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At least 271 records · Page 15
All sourcesNASA NTRSDOE OSTI
DOE OSTI2025

A multifunctional sesquiterpene synthase integrates with cytochrome P450s to reinforce the terpenoid defense network in maize

Terpenoids, the largest and most structurally diverse class of plant natural products, play essential roles in maize defense and ecological interactions. In this study, we identified and functionally characterized a sesquiterpenoid-based defense pathway in maize centered on α-santalenoic acid, a pathogen-inducible sesquiterpenoid antibiotic. Using a combination of metabolite-based genome-wide association studies (mGWAS), linkage mapping, and heterologous expression assays, we identified ZmTPS9 as a multiproduct terpene synthase that primarily produces α-santalene and β-bisabolene. Sequence analysis and site-directed mutagenesis revealed that threonine at position 413 is critical for enzyme activity, with its deletion resulting in a complete loss of enzyme activity. The sesquiterpene hydrocarbons produced by ZmTPS9 are further oxidized by three cytochrome P450 monooxygenases, ZmCYP71Z16, ZmCYP71Z18, and ZmCYP71Z19, to yield antimicrobial metabolites including α-santalenoic acid, zealexin D1 (ZD1), and zealexin D2 (ZD2). Together, these findings demonstrate a convergent biosynthetic strategy in maize, where multiproduct terpene synthases and promiscuous P450s collaboratively generate a flexible and robust terpenoid defense network.

a-santalenoic acid↗
DOE OSTI2019

Genetic and structural insights into broad neutralization of hepatitis C virus by human V H 1-69 antibodies

An effective vaccine to the antigenically diverse hepatitis C virus (HCV) must target conserved immune epitopes. Here, we investigate cross-neutralization of HCV genotypes by broadly neutralizing antibodies (bNAbs) encoded by the relatively abundant human gene family V H 1-69. We have deciphered the molecular requirements for cross-neutralization by this unique class of human antibodies from crystal structures of HCV E2 in complex with bNAbs. An unusually high binding affinity is found for germ line–reverted versions of V H 1-69 precursor antibodies, and neutralization breadth is acquired during affinity maturation. Deep sequencing analysis of an HCV-immune B cell repertoire further demonstrates the importance of the V H 1-69 gene family in the generation of HCV bNAbs. This study therefore provides critical insights into immune recognition of HCV with important implications for rational vaccine design.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2025

Elucidation of a bacterial pathway for catabolism of the β–β-linked dilignol pinoresinol

Monolignol-derived dimers containing β–β linkages are synthesized by vascular plants and can be released during lignin depolymerization. In this work, we isolated a bacterium, Novosphingobium rhizosphaerae LY, that grows with the β–β lignan (+)-pinoresinol as a sole growth substrate. Sequence analysis suggested that this strain encodes a broad range of pathways for assimilation of aromatic monomers as well as one enzyme implicated in pinoresinol catabolism but lacks other known pathways for aromatic dimer catabolism. We constructed a genome-wide barcoded transposon library and identified genes required for pinoresinol catabolism. Using feeding studies, compound isolation, targeted synthesis, and analysis of purified enzymes, we elucidated the biochemical intermediates and reaction pathway involved in pinoresinol catabolism. We demonstrated that the first enzymatic reaction is the reductive cleavage of a furan ring in (±)-pinoresinol with retention of configuration to yield lariciresinol. We additionally confirmed that the final pathway enzyme, PinU, is related to lignostilbene dioxygenases and oxidatively cleaves a diguaiacylbutadiene intermediate to yield vanillin and coniferaldehyde. Finally, based on the enzyme characterization, we demonstrated that the strain can grow with a second β–β lignan, (–)-syringaresinol, as a sole growth substrate. In combination, these results demonstrate a new biocatalytic route for transforming a widely occurring group of plant phenylpropanoid natural products.

Novosphingobium↗
DOE OSTI2024

Complete genomes of Mucilaginibacter sabulilitoris SNA2 and Mucilaginibacter sp. cycad4: microbes with the potential for plant growth promotion

Mucilaginibacter species have been isolated from various environments, often in association with plants. Here, we report the complete genomes of Mucilaginibacter sabulilitoris SNA2 and Mucilaginibacter sp. cycad4. The former is the first available for that species, and based on 16S sequence analysis, the latter strain is likely a new species.

