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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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At least 181 records · Page 10
All sourcesNASA NTRSDOE OSTI
DOE OSTI2021

Structures of Neisseria gonorrhoeae MtrR-operator complexes reveal molecular mechanisms of DNA recognition and antibiotic resistance-conferring clinical mutations

Abstract Mutations within the mtrR gene are commonly found amongst multidrug resistant clinical isolates of Neisseria gonorrhoeae, which has been labelled a superbug by the Centers for Disease Control and Prevention. These mutations appear to contribute to antibiotic resistance by interfering with the ability of MtrR to bind to and repress expression of its target genes, which include the mtrCDE multidrug efflux transporter genes and the rpoH oxidative stress response sigma factor gene. However, the DNA-recognition mechanism of MtrR and the consensus sequence within these operators to which MtrR binds has remained unknown. In this work, we report the crystal structures of MtrR bound to the mtrCDE and rpoH operators, which reveal a conserved, but degenerate, DNA consensus binding site 5′-MCRTRCRN4YGYAYGK-3′. We complement our structural data with a comprehensive mutational analysis of key MtrR-DNA contacts to reveal their importance for MtrR-DNA binding both in vitro and in vivo. Furthermore, we model and generate common clinical mutations of MtrR to provide plausible biochemical explanations for the contribution of these mutations to multidrug resistance in N. gonorrhoeae. Collectively, our findings unveil key biological mechanisms underlying the global stress responses of N. gonorrhoeae.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Improved Biofuel Production through Discovery and Engineering of Terpene Metabolism in Switchgrass

Project Objectives - Of the myriad specialized metabolites that plants deploy to adapt to environmental challenges, terpenes form the largest group. In many major crops, unique terpene blends serve as key stress defenses that directly impact plant fitness and yield. In addition, terpenes, such as bisabolene and pinene, are used for producing renewable biofuels. Essential to advancing a broader use of terpenes for biofuel feedstock engineering is a system-wide knowledge of the diverse biosynthetic machinery and defensive potential of often species-specific terpene blends. The proposed project would merge genome-wide enzyme discovery with comparative –omics, protein structural and plant microbiome studies to define the biosynthesis and stress-defensive functions of the switchgrass (Panicum virgatum) terpene network. These insights would be combined with developing and applying non-transgenic genome editing tools to design plants with desirable terpene blends for higher productivity and biofuel production on marginal lands. As a dedicated lignocellulosic feedstock for U.S. biofuel production with high net energy yield, stress tolerance, and available genome resources, switchgrass is well-suited for devising new avenues for biofuel production. Project Description – The diversity of plant terpene defenses is governed by species-specific families of terpene synthase (TPS) and cytochrome P450 monooxygenase (P450) enzymes. Mining of the switchgrass genome (genotype Alamo) identified ~100 TPS and P450 candidate genes, and combinatorial biochemical analysis of synthesized TPSs and P450s revealed more than a dozen enzymes with common and novel activities. In addition, several identified terpene metabolites and the corresponding transcripts were up-regulated in response to abiotic stressors. These findings demonstrate a unique switchgrass terpene network with probable importance to abiotic stress tolerance, thus providing a large chemical portfolio for optimizing crop resistance, yield, and biofuel composition. Leveraging these preliminary data, we propose to generate a genome-wide map of the switchgrass terpene metabolic network through multi-gene co-expression analyses that allow the efficient cross-validation of TPS and P450 functions. Key enzymes would further be applied to structure-function studies via X-ray protein crystallography, homology modeling and site-directed mutagenesis to gain mechanistic insight into the catalytic specificity of switchgrass terpene metabolism and provide gene and amino acid targets for genome editing. In tandem with terpene pathway discovery, system-wide metabolomics, transcriptomics and proteomics studies in switchgrass accessions of contrasting drought tolerance would define the role of switchgrass terpene metabolism in conferring abiotic stress resilience. Metabolic changes would be assessed in a combined approach of targeted (terpenes) and untargeted metabolite profiling using a high-resolution LC-MS/MS approach, differential gene expression analyses through multiplexed Illumina RNA sequencing, and quantitative analysis of high-priority pathway enzymes using multiple reaction monitoring (MRM). Drawing on these insights, knock-down/out mutants of stress-associated pathway nodes would be generated by optimizing transient virus-induced gene silencing (VIGS) and CRISPR/Cas9 systems under control of the Tobacco Rattle Virus (TRV). The resulting mutant lines would then be analyzed for stress susceptibility and the impact on the root microbiome to define gene functions in planta. Knowledge of terpene pathways, enzyme mechanisms and bioactivities would be applied to enhance switchgrass stress resilience and to tailor-make terpene blends for biofuel production. Here, TRV-enabled CRISPR/Cas9 genome editing, including allele-specific knock-out of redundant genes, engineering of enzyme specificity via structure-guided point mutations, and overexpression of terpene genes relevant to stress-protection or biofuel production, would be used to increase metabolic flux toward desired pathways. Broader Impacts - Integrating the system-wide discovery, mechanistic analysis and non-transgenic genome engineering of the switchgrass terpene network aligns the required steps to unlock the chemical potential of this important metabolite class to generate crops that are more resistant to stress and provide advanced biofuel production in light of rising climate pressures as foreseeable challenges for bioenergy crop cultivation. The proposed project would further offer interdisciplinary student training through active involvement in the project and integration of research concepts and outcomes into newly-developed graduate and undergraduate courses on Plant Biotechnology.

