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DOE OSTI2023

X-ray Crystallographic Study of Preferred Spacing by the NF-κB p50 Homodimer on κB DNA

Though originally characterized as an inactive or transcriptionally repressive factor, the NF-κB p50 homodimer has become appreciated as a physiologically relevant driver of specific target gene expression. By virtue of its low affinity for cytoplasmic IκB protein inhibitors, p50 accumulates in the nucleus of resting cells, where it is a binding target for the transcriptional co-activator IκBζ. In this study, we employed X-ray crystallography to analyze the structure of the p50 homodimer on κB DNA from the promoters of human interleukin-6 (IL-6) and neutrophil-gelatinase-associated lipocalin (NGAL) genes, both of which respond to IκBζ. The NF-κB p50 homodimer binds 11-bp on IL-6 κB DNA, while, on NGAL κB DNA, the spacing is 12-bp. This begs the question: what DNA binding mode is preferred by NF-κB p50 homodimer? To address this, we engineered a “Test” κB-like DNA containing the core sequence 5'-GGGGAATTCCCC-3' and determined its X-ray crystal structure in complex with p50. This revealed that, when presented with multiple options, NF-κB p50 homodimer prefers to bind 11-bp, which necessarily imposes asymmetry on the complex despite the symmetry inherent in both the protein and its target DNA, and that the p50 dimerization domain can contact DNA via distinct modes.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2023

Amphiphilic Co-Solvents Modulate the Structure of Membrane Domains

Biofuels are an increasing part of the sustainable energy picture. This makes it a societal and economic imperative to optimize biofuel production. Mitigating the toxic effects of amphiphilic co-solvents is one way to improve the efficiency of biofuel production. Amphiphiles partition into cellular membranes, leading to membrane thinning, destabilization, loss of membrane potential, and, ultimately, cell death. However, this picture of solvent toxicity misses the disruptive impact of co-solvents on lateral membrane organization, which is increasingly recognized as critical for membrane protein sorting and oligomerization. The alteration or disruption of membrane domains has deleterious effects on cellular processes. Here, in this work, we pursue the hypothesis that membrane lateral organization is disrupted by the presence of co-solvents at concentrations lower than those which lead to full membrane destabilization. The disruption occurs due to an increasing interfacial tension between the co-existing phases, resulting in conformational changes to minimize the interfacial length-to-area ratio. This represents an unrecognized mode of solvent-induced stress and a new target for interventions to improve fermentation yields.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

DOE OSTI2021

Proteins associated with the Arabidopsis thaliana plastid rhomboid-like protein RBL10

Rhomboid-like proteins are intramembrane proteases with a variety of regulatory roles in cells. Though many rhomboid-like proteins are predicted in plants, their detailed molecular mechanisms or cellular functions are not yet known. Of the 13 predicted rhomboids in Arabidopsis thaliana, one, RBL10, affects lipid metabolism in the chloroplast, because in the respective rbl10 mutant the transfer of phosphatidic acid through the inner envelope membrane is disrupted. Here we show that RBL10 is part of a high-molecular-weight complex of 250 kDa or greater in size. Nine likely components of this complex are identified by two independent methods and include Acyl Carrier Protein 4 (ACP4) and Carboxyltransferase Interactor1 (CTI1), which have known roles in chloroplast lipid metabolism. The acp4 mutant has decreased C16:3 fatty acid content of monogalactosyldiacylglycerol, similar to the rbl10 mutant, prompting us to offer a mechanistic model of how an interaction between ACP4 and RBL10 might affect chloroplast lipid assembly. We also demonstrate the presence of a seventh transmembrane domain in RBL10, refining the currently accepted topology of this protein. Taken together, the identity of possible RBL10 complex components as well as insights into RBL10 topology and distribution in the membrane provide a stepping-stone towards a deeper understanding of RBL10 function in Arabidopsis lipid metabolism.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2021

