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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 253 records · Page 14
All sourcesNASA NTRSDOE OSTI
DOE OSTI2020

Aspartate Residues in a Forisome-Forming SEO Protein Are Critical for Protein Body Assembly and Ca 2+ Responsiveness

Forisomes are protein bodies known exclusively from sieve elements of legumes. Forisomes contribute to the regulation of phloem transport due to their unique Ca 2+ -controlled, reversible swelling. The assembly of forisomes from sieve element occlusion (SEO) protein monomers in developing sieve elements and the mechanism(s) of Ca 2+ -dependent forisome contractility are poorly understood because the amino acid sequences of SEO proteins lack conventional protein–protein interaction and Ca 2+ -binding motifs. Here we selected amino acids potentially responsible for forisome-specific functions by analyzing SEO protein sequences in comparison to those of the widely distributed SEO-related (SEOR), or SEOR proteins. SEOR proteins resemble SEO proteins closely but lack any Ca 2+ responsiveness. We exchanged identified candidate residues by directed mutagenesis of the Medicago truncatula SEO1 gene, expressed the mutated genes in yeast ( Saccharomyces cerevisiae ) and studied the structural and functional phenotypes of the forisome-like bodies that formed in the transgenic cells. We identified three aspartate residues critical for Ca 2+ responsiveness and two more that were required for forisome-like bodies to assemble. The phenotypes observed further suggested that Ca 2+ -controlled and pH-inducible swelling effects in forisome-like bodies proceeded by different yet interacting mechanisms. Finally, we observed a previously unknown surface striation in native forisomes and in recombinant forisome-like bodies that could serve as an indicator of successful forisome assembly. To conclude, this study defines a promising path to the elucidation of the so-far elusive molecular mechanisms of forisome assembly and Ca 2+ -dependent contractility.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Advances in Cell Wall Matrix Research with a Focus on Mixed-Linkage Glucan

Mixed β(1,3;1,4)-linkage glucan (MLG) is commonly found in the monocot lineage, at particularly high levels in the Poaceae family, but also in the evolutionally distant genus, Equisetum. MLG has several properties that make it unique from other plant cell wall polysaccharides. It consists of β1,4-linked polymers of glucose interspersed with β1,3-linkages, but the presence of β1,3-linkages provides quite different physical properties compared to its closest form of the cell wall component, cellulose. The mechanisms of MLG biosynthesis have been investigated to understand whether single or multiple enzymes are required to build mixed linkages in the glucan chain. Currently, MLG synthesis by a single enzyme is supported by mutagenesis analyses of cellulose synthase-like F6, the major MLG synthase, but further investigation is needed to gather mechanistic insights. Because of transient accumulation of MLG in elongating cells and vegetative tissues, several hypotheses have been proposed to explain the role of MLG in the plant cell wall. Studies have been carried out to identify gene expression regulators during development and light cycles as well as enzymes involved in MLG organization in the cell wall. A role of MLG as a storage molecule in grains is evident, but the role of MLG in vegetative tissues is still not well understood. Characterization of a cell wall component is difficult due to the complex heterogeneity of the plant cell wall. Furthermore, as detailed in this review, recent exciting research has made significant impacts in the understanding of MLG biology in plants.