09 BIOMASS FUELS↗
DOE OSTI2023

Molecular and Genetic Characterization of Hepatitis B Virus (HBV) among Saudi Chronically HBV-Infected Individuals

The study aimed to characterize the genotype and subgenotypes of HBV circulating in Saudi Arabia, the presence of clinically relevant mutations possibly associated with resistance to antivirals or immune escape phenomena, and the possible impact of mutations in the structural characteristics of HBV polymerase. Plasma samples from 12 Saudi Arabian HBV-infected patients were analyzed using an in-house PCR method and direct sequencing. Saudi patients were infected with mainly subgenotype D1. A number of mutations in the RT gene (correlated to antiviral resistance) and within and outside the major hydrophilic region of the S gene (claimed to influence immunogenicity and be related to immune escape) were observed in almost all patients. Furthermore, the presence of mutations in the S region caused a change in the tertiary structure of the protein compared with the consensus region. Clinical manifestations of HBV infection may change dramatically as a result of viral and host factors: the study of mutations and protein-associated cofactors might define possible aspects relevant for the natural and therapeutic history of HBV infection.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2023

Prolonging genetic circuit stability through adaptive evolution of overlapping genes

Abstract The development of synthetic biological circuits that maintain functionality over application-relevant time scales remains a significant challenge. Here, we employed synthetic overlapping sequences in which one gene is encoded or ‘entangled’ entirely within an alternative reading frame of another gene. In this design, the toxin-encoding relE was entangled within ilvA, which encodes threonine deaminase, an enzyme essential for isoleucine biosynthesis. A functional entanglement construct was obtained upon modification of the ribosome-binding site of the internal relE gene. Using this optimized design, we found that the selection pressure to maintain functional IlvA stabilized the production of burdensome RelE for >130 generations, which compares favorably with the most stable kill-switch circuits developed to date. This stabilizing effect was achieved through a complete alteration of the allowable landscape of mutations such that mutations inactivating the entangled genes were disfavored. Instead, the majority of lineages accumulated mutations within the regulatory region of ilvA. By reducing baseline relE expression, these more ‘benign’ mutations lowered circuit burden, which suppressed the accumulation of relE-inactivating mutations, thereby prolonging kill-switch function. Overall, this work demonstrates the utility of sequence entanglement paired with an adaptive laboratory evolution campaign to increase the evolutionary stability of burdensome synthetic circuits.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Mechanistic insights explain the transforming potential of the T507K substitution in the protein-tyrosine phosphatase SHP2

