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All sourcesNASA NTRSDOE OSTI
DOE OSTI2016

Genomic analysis of three Bifidobacterium species isolated from the calf gastrointestinal tract

Ruminant animals contribute significantly to the global value of agriculture and rely on a complex microbial community for efficient digestion. However, little is known of how this microbial-host relationship develops and is maintained. To begin to address this, we have determined the ability of three Bifidobacterium species isolated from the faeces of newborn calves to grow on carbohydrates typical of a newborn ruminant diet. Genome sequences have been determined for these bacteria with analysis of the genomes providing insights into the host association and identification of several genes that may mediate interactions with the ruminant gastrointestinal tract. The present study provides a starting point from which we can define the role of potential beneficial microbes in the nutrition of young ruminants and begin to influence the interactions between the microbiota and the host. The differences observed in genomic content hint at niche partitioning among the bifidobacterial species analysed and the different strategies they employ to successfully adapt to this habitat.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2019

Volatile Memory Extraction- Based Approach for Level 0-1 CPS Forensics

Most security analyzers operate on system state that is far removed from end-point components in cyber-physical systems (CPS) identified as level 0-1 in the Purdue Architecture Reference Architecture (PERA) [1]. For example, many operate on system logs and other data dumps to disks. Tremendous value that can be gained in cyber security forensics if low level details such as dynamic changes to volatile memory can be extracted and provided to more sophisticated analysis tools. However, obtaining detailed and dynamic system state at the level of volatile memory is extremely challenging [2]. Here, we attempt to apply IT memory forensic mechanisms to CPS end-point devices and statistically evaluate them. Our focus is to extract volatile and dynamically changing internal information form CPS 0-1 level devices, and design preliminary schemes to exploit that extracted information. This new capability of generating a sequence of volatile memory snapshots for offline, detailed and sophisticated analysis opens a new class of cyber security schemes for CPS forensic analysis. As a case study for our ongoing research, we apply the proposed methodology to Modicon PLC using Modbus protocol. We extract the memory layout and subject the device to read operations at the most critical regions of memory. Similarly, write operations are initiated to carefully determine memory locations (for example, bytes that hold the firmware version number). This capability of generating a sequence of volatile memory snapshots for offline, detailed and sophisticated analysis opens a new class of cyber security schemes for CPS forensic analysis. Also, the ability to dynamically make controlled modifications to specific memory locations opens the potential for new mechanisms such as taint analysis and watermarking.

Asmar Awad, Rima↗
DOE OSTI2020

Phaseolus vulgaris SUT1.1 is a high affinity sucrose–proton co–transporter

Plant sucrose transporters are required for phloem loading, and therefore are essential for plant growth and development. In common beans (Phaseolus vulgaris) there are only two sucrose transporters functionally characterized. Through a previous RNA-seq study, we identified a putative sucrose transporter in common bean, which we hypothesize to function in import of sucrose into plant cells. In silico analysis revealed that PvSUT1.1 is a putative sucrose-proton co-transporter distinct from other characterized sucrose transporters in common bean indicating that this is a previously undescribed transporter protein in beans. Further analysis revealed that PvSUT1.1 shares high protein sequence homology to the phloem loader Arabidopsis SUC2; both have 12 transmembrane domains, a typical characteristic of plant sucrose transporters. Heterologous expression in yeast further showed PvSUT1.1 to be functional and it imported sucrose into yeast cells with a K m of 0.7 mM sucrose. Import of sucrose through PvSUT1.1 is also pH-dependent with highest uptake at pH 4.0, and activity is lost in the presence of the uncoupler carbonyl cyanide 3-chlorophenylhydrazone. Consistent with identification of PvSUT1.1 as a Type I transporter, PvSUT1.1 also transports esculin. Finally, PvSUT1.1 showed expression in multiple tissues and the protein was localized to the plasma membrane. The results show that PvSUT1.1 is a sucrose transporter that is probably involved in the uptake of sucrose into source and sink cells. The potential role of PvSUT1.1 in leaf phloem loading of sucrose in common beans and its importance in heat tolerance of reproductive tissues are further discussed.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2019

