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At least 145 records · Page 8

Identification of the core set of carbon-associated genes in a bioenergy grassland soil

Despite the central role of soil microbial communities in global carbon (C) cycling, little is known about soil microbial community structure and even less about their metabolic pathways. Efforts to characterize soil communities often focus on identifying differences in gene content across environmental gradients, but an alternative question is what genes are similar in soils. These genes may indicate critical species or potential functions that are required in all soils. Here we identified the “core” set of C cycling sequences widely present in multiple soil metagenomes from a fertilized prairie (FP). Of 226,887 sequences associated with known enzymes involved in the synthesis, metabolism, and transport of carbohydrates, 843 were identified to be consistently prevalent across four replicate soil metagenomes. This core metagenome was functionally and taxonomically diverse, representing five enzyme classes and 99 enzyme families within the CAZy database. Though it only comprised 0.4% of all CAZy-associated genes identified in FP metagenomes, the core was found to be comprised of functions similar to those within cumulative soils. The FP CAZy-associated core sequences were present in multiple publicly available soil metagenomes and most similar to soils sharing geographic proximity. In soil ecosystems, where high diversity remains a key challenge for metagenomic investigations, these core genes represent a subset of critical functions necessary for carbohydrate metabolism, which can be targeted to evaluate important C fluxes in these and other similar soils.

Howe, Adina↗

Differential CLE peptide perception by plant receptors implicated from structural and functional analyses of TDIF-TDR interactions

Tracheary Element Differentiation Inhibitory Factor (TDIF) belongs to the family of post-translationally modified CLE (CLAVATA3/embryo surrounding region (ESR)-related) peptide hormones that control root growth and define the delicate balance between stem cell proliferation and differentiation in SAM (shoot apical meristem) or RAM (root apical meristem). In Arabidopsis, Tracheary Element Differentiation Inhibitory Factor Receptor (TDR) and its ligand TDIF signaling pathway is involved in the regulation of procambial cell proliferation and inhibiting its differentiation into xylem cells. Here we present the crystal structures of the extracellular domains (ECD) of TDR alone and in complex with its ligand TDIF resolved at 2.65 Åand 2.75 Å respectively. These structures provide insights about the ligand perception and specific interactions between the CLE peptides and their cognate receptors. Our in vitro biochemical studies indicate that the interactions between the ligands and the receptors at the C-terminal anchoring site provide conserved binding. While the binding interactions occurring at the N-terminal anchoring site dictate differential binding specificities between different ligands and receptors. Our studies will open different unknown avenues of TDR-TDIF signaling pathways that will enhance our knowledge in this field highlighting the receptor ligand interaction, receptor activation, signaling network, modes of action and will serve as a structure function relationship model between the ligand and the receptor for various similar leucine-rich repeat receptor-like kinases (LRR-RLKs).

59 BASIC BIOLOGICAL SCIENCES↗

A fast comparative genome browser for diverse bacteria and archaea

Genome sequencing has revealed an incredible diversity of bacteria and archaea, but there are no fast and convenient tools for browsing across these genomes. It is cumbersome to view the prevalence of homologs for a protein of interest, or the gene neighborhoods of those homologs, across the diversity of the prokaryotes. We developed a web-based tool, fast . genomics , that uses two strategies to support fast browsing across the diversity of prokaryotes. First, the database of genomes is split up. The main database contains one representative from each of the 6,377 genera that have a high-quality genome, and additional databases for each taxonomic order contain up to 10 representatives of each species. Second, homologs of proteins of interest are identified quickly by using accelerated searches, usually in a few seconds. Once homologs are identified, fast . genomics can quickly show their prevalence across taxa, view their neighboring genes, or compare the prevalence of two different proteins. Fast . genomics is available at https://fast.genomics.lbl.gov .

