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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.

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All sourcesNASA NTRSDOE OSTI
DOE OSTI2021

SPOP mutation induces replication over-firing by impairing Geminin ubiquitination and triggers replication catastrophe upon ATR inhibition

Geminin and its binding partner Cdt1 are essential for the regulation of DNA replication. Here we show that the CULLIN3 E3 ubiquitin ligase adaptor protein SPOP binds Geminin at endogenous level and regulates DNA replication. SPOP promotes K27-linked non-degradative poly-ubiquitination of Geminin at lysine residues 100 and 127. This poly-ubiquitination of Geminin prevents DNA replication over-firing by indirectly blocking the association of Cdt1 with the MCM protein complex, an interaction required for DNA unwinding and replication. SPOP is frequently mutated in certain human cancer types and implicated in tumorigenesis. We show that cancer-associated SPOP mutations impair Geminin K27-linked poly-ubiquitination and induce replication origin over-firing and re-replication. The replication stress caused by SPOP mutations triggers replication catastrophe and cell death upon ATR inhibition. Our results reveal a tumor suppressor role of SPOP in preventing DNA replication over-firing and genome instability and suggest that SPOP-mutated tumors may be susceptible to ATR inhibitor therapy.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Section-level genome sequencing and comparative genomics of Aspergillus sections Cavernicolus and Usti.

The genus Aspergillus is diverse, including species of industrial importance, human pathogens, plant pests, and model organisms. Aspergillus includes species from sections Usti and Cavernicolus, which until recently were joined in section Usti, but have now been proposed to be non-monophyletic and were split by section Nidulantes, Aenei and Raperi. To learn more about these sections, we have sequenced the genomes of 13 Aspergillus species from section Cavernicolus (A. cavernicola, A. californicus, and A. egyptiacus), section Usti (A. carlsbadensis, A. germanicus, A. granulosus, A. heterothallicus, A. insuetus, A. keveii, A. lucknowensis, A. pseudodeflectus and A. pseudoustus), and section Nidulantes (A. quadrilineatus, previously A. tetrazonus). We compared these genomes with 16 additional species from Aspergillus to explore their genetic diversity, based on their genome content, repeat-induced point mutations (RIPs), transposable elements, carbohydrate-active enzyme (CAZyme) profile, growth on plant polysaccharides, and secondary metabolite gene clusters (SMGCs). All analyses support the split of section Usti and provide additional insights: Analyses of genes found only in single species show that these constitute genes which appear to be involved in adaptation to new carbon sources, regulation to fit new niches, and bioactive compounds for competitive advantages, suggesting that these support species differentiation in Aspergillus species. Sections Usti and Cavernicolus have mainly unique SMGCs. Section Usti contains very large and information-rich genomes, an expansion partially driven by CAZymes, as section Usti contains the most CAZyme-rich species seen in genus Aspergillus. Section Usti is clearly an underutilized source of plant biomass degraders and shows great potential as industrial enzyme producers. Citation: Nybo JL, Vesth TC, Theobald S, Frisvad JC, Larsen TO, Kjaerboelling I, Rothschild-Mancinelli K, Lyhne EK, Barry K, Clum A, Yoshinaga Y, Ledsgaard L, Daum C, Lipzen A, Kuo A, Riley R, Mondo S, LaButti K, Haridas S, Pangalinan J, Salamov AA, Simmons BA, Magnuson JK, Chen J, Drula E, Henrissat B, Wiebenga A, Lubbers RJM, Müller A, dos Santos Gomes AC, Mäkelä MR, Stajich JE, Grigoriev IV, Mortensen UH, de Vries RP, Baker SE, Andersen MR (2025). Section-level genome sequencing and comparative genomics of Aspergillus sections Cavernicolus and Usti. Studies in Mycology 111: 101-114. doi: 10.3114/sim.2025.111.03.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Adaptive Laboratory Evolution for Enhanced Performance of Cupriavidus Necator on Formic Acid