The protein-tyrosine phosphatase SHP2 is an allosteric enzyme critical for cellular events downstream of growth factor receptors. Mutations in the SHP2 gene have been linked to many different types of human diseases, including developmental disorders, leukemia, and solid tumors. Unlike most SHP2-activating mutations, the T507K substitution in SHP2 is unique in that it exhibits oncogenic Ras-like transforming activity. However, the biochemical basis of how the SHP2/T507K variant elicits transformation remains unclear. By combining kinetic and biophysical methods, X-ray crystallography, and molecular modeling, as well as using cell biology approaches, in this work we uncovered that the T507K substitution alters both SHP2 substrate specificity and its allosteric regulatory mechanism. We found that although SHP2/T507K exists in the closed, autoinhibited conformation similar to the WT enzyme, the interactions between its N-SH2 and protein-tyrosine phosphatase domains are weakened such that SHP2/T507K possesses a higher affinity for the scaffolding protein Grb2-associated binding protein 1 (Gab1). We also discovered that the T507K substitution alters the structure of the SHP2 active site, resulting in a change in SHP2 substrate preference for Sprouty1, a known negative regulator of Ras signaling and a potential tumor suppressor. Our results suggest that SHP2/T507K's shift in substrate specificity coupled with its preferential association of SHP2/T507K with Gab1 enable the mutant SHP2 to more efficiently dephosphorylate Sprouty1 at pTyr-53. This dephosphorylation hyperactivates Ras signaling, which is likely responsible for SHP2/T507K's Ras-like transforming activity.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2021

Spent enrichment media for growth of gene-matched microorganisms

A method for amplifying and detecting microorganisms, such as species of Listeria, is described. The method utilizes gene-matched enrichment media and PCR-based detection. The enrichment media is spent media produced using a modified microorganism containing a plurality of mutations in a selected gene such that the modified microorganism does not contain the PCR signature. Thus, PCR detects only the amplified microorganism of interest, not the modified microorganism. Exemplary methods and kits for amplification and detection of Listeria species are described.

Ozanich, Richard M.↗
NASA NTRS1999

Three-Dimensional, Transgenic Cell Models to Quantify Space Genotoxic Effects

The space environment contains radiation and chemical agents known to be mutagenic and carcinogenic to humans. Additionally, microgravity is a complicating factor that may modify or synergize induced genotoxic effects. Most in vitro models fail to use human cells (making risk extrapolation to humans more difficult), overlook the dynamic effect of tissue intercellular interactions on genotoxic damage, and lack the sensitivity required to measure low-dose effects. Currently a need exists for a model test system that simulates cellular interactions present in tissue, and can be used to quantify genotoxic damage induced by low levels of radiation and chemicals, and extrapolate assessed risk to humans. A state-of-the-art, three-dimensional, multicellular tissue equivalent cell culture model will be presented. It consists of mammalian cells genetically engineered to contain multiple copies of defined target genes for genotoxic assessment,. NASA-designed bioreactors were used to coculture mammalian cells into spheroids, The cells used were human mammary epithelial cells (H184135) and Stratagene's (Austin, Texas) Big Blue(TM) Rat 2 lambda fibroblasts. The fibroblasts were genetically engineered to contain -a high-density target gene for mutagenesis (60 copies of lacl/LacZ per cell). Tissue equivalent spheroids were routinely produced by inoculation of 2 to 7 X 10(exp 5) fibroblasts with Cytodex 3 beads (150 micrometers in diameter). at a 20:1 cell:bead ratio, into 50-ml HARV bioreactors (Synthecon, Inc.). Fibroblasts were cultured for 5 days, an equivalent number of epithelial cells added, and the fibroblast/epithelial cell coculture continued for 21 days. Three-dimensional spheroids with diameters ranging from 400 to 600 micrometers were obtained. Histological and immunohistochemical Characterization revealed i) both cell types present in the spheroids, with fibroblasts located primarily in the center, surrounded by epithelial cells; ii) synthesis of extracellular matrix; and iii,, mitotic cells located throughout the spheroids. Spheroidal integrity and cell viability were retained for the 30-day test period after removal of spheroids from the bioreactor. Potential utility of this three-dimensional, transgenic model for genotoxicity was initially assessed by exposure of spheroids to 0-2 Gy neon at dose rates of 0.3 to 1.5 Gy/min (National Institute of Radiological Sciences, Chiba, Japan). Quantification of mutation at the lacl gene revealed a linear dose response for mutation induction. Limited sequencing analysis of mutant clones revealed higher frequencies of deletions and multiple base sequence changes with increasing dose. These results suggest that our three-dimensional, transgenic model is applicable to a wide variety of studies involving the quantification, identification, and characterization of genotoxicity incurred in space and on Earth. This model uniquely allows investigation of the interaction of relevant factors, namely cell-to-cell interactions and the mechanistic interaction of microgravity with radiation insults and DNA repair. Using this three-dimensional model will allow us to obtain dual genotoxic information (i.e., mutation rate plus chromosome aberration data) from the same system so that one endpoint can be used to reference the other, thereby increasing the fidelity of the data set. Moreover, the tissue-equivalent nature of the three-dimensional model provides high confidence for relevance of risk assessment, i.e., the establishment of quality factors directly applicable to the microgravity environment.

