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Results for “gene network centrality analysis”

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18 records
All sourcesNASA NTRSDOE OSTI
DOE OSTI2025

Gene network centrality analysis identifies key regulators coordinating day-night metabolic transitions in Synechococcus elongatus PCC 7942 despite limited accuracy in predicting direct regulator-gene interactions

Synechococcus elongatus PCC 7942 is a model organism for studying circadian regulation and bioproduction, where precise temporal control of metabolism significantly impacts photosynthetic efficiency and CO 2 -to-bioproduct conversion. Despite extensive research on core clock components, our understanding of the broader regulatory network orchestrating genome-wide metabolic transitions remains incomplete. We address this gap by applying machine learning tools and network analysis to investigate the transcriptional architecture governing circadian-controlled gene expression. While our approach showed moderate accuracy in predicting individual transcription factor-gene interactions - a common challenge with real expression data - network-level topological analysis successfully revealed the organizational principles of circadian regulation. Our analysis identified distinct regulatory modules coordinating day-night metabolic transitions, with photosynthesis and carbon/nitrogen metabolism controlled by day-phase regulators, while nighttime modules orchestrate glycogen mobilization and redox metabolism. Through network centrality analysis, we identified potentially significant but previously understudied transcriptional regulators: HimA as a putative DNA architecture regulator, and TetR and SrrB as potential coordinators of nighttime metabolism, working alongside established global regulators RpaA and RpaB. This work demonstrates how network-level analysis can extract biologically meaningful insights despite limitations in predicting direct regulatory interactions. The regulatory principles uncovered here advance our understanding of how cyanobacteria coordinate complex metabolic transitions and may inform metabolic engineering strategies for enhanced photosynthetic bioproduction from CO 2 .

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Integrated network modeling approach defines key metabolic responses of soil microbiomes to perturbations

The soil environment is constantly changing due to shifts in soil moisture, nutrient availability and other conditions. To contend with these changes, soil microorganisms have evolved a variety of ways to adapt to environmental perturbations, including regulation of gene expression. However, it is challenging to untangle the complex phenotypic response of the soil to environmental change, partly due to the absence of predictive modeling frameworks that can mechanistically link molecular-level changes in soil microorganisms to a community’s functional phenotypes (or metaphenome). Towards filling this gap, we performed a combined analysis of metabolic and gene co-expression networks to explore how the soil microbiome responded to changes in soil moisture and nutrient conditions and to determine which genes were expressed under a given condition. Our integrated modeling approach revealed previously unknown, but critically important aspects of the soil microbiomes’ response to environmental perturbations. Incorporation of metabolomic and transcriptomic data into metabolic reaction networks identified condition-specific signature genes that are uniquely associated with dry, wet, and glycine-amended conditions. A subsequent gene co-expression network analysis revealed that drought-associated genes occupied more central positions in a network model of the soil community, compared to the genes associated with wet, and glycine-amended conditions. These results indicate the occurrence of system-wide metabolic coordination when soil microbiomes cope with moisture or nutrient perturbations. Importantly, the approach that we demonstrate here to analyze large-scale multi-omics data from a natural soil environment is applicable to other microbiome systems for which multi-omics data are available.

54 ENVIRONMENTAL SCIENCES↗
DOE OSTI2024

Transcriptomic and Proteomic Insights into Host Immune Responses in Pediatric Severe Malarial Anemia: Dysregulation in HSP60-70-TLR2/4 Signaling and Altered Glutamine Metabolism

