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At least 631 records · Page 35
All sourcesNASA NTRSDOE OSTI
DOE OSTI2024

Hepatic transcriptome analyses of juvenile white bass ( Morone chrysops ) when fed diets where fish meal is partially or totally replaced by alternative protein sources

White bass (Morone chrysops) are a popular sportfish throughout the southern United States, and one parent of the commercially-successful hybrid striped bass (M. chrysops male x M. saxatilis female). Currently, white bass are cultured using diets formulated for other carnivorous fish, such as largemouth bass (Micropterus salmoides) or hybrid striped bass and contain a significant percentage of marine fish meal. Since there are no studies regarding the utilization of alternative proteins in this species, we evaluated the global gene expression of white bass fed diets in which fish meal was partially or totally replaced by various combinations of soybean meal, poultry by-product meal, canola meal, soy protein concentrate, wheat gluten, or a commercial protein blend (Pro Cision™). Six isonitrogenous (40% protein), isolipidic (11%), and isocaloric (17.1 kJ/g) diets were formulated to meet the known nutrient and energy requirements of largemouth bass and hybrid striped bass using nutrient availability data for most of the dietary ingredients. One of the test diets consisted exclusively of plant protein sources. Juvenile white bass (40.2 g initial weight) were stocked into a flow-through aquaculture system (three tanks/diet; 10 fish/tank) and fed the test diets twice daily to satiation for 60 days. RNA sequencing and bioinformatic analyses revealed significant differentially expressed genes between all test diets when compared to fish meal control. A total of 1,260 differentially expressed genes were identified, with major ontology relating to cell cycle and metabolic processes as well as immune gene functions. This data will be useful as a resource for future refinements to moronid diet formulation, as marine fish meal becomes limiting and plant ingredients are increasingly added as a reliable protein source

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Multi-Omics Driven Metabolic Network Reconstruction and Analysis of Lignocellulosic Carbon Utilization in Rhodosporidium toruloides

An oleaginous yeast Rhodosporidium toruloides is a promising host for converting lignocellulosic biomass to bioproducts and biofuels. In this work, we performed multi-omics analysis of lignocellulosic carbon utilization in R. toruloides and reconstructed the genome-scale metabolic network of R. toruloides . High-quality metabolic network models for model organisms and orthologous protein mapping were used to build a draft metabolic network reconstruction. The reconstruction was manually curated to build a metabolic model using functional annotation and multi-omics data including transcriptomics, proteomics, metabolomics, and RB-TDNA sequencing. The multi-omics data and metabolic model were used to investigate R. toruloides metabolism including lipid accumulation and lignocellulosic carbon utilization. The developed metabolic model was validated against high-throughput growth phenotyping and gene fitness data, and further refined to resolve the inconsistencies between prediction and data. We believe that this is the most complete and accurate metabolic network model available for R. toruloides to date.

09 BIOMASS FUELS↗
DOE OSTI2023

De novo design of small beta barrel proteins

Small beta barrel proteins are attractive targets for computational design because of their considerable functional diversity despite their very small size (<70 amino acids). However, there are considerable challenges to designing such structures, and there has been little success thus far. Because of the small size, the hydrophobic core stabilizing the fold is necessarily very small, and the conformational strain of barrel closure can oppose folding; also intermolecular aggregation through free beta strand edges can compete with proper monomer folding. Here, we explore the de novo design of small beta barrel topologies using both Rosetta energy–based methods and deep learning approaches to design four small beta barrel folds: Src homology 3 (SH3) and oligonucleotide/oligosaccharide-binding (OB) topologies found in nature and five and six up-and-down-stranded barrels rarely if ever seen in nature. Both approaches yielded successful designs with high thermal stability and experimentally determined structures with less than 2.4 Å rmsd from the designed models. Using deep learning for backbone generation and Rosetta for sequence design yielded higher design success rates and increased structural diversity than Rosetta alone. The ability to design a large and structurally diverse set of small beta barrel proteins greatly increases the protein shape space available for designing binders to protein targets of interest.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

An active site loop toggles between conformations to control antibiotic hydrolysis and inhibition potency for CTX-M β-lactamase drug-resistance enzymes