Gonda, S. R.↗
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 OSTI2020

Triggering typical nemaline myopathy with compound heterozygous nebulin mutations reveals myofilament structural changes as pathomechanism

Nebulin is a giant protein that winds around the actin filaments in the skeletal muscle sarcomere. Compound-heterozygous mutations in the nebulin gene (NEB) cause typical nemaline myopathy (NM), a muscle disorder characterized by muscle weakness with limited treatment options. We created a mouse model with a missense mutation p.Ser6366Ile and a deletion of NEB exon 55, the Compound-Het model that resembles typical NM. We show that Compound-Het mice are growth-retarded and have muscle weakness. Muscles have a reduced myofibrillar fractional-area and sarcomeres are disorganized, contain rod bodies, and have longer thin filaments. In contrast to nebulin-based severe NM where haplo-insufficiency is the disease driver, Compound-Het mice express normal amounts of nebulin. X-ray diffraction revealed that the actin filament is twisted with a larger radius, that tropomyosin and troponin behavior is altered, and that the myofilament spacing is increased. The unique disease mechanism of nebulin-based typical NM reveals novel therapeutic targets.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

The molecular mechanism of sialic acid transport mediated by Sialin

Malfunction of the sialic acid transporter caused by various genetic mutations in the SLC17A5 gene encoding Sialin leads to a spectrum of neurodegenerative conditions called free sialic acid storage disorders. Unfortunately, how Sialin transports sialic acid/proton (H + ) and how pathogenic mutations impair its function are poorly defined. Here, we present the structure of human Sialin in an inward-facing partially open conformation determined by cryo–electron microscopy, representing the first high-resolution structure of any human SLC17 member. Our analysis reveals two unique features in Sialin: (i) The H + coupling/sensing requires two highly conserved Glu residues (E171 and E175) instead of one (E175) as implied in previous studies; and (ii) the normal function of Sialin requires the stabilization of a cytosolic helix, which has not been noticed in the literature. By mapping known pathogenic mutations, we provide mechanistic explanations for corresponding functional defects. We propose a structure-based mechanism for sialic acid transport mediated by Sialin.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Origin and evolution of HIV-1 subtype A6

Background: HIV outbreaks in the Former Soviet Union (FSU) countries were characterized by repeated transmission of the HIV variant AFSU, which is now classified as a distinct subtype A sub-subtype called A6. The current study used phylogenetic/phylodynamic and signature mutation analyses to determine likely evolutionary relationship between subtype A6 and other subtype A sub-subtypes. Methods: For this study, an initial Maximum Likelihood phylogenetic analysis was performed using a total of 553 full-length, publicly available, reverse transcriptase sequences, from A1, A2, A3, A4, A5, and A6 sub-subtypes of subtype A. For phylogenetic clustering and signature mutation analysis, a total of 5961 and 3959 pol and env sequences, respectively, were used. Results: Phylogenetic and signature mutation analysis showed that HIV-1 sub-subtype A6 likely originated from sub-subtype A1 of African origin. A6 and A1 pol and env genes shared several signature mutations that indicate genetic similarity between the two subtypes. For A6, tMRCA dated to 1975, 15 years later than that of A1. Conclusion: The current study provides insights into the evolution and diversification of A6 in the backdrop of FSU countries and indicates that A6 in FSU countries evolved from A1 of African origin and is getting bridged outside the FSU region.