Structural diversity in the Mycobacteria DUF3349 superfamily

A protein superfamily with a “Domain of Unknown Function,”, DUF3349 (PF11829), is present predominately in Mycobacterium and Rhodococcus bacterial species suggesting that these proteins may have a biological function unique to these bacteria. We previously reported the inaugural structure of a DUF3349 superfamily member, Mycobacterium tuberculosis Rv0543c. In this study, we report the structures determined for three additional DUF3349 proteins: Mycobacterium smegmatis MSMEG_1063 and MSMEG_1066 and Mycobacterium abscessus MAB_3403c. Like Rv0543c, the NMR solution structure of MSMEG_1063 revealed a monomeric five α-helix bundle with a similar overall topology. Conversely, the crystal structure of MSMEG_1066 revealed a five α-helix protein with a strikingly different topology and a tetrameric quaternary structure that was confirmed by size exclusion chromatography. The NMR solution structure of a fourth member of the DUF3349 superfamily, MAB_3403c, with 18 residues missing at the N-terminus, revealed a monomeric α-helical protein with a folding topology similar to the three C-terminal helices in the protomer of the MSMEG_1066 tetramer. These structures, together with a GREMLIN-based bioinformatics analysis of the DUF3349 primary amino acid sequences, suggest two subfamilies within the DUF3349 family. The division of the DUF3349 into two distinct subfamilies would have been lost if structure solution had stopped with the first structure in the DUF3349 family, highlighting the insights generated by solving multiple structures within a protein superfamily. Future studies will determine if the structural diversity at the tertiary and quaternary levels in the DUF3349 protein superfamily have functional roles in Mycobacteria and Rhodococcus species with potential implications for structure-based drug discovery.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

A Highly Ordered Nitroxide Side Chain for Distance Mapping and Monitoring Slow Structural Fluctuations in Proteins

Abstract Site-directed spin labeling electron paramagnetic resonance (SDSL-EPR) is an established tool for exploring protein structure and dynamics. Although nitroxide side chains attached to a single cysteine via a disulfide linkage are commonly employed in SDSL-EPR, their internal flexibility complicates applications to monitor slow internal motions in proteins and to structure determination by distance mapping. Moreover, the labile disulfide linkage prohibits the use of reducing agents often needed for protein stability. To enable the application of SDSL-EPR to the measurement of slow internal dynamics, new spin labels with hindered internal motion are desired. Here, we introduce a highly ordered nitroxide side chain, designated R9, attached at a single cysteine residue via a non-reducible thioether linkage. The reaction to introduce R9 is highly selective for solvent-exposed cysteine residues. Structures of R9 at two helical sites in T4 Lysozyme were determined by X-ray crystallography and the mobility in helical sequences was characterized by EPR spectral lineshape analysis, Saturation Transfer EPR, and Saturation Recovery EPR. In addition, interspin distance measurements between pairs of R9 residues are reported. Collectively, all data indicate that R9 will be useful for monitoring slow internal structural fluctuations, and applications to distance mapping via dipolar spectroscopy and relaxation enhancement methods are anticipated.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2017

Identification and molecular cloning of novel transcripts of the human kallikrein-related peptidase 10 (KLK10) gene using next-generation sequencing