Abstract β-lactamases inactivate β-lactam antibiotics leading to drug resistance. Consequently, inhibitors of β-lactamases can combat this resistance, and the β-lactamase inhibitory protein (BLIP) is a naturally occurring inhibitor. The widespread CTX-M-14 and CTX-M-15 β-lactamases have an 83% sequence identity. In this study, we show that BLIP weakly inhibits CTX-M-14 but potently inhibits CTX-M-15. The structure of the BLIP/CTX-M-15 complex reveals that binding is associated with a conformational change of an active site loop of β-lactamase. Surprisingly, the loop structure in the complex is similar to that in a drug-resistant variant (N106S) of CTX-M-14. We hypothesized that the pre-established favorable loop conformation of the N106S mutant would facilitate binding. The N106S substitution results in a ~100- and 10-fold increase in BLIP inhibition potency for CTX-M-14 and CTX-M-15, respectively. Thus, this indicates that an active site loop in β-lactamase toggles between conformations that control antibiotic hydrolysis and inhibitor susceptibility. These findings highlight the role of accessible active site conformations in controlling enzyme activity and inhibitor susceptibility as well as the influence of mutations in selectively stabilizing discrete conformations.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2026

Exploring life’s hidden majority: microbial dark matter symposium highlights

The Microbial Dark Matter Symposium held on August 28–29, 2025, in Laguna Beach, Orange County, CA, convened a multidisciplinary group of scientists to address the vast unknowns in microbial life—from uncultured taxa and uncharacterized proteins to elusive viruses and spacefaring microbes. Set against a scenic coastal backdrop, the symposium highlighted advances in single-cell genomics, proximity ligation sequencing, and artificial intelligence-ready bioinformatics, while also probing the limits of microbial persistence, metabolism, and ecological distribution. Sessions explored microbial dark matter from multiple dimensions: cultivability, where new strategies are enabling recovery of elusive microbes; functional ambiguity, where metagenomic dark zones are illuminated by computational annotation; and genomic representation, where single-cell methods bridge gaps left by shotgun community sequencing. Researchers shared breakthroughs in identifying atmospheric microbiomes, “dark oxygen” production in groundwater ecosystems, and microbial survival on the International Space Station. The symposium emphasized integration of methods, disciplines, and ecosystems, advancing a collective push to illuminate the microbial dark matter on Earth and beyond. By highlighting emerging tools, pressing questions, and cross-domain insights, the symposium underscored the need for collaborative, open, and adaptive approaches to study the microbial unknown. The meeting marks a pivotal moment in microbiology, where cultivating knowledge of the uncultivated promises transformative understanding of life, everywhere.

Podar, Mircea [ORNL] (ORCID:0000000327760205)↗
DOE OSTI2021

Developing Cost Effective Biological Removal Technology for Selenium and Nitrate from Flue Gas Desulfurization Wastewater from an Existing Power Facility

Funding from this project supported new research in environmental biotechnology at WVSU Energy and Environmental Science Institute (WVSUEESI) to develop biological treatment techniques for FGD wastewater using photosynthetic organisms algae and plants. We tested the possibility of using algae and plants in treating FGD wastewater. Results suggested that algae and plants could able to perform normal photosynthesis under low concentrations of selenium. However, higher concentrations of selenium are lethal on photosynthesis. The total protein data indicated that FGD wastewater induced the accumulation of complete proteins in the duckweeds compared to control plants. It appears that nitrate, nitrates, and other compounds in FGD wastewater promote total protein accumulation. The FGD wastewater treatment has triggered Triacylglycerols (TAG) and carbohydrate accumulation in duckweed. We studied the differential gene expression using RNA-Sequencing data. Gene expression analysis revealed that glycolysis, fatty acid, triacylglycerol biosynthesis, and starch degradation were up-regulated; on the contrary, gluconeogenesis, photosynthesis, and starch biosynthesis genes were down-regulated by FGD wastewater treatment. We confirmed the expression of some of these genes using q-RT PCR analysis. We demonstrated that when FGD gypsum was blended with the coal mine soil to grow the bioenergy crop, Camelina, it could grow and produce seed. Moreover, Camelina is a non-edible oilseed crop and has heavy metal sequestering properties, making it the right reclamation choice. Besides, this project provided the opportunity for WVSU undergraduates to integrate hands-on research and education. Funding from this grant helped us to enhance student hands-on experience in water quality and environmental science and participation in STEM research and education.