Severe malarial anemia (SMA, Hb < 6.0 g/dL) is a leading cause of childhood morbidity and mortality in holoendemic Plasmodium falciparum transmission zones. This study explored the entire expressed human transcriptome in whole blood from 66 Kenyan children with non-SMA (Hb ≥ 6.0 g/dL, n = 41) and SMA (n = 25), focusing on host immune response networks. RNA-seq analysis revealed 6862 differentially expressed genes, with equally distributed up-and down-regulated genes, indicating a complex host immune response. Deconvolution analyses uncovered leukocytic immune profiles indicative of a diminished antigenic response, reduced immune priming, and polarization toward cellular repair in SMA. Weighted gene co-expression network analysis revealed that immune-regulated processes are central molecular distinctions between non-SMA and SMA. A top dysregulated immune response signaling network in SMA was the HSP60-HSP70-TLR2/4 signaling pathway, indicating altered pathogen recognition, innate immune activation, stress responses, and antigen recognition. Validation with high-throughput gene expression from a separate cohort of Kenyan children (n = 50) with varying severities of malarial anemia (n = 38 non-SMA and n = 12 SMA) confirmed the RNA-seq findings. Proteomic analyses in 35 children with matched transcript and protein abundance (n = 19 non-SMA and n = 16 SMA) confirmed dysregulation in the HSP60-HSP70-TLR2/4 signaling pathway. Additionally, glutamine transporter and glutamine synthetase genes were differentially expressed, indicating altered glutamine metabolism in SMA. This comprehensive analysis underscores complex immune dysregulation and novel pathogenic features in SMA.

Microbiology↗
DOE OSTI2024

Transcriptomic and metabolomic changes in postharvest sugarbeet roots reveal widespread metabolic changes in storage and identify genes potentially responsible for respiratory sucrose loss

Endogenous metabolism is primarily responsible for losses in sucrose content and processing quality in postharvest sugarbeet roots. The genes responsible for this metabolism and the transcriptional changes that regulate it, however, are largely unknown. To identify genes and metabolic pathways that participate in postharvest sugarbeet root metabolism and the transcriptional changes that contribute to their regulation, transcriptomic and metabolomic profiles were generated for sugarbeet roots at harvest and after 12, 40 and 120 d storage at 5 and 12°C and gene expression and metabolite concentration changes related to storage duration or temperature were identified. During storage, 8656 genes, or 34% of all expressed genes, and 225 metabolites, equivalent to 59% of detected metabolites, were altered in expression or concentration, indicating extensive transcriptional and metabolic changes in stored roots. These genes and metabolites contributed to a wide range of cellular and molecular functions, with carbohydrate metabolism being the function to which the greatest number of genes and metabolites classified. Because respiration has a central role in postharvest metabolism and is largely responsible for sucrose loss in sugarbeet roots, genes and metabolites involved in and correlated to respiration were identified. Seventy-five genes participating in respiration were differentially expressed during storage, including two bidirectional sugar transporter SWEET17 genes that highly correlated with respiration rate. Weighted gene co-expression network analysis identified 1896 additional genes that positively correlated with respiration rate and predicted a pyruvate kinase gene to be a central regulator or biomarker for respiration rate. Overall, these results reveal the extensive and diverse physiological and metabolic changes that occur in stored sugarbeet roots and identify genes with potential roles as regulators or biomarkers for respiratory sucrose loss.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Global Network Analysis of Neisseria gonorrhoeae Identifies Coordination between Pathways, Processes, and Regulators Expressed during Human Infection

Neisseria gonorrhoeae is a Gram-negative diplococcus that is responsible for the sexually transmitted infection gonorrhea, a high-morbidity disease in the United States and worldwide. Over the past several years, N. gonorrhoeae strains resistant to antibiotics used to treat this infection have begun to emerge across the globe. Thus, new treatment strategies are needed to combat this organism. Here, we utilized N. gonorrhoeae transcriptomic data sets, including those obtained from natural infection of the human genital tract, to infer the first global gene coexpression network of this pathogen. Interrogation of this network revealed genes central to the network that are likely critical for gonococcal growth, metabolism, and virulence, including genes encoding hypothetical proteins expressed during mucosal infection. In addition, network analysis revealed overlap in the response of N. gonorrhoeae to incubation with neutrophils and exposure to hydrogen peroxide stress in vitro. Network analysis also identified new targets of the gonococcal global regulatory protein Fur, while examination of the network neighborhood of genes allowed us to assign additional putative categories to several proteins. Collectively, the characterization of the first gene coexpression network for N. gonorrhoeae described here has revealed new regulatory pathways and new categories for proteins and has shown how processes important to gonococcal infection in both men and women are linked. This information fills a critical gap in our understanding of virulence strategies of this obligate human pathogen and will aid in the development of new treatment strategies for gonorrhea.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2026

Rhythmic Mechanisms Governing CAM Photosynthesis in Kalanchoe fedtschenkoi : High-Resolution Temporal Transcriptomics