Tissue kallikrein and kallikrein-related peptidases (KLKs) form the largest group of serine proteases in the human genome, sharing many structural and functional characteristics. Multiple alternative transcripts have been reported for the most human KLK genes, while many of them are aberrantly expressed in various malignancies, thus possessing significant prognostic and/or diagnostic value. Alternative splicing of cancer-related genes is a common cellular mechanism accounting for cancer cell transcriptome complexity, as it affects cell cycle control, proliferation, apoptosis, invasion, and metastasis. In this study, we describe the identification and molecular cloning of eight novel transcripts of the human KLK10 gene using 3′ rapid amplification of cDNA ends (3′ RACE) and next-generation sequencing (NGS), as well as their expression analysis in a wide panel of cell lines, originating from several distinct cancerous and normal tissues. Bioinformatic analysis revealed that the novel KLK10 transcripts contain new alternative splicing events between already annotated exons as well as novel exons. In addition, investigation of their expression profile in a wide panel of cell lines was performed with nested RT-PCR using variant-specific pairs of primers. Since many KLK mRNA transcripts possess clinical value, these newly discovered alternatively spliced KLK10 transcripts appear as new potential biomarkers for diagnostic and/or prognostic purposes or as targets for therapeutic strategies. - Highlights: • NGS was used to identify novel transcripts of the human KLK10 gene. • 8 novel KLK10 transcripts were identified. • A novel 3′UTR was detected and characterized. • The expression profiles of all 8 novel KLK10 transcripts were identified.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2015

The microstructure and mechanical properties of Al-containing 9Cr ODS ferritic alloy

In this study, a 9Cr oxide-dispersion strengthened (ODS) alloy with additional corrosion resistant element Al was fabricated by mechanical alloying (MA) and hot pressing (HP) to explore the impact of Al on the microstructure and mechanical property of a 9Cr ODS alloy. It is found that the Al completely dissolved into the FeeCr matrix after milling for 30 h. The minor phases in the Al-containing 9Cr ODS ferritic alloy were investigated by a high-energy X-ray, and were identified to be orthorhombic-YAlO3 (YAP), bcc-Y3Al5O12 (YAG), monoclinic-Al2Y4O9 (YAM), and hexagonal-YAlO3 (YAH). These phases were further confirmed by selected area diffraction pattern (SADP), energy dispersive spectroscopy (EDS), and high resolution transmission electron microscopy (HRTEM). In addition, their volume fractions were also calculated from the integrated intensities. According to the analysis of the particles and their formation sequences, the larger particles (greater than 100 nm) are identified as mainly YAG and Al2O3 particles, while the particles with small size (less than 30 nm) are likely primarily YAM, YAH, and YAP particles. The yielding strength (YS) and ultimate tensile strength (UTS) at RT are 563 MPa and 744 MPa, respectively, while the YS and UTS at 700 C are 245 MPa and 276 MPa, respectively. Although the addition Al in ODS alloys decreases the strength at RT, the values at high temperature are similar to those obtained for 9Cr ODS alloys strengthened by fine YeTieO particles.

Microstructure↗
DOE OSTI2022

Sas20 is a highly flexible starch-binding protein in the Ruminococcus bromii cell-surface amylosome

Ruminococcus bromii is a keystone species in the human gut that has the rare ability to degrade dietary resistant starch (RS). This bacterium secretes a suite of starch-active proteins that work together within larger complexes called amylosomes that allow R. bromii to bind and degrade RS. Starch adherence system protein 20 (Sas20) is one of the more abundant proteins assembled within amylosomes, but little could be predicted about its molecular features based on amino acid sequence. Here, we performed a structure–function analysis of Sas20 and determined that it features two discrete starch-binding domains separated by a flexible linker. We show that Sas20 domain 1 contains an N-terminal β-sandwich followed by a cluster of α-helices, and the nonreducing end of maltooligosaccharides can be captured between these structural features. Furthermore, the crystal structure of a close homolog of Sas20 domain 2 revealed a unique bilobed starch-binding groove that targets the helical α1,4-linked glycan chains found in amorphous regions of amylopectin and crystalline regions of amylose. Affinity PAGE and isothermal titration calorimetry demonstrated that both domains bind maltoheptaose and soluble starch with relatively high affinity (K d ≤ 20 μM) but exhibit limited or no binding to cyclodextrins. Finally, small-angle X-ray scattering analysis of the individual and combined domains support that these structures are highly flexible, which may allow the protein to adopt conformations that enhance its starch-targeting efficiency. Taken together, we conclude that Sas20 binds distinct features within the starch granule, facilitating the ability of R. bromii to hydrolyze dietary RS.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2017

