Acid stress is a central environmental factor shaping the structure and function of microbial communities worldwide. However, there is a lack of predictive understanding of how microbial communities respond physiologically and metabolically to acid stress. Here, we find that higher acid stress favors slower-growing species, promoting population growth and coexistence. Our experiments show that acid stress influences the spatial structure of communities, wherein coexistence is ordered over centimeter-length scales and determined by growth-tolerance trade-offs. We find that interspecific interactions are highly dynamic during acid stress changes, with shifts from competition to cooperation, enhancing resilience under high-stress intensities. Slower-growing species may bolster interspecific coexistence through stress-dependent excretion and cross-feeding of public goods. We construct a resource-consumer-based mathematical model to unravel the processes experienced by species in stress-induced coexistence and their distinct physiological states. Finally, our pairwise bacterial-fungal interaction experiments elucidate universalities in stress-induced coexistence between closely related and phylogenetically distant species with complementary phenotypic profiles. Overall, our work provides insights into how acid stress affects physiological and metabolic responses, as well as overall fitness, resilience, and coexistence. Acid stress is an environmental factor shaping the structure and function of microbial communities. Here, the authors show that microbial interspecific interactions are highly dynamic during acid stress changes. They find slow-growing species promote population cooperation and coexistence under elevated acid stress.

Fecal microbiota transplantation (FMT) is a promising approach for restoring gut microbial balance in both humans and animals. However, the logistical limitations of transplanting fresh fecal samples have increased interest in freeze-dried (lyophilized) fecal material as a transplant inoculum. While lyophilization facilitates storage, it can compromise bacterial viability, which is essential for FMT effectiveness. Lyoprotectants are often used to protect bacterial cultures during freeze-drying, but their effects on a complex microbial community remain unclear, as they may preferentially preserve some taxa over others. This study investigated the impact of four lyoprotectants—mannitol, maltodextrin, trehalose, and a maltodextrin-trehalose mixture—on bacterial viability and community structure in pig fecal samples post-lyophilization. Propidium monoazide (PMA) treatment combined with 16S rRNA sequencing (PMAseq) was used to differentiate viable from non-viable bacteria. In the total community (without PMA), microbial profiles appeared similar across treatment groups. However, when focusing on the viable community (PMA-treated), lyoprotectant choice significantly influenced the post-lyophilization community composition. Gram-negative bacteria were especially susceptible to viability loss due to lyophilization. Trehalose and maltodextrin preserved bacterial viability and community structure more effectively than mannitol. Mannitol-treated samples had reduced viable bacterial cells and altered community composition, while trehalose and maltodextrin better maintained diversity and structure of the viable (PMA-treated) communities. Taken together, lyoprotectants have differential effects on microbial composition during lyophilization. Among those tested, trehalose and maltodextrin best preserved both viability and community structure, making them promising candidates for FMT applications. Future research should explore optimizing lyoprotectant formulations to enhance microbiome stability and functional outcomes.

World agriculture depends in part on the crop-associated microbiome for improved plant growth, health, and productivity. In particular, endophytic fungi (EF) with plant growth–promoting activities fulfill some of these roles and are central as bioinoculant agents. In the case of arbuscular mycorrhizal fungi (AMF), they form a symbiosis with their host plants, enhancing the uptake of water, phosphorus, nitrogen, and other micronutrients, while the plants provide them with photosynthates. This work reviews the differences in the colonization of internal plant niches between these beneficial fungi, as well as other distinctive ecological traits. It also explores mechanisms of seedborne vertical transmission in AMF and their classification. Genomic and transcriptomic advances in fungal endophytes are highlighted, shedding light on genes and expression profiles that define their lifestyle and plant associations. In addition, recent studies on their abilities to promote plant growth are analyzed, especially focusing on Trichoderma spp., Epichloë spp., Serendipita indica (formerly Piriformospora indica), and entomopathogens like Beauveria spp. and Metarhizium spp. Finally, the multiple interactions among EF, AMF, and other members of the plant microbiome—notably plant growth-promoting bacteria (PGPB)—are discussed, emphasizing how these organisms synergistically benefit the host. A deeper understanding of these fungi and their plant-beneficial effects should facilitate commercialization and help farmers achieve sustainable production, especially under challenges posed by global climate change.