Evolutionary relationships among shell proteins of carboxysomes and metabolosomes

Bacterial microcompartments (BMCs) are self-assembling prokaryotic organelles which encapsulate enzymes within a polyhedral protein shell. Additionally, the shells are comprised of only two structural modules, distinct domains that form pentagonal and hexagonal building blocks, which occupy the vertices and facets, respectively. As all BMC loci encode at least one hexamer-forming and one pentamer-forming protein, the evolutionary history of BMCs can be interrogated from the perspective of their shells. Here, we discuss how structures of intact shells and detailed phylogenies of their building blocks from a recent phylogenomic survey distinguish families of these domains and reveal clade-specific structural features. These features suggest distinct functional roles that recur across diverse BMCs. For example, it is clear that carboxysomes independently arose twice from metabolosomes, yet the principles of shell assembly are remarkably conserved.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2021

Structural basis for SARS-CoV-2 envelope protein recognition of human cell junction protein PALS1

The COVID-19 pandemic, caused by the SARS-CoV-2 virus, has created global health and economic emergencies. SARS-CoV-2 viruses promote their own spread and virulence by hijacking human proteins, which occurs through viral protein recognition of human targets. To understand the structural basis for SARS-CoV-2 viral-host protein recognition, here we use cryo-electron microscopy (cryo-EM) to determine a complex structure of the human cell junction protein PALS1 and SARS-CoV-2 viral envelope (E) protein. Our reported structure shows that the E protein C-terminal DLLV motif recognizes a pocket formed exclusively by hydrophobic residues from the PDZ and SH3 domains of PALS1. Our structural analysis provides an explanation for the observation that the viral E protein recruits PALS1 from lung epithelial cell junctions. In addition, our structure provides novel targets for peptide- and small-molecule inhibitors that could block the PALS1-E interactions to reduce E-mediated virulence.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2021

SCOPe: improvements to the structural classification of proteins – extended database to facilitate variant interpretation and machine learning

Abstract The Structural Classification of Proteins—extended (SCOPe, https://scop.berkeley.edu) knowledgebase aims to provide an accurate, detailed, and comprehensive description of the structural and evolutionary relationships amongst the majority of proteins of known structure, along with resources for analyzing the protein structures and their sequences. Structures from the PDB are divided into domains and classified using a combination of manual curation and highly precise automated methods. In the current release of SCOPe, 2.08, we have developed search and display tools for analysis of genetic variants we mapped to structures classified in SCOPe. In order to improve the utility of SCOPe to automated methods such as deep learning classifiers that rely on multiple alignment of sequences of homologous proteins, we have introduced new machine-parseable annotations that indicate aberrant structures as well as domains that are distinguished by a smaller repeat unit. We also classified structures from 74 of the largest Pfam families not previously classified in SCOPe, and we improved our algorithm to remove N- and C-terminal cloning, expression and purification sequences from SCOPe domains. SCOPe 2.08-stable classifies 106 976 PDB entries (about 60% of PDB entries).

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2022

How Distinct Structural Flexibility within SARS-CoV-2 Spike Protein Reveals Potential Therapeutic Targets

The emergence and rapid worldwide spread of the novel coronavirus disease, COVID-19, has prompted concerted efforts to find successful treatments. The causative virus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), uses its spike (S) protein to gain entry into host cells. Therefore, the S protein presents a viable target to develop a directed therapy. Here, we deployed an integrated artificial intelligence with all-atom molecular dynamics simulation approach to provide new details of the S protein structure. Based on a comprehensive structural analysis of S proteins from SARS-CoV-2 and previous human coronaviruses, we found that the protomer state of S proteins is structurally flexible. Without the presence of a stabilizing beta sheet from another protomer chain, two regions in the S2 domain and the hinge connecting the S1 and S2 subunits lose their secondary structures. Interestingly, the region in the S2 domain was previously identified as an immunodominant site in the SARS-CoV-1 S protein. We anticipate that the molecular details elucidated here will assist in effective therapeutic development for COVID-19.

