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At least 1,369 records · Page 76
All sourcesNASA NTRSDOE OSTI
DOE OSTI2022

Life and death in the soil microbiome: how ecological processes influence biogeochemistry

We report soil microorganisms shape global element cycles in life and death. Living soil microorganisms are a major engine of terrestrial biogeochemistry, driving the turnover of soil organic matter — Earth’s largest terrestrial carbon pool and the primary source of plant nutrients. Their metabolic functions are influenced by ecological interactions with other soil microbial populations, soil fauna and plants, and the surrounding soil environment. Remnants of dead microbial cells serve as fuel for these biogeochemical engines because their chemical constituents persist as soil organic matter. This non-living microbial biomass accretes over time in soil, forming one of the largest pools of organic matter on the planet. In this Review, we discuss how the biogeochemical cycling of organic matter depends on both living and dead soil microorganisms, their functional traits, and their interactions with the soil matrix and other organisms. With recent omics advances, many of the traits that frame microbial population dynamics and their ecophysiological adaptations can be deciphered directly from assembled genomes or patterns of gene or protein expression. Thus, it is now possible to leverage a trait-based understanding of microbial life and death within improved biogeochemical models and to better predict ecosystem functioning under new climate regimes.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2019

Identification and Characterization of Five Cold Stress-Related Rhododendron Dehydrin Genes: Spotlight on a FSK-Type Dehydrin With Multiple F-Segments

Dehydrins are a family of plant proteins that accumulate in response to dehydration stresses, such as low temperature, drought, high salinity, or during seed maturation. We have previously constructed cDNA libraries from Rhododendron catawbiense leaves of naturally non-acclimated (NA; leaf LT 50 , temperature that results in 50% injury of maximum, approximately -7 degrees C) and cold-acclimated (CA; leaf LT 50 approximately -50 degrees C) plants and analyzed expressed sequence tags (ESTs). Five ESTs were identified as dehydrin genes. Their full-length cDNA sequences were obtained and designated as RcDhn 1-5. To explore their functionality vis-a-vis winter hardiness, their seasonal expression kinetics was studied at two levels. Firstly, in leaves of R. catawbiense collected from the NA, CA, and de-acclimated (DA) plants corresponding to summer, winter and spring, respectively. Secondly, in leaves collected monthly from August through February, which progressively increased freezing tolerance from summer through mid-winter. The expression pattern data indicated that RcDhn 1-5 had 6- to 15-fold up-regulation during the cold acclimation process, followed by substantial down-regulation during deacclimation (even back to NA levels for some). Interestingly, our data shows RcDhn 5 contains a histidine-rich motif near N-terminus, a characteristic of metal-binding dehydrins. Equally important, RcDhn 2 contains a consensus 18 amino acid sequence (i.e., ETKDRGLFDFLGKKEEEE) near the N-terminus, with two additional copies upstream, and it is the most acidic (pI of 4.8) among the five RcDhns found. The core of this consensus 18 amino acid sequence is a 11-residue amino acid sequence (DRGLFDFLGKK), recently designated in the literature as the F-segment (based on the pair of hydrophobic F residues it contains). Furthermore, the 208 orthologs of F-segment-containing RcDhn 2 were identified across a broad range of species in GenBank database. This study expands our knowledge about the types of F-segment from the literature-reported single F-segment dehydrins (FSKn) to two or three F-segment dehydrins: Camelina sativa dehydrin ERD14 as F 2 S 2 K n type; and RcDhn 2 as F 3 SK n type identified here. Our results also indicate some consensus amino acid sequences flanking the core F-segment in dehydrins. Implications for these cold-responsive RcDhn genes in future genetic engineering efforts to improve plant cold hardiness are discussed.