60 APPLIED LIFE 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 OSTI2022

Evaluation of a parasite-density based pooled targeted amplicon deep sequencing (TADS) method for molecular surveillance of Plasmodium falciparum drug resistance genes in Haiti

Sequencing large numbers of individual samples is often needed for countrywide antimalarial drug resistance surveillance. Pooling DNA from several individual samples is an alternative cost and time saving approach for providing allele frequency (AF) estimates at a population level. Using 100 individual patient DNA samples of dried blood spots from a 2017 nationwide drug resistance surveillance study in Haiti, we compared codon coverage of drug resistance-conferring mutations in four Plasmodium falciparum genes ( crt , dhps , dhfr , and mdr1 ), for the same deep sequenced samples run individually and pooled. Samples with similar real-time PCR cycle threshold (Ct) values (+/- 1.0 Ct value) were combined with ten samples per pool. The sequencing success for samples in pools were higher at a lower parasite density than the individual samples sequence method. The median codon coverage for drug resistance-associated mutations in all four genes were greater than 3-fold higher in the pooled samples than in individual samples. The overall codon coverage distribution for pooled samples was wider than the individual samples. The sample pools with < 40 parasites/μL blood showed more discordance in AF calls for dhfr and mdr1 between the individual and pooled samples. This discordance in AF estimation may be due to low amounts of parasite DNA, which could lead to variable PCR amplification efficiencies. Grouping samples with an estimated ≥ 40 parasites/μL blood prior to pooling and deep sequencing yielded the expected population level AF. Pooling DNA samples based on estimates of > 40 parasites/μL prior to deep sequencing can be used for rapid genotyping of a large number of samples for these four genes and possibly other drug resistant markers in population-based studies. As Haiti is a low malaria transmission country with very few mixed infections and continued chloroquine sensitivity, the pooled sequencing approach can be used for routine national molecular surveillance of resistant parasites.

Louha, Swarnali (ORCID:0000000207778507)↗
NASA NTRS1994

Radiation effects in nematodes: Results from IML-1 experiments

The nematode Caenorhabditis elegans was exposed to natural space radiation using the ESA biorack facility aboard Spacelab on International Microgravity Laboratory 1, STS-42. For the major experimental objective dormant animals were suspended in buffer or on agar or immobilized next to CR-39 plastic nuclear track detectors to correlate fluence of HZE particles with genetic events. This configuration was used to isolate mutations in a set of 350 essential genes as well as in the unc-22 structural gene. From flight samples 13 mutants in the unc-22 gene were isolated along with 53 lethal mutations from autosomal regions balanced by a translocation eT1(III;V). Preliminary analysis suggests that mutants from worms correlated with specific cosmic ray tracks may have a higher proportion of rearrangements than those isolated from tube cultures on a randomly sampled basis. Flight sample mutation rate was approximately 8-fold higher than ground controls which exhibited laboratory spontaneous frequencies.

Nelson, G. A.↗
NASA NTRS2001

Drosophila Lyra mutations are gain-of-function mutations of senseless

The Lyra mutation was first described by Jerry Coyne in 1935. Lyra causes recessive pupal lethality and adult heterozygous Lyra mutants exhibit a dominant loss of the anterior and posterior wing margins. Unlike many mutations that cause loss of wing tissue (e.g., scalloped, Beadex, cut, and apterous-Xasta), Lyra wing discs do not exhibit increased necrotic or apoptotic cell death, nor do they show altered BrdU incorporation. However, during wing disc eversion, loss of the anterior and posterior wing margins is apparent. We have previously shown that senseless, a gene that is necessary and sufficient for peripheral nervous system (PNS) development, is allelic to Lyra. Here we show by several genetic criteria that Lyra alleles are neomorphic alleles of senseless that cause ectopic expression of SENSELESS in the wing pouch. Similarly, overexpression of SENSELESS in the wing disc causes loss of wing margin tissue, thereby mimicking the Lyra phenotype. Lyra mutants display aberrant expression of DELTA, VESTIGIAL, WINGLESS, and CUT. As in Lyra mutants, overexpression of SENSELESS in some areas of the wing pouch also leads to loss of WINGLESS and CUT. In summary, our data indicate that overexpression of SENSELESS causes a severe reduction in NOTCH signaling that in turn may lead to decreased transcription of several key genes required for wing development, leading to a failure in cell proliferation and loss of wing margin tissue.

