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Today, 12:26 AM
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Adaptation occurs through the selection of beneficial mutations enhancing fitness in a specific environment. However, since environments vary across time and space, mutations that are positively selected in one environment may be less beneficial or detrimental in others. Here, we investigate the evolution of microbial robustness (i.e. a consistent fitness across many diverse environments) through the adaptive evolution of two genetically distinct Saccharomyces cerevisiae populations in fluctuating conditions, followed by fitness assays and whole-genome sequencing. Our results indicate that the haploid laboratory strain S288C achieved higher average fitness than its parental strain, particularly when evolved in fluctuating environments compared to constant environments, but did not show increased robustness. In contrast, populations of the industrial diploid strain Ethanol Red failed to achieve significant fitness improvement under both fluctuating and constant evolution regimes but became more robust. Populations that adapted to fluctuating conditions acquired mutations in genes involved with cell morphology and protein degradation. Overall, our results emphasise the importance of parental traits in shaping fitness and robustness during adaptive laboratory evolution.
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December 6, 11:34 PM
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Loop engineering of enzymes remains challenging due to high flexibility and conformational complexity, posing a bottleneck for deep-learning-based design. Here, we constructed mutant libraries for three loops of TEV protease to assess combining directed evolution with deep learning. Using an M13 phagemid-based selection system, the three libraries were screened, resulting in a Loop 1 variant (HyperTEV60/L1) that significantly enhanced the Michaelis constant (Km) of the HyperTEV60 scaffold, a highly active mutant identified by ProteinMPNN. Structural modeling suggested that a single-residue deletion and substitution in Loop 1 expands the substrate binding pocket, accounting for the improved Km. Although the catalytic efficiency kcat/Km of HyperTEV60/L1 was only marginally higher than HyperTEV60, due to a kcat decrease, our results reveal that the phagemid-based selection system tended to find variants optimizing Km. This study demonstrates that combining deep-learning-based global optimization with localized directed evolution maximizes the probability of discovering distinct, high-performance enzyme variants.
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December 6, 11:23 PM
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Metagenomic taxonomic profiling is essential for characterizing microbial community composition in both environmental and clinical contexts. Existing profilers have greatly advanced community characterization; however, achieving accurate profiling across all domains of life - especially for archaea, fungi, and viruses - and for low-biomass, host-dominated samples, remains challenging. We describe Metax, a cross-domain taxonomic profiler that employs probabilistic modeling of genome coverage to distinguish true community members from artifactual signals arising from reference contamination, local genomic similarity, or reagent-derived DNA fragments. In comprehensive benchmarks across more than 500 samples, Metax demonstrated accurate species-level profiling, with consistent performance for bacteria, archaea, eukaryotes and viruses, and robustness to shallow sequencing. Applied to an oral microbiome cohort, Metax identified differentially abundant viral taxa distinguishing peri-implantitis from healthy sites, while analyses of tumor microbiome data revealed reagent-borne contaminants and potential reference misassemblies. By integrating coverage-informed statistics, Metax delivers accurate, robust, and interpretable cross-domain taxonomic profiles, maintaining stable performance across diverse sequencing depths and sample types.
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December 6, 11:02 PM
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Conventional methods for plant disease detection and management often face limitations in sensitivity, specificity, and field applicability. Aptamers, with high affinity and specificity, present a promising alternative. The review begins by elucidating the molecular mechanisms and key benefits of aptamers over traditional antibodies, highlighting their robustness and versatility. Building upon their demonstrated successes in food safety, environmental monitoring, and medical diagnostics, we systematically examine the application prospects of aptamers in plant disease management. Specifically, aptamers not only served as sensitive biosensors for detecting plant hormones and pathogens but also as active agents that interfere with pathogen effectors and modulate plant immune responses. Although emerging, aptamers hold considerable potential to transform plant disease control. Future advancements will require overcoming challenges in aptamer screening and biosensor stability through integration with emerging technologies, including microfluidics, CRISPR, and artificial intelligence, paving the way for intelligent, field-deployable diagnostics for sustainable agriculture.