Chen, Serena↗

DOE OSTI2021

Incorporation and Assembly of a Light-Emitting Enzymatic Reaction into Model Protein Condensates

Eukaryotic cells partition enzymes and other cellular components into distinct subcellular compartments to generate specialized biochemical niches. A subclass of these compartments form in the absence of lipid membranes, via liquid-liquid phase separation of proteins to form biomolecular condensates or “membraneless organelles” such as nucleoli, stress granules, and P-bodies. Because of their ability to form compartments from simple starting materials, membraneless organelles are an attractive target for engineering new functionalities in both living cells and protocells. In this work, we demonstrate incorporation of novel enzymatic activity in protein coacervates with a light-generating enzyme, NanoLuc, to produce bioluminescence. Using condensates comprised of the disordered RGG domain of C. elegans LAF-1, we show functionalization of condensates with enzymatic activity in vitro and that localization to protein coacervates enhances the assembly and activity of split enzymes. To build condensates that function as light emitting reactors, we designed a NanoLuc enzyme flanked by RGG domains. Furthermore, the resulting condensates concentrated NanoLuc by 10-fold over bulk solution and display significantly increased net reaction rates. We further show that condensate viscosity impacts light emission due to diffusion-limited behavior. By splitting NanoLuc enzyme into its constituent components, we demonstrate that NanoLuc activity can be reconstituted via co-condensation. Further, we demonstrate control of the spatial localization of enzyme within condensates by targettng NanoLuc to the surface of in vitro condensates. Collectively, this work demonstrates that membraneless organelles can be endowed with localized enzymatic activity, and that this activity can be spatially and temporally controlled via enzyme reconstitution and design of protein surfactants.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

DOE OSTI2025

Experimental and Computational Insights into the Structural Dynamics of the Fc Fragment of IgG1 Subtype from Biosimilar VEGF‐Trap

The constant fragment (Fc) of the immunoglobulin G1 (IgG1) subtype is increasingly recognized as a crucial scaffold in the development of advanced therapeutics due to its enhanced specificity, efficacy, and extended half‐life. A prime example is VEGF‐Trap (Aflibercept), a recombinant fusion protein that merges the Fc region of the IgG1 subtype with the binding domains of vascular endothelial growth factor receptors (VEGFR)‐1 and VEGFR‐2. The Fc region's role in N‐glycosylation is particularly important, as it significantly influences protein stability. Herein, the first near‐physiological temperature structures of the N‐glycan‐bound Fc fragment of IgG1 subtype from a biosimilar VEGF‐Trap are presented, determined using the SPring‐8 Angstrom Compact free electron LAser (SACLA) and the Turkish Light Source (Turkish DeLight). Comparative analysis with cryogenic structures, including existing data, reveals alternate conformations within the glycan‐binding pocket. Furthermore, molecular dynamics simulations indicate the presence of a high degree of structural plasticity, explaining how the protein adapts its structure through conformational changes. The observed structural fluctuations/conformational changes demonstrate the effect of N‐glycans on protein stability. These findings offer new insights into the molecular basis of Fc‐mediated functions and provide valuable information for the design of next‐generation therapeutics.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2024

High-Resolution Frequency-Domain Spectroscopic and Modeling Studies of Photosystem I (PSI), PSI Mutants and PSI Supercomplexes

