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At least 127 records · Page 7

The MG-RAST Metagenomics Database and Portal in 2015

MG-RAST (http://metagenomics.anl.gov) is an opensubmission data portal for processing, analyzing, sharing and disseminating metagenomic datasets. Currently, the system hosts over 200 000 datasets and is continuously updated. The volume of submissions has increased 4-fold over the past 24 months, now averaging 4 terabasepairs per month. In addition to several new features, we report changes to the analysis workflow and the technologies used to scale the pipeline up to the required throughput levels. Lastly, to show possible uses for the data from MG-RAST, we present several examples integrating data and analyses from MG-RAST into popular third-party analysis tools or sequence alignment tools.

96 KNOWLEDGE MANAGEMENT AND PRESERVATION↗

Bioinformatics and 3D Structural Analysis of the Coronavirus Main Protease Active Site Diversity

Coronaviruses (Coronaviridae) such as SARS‐CoV‐2 (severe acute respiratory syndrome coronavirus) and MERS‐CoV (Middle East respiratory syndrome coronavirus) have been the source of recent outbreaks and global health concerns. While vaccines have been essential for controlling the SARS‐CoV‐2 (COVID‐19) pandemic, it is uncertain whether they will be effective against future coronavirus strains. Therefore, identification or design of a broad‐spectrum drug that targets highly conserved regions of the main protease of multiple coronavirus strains is essential in the long term. As part of a virtual summer research experience with the RCSB PDB, bioinformatics tools were employed to predict and construct 3D models of the coronavirus main protease (MPro) using SARS‐CoV‐2 as the template, with a focus on mutational trends and active sites. This study focused on the active sites of MPro, a cysteine protease essential for viral assembly and replication. Sequence alignments and structure modeling of MPro structures has identified conserved regions across multiple coronavirus strains. Inhibition of MPro halts coronavirus replication, making it an ideal drug target, and studies of MPro may foster and accelerate the discovery of high affinity broad‐spectrum drugs.

Wu Wu, Amy↗

The structure of cytomegalovirus immune modulator UL141 highlights structural Ig-fold versatility for receptor binding

The crystal structure of Human cytomegalovirus immune modulator UL141 was solved at 3.25 Å resolution. Here, a detailed analysis of its intimate dimerization interface and the biophysical properties of its receptor (TRAIL-R2 and CD155) binding interactions are presented. Natural killer (NK) cells are critical components of the innate immune system as they rapidly detect and destroy infected cells. To avoid immune recognition and to allow long-term persistence in the host, Human cytomegalovirus (HCMV) has evolved a number of genes to evade or inhibit immune effector pathways. In particular, UL141 can inhibit cell-surface expression of both the NK cell-activating ligand CD155 as well as the TRAIL death receptors (TRAIL-R1 and TRAIL-R2). The crystal structure of unliganded HCMV UL141 refined to 3.25 Å resolution allowed analysis of its head-to-tail dimerization interface. A ‘dimerization-deficient’ mutant of UL141 (ddUL141) was further designed, which retained the ability to bind to TRAIL-R2 or CD155 while losing the ability to cross-link two receptor monomers. Structural comparison of unliganded UL141 with UL141 bound to TRAIL-R2 further identified a mobile loop that makes intimate contacts with TRAIL-R2 upon receptor engagement. Superposition of the Ig-like domain of UL141 on the CD155 ligand T-cell immunoreceptor with Ig and ITIM domains (TIGIT) revealed that UL141 can potentially engage CD155 similar to TIGIT by using the C′C′′ and GF loops. Further mutations in the TIGIT binding site of CD155 (Q63R and F128R) abrogated UL141 binding, suggesting that the Ig-like domain of UL141 is a viral mimic of TIGIT, as it targets the same binding site on CD155 using similar ‘lock-and-key’ interactions. Sequence alignment of the UL141 gene and its orthologues also showed conservation in this highly hydrophobic (L/A)X{sub 6}G ‘lock’ motif for CD155 binding as well as conservation of the TRAIL-R2 binding patches, suggesting that these host–receptor interactions are evolutionary conserved.