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December 6, 10:46 PM
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Restriction-modification (R-M) systems are one of the most widespread and, due to their often plasmid-based nature, transmittable antiphage systems bacteria have. The CfrBI R-M system studied here consists of a methyltransferase (MT) and a restriction endonuclease (RE) that are divergently expressed and share a promoter region that harbors a single CfrBI recognition site. Previously, the methylation of this site has been shown to regulate the expression of the R-M system. Here, we show that the expression dynamics of the CfrBI R-M system and its protective properties depend on the copy number of the plasmid harboring it. A higher copy number results in higher expression, but the expression on a medium-copy number plasmid interestingly conferred the highest phage resistance. After transformation of naïve cells however, expression of the RE was fastest in the high-copy plasmid background. In vivo we show that the expression strength of the MT inhibits its own expression, while enhancing RE expression. To conclude, the results indicate that for phage resistance the overall expression strength might not be the predominant factor in case of the CfrBI R-M system, while for the establishment of the system the initial expression rate of the MT seems to be the determining factor.
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December 6, 10:23 PM
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An unbiased and accurate estimation of intraspecies diversity, i.e., the extent of genetic diversity within species (or microdiversity), is crucial for clinical and environmental microbiome studies. Although it is well appreciated that sequencing depth (or coverage depth) below 10X can provide biased estimates of microdiversity, typically underestimating diversity due to the random sampling of alleles, there is a widely accepted convention that microdiversity estimates tend to be relatively stable at sequencing depth exceeding 10X. Therefore, discarding species with less than 10X or rarefying to 10-20X sequencing depth are generally used to compare microdiversity among taxa and samples. Our findings showed that these biases may persist even at depth levels above 50-200X for all popular sequencing platforms, including Illumina, PacBio, and Oxford Nanopore. The biases mostly, but not always, represent an underestimation of diversity and were attributable to the incomplete recovery of Single Nucleotide Variants (SNVs) at lower sequencing depth levels. To address this issue, we recommend using rarefaction-based approaches to standardize data at least 50X, and ideally at 200X sequencing depth, which reduces differences between observed and expected microdiversity values to less than 0.5%. Furthermore, the Average Nucleotide Identity of reads (ANIr) metric is significantly less sensitive to sequencing depth variability than nucleotide diversity (π), making it a robust alternative for estimating microdiversity at sequencing depth close or exceeding 10X, without a need to rarefying data. Therefore, the sequencing depth thresholds proposed herein provide a more standardized framework for direct comparisons of microdiversity across samples and studies.
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December 6, 3:39 PM
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Type VI secretion systems (T6SSs) are molecular machines used by bacteria to release effectors that target either host cells, competing bacteria or fungi. Regulatory mechanisms underlying antifungal T6SS activity remain unexplored. Here we show, using mouse infection with wild-type and T6SS mutant bacteria, that T6SS activity of the enteropathogen, Yersinia pseudotuberculosis (Yptb), reduces fungal prevalence in the gut microbiota and has direct activity on Candida albicans. Screening of bacterial effector mutant strains, and structural and biochemical analyses identify TfeC as an antifungal chitinase T6SS effector that can kill C. albicans. In vivo experiments confirm that TfeC expression promotes Yptb colonization and reduces C. albicans abundance. We also show that Yptb senses the fungal quorum-sensing molecule, tyrosol, through the two-component system, EnvZ–OmpR, and responds by activating T6SS4. Our findings suggest that Yptb modulates its antifungal activities by detecting changes in fungal population density cues, revealing a mechanism of fungal–bacterial interkingdom communication mediated by fungal quorum-sensing molecules. Yersinia pseudotuberculosis senses fungal tyrosol signalling through EnvZ–OmpR which triggers T6SS activation and antifungal effector release to reduce fungal competitors in the mouse gut.
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December 6, 3:16 PM
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E. coli RecBCD, a hetero-trimeric helicase and nuclease, functions in double stranded (ds) DNA break repair. RecBCD possesses ATPase motor domains within both RecB (3’ to 5’) and RecD (5’ to 3’) and a nuclease domain within RecB (RecBNuc). RecBCD binds to double stranded DNA ends and initiates DNA unwinding by first melting several DNA base pairs (bp) using only its binding free energy. The RecBNuc domain is docked ∼70 Å from the duplex DNA binding site in RecBCD-DNA structures but appears to be dynamic and able to move from its docked position. Here, we compare DNA binding of RecBCD and a variant, RecBΔNucCD, in which the 30 kDa nuclease domain has been deleted. RecBCD binding to a blunt DNA end is enthalpically unfavorable and entropically driven. Deletion of RecBNuc results in an increase in DNA binding affinity, suggesting an allosteric effect of RecBNuc. RecBΔNucCD binding to DNA possessing fully ‘pre-melted’ DNA ends is associated with a large favorable ΔHobs, but much smaller than observed for RecBCD, suggesting that deletion of RecBNuc limits bp melting from a blunt DNA. We also solved cryo-EM structures showing only 4 bp melted upon RecBΔNucCD binding to a blunt ended DNA duplex, less than the 11 bp melted upon RecBCD binding. Thus, the RecB nuclease domain regulates the extent of bp melting by RecBCD. These results suggest that RecBNuc may manifest its long-range allosteric effect on DNA binding and DNA melting via linker-linker interactions between RecB and RecC.
