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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 757 records · Page 42
All sourcesNASA NTRSDOE OSTI
DOE OSTI2017

Cas9-mediated genome editing in the methanogenic archaeon Methanosarcina acetivorans

Although Cas9-mediated genome editing has proven to be a powerful genetic tool in eukaryotes, its application in Bacteria has been limited because of inefficient targeting or repair; and its application to Archaea has yet to be reported. Here we describe the development of a Cas9-mediated genome-editing tool that allows facile genetic manipulation of the slow-growing methanogenic archaeon Methanosarcina acetivorans . Introduction of both insertions and deletions by homology-directed repair was remarkably efficient and precise, occurring at a frequency of approximately 20% relative to the transformation efficiency, with the desired mutation being found in essentially all transformants examined. Off-target activity was not observed. We also observed that multiple single-guide RNAs could be expressed in the same transcript, reducing the size of mutagenic plasmids and simultaneously simplifying their design. Cas9-mediated genome editing reduces the time needed to construct mutants by more than half (3 vs. 8 wk) and allows simultaneous construction of double mutants with high efficiency, exponentially decreasing the time needed for complex strain constructions. Furthermore, coexpression the nonhomologous end-joining (NHEJ) machinery from the closely related archaeon, Methanocella paludicola , allowed efficient Cas9-mediated genome editing without the need for a repair template. The NHEJ-dependent mutations included deletions ranging from 75 to 2.7 kb in length, most of which appear to have occurred at regions of naturally occurring microhomology. The combination of homology-directed repair-dependent and NHEJ-dependent genome-editing tools comprises a powerful genetic system that enables facile insertion and deletion of genes, rational modification of gene expression, and testing of gene essentiality.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2017

Kaposi’s sarcoma–associated herpesvirus stably clusters its genomes across generations to maintain itself extrachromosomally

Genetic elements that replicate extrachromosomally are rare in mammals; however, several human tumor viruses, including the papillomaviruses and the gammaherpesviruses, maintain their plasmid genomes by tethering them to cellular chromosomes. We have uncovered an unprecedented mechanism of viral replication: Kaposi’s sarcoma–associated herpesvirus (KSHV) stably clusters its genomes across generations to maintain itself extrachromosomally. To identify and characterize this mechanism, we developed two complementary, independent approaches: live-cell imaging and a predictive computational model. The clustering of KSHV requires the viral protein, LANA1, to bind viral genomes to nucleosomes arrayed on both cellular and viral DNA. Clustering affects both viral partitioning and viral genome numbers of KSHV. The clustering of KSHV plasmids provides it with an effective evolutionary strategy to rapidly increase copy numbers of genomes per cell at the expense of the total numbers of cells infected.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2015

MaxBin 2.0: an automated binning algorithm to recover genomes from multiple metagenomic datasets

The recovery of genomes from metagenomic datasets is a critical step to defining the functional roles of the underlying uncultivated populations. We previously developed MaxBin, an automated binning approach for high-throughput recovery of microbial genomes from metagenomes. Here, we present an expanded binning algorithm, MaxBin 2.0, which recovers genomes from co-assembly of a collection of metagenomic datasets. Tests on simulated datasets revealed that MaxBin 2.0 is highly accurate in recovering individual genomes, and the application of MaxBin 2.0 to several metagenomes from environmental samples demonstrated that it could achieve two complementary goals: recovering more bacterial genomes compared to binning a single sample as well as comparing the microbial community composition between different sampling environments. Availability and implementation: MaxBin 2.0 is freely available at http://sourceforge.net/projects/maxbin/ under BSD license. Supplementary information: Supplementary data are available at Bioinformatics online.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Into the wild: new yeast genomes from natural environments and new tools for their analysis

Genomic studies of yeasts from the wild have increased significantly in the past few years. This revolution has been fueled by advances in high-throughput sequencing technologies and a better understanding of yeast ecology and phylogeography, especially for biotechnologically important species. The present review aims to first introduce new bioinformatic tools available for the generation and analysis of yeast genomes. Furthermore, we assess the accumulated genomic data of wild isolates of industrially relevant species, such as Saccharomyces spp., which provide unique opportunities to further investigate the domestication processes associated with the fermentation industry and opportunistic pathogenesis. The availability of genome sequences of other less conventional yeasts obtained from the wild has also increased substantially, including representatives of the phyla Ascomycota (e.g. Hanseniaspora) and Basidiomycota (e.g. Phaffia). Here, we review salient examples of both fundamental and applied research that demonstrate the importance of continuing to sequence and analyze genomes of wild yeasts.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Adaptation to the dietary sugar D-tagatose via genome instability in polyploid Candida albicans cells