75 CONDENSED MATTER PHYSICS, SUPERCONDUCTIVITY AND↗

Structure of the GH76 α-mannanase homolog, BT2949, from the gut symbiont Bacteroides thetaiotaomicron

A high-resolution structure of a noncanonical α-mannanase relevant to human health and nutrition has been solved via heavy-atom phasing of a selenomethionine derivative. The large bowel microbiota, a complex ecosystem resident within the gastrointestinal tract of all human beings and large mammals, functions as an essential, nonsomatic metabolic organ, hydrolysing complex dietary polysaccharides and modulating the host immune system to adequately tolerate ingested antigens. A significant member of this community, Bacteroides thetaiotaomicron, has evolved a complex system for sensing and processing a wide variety of natural glycoproducts in such a way as to provide maximum benefit to itself, the wider microbial community and the host. The immense ability of B. thetaiotaomicron as a ‘glycan specialist’ resides in its enormous array of carbohydrate-active enzymes, many of which are arranged into polysaccharide-utilization loci (PULs) that are able to degrade sugar polymers that are often inaccessible to other gut residents, notably α-mannan. The B. thetaiotaomicron genome encodes ten putative α-mannanases spread across various PULs; however, little is known about the activity of these enzymes or the wider implications of α-mannan metabolism for the health of both the microbiota and the host. In this study, SAD phasing of a selenomethionine derivative has been used to investigate the structure of one such B. thetaiotaomicron enzyme, BT2949, which belongs to the GH76 family of α-mannanases. BT2949 presents a classical (α/α){sub 6}-barrel structure comprising a large extended surface cleft common to other GH76 family members. Analysis of the structure in conjunction with sequence alignments reveals the likely location of the catalytic active site of this noncanonical GH76.

75 CONDENSED MATTER PHYSICS, SUPERCONDUCTIVITY AND↗

Crystal structures of the naturally fused CS and cytochrome b 5 reductase ( b 5 R) domains of Ncb5or reveal an expanded CS fold, extensive CS– b 5 R interactions and productive binding of the NAD(P) + nicotinamide ring

Ncb5or (NADH-cytochrome b 5 oxidoreductase), a cytosolic ferric reductase implicated in diabetes and neurological diseases, comprises three distinct domains, cytochrome b 5 ( b 5 ) and cytochrome b 5 reductase ( b 5 R) domains separated by a CHORD–Sgt1 (CS) domain, and a novel 50-residue N-terminal region. Understanding how interdomain interactions in Ncb5or facilitate the shuttling of electrons from NAD(P)H to heme, and how the process compares with the microsomal b 5 (Cyb5A) and b 5 R (Cyb5R3) system, is of interest. A high-resolution structure of the b 5 domain (PDB entry 3lf5) has previously been reported, which exhibits substantial differences in comparison to Cyb5A. The structural characterization of a construct comprising the naturally fused CS and b 5 R domains with bound FAD and NAD + (PDB entry 6mv1) or NADP + (PDB entry 6mv2) is now reported. The structures reveal that the linker between the CS and b 5 R cores is more ordered than predicted, with much of it extending the β-sandwich motif of the CS domain. This limits the flexibility between the two domains, which recognize one another via a short β-sheet motif and a network of conserved side-chain hydrogen bonds, salt bridges and cation–Π interactions. Notable differences in FAD–protein interactions in Ncb5or and Cyb5R3 provide insight into the selectivity for docking of their respective b 5 redox partners. The structures also afford a structural explanation for the unusual ability of Ncb5or to utilize both NADH and NADPH, and represent the first examples of native, fully oxidized b 5 R family members in which the nicotinamide ring of NAD(P) + resides in the active site. Finally, the structures, together with sequence alignments, show that the b 5 R domain is more closely related to single-domain Cyb5R proteins from plants, fungi and some protists than to Cyb5R3 from animals.

