Search NASA⌕ Search

SEARCH · Search NASA

Results for “Mutational analysis”

Search indexed NASA NTRS and DOE OSTI research on propulsion, heat transfer, battery materials and energy systems. Follow report and document links to the original sources.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 217 records · Page 12
All sourcesNASA NTRSDOE OSTI
DOE OSTI2021

Predicting Antimicrobial Resistance Using Partial Genome Alignments

Antimicrobial resistance (AMR) is an important global health threat that impacts millions of people worldwide each year. Developing methods that can detect and predict AMR phenotypes can help to mitigate the spread of AMR by informing clinical decision making and appropriate mitigation strategies. Many bioinformatic methods have been developed for predicting AMR phenotypes from whole-genome sequences and AMR genes, but recent studies have indicated that predictions can be made from incomplete genome sequence data. In order to more systematically understand this, we built random forest-based machine learning classifiers for predicting susceptible and resistant phenotypes for Klebsiella pneumoniae (1,640 strains), Mycobacterium tuberculosis (2,497 strains), and Salmonella enterica (1,981 strains). We started by building models from alignments that were based on a reference chromosome for each species. We then subsampled each chromosomal alignment and built models for the resulting subalignments, finding that very small regions, representing approximately 0.1 to 0.2% of the chromosome, are predictive. In K. pneumoniae, M. tuberculosis, and S. enterica, the subalignments are able to predict multiple AMR phenotypes with at least 70% accuracy, even though most do not encode an AMR-related function. We used these models to identify regions of the chromosome with high and low predictive signals. Finally, subalignments that retain high accuracy across larger phylogenetic distances were examined in greater detail, revealing genes and intergenic regions with potential links to AMR, virulence, transport, and survival under stress conditions. IMPORTANCE Antimicrobial resistance causes thousands of deaths annually worldwide. Understanding the regions of the genome that are involved in antimicrobial resistance is important for developing mitigation strategies and preventing transmission. Machine learning models are capable of predicting antimicrobial resistance phenotypes from bacterial genome sequence data by identifying resistance genes, mutations, and other correlated features. They are also capable of implicating regions of the genome that have not been previously characterized as being involved in resistance. In this study, we generated global chromosomal alignments for Klebsiella pneumoniae, Mycobacterium tuberculosis, and Salmonella enterica and systematically searched them for small conserved regions of the genome that enable the prediction of antimicrobial resistance phenotypes. In addition to known antimicrobial resistance genes, this analysis identified genes involved in virulence and transport functions, as well as many genes with no previous implication in antimicrobial resistance.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Comparative Uptake Patterns of Radioactive Iodine and [18F]-Fluorodeoxyglucose (FDG) in Metastatic Differentiated Thyroid Cancers