Crassulacean acid metabolism (CAM) is a specialized photosynthetic pathway that enhances water-use efficiency by temporally separating nocturnal CO 2 uptake from daytime decarboxylation and carbon fixation. To uncover the regulatory mechanisms coordinating these temporal dynamics, we generated high-resolution, 48 h time-course transcriptomes for the CAM model Kalanchoe fedtschenkoi under both 12 h/12 h light/dark (LD) cycles and continuous light (LL). A rhythmicity analysis revealed that diel light cues are the dominant driver of transcript oscillations: 16,810 genes (54.3% of annotated genes) exhibited rhythmic expression only under LD, whereas just 399 genes (1.3%) remained rhythmic under LL. A smaller set of 3009 genes (9.7%) oscillated in both conditions, indicating that the intrinsic circadian clock sustains rhythmicity for a limited subset of the transcriptome. A gene co-expression network analysis revealed extensive integration between circadian clock components, core CAM pathway enzymes, and stomatal regulators, defining regulatory modules that coordinate metabolic and physiological timing. Notably, key hub genes associated with post-translational and post-transcriptional regulation, including the E3 ubiquitin ligase HUB2 and several pentatricopeptide repeat (PPR) proteins, act as central nodes in CAM-associated networks. This discovery implicates epigenetic and organellar regulation as previously unrecognized critical tiers of control in CAM. Together, our results support a regulatory model in which CAM rhythmicity is governed by both external light/dark cues and the endogenous circadian clock through multi-level control spanning transcriptional and protein-level regulation. To support community exploration, we also provide an interactive eFP (electronic Fluorescent Pictograph) browser for visualizing time-resolved gene expression profiles.

09 BIOMASS FUELS↗
DOE OSTI2022

A Path-Based Analysis of Infected Cell Line and COVID-19 Patient Transcriptome Reveals Novel Potential Targets and Drugs Against SARS-CoV-2

Most transcriptomic studies of SARS-CoV-2 infection have focused on differentially expressed genes, which do not necessarily reveal the genes mediating the transcriptomic changes. In contrast, exploiting curated biological network, our PathExt tool identifies central genes from the differentially active paths mediating global transcriptomic response. Here we apply PathExt to multiple cell line infection models of SARS-CoV-2 and other viruses, as well as to COVID-19 patient-derived PBMCs. The central genes mediating SARS-CoV-2 response in cell lines were uniquely enriched for ATP metabolic process, G1/S transition, leukocyte activation and migration. In contrast, PBMC response reveals dysregulated cell-cycle processes. In PBMC, the most frequently central genes are associated with COVID-19 severity. Importantly, relative to differential genes, PathExt-identified genes show greater concordance with several benchmark anti-COVID-19 target gene sets. We propose six novel anti-SARS-CoV-2 targets ADCY2, ADSL, OCRL, TIAM1, PBK, and BUB1, and potential drugs targeting these genes, such as Bemcentinib, Phthalocyanine, and Conivaptan.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2026

Integrative Modeling and Analysis of Fungal Central Carbon Metabolism

Over a thousand fungal genomes have been sequenced, yet manually curated genome-scale metabolic models (GEMs) are available for only a limited number of species. Moreover, these models have often been developed independently, leading to inconsistencies in namespaces, compartment definitions, and pathway representations that hinder comparative analysis, the systematic reuse of prior curation efforts, and the integration of consolidated metabolic knowledge. Here, we present the Consolidated Fungal Core Metabolism Model (CFCMM), constructed by integrating thirteen published fungal models spanning Ascomycota, Mucoromycota, and both Crabtree-positive and Crabtree-negative yeasts. We harmonized metabolites and reactions into a non-redundant shared ModelSEED ontological space, standardized compartmentalization, and refined gene–protein–reaction (GPR) rules. Using pathway-level visualization and systematic gap detection, we further improved the integrated network through literature-guided curation to correct stoichiometry, stereospecificity, and pathway architecture. Orthologous protein family reconstruction and functional annotation workflows were used to validate and inform GPR associations, with particular emphasis on ambiguous enzyme superfamilies and membrane-associated components. Using the resulting CFCMM, we built high-quality central carbon core models for each fungus and performed flux balance analysis to quantify ATP-yield variation under aerobic and anaerobic conditions, explicitly evaluating scenarios driven by differences in electron transport chain (ETC) composition. Simulations reproduced the expected fermentative yield of approximately 2 mmol ATP per mmol glucose under anaerobic conditions and separated the thirteen fungi into two bioenergetic groups under aerobic respiration based on Complex I status, with predicted yields of approximately 30 versus 22 mmol ATP per mmol glucose. Forcing flux through the alternative oxidase bypass further reduced ATP yields to approximately 12 and 4 mmol ATP per mmol glucose in Complex I-containing and Complex I-lacking fungi, respectively. Collectively, this work provides a manually curated, ModelSEED-consistent, and extensible fungal core metabolic template, deployed in DOE KBase as a resource for automated reconstruction of central carbon core models from any sequenced fungal genome. In addition, the CFCMM provides modular components for developing GEMs with more accurate energy predictions and enables robust comparative analyses of fungal bioenergetics and core metabolic diversity