Benson, David R.↗

Defining Electron Bifurcation in the Electron-Transferring Flavoprotein Family

Electron bifurcation is the coupling of exergonic and endergonic redox reactions to simultaneously generate (or utilize) low- and high-potential electrons. It is the third recognized form of energy conservation in biology and was recently described for select electron-transferring flavoproteins (Etfs). Etfs are flavin-containing heterodimers best known for donating electrons derived from fatty acid and amino acid oxidation to an electron transfer respiratory chain via Etf-quinone oxidoreductase. Canonical examples contain a flavin adenine dinucleotide (FAD) that is involved in electron transfer, as well as a non-redox-active AMP. However, Etfs demonstrated to bifurcate electrons contain a second FAD in place of the AMP. To expand our understanding of the functional variety and metabolic significance of Etfs and to identify amino acid sequence motifs that potentially enable electron bifurcation, we compiled 1,314 Etf protein sequences from genome sequence databases and subjected them to informatic and structural analyses. Etfs were identified in diverse archaea and bacteria, and they clustered into five distinct well-supported groups, based on their amino acid sequences. Gene neighborhood analyses indicated that these Etf group designations largely correspond to putative differences in functionality. Etfs with the demonstrated ability to bifurcate were found to form one group, suggesting that distinct conserved amino acid sequence motifs enable this capability. Indeed, structural modeling and sequence alignments revealed that identifying residues occur in the NADH- and FAD-binding regions of bifurcating Etfs. Altogether, a new classification scheme for Etf proteins that delineates putative bifurcating versus nonbifurcating members is presented and suggests that Etf-mediated bifurcation is associated with surprisingly diverse enzymes.

59 BASIC BIOLOGICAL SCIENCES↗

VP24-Karyopherin Alpha Binding Affinities Differ between Ebolavirus Species, Influencing Interferon Inhibition and VP24 Stability

Zaire ebolavirus (EBOV), Bundibugyo ebolavirus (BDBV), and Reston ebolavirus (RESTV) belong to the same genus but exhibit different virulence properties. VP24 protein, a structural protein present in all family members, blocks interferon (IFN) signaling and likely contributes to virulence. Inhibition of IFN signaling by EBOV VP24 (eVP24) involves its interaction with the NPI-1 subfamily of karyopherin alpha (KPNA) nuclear transporters. Here, we evaluated eVP24, BDBV VP24 (bVP24), and RESTV VP24 (rVP24) interactions with three NPI-1 subfamily KPNAs (KPNA1, KPNA5, and KPNA6). Using purified proteins, we demonstrated that each VP24 binds to each of the three NPI-1 KPNAs. bVP24, however, exhibited approximately 10-fold-lower KPNA binding affinity than either eVP24 or rVP24. Cell-based assays also indicate that bVP24 exhibits decreased KPNA interaction, decreased suppression of IFN induced gene expression, and a decreased half-life in transfected cells compared to eVP24 or rVP24. Amino acid sequence alignments between bVP24 and eVP24 also identified residues within and surrounding the previously defined eVP24-KPNA5 binding interface that decrease eVP24-KPNA affinity or bVP24-KPNA affinity. VP24 mutations that lead to reduced KPNA binding affinity also decrease IFN inhibition and shorten VP24 half-lives. Here, these data identify novel functional differences in VP24-KPNA interaction and reveal a novel impact of the VP24-KPNA interaction on VP24 stability. The interaction of Ebola virus (EBOV) VP24 protein with host karyopherin alpha (KPNA) proteins blocks type I interferon (IFN) signaling, which is a central component of the host innate immune response to viral infection. Here, we quantitatively compared the interactions of VP24 proteins from EBOV and two members of the Ebolavirus genus, Bundibugyo virus (BDBV) and Reston virus (RESTV). The data reveal lower binding affinity of the BDBV VP24 (bVP24) for KPNAs and demonstrate that the interaction with KPNA modulates inhibition of IFN signaling and VP24 stability. The effect of KPNA interaction on VP24 stability is a novel functional consequence of this virus-host interaction, and the differences identified between viral species may contribute to differences in pathogenesis.

