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At least 127 records · Page 7

The evolution of the GAL actose utilization pathway in budding yeasts

The Leloir galactose utilization or GAL pathway of budding yeasts, including that of the baker’s yeast Saccharomyces cerevisiae and the opportunistic human pathogen Candida albicans, breaks down the sugar galactose for energy and biomass production. Here, the GAL pathway has long served as a model system for understanding how eukaryotic metabolic pathways, including their modes of regulation, evolve. More recently, the physical linkage of the structural genes GAL1, GAL7, and GAL10 in diverse budding yeast genomes has been used as a model for understanding the evolution of gene clustering. In this review, we summarize exciting recent work on three different aspects of this iconic pathway’s evolution: gene cluster organization, GAL gene regulation, and the population genetics of the GAL pathway.

59 BASIC BIOLOGICAL SCIENCES↗

A giant virus infecting the amoeboflagellate Naegleria

Giant viruses (Nucleocytoviricota) are significant lethality agents of various eukaryotic hosts. Although metagenomics indicates their ubiquitous distribution, available giant virus isolates are restricted to a very small number of protist and algal hosts. Here we report on the first viral isolate that replicates in the amoeboflagellate Naegleria. This genus comprises the notorious human pathogen Naegleria fowleri, the causative agent of the rare but fatal primary amoebic meningoencephalitis. We have elucidated the structure and infection cycle of this giant virus, Catovirus naegleriensis (a.k.a. Naegleriavirus, NiV), and show its unique adaptations to its Naegleria host using fluorescence in situ hybridization, electron microscopy, genomics, and proteomics. Naegleriavirus is only the fourth isolate of the highly diverse subfamily Klosneuvirinae, and like its relatives the NiV genome contains a large number of translation genes, but lacks transfer RNAs (tRNAs). NiV has acquired genes from its Naegleria host, which code for heat shock proteins and apoptosis inhibiting factors, presumably for host interactions. Notably, NiV infection was lethal to all Naegleria species tested, including the human pathogen N. fowleri. This study expands our experimental framework for investigating giant viruses and may help to better understand the basic biology of the human pathogen N. fowleri.

59 BASIC BIOLOGICAL SCIENCES↗

Ecosystem consequences of introducing plant growth promoting rhizobacteria to managed systems and potential legacy effects

The rapidly growing industry of crop biostimulants leverages the application of plant growth promoting rhizobacteria (PGPR) to promote plant growth and health. However, introducing nonnative rhizobacteria may impact other aspects of ecosystem functioning and have legacy effects; these potential consequences are largely unexplored. Nontarget consequences of PGPR may include changes in resident microbiomes, nutrient cycling, pollinator services, functioning of other herbivores, disease suppression, and organic matter persistence. Importantly, we lack knowledge of whether these ecosystem effects may manifest in adjacent ecosystems. The introduced PGPR can leave a functional legacy whether they persist in the community or not. Legacy effects include shifts in resident microbiomes and their temporal dynamics, horizontal transfer of genes from the PGPR to resident taxa, and changes in resident functional groups and interaction networks. Ecosystem functions may be affected by legacies PGPR leave following niche construction, such as when PGPR alter soil pH that in turn alters biogeochemical cycling rates. Here, we highlight new research directions to elucidate how introduced PGPR impact resident microbiomes and ecosystem functions and their capacity for legacy effects.

59 BASIC BIOLOGICAL SCIENCES↗

The extent of multiallelic, co‐editing of LIGULELESS1 in highly polyploid sugarcane tunes leaf inclination angle and enables selection of the ideotype for biomass yield