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December 6, 2:56 PM
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Circular RNAs (circRNAs) are natural outputs of transcription and RNA processing in eukaryotes. Four subclasses of circRNAs have been identified in animal cells, and most circRNAs are generated via backsplicing. The intricate formation of circRNAs is orchestrated by various cis-regulatory elements and trans-acting factors. Previous studies have gained insights into the general factors and elements involved in backsplicing. Recently, modulation of circRNA biogenesis to generate tissue-specific expression patterns is coming into focus. We summarize various mechanisms involved in the biogenesis of distinct circRNA subclasses across multiple cell types. We also discuss the involvement of relevant mechanisms in human diseases and potential biomedical interventions that target circRNA pathways.
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December 6, 2:44 PM
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Amyloids, once viewed solely as pathological hallmarks, are now recognized as widespread and versatile functional protein assemblies. Bacterial functional amyloids (FuBAs), particularly curli (CsgA) from Escherichia coli and FapC from Pseudomonas, have emerged as paradigms for understanding amyloid structure, assembly, and function. The recent cryo-EM-based structure of FapC, together with others’ combined cryo-EM and integrative computational studies on CsgA, reveal a β-solenoid fold stabilized by imperfect repeats, producing fibrils of exceptional stability and low polymorphism, whose biogenesis is tightly controlled through dedicated accessory factors, ensuring precise secretion and nucleation. FuBAs not only scaffold biofilms but also display intrinsic catalytic activity, expanding the biochemical repertoire of extracellular matrices. They also exhibit hierarchical mechanical properties ranging from GPa stiffness at the fibril core to kPa elasticity in hydrated biofilms. FuBA operons are phylogenetically widespread, with repeat variation contributing to sequence diversity and functional adaptability. FuBAs might be seen as evolutionary intermediates between disordered peptides with significant self-interaction tendencies and highly structured globular proteins. Their simple structures make them robust platforms for biomaterial engineering. Understanding the interplay between sequence repeats, fibril architecture, and emergent functions opens avenues for harnessing amyloids as programmable nanomaterials with applications in catalysis, synthetic biology, and biofilm control.
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December 6, 2:19 PM
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Tools to edit DNA methylation in a targeted manner are vital for establishing causal relationships between DNA methylation and its function, as well as for plant breeding and gene therapy. Here, by constructing dCas9 fusions to a panel of effectors and cofactors, we develop a range of highly effective tools for editing DNA methylation in Arabidopsis, including five tools for DNA methylation and six tools for DNA demethylation. Our tools show a diversity of performance features in terms of specificity and efficiency, offering either the capacity to edit DNA methylation in a target-specific manner or the ability to edit DNA methylation genome-wide due to potent off-target effect. Importantly, DNA methylation edited by these tools is inherited in the absence of transgene. These versatile tools pave the way for diverse applications of DNA methylation editing in not only research but also epigenetic breeding of crops. Targeted DNA methylation editing is critical for establishing the causal relationship between DNA methylation and its function as well as for epigenetic crop breeding. Here, the authors develop a CRISPR/dCas9-based fusion protein strategy for DNA methylation and demethylation editing in Arabidopsis.