The threat of global warming, driven by rising carbon emissions, highlights the need to decarbonize our economy. This requires innovative solutions for managing carbon waste and its effective utilization. One promising method for CO2 capture and sequestration is the electrochemical reduction of CO2 to formic acid, a soluble C1 molecule that can be used to store carbon and energy, and as a feedstock for biological conversion. Cupriavidus necator H16, a soil bacterium capable of consuming and growing on formic acid as its sole carbon and energy source, is well positioned to upgrade CO2-derived formic acid into platform chemicals and fuel precursors. To improve the performance of C. necator on formic acid, adaptive laboratory evolution (ALE), a proven tool for improving microbial fitness, has been conducted using continuous pH-stat bioreactors. The system works on the basis that consumption of formic acid raises the pH and triggers the addition of more formic acid to maintain the pH (in this case 6.7), such that formic acid is provided at the same rate as it is consumed. This system has been coupled with level control to achieve continuous fermentation where cells acquiring mutations that improve growth on formic acid become more abundant in the population, from which they can be isolated and characterized. During developmental experiments it was discovered that formic acid accumulated to inhibitory levels. It was determined that the nitrogen source, ammonium hydroxide, must be tailored to the carbon consumption to avoid formic acid accumulation. The ALE ran in three lineages for approximately 3000 hours and more than 500 generations. Evolved isolates obtained from each lineage demonstrated an increase in growth rate in conjunction with improve formate utilization compared to the parental strain when evaluated in pH-stat bioreactors. The isolates with improved performance were then subjected to whole genome sequencing to identify potentially causative mutations. Mutations in several key genes across different lineages have been found and will be evaluated individually and in combination to identify those that improve growth on formic acid. Incorporating these mutations into production strains has the potential to greatly improve formic acid conversion and further industrial decarbonization.

adaptive laboratory evolution↗
DOE OSTI2024

Adaptive Laboratory Evolution for Enhanced Performance of Cupriavidus necator on Formic Acid

The need to decarbonize our economy is becoming ever more pressing. CO2, can be electrochemically reduced to formic acid, a soluble C1 molecule that can be used to store carbon and energy. Cupriavidus necator H16, a soil bacterium, capable of consuming and growing on formic acid as its sole carbon source, is well positioned to upgrade CO2-derived formic acid into value added chemicals such as sustainable aviation fuel. To improve the performance of C. necator on formic acid adaptive laboratory evolution (ALE), a proven tool for improving microbial fitness, has been conducted using continuous pH-stat bioreactors. The system works on the basis that consumption of formic acid raises the pH and triggers the addition of more formic acid to maintain the pH at 6.7, such that formic acid is provided at the same rate as it is consumed. This system has been coupled with level control to achieve continuous fermentation where cells acquiring mutations that improve growth on formic acid become more abundant in the population, from which they can be isolate and characterized. This configuration where the dilution rate is controlled by pH-stat has been found to seek out a dilution rate that matches the exponential growth rate of the organism. During developmental experiments it was discovered that formic acid accumulated to inhibitory levels. It was determined that the nitrogen source, ammonium hydroxide, must be tailored to the carbon consumption to avoid formic acid accumulation. ALE has run in three lineages for 2000+ hours and 300+ generations. Growth of evolved isolates derived from all lineages will be evaluated on formic acid and those with improved growth rates or biomass yield will be subjected to whole genome sequencing to identify potentially causative mutations. These mutations will be evaluated individually and in combination to identify those that improve growth on formic acid.

adaptive laboratory evolution↗
DOE OSTI2024

Describing patterns of familial cancer risk in subfertile men using population pedigree data