Background: Metastatic differentiated thyroid cancer (DTC) represents a molecularly heterogeneous group of cancers with varying radioactive iodine (RAI) and [ 18 F]-fluorodeoxyglucose (FDG) uptake patterns potentially correlated with the degree of de-differentiation through the so-called “flip-flop” phenomenon. However, it is unknown if RAI and FDG uptake patterns correlate with molecular status or metastatic site. Materials and Methods: A retrospective analysis of metastatic DTC patients (n = 46) with radioactive 131-iodine whole body scan (WBS) and FDG-PET imaging between 2008 and 2022 was performed. The inclusion criteria included accessible FDG-PET and WBS studies within 1 year of each other. Studies were interpreted by two blinded radiologists for iodine or FDG uptake in extrathyroidal sites including lungs, lymph nodes, and bone. Cases were stratified by BRAF V600E mutation status, histology, and a combination of tumor genotype and histology. The data were analyzed by McNemar’s Chi-square test. Results: Lung metastasis FDG uptake was significantly more common than iodine uptake (WBS: 52%, FDG: 84%, p = 0.04), but no significant differences were found for lymph or bone metastases. Lung metastasis FDG uptake was significantly more prevalent in the papillary pattern sub-cohort (WBS: 37%, FDG: 89%, p = 0.02) than the follicular pattern sub-cohort (WBS: 75%, FDG: 75%, p = 1.00). Similarly, BRAF V600E+ tumors with lung metastases also demonstrated a preponderance of FDG uptake (WBS: 29%, FDG: 93%, p = 0.02) than BRAF V600E- tumors (WBS: 83%, FDG: 83%, p = 1.00) with lung metastases. Papillary histology featured higher FDG uptake in lung metastasis (WBS: 39%, FDG: 89%, p = 0.03) compared with follicular histology (WBS: 69%, FDG: 77%, p = 1.00). Patients with papillary pattern disease, BRAF V600E+ mutation, or papillary histology had reduced agreement between both modalities in uptake at all metastatic sites compared with those with follicular pattern disease, BRAF V600E- mutation, or follicular histology. Low agreement in lymph node uptake was observed in all patients irrespective of molecular status or histology. Conclusions: The pattern of FDG-PET and radioiodine uptake is dependent on molecular status and metastatic site, with those with papillary histology or BRAF V600E+ mutation featuring increased FDG uptake in distant metastasis. Further study with an expanded cohort may identify which patients may benefit from specific imaging modalities to recognize and surveil metastases.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2023

Ligand dependent interaction between PC-TP and PPARδ mitigates diet-induced hepatic steatosis in male mice

Phosphatidylcholine transfer protein (PC-TP; synonym StarD2) is a soluble lipid-binding protein that transports phosphatidylcholine (PC) between cellular membranes. To better understand the protective metabolic effects associated with hepatic PC-TP, we generated a hepatocyte-specific PC-TP knockdown (L-Pctp -/- ) in male mice, which gains less weight and accumulates less liver fat compared to wild-type mice when challenged with a high-fat diet. Hepatic deletion of PC-TP also reduced adipose tissue mass and decreases levels of triglycerides and phospholipids in skeletal muscle, liver and plasma. Gene expression analysis suggest that the observed metabolic changes are related to transcriptional activity of peroxisome proliferative activating receptor (PPAR) family members. An in-cell protein complementation screen between lipid transfer proteins and PPARs uncovered a direct interaction between PC-TP and PPARδ that was not observed for other PPARs. We confirmed the PC-TP– PPARδ interaction in Huh7 hepatocytes, where it was found to repress PPARδ-mediated transactivation. Mutations of PC-TP residues implicated in PC binding and transfer reduce the PC-TP-PPARδ interaction and relieve PC-TP-mediated PPARδ repression. Reduction of exogenously supplied methionine and choline reduces the interaction while serum starvation enhances the interaction in cultured hepatocytes. Together our data points to a ligand sensitive PC-TP– PPARδ interaction that suppresses PPAR activity.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2026

A Fluorescence‐Based Transient Expression Assay for the Analysis of Upstream Open Reading Frames in Plants

Upstream open reading frames (uORFs) are regulatory elements present in the 5′ leaders of mRNA that can significantly impact downstream gene expression in eukaryotes. In crop engineering, editing of uORFs can provide an avenue to upregulate expression of native genes without the need to add persistent transgenic copies. Even with genome-wide methods to identify translated uORFs such as ribosome profiling, their functional characterization depends on validation through reporter gene assays and mutagenesis studies. Current screening methods for plants use luciferases or protoplasts to measure differential gene expression between wild-type and mutated transcript leaders, which requires tissue processing and/or substrate addition. Here, we present a time- and cost-efficient alternative to investigate transcript leaders by co-expression of two fluorescent proteins in Nicotiana benthamiana leaf tissue and test our assay on genes involved in photoprotection, editing of which could provide a pathway to increase CO 2 assimilation during sun–shade transitions.