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December 6, 1:10 PM
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Guanidine is well known as a denaturing agent. However, recent studies have demonstrated both the widespread synthesis of guanidine, e.g., in plants and mammals, as well as the widespread occurrence of guanidine metabolism in bacteria, suggesting a broader biological role. Here, we provide insights into guanidine assimilation via guanidine hydrolases (GdmH) in cyanobacteria. The gdmH gene is widespread among cyanobacteria and enables growth on guanidine as the sole nitrogen source. Consistent with this, gdmH gene expression increased under nitrogen limitation, regulated by the transcription factor NtcA. However, guanidine is toxic above 5 mM, necessitating GdmH activity and adaptive mutations activating the multidrug efflux system PrqA. The gdmH gene is frequently colocalized with ABC transporter genes (named gimABC), which are driven by an additional NtcA-regulated promoter. The corresponding substrate-binding protein GimA showed high affinity to guanidine. Consistent with a high affinity import system, disruption of genes gimA or gimB impaired guanidine-dependent growth of Synechocystis sp. PCC 6803 at low concentrations. However, in presence of >1 mM guanidine, these mutants grew like wildtype, suggesting the existence of additional uptake mechanisms for guanidine. We also demonstrate the high-affinity binding of guanidine to a previously described, conserved RNA motif located within the gdmH 5’-untranslated region, validating it as a guanidine-I riboswitch. By combining it with various promoters, we achieved precise, titratable control of heterologous gene expression in cyanobacteria in vivo. Our findings establish guanidine assimilation as an integral element of cyanobacterial nitrogen metabolism and highlight guanidine riboswitches as valuable tools for synthetic biology.
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December 6, 10:56 AM
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The escalating crisis of antimicrobial resistance demands novel therapeutics that overcome the limitations of conventional antibiotics. Human α-defensins, such as Human Neutrophil Peptide-1 (HNP-1), represent compelling candidates due to their potent, broad-spectrum antimicrobial activity and membrane-disrupting mechanism. However, clinical translation has been hindered by the absence of scalable production systems capable of delivering high yields of functional peptide. To address this challenge, we developed an efficient expression platform in the yeast Komagataella phaffii. In this system, a codon-optimized HNP-1 sequence was fused to a His6-SUMO tag downstream of the α-factor secretion signal to enhance solubility, folding, and recovery. Initial shake-flask optimization identified pH 6.0 with 96 h of induction as optimal fermentation conditions, yielding 19.75 ± 1.1 mg/L of recombinant protein. Subsequent scale-up to a controlled 5 L bioreactor significantly enhanced production, achieving 122 mg/L of the fusion protein. Following purification and precise cleavage, this process delivered 15.25 mg/L of pure, mature HNP-1 representing, to our knowledge, the highest yield of recombinant HNP-1 reported. The final product demonstrated potent antibacterial activity against Staphylococcus aureus and Escherichia coli while exhibiting excellent hemocompatibility, confirming preservation of native structure and function. This work establishes a scalable production platform for HNP-1 and provides an adaptable framework for expressing other structurally complex antimicrobial peptides with therapeutic potential.
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Today, 12:00 AM
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Rational control over diverse, ligand-responsive output networks is a foundational challenge in synthetic biology, particularly for systems based on post-translational signaling. Here, we present the design and engineering of minimal, modular protein architectures for chemically responsive molecular inverters and digital switches. Our inverter design modifies the plant-derived PYR1-HAB1 chemically inducible dimerization module by incorporating a constitutive activator, which is then competitively displaced by the ligand-bound PYR1 receptor, converting the native 'dimerization-on' mechanism into a 'signal-off' inverter. We establish key design features and demonstrate predictable tuning of the inverter's transfer function, including its maximum output, half-maximal inhibitory concentration, and minimum output, solely by adjusting protein stoichiometry. The architecture is modular, enabling plug-and-play response to diverse, user-defined drug-like small molecules. We show that an inverter biosensor for an environmental contaminant functions in engineered living cells with a low nanomolar sensitivity. Additionally, we convert the PYR1-HAB1 sensor into a digital switch by adding an engineered molecular titrant to the system. Overall, this work provides a generalizable, minimal, and tunable protein scaffold for programming complex, post-translational signaling logic, significantly expanding the toolkit for sophisticated biological circuit design.
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December 6, 11:27 PM
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Sporosarcina pasteurii is the most widely studied bacterium for microbially-induced calcium carbonate precipitation (MICP), a process of intense interest for materials and construction applications. Despite two decades of investigation, S. pasteurii has remained genetically intractable, limiting our mechanistic understanding of biomineralization pathways and constraining efforts to engineer scalable solutions. Here, we present the first genetic toolkit for S. pasteurii, including a stable replicating plasmid, a conjugation-based DNA delivery protocol, engineered inducible promoters, and methods for genome modification. Using homologous recombination, we precisely deleted 5.7 kb of the genome spanning two operons encoding urease activity and demonstrated complete loss of biocementation. We also screened a library of engineered transposon constructs for activity in S. pasteurii and generated a genome-wide mutant library with >15,000 unique insertion sites. Using this library, we identified putative genes affecting ureolytic growth, revealing previously inaccessible aspects of S. pasteurii genetics. This work establishes S. pasteurii as a genetically tractable platform for rational engineering of MICP and constitutes the first genetic modification capability within the Sporosarcina genus.