Photosystem I (PSI) is one of the two main pigment–protein complexes where the primary steps of oxygenic photosynthesis take place. This review describes low-temperature frequency-domain experiments (absorption, emission, circular dichroism, resonant and non-resonant hole-burned spectra) and modeling efforts reported for PSI in recent years. In particular, we focus on the spectral hole-burning studies, which are not as common in photosynthesis research as the time-domain spectroscopies. Experimental and modeling data obtained for trimeric cyanobacterial Photosystem I (PSI 3 ), PSI 3 mutants, and PSI 3 –IsiA 18 supercomplexes are analyzed to provide a more comprehensive understanding of their excitonic structure and excitation energy transfer (EET) processes. Detailed information on the excitonic structure of photosynthetic complexes is essential to determine the structure–function relationship. We will focus on the so-called “red antenna states” of cyanobacterial PSI, as these states play an important role in photochemical processes and EET pathways. The high-resolution data and modeling studies presented here provide additional information on the energetics of the lowest energy states and their chlorophyll (Chl) compositions, as well as the EET pathways and how they are altered by mutations. We present evidence that the low-energy traps observed in PSI are excitonically coupled states with significant charge-transfer (CT) character. The analysis presented for various optical spectra of PSI 3 and PSI 3 -IsiA 18 supercomplexes allowed us to make inferences about EET from the IsiA 18 ring to the PSI 3 core and demonstrate that the number of entry points varies between sample preparations studied by different groups. In our most recent samples, there most likely are three entry points for EET from the IsiA 18 ring per the PSI core monomer, with two of these entry points likely being located next to each other. Therefore, there are nine entry points from the IsiA 18 ring to the PSI 3 trimer. We anticipate that the data discussed below will stimulate further research in this area, providing even more insight into the structure-based models of these important cyanobacterial photosystems.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2024

Bactericidal effectors of the Stenotrophomonas maltophilia type IV secretion system: functional definition of the nuclease TfdA and structural determination of TfcB

ABSTRACT Stenotrophomonas maltophilia expresses a type IV protein secretion system (T4SS) that promotes contact-dependent killing of other bacteria and does so partly by secreting the effector TfcB. Here, we report the structure of TfcB, comprising an N-terminal domain similar to the catalytic domain of glycosyl hydrolase (GH-19) chitinases and a C-terminal domain for recognition and translocation by the T4SS. Utilizing a two-hybrid assay to measure effector interactions with the T4SS coupling protein VirD4, we documented the existence of five more T4SS substrates. One of these was protein 20845, an annotated nuclease. A S. maltophilia mutant lacking the gene for 20845 was impaired for killing Escherichia coli , Klebsiella pneumoniae , and Pseudomonas aeruginosa . Moreover, the cloned 20845 gene conferred robust toxicity, with the recombinant E. coli being rescued when 20845 was co-expressed with its cognate immunity protein. The 20845 effector was an 899 amino-acid protein, comprised of a GHH-nuclease domain in its N-terminus, a large central region of indeterminant function, and a C-terminus for secretion. Engineered variants of the 20845 gene that had mutations in the predicted catalytic site did not impede E. coli , indicating that the antibacterial effect of 20845 involves its nuclease activity. Using flow cytometry with DNA staining, we determined that 20845, but not its mutant variants, confers a loss in DNA content of target bacteria. Database searches revealed that uncharacterized homologs of 20845 occur within a range of bacteria. These data indicate that the S. maltophilia T4SS promotes interbacterial competition through the action of multiple toxic effectors, including a potent, novel DNase. IMPORTANCE Stenotrophomonas maltophilia is a multi-drug-resistant, Gram-negative bacterium that is an emerging pathogen of humans. Patients with cystic fibrosis are particularly susceptible to S. maltophilia infection. In hospital water systems and various types of infections, S. maltophilia co-exists with other bacteria, including other pathogens such as Pseudomonas aeruginosa . We previously demonstrated that S. maltophilia has a functional VirB/D4 type VI protein secretion system (T4SS) that promotes contact-dependent killing of other bacteria. Since most work on antibacterial systems involves the type VI secretion system, this observation remains noteworthy. Moreover, S. maltophilia currently stands alone as a model for a human pathogen expressing an antibacterial T4SS. Using biochemical, genetic, and cell biological approaches, we now report both the discovery of a novel antibacterial nuclease (TfdA) and the first structural determination of a bactericidal T4SS effector (TfcB).