Nicotiana benthamiana↗
DOE OSTI2024

Structural and biochemical basis for regiospecificity of the flavonoid glycosyltransferase UGT95A1

Glycosylation is a predominant strategy plants use to fine-tune the properties of small molecule metabolites to affect their bioactivity, transport, and storage. It is also important in biotechnology and medicine as many glycosides are utilized in human health. Small molecule glycosylation is largely carried out by family 1 glycosyltransferases. Here, we report a structural and biochemical investigation of UGT95A1, a family 1 GT enzyme from Pilosella officinarum that exhibits a strong, unusual regiospecificity for the 3'-O position of flavonoid acceptor substrate luteolin. We obtained an apo crystal structure to help drive the analyses of a series of binding site mutants, revealing that while most residues are tolerant to mutations, key residues M145 and D464 are important for overall glycosylation activity. Interestingly, E347 is crucial for maintaining the strong preference for 3'-O glycosylation, while R462 can be mutated to increase regioselectivity. The structural determinants of regioselectivity were further confirmed in homologous enzymes. Our study also suggests that the enzyme contains large, highly dynamic, disordered regions. We showed that while most disordered regions of the protein have little to no implication in catalysis, the disordered regions conserved among investigated homologs are important to both the overall efficiency and regiospecificity of the enzyme. This report represents a comprehensive in-depth analysis of a family 1 GT enzyme with a unique substrate regiospecificity and may provide a basis for enzyme functional prediction and engineering.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2023

Dynamic conformational switching underlies TFIIH function in transcription and DNA repair and impacts genetic diseases

Transcription factor IIH (TFIIH) is a protein assembly essential for transcription initiation and nucleotide excision repair (NER). Yet, understanding of the conformational switching underpinning these diverse TFIIH functions remains fragmentary. TFIIH mechanisms critically depend on two translocase subunits, XPB and XPD. To unravel their functions and regulation, we build cryo-EM based TFIIH models in transcription- and NER-competent states. Using simulations and graph-theoretical analysis methods, we reveal TFIIH’s global motions, define TFIIH partitioning into dynamic communities and show how TFIIH reshapes itself and self-regulates depending on functional context. Our study uncovers an internal regulatory mechanism that switches XPB and XPD activities making them mutually exclusive between NER and transcription initiation. By sequentially coordinating the XPB and XPD DNA-unwinding activities, the switch ensures precise DNA incision in NER. Mapping TFIIH disease mutations onto network models reveals clustering into distinct mechanistic classes, affecting translocase functions, protein interactions and interface dynamics.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Analysis of the ARTIC Version 3 and Version 4 SARS-CoV-2 Primers and Their Impact on the Detection of the G142D Amino Acid Substitution in the Spike Protein

The ARTIC Network provides a common resource of PCR primer sequences and recommendations for amplifying SARS-CoV-2 genomes. The initial tiling strategy was developed with the reference genome Wuhan-01, and subsequent iterations have addressed areas of low amplification and sequence drop out. Recently, a new version (V4) was released, based on new variant genome sequences, in response to the realization that some V3 primers were located in regions with key mutations. Herein, we compare the performance of the ARTIC V3 and V4 primer sets with a matched set of 663 SARS-CoV-2 clinical samples sequenced with an Illumina NovaSeq 6000 instrument. We observe general improvements in sequencing depth and quality, and improved resolution of the SNP causing the D950N variation in the spike protein. Importantly, we also find nearly universal presence of spike protein substitution G142D in Delta-lineage samples. Due to the prior release and widespread use of the ARTIC V3 primers during the initial surge of the Delta variant, it is likely that the G142D amino acid substitution is substantially underrepresented among early Delta variant genomes deposited in public repositories. In addition to the improved performance of the ARTIC V4 primer set, this study also illustrates the importance of the primer scheme in downstream analyses.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Tracking ebolavirus genomic drift with a resequencing microarray