Summary Sugarcane ( Saccharum spp. hybrid) is a prime feedstock for commercial production of biofuel and table sugar. Optimizing canopy architecture for improved light capture has great potential for elevating biomass yield. LIGULELESS1 ( LG1 ) is involved in leaf ligule and auricle development in grasses. Here, we report CRISPR/Cas9‐mediated co‐mutagenesis of up to 40 copies/alleles of the putative LG1 in highly polyploid sugarcane (2 n = 100–120, x = 10–12). Next generation sequencing revealed co‐editing frequencies of 7.4%–100% of the LG1 reads in 16 of the 78 transgenic lines. LG1 mutations resulted in a tuneable leaf angle phenotype that became more upright as co‐editing frequency increased. Three lines with loss of function frequencies of ~12%, ~53% and ~95% of lg1 were selected following a randomized greenhouse trial and grown in replicated, multi‐row field plots. The co‐edited LG1 mutations were stably maintained in vegetative progenies and the extent of co‐editing remained constant in field tested lines L26 and L35. Next generation sequencing confirmed the absence of potential off targets. The leaf inclination angle corresponded to light transmission into the canopy and tiller number. Line L35 displaying loss of function in ~12% of the lg1 NGS reads exhibited an 18% increase in dry biomass yield supported by a 56% decrease in leaf inclination angle, a 31% increase in tiller number, and a 25% increase in internode number. The scalable co‐editing of LG1 in highly polyploid sugarcane allows fine‐tuning of leaf inclination angle, enabling the selection of the ideotype for biomass yield.

59 BASIC BIOLOGICAL SCIENCES↗

Plasmid-Encoded Traits Vary across Environments

Plasmids are well known for their role in the transmission of antibiotic resistance-conferring genes. Beyond human and clinical settings, however, they disseminate many other types of genes, including those that contribute to microbially driven ecosystem processes.

59 BASIC BIOLOGICAL SCIENCES↗

PlasmidHostFinder: Prediction of Plasmid Hosts Using Random Forest

Plasmids play a major role facilitating the spread of antimicrobial resistance between bacteria. Understanding the host range and dissemination trajectories of plasmids is critical for surveillance and prevention of antimicrobial resistance. Identification of plasmid host ranges could be improved using automated pattern detection methods compared to homology-based methods due to the diversity and genetic plasticity of plasmids. In this study, we developed a method for predicting the host range of plasmids using machine learning—specifically, random forests. We trained the models with 8,519 plasmids from 359 different bacterial species per taxonomic level; the models achieved Matthews correlation coefficients of 0.662 and 0.867 at the species and order levels, respectively. Our results suggest that despite the diverse nature and genetic plasticity of plasmids, our random forest model can accurately distinguish between plasmid hosts. This tool is available online through the Center for Genomic Epidemiology (https://cge.cbs.dtu.dk/services/PlasmidHostFinder/).

59 BASIC BIOLOGICAL SCIENCES↗

Lower viral evolutionary pressure under stable versus fluctuating conditions in subzero Arctic brines

Climate change threatens Earth’s ice-based ecosystems which currently offer archives and eco-evolutionary experiments in the extreme. Arctic cryopeg brine (marine-derived, within permafrost) and sea ice brine, similar in subzero temperature and high salinity but different in temporal stability, are inhabited by microbes adapted to these extreme conditions. However, little is known about their viruses (community composition, diversity, interaction with hosts, or evolution) or how they might respond to geologically stable cryopeg versus fluctuating sea ice conditions. We used long- and short-read viromics and metatranscriptomics to study viruses in Arctic cryopeg brine, sea ice brine, and underlying seawater, recovering 11,088 vOTUs (~species-level taxonomic unit), a 4.4-fold increase of known viruses in these brines. More specifically, the long-read-powered viromes doubled the number of longer (≥25 kb) vOTUs generated and recovered more hypervariable regions by >5-fold compared to short-read viromes. Distribution assessment, by comparing to known viruses in public databases, supported that cryopeg brine viruses were of marine origin yet distinct from either sea ice brine or seawater viruses, while 94% of sea ice brine viruses were also present in seawater. A virus-encoded, ecologically important exopolysaccharide biosynthesis gene was identified, and many viruses (~half of metatranscriptome-inferred “active” vOTUs) were predicted as actively infecting the dominant microbial genera Marinobacter and Polaribacter in cryopeg and sea ice brines, respectively. Evolutionarily, microdiversity (intra-species genetic variations) analyses suggested that viruses within the stable cryopeg brine were under significantly lower evolutionary pressures than those in the fluctuating sea ice environment, while many sea ice brine virus-tail genes were under positive selection, indicating virus-host co-evolutionary arms races. Our results confirmed the benefits of long-read-powered viromics in understanding the environmental virosphere through significantly improved genomic recovery, expanding viral discovery and the potential for biological inference. Evidence of viruses actively infecting the dominant microbes in subzero brines and modulating host metabolism underscored the potential impact of viruses on these remote and underexplored extreme ecosystems. Microdiversity results shed light on different strategies viruses use to evolve and adapt when extreme conditions are stable versus fluctuating. Together, these findings verify the value of long-read-powered viromics and provide foundational data on viral evolution and virus-microbe interactions in Earth’s destabilized and rapidly disappearing cryosphere.