59 BASIC BIOLOGICAL SCIENCES↗

First report of Seville root-knot nematode, Meloidogyne hispanica (Nematoda: Meloidogynidae) in the USA and North America

A high number of second stage juveniles of the root-knot nematode were recovered from soil samples collected from a corn field, located in Pickens County, South Carolina, USA in 2019. Extracted nematodes were examined morphologically and molecularly for species identification which indicated that the specimens of root knot juveniles were Meloidogyne hispanica. The morphological examination and morphometric details from second-stage juveniles were consistent with the original description and redescriptions of this species. The ITS rRNA, D2-D3 expansion segments of 28S rRNA, intergenic COII-16S region, nad5 and COI gene sequences were obtained from the South Carolina population of M. hispanica. Phylogenetic analysis of the intergenic COII-16S region of mtDNA gene sequence alignment using statistical parsimony showed that the South Carolina population clustered with Meloidogyne hispanica from Portugal and Australia. To our best knowledge, this finding represents the first report of Meloidogyne hispanica in the USA and North America.

59 BASIC BIOLOGICAL SCIENCES↗

Final Report - Phylogenomic tools and web resources for the Systems Biology Knowledgebase

The major advance during this last reporting period (8/15/12 to present) is our release of data on the PhyloFacts website: phylogenetic trees, multiple sequence alignments and other data for protein families are now available for download from http://phylogenomics.berkeley.edu/data/. This project as a whole aimed to develop high-throughput functional annotation systems that exploit information from protein 3D structure and evolution to provide highly precise inferences of various aspects of gene function, including molecular function, biological process, pathway association, Pfam domains, cellular localization and so on. We accomplished these aims by developing and testing different systems on a database of protein family trees: the PhyloFacts Phylogenomic Encyclopedia (at http://phylogenomics.berkeley.edu/phylofacts/ ).

60 APPLIED LIFE SCIENCES↗

pH Homeostasis and Sodium Ion Pumping by Multiple Resistance and pH Antiporters in Pyrococcus furiosus

Multiple Resistance and pH (Mrp) antiporters are seven-subunit complexes that couple transport of ions across the membrane in response to a proton motive force (PMF) and have various physiological roles, including sodium ion sensing and pH homeostasis. The hyperthermophilic archaeon Pyrococcus furiosus contains three copies of Mrp encoding genes in its genome. Two are found as integral components of two respiratory complexes, membrane bound hydrogenase (MBH) and the membrane bound sulfane sulfur reductase (MBS) that couple redox activity to sodium translocation, while the third copy is a stand-alone Mrp. Sequence alignments show that this Mrp does not contain an energy-input (PMF) module but contains all other predicted functional Mrp domains. The P. furiosus Mrp deletion strain exhibits no significant changes in optimal pH or sodium ion concentration for growth but is more sensitive to medium acidification during growth. Cell suspension hydrogen gas production assays using the deletion strain show that this Mrp uses sodium as the coupling ion. Mrp likely maintains cytoplasmic pH by exchanging protons inside the cell for extracellular sodium ions. Deletion of the MBH sodium-translocating module demonstrates that hydrogen gas production is uncoupled from ion pumping and provides insights into the evolution of this Mrp-containing respiratory complex.

59 BASIC BIOLOGICAL SCIENCES↗

DeepComplex: A Web Server of Predicting Protein Complex Structures by Deep Learning Inter-chain Contact Prediction and Distance-Based Modelling

Proteins interact to form complexes. Predicting the quaternary structure of protein complexes is useful for protein function analysis, protein engineering, and drug design. However, few user-friendly tools leveraging the latest deep learning technology for inter-chain contact prediction and the distance-based modelling to predict protein quaternary structures are available. To address this gap, we develop DeepComplex, a web server for predicting structures of dimeric protein complexes. It uses deep learning to predict inter-chain contacts in a homodimer or heterodimer. The predicted contacts are then used to construct a quaternary structure of the dimer by the distance-based modelling, which can be interactively viewed and analysed. The web server is freely accessible and requires no registration. It can be easily used by providing a job name and an email address along with the tertiary structure for one chain of a homodimer or two chains of a heterodimer. The output webpage provides the multiple sequence alignment, predicted inter-chain residue-residue contact map, and predicted quaternary structure of the dimer.