Methylation of Septin9 mediated by DNMT3a enhances hepatic stellate cells activation and liver fibrogenesis

Liver fibrosis, resulting from chronic and persistent injury to the liver, is a worldwide health problem. Advanced liver fibrosis results in cirrhosis, liver failure and even hepatocellular cancer (HCC), often eventually requiring liver transplantation, poses a huge health burden on the global community. However, the specific pathogenesis of liver fibrosis remains not fully understood. Numerous basic and clinical studies have provided evidence that epigenetic modifications, especially DNA methylation, might contribute to the activation of hepatic stellate cells (HSCs), the pivotal cell type responsible for the fibrous scar in liver. Here, reduced representation bisulfite sequencing (RRBS) and bisulfite pyrosequencing PCR (BSP) analysis identified hypermethylation status of Septin9 (Sept9) gene in liver fibrogenesis. Sept9 protein was dramatically decreased in livers of CCl4-treated mice and immortalized HSC-T6 cells exposed to TGF-β1. Nevertheless, the suppression of Sept9 could be blocked by DNMT3a-siRNA and DNA methyltransferase inhibitor, 5-aza-2′-deoxycytidine (5-azadC). Overexpressed Sept9 attenuated TGF-β1-induced expression of myofibroblast markers α-SMA and Col1a1, accompanied by up-regulation of cell apoptosis-related proteins. Conversely, RNAi-mediated silencing of Sept9 enhanced accumulation of extracellular matrix. These observations suggested that Sept9 contributed to alleviate liver fibrosis might partially through promoting activated HSCs apoptosis and this anti-fibrogenesis effect might be blocked by DNMT-3a mediated methylation of Sept9. Therefore, pharmacological agents that inhibit Sept9 methylation and increase its expression could be considered as valuable treatments for liver fibrosis. - Highlights: • This is the first report of Sept9 methylation and function in liver fibrosis. • Ectopic expression of Sept9 could block the liver fibrogenesis. • DNMT3a might be responsible for the suppression of Sept9 in liver fibrosis.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2020

Development of a thermophilic coculture for corn fiber conversion to ethanol

Abstract The fiber in corn kernels, currently unutilized in the corn to ethanol process, represents an opportunity for introduction of cellulose conversion technology. We report here that Clostridium thermocellum can solubilize over 90% of the carbohydrate in autoclaved corn fiber, including its hemicellulose component glucuronoarabinoxylan (GAX). However, Thermoanaerobacterium thermosaccharolyticum or several other described hemicellulose-fermenting thermophilic bacteria can only partially utilize this GAX. We describe the isolation of a previously undescribed organism, Herbinix spp . strain LL1355, from a thermophilic microbiome that can consume 85% of the recalcitrant GAX. We sequence its genome, and based on structural analysis of the GAX, identify six enzymes that hydrolyze GAX linkages. Combinations of up to four enzymes are successfully expressed in T. thermosaccharolyticum . Supplementation with these enzymes allows T. thermosaccharolyticum to consume 78% of the GAX compared to 53% by the parent strain and increases ethanol yield from corn fiber by 24%.