Filoviruses are emerging pathogens that cause acute fever with high fatality rate and present a global public health threat. During the 2013–2016 Ebola virus outbreak, genome sequencing allowed the study of virus evolution, mutations affecting pathogenicity and infectivity, and tracing the viral spread. In 2018, early sequence identification of the Ebolavirus as EBOV in the Democratic Republic of the Congo supported the use of an Ebola virus vaccine. However, field-deployable sequencing methods are needed to enable a rapid public health response. Resequencing microarrays (RMA) are a targeted method to obtain genomic sequence on clinical specimens rapidly, and sensitively, overcoming the need for extensive bioinformatic analysis. This study presents the design and initial evaluation of an ebolavirus resequencing microarray (Ebolavirus-RMA) system for sequencing the major genomic regions of four Ebolaviruses that cause disease in humans. The design of the Ebolavirus-RMA system is described and evaluated by sequencing repository samples of three Ebolaviruses and two EBOV variants. The ability of the system to identify genetic drift in a replicating virus was achieved by sequencing the ebolavirus glycoprotein gene in a recombinant virus cultured under pressure from a neutralizing antibody. Comparison of the Ebolavirus-RMA results to the Genbank database sequence file with the accession number given for the source RNA and Ebolavirus-RMA results compared to Next Generation Sequence results of the same RNA samples showed up to 99% agreement.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Lasofoxifene as a potential treatment for therapy-resistant ER-positive metastatic breast cancer

Background: Endocrine therapy remains the mainstay of treatment for estrogen receptor-positive (ER+) breast cancer. Constitutively active mutations in the ligand binding domain of ERα render tumors resistant to endocrine agents. Breast cancers with the two most common ERα mutations, Y537S and D538G, have low sensitivity to fulvestrant inhibition, a typical second-line endocrine therapy. Lasofoxifene is a selective estrogen receptor modulator with benefits on bone health and breast cancer prevention potential. This study investigated the anti-tumor activity of lasofoxifene in breast cancer xenografts expressing Y537S and D538G ERα mutants. The combination of lasofoxifene with palbociclib, a CDK4/6 inhibitor, was also evaluated. Methods: Luciferase-GFP tagged MCF7 cells bearing wild-type, Y537S, or D538G ERα were injected into the mammary ducts of NSG mice (MIND model), which were subsequently treated with lasofoxifene or fulvestrant as single agents or in combination with palbociclib. Tumor growth and metastasis were monitored with in vivo and ex vivo luminescence imaging, terminal tumor weight measurements, and histological analysis. Results: As a monotherapy, lasofoxifene was more effective than fulvestrant at inhibiting primary tumor growth and reducing metastases. Adding palbociclib improved the effectiveness of both lasofoxifene and fulvestrant for tumor suppression and metastasis prevention at four distal sites (lung, liver, bone, and brain), with the combination of lasofoxifene/palbociclib being generally more potent than that of fulvestrant/palbociclib. X-ray crystallography of the ERα ligand binding domain (LBD) shows that lasofoxifene stabilizes an antagonist conformation of both wild-type and Y537S LBD. The ability of lasofoxifene to promote an antagonist conformation of Y537S, combined with its long half-life and bioavailability, likely contributes to the observed potent inhibition of primary tumor growth and metastasis of MCF7 Y537S cells. Conclusions: We report for the first time the anti-tumor activity of lasofoxifene in mouse models of endocrine therapy-resistant breast cancer. The results demonstrate the potential of using lasofoxifene as an effective therapy for women with advanced or metastatic ER+ breast cancers expressing the most common constitutively active ERα mutations.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2023