The opportunistic fungal pathogen Candida albicans undergoes an unusual parasexual cycle wherein diploid cells mate to form tetraploid cells that can generate genetically diverse progeny via a nonmeiotic program of chromosome loss. The genetic diversity afforded by parasex impacts clinically relevant features including drug resistance and virulence, and yet the factors influencing genome instability in C. albicans are not well defined. To understand how environmental cues impact genome instability, we monitored ploidy change following tetraploid cell growth in a panel of different carbon sources. We found that growth in one carbon source, D-tagatose, led to high levels of genomic instability and chromosome loss in tetraploid cells. This sugar is a stereoisomer of L-sorbose which was previously shown to promote karyotypic changes in C. albicans. However, while expression of the SOU1 gene enabled utilization of L-sorbose, overexpression of this gene did not promote growth in D-tagatose, indicating differences in assimilation of the two sugars. In addition, genome sequencing of multiple progenies recovered from D-tagatose cultures revealed increased relative copy numbers of chromosome 4, suggestive of chromosome-level regulation of D-tagatose metabolism. Together, these studies identify a novel environmental cue that induces genome instability in C. albicans, and further implicate chromosomal changes in supporting metabolic adaptation in this species.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2019

Box, stalked, and upside-down? Draft genomes from diverse jellyfish (Cnidaria, Acraspeda) lineages: Alatina alata (Cubozoa), Calvadosia cruxmelitensis (Staurozoa), and Cassiopea xamachana (Scyphozoa)

Abstract Background Anthozoa, Endocnidozoa, and Medusozoa are the 3 major clades of Cnidaria. Medusozoa is further divided into 4 clades, Hydrozoa, Staurozoa, Cubozoa, and Scyphozoa—the latter 3 lineages make up the clade Acraspeda. Acraspeda encompasses extraordinary diversity in terms of life history, numerous nuisance species, taxa with complex eyes rivaling other animals, and some of the most venomous organisms on the planet. Genomes have recently become available within Scyphozoa and Cubozoa, but there are currently no published genomes within Staurozoa and Cubozoa. Findings Here we present 3 new draft genomes of Calvadosia cruxmelitensis (Staurozoa), Alatina alata (Cubozoa), and Cassiopea xamachana (Scyphozoa) for which we provide a preliminary orthology analysis that includes an inventory of their respective venom-related genes. Additionally, we identify synteny between POU and Hox genes that had previously been reported in a hydrozoan, suggesting this linkage is highly conserved, possibly dating back to at least the last common ancestor of Medusozoa, yet likely independent of vertebrate POU-Hox linkages. Conclusions These draft genomes provide a valuable resource for studying the evolutionary history and biology of these extraordinary animals, and for identifying genomic features underlying venom, vision, and life history traits in Acraspeda.

Ohdera, Aki↗
DOE OSTI2023

Computer-aided, resistance gene-guided genome mining for proteasome and HMG-CoA reductase inhibitors

Secondary metabolites (SMs) are biologically active small molecules, many of which are medically valuable. Fungal genomes contain vast numbers of SM biosynthetic gene clusters (BGCs) with unknown products, suggesting that huge numbers of valuable SMs remain to be discovered. It is challenging, however, to identify SM BGCs, among the millions present in fungi, that produce useful compounds. One solution is resistance gene-guided genome mining, which takes advantage of the fact that some BGCs contain a gene encoding a resistant version of the protein targeted by the compound produced by the BGC. The bioinformatic signature of such BGCs is that they contain an allele of an essential gene with no SM biosynthetic function, and there is a second allele elsewhere in the genome. We have developed a computer-assisted approach to resistance gene-guided genome mining that allows users to query large databases for BGCs that putatively make compounds that have targets of therapeutic interest. Working with the MycoCosm genome database, we have applied this approach to look for SM BGCs that target the proteasome β6 subunit, the target of the proteasome inhibitor fellutamide B, or HMG-CoA reductase, the target of cholesterol reducing therapeutics such as lovastatin. Our approach proved effective, finding known fellutamide and lovastatin BGCs as well as fellutamide- and lovastatin-related BGCs with variations in the SM genes that suggest they may produce structural variants of fellutamides and lovastatin. Gratifyingly, we also found BGCs that are not closely related to lovastatin BGCs but putatively produce novel HMG-CoA reductase inhibitors.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