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Developing stable, simplified, functional consortia from Brachypodium rhizosphere for microbial application in sustainable agriculture

The rhizosphere microbiome plays a crucial role in supporting plant productivity and ecosystem functioning by regulating nutrient cycling, soil integrity, and carbon storage. However, deciphering the intricate interplay between microbial relationships within the rhizosphere is challenging due to the overwhelming taxonomic and functional diversity. Here we present our systematic design framework built on microbial colocalization and microbial interaction, toward successful assembly of multiple rhizosphere-derived Reduced Complexity Consortia (RCC). We enriched co-localized microbes from Brachypodium roots grown in field soil with carbon substrates mimicking Brachypodium root exudates, generating 768 enrichments. By transferring the enrichments every 3 or 7 days for 10 generations, we developed both fast and slow-growing reduced complexity microbial communities. Most carbon substrates led to highly stable RCC just after a few transfers. 16S rRNA gene amplicon analysis revealed distinct community compositions based on inoculum and carbon source, with complex carbon enriching slow growing yet functionally important soil taxa like Acidobacteria and Verrucomicrobia. Network analysis showed that microbial consortia, whether differentiated by growth rate (fast vs. slow) or by succession (across generations), had significantly different network centralities. Besides, the keystone taxa identified within these networks belong to genera with plant growth-promoting traits, underscoring their critical function in shaping rhizospheric microbiome networks. Furthermore, tested consortia demonstrated high stability and reproducibility, assuring successful revival from glycerol stocks for long-term viability and use. Our study represents a significant step toward developing a framework for assembling rhizosphere consortia based on microbial colocalization and interaction, with future implications for sustainable agriculture and environmental management.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2017

Germline Chd8 haploinsufficiency alters brain development in mouse

The chromatin remodeling gene CHD8 represents a central node in neurodevelopmental gene networks implicated in autism. In this paper, we examined the impact of germline heterozygous frameshift Chd8 mutation on neurodevelopment in mice. Chd8 + / del5 mice displayed normal social interactions with no repetitive behaviors but exhibited cognitive impairment correlated with increased regional brain volume, validating that phenotypes of Chd8 + / del5 mice overlap pathology reported in humans with CHD8 mutations. We applied network analysis to characterize neurodevelopmental gene expression, revealing widespread transcriptional changes in Chd8 + / del5 mice across pathways disrupted in neurodevelopmental disorders, including neurogenesis, synaptic processes and neuroimmune signaling. We identified a co-expression module with peak expression in early brain development featuring dysregulation of RNA processing, chromatin remodeling and cell-cycle genes enriched for promoter binding by Chd8, and we validated increased neuronal proliferation and developmental splicing perturbation in Chd8 + / del5 mice. Finally, this integrative analysis offers an initial picture of the consequences of Chd8 haploinsufficiency for brain development.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

The rice CONSTANS-like protein OsCOL15 suppresses flowering by promoting Ghd7 and repressing RID1