Mucilaginibacter↗
DOE OSTI2022

Temporal variation in skin microbiota of cohabitating amphibians

Temporal changes and transmission patterns in host-associated microbial communities have important implications for host health. The diversity of amphibian skin microbial communities is associated with disease outcome in amphibians exposed to the fungal pathogen Batrachochytrium dendrobatidis (Bd). To successfully develop conservation strategies against Bd, we need a comprehensive understanding of how skin microbes are maintained and transmitted over time within populations. Here we used 16S rRNA sequence analysis to compare Epipedobates anthonyi frogs housed with one conspecific to frogs housed singly at four time points over the course of 1 year. We found that both α and β diversity of frog skin bacterial communities changed significantly over the course of the experiment. Specifically, we found that bacterial communities of cohabitating frogs became more similar over time. We also observed that some bacterial taxa were differentially abundant between frogs housed singly and frogs housed with a conspecific. These results suggest that conspecific contact may play a role in mediating amphibian skin microbial diversity and that turnover of skin microbial communities can occur across time. Our findings provide rationale for future studies exploring horizontal transmission as a potential mechanism of host-associated microbial maintenance in amphibians.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

The TLK-ASF1 histone chaperone pathway plays a critical role in IL-1β–mediated AML progression

Identifying and targeting microenvironment-driven pathways that are active across acute myeloid leukemia (AML) genetic subtypes should allow the development of more broadly effective therapies. The proinflammatory cytokine interleukin-1β (IL-1β) is abundant in the AML microenvironment and promotes leukemic growth. Through RNA-sequencing analysis, we identify that IL-1β–upregulated ASF1B (antisilencing function-1B), a histone chaperone, in AML progenitors compared with healthy progenitors. ASF1B, along with its paralogous protein ASF1A, recruits H3-H4 histones onto the replication fork during S-phase, a process regulated by Tousled-like kinase 1 and 2 (TLKs). Although ASF1s and TLKs are known to be overexpressed in multiple solid tumors and associated with poor prognosis, their functional roles in hematopoiesis and inflammation-driven leukemia remain unexplored. In this study, we identify that ASF1s and TLKs are overexpressed in multiple genetic subtypes of AML. We demonstrate that depletion of ASF1s significantly reduces leukemic cell growth in both in vitro and in vivo models using human cells. Using a murine model, we show that overexpression of ASF1B accelerates leukemia progression. Moreover, Asf1b or Tlk2 deletion delayed leukemia progression, whereas these proteins are dispensable for normal hematopoiesis. Through proteomics and phosphoproteomics analyses, we uncover that the TLK-ASF1 pathway promotes leukemogenesis by affecting the cell cycle and DNA damage pathways. Collectively, our findings identify the TLK1-ASF1 pathway as a novel mediator of inflammatory signaling and a promising therapeutic target for AML treatment across diverse genetic subtypes. Finally, selective inhibition of this pathway offers potential opportunities to intervene effectively, address intratumoral heterogeneity, and ultimately improve clinical outcomes in AML.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2012

HIV-1 subtype C superinfected individuals mount low autologous neutralizing antibody responses prior to intrasubtype superinfection

Background: The potential role of antibodies in protection against intra-subtype HIV-1 superinfection remains to be understood. We compared the early neutralizing antibody (NAb) responses in three individuals, who were superinfected within one year of primary infection, to ten matched non-superinfected controls from a Zambian cohort of subtype C transmission cases. Sequence analysis of single genome amplified full-length envs from a previous study showed limited diversification in the individuals who became superinfected with the same HIV-1 subtype within year one post-seroconversion. We hypothesized that this reflected a blunted NAb response, which may have made these individuals more susceptible to superinfection. Results: Neutralization assays showed that autologous plasma NAb responses to the earliest, and in some cases transmitted/founder, virus were delayed and had low to undetectable titers in all three superinfected individuals prior to superinfection. In contrast, NAbs with a median IC50 titer of 1896 were detected as early as three months post-seroconversion in non-superinfected controls. Early plasma NAbs in all subjects showed limited but variable levels of heterologous neutralization breadth. Superinfected individuals also exhibited a trend toward lower levels of gp120- and V1V2-specific IgG binding antibodies but higher gp120-specific plasma IgA binding antibodies. Conclusions: These data suggest that the lack of development of IgG antibodies, as reflected in autologous NAbs as well as gp120 and V1V2 binding antibodies to the primary infection virus, combined with potentially competing, non-protective IgA antibodies, may increase susceptibility to superinfection in the context of settings where a single HIV-1 subtype predominates.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2012