STUDY QUESTION Can we simultaneously assess risk for multiple cancers to identify familial multicancer patterns in families of azoospermic and severely oligozoospermic men? SUMMARY ANSWER Here, distinct familial cancer patterns were observed in the azoospermia and severe oligozoospermia cohorts, suggesting heterogeneity in familial cancer risk by both type of subfertility and within subfertility type. WHAT IS KNOWN ALREADY Subfertile men and their relatives show increased risk for certain cancers including testicular, thyroid, and pediatric. STUDY DESIGN, SIZE, DURATION A retrospective cohort of subfertile men (N = 786) was identified and matched to fertile population controls (N = 5674). Family members out to third-degree relatives were identified for both subfertile men and fertile population controls (N = 337 754). The study period was 1966–2017. Individuals were censored at death or loss to follow-up, loss to follow-up occurred if they left Utah during the study period. PARTICIPANTS/MATERIALS, SETTING, METHODS Azoospermic (0 × 10 6 /mL) and severely oligozoospermic (<1.5 × 10 6 /mL) men were identified in the Subfertility Health and Assisted Reproduction and the Environment cohort (SHARE). Subfertile men were age- and sex-matched 5:1 to fertile population controls and family members out to third-degree relatives were identified using the Utah Population Database (UPDB). Cancer diagnoses were identified through the Utah Cancer Registry. Families containing ≥10 members with ≥1 year of follow-up 1966–2017 were included (azoospermic: N = 426 families, 21 361 individuals; oligozoospermic: N = 360 families, 18 818 individuals). Unsupervised clustering based on standardized incidence ratios for 34 cancer phenotypes in the families was used to identify familial multicancer patterns; azoospermia and severe oligospermia families were assessed separately. MAIN RESULTS AND THE ROLE OF CHANCE Compared to control families, significant increases in cancer risks were observed in the azoospermia cohort for five cancer types: bone and joint cancers hazard ratio (HR) = 2.56 (95% CI = 1.48–4.42), soft tissue cancers HR = 1.56 (95% CI = 1.01–2.39), uterine cancers HR = 1.27 (95% CI = 1.03–1.56), Hodgkin lymphomas HR = 1.60 (95% CI = 1.07–2.39), and thyroid cancer HR = 1.54 (95% CI = 1.21–1.97). Among severe oligozoospermia families, increased risk was seen for three cancer types: colon cancer HR = 1.16 (95% CI = 1.01–1.32), bone and joint cancers HR = 2.43 (95% CI = 1.30–4.54), and testis cancer HR = 2.34 (95% CI = 1.60–3.42) along with a significant decrease in esophageal cancer risk HR = 0.39 (95% CI = 0.16–0.97). Thirteen clusters of familial multicancer patterns were identified in families of azoospermic men, 66% of families in the azoospermia cohort showed population-level cancer risks, however, the remaining 12 clusters showed elevated risk for 2-7 cancer types. Several of the clusters with elevated cancer risks also showed increased odds of cancer diagnoses at young ages with six clusters showing increased odds of adolescent and young adult (AYA) diagnosis [odds ratio (OR) = 1.96–2.88] and two clusters showing increased odds of pediatric cancer diagnosis (OR = 3.64–12.63). Within the severe oligozoospermia cohort, 12 distinct familial multicancer clusters were identified. All 12 clusters showed elevated risk for 1–3 cancer types. An increase in odds of cancer diagnoses at young ages was also seen in five of the severe oligozoospermia familial multicancer clusters, three clusters showed increased odds of AYA diagnosis (OR = 2.19–2.78) with an additional two clusters showing increased odds of a pediatric diagnosis (OR = 3.84–9.32). LIMITATIONS, REASONS FOR CAUTION Although this study has many strengths, including population data for family structure, cancer diagnoses and subfertility, there are limitations. First, semen measures are not available for the sample of fertile men. Second, there is no information on medical comorbidities or lifestyle risk factors such as smoking status, BMI, or environmental exposures. Third, all of the subfertile men included in this study were seen at a fertility clinic for evaluation. These men were therefore a subset of the overall population experiencing fertility problems and likely represent those with the socioeconomic means for evaluation by a physician. WIDER IMPLICATIONS OF THE FINDINGS This analysis leveraged unique population-level data resources, SHARE and the UPDB, to describe novel multicancer clusters among the families of azoospermic and severely oligozoospermic men. Distinct overall multicancer risk and familial multicancer patterns were observed in the azoospermia and severe oligozoospermia cohorts, suggesting heterogeneity in cancer risk by type of subfertility and within subfertility type. Describing families with similar cancer risk patterns provides a new avenue to increase homogeneity for focused gene discovery and environmental risk factor studies. Such discoveries will lead to more accurate risk predictions and improved counseling for patients and their families. STUDY FUNDING/COMPETING INTEREST(S) This work was funded by GEMS: Genomic approach to connecting Elevated germline Mutation rates with male infertility and Somatic health (Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD): R01 HD106112). The authors have no conflicts of interest relevant to this work.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2026

Multilayered regulation by RNA thermometers enables precise control of Cas9 expression in E. coli

