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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 OSTI2013

Switchgrass Genomic Diversity, Ploidy, and Evolution: Novel Insights from a Network-Based SNP Discovery Protocol

Switchgrass (Panicum virgatum L.) is a perennial grass that has been designated as an herbaceous model biofuel crop for the United States of America. To facilitate accelerated breeding programs of switchgrass, we developed both an association panel and linkage populations for genome-wide association study (GWAS) and genomic selection (GS). All of the 840 individuals were then genotyped using genotyping by sequencing (GBS), generating 350 GB of sequence in total. As a highly heterozygous polyploid (tetraploid and octoploid) species lacking a reference genome, switchgrass is highly intractable with earlier methodologies of single nucleotide polymorphism (SNP) discovery. To access the genetic diversity of species like switchgrass, we developed a SNP discovery pipeline based on a network approach called the Universal Network-Enabled Analysis Kit (UNEAK). Complexities that hinder single nucleotide polymorphism discovery, such as repeats, paralogs, and sequencing errors, are easily resolved with UNEAK. Here, 1.2 million putative SNPs were discovered in a diverse collection of primarily upland, northern-adapted switchgrass populations. Further analysis of this data set revealed the fundamentally diploid nature of tetraploid switchgrass. Taking advantage of the high conservation of genome structure between switchgrass and foxtail millet (Setaria italica (L.) P. Beauv.), two parent-specific, synteny-based, ultra high-density linkage maps containing a total of 88,217 SNPs were constructed. Also, our results showed clear patterns of isolation-by-distance and isolation-by-ploidy in natural populations of switchgrass. Phylogenetic analysis supported a general south-to-north migration path of switchgrass. In addition, this analysis suggested that upland tetraploid arose from upland octoploid. All together, this study provides unparalleled insights into the diversity, genomic complexity, population structure, phylogeny, phylogeography, ploidy, and evolutionary dynamics of switchgrass.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2012

Genome-Wide Study of the Defective Sucrose Fermenter Strain of Vibrio cholerae from the Latin American Cholera Epidemic

The 7th cholera pandemic reached Latin America in 1991, spreading from Peru to virtually all Latin American countries. During the late epidemic period, a strain that failed to ferment sucrose dominated cholera outbreaks in the Northern Brazilian Amazon region. In order to understand the genomic characteristics and the determinants of this altered sucrose fermenting phenotype, the genome of the strain IEC224 was sequenced. This paper reports a broad genomic study of this strain, showing its correlation with the major epidemic lineage. The potentially mobile genomic regions are shown to possess GC content deviation, and harbor the main V. cholera virulence genes. A novel bioinformatic approach was applied in order to identify the putative functions of hypothetical proteins, and was compared with the automatic annotation by RAST. The genome of a large bacteriophage was found to be integrated to the IEC224’s alanine aminopeptidase gene. The presence of this phage is shown to be a common characteristic of the El Tor strains from the Latin American epidemic, as well as its putative ancestor from Angola. The defective sucrose fermenting phenotype is shown to be due to a single nucleotide insertion in the V. cholerae sucrose-specific transportation gene. This frame-shift mutation truncated a membrane protein, altering its structural pore-like conformation. Further, the identification of a common bacteriophage reinforces both the monophyletic and African-Origin hypotheses for the main causative agent of the 1991 Latin America cholera epidemics.

59 BASIC BIOLOGICAL 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 OSTI2021

Population genomics of the pathogenic yeast Candida tropicalis identifies hybrid isolates in environmental samples

Candida tropicalis is a human pathogen that primarily infects the immunocompromised. Whereas the genome of one isolate, C. tropicalis MYA-3404, was originally sequenced in 2009, there have been no large-scale, multi-isolate studies of the genetic and phenotypic diversity of this species. Here, we used whole genome sequencing and phenotyping to characterize 77 isolates of C. tropicalis from clinical and environmental sources from a variety of locations. We show that most C. tropicalis isolates are diploids with approximately 2–6 heterozygous variants per kilobase. The genomes are relatively stable, with few aneuploidies. However, we identified one highly homozygous isolate and six isolates of C. tropicalis with much higher heterozygosity levels ranging from 36–49 heterozygous variants per kilobase. Our analyses show that the heterozygous isolates represent two different hybrid lineages, where the hybrids share one parent (A) with most other C. tropicalis isolates, but the second parent (B or C) differs by at least 4% at the genome level. Four of the sequenced isolates descend from an AB hybridization, and two from an AC hybridization. The hybrids are MTLa/α heterozygotes. Hybridization, or mating, between different parents is therefore common in the evolutionary history of C. tropicalis. The new hybrids were predominantly found in environmental niches, including from soil. Hybridization is therefore unlikely to be associated with virulence. In addition, we used genotype-phenotype correlation and CRISPR-Cas9 editing to identify a genome variant that results in the inability of one isolate to utilize certain branched-chain amino acids as a sole nitrogen source.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2018

