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134 records · Page 8
All sourcesNASA NTRSDOE OSTI
DOE OSTI2024

Identification of glycosyltransferases mediating 2‐ O ‐arabinopyranosyl and 2‐ O ‐galactosyl substitutions of glucuronosyl side chains of xylan

SUMMARY Xylan is one of the major hemicelluloses in plant cell walls and its xylosyl backbone is often decorated at O ‐2 with glucuronic acid (GlcA) and/or methylglucuronic acid (MeGlcA) residues. The GlcA/MeGlcA side chains may be further substituted with 2‐ O ‐arabinopyranose (Ara p ) or 2‐ O ‐galactopyranose (Gal) residues in some plant species, but the enzymes responsible for these substitutions remain unknown. During our endeavor to investigate the enzymatic activities of Arabidopsis MUR3‐clade members of the GT47 glycosyltransferase family, we found that one of them was able to transfer Ara p from UDP‐Ara p onto O ‐2 of GlcA side chains of xylan, and thus it was named xylan 2‐ O ‐arabinopyranosyltransferase 1 (AtXAPT1). The function of AtXAPT1 was verified in planta by its T‐DNA knockout mutation showing a loss of the Ara p substitution on xylan GlcA side chains. Further biochemical characterization of XAPT close homologs from other plant species demonstrated that while the poplar ones had the same catalytic activity as AtXAPT1, those from Eucalyptus , lemon‐scented gum, sea apple, 'Ohi'a lehua, duckweed and purple yam were capable of catalyzing both 2‐ O ‐Ara p and 2‐ O ‐Gal substitutions of xylan GlcA side chains albeit with differential activities. Sequential reactions with XAPTs and glucuronoxylan methyltransferase 3 (GXM3) showed that XAPTs acted poorly on MeGlcA side chains, whereas GXM3 could efficiently methylate arabinosylated or galactosylated GlcA side chains of xylan. Furthermore, molecular docking and site‐directed mutagenesis analyses of Eucalyptus XAPT1 revealed critical roles of several amino acid residues at the putative active site in its activity. Together, these findings establish that XAPTs residing in the MUR3 clade of family GT47 are responsible for 2‐ O ‐arabinopyranosylation and 2‐ O ‐galactosylation of GlcA side chains of xylan.

Zhong, Ruiqin↗
DOE OSTI2025

Sulfide stress tolerance as a controller of methane production in temperate wetlands

Abstract Wetlands are a major source of methane emissions and contribute to the observed increase in atmospheric methane over the last 20 years. Methane production in wetlands is the final step of carbon decomposition performed by anaerobic archaea. Although hydrogen/carbon dioxide and acetate are the substrates most often attributed to methanogenesis, other substrates—such as methylated compounds—may additionally play important roles in driving methane production in wetland systems. Here we conducted mesocosm experiments combined with genome-resolved metatranscriptomics to investigate the impact of diverse methanogenic substrate amendment on methanogenesis in two high methane-emitting wetlands with distinct geochemistry, termed P7 and P8. Methanol amendment resulted in high methane production at both sites, whereas acetate and formate amendment only stimulated methanogenesis in P7 mesocosms, where aqueous sulfide concentrations were lower. In P7 sediments, formate amendment fueled acetogenic microbes that produced acetate, which was subsequently utilized by acetoclastic methanogens. In contrast to expression profiles in P7 mesocosms, active methylotrophic methanogen genomes from P8 showed increased expression of genes related to membrane remodeling and DNA damage repair, indicative of stress tolerance mechanisms to counter sulfide toxicity. Methylotrophic methanogenesis generates higher free energy yields than acetoclastic methanogenesis, which likely enables allocation of more energy toward stress responses. These findings contribute to the growing body of literature highlighting methylotrophic methanogenesis as an important methane production pathway in wetlands. By using less competitive substrates like methanol that provide greater energy yields, methylotrophic methanogens may invest in physiological strategies that provide competitive advantages across a range of environmental stresses.

Environmental Sciences & Ecology↗
DOE OSTI2022

Borgs are giant genetic elements with potential to expand metabolic capacity

Anaerobic methane oxidation exerts a key control on greenhouse gas emissions1, yet factors that modulate the activity of microorganisms performing this function remain poorly understood. Here we discovered extraordinarily large, diverse DNA sequences that primarily encode hypothetical proteins through studying groundwater, sediments and wetland soil where methane production and oxidation occur. Four curated, complete genomes are linear, up to approximately 1 Mb in length and share genome organization, including replichore structure, long inverted terminal repeats and genome-wide unique perfect tandem direct repeats that are intergenic or generate amino acid repeats. We infer that these are highly divergent archaeal extrachromosomal elements with a distinct evolutionary origin. Gene sequence similarity, phylogeny and local divergence of sequence composition indicate that many of their genes were assimilated from methane-oxidizing Methanoperedens archaea. We refer to these elements as ‘Borgs’. We identified at least 19 different Borg types coexisting with Methanoperedens spp. in four distinct ecosystems. Borgs provide methane-oxidizing Methanoperedens archaea access to genes encoding proteins involved in redox reactions and energy conservation (for example, clusters of multihaem cytochromes and methyl coenzyme M reductase). These data suggest that Borgs might have previously unrecognized roles in the metabolism of this group of archaea, which are known to modulate greenhouse gas emissions, but further studies are now needed to establish their functional relevance.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