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December 6, 11:06 PM
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Fluorescent proteins (FPs) are widely used reporters for visualizing cellular structures and processes. Traditional wet-lab strategies for FP engineering (rational design and directed evolution) have enabled substantial improvements in photophysical performance but are limited by their requirement for deep expert knowledge or labor-intensive screening. AI-driven approaches have recently gained traction for engineering variants of green FPs, yet applications to red fluorescent proteins (RFPs) remain scarce. Here, we applied machine learning models to an RFP sequence-function dataset and trained these models to predict functional single-mutation variants of the state-of-the-art RFP mScarlet-I3. Guided by model predictions, we identified variants exhibiting red-shifted emission peaks, large Stokes shifts, or brightness comparable to the parental protein. Our findings show that even lightweight, data-efficient models can extract actionable design principles for improving RFPs. This work demonstrates the feasibility of AI-guided design for RFPs and provides a reliable benchmark for future development of more powerful AI-driven strategies for FP engineering.
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December 6, 10:53 PM
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This study presents a LoRaWAN-based IoT system developed for real-time monitoring of ammonia (NH₃) emissions in cereal crop fields. Sustainable agriculture increasingly demands on-farm greenhouse gas (GHG) tracking linked to environmental variables. IoT offers efficient real-time monitoring of soil NH₃ emissions and associated factors. Our research introduces a unique Field Monitoring Laboratory: a LoRaWAN-connected IoT system integrating soil, crop, and microclimate sensors to observe NH₃⁺, air temperature, rainfall, humidity, soil temperature, and moisture content. The system comprises a field lab, data server, and custom dashboard with analytics capabilities. NH₃ fluxes were measured in autumn-sown cereals across three growing seasons (2020–2023). Tukey’s Kramer test revealed significant (p < 0.05, p < 0.001) differences in NH₃ emissions and environmental variables between years. Highest NH₃ emissions (1.94 ppm in 2020, 1.71 ppm in 2021) coincided with elevated air (25–31 °C) and soil (21–23 °C) temperatures, and higher mean and peak rainfall (0.40–0.48 mm average; max 9–31.6 mm). Principal Component Analysis showed 65.8% variance explained by PC1 and PC2, with high loadings from temperature and soil moisture. Spearman’s correlation indicated moderate positive associations (r = 0.38–0.4, p < 0.05) of NH₃ with soil moisture at 20 cm and 40 cm of soil depth, and a weak negative correlation (r = -0.16 and − 0.17) with soil temperature at 20 cm and 40 cm. The study underscores the potential of IoT technology using calibrated gas sensors and LoRaWAN for real-time NH₃ and environmental monitoring, enabling informed decision-making in smart agriculture.
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December 6, 10:41 PM
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The methylotrophic yeast Pichia pastoris (also known as Komagataella phaffii) is a prominent platform for recombinant protein production, offering benefits such as thermo- and osmotolerance, high-density growth, and efficient protein secretion. Its ability to metabolize methanol, an increasingly available carbon source, enhances its cost-effectiveness and sustainability for industrial use. As a eukaryotic host, P. pastoris ensures proper protein folding and post-translational modifications (PTMs), including glycosylation, which is essential for correct folding and endoplasmic reticulum (ER) quality control. While ER-transferred glycans are critical for maturation, additional modification in the Golgi apparatus can yield larger glycans whose impact on stability, solubility, and bioactivity may be either beneficial or undesirable, depending on the application of the heterologous protein. The impact of induction conditions on glycosylation of proteins secreted by P. pastoris SuperMan5 was examined, using the DS-1 (G2P[4]) and WA (G1P[8]) VP8* rotavirus capsid proteins as a model. An ELISA-based screening system was employed for clone selection and media optimization, with results showing easy integration into automated workflows. Methanol concentration was found to impact both N- and O-linked glycosylation complexity, shaping the glycosylation profile of the target protein as well as the P. pastoris secretome. This study underscores the importance of optimizing cultivation conditions to enhance protein yield, refine glycosylation, and minimise impurities, all of which are crucial for large-scale production and efficient downstream processing. It also suggests a method for easy modulation of glycosylation depending on the target application and the desired level of glycosylation.