09 BIOMASS FUELS↗
DOE OSTI2021

MULTICOM2 open-source protein structure prediction system powered by deep learning and distance prediction

Protein structure prediction is an important problem in bioinformatics and has been studied for decades. However, there are still few open-source comprehensive protein structure prediction packages publicly available in the field. In this paper, we present our latest open-source protein tertiary structure prediction system—MULTICOM2, an integration of template-based modeling (TBM) and template-free modeling (FM) methods. The template-based modeling uses sequence alignment tools with deep multiple sequence alignments to search for structural templates, which are much faster and more accurate than MULTICOM1. The template-free (ab initio or de novo) modeling uses the inter-residue distances predicted by DeepDist to reconstruct tertiary structure models without using any known structure as template. In the blind CASP14 experiment, the average TM-score of the models predicted by our server predictor based on the MULTICOM2 system is 0.720 for 58 TBM (regular) domains and 0.514 for 38 FM and FM/TBM (hard) domains, indicating that MULTICOM2 is capable of predicting good tertiary structures across the board. It can predict the correct fold for 76 CASP14 domains (95% regular domains and 55% hard domains) if only one prediction is made for a domain. The success rate is increased to 3% for both regular and hard domains if five predictions are made per domain. Moreover, the prediction accuracy of the pure template-free structure modeling method on both TBM and FM targets is very close to the combination of template-based and template-free modeling methods. This demonstrates that the distance-based template-free modeling method powered by deep learning can largely replace the traditional template-based modeling method even on TBM targets that TBM methods used to dominate and therefore provides a uniform structure modeling approach to any protein. Finally, on the 38 CASP14 FM and FM/TBM hard domains, MULTICOM2 server predictors (MULTICOM-HYBRID, MULTICOM-DEEP, MULTICOM-DIST) were ranked among the top 20 automated server predictors in the CASP14 experiment. After combining multiple predictors from the same research group as one entry, MULTICOM-HYBRID was ranked no. 5. The source code of MULTICOM2 is freely available at https://github.com/multicom-toolbox/multicom/tree/multicom_v2.0 .

97 MATHEMATICS AND COMPUTING↗
DOE OSTI2025

Decoding substrate specificity determining factors in glycosyltransferase-B enzymes – insights from machine learning models

Substrate specificity is an essential characteristic of any enzyme's function and an understanding of the factors that determine this specificity is crucial for enzyme engineering. Unlike the structure of an enzyme which is directly impacted by its sequence, substrate specificity as an enzyme attribute involves a rather indirect relationship with sequence as it also depends on structural aspects that dictate substrate accessibility and active site dynamics. In this study, we explore the performance of classifier-based machine learning models trained on curated sequence and structural data for a class of glycosyltransferases (GTs), namely GT-Bs, to understand their substrate specificity determining factors. GTs enable the transfer of sugar moieties to other biomolecules such as oligosaccharides or proteins and are found in all kingdoms of life. In plants, GTs participate in the biosynthesis of plant cell wall biopolymers (e.g.: hemicelluloses and pectins) and are an integral part of the enzymatic machinery that enables the storage of carbon and energy as plant biomass. To elucidate the substrate specificity of uncharacterized GT-Bs, we constructed multi-label machine learning models (Support Vector Classifier, K-Nearest Neighbors, Gaussian Naïve-Bayes, Random Forest) that incorporate both sequence and structural features. These models achieve good predictive accuracies on test datasets. However, despite our use of structural information, we highlight that there is further scope for improvement in training these models to draw interpretable relationships between sequence, structure and substrate specificity determining motifs in GT-Bs.