59 BASIC BIOLOGICAL SCIENCES↗

Homology Modeling and Analysis of Structure Predictions of the Bovine Rhinitis B Virus RNA Dependent RNA Polymerase (RdRp)

Bovine Rhinitis B Virus (BRBV) is a picornavirus responsible for mild respiratory infection of cattle. It is probably the least characterized among the aphthoviruses. BRBV is the closest relative known to Foot and Mouth Disease virus (FMDV) with a ~43% identical polyprotein sequence and as much as 67% identical sequence for the RNA dependent RNA polymerase (RdRp), which is also known as 3D polymerase (3D pol ). In the present study we carried out phylogenetic analysis, structure based sequence alignment and prediction of three-dimensional structure of BRBV 3D pol using a combination of different computational tools. Model structures of BRBV 3D pol were verified for their stereochemical quality and accuracy. The BRBV 3D pol structure predicted by SWISS-MODEL exhibited highest scores in terms of stereochemical quality and accuracy, which were in the range of 2Å resolution crystal structures. The active site, nucleic acid binding site and overall structure were observed to be in agreement with the crystal structure of unliganded as well as template/primer (T/P), nucleotide tri-phosphate (NTP) and pyrophosphate (PPi) bound FMDV 3D pol (PDB, 1U09 and 2E9Z). The closest proximity of BRBV and FMDV 3D pol as compared to human rhinovirus type 16 (HRV-16) and rabbit hemorrhagic disease virus (RHDV) 3D pols is also substantiated by phylogeny analysis and root-mean square deviation (RMSD) between C-α traces of the polymerase structures. The absence of positively charged α-helix at C terminal, significant differences in non-covalent interactions especially salt bridges and CH-pi interactions around T/P channel of BRBV 3D pol compared to FMDV 3D pol , indicate that despite a very high homology to FMDV 3D pol , BRBV 3D pol may adopt a different mechanism for handling its substrates and adapting to physiological requirements. Our findings will be valuable in the design of structure-function interventions and identification of molecular targets for drug design applicable to Aphthovirus RdRps.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

Zam Is a Redox-Regulated Member of the RNB-Family Required for Optimal Photosynthesis in Cyanobacteria

The zam gene mediating resistance to acetazolamide in cyanobacteria was discovered thirty years ago during a drug tolerance screen. We use phylogenetics to show that Zam proteins are distributed across cyanobacteria and that they form their own unique clade of the ribonuclease II/R (RNB) family. Despite being RNB family members, multiple sequence alignments reveal that Zam proteins lack conservation and exhibit extreme degeneracy in the canonical active site—raising questions about their cellular function(s). Several known phenotypes arise from the deletion of zam, including drug resistance, slower growth, and altered pigmentation. Using room-temperature and low-temperature fluorescence and absorption spectroscopy, we show that deletion of zam results in decreased phycocyanin synthesis rates, altered PSI:PSII ratios, and an increase in coupling between the phycobilisome and PSII. Conserved cysteines within Zam are identified and assayed for function using in vitro and in vivo methods. We show that these cysteines are essential for Zam function, with mutation of either residue to serine causing phenotypes identical to the deletion of Zam. Redox regulation of Zam activity based on the reversible oxidation-reduction of a disulfide bond involving these cysteine residues could provide a mechanism to integrate the ‘central dogma’ with photosynthesis in cyanobacteria.

59 BASIC BIOLOGICAL SCIENCES↗

The 1.6-Å crystal structure of the class of chaperones represented by Escherichia coli Hsp31 reveals a putative catalytic triad

Heat shock proteins (Hsps) play essential protective roles under stress conditions by preventing the formation of protein aggregates and degrading misfolded proteins. EcHsp31, the yedU (hchA) gene product, is a representative member of a family of chaperones that alleviates protein misfolding by interacting with early unfolding intermediates. The 1.6-{angstrom} crystal structure of the EcHsp31 dimer reveals a system of hydrophobic patches, canyons, and grooves, which may stabilize partially unfolded substrate. The presence of a well conserved, yet buried, triad in each two-domain subunit suggests a still unproven hydrolytic function of the protein. A flexible extended linker between the A and P domains may play a role in conformational flexibility and substrate binding. The {alpha}-{beta} sandwich of the EcHsp31 monomer shows structural similarity to PhPI, a protease belonging to the DJ-1 superfamily. The structure-guided sequence alignment indicates that Hsp31 homologs can be divided in three classes based on variations in the P domain that dramatically affect both oligomerization and catalytic triad formation.