Highlights: • OsCOL15 encodes a CONSTANS-like transcription factor. • OsCOL15 localized to the nucleus and had transcriptional activation activity. • OsCOL15 is most highly expressed in young organs and exhibits a diurnal expression pattern. • Overexpression of OsCOL15 resulted in a delayed flowering phenotype under both SD and LD conditions. • OsCOL15 delays heading by up-regulating Ghd7 and down-regulating RID1, leading to down-regulation of Ehd1, Hd3a, and RFT1. The photoperiodic flowering pathway is one of the most important regulatory networks controlling flowering time in rice (Oryza sativa L.). Rice is a facultative short-day (SD) plant; flowering is promoted under inductive SD conditions and delayed under non-inductive long-day (LD) conditions. In rice, flowering inhibitor genes play an important role in maintaining the trade-off between reproduction and yield. In this study, we identified a novel floral inhibitor, OsCOL15, which encodes a CONSTANS-like transcription factor. Consistent with a function in transcriptional regulation, OsCOL15 localized to the nucleus. Moreover, OsCOL15 had transcriptional activation activity, and the central region of the protein between the B-box and CCT domains was required for this activity. We determined that OsCOL15 is most highly expressed in young organs and exhibits a diurnal expression pattern typical of other floral regulators. Overexpression of OsCOL15 resulted in a delayed flowering phenotype under both SD and LD conditions. Real-time quantitative RT-PCR analysis of flowering regulator gene expression suggested that OsCOL15 suppresses flowering by up-regulating the flowering repressor Grain number, plant height and heading date 7 (Ghd7) and down-regulating the flowering activator Rice Indeterminate 1 (RID1), thus leading to the down-regulation of the flowering activators Early heading date 1, Heading date 3a, and RICE FLOWERING LOCUS T1. These results demonstrate that OsCOL15 is an important floral regulator acting upstream of Ghd7 and RID1 in the rice photoperiodic flowering-time regulatory network.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2022

Deeply conserved synteny and the evolution of metazoan chromosomes

Animal genomes show networks of deeply conserved gene linkages whose phylogenetic scope and chromosomal context remain unclear. Here, we report chromosome-scale conservation of synteny among bilaterians, cnidarians, and sponges and use comparative analysis to reconstruct ancestral chromosomes across major animal groups. Comparisons among diverse metazoans reveal the processes of chromosome evolution that produced contemporary karyotypes from their Precambrian progenitors. On the basis of these findings, we introduce a simple algebraic representation of chromosomal change and use it to establish a unified systematic framework for metazoan chromosome evolution. We find that fusion-with-mixing, a previously unappreciated mode of chromosome change, has played a central role. We find that relicts of several metazoan chromosomal units are preserved in unicellular eukaryotes. These conserved pre-metazoan linkages include the chromosomal unit that encodes the most diverse set of metazoan homeobox genes, suggesting a candidate genomic context for the early diversification of this key gene family.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Multi-Omics Reveals Temporal Scales of Carbon Metabolism in Synechococcus Elongatus PCC 7942 Under Light Disturbance

Central carbon metabolism in model cyanobacteria involves multiple pathways to adapt to energy-light limitations across diel cycles. However, the success in mechanistic modeling for phenotypic prediction of the protein regulators in the metabolic state depends on capturing the vast possibilities emerging from multiple regulatory pathways in complex biological processes. Here, we developed a physics-informed machine learning approach based on energy-landscape concepts to predict regulatory proteins responding to cyclic circadian and unforeseen light perturbations in cyanobacterial metabolic networks. Our approach provides interpretable de novo models for inferring gene expression dynamics from Synechococcus elongatus over diel cycles and using redox proteome analysis to distinguish immediate light-responsive elements from circadian-regulated processes in carbon metabolism pathways. We identified distinct temporal signatures with the analysis of the redox proteome: there was an immediate shift in cysteine redox states accompanied by a limited change in protein abundance under constant illumination and after 2 hours of darkness. This discovery indicates that the generation of reductants coordinates photoinduced electron transport with redox metabolic pathways in two discernable molecular mechanisms: fast redox-based protein modifications occur immediately after the light disturbance, followed by slow transcriptional regulations across networks. This temporal regulation reveals how metabolic networks integrate rapid light responses with programmed circadian rhythms to maintain cellular homeostasis under the light-energy limitations over the diel cycle.