Cas9-based genome editing technologies can rapidly generate mutations to probe a diverse array of mutant genotypes. However, aberrant Cas9 nuclease translation and activity can occur despite the use of inducible promoters to control expression, leading to extensive cell death. This background killing caused by promoter leakiness severely limits the application of Cas9 for generating mutant libraries because of the potential for population skew. We demonstrate the utility of temperature sensitive RNA elements as a layer of post-transcriptional regulation to reduce the impact of promoter leak. We observe significant temperature-dependent increases in cell survival when certain RNA thermometers (RNATs) are placed upstream of the cas9 coding sequence. We also show that the most highly repressing RNAT, hsp17rep, significantly reduces population skew with a library of characterized guide RNAs in Escherichia coli. This strategy should be applicable to all bacterial Cas9-based methods and technologies.

Kammerdiener, Elise K. [Oak Ridge National Laborat↗
DOE OSTI2025

Evaluation of Cross-Protection of African Swine Fever Vaccine ASFV-G-ΔI177L Between ASFV Biotypes

Background/Objectives: Vaccine development for the prevention of ASF has been very challenging due to the extensive genetic and largely unknown antigenic diversity. Inactivated vaccines, using different inactivation methods and a variety of adjuvants, have been consistently inefficacious. Historically, animals recovering from an infection with an attenuated virus became protected from the development of a clinical disease caused by an antigenically related strain. Therefore, immunization of susceptible animals with attenuathe ted virus strains has become a common method of vaccination with the first two commercially available vaccines based on recombinant live-attenuated viruses (LAVs). An important limitation is that the efficacy of the LAV is restricted to those strains that are antigenically related and, in most cases, only provide protection against homologous strains. Due to the unknown antigenic heterogeneity among all ASFV field isolates, the development of broad-spectrum vaccines is a challenge. Besides the anecdotal data, there is not a large amount of information describing patterns of cross-protection between different ASFV strains. Methods: We evaluated the cross-protection induced by the ASFV live-attenuated vaccine ASFV-G-ΔI177L against different biotypes of ASFV and compared their genomic sequences to determine potential genetic mutations that could cause the lack of cross-protection. Results: Results presented here demonstrate different patterns of protection when ASFV-G-ΔI177L vaccinated pigs were challenged with six different ASFV field isolates belonging to different biotypes. Conclusions: The presence of cross-protection cannot be predicted solely by the classical methodology for genotyping-based B646L ORF only. Biotyping, considering the entire virus proteome, appears to be a more promising prediction tool, although additional gathering of experimental data will be necessary to fully validate it; until then, the presence of cross-protection needs to be confirmed in efficacy trials challenging vaccinated animals.

Immunology↗
NASA NTRS2011

Early and Late Damages in Chromosome 3 of Human Lymphocytes After Radiation Exposure

Tumor formation in humans or animals is a multi-step process. An early stage of cancer development is believed to be genomic instability (GI) which accelerates the mutation rate in the descendants of the cells surviving radiation exposure. GI is defined as elevated or persistent genetic damages occurring many generations after the cells are exposed. While early studies have demonstrated radiation-induced GI in several cell types as detected in endpoints such as mutation, apoptosis and damages in chromosomes, the dependence of GI on the quality of radiation remains uncertain. To investigate GI in human lymphocytes induced by both low- and high-LET radiation, we initially exposed white blood cells collected from healthy subjects to gamma rays in vitro, and cultured the cells for multiple generations. Chromosome aberrations were analyzed in cells collected at first mitosis post irradiation and at several intervals during the culture period. Among a number of biological endpoints planned for the project, the multi-color banding fluorescent in situ hybridization (mBAND) allows identification of inversions that were expected to be stable. We present here early and late chromosome aberrations detected with mBAND in chromosome 3 after gamma exposure. Comparison of chromosome damages in between human lymphocytes and human epithelial cells is also discussed

Sunagawa, Mayumi↗
NASA NTRS2016

Proton and Fe Ion-Induced Early and Late Chromosome Aberrations in Different Cell Types