Intrasubtype C superinfected individuals mount delayed and low-titer autologous neutralizing antibody responses prior to superinfection

The potential role of neutralizing antibody in protecting against intra-subtype HIV-1 superinfection remains to be understood. We compared the early neutralizing antibody responses in three individuals, who were superinfected within one year of primary infection, to ten case-matched non-superinfected controls from a Zambian cohort of subtype C transmission cases. Sequence analysis of single genome amplified full-length env showed minimal diversification in the individuals who became superinfected with the same subtype of HIV-1 within year one postseroconversion. We hypothesized that these superinfected individuals had a muted neutralizing antibody response that elicited little pressure on the founder virus to escape.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2017

TSSK6 is required for γH2AX formation and the histone-to-protamine transition during spermiogenesis

ABSTRACT Spermiogenesis includes transcriptional silencing, chromatin condensation and extensive morphological changes as spermatids transform into sperm. Chromatin condensation involves histone hyperacetylation, transitory DNA breaks, histone H2AX (also known as H2AFX) phosphorylation at Ser139 (γH2AX), and replacement of histones by protamines. Previously, we have reported that the spermatid protein kinase TSSK6 is essential for fertility in mice, but its specific role in spermiogenesis is unknown. Here, we show that TSSK6 expression is spatiotemporally coincident with γH2AX formation in the nuclei of developing mouse spermatids. RNA-sequencing analysis demonstrates that genetic ablation of Tssk6 does not impact gene expression or silencing in spermatids. However, loss of TSSK6 blocks γH2AX formation, even though the timing and level of the transient DNA breaks is unaltered. Further, Tssk6-knockout sperm contained increased levels of histones H3 and H4, and protamine 2 precursor and intermediate(s) indicative of a defective histone-to-protamine transition. These results demonstrate that TSSK6 is required for γH2AX formation during spermiogenesis, and also link γH2AX to the histone-to-protamine transition and male fertility.

Jha, Kula N. (ORCID:0000000222181864)↗
DOE OSTI2016

Evolutionary genomics suggests that CheV is an additional adaptor for accommodating specific chemoreceptors within the chemotaxis signaling complex

Escherichia coli and Salmonella enterica are models for many experiments in molecular biology including chemotaxis, and most of the results obtained with one organism have been generalized to another. While most components of the chemotaxis pathway are strongly conserved between the two species, Salmonella genomes contain some chemoreceptors and an additional protein, CheV, that are not found in E. coli. The role of CheV was examined in distantly related species Bacillus subtilis and Helicobacter pylori , but its role in bacterial chemotaxis is still not well understood. We tested a hypothesis that in enterobacteria CheV functions as an additional adaptor linking the CheA kinase to certain types of chemoreceptors that cannot be effectively accommodated by the universal adaptor CheW. Phylogenetic profiling, genomic context and comparative protein sequence analyses suggested that CheV interacts with specific domains of CheA and chemoreceptors from an orthologous group exemplified by the Salmonella McpC protein. Structural consideration of the conservation patterns suggests that CheV and CheW share the same binding spot on the chemoreceptor structure, but have some affinity bias towards chemoreceptors from different orthologous groups. Finally, published experimental results and data newly obtained via comparative genomics support the idea that CheV functions as a "phosphate sink" possibly to off-set the over-stimulation of the kinase by certain types of chemoreceptors. Altogether, our results strongly suggest that CheV is an additional adaptor for accommodating specific chemoreceptors within the chemotaxis signaling complex.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2013