59 BASIC BIOLOGICAL SCIENCES↗

Multiallelic, Targeted Mutagenesis of Magnesium Chelatase With CRISPR/Cas9 Provides a Rapidly Scorable Phenotype in Highly Polyploid Sugarcane

Genome editing with sequence-specific nucleases, such as clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9), is revolutionizing crop improvement. Developing efficient genome-editing protocols for highly polyploid crops, including sugarcane ( x = 10–13), remains challenging due to the high level of genetic redundancy in these plants. Here, we report the efficient multiallelic editing of magnesium chelatase subunit I ( MgCh ) in sugarcane. Magnesium chelatase is a key enzyme for chlorophyll biosynthesis. CRISPR/Cas9-mediated targeted co-mutagenesis of 49 copies/alleles of magnesium chelatase was confirmed via Sanger sequencing of cloned PCR amplicons. This resulted in severely reduced chlorophyll contents, which was scorable at the time of plant regeneration in the tissue culture. Heat treatment following the delivery of genome editing reagents elevated the editing frequency 2-fold and drastically promoted co-editing of multiple alleles, which proved necessary to create a phenotype that was visibly distinguishable from the wild type. Despite their yellow leaf color, the edited plants were established well in the soil and did not show noticeable growth retardation. This approach will facilitate the establishment of genome editing protocols for recalcitrant crops and support further optimization, including the evaluation of alternative RNA-guided nucleases to overcome the limitations of the protospacer adjacent motif (PAM) site or to develop novel delivery strategies for genome editing reagents.

59 BASIC BIOLOGICAL SCIENCES↗

Evolutionary Divergence of Marinobacter Strains in Cryopeg Brines as Revealed by Pangenomics

Marinobacter spp. are cosmopolitan in saline environments, displaying a diverse set of metabolisms that allow them to competitively occupy these environments, some of which can be extreme in both salinity and temperature. Here, we introduce a distinct cluster of Marinobacter genomes, composed of novel isolates and in silico assembled genomes obtained from subzero, hypersaline cryopeg brines, relic seawater-derived liquid habitats within permafrost sampled near Utqiaġvik, Alaska. Using these new genomes and 45 representative publicly available genomes of Marinobacter spp. from other settings, we assembled a pangenome to examine how the new extremophile members fit evolutionarily and ecologically, based on genetic potential and environmental source. This first genus-wide genomic analysis revealed that Marinobacter spp. in general encode metabolic pathways that are thermodynamically favored at low temperature, cover a broad range of organic compounds, and optimize protein usage, e.g., the Entner–Doudoroff pathway, the glyoxylate shunt, and amino acid metabolism. The new isolates contributed to a distinct clade of subzero brine-dwelling Marinobacter spp. that diverged genotypically and phylogenetically from all other Marinobacter members. The subzero brine clade displays genomic characteristics that may explain competitive adaptations to the extreme environments they inhabit, including more abundant membrane transport systems (e.g., for organic substrates, compatible solutes, and ions) and stress-induced transcriptional regulatory mechanisms (e.g., for cold and salt stress) than in the other Marinobacter clades. We also identified more abundant signatures of potential horizontal transfer of genes involved in transcription, the mobilome, and a variety of metabolite exchange systems, which led to considering the importance of this evolutionary mechanism in an extreme environment where adaptation via vertical evolution is physiologically rate limited. Assessing these new extremophile genomes in a pangenomic context has provided a unique view into the ecological and evolutionary history of the genus Marinobacter , particularly with regard to its remarkable diversity and its opportunism in extremely cold and saline environments.