36 MATERIALS SCIENCE↗

A Comparative Biochemical and Structural Analysis of the Intracellular chorismate mutase (Rv0948c) from Mycobacterium tuberculosis H(37)R(v) and the Secreted chorismate mutase (y2828) from Yersinia pestis

The Rv0948c gene from Mycobacterium tuberculosis H{sub 37}R{sub v} encodes a 90 amino acid protein as the natural gene product with chorismate mutase (CM) activity. The protein, 90-MtCM, exhibits Michaelis-Menten kinetics with a k{sub cat} of 5.5 {+-} 0.2 s{sup -1} and a K{sub m} of 1500 {+-} 100 {micro}m at 37 C and pH 7.5. The 2.0 {angstrom} X-ray structure shows that 90-MtCM is an all {alpha}-helical homodimer (Protein Data Bank ID: 2QBV) with the topology of Escherichia coli CM (EcCM), and that both protomers contribute to each catalytic site. Superimposition onto the structure of EcCM and the sequence alignment shows that the C-terminus helix 3 is shortened. The absence of two residues in the active site of 90-MtCM corresponding to Ser84 and Gln88 of EcCM appears to be one reason for the low k{sub cat}. Hence, 90-MtCM belongs to a subfamily of {alpha}-helical AroQ CMs termed AroQ{delta}. The CM gene (y2828) from Yersinia pestis encodes a 186 amino acid protein with an N-terminal signal peptide that directs the protein to the periplasm. The mature protein, *YpCM, exhibits Michaelis-Menten kinetics with a k{sub cat} of 70 {+-} 5 s{sup -1} and Km of 500 {+-} 50 {micro}m at 37 C and pH 7.5. The 2.1 {angstrom} X-ray structure shows that *YpCM is an all {alpha}-helical protein, and functions as a homodimer, and that each protomer has an independent catalytic unit (Protein Data Bank ID: 2GBB). *YpCM belongs to the AroQ{gamma} class of CMs, and is similar to the secreted CM (Rv1885c, *MtCM) from M. tuberculosis.

59 BASIC BIOLOGICAL SCIENCES↗

Structure and Mechanism of the S Component of a Bacterial ECF Transporter

The energy-coupling factor (ECF) transporters, responsible for vitamin uptake in prokaryotes, are a unique family of membrane transporters. Each ECF transporter contains a membrane-embedded, substrate-binding protein (known as the S component), an energy-coupling module that comprises two ATP-binding proteins (known as the A and A' components) and a transmembrane protein (known as the T component). The structure and transport mechanism of the ECF family remain unknown. Here we report the crystal structure of RibU, the S component of the ECF-type riboflavin transporter from Staphylococcus aureus at 3.6-{angstrom} resolution. RibU contains six transmembrane segments, adopts a previously unreported transporter fold and contains a riboflavin molecule bound to the L1 loop and the periplasmic portion of transmembrane segments 4-6. Structural analysis reveals the essential ligand-binding residues, identifies the putative transport path and, with sequence alignment, uncovers conserved structural features and suggests potential mechanisms of action among the ECF transporters.