Non-NASA Center↗
DOE OSTI2021

Drosophila models to study causative genes for human rare intractable neurological diseases

Highlights: • Drosophila models targeting some ALS-causing genes are described. • Drosophila models targeting some CMT-causing genes are described. • Drosophila models targeting some Sotos syndrome-causing genes are described. • Genes and pathways that commonly interact with ALS- and/or CMT-causing genes are described. Drosophila is emerging as a convenient model for investigating human diseases. Functional homologues of almost 75% of human disease-related genes are found in Drosophila. Amyotrophic lateral sclerosis (ALS) is a severe neurodegenerative disease that causes defects in motoneurons. Charcot-Marie-Tooth disease (CMT) is one of the most commonly found inherited neuropathies affecting both motor and sensory neurons. No effective therapy has been established for either of these diseases. In this review, after overviewing ALS, Drosophila models targeting several ALS-causing genes, including TDP-43, FUS and Ubiquilin2, are described with their genetic interactants. Then, after overviewing CMT, examples of Drosophila models targeting several CMT-causing genes, including mitochondria-related genes and FIG 4, are also described with their genetic interactants. In addition, we introduce Sotos syndrome caused by mutations in the epigenetic regulator gene NSD1. Lastly, several genes and pathways that commonly interact with ALS- and/or CMT-causing genes are described. In the case of ALS and CMT that have many causative genes, it may be not practical to perform gene therapy for each of the many disease-causing genes. The possible uses of the common genes and pathways as novel diagnosis markers and effective therapeutic targets are discussed.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2023

Strategies to identify and edit improvements in synthetic genome segments episomally

Genome engineering projects often utilize bacterial artificial chromosomes (BACs) to carry multi-kilobase DNA segments at low copy number. However, all stages of whole-genome engineering have the potential to impose mutations on the synthetic genome that can reduce or eliminate the fitness of the final strain. Here, we describe improvements to a multiplex automated genome engineering (MAGE) protocol to improve recombineering frequency and multiplexability. This protocol was applied to recoding an Escherichia coli strain to replace seven codons with synonymous alternatives genome wide. Ten 44 402–47 179 bp de novo synthesized DNA segments contained in a BAC from the recoded strain were unable to complement deletion of the corresponding 33–61 wild-type genes using a single antibiotic resistance marker. Next-generation sequencing (NGS) was used to identify 1–7 non-recoding mutations in essential genes per segment, and MAGE in turn proved a useful strategy to repair these mutations on the recoded segment contained in the BAC when both the recoded and wild-type copies of the mutated genes had to exist by necessity during the repair process. Finally, two web-based tools were used to predict the impact of a subset of non-recoding missense mutations on strain fitness using protein structure and function calls.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Comparison of phage-derived recombinases for genetic manipulation of Pseudomonas species

ABSTRACT Several strains in the Pseudomonas genus are categorized as plant growth-promoting rhizobacteria (PGPR). Although several of these strains are strong candidates for applications as biofertilizers or biopesticides, genome editing approaches are generally limited and require further development. Editing genomes in PGPR could enable more robust agricultural applications, persistence, and biosafety measures. In this study, we investigate the use of five phage-encoded recombinases to develop a recombineering workflow in three PGPR strains: Pseudomonas protegens Pf-5, Pseudomonas protegens CHA0, and Pseudomonas putida KT2440. Using point mutations in the rpoB gene, we reach maximum recombineering efficiencies of 1.5 × 10 −4 , 3 × 10 −4 , and 5 × 10 −5 , respectively, in these strains using λ-Red Beta recombinase from Escherichia coli . We further examine recombineering efficiencies across these strains as a function of selected mutation, editing template concentration, and phosphorothiolate bond protection. This work validates the use of these tools across several environmentally and biotechnologically relevant strains to expand the possibilities of genetic manipulation in the Pseudomonas genus. IMPORTANCE The Pseudomonas genus contains many members currently being investigated for applications in biodegradation, biopesticides, biocontrol, and synthetic biology. Though several strains have been identified with beneficial properties, chromosomal manipulations to further improve these strains for commercial applications have been limited due to the lack of efficient genetic tools that have been tested across this genus. Here, we test the recombineering efficiencies of five phage-derived recombinases across three biotechnologically relevant Pseudomonas strains: P. putida KT2440, P. protegens Pf-5, and P. protegens CHA0. These results demonstrate a method to generate targeted mutations quickly and efficiently across these strains, ideally introducing a method that can be implemented across the Pseudomonas genus and a strategy that may be applied to develop analogous systems in other nonmodel bacteria.

59 BASIC BIOLOGICAL SCIENCES↗