59 BASIC BIOLOGICAL SCIENCES↗

Coming-of-Age Characterization of Soil Viruses: A User’s Guide to Virus Isolation, Detection within Metagenomes, and Viromics

The study of soil viruses, though not new, has languished relative to the study of marine viruses. This is particularly due to challenges associated with separating virions from harboring soils. Generally, three approaches to analyzing soil viruses have been employed: (1) Isolation, to characterize virus genotypes and phenotypes, the primary method used prior to the start of the 21st century. (2) Metagenomics, which has revealed a vast diversity of viruses while also allowing insights into viral community ecology, although with limitations due to DNA from cellular organisms obscuring viral DNA. (3) Viromics (targeted metagenomics of virus-like-particles), which has provided a more focused development of ‘virus-sequence-to-ecology’ pipelines, a result of separation of presumptive virions from cellular organisms prior to DNA extraction. This separation permits greater sequencing emphasis on virus DNA and thereby more targeted molecular and ecological characterization of viruses. Employing viromics to characterize soil systems presents new challenges, however. Ones that only recently are being addressed. Here we provide a guide to implementing these three approaches to studying environmental viruses, highlighting benefits, difficulties, and potential contamination, all toward fostering greater focus on viruses in the study of soil ecology.

59 BASIC BIOLOGICAL SCIENCES↗

Files_used_for_figures

This folder includes all the relevant files used to generate the main Figures in the paper.

DNA repair↗

Productivity and bioproduct formation in phototropic knock/out mutants in micro algae

Phototropin is a blue light receptor, which mediates a variety of blue-light elicited physiological processes in plants and algae. In higher plants these processes include phototropism, chloroplast movement and stomatal opening. In the green alga Chlamydomonas reinhardtii, phototropin plays a vital role in progression of the sexual life cycle and in the control of the eye spot size and light sensitivity. Phototropin is also involved in blue-light mediated changes in the synthesis of chlorophylls, carotenoids, chlorophyll binding proteins. We compared the transcriptome of phototropin knock out (PHOT KO) mutant and wild-type parent to analyze differences in gene expression in high light grown cultures (500 μmol photons m⁻²s⁻¹). Our results indicate the up-regulation of genes involved in photosynthetic electron transport chain, carbon fixation pathway, starch, lipid, and cell cycle control genes. With respect to photosynthetic electron transport genes, genes encoding proteins of the cytochrome b6f and ATP synthase complex were up regulated potentially facilitating proton-coupled electron transfer. In addition genes involved in limiting steps in the Calvin cycle Ribulose-1,5-biphosphate carboxylase/oxygenase (RuBisCO), Sidoheptulose 1,7 biophosphatase (SBPase), Glyceraldehyde-3-phosphate dehydrogenase (3PGDH) and that mediate cell-cycle control (CDK) were also up regulated along with starch synthase and fatty acid biosynthesis genes involved in starch and lipid synthesis. In addition, transmission electron micrographs show increased accumulation of starch granules in PHOT mutant compared to wild type, which is consistent with the higher expression of starch synthase genes. Collectively, the altered patterns of gene expression in the PHOT mutants were associated with a two-fold increase in growth and biomass accumulation compared to wild type when grown in environmental photobioreactors (Phenometrics) that simulate a pond environment. In conclusion, our studies suggest that phototropin may be a master gene regulator that suppresses rapid cell growth and promotes gametogenesis and sexual recombination in wild type strains.

59 BASIC BIOLOGICAL SCIENCES↗

A colorimetric method to measure in vitro nitrogenase functionality for engineering nitrogen fixation