Biomolecular & subcellular processes↗
DOE OSTI2026

From microbial diversity to functional potential using dimensionality reduction

The high dimensionality of microbial diversity data from ‘omics observations can be reduced using Machine Learning, with many recent studies showcasing ML utility for exploratory ecological feature finding and process prediction. Here, we compare the Self Organizing Map (SOM) dimensionality reduction method to the well-documented sample-based Principal Coordinate Analysis (PCoA) and taxa-based Weighted Gene Correlation Network Analysis (WGCNA) using near daily 16S rRNA gene amplicon sequencing data from the 2019 to 2020 MOSAiC International Arctic Drift Expedition. We then map k-means clustering outputs from each method to available metagenomes, extracting functionally distinct seasonal microbial ecotypes in the surface Arctic Ocean. Our results indicate the SOM method better represented expected seasonal transitions and identified a greater number of metabolically distinct functional groups than the more traditional PCoA ordination. Ultimately, we identified four community ecotypes with distinct taxonomic and functional cut-offs driven by seasonality, water mass, and substrate turnover, highlighting the importance of succession in functional diversity for the central Arctic Ocean. These results reinforce ML dimensionality reduction as a meaningful translator in the mining of historical amplicon datasets to address modern mechanistic questions and potentially provide ’omics informed ecotype diversity to leverage in mechanistic biogeochemical models.

Arctic Ocean↗
DOE OSTI2017

InvS Coordinates Expression of PrgH and FimZ and Is Required for Invasion of Epithelial Cells by Salmonella enterica serovar Typhimurium

ABSTRACT Deep sequencing has revolutionized our understanding of the bacterial RNA world and has facilitated the identification of 280 small RNAs (sRNAs) in Salmonella . Despite the suspicions that sRNAs may play important roles in Salmonella pathogenesis, the functions of most sRNAs remain unknown. To advance our understanding of RNA biology in Salmonella virulence, we searched for sRNAs required for bacterial invasion into nonphagocytic cells. After screening 75 sRNAs, we discovered that the ablation of InvS caused a significant decrease of Salmonella invasion into epithelial cells. A proteomic analysis showed that InvS modulated the levels of several type III secreted Salmonella proteins. The level of PrgH, a type III secretion apparatus protein, was significantly lower in the absence of InvS, consistent with the known roles of PrgH in effector secretion and bacterial invasion. We discovered that InvS modulates fimZ expression and hence flagellar gene expression and motility. We propose that InvS coordinates the increase of PrgH and decrease in FimZ that promote efficient Salmonella invasion into nonphagocytic cells. IMPORTANCE Salmonellosis continues to be the most common foodborne infection reported by the CDC in the United States. Central to Salmonella pathogenesis is the ability to invade nonphagocytic cells and to replicate inside host cells. Invasion genes are known to be regulated by protein transcriptional networks, but little is known about the role played by small RNAs (sRNAs) in this process. We have identified a novel sRNA, InvS, that is involved in Salmonella invasion. Our result will likely provide an opportunity to better understand the fundamental question of how Salmonella regulates invasion gene expression and may inform strategies for therapeutic intervention.

Environmental Molecular Sciences Laboratory↗
DOE OSTI2023

A glimpse into the fungal metabolomic abyss: Novel network analysis reveals relationships between exogenous compounds and their outputs

Fungal specialized metabolites are a major source of beneficial compounds that are routinely isolated, characterized, and manufactured as pharmaceuticals, agrochemical agents, and industrial chemicals. The production of these metabolites is encoded by biosynthetic gene clusters that are often silent under standard growth conditions. There are limited resources for characterizing the direct link between abiotic stimuli and metabolite production. Herein, we introduce a network analysis-based, data-driven algorithm comprising two routes to characterize the production of specialized fungal metabolites triggered by different exogenous compounds: the direct route and the auxiliary route. Both routes elucidate the influence of treatments on the production of specialized metabolites from experimental data. The direct route determines known and putative metabolites induced by treatments and provides additional insight over traditional comparison methods. The auxiliary route is specific for discovering unknown analytes, and further identification can be curated through online bioinformatic resources. We validated our algorithm by applying chitooligosaccharides and lipids at two different temperatures to the fungal pathogen Aspergillus fumigatus. After liquid chromatography–mass spectrometry quantification of significantly produced analytes, we used network centrality measures to rank the treatments’ ability to elucidate these analytes and confirmed their identity through fragmentation patterns or in silico spiking with commercially available standards. Later, we examined the transcriptional regulation of these metabolites through real-time quantitative polymerase chain reaction. Our data-driven techniques can complement existing metabolomic network analysis by providing an approach to track the influence of any exogenous stimuli on metabolite production. Our experimental-based algorithm can overcome the bottlenecks in elucidating novel fungal compounds used in drug discovery.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Mechanisms of metabolic adaptation in the duckweed Lemna gibba: an integrated metabolic, transcriptomic and flux analysis