97 MATHEMATICS AND COMPUTING↗
DOE OSTI2024

Labile assembly of a tardigrade protein induces biostasis

Abstract Tardigrades are microscopic animals that survive desiccation by inducing biostasis. To survive drying tardigrades rely on intrinsically disordered CAHS proteins, which also function to prevent perturbations induced by drying in vitro and in heterologous systems. CAHS proteins have been shown to form gels both in vitro and in vivo, which has been speculated to be linked to their protective capacity. However, the sequence features and mechanisms underlying gel formation and the necessity of gelation for protection have not been demonstrated. Here we report a mechanism of fibrillization and gelation for CAHS D similar to that of intermediate filament assembly. We show that in vitro, gelation restricts molecular motion, immobilizing and protecting labile material from the harmful effects of drying. In vivo, we observe that CAHS D forms fibrillar networks during osmotic stress. Fibrillar networking of CAHS D improves survival of osmotically shocked cells. We observe two emergent properties associated with fibrillization; (i) prevention of cell volume change and (ii) reduction of metabolic activity during osmotic shock. We find that there is no significant correlation between maintenance of cell volume and survival, while there is a significant correlation between reduced metabolism and survival. Importantly, CAHS D's fibrillar network formation is reversible and metabolic rates return to control levels after CAHS fibers are resolved. This work provides insights into how tardigrades induce reversible biostasis through the self‐assembly of labile CAHS gels.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Room-temperature structural studies of SARS-CoV-2 protein NendoU with an X-ray free-electron laser

NendoU from SARS-CoV-2 is responsible for the virus’s ability to evade the innate immune system by cleaving the polyuridine leader sequence of antisense viral RNA. Here we report the room-temperature structure of NendoU, solved by serial femtosecond crystallography at an X-ray free-electron laser to 2.6 Å resolution. The room-temperature structure provides insight into the flexibility, dynamics, and other intrinsic properties of NendoU, with indications that the enzyme functions as an allosteric switch. Functional studies examining cleavage specificity in solution and in crystals support the uridine-purine cleavage preference, and we demonstrate that enzyme activity is fully maintained in crystal form. Optimizing the purification of NendoU and identifying suitable crystallization conditions set the benchmark for future time-resolved serial femtosecond crystallography studies. This could advance the design of antivirals with higher efficacy in treating coronaviral infections, since drugs that block allosteric conformational changes are less prone to drug resistance.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Spacer2PAM: A computational framework to guide experimental determination of functional CRISPR-Cas system PAM sequences

Abstract RNA-guided nucleases from CRISPR-Cas systems expand opportunities for precise, targeted genome modification. Endogenous CRISPR-Cas systems in many prokaryotes are attractive to circumvent expression, functionality, and unintended activity hurdles posed by heterologous CRISPR-Cas effectors. However, each CRISPR-Cas system recognizes a unique set of protospacer adjacent motifs (PAMs), which requires identification by extensive screening of randomized DNA libraries. This challenge hinders development of endogenous CRISPR-Cas systems, especially those based on multi-protein effectors and in organisms that are slow-growing or have transformation idiosyncrasies. To address this challenge, we present Spacer2PAM, an easy-to-use, easy-to-interpret R package built to predict and guide experimental determination of functional PAM sequences for any CRISPR-Cas system given its corresponding CRISPR array as input. Spacer2PAM can be used in a ‘Quick’ method to generate a single PAM prediction or in a ‘Comprehensive’ method to inform targeted PAM libraries small enough to screen in difficult to transform organisms. We demonstrate Spacer2PAM by predicting PAM sequences for industrially relevant organisms and experimentally identifying seven PAM sequences that mediate interference from the Spacer2PAM-informed PAM library for the type I-B CRISPR-Cas system from Clostridium autoethanogenum. We anticipate that Spacer2PAM will facilitate the use of endogenous CRISPR-Cas systems for industrial biotechnology and synthetic biology.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Analysis of the molecular determinants for furin cleavage of the spike protein S1/S2 site in defined strains of the prototype coronavirus murine hepatitis virus (MHV)

We analyzed the spike protein S1/S2 cleavage of selected strains of a prototype coronavirus, mouse hepatitis virus (MHV) by the cellular protease furin, in order to understand the structural requirements underlying the sequence selectivity of the scissile segment. The probability of cleavage of selected MHV strains was first evaluated from furin cleavage scores predicted by the ProP computer software, and then cleavage was measured experimentally with a fluorogenic peptide cleavage assay consisting of S1/S2 peptide mimics and purified furin. We found that in vitro cleavability varied across MHV strains in line with predicted results—but with the notable exception of MHV-A59, which was not cleaved despite a high score predicted for its sequence. Using the known X-Ray structure of furin in complex with a substrate-like inhibitor as an initial structural reference, we carried out molecular dynamics (MD) simulations to learn the modes of binding of the peptides in the furin active site, and the suitability of the complex for initiation of the enzymatic cleavage. We identified the 3D structural requirements of the furin active site configuration that enable bound peptides to undergo cleavage, and the way in which the various strains tested experimentally are fulfilling these requirements. We find that despite some flexibility in the organization of the peptide bound to the active site of the enzyme, the presence of a histidine at P2 of MHV-A59 fails to properly orient the sidechain of His194 of the furin catalytic triad and therefore produces a distortion that renders the peptide/complex structural configuration in the active site incompatible with requirements for cleavage initiation. The Ser/Thr in P1 of MHV-2 and MHV-S has a similar effect of distorting the conformation of the furin active site residues produced by the elimination of the canonical salt-bridge formed by arginine in P1 position. This work informs a study of coronavirus infection and pathogenesis with respect to the function of the viral spike protein, and suggests an important process of viral adaptation and evolution within the spike S1/S2 structural loop.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2021

