Metals are essential trace elements for almost all organisms including bacteria. Yet, metals are toxic at high concentrations, requiring fine-tuned regulatory mechanisms to steer metal homeostasis inside cells. In this primer, we explain how bacterial metallophores – small secreted secondary metabolites – act as gatekeepers by carefully orchestrating the scavenging and uptake of essential metals whilst preventing intracellular toxicity and keeping toxic metals outside the cell. We further introduce metallophore diversity together with main synthesis, secretion and uptake mechanisms. Finally, we show how secreted metallophores shape ecological interactions between bacteria and with eukaryotic organisms and how fundamental research on metallophores opens promising avenues for therapeutic and biotechnological applications.

Coral diseases are increasing in prevalence, accelerating the global decline of tropical reefs, which threatens over 25% of marine biodiversity and vital ecosystem services for human societies. While outbreaks are frequently linked to environmental change, including heat stress, sedimentation, and reduced water quality, the mechanisms by which such factors promote disease remain poorly understood. Here we show that nutrient stress, caused by skewed seawater nitrogen-to-phosphorus (N:P) stoichiometry, promotes the onset of Black Band Disease (BBD), a common and easily recognisable syndrome that affects corals around the globe. Using Turbinaria reniformis as a model system, controlled laboratory experiments demonstrate that skewed N:P ratios disrupt the functional integrity of coral-associated microbial networks while favouring opportunists that exploit dysfunctional host–symbiont interactions. Disease lesion-associated microbial mats are dominated by cyanobacteria and include sulphur-metabolising bacteria, hallmarks of natural BBD communities. Strikingly, similar cyanobacterial taxa are also detected in the visually healthy coral tissue ahead of the expanding lesions, suggesting an opportunistic recruitment of disease-associated members from the resident microbiome. Global analyses of BBD outbreaks reveal that over 88% occurred in regions with skewed N:P ratios, compared with only 16% that were linked to prior heat stress. Together, our findings identify nutrient-driven microbiome destabilisation as a key pathway to coral disease, reinforcing nutrient management as a major lever for reef conservation and restoration practice. Coral diseases contribute to the decline of reefs around the globe. This study reveals that disruptions of the nutrient balance in seawater can change coral-associated microbial communities leading to disease.

The biological significance of the transition metal molybdenum (Mo) lies in its function at the catalytic center of several enzymes that drive a wide spectrum of redox reactions underlying global biogeochemical cycles, yet a paradox persists. While modern life ubiquitously relies on Mo, geochemical evidence suggests that its availability in early Earth’s anoxic oceans was extremely limited. Modern organisms can use Mo down to trace levels; however, the rates of Mo-dependent metabolisms slow down when Mo availability decreases, posing fundamental questions about the extent to which changing Mo abundances shaped the evolution of molybdoenzymes, and when early life began harnessing Mo. Here, we confront this evolutionary enigma by reconstructing the temporal and ecological emergence of molybdoenzymes, their transport systems, and biosynthetic pathways. In parallel, we examine biological tungsten (W) usage due to shared chemical properties and cofactor biosynthetic pathways with Mo. We provide molecular dating evidence of Mo/W utilization back to the Eo- to Mesoarchean (~3.7–3.1 Ga). These findings challenge prevailing assumptions about trace metal availability on the early Earth and underscore the profound antiquity and adaptability of Mo-based biochemistry in shaping early microbial evolution. The study shows that life began using molybdenum and tungsten enzymes as early as 3.7–3.1 billion years ago. Study reveals that key metabolic processes arose despite scarce metals and highlighting the early adaptability of microbial life.

The environmental and therapeutic application of genetically engineered microorganisms necessitates the development of robust, irreversible biocontainment systems. In this study, we present an eEGM (editing-driven essential gene multiplex inactivation) module that utilizes CRISPR-mediated cytidine base editing to induce permanent self-killing via a single transient induction. By targeting the start codons of essential genes, we achieved an irreversible translational blockade that avoids the fitness costs associated with basal toxicity in nuclease-based systems. Multiplexed targeting of non-redundant essential loci (holA, ftsB, and dfp) yielded escape frequencies at or below the NIH guideline criterion (10−8) within 1 h of pulse induction. Furthermore, the eEGM system exhibited robust functional orthogonality and portability across laboratory, industrial, and therapeutic E. coli strains, including MG1655, W3110, and Nissle 1917, without detectable interference with heterologous protein expression. This work establishes base editing as a cleavage-free CRISPR effector for pulse-activated, irreversible biocontainment and provides a practical framework for safer deployment of engineered microbes.