60 APPLIED LIFE SCIENCES↗

Structure-Based Analysis of the Interaction between the Simian Virus 40 T-Antigen Origin Binding Domain and Single-Stranded DNA

The origin-binding domain (OBD) of simian virus 40 (SV40) large T-antigen (T-Ag) is essential for many of T-Ag's interactions with DNA. Nevertheless, many important issues related to DNA binding, for example, how single-stranded DNA (ssDNA) transits along the T-Ag OBD, have yet to be established. Therefore, X-ray crystallography was used to determine the costructure of the T-Ag OBD bound to DNA substrates such as the single-stranded region of a forked oligonucleotide. A second structure of the T-Ag OBD crystallized in the presence of poly(dT){sub 12} is also reported. To test the conclusions derived from these structures, residues identified as being involved in binding to ssDNA by crystallography or by an earlier nuclear magnetic resonance study were mutated, and their binding to DNA was characterized via fluorescence anisotropy. In addition, these mutations were introduced into full-length T-Ag, and these mutants were tested for their ability to support replication. When considered in terms of additional homology-based sequence alignments, our studies refine our understanding of how the T-Ag OBDs encoded by the polyomavirus family interact with ssDNA, a critical step during the initiation of DNA replication.

59 BASIC BIOLOGICAL SCIENCES↗

biodataganache/SIEVE-Ub

Code and data for the SIEVE-Ub prediction algorithm for prediction of E3 ubiquitin ligase mimics The SIEVEUb approach is a novel approach to determination of sequence similarity that retains flexibility in sequence via a reduced amino acid alphabet and does not require sequence alignment, a prohibitive step for many applications.

McDermott, Jason↗

Cache domains that are homologous to, but different from PAS domains comprise the largest superfamily of extracellular sensors in prokaryotes

Cellular receptors usually contain a designated sensory domain that recognizes the signal. Per/Arnt/Sim (PAS) domains are ubiquitous sensors in thousands of species ranging from bacteria to humans. Although PAS domains were described as intracellular sensors, recent structural studies revealed PAS-like domains in extracytoplasmic regions in several transmembrane receptors. However, these structurally defined extracellular PAS-like domains do not match sequence-derived PAS domain models, and thus their distribution across the genomic landscape remains largely unknown. Here we show that structurally defined extracellular PAS-like domains belong to the Cache superfamily, which is homologous to, but distinct from the PAS superfamily. Our newly built computational models enabled identification of Cache domains in tens of thousands of signal transduction proteins including those from important pathogens and model organisms.Moreover, we show that Cache domains comprise the dominant mode of extracellular sensing in prokaryotes.

59 BASIC BIOLOGICAL SCIENCES↗

Machine learning identifies novel signatures of antifungal drug resistance in Saccharomycotina yeasts

Antifungal drug resistance is a major challenge in fungal infection management. Numerous genomic changes are known to contribute to acquired drug resistance in clinical isolates of specific pathogens, but whether they broadly explain natural resistance across entire lineages is unknown. We leveraged genomic, ecological, and phenotypic trait data from naturally sampled strains from nearly all known species in subphylum Saccharomycotina to examine the evolution of resistance to eight antifungal drugs. The phylogenetic distribution of drug resistance varied by drug; fluconazole resistance was widespread, while 5-fluorocytosine resistance was rare, except in Lipomycetales. A random forest algorithm trained on genomic data predicted drug-resistant yeasts with 54–75% accuracy. Fluconazole resistance was consistently predicted with the highest accuracy (75.2%). Furthermore, fluconazole resistance prediction accuracy was similar between models trained on genome-wide variation in the presence and number of InterPro protein annotations across Saccharomycotina (75.2%) and those trained on amino acid sequence alignment data of Erg11, a protein known to be involved in fluconazole resistance (74.3-74.9%). Interestingly, the top Erg11 residues for predicting fluconazole resistance across Saccharomycotina do not overlap with, are not spatially close to, and are less conserved than those previously linked to resistance in clinical isolates of Candida albicans. In silico deep mutational scanning of the C. albicans Erg11 protein reveals that amino acid variants implicated in clinical cases of resistance are almost universally destabilizing while variants in our most informative residues are energetically more neutral, explaining why the latter are much more common than the former in natural populations. Importantly, previous experimental analyses of C. albicans Erg11 have shown that amino acid variation in our most informative residues, despite having never been directly implicated in clinical cases, can directly contribute to resistance. Our results suggest that studies of natural resistance in yeast species never encountered in the clinic will yield a fuller understanding of antifungal drug resistance.