09 BIOMASS FUELS↗
DOE OSTI2022

Diploid-dominant life cycles characterize the early evolution of Fungi

Most of the described species in kingdom Fungi are contained in two phyla, the Ascomycota and the Basidiomycota (subkingdom Dikarya). As a result, our understanding of the biology of the kingdom is heavily influenced by traits observed in Dikarya, such as aerial spore dispersal and life cycles dominated by mitosis of haploid nuclei. We now appreciate that Fungi comprises numerous phylum-level lineages in addition to those of Dikarya, but the phylogeny and genetic characteristics of most of these lineages are poorly understood due to limited genome sampling. Here, we addressed major evolutionary trends in the non-Dikarya fungi by phylogenomic analysis of 69 newly generated draft genome sequences of the zoosporic (flagellated) lineages of true fungi. Our phylogeny indicated five lineages of zoosporic fungi and placed Blastocladiomycota, which has an alternation of haploid and diploid generations, as branching closer to the Dikarya than to the Chytridiomyceta. Our estimates of heterozygosity based on genome sequence data indicate that the zoosporic lineages plus the Zoopagomycota are frequently characterized by diploid-dominant life cycles. We mapped additional traits, such as ancestral cell-cycle regulators, cell-membrane– and cell-wall–associated genes, and the use of the amino acid selenocysteine on the phylogeny and found that these ancestral traits that are shared with Metazoa have been subject to extensive parallel loss across zoosporic lineages. Together, our results indicate a gradual transition in the genetics and cell biology of fungi from their ancestor and caution against assuming that traits measured in Dikarya are typical of other fungal lineages.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Mechanism-guided engineering of a minimal biological particle for genome editing

The widespread application of genome editing to treat and cure disease requires the delivery of genome editors into the nucleus of target cells. Enveloped delivery vehicles (EDVs) are engineered virally derived particles capable of packaging and delivering CRISPR-Cas9 ribonucleoproteins (RNPs). However, the presence of lentiviral genome encapsulation and replication proteins in EDVs has obscured the underlying delivery mechanism and precluded particle optimization. Here, we show that Cas9 RNP nuclear delivery is independent of the native lentiviral capsid structure. Instead, EDV-mediated genome editing activity corresponds directly to the number of nuclear localization sequences on the Cas9 enzyme. EDV structural analysis using cryo-electron tomography and small molecule inhibitors guided the removal of ~80% of viral residues, creating a minimal EDV (miniEDV) that retains full RNP delivery capability. MiniEDVs are 25% smaller yet package equivalent amounts of Cas9 RNPs relative to the original EDVs and demonstrated increased editing in cell lines and therapeutically relevant primary human T cells. These results show that virally derived particles can be streamlined to create efficacious genome editing delivery vehicles with simpler production and manufacturing.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Niche Partitioning of Microbial Communities at an Ancient Vitrified Hillfort: Implications for Vitrified Radioactive Waste Disposal

A pre-Viking era vitrified hillfort, Broborg, provides a habitat analogue for disposed radioactive waste glass and shows strong niche partitioning among the organisms involved in glass alteration. Microbes cannot be eliminated from radioactive waste disposal facilities and the consequences of bio-colonization must be understood. We use Broborg as a model system to inform what microbial processes might influence long-term radioactive waste glass durability by examining anthropogenic glass that has been subjected to bio-colonization for over 1,500 years. Scanning electron microscopy (SEM) images reveal the surficial biofilm structure, and chemical/mineralogy analysis in combination with deoxyribonucleic acid (DNA) sequencing of samples from the vitrified substrate, the adjacent soil, and the general topsoil provide insight into niche partitioning. The ancient glass niche supports a unique microbial community of bacteria, fungi, and protists that manifests the species response to local geochemical and mineralogical conditions. Communities from the geochemical niche associated with the glass are distinct and less diverse than soil communities. The microbiome of the glass and adjacent soil are dominated by lichens, lichen-associated microorganisms, and other epilithic, endolithic, and epigeic organisms. Pseudomonads dominate the prokaryotic communities on the vitrified material, but not the adjacent soil. In contrast, the general topsoil communities are enriched in plant rhizosphere organisms. Taxa associated with vitrification have bio-corrosive properties that could be detrimental to glass durability, including silicate mineral dissolution, extraction of essential elements, secretion of geochemically reactive organic acids, and dissolution induced by improved water retention. However, these stable long-term biofilms also possess a homeostatic function that could limit glass alteration. Overall, this study demonstrates the potential impacts that microbial colonization and niche partitioning can have on glass alteration and subsequent release of radionuclides from a disposal facility for vitrified radioactive waste.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2015