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December 6, 3:52 PM
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Protein glycosylation is a very common post-translational modification PTM seen in all branches of biology. The functional roles for protein glycosylation are many and varied, essential in eukaryotes but seemingly dispensable in bacteria. One group of bacteria where protein glycosylation has been looked at for at least 50 years are the actinobacteria, a large and diverse group of bacteria which include well know pathogens like Mycobacteria tuberculosis, Corynebacterium diphtheriae, and well know species important in biotechnology like Streptomyces lividans and Corynebacterium glutamicum. Actinobacterial protein glycosylation is a form of protein O-mannosylation which is found widely in eukaryotes from yeast on up the evolutionary ladder but is much less understood at the functional level. Very few direct roles for protein O-mannosylation have been described in the literature. This review examines newer findings from the actinobacterial world which with the help of glycoprotein models suggests how the glycans might play a role in actinobacterial biology.
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December 6, 3:26 PM
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Ammonia-oxidizing archaea (AOA) are among the most abundant microorganisms in the ocean and play a critical role in marine nitrogen cycling. Recently, urea has been shown to serve as an additional substrate for marine AOA, with substantial urea use in the ammonium-depleted open-ocean. Yet, the mechanisms that control urea use and potentially maintain high AOA abundances remain unclear. Here, we investigate urea and ammonia use by AOA in three contrasting marine environments, from coastal, ammonium-rich to open-ocean, ammonium-poor waters. Our combined results indicate that distinct substrate utilization strategies of Nitrosopumilus and Nitrosopelagicus control their environmental distribution. The more coastal AOA genus, Nitrosopumilus, primarily uses ammonium. In contrast, enhanced urea utilization in ammonium-limited waters is linked to the activity and growth of Nitrosopelagicus. Thus, the use of urea, and potentially other organic-N compounds by Nitrosopelagicus plays a major role in fueling open-ocean nitrification and sustaining primary productivity in these vast regions. Two groups of ammonia-oxidizing archaea drive marine nitrification. Stuehrenberg et al. reveal that their distribution reflects substrate use, with one relying on urea and the other on ammonia to maintain nitrification in open-ocean waters.
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December 6, 3:00 PM
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Plant protein production systems are scalable and sustainable platforms capable of meeting the growing demand for functional proteins in nutrition, pharmaceuticals, and industry. Recent advances in essential amino acid (EAA) biosynthesis, gene regulation, and subcellular targeting have enhanced protein yields and stability, but are yet to be integrated into holistic engineering approaches. Metabolic engineering can improve amino acid (AA) metabolism and energy efficiency, while genetic engineering enables finetuned, spatiotemporal expression of target proteins. Coupled with in silico tools for protein design, novel proteins with enhanced stability and functionality can be developed. Integrating these strategies would enable the fine-tuning of protein synthesis while balancing cellular energy costs, offering context-dependent opportunities to advance protein production in plant systems.
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December 6, 2:51 PM
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Reactive oxygen species (ROS) are a promising alternative bactericide. However, it is questioned that bacteria can potentially develop resistance to ROS, similar to their resistance against antibiotics and silver. Herein, it is reported that Gram-negative bacteria, including Pseudomonas aeruginosa, Escherichia coli, and Klebsiella pneumoniae, develop resistance to ROS after six repeated exposures. Notably, ROS minimum inhibitory concentration of P. aeruginosa significantly increases to 256-fold after ten passages. The resistance mechanism predominantly originates from the intensified biosynthesis of the highly reductive hydrogen sulfide (H2S) and pyoverdine (PVD) siderophores, effectively neutralizing ROS. Simultaneously, PVD transports Fe3+ from the extracellular space into the bacteria, releasing H2S bound to Fe3+ and enhancing ROS scavenging. Additionally, the enhanced outer membrane (OM) biogenesis establishes a robust OM barrier, impeding ROS penetration. The acquired resistance to ROS can be significantly reduced by incorporating additional Fe3+ into the culture medium or disrupting the H2S biosynthetic gene. These observations suggest that careful consideration is required when utilizing ROS against Gram-negative bacteria. It is anticipated that understanding this resistance mechanism can inform the development of future antimicrobial agents, particularly for Gram-negative bacteria.