An early stage of cancer development is believed to be genomic instability (GI) which accelerates the mutation rate in the descendants of the cells surviving radiation exposure. To investigate GI induced by charged particles, we exposed human lymphocytes, human fibroblast cells, and human mammary epithelial cells to high energy protons and Fe ions. In addition, we also investigated GI in bone marrow cells isolated from CBA/CaH (CBA) and C57BL/6 (C57) mice, by analyzing cell survival and chromosome aberrations in the cells after multiple cell divisions. Results analyzed so far from the experiments indicated different sensitivities to charged particles between CBA/CaH (CBA) and C57BL/6 (C57) mouse strains, suggesting that there are two main types of response to irradiation: 1) responses associated with survival of damaged cells and 2) responses associated with the induction of non-clonal chromosomal instability in the surviving progeny of stem cells. Previously, we reported that the RBE for initial chromosome damages was high in human lymphocytes exposed to Fe ions. Our results with different cell types demonstrated different RBE values between different cell types and between early and late chromosomal damages. This study also attempts to offer an explanation for the varying RBE values for different cancer types.

Wu, Honglu↗
NASA NTRS2001

Rapid evolution of cis-regulatory sequences via local point mutations

Although the evolution of protein-coding sequences within genomes is well understood, the same cannot be said of the cis-regulatory regions that control transcription. Yet, changes in gene expression are likely to constitute an important component of phenotypic evolution. We simulated the evolution of new transcription factor binding sites via local point mutations. The results indicate that new binding sites appear and become fixed within populations on microevolutionary timescales under an assumption of neutral evolution. Even combinations of two new binding sites evolve very quickly. We predict that local point mutations continually generate considerable genetic variation that is capable of altering gene expression.

Non-NASA Center↗
DOE OSTI2022

Intrahost SARS-CoV-2 k-mer Identification Method (iSKIM) for Rapid Detection of Mutations of Concern Reveals Emergence of Global Mutation Patterns

Despite unprecedented global sequencing and surveillance of SARS-CoV-2, timely identification of the emergence and spread of novel variants of concern (VoCs) remains a challenge. Several million raw genome sequencing runs are now publicly available. We sought to survey these datasets for intrahost variation to study emerging mutations of concern. We developed iSKIM (“intrahost SARS-CoV-2 k-mer identification method”) to relatively quickly and efficiently screen the many SARS-CoV-2 datasets to identify intrahost mutations belonging to lineages of concern. Certain mutations surged in frequency as intrahost minor variants just prior to, or while lineages of concern arose. The Spike N501Y change common to several VoCs was found as a minor variant in 834 samples as early as October 2020. This coincides with the timing of the first detected samples with this mutation in the Alpha/B.1.1.7 and Beta/B.1.351 lineages. Using iSKIM, we also found that Spike L452R was detected as an intrahost minor variant as early as September 2020, prior to the observed rise of the Epsilon/B.1.429/B.1.427 lineages in late 2020. iSKIM rapidly screens for mutations of interest in raw data, prior to genome assembly, and can be used to detect increases in intrahost variants, potentially providing an early indication of novel variant spread.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2024

Adaptive laboratory evolution and genetic engineering improved terephthalate utilization in Pseudomonas putida KT2440

Poly(ethylene terephthalate) (PET) is one of the most ubiquitous plastics and can be depolymerized through biological and chemo-catalytic routes to its constituent monomers, terephthalic acid (TPA) and ethylene glycol (EG). TPA and EG can be re-synthesized into PET for closed-loop recycling or microbially converted into higher-value products for open-loop recycling. Here, in this study, we expand on our previous efforts engineering and applying Pseudomonas putida KT2440 for PET conversion by employing adaptive laboratory evolution (ALE) to improve TPA catabolism. Three P. putida strains with varying degrees of metabolic engineering for EG catabolism underwent an automation-enabled ALE campaign on TPA, a TPA and EG mixture, and glucose as a control. ALE increased the growth rate on TPA and TPA-EG mixtures by 4.1- and 3.5-fold, respectively, in approximately 350 generations. Evolved isolates were collected at the midpoints and endpoints of 39 independent ALE experiments, and growth rates were increased by 0.15 and 0.20 h -1 on TPA and a TPA-EG, respectively, in the best performing isolates. Whole-genome re-sequencing identified multiple converged mutations, including loss-of-function mutations to global regulators gacS, gacA, and turA along with large duplication and intergenic deletion events that impacted the heterologously-expressed tphAB II catabolic genes. Reverse engineering of these targets confirmed causality, and a strain with all three regulators deleted and second copies of tphAB II and tpaK displayed improved TPA utilization compared to the base strain. Taken together, an iterative strain engineering process involving heterologous pathway engineering, ALE, whole genome sequencing, and genome editing identified five genetic interventions that improve P. putida growth on TPA, aimed at developing enhanced whole-cell biocatalysts for PET upcycling.