Thermocrinis jamiesonii sp. nov., a thiosulfate-oxidizing, autotropic thermophile isolated from a geothermal spring

An obligately thermophilic, chemolithotrophic, microaerophilic bacterium, designated strain GBS1T, was isolated from the water column of Great Boiling Spring, Nevada, USA. Thiosulfate was required for growth. Although capable of autotrophy, growth of GBS1T was enhanced in the presence of acetate, peptone, or Casamino acids. Growth occurred at 70-85 °C with an optimum at 80 °C, at pH 6.5-7.75 with an optimum at pH 7.25, at 0.5-8% oxygen with an optimum at 1-2%, and at ≤200 mM sodium chloride. The doubling time under optimal growth conditions was 1.3 hrs, with a final cell density of 6.2±0.5 x 107 cells/mL. Non-motile, rod-shaped cells 1.4-2.4 x 0.4-0.6 µm occurred singly or in pairs. Major cellular fatty acids (>5% of total) were C20:1ω9c (44.8%), C18:0 (26.0%), C16:0 (9.9%) and C20:0 (5.4%). Phylogenetic analysis of the GBS1T 16S rRNA gene sequence indicated an affiliation with Thermocrinis ruber and other Thermocrinis spp., but comparisons of 16S rRNA gene identity (≤97.10%) and in silico estimated DNA-DNA hybridization values (≤18.4%) with Thermocrinis spp. indicate that his strain is distinct from described species. Based on phenotypic, genotypic, and phylogenetic characteristics, the name Thermocrinis jamiesonii sp. nov. is proposed, with GBS1T (= JCM 19133T = DSM 27162T) as the type strain.

Environmental Molecular Sciences Laboratory↗
DOE OSTI2019

Transposon insertional mutagenesis in Saccharomyces uvarum reveals trans-acting effects influencing species-dependent essential genes

To understand how complex genetic networks perform and regulate diverse cellular processes, the function of each individual component must be defined. Comprehensive phenotypic studies of mutant alleles have been successful in model organisms in determining what processes depend on the normal function of a gene. These results are often ported to newly sequenced genomes by using sequence homology. However, sequence similarity does not always mean identical function or phenotype, suggesting that new methods are required to functionally annotate newly sequenced species. We have implemented here comparative analysis by high-throughput experimental testing of gene dispensability in Saccharomyces uvarum, a sister species of Saccharomyces cerevisiae. We created haploid and heterozygous diploid Tn7 insertional mutagenesis libraries in S. uvarum to identify species-dependent essential genes, with the goal of detecting genes with divergent functions and/or different genetic interactions. Comprehensive gene dispensability comparisons with S. cerevisiae predicted diverged dispensability at 12% of conserved orthologs, and validation experiments confirmed 22 differentially essential genes. Despite their differences in essentiality, these genes were capable of cross-species complementation, demonstrating that trans-acting factors that are background-dependent contribute to differential gene essentiality. This study shows that direct experimental testing of gene disruption phenotypes across species can inform comparative genomic analyses and improve gene annotations. Our method can be widely applied in microorganisms to further our understanding of genome evolution.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2019

Construction and comparison of three reference-quality genome assemblies for soybean