Genome-Based Taxonomic Classification of the Phylum Actinobacteria

The application of phylogenetic taxonomic procedures led to improvements in the classification of bacteria assigned to the phylum Actinobacteria but even so there remains a need to further clarify relationships within a taxon that encompasses organisms of agricultural, biotechnological, clinical, and ecological importance. Classification of the morphologically diverse bacteria belonging to this large phylum based on a limited number of features has proved to be difficult, not least when taxonomic decisions rested heavily on interpretation of poorly resolved 16S rRNA gene trees. Here, draft genome sequences of a large collection of actinobacterial type strains were used to infer phylogenetic trees from genome-scale data using principles drawn from phylogenetic systematics. The majority of taxa were found to be monophyletic but several orders, families, and genera, as well as many species and a few subspecies were shown to be in need of revision leading to proposals for the recognition of 2 orders, 10 families, and 17 genera, as well as the transfer of over 100 species to other genera. In addition, emended descriptions are given for many species mainly involving the addition of data on genome size and DNA G+C content, the former can be considered to be a valuable taxonomic marker in actinobacterial systematics. Many of the incongruities detected when the results of the present study were compared with existing classifications had been recognized from 16S rRNA gene trees though whole-genome phylogenies proved to be much better resolved. The few significant incongruities found between 16S/23S rRNA and whole genome trees underline the pitfalls inherent in phylogenies based upon single gene sequences. Similarly good congruence was found between the discontinuous distribution of phenotypic properties and taxa delineated in the phylogenetic trees though diverse non-monophyletic taxa appeared to be based on the use of plesiomorphic character states as diagnostic features.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Comparative Virulence and Genomic Analysis of Streptococcus suis Isolates

Streptococcus suis is a zoonotic bacterial swine pathogen causing substantial economic and health burdens to the pork industry. Mechanisms used by S. suis to colonize and cause disease remain unknown and vaccines and/or intervention strategies currently do not exist. Studies addressing virulence mechanisms used by S. suis have been complicated because different isolates can cause a spectrum of disease outcomes ranging from lethal systemic disease to asymptomatic carriage. The objectives of this study were to evaluate the virulence capacity of nine United States S. suis isolates following intranasal challenge in swine and then perform comparative genomic analyses to identify genomic attributes associated with swine-virulent phenotypes. No correlation was found between the capacity to cause disease in swine and the functional characteristics of genome size, serotype, sequence type (ST), or in vitro virulence-associated phenotypes. A search for orthologs found in highly virulent isolates and not found in non-virulent isolates revealed numerous predicted protein coding sequences specific to each category. While none of these predicted protein coding sequences have been previously characterized as potential virulence factors, this analysis does provide a reliable one-to-one assignment of specific genes of interest that could prove useful in future allelic replacement and/or functional genomic studies. Collectively, this report provides a framework for future allelic replacement and/or functional genomic studies investigating genetic characteristics underlying the spectrum of disease outcomes caused by S. suis isolates.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Genomics, Exometabolomics, and Metabolic Probing Reveal Conserved Proteolytic Metabolism of Thermoflexus hugenholtzii and Three Candidate Species From China and Japan