Transglucosylase/hydrolase (XTH)↗
DOE OSTI2020

A temperature-sensitive FERONIA mutant allele that alters root hair growth

Abstract In plants, root hairs undergo a highly polarized form of cell expansion called tip-growth, in which cell wall deposition is restricted to the root hair apex. In order to identify essential cellular components that might have been missed in earlier genetic screens, we identified conditional temperature-sensitive (ts) root hair mutants by ethyl methanesulfonate mutagenesis in Arabidopsis thaliana. Here, we describe one of these mutants, feronia-temperature sensitive (fer-ts). Mutant fer-ts seedlings were unaffected at normal temperatures (20°C), but failed to form root hairs at elevated temperatures (30°C). Map based-cloning and whole-genome sequencing revealed that fer-ts resulted from a G41S substitution in the extracellular domain of FERONIA (FER). A functional fluorescent fusion of FER containing the fer-ts mutation localized to plasma membranes, but was subject to enhanced protein turnover at elevated temperatures. While tip-growth was rapidly inhibited by addition of rapid alkalinization factor 1 (RALF1) peptides in both wild-type and fer-ts mutants at normal temperatures, root elongation of fer-ts seedlings was resistant to added RALF1 peptide at elevated temperatures. Additionally, at elevated temperatures fer-ts seedlings displayed altered reactive oxygen species (ROS) accumulation upon auxin treatment and phenocopied constitutive fer mutant responses to a variety of plant hormone treatments. Molecular modeling and sequence comparison with other Catharanthus roseus receptor-like kinase 1L (CrRLK1L) receptor family members revealed that the mutated glycine in fer-ts is highly conserved, but is not located within the recently characterized RALF23 and LORELI-LIKE-GLYCOPROTEIN 2 binding domains, perhaps suggesting that fer-ts phenotypes may not be directly due to loss of binding to RALF1 peptides.

Plant Sciences↗
DOE OSTI2021

The SvFUL2 transcription factor is required for inflorescence determinacy and timely flowering in Setaria viridis

Inflorescence architecture in cereal crops directly impacts yield potential through regulation of seed number and harvesting ability. Extensive architectural diversity found in inflorescences of grass species is due to spatial and temporal activity and determinacy of meristems, which control the number and arrangement of branches and flowers, and underlie plasticity. Timing of the floral transition is also intimately associated with inflorescence development and architecture, yet little is known about the intersecting pathways and how they are rewired during development. Here, we show that a single mutation in a gene encoding an AP1/FUL-like MADS-box transcription factor significantly delays flowering time and disrupts multiple levels of meristem determinacy in panicles of the C 4 model panicoid grass, Setaria viridis. Previous reports of AP1/FUL-like genes in cereals have revealed extensive functional redundancy, and in panicoid grasses, no associated inflorescence phenotypes have been described. In S. viridis, perturbation of SvFul2, both through chemical mutagenesis and gene editing, converted a normally determinate inflorescence habit to an indeterminate one, and also repressed determinacy in axillary branch and floral meristems. Our analysis of gene networks connected to disruption of SvFul2 identified regulatory hubs at the intersection of floral transition and inflorescence determinacy, providing insights into the optimization of cereal crop architecture.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Multiplex knockout of trichome-regulating MYB duplicates in hybrid poplar using a single gRNA

As the focus for CRISPR/Cas-edited plants moves from proof-of-concept to real-world applications, precise gene manipulation will increasingly require concurrent multiplex editing for polygenic traits. A common approach for editing across multiple sites is to design one guide RNA (gRNA) per target; however, this complicates construct assembly and increases the possibility of off-target mutations. In this study, we utilized one gRNA to target MYB186, a known positive trichome regulator, as well as its paralogs MYB138 and MYB38 at a consensus site for mutagenesis in hybrid poplar (Populus tremula × P. alba INRA 717-1B4). Unexpected duplications of MYB186 and MYB138 resulted in eight alleles for the three targeted genes in the hybrid poplar. Deep sequencing and polymerase chain reaction analyses confirmed editing across all eight targets in nearly all of the resultant glabrous mutants, ranging from small indels to large genomic dropouts, with no off-target activity detected at four potential sites. This highlights the effectiveness of a single gRNA targeting conserved exonic regions for multiplex editing. Additionally, cuticular wax and whole-leaf analyses showed a complete absence of triterpenes in the trichomeless mutants, hinting at a previously undescribed role for the nonglandular trichomes of poplar.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Essential amino acids in the Plant-Conserved and Class-Specific Regions of cellulose synthases