An optimized ChIP-Seq framework for profiling histone modifications in Chromochloris zofingiensis

The eukaryotic green alga Chromochloris zofingiensis is a reference organism for studying carbon partitioning and a promising candidate for the production of biofuel precursors. Recent transcriptome profiling transformed our understanding of its biology and generally algal biology, but epigenetic regulation remains understudied and represents a fundamental gap in our understanding of algal gene expression. Chromatin immunoprecipitation followed by deep sequencing (ChIP-Seq) is a powerful tool for the discovery of such mechanisms, by identifying genome-wide histone modification patterns and transcription factor-binding sites alike. Here, we established a ChIP-Seq framework for Chr. zofingiensis yielding over 20 million high-quality reads per sample. The most critical steps in a ChIP experiment were optimized, including DNA shearing to obtain an average DNA fragment size of 250 bp and assessment of the recommended formaldehyde concentration for optimal DNA-protein cross-linking. We used this ChIP-Seq framework to generate a genome-wide map of the H3K4me3 distribution pattern and to integrate these data with matching RNA-Seq data. In line with observations from other organisms, H3K4me3 marks predominantly transcription start sites of genes. Our H3K4me3 ChIP-Seq data will pave the way for improved genome structural annotation in the emerging reference alga Chr. zofingiensis.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Pseudodesulfovibrio mercurii sp. nov., a mercury-methylating bacterium isolated from sediment

The sulfate-reducing, mercury-methylating strain ND132 T was isolated from the brackish anaerobic bottom sediments of Chesapeake Bay, USA. Capable of high levels of mercury (Hg) methylation, ND132 T has been widely used as a model strain to study the process and to determine the genetic basis of Hg methylation. Originally called Desulfovibrio desulfuricans ND132 T on the basis of an early partial 16S rRNA sequence, the strain has never been formally described. Phylogenetic and physiological traits place this strain within the genus Pseudodesulfovibrio, in the recently reclassified phylum Desulfobacterota (formerly Deltaproteobacteria ). ND132 T is most closely related to Pseudodesulfovibrio hydrargyri BerOc1 T and Pseudodesulfovibrio indicus J2T. Analysis of average nucleotide identity (ANI) of whole-genome sequences showed roughly 88 % ANI between P. hydrargyri BerOc1T and ND132T, and 84 % similarity between ND132T and P. indicus J2 T . These cut-off scores <95 %, along with a multi-gene phylogenetic analysis of members of the family Desulfovibrionacea, and differences in physiology indicate that all three strains represent separate species. The Gram-stain-negative cells are vibrio-shaped, motile and not sporulated. ND132 T is a salt-tolerant mesophile with optimal growth in the laboratory at 32 °C, 2 % salinity, and pH 7.8. The DNA G+C content of the genomic DNA is 65.2 %. It is an incomplete oxidizer of short chain fatty acids, using lactate, pyruvate and fumarate with sulfate or sulfite as the terminal electron acceptors. ND132 T can respire fumarate using pyruvate as an electron donor. The major fatty acids are iso-C 15 :0 , anteiso-C 15 : 0 , iso-C 17 : 0 , iso-C 17 : 1 ω9c and anteiso-C 17 : 0 . We propose the classification of strain ND132 T (DSM 110689, ATCC TSD-224) as the type strain Pseudodesulfovibrio mercurii sp. nov.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Rev1 overexpression accelerates N -methyl- N -nitrosourea (MNU)-induced thymic lymphoma by increasing mutagenesis