Gram-negative bacteria are equipped with a unique cell envelope structure that includes an outer membrane populated by diverse outer membrane proteins (OMPs). These OMPs are not only essential for bacterial survival, mediating critical functions such as nutrient transport, antibiotic resistance, and structural integrity, but they also play pivotal roles as virulence factors during host-pathogen interactions. Recent research highlights the ability of OMPs to manipulate host cellular processes, often targeting mitochondria to induce cell death or modulate immune responses. This review explores the multifunctional roles of bacterial OMPs, emphasizing their structural features, biogenesis, and pathogenic mechanisms. Furthermore, it delves into how bacterial OMPs exploit host cell machinery, particularly mitochondria, to promote infection, as well as their potential as targets for innovative antimicrobial strategies. Specifically, this review focuses on β-barrel OMPs that reach host mitochondria, detailing their delivery routes and mechanisms of organelle manipulation, while excluding non-β-barrel toxins and secretion-system effectors, to provide a defined perspective on mitochondria-targeting OMP virulence mechanisms. omv

Multiplexed nucleic-acid detection is essential for molecular diagnostics and spatial genomics, but conventional fluorescence methods are often limited by spectral overlap, nonspecific signals, and restricted encoding capacity. We present a spatial fluorescence barcode (SFB) platform based on transiently luminescent DNA beads (TLDBs) that enables single-color, high-plex readout. In this method, targets are encoded through the spatial arrangement of DNA-functionalized beads, eliminating the need for multicolor labeling or spectral unmixing. Target recognition is achieved through toehold-mediated strand displacement, and built-in nucleases enable autonomous enzymatic resetting for repeated use of probes. The system employs monochromatic spatial encoding, decoupling encoding capacity from spectral channels, and features a simplified probe design and decoding workflow. Self-resetting probes not only streamline the encoding process but also enhance practicality by allowing repeated assays without the need to re-prepare costly probe combinations. We demonstrate robust detection of pathogen-derived nucleic acids in infected blood and cancer-associated microRNAs in tissue samples, validating the platform’s clinical applicability. Compared to existing barcoding strategies, SFB integrates monochromatic spatial encoding, simplified design, and autonomous reusability, offering a practical, scalable, and cost-effective solution for high-throughput nucleic acid analysis. Fluorescence methods of multiplexed nucleic-acid detection are often limited by spectral overlap, nonspecific signals, and encoding capacity. Here the authors design a spatial fluorescence barcode platform which uses DNA beads for reusable multiplexed pathogen and cancer biomarker detection.

It is essential to determine glycan structures in complex biological samples to understand their biology and exploit their diagnostics, therapeutics and nutraceuticals potential. An unresolved analytical challenge is the identification of isomeric glycan structures in complex biological samples. Ion mobility (IM) combined with MS enables separation of isomeric glycans and identification by comparing their intrinsic collision cross section (CCS) values with similar data of synthetic standards. To identify glycans without the need to synthesize all biologically occurring glycans, we describe here an IM-MS de novo sequencing method based on fragment identification and sequence assembly. CCS values of additional fragments from glycans in biological samples result in a self-expanding reference database, gradually facilitating the sequencing of glycans of increasing complexity and expanding the database from an initial 19 standards to 332 unique entries. The methodology is employed to determine structures of human milk oligosaccharides and N-glycans of biotherapeutics. This study introduces an ion mobility–mass spectrometry method for de novo glycan structure identification, using a self-expanding database that reduces reliance on synthetic reference standards.

Ancestral recombination graphs (ARGs) are a complete representation of the genetic relationships between recombining lineages and are of central importance in population genetics. Recent breakthroughs in simulation and inference methods have led to a surge of interest in ARGs. However, understanding how best to take advantage of the graphical structure of ARGs remains an open question for researchers. Here, we introduce tskit_arg_visualizer, a Python package for programmatically drawing ARGs using the interactive D3.js visualization library.  We highlight the usefulness of this visualization tool for both teaching ARG concepts and exploring ARGs inferred from empirical datasets.

Ethanol is an attractive C2 feedstock for microbial biomanufacturing because it is directly oxidized to acetyl-CoA with favorable redox balance. 3-Hydroxypropionic acid (3-HP), a versatile platform chemical, can be synthesized via the malonyl-CoA and β-alanine pathways. An inducer-free ethanol-to-3-HP process in Pseudomonas putida KT2440 was developed by enabling constitutive expression of both pathways and deleting native 3-HP catabolism and polyhydroxyalkanoate (PHA) synthesis to redirect flux toward the target product. Each route and their co-activation were evaluated, and a genome-scale model constrained with transcriptomic data was applied to identify metabolic nodes governing pathway choice and performance. Shake-flask experiments with 1% (v/v) ethanol showed that the malonyl-CoA pathway yielded higher 3-HP titers than the β-alanine route. Co-activation of both pathways in a PHA-deficient strain improved production to 15.9 mM (1.42 g/L; 179 mg/g ethanol) while reducing acetate overflow. In a fed-batch with continuous ethanol feeding, the engineered strain reached 43.7 mM (3.92 g/L; 154 mg/g ethanol) 3-HP. Deletion of endogenous 3-HP catabolism and PHA synthesis redirected carbon flux toward the product, and additional acetyl-CoA supply was achieved by introducing a heterologous acetaldehyde dehydrogenase. Genome-scale modeling constrained with transcriptomic data revealed dominant flux routing through the glyoxylate shunt and limited oxaloacetate regeneration, explaining the advantage of the malonyl-CoA pathway and the acetate accumulation associated with β-alanine operation. An inducer-free ethanol-to-3-HP platform was established in P. putida KT2440 by co-activating the malonyl-CoA and β-alanine pathways while eliminating competing sinks such as 3-HP catabolism and PHA synthesis. This approach enhanced yield, reduced acetate overflow, and systems-level analysis identified the glyoxylate shunt and oxaloacetate limitation as key control points. Overall, the study demonstrates an efficient and scalable route for 3-HP production from ethanol.