09 BIOMASS FUELS↗
DOE OSTI2018

Olfactomedin-like protein OLFML1 inhibits Hippo signaling and mineralization in osteoblasts

Highlights: • A de novo mutation was identified in OLFML1 in a patient with congenital scoliosis. • Olfml1 is strongly expressed in calvarial osteoblasts in mice. • Knockdown of Olfml1 accelerates osteoblast mineralization. • Olfml1 knockdown induces nuclear translocation of YAP and activates Hippo signaling. • Olfml1 expression vector transfection retains YAP in the cytoplasm. Congenital scoliosis is a lateral curvature of the spine that is due to the presence of vertebral anomalies. Although genetic and environmental factors are involved in the pathogenesis of congenital scoliosis, the specific cause of only a small number of individuals has been identified to date. We identified a de novo missense mutation in the olfactomedin-like 1 (OLFML1) gene by whole-exome sequencing of a patient with congenital scoliosis. Then, we carried out further functional investigation in mice. An assessment of the tissue distribution of Olfml1 revealed it to be prominently expressed in developing skeletal tissues, specifically osteoblasts. Short hairpin RNA-mediated knockdown of Olfml1 in osteoblasts induced the translocation of Yes-associated protein (YAP) transcriptional coactivator from the cytoplasm to the nucleus, which accelerated the Hippo signaling pathway to promote osteoblast mineralization. In contrast, experimentally induced gain of function of Olfml1 retained YAP in the cytoplasm. There appears to exist a novel cell-autonomous mechanism by which osteoblasts avoid excess mineralization through Olfml1. Our results also indicate that mutation of OLFML1 leads to impaired osteoblast differentiation and abnormal development of bone tissue.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2023

Structure of VanS from vancomycin-resistant enterococci: A sensor kinase with weak ATP binding

The VanRS two-component system regulates the resistance phenotype of vancomycin-resistant enterococci. VanS is a sensor histidine kinase that responds to the presence of vancomycin by autophosphorylating and subsequently transferring the phosphoryl group to the response regulator, VanR. The phosphotransfer activates VanR as a transcription factor, which initiates the expression of resistance genes. Structural information about VanS proteins has remained elusive, hindering the molecular-level understanding of their function. Here, we present X-ray crystal structures for the catalytic and ATP-binding (CA) domains of two VanS proteins, derived from vancomycin-resistant enterococci types A and C. Both proteins adopt the canonical Bergerat fold that has been observed for CA domains of other prokaryotic histidine kinases. We attempted to determine structures for the nucleotide-bound forms of both proteins; however, despite repeated efforts, these forms could not be crystallized, prompting us to measure the proteins' binding affinities for ATP. Unexpectedly, both CA domains displayed low affinities for the nucleotide, with K D values in the low millimolar range. Since these K D values are comparable to intracellular ATP concentrations, this weak substrate binding could reflect a way of regulating expression of the resistance phenotype.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2018

Resveratrol promotes oxidative stress to drive DLC1 mediated cellular senescence in cancer cells

Highlights: • Resveratrol stimulates senescence-associated proteins to induce cellular senescence. • Resveratrol causes mitochondrial dysfunction to promote ROS production. • DLC1 is essential for resveratrol induced senescence by promoting ROS production. • DLC1 induces cellular senescence through FoxO3a/NF-κB signaling mediated by SIRT1. • ROS production as the cause of resveratrol to induce DNA damage in cancer cells. Induction of cellular senescence represents a novel strategy to inhibit aberrant proliferation of cancer cells. Resveratrol is gaining attention for its cancer preventive and suppressive properties. Tumor suppressor gene DLC1 is shown to induce apoptosis, suppress migration and invasion in various cancer cells. However, the function of DLC1 in cancer cellular senescence is unclear. This study was designed to investigate the biological role of DLC1 in resveratrol induced cancer cellular senescence. Our results showed that resveratrol inhibited proliferation of cancer cell lines (MCF-7, MDA-MB-231 and H1299) and induced senescence along with increase of SA-β-gal activity and regulation of senescence-associated molecular markers p38MAPK, p-p38MAPK, p27, p21, Rb and p-Rb protein. The underlying mechanism was that resveratrol induced mitochondrial dysfunction with reduction of mitochondrial membrane potential, down-regulation of MT-ND1, MT-ND6 and ATPase8 in transcript level and down-regulation of PGC-1α in protein level to result in ROS production. With ROS elevation, resveratrol decreased DNMT1 and increased DLC1 expression significantly. However, after ROS scavenger NAC was added to the cancer cells treated by resveratrol, DNMT1, DLC1 and senescence-associated molecular markers were reversed. This reveals that resveratrol induced cancer cellular senescence through DLC1 in a ROS-dependent manner. Silencing DLC1 markedly attenuated SA-β-gal activity and p38MAPK, p27 and p21 protein levels, and increased Rb expression, indicating that resveratrol promoted senescence via targeting DLC1. Moreover, DLC1 promoted senescence through FoxO3a/NF-κB signaling mediated by SIRT1 after resveratrol treatment. Finally, resveratrol increased ROS production to induce DNA damage with p-CHK1 up-regulation and result in cancer cellular senescence. This is the first time to investigate resveratrol induced cancer cellular senescence by primarily targeting DLC1. Induction of cellular senescence by resveratrol may represent a novel anticancer mechanism.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2023