Thermoflexus hugenholtzii JAD2 T , the only cultured representative of the Chloroflexota order Thermoflexales, is abundant in Great Boiling Spring (GBS), NV, United States, and close relatives inhabit geothermal systems globally. However, no defined medium exists for T. hugenholtzii JAD2 T and no single carbon source is known to support its growth, leaving key knowledge gaps in its metabolism and nutritional needs. Here, we report comparative genomic analysis of the draft genome of T. hugenholtzii JAD2 T and eight closely related metagenome-assembled genomes (MAGs) from geothermal sites in China, Japan, and the United States, representing “Candidatus Thermoflexus japonica,” “Candidatus Thermoflexus tengchongensis,” and “Candidatus Thermoflexus sinensis.” Genomics was integrated with targeted exometabolomics and 13 C metabolic probing of T. hugenholtzii. The Thermoflexus genomes each code for complete central carbon metabolic pathways and an unusually high abundance and diversity of peptidases, particularly Metallo- and Serine peptidase families, along with ABC transporters for peptides and some amino acids. The T. hugenholtzii JAD2 T exometabolome provided evidence of extracellular proteolytic activity based on the accumulation of free amino acids. However, several neutral and polar amino acids appear not to be utilized, based on their accumulation in the medium and the lack of annotated transporters. Adenine and adenosine were scavenged, and thymine and nicotinic acid were released, suggesting interdependency with other organisms in situ. Metabolic probing of T. hugenholtzii JAD2 T using 13 C-labeled compounds provided evidence of oxidation of glucose, pyruvate, cysteine, and citrate, and functioning glycolytic, tricarboxylic acid (TCA), and oxidative pentose-phosphate pathways (PPPs). However, differential use of position-specific 13 C-labeled compounds showed that glycolysis and the TCA cycle were uncoupled. Thus, despite the high abundance of Thermoflexus in sediments of some geothermal systems, they appear to be highly focused on chemoorganotrophy, particularly protein degradation, and may interact extensively with other microorganisms in situ.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2018

Physiological and Comparative Genomic Analysis of Arthrobacter sp. SRS-W-1-2016 Provides Insights on Niche Adaptation for Survival in Uraniferous Soils

Arthrobacter sp. strain SRS-W-1-2016 was isolated on high concentrations of uranium (U) from the Savannah River Site (SRS) that remains co-contaminated by radionuclides, heavy metals, and organics. SRS is located on the northeast bank of the Savannah River (South Carolina, USA), which is a U.S. Department of Energy (DOE) managed ecosystem left historically contaminated from decades of nuclear weapons production activities. Predominant contaminants within the impacted SRS environment include U and Nickel (Ni), both of which can be transformed microbially into less toxic forms via metal complexation mechanisms. Strain SRS-W-1-2016 was isolated from the uraniferous SRS soils on high concentrations of U (4200 μM) and Ni (8500 μM), but rapid growth was observed at much lower concentrations of 500 μM U and 1000 μM Ni, respectively. Microcosm studies established with strain SRS-W-1-2016 revealed a rapid decline in the concentration of spiked U such that it was almost undetectable in the supernatant by 72 h of incubation. Conversely, Ni concentrations remained unchanged, suggesting that the strain removed U but not Ni under the tested conditions. To obtain a deeper understanding of the metabolic potential, a draft genome sequence of strain SRS-W-1-2016 was obtained at a coverage of 90×, assembling into 93 contigs with an N50 contig length of 92,788 bases. The genomic size of strain SRS-W-1-2016 was found to be 4,564,701 bases with a total number of 4327 putative genes. An in-depth, genome-wide comparison between strain SRS-W-1-2016 and its four closest taxonomic relatives revealed 1159 distinct genes, representing 26.7% of its total genome; many associating with metal resistance proteins (e.g., for cadmium, cobalt, and zinc), transporter proteins, stress proteins, cytochromes, and drug resistance functions. Additionally, several gene homologues coding for resistance to metals were identified in the strain, such as outer membrane efflux pump proteins, peptide/nickel transport substrate and ATP-binding proteins, a high-affinity nickel-transport protein, and the spoT gene, which was recently implicated in bacterial resistance towards U. Detailed genome mining analysis of strain SRS-W-1-2016 also revealed the presence of a plethora of secondary metabolite biosynthetic gene clusters likely facilitating resistance to antibiotics, biocides, and metals. Additionally, several gene homologous for the well-known oxygenase enzyme system were also identified, potentially functioning to generate energy via the breakdown of organic compounds and thus enabling the successful colonization and natural attenuation of contaminants by Arthrobacter sp. SRS-W-1-2016 at the SRS site.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