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December 6, 2:31 PM
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JBrowse 2 is an open-source genome browser that provides unique features for visualizing syntenic relationships between multiple genomes. This article describes a protocol for setting up synteny views in JBrowse 2, using an assembly-to-assembly whole-genome alignment example. We detail data preparation steps, including the generation and formatting of whole-genome alignment data into formats compatible with JBrowse 2's synteny visualization capabilities, and show the GUI-driven process for setting up interactive synteny views and generating publication-quality figures. This protocol establishes methods for using JBrowse 2 to explore conserved sequences across multiple genomes.
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December 6, 2:09 PM
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Despite the importance of the cervicovaginal microbiome, the mechanisms that govern its composition and drive its impact on host physiology remain poorly understood. With the aim to expand our understanding of the function and ecology of the vaginal microbiome, we present VIRGO2, an enhanced non-redundant gene catalog comprising over 1.7 million well-annotated genes from body-site specific microbes and viruses. Analyses using VIRGO2 reveal insights such as including the identification of previously uncharacterized vaginal bacteria, features of the vaginal mycobiome and phageome, and differential expression of bacterial carbohydrate catabolic genes. Constructed from over 2500 metagenomes and 4000 bacterial genomes, VIRGO2 broadens geographic representation and microbial diversity compared to its predecessor. This updated catalog enables more precise profiling of taxonomic and functional composition from metagenomic and metatranscriptomic datasets. VIRGO2 is a critical resource for integrative analyses of vaginal microbial communities and their interactions with host tissues, thereby enhancing our mechanistic understanding of vaginal health and disease. The vaginal microbiome is a critical determinant of health. Here, the authors present VIRGO2, a gene catalog describing these microbial communities and analyze vaginal metagenomes and metatranscriptomes to derive insights into their function and ecology.
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December 6, 12:50 PM
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Prokaryotes possess a remarkably diverse and dynamic repertoire of antiviral defense systems, enabling them to withstand phage predation. However, their frequent horizontal gene transfer, extensive sequence diversity, modular genomic organization, and rapid evolution make purely experimental discovery challenging. Coupled with the massive influx of microbial genomes from high-throughput sequencing, computational strategies have become indispensable complementary tools that can enhance the efficiency and scope of defense systems discovery. In this review, we categorize computational approaches into four major strategies: (i) Sequence homology-based methods, which reliably annotate known defense systems through protein sequence similarity but are limited in detecting highly divergent or novel systems; (ii) Structure-guided approaches, which leverage conserved protein folds to uncover remote homologs and single-gene defense proteins, providing sensitivity beyond sequence-based identification, albeit at high computational cost; (iii) Genomic context-based strategies, which exploit gene co-localization and defense islands to uncover multi-gene defense clusters and previously uncharacterized defense modules; and (iv) Artificial intelligence-powered methods, which integrate sequence-derived embeddings with genomic context information to predict low-homology proteins and reconstruct candidate defense systems at scale, enabling discovery of novel systems beyond the reach of conventional approaches. We further discuss emerging tools and frameworks, such as the conserved gene cluster discovery tool and genomic foundation models, which hold strong potential to extend conventional approaches for identifying novel defense systems and supporting the generative design of synthetic modules. By comparing methodological principles, strengths, and limitations, this review provides a practical framework for the systematic exploration of microbial immune systems, guiding applications such as rational phage therapy, microbiome engineering, and synthetic biology.
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The pre-trained model was fine-tuned on 4 biologically relevant tasks: (1) plasmid host prediction based on encoded product profiles; (2) prediction of masked gene products from replicon product lists; (3) context-aware contig ordering for genome assembly; and (4) gene essentiality prediction. Model parameters were continuously updated during fine-tuning.
Each panel illustrates prediction accuracies for a different phenotype: (a) distinguishing pathogenic from non-pathogenic strains, (b) identifying whether a genome carries plasmids, (c) classifying cell shape (e.g., coccus, rod, spiral), (d) Gram-staining reaction (positive vs. negative), (e) indole test outcome (positive vs. negative), (f) cell motility (mobile vs. nonmobile), (g) ability to form multicellular complexes (positivevs. negative), (h) oxygen tolerance (e.g., anaerobe, aerobe, facultative anaerobe), (i) Voges–Proskauer test outcome (positive vs. negative), (j) preferred growth temperature (<20 °C, 20–40 °C, or >40 °C), (k) cell length (≤2 µm vs. >2 µm), and (l) cell width (≤0.5 µm vs. >0.5 µm).