Engineering Rhodosporidium toruloides for production of 3-hydroxypropionic acid from lignocellulosic hydrolysate

Microbial production of valuable bioproducts is a promising route towards green and sustainable manufacturing. The oleaginous yeast, Rhodosporidium toruloides, has emerged as an attractive host for the production of biofuels and bioproducts from lignocellulosic hydrolysates. 3-hydroxypropionic acid (3HP) is an attractive platform molecule that can be used to produce a wide range of commodity chemicals. This study focuses on establishing and optimizing the production of 3HP in R. toruloides. As R. toruloides naturally has a high metabolic flux towards malonyl-CoA, we exploited this pathway to produce 3HP. Upon finding the yeast capable of catabolizing 3HP, we then implemented functional genomics and metabolomic analysis to identify the catabolic pathways. Deletion of a putative malonate semialdehyde dehydrogenase gene encoding an oxidative 3HP pathway was found to significantly reduce 3HP degradation. We further explored monocarboxylate transporters to promote 3HP transport and identified a novel 3HP transporter in Aspergillus pseudoterreus by RNA-seq and proteomics. Combining these engineering efforts with media optimization in a fed-batch fermentation resulted in 45.4 g/L 3HP production. This represents one of the highest 3HP titers reported, and the highest titer produced from lignocellulosic hydrolysate to the best of our knowledge. This work demonstrates microbial production of 3HP in R. toruloides from lignocellulosic hydrolysate at high titers, and it represents a significant step toward enabling industrial production of 3HP in the future.

3-Hydroxypropionic acid↗
DOE OSTI2023

Detecting macroevolutionary genotype–phenotype associations using error-corrected rates of protein convergence

On macroevolutionary timescales, extensive mutations and phylogenetic uncertainty mask the signals of genotype–phenotype associations underlying convergent evolution. To overcome this problem, we extended the widely used framework of non-synonymous to synonymous substitution rate ratios and developed the novel metric ω C , which measures the error-corrected convergence rate of protein evolution. While ω C distinguishes natural selection from genetic noise and phylogenetic errors in simulation and real examples, its accuracy allows an exploratory genome-wide search of adaptive molecular convergence without phenotypic hypothesis or candidate genes. Using gene expression data, we explored over 20 million branch combinations in vertebrate genes and identified the joint convergence of expression patterns and protein sequences with amino acid substitutions in functionally important sites, providing hypotheses on undiscovered phenotypes. We further extended our method with a heuristic algorithm to detect highly repetitive convergence among computationally non-trivial higher-order phylogenetic combinations. Our approach allows bidirectional searches for genotype–phenotype associations, even in lineages that diverged for hundreds of millions of years.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Lanthanide-dependent alcohol dehydrogenases require an essential aspartate residue for metal coordination and enzymatic function