A metagenomic perspective on the microbial prokaryotic genome census

Following 30 years of sequencing, we assessed the phylogenetic diversity (PD) of >1.5 million microbial genomes in public databases, including metagenome-assembled genomes (MAGs) of uncultivated microbes. As compared to the vast diversity uncovered by metagenomic sequences, cultivated taxa account for a modest portion of the overall diversity, 9.73% in bacteria and 6.55% in archaea, while MAGs contribute 48.54% and 57.05%, respectively. Therefore, a substantial fraction of bacterial (41.73%) and archaeal PD (36.39%) still lacks any genomic representation. This unrepresented diversity manifests primarily at lower taxonomic ranks, exemplified by 134,966 species identified in 18,087 metagenomic samples. Our study exposes diversity hotspots in freshwater, marine subsurface, sediment, soil, and other environments, whereas human samples yielded minimal novelty within the context of existing datasets. These results offer a roadmap for future genome recovery efforts, delineating uncaptured taxa in underexplored environments and underscoring the necessity for renewed isolation and sequencing.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2012

The “Most Wanted” Taxa from the Human Microbiome for Whole Genome Sequencing

The goal of the Human Microbiome Project (HMP) is to generate a comprehensive catalog of human-associated microorganisms including reference genomes representing the most common species. Toward this goal, the HMP has characterized the microbial communities at 18 body habitats in a cohort of over 200 healthy volunteers using 16S rRNA gene (16S) sequencing and has generated nearly 1,000 reference genomes from human-associated microorganisms. To determine how well current reference genome collections capture the diversity observed among the healthy microbiome and to guide isolation and future sequencing of microbiome members, we compared the HMP’s 16S data sets to several reference 16S collections to create a ‘most wanted’ list of taxa for sequencing. Our analysis revealed that the diversity of commonly occurring taxa within the HMP cohort microbiome is relatively modest, few novel taxa are represented by these OTUs and many common taxa among HMP volunteers recur across different populations of healthy humans. Taken together, these results suggest that it should be possible to perform whole-genome sequencing on a large fraction of the human microbiome, including the ‘most wanted’, and that these sequences should serve to support microbiome studies across multiple cohorts. Also, in stark contrast to other taxa, the ‘most wanted’ organisms are poorly represented among culture collections suggesting that novel culture- and single-cell-based methods will be required to isolate these organisms for sequencing.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2019

Comprehensive genomic and transcriptomic analysis of polycyclic aromatic hydrocarbon degradation by a mycoremediation fungus, sp. KUC8613

The environmental accumulation of polycyclic aromatic hydrocarbons (PAHs) is of great concern due to potential carcinogenic and mutagenic risks, as well as their resistance to remediation. While many fungi have been reported to break down PAHs in environments, the details of gene-based metabolic pathways are not yet comprehensively understood. Specifically, the genome-scale transcriptional responses of fungal PAH degradation have rarely been reported. In this study, we report the genomic and transcriptomic basis of PAH bioremediation by a potent fungal degrader, Dentipellis sp. KUC8613. The genome size of this fungus was 36.71 Mbp long encoding 14,320 putative protein-coding genes. The strain efficiently removed more than 90% of 100 mg/l concentration of PAHs within 10 days. The genomic and transcriptomic analysis of this white rot fungus highlights that the strain primarily utilized non-ligninolytic enzymes to remove various PAHs, rather than typical ligninolytic enzymes known for playing important roles in PAH degradation. PAH removal by non-ligninolytic enzymes was initiated by both different PAH-specific and common upregulation of P450s, followed by downstream PAH-transforming enzymes such as epoxide hydrolases, dehydrogenases, FAD-dependent monooxygenases, dioxygenases, and glycosyl- or glutathione transferases. Among the various PAHs, phenanthrene induced a more dynamic transcriptomic response possibly due to its greater cytotoxicity, leading to highly upregulated genes involved in the translocation of PAHs, a defense system against reactive oxygen species, and ATP synthesis. Finally, our genomic and transcriptomic data provide a foundation of understanding regarding the mycoremediation of PAHs and the application of this strain for polluted environments.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2019

Nuclear genome shuffling significantly increases production of chloroplast-based recombinant protein in Chlamydomonas reinhardtii