Structural and Histone Binding Ability Characterizations of Human PWWP Domains

The PWWP domain was first identified as a structural motif of 100-130 amino acids in the WHSC1 protein and predicted to be a protein-protein interaction domain. It belongs to the Tudor domain 'Royal Family', which consists of Tudor, chromodomain, MBT and PWWP domains. While Tudor, chromodomain and MBT domains have long been known to bind methylated histones, PWWP was shown to exhibit histone binding ability only until recently. The PWWP domain has been shown to be a DNA binding domain, but sequence analysis and previous structural studies show that the PWWP domain exhibits significant similarity to other 'Royal Family' members, implying that the PWWP domain has the potential to bind histones. In order to further explore the function of the PWWP domain, we used the protein family approach to determine the crystal structures of the PWWP domains from seven different human proteins. Our fluorescence polarization binding studies show that PWWP domains have weak histone binding ability, which is also confirmed by our NMR titration experiments. Furthermore, we determined the crystal structures of the BRPF1 PWWP domain in complex with H3K36me3, and HDGF2 PWWP domain in complex with H3K79me3 and H4K20me3. PWWP proteins constitute a new family of methyl lysine histone binders. The PWWP domain consists of three motifs: a canonical {beta}-barrel core, an insertion motif between the second and third {beta}-strands and a C-terminal {alpha}-helix bundle. Both the canonical {beta}-barrel core and the insertion motif are directly involved in histone binding. The PWWP domain has been previously shown to be a DNA binding domain. Therefore, the PWWP domain exhibits dual functions: binding both DNA and methyllysine histones.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2012

Structure of a Novel Winged-Helix Like Domain from Human NFRKB Protein

The human nuclear factor related to kappa-B-binding protein (NFRKB) is a 1299-residue protein that is a component of the metazoan INO80 complex involved in chromatin remodeling, transcription regulation, DNA replication and DNA repair. Although full length NFRKB is predicted to be around 65% disordered, comparative sequence analysis identified several potentially structured sections in the N-terminal region of the protein. These regions were targeted for crystallographic studies, and the structure of one of these regions spanning residues 370–495 was determined using the JCSG highthroughput structure determination pipeline. The structure reveals a novel, mostly helical domain reminiscent of the winged-helix fold typically involved in DNA binding. However, further analysis shows that this domain does not bind DNA, suggesting it may belong to a small group of winged-helix domains involved in protein-protein interactions.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2014

The functional potential of microbial communities in hydraulic fracturing source water and produced water from natural gas extraction characterized by metagenomic sequencing

Microbial activity in produced water from hydraulic fracturing operations can lead to undesired environmental impacts and increase gas production costs. However, the metabolic profile of these microbial communities is not well understood. Here, for the first time, we present results from a shotgun metagenome of microbial communities in both hydraulic fracturing source water and wastewater produced by hydraulic fracturing. Taxonomic analyses showed an increase in anaerobic/facultative anaerobic classes related to Clostridia , Gammaproteobacteria , Bacteroidia and Epsilonproteobacteria in produced water as compared to predominantly aerobic Alphaproteobacteria in the fracturing source water. Thus, the metabolic profile revealed a relative increase in genes responsible for carbohydrate metabolism, respiration, sporulation and dormancy, iron acquisition and metabolism, stress response and sulfur metabolism in the produced water samples. These results suggest that microbial communities in produced water have an increased genetic ability to handle stress, which has significant implications for produced water management, such as disinfection.

54 ENVIRONMENTAL SCIENCES↗
DOE OSTI2015

Spatial distribution of an uranium-respiring betaproteobacterium at the Rifle, CO field research site