The Plant-Conserved Region (P-CR) and the Class-Specific Region (CSR) are two plant-unique sequences in the catalytic core of cellulose synthases (CESAs) for which specific functions have not been established. Here, we used site-directed mutagenesis to replace amino acids and motifs within these sequences predicted to be essential for assembly and function of CESAs. We developed an in vivo method to determine the ability of mutated CesA1 transgenes to complement an Arabidopsis (Arabidopsis thaliana) temperature-sensitive root-swelling1 (rsw1) mutant. Replacement of a Cys residue in the CSR, which blocks dimerization in vitro, rendered the AtCesA1 transgene unable to complement the rsw1 mutation. Examination of the CSR sequences from 33 diverse angiosperm species showed domains of high-sequence conservation in a class-specific manner but with variation in the degrees of disorder, indicating a nonredundant role of the CSR structures in different CESA isoform classes. The Cys residue essential for dimerization was not always located in domains of intrinsic disorder. Expression of AtCesA1 transgene constructs, in which Pro417 and Arg453 were substituted for Ala or Lys in the coiled-coil of the P-CR, were also unable to complement the rsw1 mutation. Despite an expected role for Arg457 in trimerization of CESA proteins, AtCesA1 transgenes with Arg457Ala mutations were able to fully restore the wild-type phenotype in rsw1. Our data support that Cys662 within the CSR and Pro417 and Arg453 within the P-CR of Arabidopsis CESA1 are essential residues for functional synthase complex formation, but our data do not support a specific role for Arg457 in trimerization in native CESA complexes.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Disruption of p -coumaroyl-CoA:monolignol transferases in rice drastically alters lignin composition

Grasses are abundant feedstocks that can supply lignocellulosic biomass for production of cell-wall-derived chemicals. In grass cell walls, lignin is acylated with p-coumarate. These p-coumarate decorations arise from the incorporation of monolignol p-coumarate conjugates during lignification. A previous biochemical study identified a rice (Oryza sativa) BAHD acyltransferase (AT) with p-coumaroyl-CoA:monolignol transferase (PMT) activity in vitro. In this study, we determined that that enzyme, which we name OsPMT1 (also known as OsAT4), and the closely related OsPMT2 (OsAT3) harbor similar catalytic activity toward monolignols. We generated rice mutants deficient in either or both OsPMT1 and OsPMT2 by CRISPR/Cas9-mediated mutagenesis and subjected the mutants’ cell walls to analysis using chemical and nuclear magnetic resonance methods. Our results demonstrated that OsPMT1 and OsPMT2 both function in lignin p-coumaroylation in the major vegetative tissues of rice. Notably, lignin-bound p-coumarate units were undetectable in the ospmt1 ospmt2-2 double-knockout mutant. Further, in-depth structural analysis of purified lignins from the ospmt1 ospmt2-2 mutant compared with control lignins from wild-type rice revealed stark changes in polymer structures, including alterations in syringyl/guaiacyl aromatic unit ratios and inter-monomeric linkage patterns, and increased molecular weights. Furthermore, our results provide insights into lignin polymerization in grasses that will be useful for the optimization of bioengineering approaches for the effective use of biomass in biorefineries.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Propelling sustainable energy: Multi-omics analysis of pennycress FATTY ACID ELONGATION1 knockout for biofuel production