Here, we report reference-quality genome assemblies and annotations for two accessions of soybean (Glycine max) and for one accession of Glycine soja, the closest wild relative of G. max. The G. max assemblies provided are for widely used US cultivars: the northern line Williams 82 (Wm82) and the southern line Lee. The Wm82 assembly improves the prior published assembly, and the Lee and G. soja assemblies are new for these accessions. Comparisons among the three accessions show generally high structural conservation, but nucleotide difference of 1.7 single-nucleotide polymorphisms (snps) per kb between Wm82 and Lee, and 4.7 snps per kb between these lines and G. soja. snp distributions and comparisons with genotypes of the Lee and Wm82 parents highlight patterns of introgression and haplotype structure. Comparisons against the US germplasm collection show placement of the sequenced accessions relative to global soybean diversity. Analysis of a pan-gene collection shows generally high conservation, with variation occurring primarily in genomically clustered gene families. We found approximately 40-42 inversions per chromosome between either Lee or Wm82v4 and G. soja, and approximately 32 inversions per chromosome between Wm82 and Lee. We also investigated five domestication loci. For each locus, we found two different alleles with functional differences between G. soja and the two domesticated accessions. The genome assemblies for multiple cultivated accessions and for the closest wild ancestor of soybean provides a valuable set of resources for identifying causal variants that underlie traits for the domestication and improvement of soybean, serving as a basis for future research and crop improvement efforts for this important crop species.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Association of Diet and Antimicrobial Resistance in Healthy U.S. Adults

Antimicrobial resistance (AMR) represents a significant source of morbidity and mortality worldwide, with expectations that AMR-associated consequences will continue to worsen throughout the coming decades. Since resistance to antibiotics is encoded in the microbiome, interventions aimed at altering the taxonomic composition of the gut might allow us to prophylactically engineer microbiomes that harbor fewer antibiotic resistant genes (ARGs). Diet is one method of intervention, and yet little is known about the association between diet and antimicrobial resistance. To address this knowledge gap, we examined diet using the food frequency questionnaire (FFQ; habitual diet) and 24-h dietary recalls (Automated Self-Administered 24-h [ASA24 ® ] tool) coupled with an analysis of the microbiome using shotgun metagenome sequencing in 290 healthy adult participants of the United States Department of Agriculture (USDA) Nutritional Phenotyping Study. We found that aminoglycosides were the most abundant and prevalent mechanism of AMR in these healthy adults and that aminoglycoside-O-phosphotransferases (aph3-dprime) correlated negatively with total calories and soluble fiber intake. Individuals in the lowest quartile of ARGs (low-ARG) consumed significantly more fiber in their diets than medium- and high-ARG individuals, which was concomitant with increased abundances of obligate anaerobes, especially from the family Clostridiaceae, in their gut microbiota. Finally, we applied machine learning to examine 387 dietary, physiological, and lifestyle features for associations with antimicrobial resistance, finding that increased phylogenetic diversity of diet was associated with low-ARG individuals. These data suggest diet may be a potential method for reducing the burden of AMR.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2018

Reevaluating the Salty Divide: Phylogenetic Specificity of Transitions between Marine and Freshwater Systems

Marine and freshwater microbial communities are phylogenetically distinct, and transitions between habitat types are thought to be infrequent. We compared the phylogenetic diversity of marine and freshwater microorganisms and identified specific lineages exhibiting notably high or low similarity between marine and freshwater ecosystems using a meta-analysis of 16S rRNA gene tag-sequencing data sets. As expected, marine and freshwater microbial communities differed in the relative abundance of major phyla and contained habitat-specific lineages. At the same time, and contrary to expectations, many shared taxa were observed in both habitats. Based on several metrics, we found that Gammaproteobacteria, Alphaproteobacteria, Bacteroidetes, and Betaproteobacteria contained the highest number of closely related marine and freshwater sequences, suggesting comparatively recent habitat transitions in these groups. Using the abundant alphaproteobacterial group SAR11 as an example, we found evidence that new lineages, beyond the recognized LD12 clade, are detected in freshwater at low but reproducible abundances; this evidence extends beyond the 16S rRNA locus to core genes throughout the genome. Our results suggest that shared taxa are numerous, but tend to occur sporadically and at low relative abundance in one habitat type, leading to an underestimation of transition frequency between marine and freshwater habitats. Rare taxa with abundances near or below detection, including lineages that appear to have crossed the salty divide relatively recently, may possess adaptations enabling them to exploit opportunities for niche expansion when environments are disturbed or conditions change.

16S rRNA↗