The Department of Energy’s Integrated Field-Scale Subsurface Research Challenge Site (IFRC) at Rifle, Colorado was created to address the gaps in knowledge on the mechanisms and rates of U(VI) bioreduction in alluvial sediments. Previous studies at the Rifle IFRC have linked microbial processes to uranium immobilization during acetate amendment. Several key bacteria believed to be involved in radionuclide containment have been described; however, most of the evidence implicating uranium reduction with specific microbiota has been indirect. Here, we report on the cultivation of a microorganism from the Rifle IFRC that reduces uranium and appears to utilize it as a terminal electron acceptor for respiration with acetate as electron donor. Furthermore, this bacterium constitutes a significant proportion of the subsurface sediment community prior to biostimulation based on TRFLP profiling of 16S rRNA genes. 16S rRNA gene sequence analysis indicates that the microorganism is a betaproteobacterium with a high similarity to Burkholderia fungorum . This is, to our knowledge, the first report of a betaproteobacterium capable of uranium respiration. Our results indicate that this microorganism occurs commonly in alluvial sediments located between 3-6 m below ground surface at Rifle and may play a role in the initial reduction of uranium at the site.

12 MANAGEMENT OF RADIOACTIVE AND NON-RADIOACTIVE W↗
DOE OSTI2015

Evaluating models of cellulose degradation by Fibrobacter succinogenes S85

Fibrobacter succinogenes S85 is an anaerobic non-cellulosome utilizing cellulolytic bacterium originally isolated from the cow rumen microbial community. Efforts to elucidate its cellulolytic machinery have resulted in the proposal of numerous models which involve a combination of cell-surface attachment via a combination of cellulose-binding fibro-slime proteins and pili, the production of cellulolytic vesicles, and the entry of cellulose fibers into the periplasmic space. Here, we used a combination of RNA-sequencing, proteomics, and transmission electron microscopy (TEM) to further elucidate the cellulolytic mechanism of F. succinogenes . Our RNA-sequence analysis shows that genes encoding Type II and III secretion systems, fibro-slime proteins, and pili are differentially expressed on cellulose, relative to glucose. A subcellular fractionation of cells grown on cellulose revealed that carbohydrate active enzymes associated with cellulose deconstruction and fibro-slime proteins were greater in the extracellular media, as compared to the periplasm and outer membrane fractions. TEMs of samples harvested at mid-exponential and stationary phases of growth on cellulose and glucose showed the presence of grooves in the cellulose between the bacterial cells and substrate, suggesting enzymes work extracellularly for cellulose degradation. Membrane vesicles were only observed in stationary phase cultures grown on cellulose. Furthermore, these results provide evidence that F. succinogenes attaches to cellulose fibers using fibro-slime and pili, produces cellulases, such as endoglucanases, that are secreted extracellularly using type II and III secretion systems, and degrades the cellulose into cellodextrins that are then imported back into the periplasm for further digestion by β-glucanases and other cellulases.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2016

Impact of Organic Carbon Electron Donors on Microbial Community Development under Iron- and Sulfate-Reducing Conditions

Although iron- and sulfate-reducing bacteria in subsurface environments have crucial roles in biogeochemical cycling of C, Fe, and S, how specific electron donors impact the compositional structure and activity of native iron- and/or sulfate-reducing communities is largely unknown. To understand this better, we created bicarbonate-buffered batch systems in duplicate with three different electron donors (acetate, lactate, or glucose) paired with ferrihydrite and sulfate as the electron acceptors and inoculated them with subsurface sediment as the microbial inoculum. Sulfate and ferrihydrite reduction occurred simultaneously and were faster with lactate than with acetate. 16S rRNA-based sequence analysis of the communities over time revealed that Desulfotomaculum was the major driver for sulfate reduction coupled with propionate oxidation in lactate-amended incubations. The reduction of sulfate resulted in sulfide production and subsequent abiotic reduction of ferrihydrite. In contrast, glucose promoted faster reduction of ferrihydrite, but without reduction of sulfate. Interestingly, the glucose-amended incubations led to two different biogeochemical trajectories among replicate bottles that resulted in distinct coloration (white and brown). The two outcomes in geochemical evolution might be due to the stochastic evolution of the microbial communities or subtle differences in the initial composition of the fermenting microbial community and its development via the use of different glucose fermentation pathways available within the community. Synchrotron-based x-ray analysis indicated that siderite and amorphous Fe(II) were formed in the replicate bottles with glucose, while ferrous sulfide and vivianite were formed with lactate or acetate. As a result, these data sets reveal that use of different C utilization pathways projects significant changes in microbial community composition over time that uniquely impact both the geochemistry and mineralogy of subsurface environments.