Identification of ancestral gnathostome Gli3 enhancers with activity in mammals

Abnormal expression of the transcriptional regulator and hedgehog (Hh) signaling pathway effector Gli3 is known to trigger congenital disease, most frequently affecting the central nervous system (CNS) and the limbs. Accurate delineation of the genomic cis-regulatory landscape controlling Gli3 transcription during embryonic development is critical for the interpretation of noncoding variants associated with congenital defects. Here, we employed a comparative genomic analysis on fish species with a slow rate of molecular evolution to identify seven previously unknown conserved noncoding elements (CNEs) in Gli3 intronic intervals (CNE15–21). Transgenic assays in zebrafish revealed that most of these elements drive activities in Gli3 expressing tissues, predominantly the fins, CNS, and the heart. Intersection of these CNEs with human disease associated SNPs identified CNE15 as a putative mammalian craniofacial enhancer, with conserved activity in vertebrates and potentially affected by mutation associated with human craniofacial morphology. Finally, comparative functional dissection of an appendage-specific CNE conserved in slowly evolving fish (elephant shark), but not in teleost (CNE14/hs1586) indicates co-option of limb specificity from other tissues prior to the divergence of amniotes and lobe-finned fish. In conclusion, these results uncover a novel subset of intronic Gli3 enhancers that arose in the common ancestor of gnathostomes and whose sequence components were likely gradually modified in other species during the process of evolutionary diversification.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Structure of Rhizobium sp. 4-9 histamine dehydrogenase and analysis of the electron transfer pathway to an abiological electron acceptor

Histamine dehydrogenase from the gram-negative bacterium Rhizobium sp. 4-9 (HaDHR) is a member of a small family of dehydrogenases containing a covalently attached FMN, and the only member so far identified to date that does not exhibit substrate inhibition. Here, in this study, we present the 2.1 Å resolution crystal structure of HaDHR. This new structure allowed for the identification of the internal electron transfer pathway to abiological ferrocene-based mediators. Alanine 437 was identified as the exit point of electrons from the Fe 4 S 4 cluster. The enzyme was modified with a Ser436Cys mutation to facilitate covalent attachment of a ferrocene moiety. When modified with Fc-maleimide, this new construct demonstrated direct electron transfer from the enzyme to a gold electrode in a histamine concentration-dependent manner without the need for any additional electron mediators.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Soybean ( Glycine max ) Haplotype Map (GmHapMap): a universal resource for soybean translational and functional genomics

Here, we describe a worldwide haplotype map for soybean (GmHapMap) constructed using whole-genome sequence data for 1007 Glycine max accessions and yielding 14.9 million variants as well as 4.3 M tag single-nucleotide polymorphisms (SNPs). When sampling random subsets of these accessions, the number of variants and tag SNPs plateaued beyond approximately 800 and 600 accessions, respectively. This suggests extensive coverage of diversity within the cultivated soybean. GmHapMap variants were imputed onto 21 618 previously genotyped accessions with up to 96% success for common alleles. A local association analysis was performed with the imputed data using markers located in a 1-Mb region known to contribute to seed oil content and enabled us to identify a candidate causal SNP residing in the NPC1 gene. We determined gene-centric haplotypes (407 867 GCHs) for the 55 589 genes and showed that such haplotypes can help to identify alleles that differ in the resulting phenotype. Finally, we predicted 18 031 putative loss-of-function (LOF) mutations in 10 662 genes and illustrated how such a resource can be used to explore gene function. The GmHapMap provides a unique worldwide resource for applied soybean genomics and breeding.

54 ENVIRONMENTAL SCIENCES↗
DOE OSTI2022

Structure and conformational dynamics of Clostridioides difficile toxin A

Clostridioides difficile toxin A and B (TcdA and TcdB) are two major virulence factors responsible for diseases associated with C. difficile infection (CDI). Here, we report the 3.18-Å resolution crystal structure of a TcdA fragment (residues L843–T2481), which advances our understanding of the complete structure of TcdA holotoxin. Our structural analysis, together with complementary single molecule FRET and limited proteolysis studies, reveal that TcdA adopts a dynamic structure and its CROPs domain can sample a spectrum of open and closed conformations in a pH-dependent manner. Furthermore, a small globular subdomain (SGS) and the CROPs protect the pore-forming region of TcdA in the closed state at neutral pH, which could contribute to modulating the pH-dependent pore formation of TcdA. A rationally designed TcdA mutation that trapped the CROPs in the closed conformation showed drastically reduced cytotoxicity. Taken together, these studies shed new lights into the conformational dynamics of TcdA and its roles in TcdA intoxication.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Secure biosystems design in Saccharomyces cerevisiae establishes effective biocontainment strategies and mechanisms of escape

