which can be found at http://y1000plus.org/blast.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Dual incorporation of non-canonical amino acids enables production of post-translationally modified selenoproteins

Post-translational modifications (PTMs) can occur on almost all amino acids in eukaryotes as a key mechanism for regulating protein function. The ability to study the role of these modifications in various biological processes requires techniques to modify proteins site-specifically. One strategy for this is genetic code expansion (GCE) in bacteria. The low frequency of post-translational modifications in bacteria makes it a preferred host to study whether the presence of a post-translational modification influences a protein’s function. Genetic code expansion employs orthogonal translation systems engineered to incorporate a modified amino acid at a designated protein position. Selenoproteins, proteins containing selenocysteine, are also known to be post-translationally modified. Selenoproteins have essential roles in oxidative stress, immune response, cell maintenance, and skeletal muscle regeneration. Their complicated biosynthesis mechanism has been a hurdle in our understanding of selenoprotein functions. As technologies for selenocysteine insertion have recently improved, we wanted to create a genetic system that would allow the study of post-translational modifications in selenoproteins. By combining genetic code expansion techniques and selenocysteine insertion technologies, we were able to recode stop codons for insertion of N ε -acetyl- l -lysine and selenocysteine, respectively, into multiple proteins. The specificity of these amino acids for their assigned position and the simplicity of reverting the modified amino acid via mutagenesis of the codon sequence demonstrates the capacity of this method to study selenoproteins and the role of their post-translational modifications. Moreover, the evidence that Sec insertion technology can be combined with genetic code expansion tools further expands the chemical biology applications.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

YPK9 and WHI2 Negatively Interact during Oxidative Stress

Yeast PARK9 (YPK9) shares homology with human ATP13A2, which encodes a polyamine transporter implicated in juvenile forms of Parkinson’s disease. We used YPK9 to gain insight into how ATP13A2 affects cell growth and sensitivity to oxidative stress. Surprisingly, the YPK9 deletion strain from the Saccharomyces cerevisiae deletion collection (YKO) in wildtype BY4741 (mating type a) grew faster and was more resistant to hydrogen peroxide than a commercial, putative parental BY4741 wildtype strain (BY4741 COM ). In contrast, deleting YPK9 from BY4741 COM rendered it very sensitive to hydrogen peroxide, suggesting its background is different from that of the deletion collection. Whole-genome sequencing revealed that BY4741 COM and BY4741 COM ypk9Δ contain a novel premature stop codon near the 3' end of WHI2 (WHI2 G1324T ), whereas the collection’s YPK9 deletion strain contains WHI2, which encodes a 486 amino acid protein, Whi2p. Replacing full-length WHI2 with the sequence coding for the predicted truncation (Whi2p E442* ) rendered strains more sensitive to hydrogen peroxide, whereas the converse replacement rendered them more resistant. The sequences of WHI2 in 20 randomly chosen strains from the collection encode the full-length protein, indicating that the putative parental BY4741 WHI2 G1324T strain’s genetic background differs from that of the deletion collection. Examination of WHI2 sequences in several commonly used wildtype S. cerevisiae strains and isolates revealed other Whi2p truncations that might yield altered phenotypes. Together, these results demonstrate a novel premature stop codon in WHI2 that renders yeast sensitive to hydrogen peroxide; they also reveal a negative genetic interaction between WHI2 and YPK9 in the presence of hydrogen peroxide in the BY4741 background.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2019

Mitovirus and Mitochondrial Coding Sequences from Basal Fungus Entomophthora muscae

Fungi constituting the Entomophthora muscae species complex (members of subphylum Entomophthoromycotina, phylum Zoopagamycota) commonly kill their insect hosts and manipulate host behaviors in the process. In this study, we made use of public transcriptome data to identify and characterize eight new species of mitoviruses associated with several different E. muscae isolates. Mitoviruses are simple RNA viruses that replicate in host mitochondria and are frequently found in more phylogenetically apical fungi (members of subphylum Glomeromyoctina, phylum Mucoromycota, phylum Basidiomycota and phylum Ascomycota) as well as in plants. E. muscae is the first fungus from phylum Zoopagomycota, and thereby the most phylogenetically basal fungus, found to harbor mitoviruses to date. Multiple UGA (Trp) codons are found not only in each of the new mitovirus sequences from E. muscae but also in mitochondrial core-gene coding sequences newly assembled from E. muscae transcriptome data, suggesting that UGA (Trp) is not a rarely used codon in the mitochondria of this fungus. The presence of mitoviruses in these basal fungi has possible implications for the evolution of these viruses.

59 BASIC BIOLOGICAL SCIENCES↗