Algae biotechnology holds great promise for the production of sustainable and environmentally friendly products, however more robust strains of microalgae must be developed before algae can be used as a commercially relevant production platform. Here we demonstrate a timely and effective approach to significantly improve a desired phenotype in algae through a combination of mutagenesis and genome shuffling. In this study, we engineered a strain of Chlamydomonas reinhardtii to produce recombinant green fluorescent protein in the chloroplast and then subjected the nuclear genome to various rounds of UV-induced mutagenesis and genome shuffling via sexual recombination, followed by isolation of strains with enhanced recombinant protein expression using fluorescence-activated cell sorting (FACS). Here, the greatest success came from first UV-mutagenizing the host strain to create mutations in the nuclear genome, followed by FACS to isolate the best GFP-expressing mutant, then shuffling the nuclear genome of that mutant by mating it with selected wild-type strains, and then again isolating the best progeny via FACS. Using this strategy, a novel strain was evolved in <3 months to express GFP over 2% total soluble protein (TSP), a 15-fold increase from the initial recombinant strain which expressed GFP at 0.14% TSP. Furthermore, this significant increase in GFP expression from the chloroplast was accomplished without altering the GFP gene itself or reducing the growth rate of the strain, demonstrating the effectiveness of this strategy to enhance a specific phenotype and significantly improve product production within an algal species in a commercially relevant timeframe.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Genome sequence, phylogenetic analysis, and structure-based annotation reveal metabolic potential of Chlorella sp. SLA-04

Algae are a broad class of photosynthetic eukaryotes that are phylogenetically and physiologically diverse. Most of the phylogenetic diversity has been inferred from 18S rDNA sequencing since there are only a few complete genomes available in public databases. Here we use ultra-long-read Nanopore sequencing to determine a gapless, telomere-to-telomere complete genome sequence of Chlorella sp. SLA-04, previously described as Chlorella sorokiniana SLA-04. Chlorella sp. SLA-04 is a green alga that grows to high cell density in a wide variety of environments - high and neutral pH, high and low alkalinity, and high and low salinity. SLA-04's ability to grow in high pH and high alkalinity media without external CO 2 supply is favorable for large-scale algal biomass production. Phylogenetic analysis performed using ribosomal DNA and conserved protein sequences consistently reveal that Chlorella sp. SLA-04 forms a distinct lineage from other strains of Chlorella sorokiniana. We complement traditional genome annotation methods with high throughput structural predictions and demonstrate that this approach expands functional prediction of the SLA-04 proteome. Genomic analysis of the SLA-04 genome identifies the genes capable of utilizing TCA cycle intermediates to replenish cytosolic acetyl-CoA pools for lipid production. We also identify a complete metabolic pathway for sphingolipid anabolism that may allow SLA-04 to readily adapt to changing environmental conditions and facilitate robust cultivation in mass production systems. Altogether, this work clarifies the phylogeny of Chlorella sp. SLA-04 within Trebouxiophyceae and demonstrates how structural predictions can be used to improve annotation beyond sequencebased methods.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2017

Draft genome sequence of Chryseobacterium limigenitum SUR2 T (LMG 28734 T ) isolated from dehydrated sludge

The type strain SUR2 of the novel species Chryseobacterium limigenitum was isolated from a dehydrated sludge of the municipal sewage treatment plant in Dogoše near Maribor in Slovenia. The draft genome, with 60 contigs, 4,697,725 bp, 34.4% of G+C content, was obtained using the Illumina HiSeq 2500-1 platform. Joint Genome Institute Microbial Genome Annotation Pipeline (MGAP v.4) has identified 4322 protein-coding sequences including resistance genes against arsenic and other heavy metals. In addition, a subclass B3 metallo-β-lactamase, which confers resistance to penicillins, cephalosporins and carbapenems, was also present in the genome. The genome sequence provides important information regarding bioremediation potential and pathogenic properties of this newly identified species.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2026

Discovery of additional ancient genome duplications in yeasts

Whole-genome duplication (WGD) has had profound macroevolutionary impacts on diverse lineages, preceding adaptive radiations in vertebrates, teleost fish, and angiosperms. In contrast to the many known ancient WGDs in animals, and especially plants, we are aware of evidence for only four WGDs in fungi. The oldest of these occurred ∼100 million years ago (mya) and is shared by ∼60 extant Saccharomycetales species, including the baker’s yeast Saccharomyces cerevisiae. Notably, this is the only known ancient WGD event in the yeast subphylum Saccharomycotina. The dearth of ancient WGD events in fungi remains a mystery. Some studies have suggested that fungal lineages that experience chromosome and genome duplication quickly go extinct, leaving no trace in the genomic record, while others contend that the lack of known WGDs is due to an absence of data. Under the second hypothesis, additional sampling and deeper sequencing of fungal genomes should lead to the discovery of more WGD events. Coupling hundreds of recently published genomes from nearly every described Saccharomycotina species, with three additional long-read assemblies, we discovered three novel WGD events. Although the functions of retained duplicate genes originating from these events are broad, they bear similarities to the well-known WGD that occurred in the Saccharomycetales. In conclusion, our results suggest that WGD may be a more common evolutionary force in fungi than previously believed.