36 MATERIALS SCIENCE↗
DOE OSTI2023

A large sequenced mutant library – valuable reverse genetic resource that covers 98% of sorghum genes

SUMMARY Mutant populations are crucial for functional genomics and discovering novel traits for crop breeding. Sorghum , a drought and heat‐tolerant C4 species, requires a vast, large‐scale, annotated, and sequenced mutant resource to enhance crop improvement through functional genomics research. Here, we report a sorghum large‐scale sequenced mutant population with 9.5 million ethyl methane sulfonate (EMS)‐induced mutations that covered 98% of sorghum's annotated genes using inbred line BTx623. Remarkably, a total of 610 320 mutations within the promoter and enhancer regions of 18 000 and 11 790 genes, respectively, can be leveraged for novel research of cis ‐regulatory elements. A comparison of the distribution of mutations in the large‐scale mutant library and sorghum association panel (SAP) provides insights into the influence of selection. EMS‐induced mutations appeared to be random across different regions of the genome without significant enrichment in different sections of a gene, including the 5′ UTR, gene body, and 3′‐UTR. In contrast, there were low variation density in the coding and UTR regions in the SAP. Based on the K a / K s value, the mutant library (~1) experienced little selection, unlike the SAP (0.40), which has been strongly selected through breeding. All mutation data are publicly searchable through SorbMutDB ( https://www.depts.ttu.edu/igcast/sorbmutdb.php ) and SorghumBase ( https://sorghumbase.org/ ). This current large‐scale sequence‐indexed sorghum mutant population is a crucial resource that enriched the sorghum gene pool with novel diversity and a highly valuable tool for the Poaceae family, that will advance plant biology research and crop breeding.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Synergistic epistasis enhances the co-operativity of mutualistic interspecies interactions

Abstract Early evolution of mutualism is characterized by big and predictable adaptive changes, including the specialization of interacting partners, such as through deleterious mutations in genes not required for metabolic cross-feeding. We sought to investigate whether these early mutations improve cooperativity by manifesting in synergistic epistasis between genomes of the mutually interacting species. Specifically, we have characterized evolutionary trajectories of syntrophic interactions of Desulfovibrio vulgaris (Dv) with Methanococcus maripaludis (Mm) by longitudinally monitoring mutations accumulated over 1000 generations of nine independently evolved communities with analysis of the genotypic structure of one community down to the single-cell level. We discovered extensive parallelism across communities despite considerable variance in their evolutionary trajectories and the perseverance within many evolution lines of a rare lineage of Dv that retained sulfate-respiration (SR+) capability, which is not required for metabolic cross-feeding. An in-depth investigation revealed that synergistic epistasis across pairings of Dv and Mm genotypes had enhanced cooperativity within SR− and SR+ assemblages, enabling their coexistence within the same community. Thus, our findings demonstrate that cooperativity of a mutualism can improve through synergistic epistasis between genomes of the interacting species, enabling the coexistence of mutualistic assemblages of generalists and their specialized variants.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Sodium azide mutagenesis induces a unique pattern of mutations

The nature and effect of mutations are of fundamental importance to the evolutionary process. The generation of mutations with mutagens has also played important roles in genetics. Applications of mutagens include dissecting the genetic basis of trait variation, inducing desirable traits in crops, and understanding the nature of genetic load. Previous studies of sodium azide-induced mutations have reported single nucleotide variants (SNVs) found in individual genes. To characterize the nature of mutations induced by sodium azide, we analyze whole-genome sequencing (WGS) of 11 barley lines derived from sodium azide mutagenesis, where all lines were selected for diminution of plant fitness owing to induced mutations. We contrast observed mutagen-induced variants with those found in standing variation in WGS of 13 barley landraces. Here, we report indels that are two orders of magnitude more abundant than expected based on nominal mutation rates. We found induced SNVs are very specific, with C → T changes occurring in a context followed by another C on the same strand (or the reverse complement). The codons most affected by the mutagen include the sodium azide-specific CC motif (or the reverse complement), resulting in a handful of amino acid changes and few stop codons. The specific nature of induced mutations suggests that mutagens could be chosen based on experimental goals. Sodium azide would not be ideal for gene knockouts but will create many missense mutations with more subtle effects on protein function.