PhycoCosm, a comparative algal genomics resource

Abstract Algae are a diverse, polyphyletic group of photosynthetic eukaryotes spanning nearly all eukaryotic lineages of life and collectively responsible for ∼50% of photosynthesis on Earth. Sequenced algal genomes, critical to understanding their complex biology, are growing in number and require efficient tools for analysis. PhycoCosm (https://phycocosm.jgi.doe.gov) is an algal multi-omics portal, developed by the US Department of Energy Joint Genome Institute to support analysis and distribution of algal genome sequences and other ‘omics’ data. PhycoCosm provides integration of genome sequence and annotation for >100 algal genomes with available multi-omics data and interactive web-based tools to enable algal research in bioenergy and the environment, encouraging community engagement and data exchange, and fostering new sequencing projects that will further these research goals.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Conserved unique peptide patterns (CUPP) online platform 2.0: implementation of +1000 JGI fungal genomes

Carbohydrate-processing enzymes, CAZymes, are classified into families based on sequence and three-dimensional fold. Because many CAZyme families contain members of diverse molecular function (different EC-numbers), sophisticated tools are required to further delineate these enzymes. Such delineation is provided by the peptide-based clustering method CUPP, Conserved Unique Peptide Patterns. CUPP operates synergistically with the CAZy family/subfamily categorizations to allow systematic exploration of CAZymes by defining small protein groups with shared sequence motifs. The updated CUPP library contains 21,930 of such motif groups including 3,842,628 proteins. The new implementation of the CUPP-webserver, https://cupp.info/, now includes all published fungal and algal genomes from the Joint Genome Institute (JGI), genome resources MycoCosm and PhycoCosm, dynamically subdivided into motif groups of CAZymes. This allows users to browse the JGI portals for specific predicted functions or specific protein families from genome sequences. Thus, a genome can be searched for proteins having specific characteristics. All JGI proteins have a hyperlink to a summary page which links to the predicted gene splicing including which regions have RNA support. The new CUPP implementation also includes an update of the annotation algorithm that uses only a fourth of the RAM while enabling multi-threading, providing an annotation speed below 1 ms/protein.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Strategies to identify and edit improvements in synthetic genome segments episomally

Genome engineering projects often utilize bacterial artificial chromosomes (BACs) to carry multi-kilobase DNA segments at low copy number. However, all stages of whole-genome engineering have the potential to impose mutations on the synthetic genome that can reduce or eliminate the fitness of the final strain. Here, we describe improvements to a multiplex automated genome engineering (MAGE) protocol to improve recombineering frequency and multiplexability. This protocol was applied to recoding an Escherichia coli strain to replace seven codons with synonymous alternatives genome wide. Ten 44 402–47 179 bp de novo synthesized DNA segments contained in a BAC from the recoded strain were unable to complement deletion of the corresponding 33–61 wild-type genes using a single antibiotic resistance marker. Next-generation sequencing (NGS) was used to identify 1–7 non-recoding mutations in essential genes per segment, and MAGE in turn proved a useful strategy to repair these mutations on the recoded segment contained in the BAC when both the recoded and wild-type copies of the mutated genes had to exist by necessity during the repair process. Finally, two web-based tools were used to predict the impact of a subset of non-recoding missense mutations on strain fitness using protein structure and function calls.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Directed evolution expands CRISPR–Cas12a genome-editing capacity

CRISPR-Cas12a enzymes are versatile RNA-guided genome-editing tools with applications encompassing viral diagnosis, agriculture, and human therapeutics. However, their dependence on a 5'-TTTV-3' protospacer adjacent motif (PAM) next to DNA target sequences restricts Cas12a's gene targeting capability to only ∼1% of a typical genome. To mitigate this constraint, we used a bacterial-based directed evolution assay combined with rational engineering to identify variants of Lachnospiraceae bacterium Cas12a with expanded PAM recognition. The resulting Cas12a variants use a range of noncanonical PAMs while retaining recognition of the canonical 5'-TTTV-3' PAM. In particular, biochemical and cell-based assays show that the variant Flex-Cas12a utilizes 5'-NYHV-3' PAMs that expand DNA recognition sites to ∼25% of the human genome. With enhanced targeting versatility, Flex-Cas12a unlocks access to previously inaccessible genomic loci, providing new opportunities for both therapeutic and agricultural genome engineering.