Abstract The aviation industry’s growing interest in renewable jet fuel has encouraged the exploration of alternative oilseed crops. Replacing traditional fossil fuels with a sustainable, domestically sourced crop can substantially reduce carbon emissions, thus mitigating global climate instability. Pennycress (Thlaspi arvense L.) is an emerging oilseed intermediate crop that can be grown during the offseason between maize (Zea mays) and soybean (Glycine max) to produce renewable biofuel. Pennycress is being domesticated through breeding and mutagenesis, providing opportunities for trait enhancement. Here, we employed metabolic engineering strategies to improve seed oil composition and bolster the plant's economic competitiveness. FATTY ACID ELONGATION1 (FAE1) was targeted using CRISPR-Cas 9 gene editing to eliminate very long chain fatty acids (VLCFAs) from pennycress seed oil, thereby enhancing its cold flow properties. Through an integrated multiomics approach, we investigated the impact of eliminating VLCFAs in developing and mature plant embryos. Our findings revealed improved cold-germination efficiency in fae1, with seedling emergence occurring up to 3 d earlier at 10 °C. However, these alterations led to a tradeoff between storage oil content and composition. Additionally, these shifts in lipid biosynthesis were accompanied by broad metabolic changes, such as the accumulation of glucose and ADP-glucose quantities consistent with increased starch production. Furthermore, shifts to shorter FA chains triggered the upregulation of heat shock proteins, underscoring the importance of VLCFAs in stress signaling pathways. Overall, this research provides crucial insights for optimizing pennycress seed oil while preserving essential traits for biofuel applications.

Rasoul, Amira (ORCID:0009000714290175)↗
DOE OSTI2025

DNA adducts form in mouse lung and liver after oral naphthalene exposure

Naphthalene is a ubiquitous combustion product and environmental contaminant with known human exposure. Chronic exposure to naphthalene vapor leads to respiratory tumor formation in rodents. Naphthalene forms DNA adducts (precursors to genotoxicity) in tissue explants but it is unclear if this occurs in vivo. Wild-type C57BL/6 mice were orally exposed to 50 mg/kg 14 C-naphthalene. Naphthalene-DNA adducts were detected by accelerator mass spectrometry at 2- to 72-h post-exposure in both lung and liver, with decreasing abundance over time. Adducts persisted even at 72-h after exposure, which indicates possible evasion of DNA repair and potential to contribute to mutagenesis.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2022

Transient local secondary structure in the intrinsically disordered C‐term of the Albino3 insertase

Albino3 (Alb3) is an integral membrane protein fundamental to the targeting and insertion of light‐harvesting complex (LHC) proteins into the thylakoid membrane. Alb3 contains a stroma‐exposed C‐terminus (Alb3‐Cterm) that is responsible for binding the LHC‐loaded transit complex before LHC membrane insertion. Alb3‐Cterm has been reported to be intrinsically disordered, but precise mechanistic details underlying how it recognizes and binds to the transit complex are lacking, and the functional roles of its four different motifs have been debated. Using a novel combination of experimental and computational techniques such as single‐molecule fluorescence resonance energy transfer, circular dichroism with deconvolution analysis, site‐directed mutagenesis, trypsin digestion assays, and all‐atom molecular dynamics simulations in conjunction with enhanced sampling techniques, we show that Alb3‐Cterm contains transient secondary structure in motifs I and II. The excellent agreement between the experimental and computational data provides a quantitatively consistent picture and allows us to identify a heterogeneous structural ensemble that highlights the local and transient nature of the secondary structure. This structural ensemble was used to predict both the inter‐residue distance distributions of single molecules and the apparent unfolding free energy of the transient secondary structure, which were both in excellent agreement with those determined experimentally. We hypothesize that this transient local secondary structure may play an important role in the recognition of Alb3‐Cterm for the LHC‐loaded transit complex, and these results should provide a framework to better understand protein targeting by the Alb3‐Oxa1‐YidC family of insertases.