The widespread application of recombinant DNA and synthetic biology approaches for microbial metabolic engineering pursuits has motivated the development of biocontainment strategies, targeting safe and secure deployment of genetically modified microorganisms (GMMs). However, the design rules and mechanistic drivers governing biocontainment efficacy, as well as impacts of biocontainment upon microbial fitness, remain to be comprehensively evaluated, hindering predictive design and application of these strategies. We have developed a platform for high-resolution analysis of a transactivated kill switch in laboratory and industrial strains of Saccharomyces cerevisiae to assess modes of biocontainment escape and establish design rules for development of kill switch systems in diverse microbes. A camphor-regulated, RelE toxin system was systematically deployed to assess the impacts of differential kill switch copy number and ploidy in laboratory vs industrial strains. CRISPR-mediated integration of the biocontainment system at various loci revealed rapid escape events driven, in part, by mutations to both the Cam-transactivator (cam-TA) and RelE toxin. Genetic engineering enabled recapitulation of escape phenotypes, confirming mechanisms of escape and establishing structure-function relationships in the cam-TA system. Interestingly, genomic resequencing of escape mutants also revealed a series of off-target mutations, implicating additional modes of kill switch escape. Multi-copy integration of the kill switch system mitigated these effects by orders of magnitude, without compromising the biosynthetic capacity of the microbes, but proved insufficient to establish sustained biocontainment. The resultant data define a series of key design rules for next-generation biocontainment strategies and add to a growing foundational knowledge base targeting establishment of secure biosystems designs.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Bacteriophage-Induced Lipopolysaccharide Mutations in Escherichia coli Lead to Hypersensitivity to Food Grade Surfactant Sodium Dodecyl Sulfate

Bacteriophages (phages) are considered as one of the most promising antibiotic alternatives in combatting bacterial infectious diseases. However, one concern of employing phage application is the emergence of bacteriophage-insensitive mutants (BIMs). Here, we isolated six BIMs from E. coli B in the presence of phage T4 and characterized them using genomic and phenotypic methods. Of all six BIMs, a six-amino acid deletion in glucosyltransferase WaaG likely conferred phage resistance by deactivating the addition of T4 receptor glucose to the lipopolysaccharide (LPS). This finding was further supported by the impaired phage adsorption to BIMs and glycosyl composition analysis which quantitatively confirmed the absence of glucose in the LPS of BIMs. Since LPSs actively maintain outer membrane (OM) permeability, phage-induced truncations of LPSs destabilized the OM and sensitized BIMs to various substrates, especially to the food-grade surfactant sodium dodecyl sulfate (SDS). This hypersensitivity to SDS was exploited to design a T4–SDS combination which successfully prevented the generation of BIMs and eliminated the inoculated bacteria. Collectively, phage-driven modifications of LPSs immunized BIMs from T4 predation but increased their susceptibilities as a fitness cost. The findings of this study suggest a novel strategy to enhance the effectiveness of phage-based food safety interventions.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2025

The genome of the polyextremophilic yeast, Naganishia friedmannii, reveals adaptations involved in stress response pathways, carbohydrate metabolism expansion, and a limited DNA repair repertoire