Quantifying the Long‐Range Coupling of Electronic Properties in Proteins with ab initio Molecular Dynamics**

Abstract The delicate interplay of covalent and non‐covalent interactions in proteins is inherently quantum mechanical and highly dynamic in nature. To directly interrogate the evolving nature of the electronic structure of proteins, we carry out 100‐ps‐scale ab initio molecular dynamics simulations of three representative small proteins with range‐separated hybrid density functional theory. We quantify the nature and length‐scale of the coupling of residue‐specific charge probability distributions in these proteins. While some nonpolar residues exhibit expectedly narrow charge distributions, most polar and charged residues exhibit broad, multimodal distributions. Even for nonpolar residues, we observe sequence‐specific deviations corresponding to charge accumulation or depletion that would be challenging to capture in a fixed charge force field. We quantify the effect of residue‐residue interactions on charge distributions first with linear cross‐correlations. We then show how additional insight can be gained from evaluating the mutual information of charge distributions. We show that a significant number of residues couple most strongly with residues that are distant in both sequence and space over a range of secondary structures including α‐helical, β‐sheet, disulfide bridging, and lasso motifs. The mutual information analysis is necessary to capture coupling between some polar and charged residues that would be otherwise missed.

Yang, Zhongyue↗
DOE OSTI2021

Polarity-Tolerant Chloride Binding in Foldamer Capsules by Programmed Solvent-Exclusion

Persistent anion binding in a wide range of solution environments is a key challenge that continues to motivate and demand new strategies in synthetic receptor design. Though strong binding in low-polarity solvents has become routine, our ability to maintain high affinities in high-polarity solvents has not yet reached the standard set by nature. Anions are bound and transported regularly in aqueous environments by proteins that use secondary and tertiary structure to isolate anion binding sites from water. Inspired by this principle of solvent exclusion, we created a sequence-defined foldameric capsule whose global minimum conformation displays a helical folded state and is preorganized for 1:1 anion complexation. The high stability of the folded geometry and its ability to exclude solvent were supported by solid-state and solution phase studies. This capsule then withstood a 4-fold increase in solvent dielectric constant (εr) from dichloromethane (9) to acetonitrile (36) while maintaining a high and solvent-independent affinity of 10 5 M –1 ; ΔG ~ 28 kJ mol –1 . This behavior is unusual. More typical of solvent-dependent behavior, Cl – affinities were seen to plummet in control compounds, such as aryl-triazole macrocycles and pentads, with their solvent-exposed binding cavities susceptible to dielectric screening. Finally, dimethyl sulfoxide denatures the foldamer by putative solvent binding, which then lowers the foldamer’s Cl – affinity to normal levels. Furthermore, the design of this capsule demonstrates a new prototype for the development of potent receptors that can operate in polar solvents and has the potential to help manage hydrophilic anions present in the hydrosphere and biosphere.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2024

Structural basis for intermodular communication in assembly-line polyketide biosynthesis

Assembly-line polyketide synthases (PKSs) are modular multi-enzyme systems with considerable potential for genetic reprogramming. Understanding how they selectively transport biosynthetic intermediates along a defined sequence of active sites could be harnessed to rationally alter PKS product structures. Here, to investigate functional interactions between PKS catalytic and substrate acyl carrier protein (ACP) domains, we employed a bifunctional reagent to crosslink transient domain–domain interfaces of a prototypical assembly line, the 6-deoxyerythronolide B synthase, and resolved their structures by single-particle cryogenic electron microscopy (cryo-EM). Together with statistical per-particle image analysis of cryo-EM data, we uncovered interactions between ketosynthase (KS) and ACP domains that discriminate between intra-modular and inter-modular communication while reinforcing the relevance of conformational asymmetry during the catalytic cycle. Our findings provide a foundation for the structure-based design of hybrid PKSs comprising biosynthetic modules from different naturally occurring assembly lines.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Peripheral positions encode transport specificity in the small multidrug resistance exporters