The lanthanide elements (Ln 3+ ), those with atomic numbers 57–63 (excluding promethium, Pm 3+ ), form a cofactor complex with pyrroloquinoline quinone (PQQ) in bacterial XoxF methanol dehydrogenases (MDHs) and ExaF ethanol dehydrogenases (EDHs), expanding the range of biological elements and opening novel areas of metabolism and ecology. Other MDHs, known as MxaFIs, are related in sequence and structure to these proteins, yet they instead possess a Ca 2+ -PQQ cofactor. An important missing piece of the Ln 3+ puzzle is defining what features distinguish enzymes that use Ln 3+ -PQQ cofactors from those that do not. In this work, using XoxF1 MDH from the model methylotrophic bacterium Methylorubrum extorquens AM1, we investigated the functional importance of a proposed lanthanide-coordinating aspartate residue. We report two crystal structures of XoxF1, one with and another without PQQ, both with La 3+ bound in the active-site region and coordinated by Asp 320 . Using constructs to produce either recombinant XoxF1 or its D320A variant, we show that Asp 320 is needed for in vivo catalytic function, in vitro activity, and La 3+ coordination. XoxF1 and XoxF1 D320A, when produced in the absence of La 3+ , coordinated Ca 2+ but exhibited little or no catalytic activity. We also generated the parallel substitution in ExaF to produce ExaF D319S and found that this variant loses the capacity for efficient ethanol oxidation with La 3+ . These results provide evidence that a Ln 3+ -coordinating aspartate is essential for the enzymatic functions of XoxF MDHs and ExaF EDHs, supporting the notion that sequences of these enzymes, and the genes that encode them, are markers for Ln 3+ metabolism.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2024

Novel order-level lineage of ammonia-oxidizing archaea widespread in marine and terrestrial environments

Ammonia-oxidizing archaea (AOA) are among the most ubiquitous and abundant archaea on Earth, widely distributed in marine, terrestrial, and geothermal ecosystems. However, the genomic diversity, biogeography, and evolutionary process of AOA populations in subsurface environments are vastly understudied compared to those in marine and soil systems. Here, we report a novel AOA order Candidatus (Ca.) Nitrosomirales which forms a sister lineage to the thermophilic Ca. Nitrosocaldales. Metagenomic and 16S rRNA gene-read mapping demonstrates the abundant presence of Nitrosomirales AOA in various groundwater environments and their widespread distribution across a range of geothermal, terrestrial, and marine habitats. Terrestrial Nitrosomirales AOA show the genetic capacity of using formate as a source of reductant and using nitrate as an alternative electron acceptor. Nitrosomirales AOA appear to have acquired key metabolic genes and operons from other mesophilic populations via horizontal gene transfer, including genes encoding urease, nitrite reductase, and V-type ATPase. The additional metabolic versatility conferred by acquired functions may have facilitated their radiation into a variety of subsurface, marine, and soil environments. We also provide evidence that each of the four AOA orders spans both marine and terrestrial habitats, which suggests a more complex evolutionary history for major AOA lineages than previously proposed. Together, these findings establish a robust phylogenomic framework of AOA and provide new insights into the ecology and adaptation of this globally abundant functional guild.

54 ENVIRONMENTAL SCIENCES↗
DOE OSTI2024

Major impacts of widespread structural variation on sorghum

Genetic diversity is critical to crop breeding and improvement, and dissection of the genomic variation underlying agronomic traits can both assist breeding and give insight into basic biological mechanisms. Although recent genome analyses in plants reveal many structural variants (SVs), most current studies of crop genetic variation are dominated by single-nucleotide polymorphisms (SNPs). The extent of the impact of SVs on global trait variation, as well as their utility in genome-wide selection, is not yet understood. In this study, we built an SV data set based on whole-genome resequencing of diverse sorghum lines (n = 363), validated the correlation of photoperiod sensitivity and variety type, and identified SV hotspots underlying the divergent evolution of cellulosic and sweet sorghum. In addition, we showed the complementary contribution of SVs for heritability of traits related to sorghum adaptation. Importantly, inclusion of SV polymorphisms in association studies revealed genotype–phenotype associations not observed with SNPs alone. Three-way genome-wide association studies (GWAS) based on whole-genome SNP, SV, and integrated SNP + SV data sets showed substantial associations between SVs and sorghum traits. The addition of SVs to GWAS substantially increased heritability estimates for some traits, indicating their important contribution to functional allelic variation at the genome level. Our discovery of the widespread impacts of SVs on heritable gene expression variation could render a plausible mechanism for their disproportionate impact on phenotypic variation. This study expands our knowledge of SVs and emphasizes the extensive impacts of SVs on sorghum.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Populus PtrbHLH011 Is a Transcriptional Co‐Regulator Involved in the Activation of Cell Wall Biosynthesis by Iron Deprivation