This study presents an integrated waste-to-energy strategy for the sustainable conversion of the biodegradable organic fraction of municipal solid waste (BOFMSW) into biohydrogen (Bio.H2) and biomethane (Bio.CH4) through a two-stage continuous stirred dark fermentation (DF) process. The first-stage bioreactor was inoculated with Clostridium Thermocellum selectively enriched in a 2-bromoethanesulfonic acid (BESA) medium, and the influence of bimetallic ion catalysts NiCl2 + FeCl2 and NiCl2 + FeSO4 was evaluated at various concentrations (25, 50, 75, and 100 mg/L). The catalyst combination NiCl2 + FeCl2 at 75 mg/L produced the maximum Bio.H2 yield of 3162 L, representing a 69% enhancement compared with the catalyst-free substrate. At this optimal catalytic concentration, the percentage of H2 in the gas composition was 69.26%. The second-stage bioreactor utilized the effluent from the first stage for Bio.CH4 generation, achieving the highest cumulative yield of 729 L at 50 mg/L NiCl2 + FeCl2, which was 59% higher than that of the catalyst-free substrate. The two-stage process achieved an overall COD removal efficiency of 93.18%, demonstrating the system’s effective capacity for energy recovery. Fourier Transform Infrared (FTIR) and Field Emission Scanning Electron Microscopy (FESEM) analyses confirmed the biochemical and morphological degradation of complex organics into volatile fatty acids, illustrating efficient substrate conversion and microbial proliferation. The final digested slurry, rich in nitrogen, phosphorus, and potassium, was found suitable for use as a bio-fertilizer, supporting nutrient recycling and soil enrichment. This integrated process not only improved energy recovery efficiency but also achieved near-zero waste discharge, combining waste-to-energy and waste-to-resource approaches. The developed system demonstrates strong potential for pilot-scale implementation of Bio.H2, and Bio.CH4 for co-production from municipal solid waste (MSW), offering a circular bioeconomy pathway toward low-carbon, sustainable urban waste management.

xBind is an interactive, freely accessible, and fully configurable webserver for large language model (LLM)-enabled cross-molecular protein binding-site prediction. xBind leverages LLM embeddings from the ESM-2 model together with sequence- and structure-derived features to predict protein–protein, protein–DNA, and protein–RNA binding sites using symmetry-aware deep graph neural networks. The input to xBind is either a single-chain protein sequence in FASTA format or a monomer protein structure in PDB or mmCIF format and it outputs predicted residue-level binding sites of the input protein with its pre-selected interaction partner. The customizable xBind web interface provides: (i) choice of interaction partners including protein–protein, protein–DNA, and protein–RNA; (ii) on-the-fly AlphaFold-based protein structure prediction for sequence-only inputs; (iii) on-demand selection of the likelihood threshold for calibrating structure-aware binding site annotations; (iv) interactive and interpretable web-based results, including sequence and structural visualizations and plots of residue-level binding likelihoods with user-adjustable threshold calibration; and (v) extensive help information for usage and results interpretation through a web-based tutorial and guide. xBind is freely available at https://fusion.cs.vt.edu/xBind.

Elucidating gene function in highly redundant genetic programs such as signaling pathways is challenging in model and nonmodel plants with current whole-plant genetic screening tools. Many of these challenges could be overcome if screens were instead carried out using individual cells harboring genetic perturbations. Here we report a single-cell screening platform, PIVOT (protoplast isolation after virus overexpression in planta), to accelerate identification and functional characterization of plant genes. We use Nicotiana benthamiana as a heterologous host to test gene libraries arrayed in a single leaf. PIVOT harnesses viral superinfection exclusion to ensure single multiplicity of infection per cell during pooled library delivery. Additionally, we engineer a cell-surface protein as a phenotypic marker for isolating cells of interest from a heterogeneous population. Using this system, we recover regulators of cytokinin signaling from an Arabidopsis open reading frame library. We anticipate PIVOT will be broadly applicable for high-throughput, single-cell functional genetic screening across the plant kingdom. A pooled, cell-based, genetic screening platform in plants is used for the functional analysis of cytokinin signaling proteins.