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2023

The N‐terminal intrinsically disordered region of Ncb5or docks with the cytochrome b 5 core to form a helical motif that is of ancient origin

Abstract NADH cytochromeb 5 oxidoreductase (Ncb5or) is a cytosolic ferric reductase implicated in diabetes and neurological conditions. Ncb5or comprises cytochromeb 5 (b 5 ) and cytochromeb 5 reductase (b 5 R) domains separated by a CHORD‐Sgt1 (CS) linker domain. Ncb5or redox activity depends on proper inter‐domain interactions to mediate electron transfer from NADH or NADPH via FAD to heme. While full‐length human Ncb5or has proven resistant to crystallization, we have succeeded in obtaining high‐resolution atomic structures of theb 5 domain and a construct containing the CS andb 5 R domains (CS/b 5 R). Ncb5or also contains an N‐terminal intrinsically disordered region of 50 residues that has no homologs in other protein families in animals but features a distinctive, conserved L 34 MDWIRL 40 motif also present in reduced lateral root formation (RLF) protein in rice and increased recombination center 21 in baker's yeast, all attaching to ab 5 domain. After unsuccessful attempts at crystallizing a human Ncb5or construct comprising the N‐terminal region naturally fused to theb 5 domain, we were able to obtain a high‐resolution atomic structure of a recombinant rice RLF construct corresponding to residues 25–129 of human Ncb5or (52% sequence identity; 74% similarity). The structure reveals Trp 120 (corresponding to invariant Trp 37 in Ncb5or) to be part of an 11‐residue α‐helix (S 116 QMDWLKLTRT 126 ) packing against two of the four helices in theb 5 domain that surround heme (α2 and α5). The Trp 120 side chain forms a network of interactions with the side chains of four highly conserved residues corresponding to Tyr 85 and Tyr 88 (α2), Cys 124 (α5), and Leu 47 in Ncb5or. Circular dichroism measurements of human Ncb5or fragments further support a key role of Trp 37 in nucleating the formation of the N‐terminal helix, whose location in the N/b 5 module suggests a role in regulating the function of this multi‐domain redox enzyme. This study revealed for the first time an ancient origin of a helical motif in the N/b 5 module as reflected by its existence in a class of cytochromeb 5 proteins from three kingdoms among eukaryotes.

Biochemistry & Molecular Biology↗

DOE OSTI2021

PLEKHA7 signaling is necessary for the growth of mutant KRAS driven colorectal cancer

Plekha7 (Pleckstrin homology [PH] domain containing, family A member 7) regulates the assembly of proteins of the cytoplasmic apical zonula adherens junction (AJ), thus ensuring cell-cell adhesion and tight-junction barrier integrity. Little is known of Plekha7 function in cancer. In colorectal cancer (CRC) Plekha7 expression is elevated compared to adjacent normal tissue levels, increasing with clinical stage. Plekha7 was present at plasma membrane AJ with wild-type KRas (wt-KRas) but was dispersed in cells expressing mutant KRas (mut-KRas). Fluorescence lifetime imaging microscopy (FLIM) indicated a direct Plekha7 interaction with wt-KRas but scantily with mut-KRas. Inhibiting Plekha7 specifically decreased mut-KRas cell signaling, proliferation, attachment, migration, and retarded mut-KRAS CRC tumor growth. Binding of diC8-phosphoinositides (PI) to the PH domain of Plekha7 was relatively low affinity. This may be because a D175 amino acid residue plays a “sentry” role preventing PI(3,4)P{sub 2} and PI(3,4,5)P{sub 3} binding. Molecular or pharmacological inhibition of the Plekha7 PH domain prevented the growth of mut-KRas but not wt-KRas cells. Taken together the studies suggest that Plekha7, in addition to maintaining AJ structure plays a role in mut-KRas signaling and phenotype through interaction of its PH domain with membrane mut-KRas, but not wt-KRas, to increase the efficiency of mut-KRas downstream signaling.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2025