The lack of a mechanistic understanding of the environmental plasticity of secondary cell wall (SCW) biosynthesis restricts large-scale biomass and bioenergy production on marginal lands. Using Populus (poplar), a key bioenergy crop, we discovered that iron deprivation, a prevalent abiotic stress on marginal lands, stimulates SCW biosynthesis in stems. We identified the transcription factor PtrbHLH011 as a critical regulator underlying this response. Through integrated analyses involving phenotypic characterisation of PtrbHLH011 knockout and overexpression plants, functional genomics and molecular investigations, we established that PtrbHLH011 functions as a central regulator of SCW biosynthesis, iron homeostasis and flavonoid biosynthesis by directly repressing essential genes in these pathways. Iron deprivation downregulates PtrbHLH011 expression, subsequently activating these biosynthetic pathways. Notably, cytosine base editing-based knockout of PtrbHLH011 significantly enhanced plant growth, yielding up to a 110% increase in stem diameter and a 300% increase in leaf iron content. These findings present a novel regulatory mechanism linking environmental iron availability to SCW biosynthesis and illustrate a practical strategy to improve biomass yield on iron-deficient marginal lands. Furthermore, our mechanistic insights into PtrbHLH011 target recognition and regulation provide a valuable foundation for precise manipulation of gene regulatory networks, facilitating the development of high-performance bioenergy crops adapted to marginal environments.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2019

Metaphenomic Responses of a Native Prairie Soil Microbiome to Moisture Perturbations

Climate change is causing shifts in precipitation patterns in the central grasslands of the United States, with largely unknown consequences on the collective physiological responses of the soil microbial community, i.e., the metaphenome. Here, we used an untargeted omics approach to determine the soil microbial community’s metaphenomic response to soil moisture and to define specific metabolic signatures of the response. Specifically, we aimed to develop the technical approaches and metabolic mapping framework necessary for future systematic ecological studies. We collected soil from three locations at the Konza Long-Term Ecological Research (LTER) field station in Kansas, and the soils were incubated for 15 days under dry or wet conditions and compared to field-moist controls. The microbiome response to wetting or drying was determined by 16S rRNA amplicon sequencing, metatranscriptomics, and metabolomics, and the resulting shifts in taxa, gene expression, and metabolites were assessed. Soil drying resulted in significant shifts in both the composition and function of the soil microbiome. In contrast, there were few changes following wetting. The combined metabolic and metatranscriptomic data were used to generate reaction networks to determine the metaphenomic response to soil moisture transitions. Site location was a strong determinant of the response of the soil microbiome to moisture perturbations. However, some specific metabolic pathways changed consistently across sites, including an increase in pathways and metabolites for production of sugars and other osmolytes as a response to drying. Using this approach, we demonstrate that despite the high complexity of the soil habitat, it is possible to generate insight into the effect of environmental change on the soil microbiome and its physiology and functions, thus laying the groundwork for future, targeted studies. Climate change is predicted to result in increased drought extent and intensity in the highly productive, former tallgrass prairie region of the continental United States. These soils store large reserves of carbon. The decrease in soil moisture due to drought has largely unknown consequences on soil carbon cycling and other key biogeochemical cycles carried out by soil microbiomes. In this study, we found that soil drying had a significant impact on the structure and function of soil microbial communities, including shifts in expression of specific metabolic pathways, such as those leading toward production of osmoprotectant compounds. This study demonstrates the application of an untargeted multi-omics approach to decipher details of the soil microbial community’s metaphenotypic response to environmental perturbations and should be applicable to studies of other complex microbial systems as well.

54 ENVIRONMENTAL SCIENCES↗
DOE OSTI2023

Precision engineering of biological function with large-scale measurements and machine learning

As synthetic biology expands and accelerates into real-world applications, methods for quantitatively and precisely engineering biological function become increasingly relevant. This is particularly true for applications that require programmed sensing to dynamically regulate gene expression in response to stimuli. However, few methods have been described that can engineer biological sensing with any level of quantitative precision. Here, we present two complementary methods for precision engineering of genetic sensors: in silico selection and machine-learning-enabled forward engineering. Both methods use a large-scale genotype-phenotype dataset to identify DNA sequences that encode sensors with quantitatively specified dose response. First, we show that in silico selection can be used to engineer sensors with a wide range of dose-response curves. To demonstrate in silico selection for precise, multi-objective engineering, we simultaneously tune a genetic sensor’s sensitivity (EC 50 ) and saturating output to meet quantitative specifications. In addition, we engineer sensors with inverted dose-response and specified EC 50 . Second, we demonstrate a machine-learning-enabled approach to predictively engineer genetic sensors with mutation combinations that are not present in the large-scale dataset. We show that the interpretable machine learning results can be combined with a biophysical model to engineer sensors with improved inverted dose-response curves.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2018