In secondary active transporters, a relatively limited set of protein folds have evolved diverse solute transport functions. Because of the conformational changes inherent to transport, altering substrate specificity typically involves remodeling the entire structural landscape, limiting our understanding of how novel substrate specificities evolve. In the current work, we examine a structurally minimalist family of model transport proteins, the small multidrug resistance (SMR) transporters, to understand the molecular basis for the emergence of a novel substrate specificity. We engineer a selective SMR protein to promiscuously export quaternary ammonium antiseptics, similar to the activity of a clade of multidrug exporters in this family. Using combinatorial mutagenesis and deep sequencing, we identify the necessary and sufficient molecular determinants of this engineered activity. Using X-ray crystallography, solid-supported membrane electrophysiology, binding assays, and a proteoliposome-based quaternary ammonium antiseptic transport assay that we developed, we dissect the mechanistic contributions of these residues to substrate polyspecificity. We find that substrate preference changes not through modification of the residues that directly interact with the substrate but through mutations peripheral to the binding pocket. Our work provides molecular insight into substrate promiscuity among the SMRs and can be applied to understand multidrug export and the evolution of novel transport functions more generally.

Science & Technology - Other Topics↗
DOE OSTI2023

Dual incorporation of non-canonical amino acids enables production of post-translationally modified selenoproteins

Post-translational modifications (PTMs) can occur on almost all amino acids in eukaryotes as a key mechanism for regulating protein function. The ability to study the role of these modifications in various biological processes requires techniques to modify proteins site-specifically. One strategy for this is genetic code expansion (GCE) in bacteria. The low frequency of post-translational modifications in bacteria makes it a preferred host to study whether the presence of a post-translational modification influences a protein’s function. Genetic code expansion employs orthogonal translation systems engineered to incorporate a modified amino acid at a designated protein position. Selenoproteins, proteins containing selenocysteine, are also known to be post-translationally modified. Selenoproteins have essential roles in oxidative stress, immune response, cell maintenance, and skeletal muscle regeneration. Their complicated biosynthesis mechanism has been a hurdle in our understanding of selenoprotein functions. As technologies for selenocysteine insertion have recently improved, we wanted to create a genetic system that would allow the study of post-translational modifications in selenoproteins. By combining genetic code expansion techniques and selenocysteine insertion technologies, we were able to recode stop codons for insertion of N ε -acetyl- l -lysine and selenocysteine, respectively, into multiple proteins. The specificity of these amino acids for their assigned position and the simplicity of reverting the modified amino acid via mutagenesis of the codon sequence demonstrates the capacity of this method to study selenoproteins and the role of their post-translational modifications. Moreover, the evidence that Sec insertion technology can be combined with genetic code expansion tools further expands the chemical biology applications.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

A randomized multiplex CRISPRi-Seq approach for the identification of critical combinations of genes

Identifying virulence-critical genes from pathogens is often limited by functional redundancy. To rapidly interrogate the contributions of combinations of genes to a biological outcome, we have developed a mu ltiplex, r andomized C RISPR i nterference s equencing (MuRCiS) approach. At its center is a new method for the randomized self-assembly of CRISPR arrays from synthetic oligonucleotide pairs. When paired with PacBio long-read sequencing, MuRCiS allowed for near-comprehensive interrogation of all pairwise combinations of a group of 44 Legionella pneumophila virulence genes encoding highly conserved transmembrane proteins for their role in pathogenesis. Both amoeba and human macrophages were challenged with L. pneumophila bearing the pooled CRISPR array libraries, leading to the identification of several new virulence-critical combinations of genes. lpg2888 and lpg3000 were particularly fascinating for their apparent redundant functions during L. pneumophila human macrophage infection, while lpg3000 alone was essential for L. pneumophila virulence in the amoeban host Acanthamoeba castellanii . Thus, MuRCiS provides a method for rapid genetic examination of even large groups of redundant genes, setting the stage for application of this technology to a variety of biological contexts and organisms.

59 BASIC BIOLOGICAL SCIENCES↗