Ma, Enbo↗
DOE OSTI2014

Strain/species identification in metagenomes using genome-specific markers

Shotgun metagenome sequencing has become a fast, cheap and high-throughput technology for characterizing microbial communities in complex environments and human body sites. However, accurate identification of microorganisms at the strain/species level remains extremely challenging. We present a novel k-mer-based approach, termed GSMer, that identifies genome-specific markers (GSMs) from currently sequenced microbial genomes, which were then used for strain/species-level identification in metagenomes. Using 5390 sequenced microbial genomes, 8 770 321 50-mer strain-specific and 11 736 360 species-specific GSMs were identified for 4088 strains and 2005 species (4933 strains), respectively. The GSMs were first evaluated against mock community metagenomes, recently sequenced genomes and real metagenomes from different body sites, suggesting that the identified GSMs were specific to their targeting genomes. Sensitivity evaluation against synthetic metagenomes with different coverage suggested that 50 GSMs per strain were sufficient to identify most microbial strains with ≥0.25× coverage, and 10% of selected GSMs in a database should be detected for confident positive callings. Application of GSMs identified 45 and 74 microbial strains/species significantly associated with type 2 diabetes patients and obese/lean individuals from corresponding gastrointestinal tract metagenomes, respectively. Our result agreed with previous studies but provided strain-level information. The approach can be directly applied to identify microbial strains/species from raw metagenomes, without the effort of complex data pre-processing.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2015

BiGG Models: A platform for integrating, standardizing and sharing genome-scale models

In this study, genome-scale metabolic models are mathematically structured knowledge bases that can be used to predict metabolic pathway usage and growth phenotypes. Furthermore, they can generate and test hypotheses when integrated with experimental data. To maximize the value of these models, centralized repositories of high-quality models must be established, models must adhere to established standards and model components must be linked to relevant databases. Tools for model visualization further enhance their utility. To meet these needs, we present BiGG Models (http://bigg.ucsd.edu), a completely redesigned Biochemical, Genetic and Genomic knowledge base. BiGG Models contains more than 75 high-quality, manually-curated genome-scale metabolic models. On the website, users can browse, search and visualize models. BiGG Models connects genome-scale models to genome annotations and external databases. Reaction and metabolite identifiers have been standardized across models to conform to community standards and enable rapid comparison across models. Furthermore, BiGG Models provides a comprehensive application programming interface for accessing BiGG Models with modeling and analysis tools. As a resource for highly curated, standardized and accessible models of metabolism, BiGG Models will facilitate diverse systems biology studies and support knowledge-based analysis of diverse experimental data.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Congruity of genomic and epidemiological data in modelling of local cholera outbreaks

Cholera continues to be a global health threat. Understanding how cholera spreads between locations is fundamental to the rational, evidence-based design of intervention and control efforts. Traditionally, cholera transmission models have used cholera case-count data. More recently, whole-genome sequence data have qualitatively described cholera transmission. Integrating these data streams may provide much more accurate models of cholera spread; however, no systematic analyses have been performed so far to compare traditional case-count models to the phylodynamic models from genomic data for cholera transmission. Here, we use high-fidelity case-count and whole-genome sequencing data from the 1991 to 1998 cholera epidemic in Argentina to directly compare the epidemiological model parameters estimated from these two data sources. We find that phylodynamic methods applied to cholera genomics data provide comparable estimates that are in line with established methods. Our methodology represents a critical step in building a framework for integrating case-count and genomic data sources for cholera epidemiology and other bacterial pathogens.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Topologically Associating Domain Boundaries are Commonly Required for Normal Genome Function

Topologically associating domain (TAD) boundaries are thought to partition the genome into distinct regulatory territories. Anecdotal evidence suggests that their disruption may interfere with normal gene expression and cause disease phenotype, but the overall extent to which this occurs remains unknown. Here we show that TAD boundary deletions commonly disrupt normal genome function in vivo . We used CRISPR genome editing in mice to individually delete eight TAD boundaries (11-80kb in size) from the genome in mice. All deletions examined resulted in at least one detectable molecular or organismal phenotype, which included altered chromatin interactions or gene expression, reduced viability, and anatomical phenotypes. For 5 of 8 (62%) loci examined, boundary deletions were associated with increased embryonic lethality or other developmental phenotypes. For example, a TAD boundary deletion near Smad3/Smad6 caused complete embryonic lethality, while a deletion near Tbx5/Lhx5 resulted in a severe lung malformation. Our findings demonstrate the importance of TAD boundary sequences for in vivo genome function and suggest that noncoding deletions affecting TAD boundaries should be carefully considered for potential pathogenicity in clinical genetics screening.