Computer Vision Pipeline for Image Analysis for Freeze‐Fracture Electron Microscopy: Rosette Cellulose Synthase Complexes Case

In materials science, plant biology, agriculture, and environmental research, the automated analysis of high-magnification, complex microscopy images, such as those generated by freeze-fracture electron microscopy (FF-TEM), remains a critical challenge that limits the scalability of data interpretation. We present a deep learning computer vision pipeline for high-throughput detection and morphological characterization analysis of cellulose synthase complexes (CSCs, or rosettes) in FF-TEM images. The pipeline integrates preprocessing, detection, human-in-the-loop verification, and semantic segmentation to quantify features such as rosette diameter and inter-lobe spacing. The approach was trained and tested on a curated dataset of high-resolution FF-TEM micrographs of Physcomitrium patens, expanded via strategic tiling and augmentation to over 650 images. We compare YOLOv8 and YOLOv9 architectures and demonstrate that YOLOv9 achieves superior performance in both localization accuracy (mAP50-95 = 0.854) and inference speed. The resulting distributions revealed biological variability consistent with prior manual studies, validating the approach for high-throughput applications. Our results show that the pipeline achieves human-expert level accuracy while dramatically reducing analysis time, enabling scalable, reproducible structural characterization of intramembrane protein complexes. The pipeline is broadly applicable to other domains requiring precise interpretation of complex microscopy data and establishes a foundation for future artificial intelligence (AI)-assisted workflows in biological imaging.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2021

Dual arginine recognition of LRRK2 phosphorylated Rab GTPases

Parkinson’s-disease-associated LRRK2 is a multidomain Ser/Thr kinase that phosphorylates a subset of Rab GTPases to control their effector functions. Rab GTPases are the prime regulators of membrane trafficking in eukaryotic cells. Rabs exert their biological effects by recruitment of effector proteins to subcellular compartments via their Rab-binding domain (RBD). Effectors are modular and typically contain additional domains that regulate various aspects of vesicle formation, trafficking, fusion, and organelle dynamics. The RBD of effectors is typically an α-helical coiled coil that recognizes the GTP conformation of the switch 1 and switch 2 motifs of Rabs. LRRK2 phosphorylates Rab8a at T72 (pT72) of its switch 2 α-helix. This post-translational modification enables recruitment of RILPL2, an effector that regulates ciliogenesis in model cell lines. A newly identified RBD motif of RILPL2, termed the X-cap, has been shown to recognize the phosphate via direct interactions between an arginine residue (R132) and pT72 of Rab8a. Here, we show that a second distal arginine (R130) is also essential for phospho-Rab binding by RILPL2. Through structural, biophysical, and cellular studies, we find that R130 stabilizes the primary R132:pT72 salt bridge through favorable enthalpic contributions to the binding affinity. These findings may have implications for the mechanism by which LRRK2 activation leads to assembly of phospho-Rab complexes and subsequent control of their membrane trafficking functions in cells.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2022

Recombinant FimH Adhesin Demonstrates How the Allosteric Catch Bond Mechanism Can Support Fast and Strong Bacterial Attachment in the Absence of Shear

The FimH protein of Escherichia coli is a model two-domain adhesin that is able to mediate an allosteric catch bond mechanism of bacterial cell attachment, where the mannose-binding lectin domain switches from an ‘inactive’ conformation with fast binding to mannose to an ‘active’ conformation with slow detachment from mannose. Because mechanical tensile force favors separation of the domains and, thus, FimH activation, it has been thought that the catch bonds can only be manifested in a fluidic shear-dependent mode of adhesion. Here, we used recombinant FimH variants with a weakened inter-domain interaction and show that a fast and sustained allosteric activation of FimH can also occur under static, non-shear conditions. Moreover, it appears that lectin domain conformational activation happens intrinsically at a constant rate, independently from its ability to interact with the pilin domain or mannose. However, the latter two factors control the rate of FimH deactivation. Thus, the allosteric catch bond mechanism can be a much broader phenomenon involved in both fast and strong cell-pathogen attachments under a broad range of hydrodynamic conditions. In conclusion, this concept that allostery can enable more effective receptor-ligand interactions is fundamentally different from the conventional wisdom that allostery provides a mechanism to turn binding off under specific conditions.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2023