Genetics & Heredity↗
DOE OSTI2022

Improving growth of Cupriavidus necator H16 on formate using adaptive laboratory evolution-informed engineering

Conversion of CO 2 to value-added products presents an opportunity to reduce GHG emissions while generating revenue. Formate, which can be generated by the electrochemical reduction of CO 2 , has been proposed as a promising intermediate compound for microbial upgrading. Here we present progress towards improving the soil bacterium Cupriavidus necator H16, which is capable of growing on formate as its sole source of carbon and energy using the Calvin-Benson-Bassham (CBB) cycle, as a host for formate utilization. Using adaptive laboratory evolution, we generated several isolates that exhibited faster growth rates on formate. The genomes of these isolates were sequenced, and resulting mutations were systematically reintroduced by metabolic engineering, to identify those that improved growth. Further, the metabolic impact of several mutations was investigated further using RNA-seq transcriptomics. We found that deletion of a transcriptional regulator implicated in quorum sensing, PhcA, reduced expression of several operons and led to improved growth on formate. Growth was also improved by deleting large genomic regions present on the extrachromosomal megaplasmid pHG1, particularly two hydrogenase operons and the megaplasmid CBB operon, one of two copies present in the genome. Based on these findings, we generated a rationally engineered ..delta..phcA and megaplasmid-deficient strain that exhibited a 24% faster maximum growth rate on formate. Moreover, this strain achieved a 7% growth rate improvement on succinate and a 19% increase on fructose, demonstrating the broad utility of microbial genome reduction. This strain has the potential to serve as an improved microbial chassis for biological conversion of formate to value-added products.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Genome-wide identification and functional prediction of silicon (Si) transporters in poplar (Populus trichocarpa)

Abstract Silicon (Si) enhances plant tolerance to various biotic and abiotic stressors such as salinity, drought, and heat. In addition, Si can be biomineralized within plants to form organic carbon-containing phytoliths that can have ecosystem-level consequences by contributing to long-term carbon sequestration. Si is taken up and transported in plants via different transporter proteins such as influx transporters (e.g., Lsi1, Lsi6) and efflux transporters (e.g., Lsi2). Additionally, the imported Si can be deposited in plant leaves via silicification process using the Siliplant 1 (e.g., Slp1) protein. Functional homologs of these proteins have been reported in different food crops. Here, we performed a genome-wide analysis to identify different Si transporters and Slp1 homologs in the bioenergy crop poplar ( Populus trichocarpa Torr. and A. Gray ex W. Hook). We identified one channel-type Si influx transporter (PtLsi1; Potri.017G083300), one Si efflux transporter (PtLsi2; Potri.012G144000) and two proteins like Slp1 (PtSlp1a; Potri.004G168600 and PtSlp1b; Potri.009G129900 ) in the P. trichocarpa genome. We found a unique sequence (KPKPPVFKPPPVPI) in PtSlp1a which is repeated six times. Repeated presence of this sequence in PtSlp1a indicates that this protein might be important for silicification processes in P. trichocarpa. The mutation profiles of different Si transporters in a P. trichocarpa genome-wide association study population identified significant and impactful mutations in Potri.004G168600 and Potri.009G129900 . Using a publically accessible database ( http://bar.utoronto.ca/eplant_poplar/ ), digital expression analysis of the putative Si transporters in P. trichocarpa found low to moderate expression in the anticipated tissues, such as roots and leaves. Subcellular localization analysis found that PtLsi1/PtLsi2 are localized in the plasma membrane, whereas PtSlp1a/PtSlp1b are found in the extracellular spaces. Protein–Protein interaction analysis of PtLsi1/PtLsi2 identified Delta-1-pyrroline-5-carboxylate synthase (P5CS) as one of the main interacting partners of PtLsi2, which plays a key role in proline biosynthesis. Proline is a well-known participant in biotic and abiotic stress tolerance in plants. These findings will reinforce future efforts to modify Si accumulation for enhancing plant stress tolerance and carbon sequestration in poplar.

59 BASIC BIOLOGICAL SCIENCES↗