59 BASIC BIOLOGICAL SCIENCES↗

Pairwise alignment for very long nucleic acid sequences

Highlights: • The PAAVLS algorithm can be employed for alignment of very long sequences. • The PAAVLS algorithm largely reduces memory demand as the Smith–Waterman algorithm. • The running times of the PAAVLS and Smith–Waterman algorithms are comparable. Sequence alignment is one of the fundamental problems in computational biology and has numerous applications. The Smith–Waterman algorithm generates optimal local alignment for pairwise alignment task and has become a standard algorithm in its field. However, the current version of the Smith–Waterman algorithm demands a significant amount of memory and is not suitable for alignment of very long sequences. On the hand, the recent DNA sequencing technologies have produced vast amounts of biological sequences. Some nucleic acid sequences are very long and cannot employ the Smith–Waterman algorithm. To this end, this study proposes the PAAVLS algorithm that follows the dynamic programming technique employed by the Smith–Waterman algorithm and largely reduces the demand of memory. The proposed PAAVLS algorithm can be employed for alignment of very long sequences, i.e., sequences contain more than 100,000,000 nucleotides, on a personal computer. Additionally, the running time of the proposed PAAVLS algorithm is comparable with the running time of the standard Smith–Waterman algorithm.

60 APPLIED LIFE SCIENCES↗

Deciphering Natural Allelic Variation in Switchgrass for Biomass Yield and Quality Using a Nested Association Mapping Population

Switchgrass (Panicum virgatum L.) is a C4 grass with high biomass yield potential and a model species for bioenergy feedstock development. Understanding the genetic basis of quantitative traits is essential to facilitate genome-enabled breeding programs. The nested association mapping (NAM) analysis combines the best features of both bi-parental and association analyses and can provide high power and high resolution in QTL detection and will ensure significant improvements in biomass yield and quality. To develop a NAM population of switchgrass, 15 highly diverse genotypes with specific characteristics were selected from a diversity panel and crossed to a recurrent parent, AP13, a genotype selected for whole genome sequencing and parent of a mapping population. Ten genotypes from each of the 15 F1 families were then chain crossed. Progenies form each family were randomly selected to develop the NAM population. The switchgrass NAM population consists of a total of 2000 genotypes from 15 families. All the progenies, founder parents, F1 parents (n=2350) were evaluated in replicated field trials at Ardmore, OK and Knoxville, TN. Phenotypic data on plant height, tillering ability, regrowth, flowering time, and biomass yield were collected. Dried biomass samples were also analyzed using prediction equations of NIRS at the Noble Foundation and for lignin content, S/G ratio, and sugar release characteristics at the NREL. Genomic shotgun sequencing of 15 switchgrass NAM founder parental genomes at JGI produced 28-66 Gb high-quality sequence data. Alignment of these sequences with the reference genome, AP13 (v3.0), revealed that up to 99% of the genomic sequences mapped to the reference genome. A total of 2,149 individuals from NAM populations were sequenced by exome capture and two sets of 15 SNP matrices (one for each family) were generated. QTL associated with important traits have been identified and verified in breeding populations. The QTL detected and their associated markers can be used in molecular breeding programs to facilitate development of improved switchgrass cultivars for biofuel production.

09 BIOMASS FUELS↗

Enzyme sequence similarity improves the reaction alignment method for cross-species pathway comparison

Pathway-based information has become an important source of information for both establishing evolutionary relationships and understanding the mode of action of a chemical or pharmaceutical among species. Cross-species comparison of pathways can address two broad questions: comparison in order to inform evolutionary relationships and to extrapolate species differences used in a number of different applications including drug and toxicity testing. Cross-species comparison of metabolic pathways is complex as there are multiple features of a pathway that can be modeled and compared. Among the various methods that have been proposed, reaction alignment has emerged as the most successful at predicting phylogenetic relationships based on NCBI taxonomy. We propose an improvement of the reaction alignment method by accounting for sequence similarity in addition to reaction alignment method. Using nine species, including human and some model organisms and test species, we evaluate the standard and improved comparison methods by analyzing glycolysis and citrate cycle pathways conservation. In addition, we demonstrate how organism comparison can be conducted by accounting for the cumulative information retrieved from nine pathways in central metabolism as well as a more complete study involving 36 pathways common in all nine species. Our results indicate that reaction alignment with enzyme sequence similarity results in a more accurate representation of pathway specific cross-species similarities and differences based on NCBI taxonomy.

60 APPLIED LIFE SCIENCES↗