Flux, Impact, and Fate of Halogenated Xenobiotic Compounds in the Gut

Humans and their associated microbiomes are exposed to numerous xenobiotics through drugs, dietary components, personal care products as well as environmental chemicals. Most of the reciprocal interactions between the microbiota and xenobiotics, such as halogenated compounds, occur within the human gut harboring diverse and dense microbial communities. Here, we provide an overview of the flux of halogenated compounds in the environment, and diverse exposure routes of human microbiota to these compounds. Subsequently, we review the impact of halogenated compounds in perturbing the structure and function of gut microbiota and host cells. In turn, cultivation-dependent and metagenomic surveys of dehalogenating genes revealed the potential of the gut microbiota to chemically alter halogenated xenobiotics and impact their fate. Finally, we provide an outlook for future research to draw attention and attract interest to study the bidirectional impact of halogenated and other xenobiotic compounds and the gut microbiota.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2026

Decreases in polyunsaturated fatty acid content improve heat stress tolerance during flowering and silicle development in pennycress (Thlaspi arvense L.)

Introduction: Pennycress (Thlaspi arvense L.) is an emerging intermediate oilseed crop grown in the offseason between primary summer crops to produce three cash crops in two years. Previous efforts to improve seed oil quality produced Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) genome-edited lines with decreased polyunsaturated fatty acids (PUFAs) levels, through loss of function of the FATTY ACID DESATURASE 2 (FAD2), REDUCED OLEATE DESATURATION1 (ROD1), and FATTY ACID ELONGASE1 (FAE1) genes. While seed oil compositions were previously characterized, it remains unknown how vegetative and reproductive tissue compositions might differ and affect tolerance to high temperature (HT) conditions.Methods: In four growth chamber experiments, we explored HT tolerance during flowering and silicle development. Plants were subjected to a 34 °C day/28 °C night regime and compared to control plants maintained at 20 °C. Pollen grain viability at a range of temperatures, lipid peroxidation and proline content in leaves and silicles following HT, and seed yield were measured.Results: Both fad2 and rod1 mutant lines had relatively higher pollen viability (71% and 54% respectively) under moderately elevated temperature (28 °C) compared to wild-type controls (37%). They also showed smaller decreases in seed yield (0% and 40% for fad2 and rod1 respectively, compared to 61% for wild type), following HT exposure during late flowering and early silicle development. Silicles of fad2 plants experienced 65% less lipid peroxidation under HT and 55% less buildup of proline, signifying less stress.Discussion: The differential results of fad2 and rod1 are likely due to the role of FAD2 in membranes in all tissues, whereas ROD1 predominantly affects triacylglycerol (TAG) composition in oil-accumulating tissues including pollen. Our results indicate that decreasing PUFAs, through gene editing, can increase heat tolerance in reproductive tissues as an auxiliary benefit accompanying improved seed oil quality.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2018

Proteogenomic Analysis of Burkholderia Species Strains 25 and 46 Isolated from Uraniferous Soils Reveals Multiple Mechanisms to Cope with Uranium Stress