Okoto, Patience S.↗
DOE OSTI2020

Molecular mechanism for the interaction between human CPSF30 and hFip1

Most eukaryotic pre-mRNAs must undergo 3'-end cleavage and polyadenylation prior to their export from the nucleus. A large number of proteins in several complexes participate in this 3'-end processing, including cleavage and polyadenylation specificity factor (CPSF) in mammals. The CPSF30 subunit contains five CCCH zinc fingers (ZFs), with ZF2–ZF3 being required for the recognition of the AAUAAA poly(A) signal. ZF4–ZF5 recruits the hFip1 subunit of CPSF, although the details of this interaction have not been characterized. Here we report the crystal structure of human CPSF30 ZF4–ZF5 in complex with residues 161–200 of hFip1 at 1.9 Å resolution, illuminating the molecular basis for their interaction. Unexpectedly, the structure reveals one hFip1 molecule binding to each ZF4 and ZF5, with a conserved mode of interaction. Our mutagenesis studies confirm that the CPSF30–hFip1 complex has 1:2 stoichiometry in vitro. Mutation of each binding site in CPSF30 still allows one copy of hFip1 to bind, while mutation of both sites abrogates binding. Our fluorescence polarization binding assays show that ZF4 has higher affinity for hFip1, with a K d of 1.8 nM. We also demonstrate that two copies of the catalytic module of poly(A) polymerase (PAP) are recruited by the CPSF30–hFip1 complex in vitro, and both hFip1 binding sites in CPSF30 can support polyadenylation.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Molecular Mechanism of Polysaccharide Acetylation by the Arabidopsis Xylan O-acetyltransferase XOAT1

Xylans are a major component of plant cell walls. O-Acetyl moieties are the dominant backbone substituents of glucuronoxylan in dicots and play a major role in the polymer-polymer interactions that are crucial for wall architecture and normal plant development. Here, we describe the biochemical, structural, and mechanistic characterization of Arabidopsis (Arabidopsis thaliana) xylan O-acetyltransferase 1 (XOAT1), a member of the plant-specific Trichome Birefringence Like (TBL) family. Detailed characterization of XOAT1-catalyzed reactions by real-time NMR confirms that it exclusively catalyzes the 2-O-acetylation of xylan, followed by nonenzymatic acetyl migration to the O-3 position, resulting in products that are monoacetylated at both O-2 and O-3 positions. In addition, we report the crystal structure of the catalytic domain of XOAT1, which adopts a unique conformation that bears some similarities to the a/ß/a topology of members of the GDSL-like lipase/acylhydrolase family. Finally, we use a combination of biochemical analyses, mutagenesis, and molecular simulations to show that XOAT1 catalyzes xylan acetylation through formation of an acyl-enzyme intermediate, Ac–Ser-216, by a double displacement bi-bi mechanism involving a Ser-His-Asp catalytic triad and unconventionally uses an Arg residue in the formation of an oxyanion hole.

acetylation↗
DOE OSTI2021

Integrating continuous hypermutation with high–throughput screening for optimization of cis,cis –muconic acid production in yeast

Directed evolution is a powerful method to optimize proteins and metabolic reactions towards user-defined goals. It usually involves subjecting genes or pathways to iterative rounds of mutagenesis, selection and amplification. While powerful, systematic searches through large sequence-spaces is a labour-intensive task, and can be further limited by a priori knowledge about the optimal initial search space, and/or limits in terms of screening throughput. Here, we demonstrate an integrated directed evolution workflow for metabolic pathway enzymes that continuously generate enzyme variants using the recently developed orthogonal replication system, OrthoRep and screens for optimal performance in high-throughput using a transcription factor-based biosensor. We demonstrate the strengths of this workflow by evolving a rate-limiting enzymatic reaction of the biosynthetic pathway for cis,cis-muconic acid (CCM), a precursor used for bioplastic and coatings, in Saccharomyces cerevisiae. After two weeks of simply iterating between passaging of cells to generate variant enzymes via OrthoRep and high-throughput sorting of best-performing variants using a transcription factor-based biosensor for CCM, we ultimately identified variant enzymes improving CCM titers > 13-fold compared with reference enzymes. Taken together, the combination of synthetic biology tools as adopted in this study is an efficient approach to debottleneck repetitive workflows associated with directed evolution of metabolic enzymes.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Utilization of lasso peptides for biodegradation of polycyclic aromatic hydrocarbons