Chemically inducible DNA recombination systems are a very attractive tool for implementing potent genetic applications. However, conventional systems still suffer from leaky properties in the absence of chemicals. Here we describe a chemically inducible, leakless destabilized Cre recombinase (SPEED-Cre) based on a new approach, split-protein-based efficient and enhanced degradation (SPEED), that consists of a self-assembling split-Cre tagged with a destabilizing domain (DD) mutant from the Escherichia coli dihydrofolate reductase that is stabilized by the antibiotic ligand trimethoprim (TMP). We demonstrate that SPEED-Cre has no significant leak activity of background DNA recombination in the absence of TMP; nevertheless, it enables full induction of TMP-dependent Cre-loxP recombination in human cells and living mice. We also demonstrate the general applicability of the SPEED approach, which can be widely applied to other proteins, by showing high TMP-dependent recombination performances of destabilized Flp, VCre and Dre recombinases based on the SPEED approach. This robust platform technology will greatly enhance chemogenetic applications for genome engineering in living systems. A combination of self-assembling split-Cre with a destabilizing domain system is found to be an effective way to improve the degradation efficiency of destabilized Cre in the absence of Trimethoprim (Tmp) while maintaining efficient TMP-inducible DNA recombination.

Antimicrobial resistance (AMR) poses a pressing global health challenge in the 21st century. The rapid increase and prevalence of multidrug-resistant bacteria will require novel approaches to develop new antibiotics. Major advances in nucleic acid-based therapeutics, particularly antisense technologies, could be one solution for developing precision antibiotics. The selectivity and specificity in the drug design of antibacterial antisense oligomers (ASOs) allows precise gene-specific silencing and ultimately enables targeting of currently undruggable gene products. Our goal here is to comprehensively review the advances in asobiotics (antisense oligomer biotics) leading to therapeutic success, including: modifications in the nucleic acid backbone of ASOs which have improved their properties and advances in delivery. We will discuss utilization of ASOs against several pathogens, strategies to overcome resistance, and finally future scenarios and prospects for asobiotics as pathogen-specific therapy in the clinic.

Advances in genome engineering have improved our ability to perturb microbial metabolic networks, yet bioproduction campaigns often struggle with parsing complex metabolic datasets to efficiently enhance product titers. We address this challenge by coupling laboratory automation with machine learning to systematically optimize the production of isoprenol, a sustainable aviation fuel precursor, in Pseudomonas putida. The simultaneous downregulation through CRISPR interference of combinations of up to four gene targets, guided by machine learning, permitted us to increase isoprenol titer 5-fold in six consecutive design-build-test-learn cycles. Moreover, machine learning enabled us to swiftly explore a vast experimental design space of 800,000 possible combinations by strategically recommending approximately 400 priority constructs. High-throughput proteomics allowed us to validate CRISPRi downregulation and identify biological mechanisms driving production increases. Our work demonstrates that ML-driven automated design-build-test-learn cycles, when combined with rigorous data validation, can rapidly enhance titers without specific biological knowledge, suggesting that it can be applied to any host, product, or pathway. Laboratory automation, machine learning, and metabolic engineering may be combined to quickly and efficiently build productive microbial strains. Here the authors used these techniques in P. putida to boost isoprenol titers 5-fold over six DBTL cycles while sampling a reduced design space.

Efficient screening of microalgae is critical for their research and industrial applications. However, conventional methods of microalgae screening are often considered inefficient and are applicable to a narrow range of species. We conducted a proof-of-concept study on the applicability of a water-in-oil droplet (WODL)-based microfluidic system to high-throughput screening of microalgae and effect of microfluidic droplet cultivation on the species diversity within microalgal samples. First, we confirmed that microalgae with diverse morphologies could be successfully cultured within droplets. Second, we conducted culture tests using a mock community derived from monocultured microalgal strains and a field community derived from the natural environment. Subsequent long-read metabarcoding targeting the 16 S rRNA gene, followed by diversity analysis, revealed that droplet culture is more effective than bulk mixed-species culture in maintaining species diversity. Our results lay the groundwork for the future development of WODL-based high-throughput screening systems, allowing access to a richer microalgal biodiversity.