While nanopore direct RNA sequencing has substantially advanced transcriptomics, its detection of RNA modifications remains primarily focused on abundant biological base modifications. However, therapeutic RNAs employ a diverse catalog of modifications, including base, sugar, and backbone modifications, to enhance stability and pharmacological properties. To address this gap, we systematically evaluated a set of therapeutically relevant modifications [phosphorothioate (PS)], sugar [2′-O-methylation (2′OMe), 2′-Fluoro (2′F), locked nucleic acid (LNA), 2′-O-(2′-methoxyethyl) (2′MOE)], and base [N1-methylpseudouridine (m1Ψ), 5-methylcytidine (m5C), 5-methoxyuridine (5moU), and 5-iodocytidine (5iodoC)] using direct RNA nanopore sequencing. Modifications were systematically analyzed using basecall errors, raw current signals, and modification-aware basecalling models. Ribose modifications, m1Ψ, and 5moU induced significant error rate increases and noticeable current alterations, whereas 2′OMe and 2′MOE affected dwell time adjacent to the pore. In contrast, PS linkages produced only slight current alterations without increasing basecalling errors. We further evaluated modification-aware basecallers for 2′OMe and m5C. While these tools can distinguish modification types, they are limited by poor quantification accuracy and high local error rates, especially for 2′OMe. This study establishes a critical performance baseline, clarifying the current capability and limitations of nanopore technology for the analysis of therapeutically relevant RNA modifications.

Recent advances in in silico protein design and bioinformatics have enabled the rapid generation of candidate sequences for functional proteins. However, experimental validation remains a bottleneck, largely due to time-consuming DNA assembly and cell-based cloning processes. Technologies that reduce the time required to convert synthetic oligonucleotides (oligos) into expressed proteins are therefore of considerable interest. Here, we demonstrate that phase-separated droplets formed by the intrinsically disordered protein (Ddx4N1) concentrate both oligos and ligation enzymes, enabling efficient oligo assembly at nanomolar to sub-nanomolar concentrations that are typically inaccessible to conventional ligation-based methods. The assembled products can be directly introduced into femtoliter-scale microreactors for digital cell-free gene expression, allowing protein expression from single assembled DNA molecules without polymerase chain reaction amplification or cellular cloning. Simultaneous expression of two distinct proteins from separately assembled DNA templates in a one-pot reaction was also demonstrated. The complete workflow—from oligo assembly to detectable protein expression—can be performed within half a day. While further development will be required to enhance reaction parallelization and enable systematic retrieval of sequence information from expressed products, this amplification-free, low-input system establishes a technical foundation for integrating oligo-pool-based gene assembly with digital protein prototyping platforms.

While the impact of non-antibiotic drugs on gut bacteria is well-known, their mechanisms of action remain poorly characterized, and effective mitigation strategies for drug-induced dysbiosis are still limited. Here, we screened bacteria-derived drug-target protein homologs (BDTPHs) mapped to 63 target proteins and 107 associated drugs to quantify “drug–BDTPH–bacterium” interactions. These interactions were validated by co-culture experiments using 10 drugs and 25 strains, enzyme assays, and genetic perturbations in Escherichia coli. Ex vivo and in vivo testing with six drugs showed that over 50% of affected genera exhibited high affinity, indicating microbiota alterations through the “drug–BDTPH–bacterium” axis. Leveraging this quantitative interaction framework, we identified a strain of Bifidobacterium animalis that can competitively bind methotrexate through high-affinity BDTPH, thereby effectively alleviating gut microbiota dysbiosis in vivo. Our findings elucidate a mechanism by which non-antibiotic drug effects on bacterial growth, and suggest a universal homology-based competition strategy to restore drug-disrupted microbiota. Here, the authors uncover a mechanism by which drug-target homologs mediate non-antibiotic drug effects on bacterial growth, and suggest a universal homology-based competition strategy to reverse MTX-induced microbiota dysbiosis.