Rev1 has two important functions in the translesion synthesis pathway, including dCMP transferase activity, and acts as a scaffolding protein for other polymerases involved in translesion synthesis. However, the role of Rev1 in mutagenesis and tumorigenesis in vivo remains unclear. We previously generated Rev1-overexpressing (Rev1-Tg) mice and reported that they exhibited a significantly increased incidence of intestinal adenoma and thymic lymphoma (TL) after N-methyl-N-nitrosourea (MNU) treatment. In this study, we investigated mutagenesis of MNU-induced TL tumorigenesis in wild-type (WT) and Rev1-Tg mice using diverse approaches, including whole-exome sequencing (WES). In Rev1-Tg TLs, the mutation frequency was higher than that in WT TL in most cases. However, no difference in the number of nonsynonymous mutations in the Catalogue of Somatic Mutations in Cancer (COSMIC) genes was observed, and mutations involved in Notch1 and MAPK signaling were similarly detected in both TLs. Mutational signature analysis of WT and Rev1-Tg TLs revealed cosine similarity with COSMIC mutational SBS5 (aging-related) and SBS11 (alkylation-related). Interestingly, the total number of mutations, but not the genotypes of WT and Rev1-Tg, was positively correlated with the relative contribution of SBS5 in individual TLs, suggesting that genetic instability could be accelerated in Rev1-Tg TLs. Finally, we demonstrated that preleukemic cells could be detected earlier in Rev1-Tg mice than in WT mice, following MNU treatment. In conclusion, Rev1 overexpression accelerates mutagenesis and increases the incidence of MNU-induced TL by shortening the latency period, which may be associated with more frequent DNA damage-induced genetic instability.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2024

Genome-wide profiling of histone (H3) lysine 4 (K4) tri-methylation (me3) under drought, heat, and combined stresses in switchgrass

Background: Switchgrass (Panicum virgatum L.) is a warm-season perennial (C4) grass identified as an important biofuel crop in the United States. It is well adapted to the marginal environment where heat and moisture stresses predominantly affect crop growth. However, the underlying molecular mechanisms associated with heat and drought stress tolerance still need to be fully understood in switchgrass. The methylation of H3K4 is often associated with transcriptional activation of genes, including stress-responsive. Therefore, this study aimed to analyze genome-wide histone H3K4-tri-methylation in switchgrass under heat, drought, and combined stress. Results: In total, ~ 1.3 million H3K4me3 peaks were identified in this study using SICER. Among them, 7,342; 6,510; and 8,536 peaks responded under drought (DT), drought and heat (DTHT), and heat (HT) stresses, respectively. Most DT and DTHT peaks spanned 0 to + 2000 bases from the transcription start site [TSS]. By comparing differentially marked peaks with RNA-Seq data, we identified peaks associated with genes: 155 DT-responsive peaks with 118 DT-responsive genes, 121 DTHT-responsive peaks with 110 DTHT-responsive genes, and 175 HT-responsive peaks with 136 HT-responsive genes. We have identified various transcription factors involved in DT, DTHT, and HT stresses. Gene Ontology analysis using the AgriGO revealed that most genes belonged to biological processes. Most annotated peaks belonged to metabolite interconversion, RNA metabolism, transporter, protein modifying, defense/immunity, membrane traffic protein, transmembrane signal receptor, and transcriptional regulator protein families. Further, we identified significant peaks associated with TFs, hormones, signaling, fatty acid and carbohydrate metabolism, and secondary metabolites. qRT-PCR analysis revealed the relative expressions of six abiotic stress-responsive genes (transketolase, chromatin remodeling factor-CDH3, fatty-acid desaturase A, transmembrane protein 14C, beta-amylase 1, and integrase-type DNA binding protein genes) that were significantly (P < 0.05) marked during drought, heat, and combined stresses by comparing stress-induced against un-stressed and input controls. Conclusion: Our study provides a comprehensive and reproducible epigenomic analysis of drought, heat, and combined stress responses in switchgrass. Significant enrichment of H3K4me3 peaks downstream of the TSS of protein-coding genes was observed. In addition, the cost-effective experimental design, modified ChIP-Seq approach, and analyses presented here can serve as a prototype for other non-model plant species for conducting stress studies.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

An autoinhibited state of 53BP1 revealed by small molecule antagonists and protein engineering

The recruitment of 53BP1 to chromatin, mediated by its recognition of histone H4 dimethylated at lysine 20 (H4K20me2), is important for DNA double-strand break repair. Using a series of small molecule antagonists, we demonstrate a conformational equilibrium between an open and a pre-existing lowly populated closed state of 53BP1 in which the H4K20me2 binding surface is buried at the interface between two interacting 53BP1 molecules. In cells, these antagonists inhibit the chromatin recruitment of wild type 53BP1, but do not affect 53BP1 variants unable to access the closed conformation despite preservation of the H4K20me2 binding site. Thus, this inhibition operates by shifting the conformational equilibrium toward the closed state. Our work therefore identifies an auto-associated form of 53BP1—autoinhibited for chromatin binding—that can be stabilized by small molecule ligands encapsulated between two 53BP1 protomers. Such ligands are valuable research tools to study the function of 53BP1 and have the potential to facilitate the development of new drugs for cancer therapy.

59 BASIC BIOLOGICAL SCIENCES↗