Biological nitrogen fixation (BNF) is the reduction of N 2 into NH 3 in a group of prokaryotes by an extremely O 2 -sensitive protein complex called nitrogenase. Transfer of the BNF pathway directly into plants, rather than by association with microorganisms, could generate crops that are less dependent on synthetic nitrogen fertilizers and increase agricultural productivity and sustainability. In the laboratory, nitrogenase activity is commonly determined by measuring ethylene produced from the nitrogenase-dependent reduction of acetylene (ARA) using a gas chromatograph. The ARA is not well suited for analysis of large sample sets nor easily adapted to automated robotic determination of nitrogenase activities. Here, we show that a reduced sulfonated viologen derivative (S 2 V red ) assay can replace the ARA for simultaneous analysis of isolated nitrogenase proteins using a microplate reader. We used the S 2 V red to screen a library of NifH nitrogenase components targeted to mitochondria in yeast. Two NifH proteins presented properties of great interest for engineering of nitrogen fixation in plants, namely NifM independency, to reduce the number of genes to be transferred to the eukaryotic host; and O 2 resistance, to expand the half-life of NifH iron-sulfur cluster in a eukaryotic cell. This study established that NifH from Dehalococcoides ethenogenes did not require NifM for solubility, [Fe-S] cluster occupancy or functionality, and that NifH from Geobacter sulfurreducens was more resistant to O 2 exposure than the other NifH proteins tested. It demonstrates that nitrogenase components with specific biochemical properties such as a wider range of O 2 tolerance exist in Nature, and that their identification should be an area of focus for the engineering of nitrogen-fixing crops.

59 BASIC BIOLOGICAL SCIENCES↗

Transport of metformin metabolites by guanidinium exporters of the small multidrug resistance family

Proteins from the small multidrug resistance (SMR) family are frequently associated with horizontally transferred multidrug resistance gene arrays found in bacteria from wastewater and the human-adjacent biosphere. Recent studies suggest that a subset of SMR transporters might participate in the metabolism of the common pharmaceutical metformin by bacterial consortia. Here, we show that both genomic and plasmid-associated transporters of the SMRGdx functional subtype export byproducts of microbial metformin metabolism, with particularly high export efficiency for guanylurea. We use solid-supported membrane electrophysiology to evaluate the transport kinetics for guanylurea and native substrate guanidinium by four representative SMRGdx homologs. Using an internal reference to normalize independent electrophysiology experiments, we show that transport rates are comparable for genomic and plasmid-associated SMRGdx homologs, and using a proteoliposome-based transport assay, we show that 2 proton:1 substrate transport stoichiometry is maintained. Additional characterization of guanidinium and guanylurea export properties focuses on the structurally characterized homolog, Gdx-Clo, for which we examined the pH dependence and thermodynamics of substrate binding and solved an x-ray crystal structure with guanylurea bound. Together, these experiments contribute in two main ways. By providing the first detailed kinetic examination of the structurally characterized SMRGdx homolog Gdx-Clo, they provide a functional framework that will inform future mechanistic studies of this model transport protein. Second, this study casts light on a potential role for SMRGdx transporters in microbial handling of metformin and its microbial metabolic byproducts, providing insight into how native transport physiologies are co-opted to contend with new selective pressures.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

Deeplasmid: deep learning accurately separates plasmids from bacterial chromosomes

Plasmids are mobile genetic elements that play a key role in microbial ecology and evolution by mediating horizontal transfer of important genes, such as antimicrobial resistance genes. Many microbial genomes have been sequenced by short read sequencers and have resulted in a mix of contigs that derive from plasmids or chromosomes. New tools that accurately identify plasmids are needed to elucidate new plasmid-borne genes of high biological importance. We have developed Deeplasmid, a deep learning tool for distinguishing plasmids from bacterial chromosomes based on the DNA sequence and its encoded biological data. It requires as input only assembled sequences generated by any sequencing platform and assembly algorithm and its runtime scales linearly with the number of assembled sequences. Deeplasmid achieves an AUC–ROC of over 89%, and it was more accurate than five other plasmid classification methods. Finally, as a proof of concept, we used Deeplasmid to predict new plasmids in the fish pathogen Yersinia ruckeri ATCC 29473 that has no annotated plasmids. Deeplasmid predicted with high reliability that a long assembled contig is part of a plasmid. Using long read sequencing we indeed validated the existence of a 102 kb long plasmid, demonstrating Deeplasmid's ability to detect novel plasmids.