Abstract Many microbial genes involved in degrading recalcitrant environmental contaminants such as polycyclic aromatic hydrocarbons (PAHs) have been identified and characterized. However, all molecular mechanisms required for PAH utilization have not yet been elucidated. In this work, we demonstrate the proposed involvement of lasso peptides in the utilization of the PAH phenanthrene in Sphingomonas BPH. Transpositional mutagenesis of Sphingomonas BPH with the miniTn5 transposon yielded 3 phenanthrene utilization deficient mutants, #257, #1778, and #1782. In mutant #1782, Tn5 had inserted into the large subunit of the naph/bph dioxygenase gene. In mutant #1778, Tn5 had inserted into the B2 protease gene of a lasso peptide cluster. This finding is the first report on the role of lasso peptides in PAH utilization. Our studies also demonstrate that interruption of the lasso peptide cluster resulted in a significant increase in the amount of biosurfactant produced in the presence of glucose when compared to the wild‐type strain. Collectively, these results suggest that the mechanisms Sphingomonas BPH utilizes to degrade phenanthrene are far more complex than previously understood and that the #1778 mutant may be a good candidate for bioremediation when glucose is applied as an amendment due to its higher biosurfactant production.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Design of an Ara h 2 hypoallergen from conformational epitopes

Abstract Introduction Adverse reactions are relatively common during peanut oral immunotherapy. To reduce the risk to the patient, some researchers have proposed modifying the allergen to reduce IgE reactivity, creating a putative hypoallergen. Analysis of recently cloned human IgG from patients treated with peanut immunotherapy suggested that there are three common conformational epitopes for the major peanut allergen Ara h 2. We sought to test if structural information on these epitopes could indicate mutagenesis targets for designing a hypoallergen and evaluated the reduction in IgE binding via immunochemistry and a mouse model of passive cutaneous anaphylaxis (PCA). Methods X‐ray crystallography characterized the conformational epitopes in detail, followed by mutational analysis of key residues to modify monoclonal antibody (mAb) and serum IgE binding, assessed by ELISA and biolayer interferometry. A designed Ara h 2 hypoallergen was tested for reduced vascularization in mouse PCA experiments using pooled peanut allergic patient serum. Results A ternary crystal structure of Ara h 2 in complex with patient antibodies 13T1 and 13T5 was determined. Site‐specific mutants were designed that reduced 13T1, 13T5, and 22S1 mAbs binding by orders of magnitude. By combining designed mutations from the three major conformational bins, a hexamutant (Ara h 2 E46R, E89R, E97R, E114R, Q146A, R147E) was created that reduced IgE binding in serum from allergic patients. Further, in the PCA model where mice were primed with peanut allergic patient serum, reactivity upon allergen challenge was significantly decreased using the hexamutant. Conclusion These studies demonstrate that prior knowledge of common conformational epitopes can be used to engineer reduced IgE reactivity, an important first step in hypoallergen design.

Allergy↗
DOE OSTI2023

Structural insights into hydrolytic defluorination of difluoroacetate by microbial fluoroacetate dehalogenases