Rajderkar, Sudha↗
DOE OSTI2019

Genomics insights into ecotype formation of ammonia‐oxidizing archaea in the deep ocean

Summary Various lineages of ammonia‐oxidizing archaea (AOA) are present in deep waters, but the mechanisms that determine ecotype formation are obscure. We studied 18 high‐quality genomes of the marine group I AOA lineages (alpha, gamma and delta) from the Mariana and Ogasawara trenches. The genomes of alpha AOA resembled each other, while those of gamma and delta lineages were more divergent and had even undergone insertion of some phage genes. The instability of the gamma and delta AOA genomes could be partially due to the loss of DNA polymerase B ( polB) and methyladenine DNA glycosylase ( tag ) genes responsible for the repair of point mutations. The alpha AOA genomes harbour genes encoding a thrombospondin‐like outer membrane structure that probably serves as a barrier to gene flow. Moreover, the gamma and alpha AOA lineages rely on vitamin B 12 ‐independent MetE and B 12 ‐dependent MetH, respectively, for methionine synthesis. The delta AOA genome contains genes involved in uptake of sugar and peptide perhaps for heterotrophic lifestyle. Our study provides insights into co‐occurrence of cladogenesis and anagenesis in the formation of AOA ecotypes that perform differently in nitrogen and carbon cycling in dark oceans.

Wang, Yong↗
DOE OSTI2018

Physiological and genomic signatures of evolutionary thermal adaptation in redband trout from extreme climates

Abstract Temperature is a master environmental factor that limits the geographical distribution of species, especially in ectotherms. To address challenges in biodiversity conservation under ongoing climate change, it is essential to characterize relevant functional limitations and adaptive genomic content at population and species levels. Here, we present evidence for adaptive divergence in cardiac function and genomic regions in redband trout ( Oncorhynchus mykiss gairdneri ) populations from desert and montane streams. Cardiac phenotypes of individual fish were measured in the field with a custom‐built electrocardiogram apparatus. Maximum heart rate and its rate limiting temperature during acute warming were significantly higher in fish that have evolved in the extreme of a desert climate compared to a montane climate. Association mapping with 526,301 single nucleotide polymorphisms ( SNP s) across the genome revealed signatures of thermal selection both within and among ecotypes. Among desert and montane populations, 435 SNP s were identified as putative outliers under natural selection and 20 of these loci showed significant association with average summer water temperatures among populations. Phenotypes for cardiac performance were variable within each ecotype, and 207 genomic regions were strongly associated with either maximum heart rate or rate limiting temperatures among individuals. Annotation of significant loci provided candidate genes that underlie thermal adaptation, including pathways associated with cardiac function ( IRX 5, CASQ 1, CAC 1D, and TITIN ), neuroendocrine system ( GPR 17 and NOS ), and stress response ( SERPH ). By integrating comparative physiology and population genomics, results here advance our knowledge on evolutionary processes of thermal adaptation in aquatic ectotherms.

Chen, Zhongqi↗
DOE OSTI2017

Comparative plastome genomics and phylogenomics of Brachypodium : flowering time signatures, introgression and recombination in recently diverged ecotypes

Few pan-genomic studies have been conducted in plants, and none of them have focused on the intraspecific diversity and evolution of their plastid genomes. We address this issue in Brachypodium distachyon and its close relatives B. stacei and B. hybridum, for which a large genomic data set has been compiled. We analyze inter- and intraspecific plastid comparative genomics and phylogenomic relationships within a family-wide framework. Major indel differences were detected between Brachypodium plastomes. Within B. distachyon, we detected two main lineages, a mostly Extremely Delayed Flowering (EDF+) clade and a mostly Spanish (S+) – Turkish (T+) clade, plus nine chloroplast capture and two plastid DNA (ptDNA) introgression and micro-recombination events. Early Oligocene (30.9 million yr ago (Ma)) and Late Miocene (10.1 Ma) divergence times were inferred for the respective stem and crown nodes of Brachypodium and a very recent Mid-Pleistocene(0.9 Ma) time for the B. distachyon split. Flowering time variation is a main factor driving rapid intraspecific divergence in B. distachyon, although it is counterbalanced by repeated introgression between previously isolated lineages. Swapping of plastomes between the three different genomic groups, EDF+,T+, S+, probably resulted from random backcrossing followed by stabilization through selection pressure.

Sancho, Rubén↗