59 BASIC BIOLOGICAL SCIENCES↗

Developing stable, simplified, functional consortia from Brachypodium rhizosphere for microbial application in sustainable agriculture

The rhizosphere microbiome plays a crucial role in supporting plant productivity and ecosystem functioning by regulating nutrient cycling, soil integrity, and carbon storage. However, deciphering the intricate interplay between microbial relationships within the rhizosphere is challenging due to the overwhelming taxonomic and functional diversity. Here we present our systematic design framework built on microbial colocalization and microbial interaction, toward successful assembly of multiple rhizosphere-derived Reduced Complexity Consortia (RCC). We enriched co-localized microbes from Brachypodium roots grown in field soil with carbon substrates mimicking Brachypodium root exudates, generating 768 enrichments. By transferring the enrichments every 3 or 7 days for 10 generations, we developed both fast and slow-growing reduced complexity microbial communities. Most carbon substrates led to highly stable RCC just after a few transfers. 16S rRNA gene amplicon analysis revealed distinct community compositions based on inoculum and carbon source, with complex carbon enriching slow growing yet functionally important soil taxa like Acidobacteria and Verrucomicrobia. Network analysis showed that microbial consortia, whether differentiated by growth rate (fast vs. slow) or by succession (across generations), had significantly different network centralities. Besides, the keystone taxa identified within these networks belong to genera with plant growth-promoting traits, underscoring their critical function in shaping rhizospheric microbiome networks. Furthermore, tested consortia demonstrated high stability and reproducibility, assuring successful revival from glycerol stocks for long-term viability and use. Our study represents a significant step toward developing a framework for assembling rhizosphere consortia based on microbial colocalization and interaction, with future implications for sustainable agriculture and environmental management.

59 BASIC BIOLOGICAL SCIENCES↗

Adaptive_Evolution_2026

Thermoanaerobacterium saccharolyticum, an anaerobic and thermophilic bacterium capable of metabolizing sugar monomers and soluble oligomers into ethanol, has been proposed for use in consolidated bioprocessing in coculture with compatible cellulolytic bacteria such asClostridium thermocellum. Although the mixed acid fermentation of both of these strains has been engineered to produce ethanol as the major fermentation product, the maximum titer produced thus far byC. thermocellum, about 3% (w/v) ethanol, is half that produced byT. saccharolyticum. There is thus motivation to understand the mechanistic basis of the robust ethanol pathway inT. saccharolyticumso that key features can be recapitulated inC. thermocellumand other organisms.Previously, we characterized theindividualrole of the main genes responsible for electron transfer in the ethanol production ofT. saccharolyticum. However, the consequences of thecombinedloss of function of all these genes have not been investigated, nor has the way in which fermentative metabolism adapts to such constraints. In this work, we combined knockouts of ferredoxin nicotinamide oxidoreductase (fnor) genes (nfnAandnfnB) and hydrogenase genes (hydAandhfsD) and studied their effects on fermentation and grow. We showed that these genetic modifications together impair growth and decrease electron transfer from reduced ferredoxin, thereby redirecting flux from the pyruvate ferredoxin oxidoreductase enzyme to the pyruvate formate lyase enzyme. We also performed adaptive evolution of these mutants to rescue their growth, and most notably, we observed a single nucleotide variation in the alcohol dehydrogenaseadhAgene. Through molecular dynamics simulations and enzymatic assays, we determined that this point mutation causes a structural change that impairs the AdhA specificity for the NADPH cofactor and increases NADH-linked activity to restore redox balance. These findings consolidate our understanding of the functioning of electron transfer pathways in this organism.

alcohol dehydrogenase↗

An orphan gene BOOSTER enhances photosynthetic efficiency and plant productivity

Organelle-to-nucleus DNA transfer is an ongoing process playing an important role in the evolution of eukaryotic life. Here, genome-wide association studies (GWAS) of non-photochemical quenching parameters in 743 Populus trichocarpa accessions identified a nuclear-encoded genomic region associated with variation in photosynthesis under fluctuating light. The identified gene, BOOSTER (BSTR), comprises three exons, two with apparent endophytic origin and the third containing a large fragment of plastid-encoded Rubisco large subunit. Higher expression of BSTR facilitated anterograde signaling between nucleus and plastid, which corresponded to enhanced expression of Rubisco, increased photosynthesis, and up to 35% greater plant height and 88% biomass in poplar accessions under field conditions. Overexpression of BSTR in Populus tremula × P. alba achieved up to a 200% in plant height. Similarly, Arabidopsis plants heterologously expressing BSTR gained up to 200% in biomass and up to 50% increase in seed.

60 APPLIED LIFE SCIENCES↗