Harrison, Marie-Claire [Vanderbilt Univ., Nashvill↗

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↗

Arrangement of the Clostridium baratii F7 Toxin Gene Cluster with Identification of a σ Factor That Recognizes the Botulinum Toxin Gene Cluster Promoters

Botulinum neurotoxin (BoNT) is the most poisonous substances known and its eight toxin types (A to H) are distinguished by the inability of polyclonal antibodies that neutralize one toxin type to neutralize any of the other seven toxin types. Infant botulism, an intestinal toxemia orphan disease, is the most common form of human botulism in the United States. It results from swallowed spores of Clostridium botulinum (or rarely, neurotoxigenic Clostridium butyricum or Clostridium baratii) that germinate and temporarily colonize the lumen of the large intestine, where, as vegetative cells, they produce botulinum toxin. Botulinum neurotoxin is encoded by the bont gene that is part of a toxin gene cluster that includes several accessory genes. In this paper, we sequenced for the first time the complete botulinum neurotoxin gene cluster of nonproteolytic C. baratii type F7. Like the type E and the nonproteolytic type F6 botulinum toxin gene clusters, the C. baratii type F7 had an orfX toxin gene cluster that lacked the regulatory botR gene which is found in proteolytic C. botulinum strains and codes for an alternative σ factor. In the absence of botR, we identified a putative alternative regulatory gene located upstream of the C. baratii type F7 toxin gene cluster. This putative regulatory gene codes for a predicted σ factor that contains DNA-binding-domain homologues to the DNA-binding domains both of BotR and of other members of the TcdR-related group 5 of the σ 70 family that are involved in the regulation of toxin gene expression in clostridia. We showed that this TcdR-related protein in association with RNA polymerase core enzyme specifically binds to the C. baratii type F7 botulinum toxin gene cluster promoters. Finally, this TcdR-related protein may therefore be involved in regulating the expression of the genes of the botulinum toxin gene cluster in neurotoxigenic C. baratii.

59 BASIC BIOLOGICAL SCIENCES↗

High-throughput analysis of T-DNA location and structure using sequence capture

Agrobacterium-mediated transformation of plants with T-DNA is used both to introduce transgenes and for mutagenesis. Conventional approaches used to identify the genomic location and the structure of the inserted T-DNA are laborious and high-throughput methods using next-generation sequencing are being developed to address these problems. Here, we present a cost-effective approach that uses sequence capture targeted to the T-DNA borders to select genomic DNA fragments containing T-DNA—genome junctions, followed by Illumina sequencing to determine the location and junction structure of T-DNA insertions. Multiple probes can be mixed so that transgenic lines transformed with different T-DNA types can be processed simultaneously, using a simple, index-based pooling approach. We also developed a simple bioinformatic tool to find sequence read pairs that span the junction between the genome and T-DNA or any foreign DNA. We analyzed 29 transgenic lines of Arabidopsis thaliana, each containing inserts from 4 different T-DNA vectors. We determined the location of T-DNA insertions in 22 lines, 4 of which carried multiple insertion sites. Additionally, our analysis uncovered a high frequency of unconventional and complex T-DNA insertions, highlighting the needs for high-throughput methods for T-DNA localization and structural characterization. Transgene insertion events have to be fully characterized prior to use as commercial products. As a result, our method greatly facilitates the first step of this characterization of transgenic plants by providing an efficient screen for the selection of promising lines.