54 ENVIRONMENTAL SCIENCES↗
DOE OSTI2017

Variations of five eIF4E genes across cassava accessions exhibiting tolerant and susceptible responses to cassava brown streak disease

Cassava (Manihot esculenta) is an important tropical subsistence crop that is severely affected by cassava brown streak disease (CBSD) in East Africa. The disease is caused by Cassava brown streak virus (CBSV) and Ugandan cassava brown streak virus (UCBSV). Both have a (+)-sense single-stranded RNA genome with a 5’ covalently-linked viral protein, which functionally resembles the cap structure of mRNA, binds to eukaryotic translation initiation factor 4E (eIF4E) or its analogues, and then enable the translation of viral genomic RNA in host cells. To characterize cassava eIF4Es and their potential role in CBSD tolerance and susceptibility, we cloned five eIF4E transcripts from cassava (accession TMS60444). Sequence analysis indicated that the cassava eIF4E family of proteins consisted of one eIF4E, two eIF(iso)4E, and two divergent copies of novel cap-binding proteins (nCBPs). Our data demonstrated experimentally the coding of these five genes as annotated in the published cassava genome and provided additional evidence for refined annotations. Illumina resequencing data of the five eIF4E genes were analyzed from 14 cassava lines tolerant or susceptible to CBSD. Abundant single nucleotide polymorphisms (SNP) and biallelic variations were observed in the eIF4E genes; however, most of the SNPs were located in the introns and non-coding regions of the exons. Association studies of non-synonymous SNPs revealed no significant association between any SNP of the five eIF4E genes and the tolerance or susceptibility to CBSD. However, two SNPs in two genes were weakly associated with the CBSD responses but had no direct causal-effect relationship. SNPs in an intergenic region upstream of eIF4E_me showed a surprising strong association with CBSD responses. Digital expression profile analysis showed differential expression of different eIF4E genes but no significant difference in gene expression was found between susceptible and tolerant cassava accessions despite the association of the intergenic SNPs with CBSD responses.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2017

Draft genome sequence of Actinotignum schaalii DSM 15541 T : Genetic insights into the lifestyle, cell fitness and virulence

The permanent draft genome sequence of Actinotignum schaalii DSM 15541 T is presented. The annotated genome includes 2,130,987 bp, with 1777 protein-coding and 58 rRNA-coding genes. Genome sequence analysis revealed absence of genes encoding for: components of the PTS systems, enzymes of the TCA cycle, glyoxylate shunt and gluconeogensis. Genomic data revealed that A. schaalii is able to oxidize carbohydrates via glycolysis, the nonoxidative pentose phosphate and the Entner-Doudoroff pathways. Besides, the genome harbors genes encoding for enzymes involved in the conversion of pyruvate to lactate, acetate and ethanol, which are found to be the end products of carbohydrate fermentation. The genome contained the gene encoding Type I fatty acid synthase required for de novo FAS biosynthesis. The plsY and plsX genes encoding the acyltransferases necessary for phosphatidic acid biosynthesis were absent from the genome. The genome harbors genes encoding enzymes responsible for isoprene biosynthesis via the mevalonate (MVA) pathway. Genes encoding enzymes that confer resistance to reactive oxygen species (ROS) were identified. In addition, A. schaalii harbors genes that protect the genome against viral infections. These include restriction-modification (RM) systems, type II toxin-antitoxin (TA), CRISPR-Cas and abortive infection system. A. schaalii genome also encodes several virulence factors that contribute to adhesion and internalization of this pathogen such as the tad genes encoding proteins required for pili assembly, the nanI gene encoding exo-alpha-sialidase, genes encoding heat shock proteins and genes encoding type VII secretion system. These features are consistent with anaerobic and pathogenic lifestyles. Finally, resistance to ciprofloxacin occurs by mutation in chromosomal genes that encode the subunits of DNA-gyrase (GyrA) and topisomerase IV (ParC) enzymes, while resistant to metronidazole was due to the frxA gene, which encodes NADPH-flavin oxidoreductase.

59 BASIC BIOLOGICAL SCIENCES↗