Fluorine forms the strongest single bond to carbon with the highest bond dissociation energy among natural products. However, fluoroacetate dehalogenases (FADs) have been shown to hydrolyze this bond in fluoroacetate under mild reaction conditions. Furthermore, two recent studies demonstrated that the FAD RPA1163 fromRhodopseudomonas palustriscan also accept bulkier substrates. In this study, we explored the substrate promiscuity of microbial FADs and their ability to defluorinate polyfluorinated organic acids. Enzymatic screening of eight purified dehalogenases with reported fluoroacetate defluorination activity revealed significant hydrolytic activity against difluoroacetate in three proteins. Product analysis using liquid chromatography‐mass spectrometry identified glyoxylic acid as the final product of enzymatic DFA defluorination. The crystal structures of DAR3835 fromDechloromonas aromaticaand NOS0089 fromNostocsp. were determined in the apo‐state along with the DAR3835 H274N glycolyl intermediate. Structure‐based site‐directed mutagenesis of DAR3835 demonstrated a key role for the catalytic triad and other active site residues in the defluorination of both fluoroacetate and difluoroacetate. Computational analysis of the dimer structures of DAR3835, NOS0089, and RPA1163 indicated the presence of one substrate access tunnel in each protomer. Moreover, protein‐ligand docking simulations suggested similar catalytic mechanisms for the defluorination of both fluoroacetate and difluoroacetate, with difluoroacetate being defluorinated via two consecutive defluorination reactions producing glyoxylate as the final product. Thus, our findings provide molecular insights into substrate promiscuity and catalytic mechanism of FADs, which are promising biocatalysts for applications in synthetic chemistry and bioremediation of fluorochemicals.

Biochemistry & Molecular Biology↗
DOE OSTI2026

Elongation Factor Tu Acts as a Chaperone to Activate an Antibacterial RNase Toxin

Many Gram-negative bacterial species use contact-dependent growth inhibition (CDI) systems to deliver toxic proteins into neighboring competitors. CDI + strains deploy CdiA effector proteins, which translocate their C-terminal toxin (CT) domains into target bacteria through a receptor-mediated delivery pathway. To protect against auto-intoxication, CDI + bacteria also produce CdiI immunity proteins that neutralize CT toxin activity. Here, we present the crystal structure of the CT·CdiI O32:H37 complex from Escherichia coli O32:H37. CT O32:H37 adopts the same fold as the tRNase domain of colicin D, and the nucleases share similar catalytic centers. However, unlike colicin D, which cleaves the anticodon loops of tRNA Arg isoacceptors, CT O32:H37 exhibits nonspecific RNase activity. Notably, we find that endogenous elongation factor Tu (EF-Tu) co-purifies with the over-produced CT·CdiI O32:H37 complex. Although EF-Tu does not bind stably to CT O32:H37 in the absence of CdiI O32:H37 , the translation factor is required for toxic RNase activity in vitro. AlphaFold 3 modeling and site-directed mutagenesis indicate that CT O32:H37 interacts with the N-terminal GTPase domain of EF-Tu. EF-Tu appears to stabilize residue Trp52 within the hydrophobic core of the toxin, which in turn supports the RNase active site through an unusual hydrogen-bonding interaction with the catalytic His67 residue. Furthermore, EF-Tu is hijacked as an essential co-factor to organize the toxin's catalytic center.

Nhan, Dinh Quan [University of California, Santa B↗
DOE OSTI2025

Bacterial fitness for plant colonization is influenced by plant growth substrate

Despite advances in our understanding of bacterial plant colonization, the extent to which growth substrate influences the molecular mechanisms enabling bacteria to efficiently colonize plants remains poorly understood. To address this, we used randomly barcoded transposon mutagenesis sequencing (RB-TnSeq) in Paraburkholderia graminis OAS925, an efficient rhizosphere colonizer, and Brachypodium distachyon grown in six different substrates. Of the 382 rhizosphere colonization genes that we identified in OAS925, 348 genes (91.1%) are dependent on the growth substrate evaluated, and 34 genes (8.9%) are shared across all the substrates. Both the core and substrate-dependent colonization genes are from multiple functional categories, demonstrating the multifaceted and major impact that plant growth substrate has on bacterial colonization. The identified colonization genes and their varied importance across plant growth substrates could not be readily explained by differences in root exudate profiles, suggesting that the substrate environment itself plays an outsized role in the ability of a bacterium to colonize the rhizosphere. Our data confirm that bacterial fitness for plant colonization is strongly influenced by plant growth substrate type and highlights the importance of taking this parameter into consideration when engineering bacterial strains for improved host colonization.

Paraburkholderia↗