Abstract Background Duckweeds are small, rapidly growing aquatic flowering plants. Due to their ability for biomass production at high rates they represent promising candidates for biofuel feedstocks. Duckweeds are also excellent model organisms because they can be maintained in well-defined liquid media, usually reproduce asexually, and because genomic resources are becoming increasingly available. To demonstrate the utility of duckweed for integrated metabolic studies, we examined the metabolic adaptation of growing Lemna gibba cultures to different nutritional conditions. Results To establish a framework for quantitative metabolic research in duckweeds we derived a central carbon metabolism network model of Lemna gibba based on its draft genome. Lemna gibba fronds were grown with nitrate or glutamine as nitrogen source. The two conditions were compared by quantification of growth kinetics, metabolite levels, transcript abundance, as well as by 13 C-metabolic flux analysis. While growing with glutamine, the fronds grew 1.4 times faster and accumulated more protein and less cell wall components compared to plants grown on nitrate. Characterization of photomixotrophic growth by 13 C-metabolic flux analysis showed that, under both metabolic growth conditions, the Calvin-Benson-Bassham cycle and the oxidative pentose-phosphate pathway are highly active, creating a futile cycle with net ATP consumption. Depending on the nitrogen source, substantial reorganization of fluxes around the tricarboxylic acid cycle took place, leading to differential formation of the biosynthetic precursors of the Asp and Gln families of proteinogenic amino acids. Despite the substantial reorganization of fluxes around the tricarboxylic acid cycle, flux changes could largely not be associated with changes in transcripts. Conclusions Through integrated analysis of growth rate, biomass composition, metabolite levels, and metabolic flux, we show that Lemna gibba is an excellent system for quantitative metabolic studies in plants. Our study showed that Lemna gibba adjusts to different nitrogen sources by reorganizing central metabolism. The observed disconnect between gene expression regulation and metabolism underscores the importance of metabolic flux analysis as a tool in such studies.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2016

MAX is an epigenetic sensor of 5-carboxylcytosine and is altered in multiple myeloma

The oncogenic transcription factor MYC and its binding partner MAX regulate gene expression by binding to DNA at enhancer-box (E-box) elements 5'-CACGTG-3'. In mammalian genomes, the central E-box CpG has the potential to be methylated at the 5-position of cytosine (5mC), or to undergo further oxidation to the 5-hydroxymethyl (5hmC), 5-formyl (5fC), or 5-carboxyl (5caC) forms. We find that MAX exhibits the greatest affinity for a 5caC or unmodified C-containing E-box, and much reduced affinities for the corresponding 5mC, 5hmC or 5fC forms. Crystallization of MAX with a 5caC modified E-box oligonucleotide revealed that MAX Arg36 recognizes 5caC using a 5caC–Arg–Guanine triad, with the next nearest residue to the carboxylate group being Arg60. In an analysis of >800 primary multiple myelomas, MAX alterations occurred at a frequency of ~3%, more than half of which were single nucleotide substitutions affecting a basic clamp-like interface important for DNA interaction. Among these, arginines 35, 36 and 60 were the most frequently altered. In vitro binding studies showed that whereas mutation of Arg36 (R36W) or Arg35 (R35H/L) completely abolished DNA binding, mutation of Arg60 (R60Q) significantly reduced DNA binding, but retained a preference for the 5caC modified E-box. Interestingly, MAX alterations define a subset of myeloma patients with lower MYC expression and a better overall prognosis. Together these data indicate that MAX can act as a direct epigenetic sensor of E-box cytosine modification states and that local CpG modification and MAX variants converge to modulate the MAX-MYC transcriptional network.

59 BASIC BIOLOGICAL SCIENCES↗