59 BASIC BIOLOGICAL SCIENCES↗

The influence of age and gender on skin-associated microbial communities in urban and rural human populations

Differences in the bacterial community structure associated with 7 skin sites in 71 healthy people over five days showed significant correlations with age, gender, physical skin parameters, and whether participants lived in urban or rural locations in the same city. While body site explained the majority of the variance in bacterial community structure, the composition of the skin-associated bacterial communities were predominantly influenced by whether the participants were living in an urban or rural environment, with a significantly greater relative abundance of Trabulsiella in urban populations. Adults maintained greater overall microbial diversity than adolescents or the elderly, while the intragroup variation among the elderly and rural populations was significantly greater. Lastly, skin-associated bacterial community structure and composition could predict whether a sample came from an urban or a rural resident ~ 5x greater than random.

59 BASIC BIOLOGICAL SCIENCES↗

Comparing residue clusters from thermophilic and mesophilic enzymes reveals adaptive mechanisms

Understanding how proteins adapt to function at high temperatures is important for deciphering the energetics that dictate protein stability and folding. While multiple principles important for thermostability have been identified, we lack a unified understanding of how internal protein structural and chemical environment determine qualitative or quantitative impact of evolutionary mutations. In this work we compare equivalent clusters of spatially neighboring residues between paired thermophilic and mesophilic homologues to evaluate adaptations under the selective pressure of high temperature. We find the residue clusters in thermophilic enzymes generally display improved atomic packing compared to mesophilic enzymes, in agreement with previous research. Unlike residue clusters from mesophilic enzymes, however, thermophilic residue clusters do not have significant cavities. In addition, anchor residues found in many clusters are highly conserved with respect to atomic packing between both thermophilic and mesophilic enzymes. As a result, the improvements in atomic packing observed in thermophilic homologues are not derived from these anchor residues but from neighboring positions, which may serve to expand optimized protein core regions.

09 BIOMASS FUELS↗

Rapid discovery and functional characterization of terpene synthases from four endophytic xylariaceae

Endophytic fungi are ubiquitous plant endosymbionts that establish complex and poorly understood relationships with their host organisms. Many endophytic fungi are known to produce a wide spectrum of volatile organic compounds (VOCs) with potential energy applications, which have been described as "mycodiesel". Many of these mycodiesel hydrocarbons are terpenes, a chemically diverse class of compounds produced by many plants, fungi, and bacteria. Due to their high energy densities, terpenes, such as pinene and bisabolene, are actively being investigated as potential "drop-in" biofuels for replacing diesel and aviation fuel. In this study, we rapidly discovered and characterized 26 terpene synthases (TPSs) derived from four endophytic fungi known to produce mycodiesel hydrocarbons. The TPS genes were expressed in an E. coli strain harboring a heterologous mevalonate pathway designed to enhance terpene production, and their product profiles were determined using Solid Phase Micro-Extraction (SPME) and GC-MS. Lastly, out of the 26 TPS’s profiled, 12 TPS’s were functional, with the majority of them exhibiting both monoterpene and sesquiterpene synthase activity.

59 BASIC BIOLOGICAL SCIENCES↗

A novel analysis method for paired-sample microbial ecology experiments

Many microbial ecology experiments use sequencing data to measure a community s response to an experimental treatment. In a common experimental design, two units, one control and one experimental, are sampled before and after the treatment is applied to the experimental unit. The four resulting samples contain information about the dynamics of organisms that respond to the treatment, but there are no analytical methods designed to extract exactly this type of information from this configuration of samples. Here we present an analytical method specifically designed to visualize and generate hypotheses about microbial community dynamics in experiments that have paired samples and few or no replicates. The method is based on the Poisson lognormal distribution, long studied in macroecology, which we found accurately models the abundance distribution of taxa counts from 16S rRNA surveys. To demonstrate the method s validity and potential, we analyzed an experiment that measured the effect of crude oil on ocean microbial communities in microcosm. Our method identified known oil degraders as well as two clades, Maricurvus and Rhodobacteraceae, that responded to amendment with oil but do not include known oil degraders. Furthermore, our approach is sensitive to organisms that increased in abundance only in the experimental unit but less sensitive to organisms that increased in both control and experimental units, thus mitigating the role of bottle effects .

59 BASIC BIOLOGICAL SCIENCES↗