Two Burkholderia spp. (strains SRS-25 and SRS-46) were isolated from high concentrations of uranium (U) from the U.S. Department of Energy (DOE)-managed Savannah River Site (SRS). SRS contains soil gradients that remain co-contaminated by heavy metals from previous nuclear weapons production activities. Uranium (U) is one of the dominant contaminants within the SRS impacted soils, which can be microbially transformed into less toxic forms. We established microcosms containing strains SRS-25 and SRS-46 spiked with U and evaluated the microbially-mediated depletion with concomitant genomic and proteomic analysis. Both strains showed a rapid depletion of U; draft genome sequences revealed SRS-25 genome to be of approximately 8,152,324 bp, a G + C content of 66.5, containing a total 7604 coding sequences with 77 total RNA genes. Similarly, strain SRS-46 contained a genome size of 8,587,429 bp with a G + C content of 67.1, 7895 coding sequences, with 73 total RNA genes, respectively. An in-depth, genome-wide comparisons between strains 25, 46 and a previously isolated strain from our research (Burkholderia sp. strain SRS-W-2-2016), revealed a common pool of 3128 genes; many were found to be homologues to previously characterized metal resistance genes (e.g., for cadmium, cobalt, and zinc), as well as for transporter, stress/detoxification, cytochromes, and drug resistance functions. Furthermore, proteomic analysis of strains with or without U stress, revealed the increased expression of 34 proteins from strain SRS-25 and 52 proteins from strain SRS-46; similar to the genomic analyses, many of these proteins have previously been shown to function in stress response, DNA repair, protein biosynthesis and metabolism. Overall, this comparative proteogenomics study confirms the repertoire of metabolic and stress response functions likely rendering the ecological competitiveness to the isolated strains for colonization and survival in the heavy metals contaminated SRS soil habitat.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Unveiling the Arsenal of Apple Bitter Rot Fungi: Comparative Genomics Identifies Candidate Effectors, CAZymes, and Biosynthetic Gene Clusters in Colletotrichum Species

The bitter rot of apple is caused by Colletotrichum spp. and is a serious pre-harvest disease that can manifest in postharvest losses on harvested fruit. In this study, we obtained genome sequences from four different species, C. chrysophilum, C. noveboracense, C. nupharicola, and C. fioriniae, that infect apple and cause diseases on other fruits, vegetables, and flowers. Our genomic data were obtained from isolates/species that have not yet been sequenced and represent geographic-specific regions. Genome sequencing allowed for the construction of phylogenetic trees, which corroborated the overall concordance observed in prior MLST studies. Bioinformatic pipelines were used to discover CAZyme, effector, and secondary metabolic (SM) gene clusters in all nine Colletotrichum isolates. We found redundancy and a high level of similarity across species regarding CAZyme classes and predicted cytoplastic and apoplastic effectors. SM gene clusters displayed the most diversity in type and the most common cluster was one that encodes genes involved in the production of alternapyrone. Our study provides a solid platform to identify targets for functional studies that underpin pathogenicity, virulence, and/or quiescence that can be targeted for the development of new control strategies. With these new genomics resources, exploration via omics-based technologies using these isolates will help ascertain the biological underpinnings of their widespread success and observed geographic dominance in specific areas throughout the country.

59 BASIC BIOLOGICAL SCIENCES↗
NASA NTRS2000

Developmental evolutionary biology of the vertebrate ear: conserving mechanoelectric transduction and developmental pathways in diverging morphologies

This brief overview shows that a start has been made to molecularly dissect vertebrate ear development and its evolutionary conservation to the development of the insect hearing organ. However, neither the patterning process of the ear nor the patterning process of insect sensory organs is sufficiently known at the moment to provide more than a first glimpse. Moreover, hardly anything is known about otocyst development of the cephalopod molluscs, another triploblast lineage that evolved complex 'ears'. We hope that the apparent conserved functional and cellular components present in the ciliated sensory neurons/hair cells will also be found in the genes required for vertebrate ear and insect sensory organ morphogenesis (Fig. 3). Likewise, we expect that homologous pre-patterning genes will soon be identified for the non-sensory cell development, which is more than a blocking of neuronal development through the Delta/Notch signaling system. Generation of the apparently unique ear could thus represent a multiplication of non-sensory cells by asymmetric and symmetric divisions as well as modification of existing patterning process by implementing novel developmental modules. In the final analysis, the vertebrate ear may come about by increasing the level of gene interactions in an already existing and highly conserved interactive cascade of bHLH genes. Since this was apparently achieved in all three lineages of triploblasts independently (Fig. 3), we now need to understand how much of the morphogenetic cascades are equally conserved across phyla to generate complex ears. The existing mutations in humans and mice may be able to point the direction of future research to understand the development of specific cell types and morphologies in the formation of complex arthropod, cephalopod, and vertebrate 'ears'.

Non-NASA Center↗