Here we report the draft genome sequence of Naganishia friedmannii (formerly Cryptococcus friedmannii) isolate, a Basidiomycota yeast commonly found in some of the most extreme environments of the Earth's cryosphere. We isolated N. friedmannii strain Llullensis from soils at 6000 m above sea level on Volcán Llullaillaco, Argentina. The genome was 22.2 Mb with 6251 identified protein coding genes. Proteins known to be associated with thermal, osmotic, and radiation stress were identified in the genome. Comparative analysis with seven other Naganishia genomes revealed unique features underlying its polyextremophilic lifestyle. Naganishia friedmannii showed an expansion of genes involved in breaking down plant-derived carbohydrates, supporting the hypothesis that it survives at high elevations by metabolizing wind-deposited organic matter. Surprisingly, many genes involved in cell-cycle checkpoints and DNA repair were missing, as in several other Naganishia species. This extensive loss may be adaptive in extreme environments prone to abiotic stress, where a high mutation rate could generate advantageous traits, and reduced cell-cycle control may allow for faster reproduction that would be advantageous for rapid growth during brief periods of soil wetting following rare snow events.

Vimercati, Lara↗
DOE OSTI2021

Identification and analysis of sugar transporters capable of co–transporting glucose and xylose simultaneously

Simultaneous co-fermentation of glucose and xylose is a key desired trait of engineered Saccharomyces cerevisiae for efficient and rapid production of biofuels and chemicals. However, glucose strongly inhibits xylose transport by endogenous hexose transporters of S. cerevisiae. We identified structurally distant sugar transporters (Lipomyces starkeyi LST1_205437 and Arabidopsis thaliana AtSWEET7) capable of co-transporting glucose and xylose from previously unexplored oleaginous yeasts and plants. Kinetic analysis showed that LST1_205437 had lenient glucose inhibition on xylose transport and AtSWEET7 transported glucose and xylose simultaneously with no inhibition. Modelling studies of LST1_205437 revealed that Ala335 residue at sugar binding site can accommodates both glucose and xylose. Docking studies with AtSWEET7 revealed that Trp59, Trp183, Asn145, and Asn179 residues stabilized the interactions with sugars, allowing both xylose and glucose to be co-transported. In addition, we altered sugar preference of LST1_205437 by single amino acid mutation at Asn365. Furthermore, our findings provide a new mechanistic insight on glucose and xylose transport mechanism of sugar transporters and the identified sugar transporters can be employed to develop engineered yeast strains for producing cellulosic biofuels and chemicals.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

INDETERMINATE1 -mediated expression of FT family genes is required for proper timing of flowering in Brachypodium distachyon

The transition to flowering is a major developmental switch in plants. In many temperate grasses, perception of indicators of seasonal change, such as changing day-length and temperature, leads to expression of FLOWERING LOCUS T1 ( FT1 ) and FT-Like ( FTL ) genes that are essential for promoting the transition to flowering. However, little is known about the upstream regulators of FT1 and FTL genes in temperate grasses. Here, we characterize the monocot-specific gene INDETERMINATE1 ( BdID1 ) in Brachypodium distachyon and demonstrate that BdID1 is a regulator of FT family genes. Mutations in ID1 impact the ability of the short-day (SD) vernalization, cold vernalization, and long-day (LD) photoperiod pathways to induce certain FTL genes. BdID1 is required for upregulation of FTL9 ( FT-LIKE9 ) expression by the SD vernalization pathway, and overexpression of FTL9 in an id1 background can partially restore the delayed flowering phenotype of id1 . We show that BdID1 binds in vitro to the promoter region of FTL genes suggesting that ID1 directly activates FTL expression. Transcriptome analysis shows that BdID1 is required for FT1 , FT2 , FTL12 , and FTL13 expression under inductive LD photoperiods, indicating that BdID1 is a regulator of the FT gene family. Moreover, overexpression of FT1 in the id1 background results in rapid flowering similar to overexpressing FT1 in the wild type, demonstrating that BdID1 is upstream of FT family genes. Interestingly, ID1 negatively regulates a previously uncharacterized FTL gene, FTL4, and we show that FTL4 is a repressor of flowering. Thus, BdID1 is critical for proper timing of flowering in temperate grasses.

59 BASIC BIOLOGICAL SCIENCES↗