convergent evolution↗
DOE OSTI2018

Whole-genome sequencing and single nucleotide polymorphisms in multidrug-resistant clinical isolates of Mycobacterium tuberculosis from the Philippines

Thousands of cases of multidrug-resistant tuberculosis (TB) have been observed in the Philippines, but studies on the Mycobacterium tuberculosis (MTB) genotypes that underlie the observed drug resistance profiles are lacking. This study aimed to analyse the whole genomes of clinical MTB isolates representing various resistance profiles to identify single nucleotide polymorphisms (SNPs) in resistance-associated genes. The genomes of ten MTB isolates cultured from banked sputum sources were sequenced. Bioinformatics analysis consisted of assembly, annotation and SNP identification in genes reported to be associated with resistance to isoniazid (INH), rifampicin (RIF), ethambutol (ETH), streptomycin, pyrazinamide (PZA) and fluoroquinolones (FQs). The draft assemblies covered an average of 97.08% of the expected genome size. Seven of the ten isolates belonged to the Indo-Oceanic lineage/EA12-Manila clade. Two isolates were classified into the Euro-American lineage, whilst the pre-XDR (pre-extensively drug-resistant) isolate was classified under the East Asian/Beijing clade. The SNPs katG Ser315Thr, rpoB Ser450Leu and embB Met306Val were found in INH- (4/7), RIF- (3/6) and ETH-resistant (2/6) isolates, respectively, but not in susceptible isolates. Mutations in the inhA promoter and in the pncA and gyrA genes known to be involved in resistance to INH, PZA and FQs, respectively, were also identified. This study represents the first effort to investigate the whole genomes of Philippine clinical strains of MTB exhibiting various multidrug resistance profiles. Whole-genome data can provide valuable insights to the mechanistic and epidemiological qualities of TB in a high-burden setting such as the Philippines.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Iterative genome editing of Escherichia coli for 3-hydroxypropionic acid production

Synthetic biology requires strategies for the targeted, efficient, and combinatorial engineering of biological sub-systems at the molecular level. Here, we report the use of the iterative CRISPR EnAbled Trackable genome Engineering (iCREATE) method for the rapid construction of combinatorially modified genomes. We coupled this genome engineering strategy with high-throughput phenotypic screening and selections to recursively engineer multiple traits in Escherichia coli for improved production of the platform chemical 3- hydroxypropionic acid (3HP). Particularly, we engineered i) central carbon metabolism, ii) 3HP synthesis, and (iii) 3HP tolerance through design, construction and testing of ~162,000 mutations across 115 genes spanning global regulators, transcription factors, and enzymes involved in 3HP synthesis and tolerance. The iCREATE process required ~1 month to perform 13 rounds of combinatorial genome modifications with targeted gene knockouts, expression modification by ribosomal binding site (RBS) engineering, and genome-level site-saturation mutagenesis. Specific mutants conferring increased 3HP titer, yield, and productivity were identified and then combined to produce 3HP at a yield and concentration ~60-fold higher than the wild-type strain.

3-hydroxypropionic acid↗
DOE OSTI2017

Single-virus genomics reveals hidden cosmopolitan and abundant viruses

Microbes drive ecosystems under constraints imposed by viruses. However, a lack of virus genome information hinders our ability to answer fundamental, biological questions concerning microbial communities. Here we apply single-virus genomics (SVGs) to assess whether portions of marine viral communities are missed by current techniques. The majority of the here-identified 44 viral single-amplified genomes (vSAGs) are more abundant in global ocean virome data sets than published metagenome-assembled viral genomes or isolates. This indicates that vSAGs likely best represent the dsDNA viral populations dominating the oceans. Species-specific recruitment patterns and virome simulation data suggest that vSAGs are highly microdiverse and that microdiversity hinders the metagenomic assembly, which could explain why their genomes have not been identified before. Altogether, SVGs enable the discovery of some of the likely most abundant and ecologically relevant marine viral species, such as vSAG 37-F6, which were overlooked by other methodologies.

59 BASIC BIOLOGICAL SCIENCES↗