Efficacy of antivirals and mRNA vaccination against an XBF clinical isolate

Recombination events occur frequently in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), resulting in genetic diversity. Because these events contribute to altered host immune evasion and antiviral susceptibility, it is crucial to evaluate the efficacy of COVID-19 vaccines and antivirals against recombinant variants. As of March 2023, XBB.1.5, a recombinant sublineage of XBB, is currently the dominant form globally (Fig. S1A, Supplementary Appendix). XBB emerged as a result of recombination between two BA.2 descendants, BJ.1 and BM.1.1.1 (a progeny of BA.2.75). We and other groups have shown that XBB.1.5 is resistant to several therapeutic monoclonal antibodies and effectively evades humoral immunity elicited by natural infection or COVID-19 vaccination.1, 2, 3 By March 30, 2023, an additional recombinant variant, XBF, had been sampled 8966 times in 47 countries and territories in GISAID, reaching it highest prevalence in Australia and New Zealand (Fig. S2, Supplementary Appendix). XBF is still increasing in frequency, although the prevalence of XBB.1.5 appears to be increasing at a faster pace in most regions (Fig. S1B, Supplementary Appendix). XBF is a recombinant of BA.5.2.3 (a descendant of BA.5) and CJ.1 (a descendant of BA.2.75) and, like CJ.1, has an additional three substitutions (R346T, F486P, and F490S) in the receptor-binding domain (RBD) of the consensus form of its spike protein compared to baseline BA.2.75 (Fig. S3A, Supplementary Appendix). CJ.1 and its related sublineage CJ.1.1 did not expand as extensively as XBF, being sampled in GISAID only 1589 and 142 times respectively. CJ.1 was found circulating in many countries, but most commonly sampled in South Korea, where it is still increasing and has currently reached about 5% of the sampled population. CJ.1.1 was most frequently sampled in Malaysia and Singapore, but remained rare in both nations, peaking at about 1% of the sample in December of 2022. Despite the importance of these related variants, we have no information about the antiviral efficacy and immunity induced by COVID-19 vaccines against a clinical isolate of XBF.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2021

Diploid genomic architecture of Nitzschia inconspicua, an elite biomass production diatom

Abstract A near-complete diploid nuclear genome and accompanying circular mitochondrial and chloroplast genomes have been assembled from the elite commercial diatom species Nitzschia inconspicua . The 50 Mbp haploid size of the nuclear genome is nearly double that of model diatom Phaeodactylum tricornutum , but 30% smaller than closer relative Fragilariopsis cylindrus . Diploid assembly, which was facilitated by low levels of allelic heterozygosity (2.7%), included 14 candidate chromosome pairs composed of long, syntenic contigs, covering 93% of the total assembly. Telomeric ends were capped with an unusual 12-mer, G-rich, degenerate repeat sequence. Predicted proteins were highly enriched in strain-specific marker domains associated with cell-surface adhesion, biofilm formation, and raphe system gliding motility. Expanded species-specific families of carbonic anhydrases suggest potential enhancement of carbon concentration efficiency, and duplicated glycolysis and fatty acid synthesis pathways across cytosolic and organellar compartments may enhance peak metabolic output, contributing to competitive success over other organisms in mixed cultures. The N. inconspicua genome delivers a robust new reference for future functional and transcriptomic studies to illuminate the physiology of benthic pennate diatoms and harness their unique adaptations to support commercial algae biomass and bioproduct production.

09 BIOMASS FUELS↗