Diversity-generating retroelements (DGRs) are natural systems that accelerate the evolution of diverse bacterial functions through targeted hypermutation. We establish a method using DGRs coupled to recombineering (DGRec), which enables the diversification of any sequence of interest in E. coli. Detailed characterization of reverse transcriptase sequence biases demonstrates how it maximizes the exploration of the sequence space while avoiding nonsense mutations. By leveraging the high error rate of the DGR reverse transcriptase at adenines, DGRec can efficiently diversify user-defined sequence windows of 50–200 bp. Mutations can be focused at specific positions, with rates reaching up to 1.38 × 10−2 per base per generation, allowing up to 24 mutations to accumulate within a single target sequence after 48 h. We apply DGRec to phage λ host-range engineering, to the evolution of dCas9 variants and to accelerated evolution of specific nanobodies through a bacterial display setup. Lastly, we establish the feasibility of DGR-mediated mutagenesis in yeast by adapting a recombination and selection strategy previously developed for retrons. Diversity-generating retroelements are engineered for directed evolution in E.coli.

The fitness and virulence of Pseudomonas aeruginosa rely on its ability to maintain a functional pool of ribosomes, which are essential for protein synthesis. This review explores the intricate ways of ribosome protection, rescue and hibernation, by which P. aeruginosa preserves ribosome functionality under stress. These processes enhance the adaptability and resistance of this pathogen to ribosome-targeting antibiotics and present significant challenges to current therapeutic strategies. By highlighting recent discoveries and identifying promising directions for future research, this review aims to explore potential targets for innovative drug discovery.

Internal chemical modifications of mRNA constitute a key epitranscriptomic layer of gene regulation in eukaryotes. Although N6-methyladenosine (m6A) has been the most intensively studied, accumulating evidence reveals important roles of non-m6A mRNA modifications, such as 5-methylcytosine, N4-acetylcytidine and pseudouridine, in plants. These modifications modulate diverse aspects of mRNA metabolism, including alternative splicing, stability, translation and long-distance transport, and thereby shape plant development and environmental adaptation. Here we summarize current advances in understanding non-m6A mRNA modifications in plants, emphasizing their regulatory roles in mRNA metabolism and their effects on plant development and stress resilience. We also review the detection technologies for non-m6A modifications and discuss key challenges and future directions towards elucidating their regulatory functions. This Review highlights emerging roles of non-m6A modifications, such as 5-methylcytosine, N4-acetylcytidine and pseudouridine, in plant mRNAs, outlining their effects on mRNA processing, development and stress adaptation, as well as advances in detection methods and future directions.

Protein allostery underlies most information and energy processing in biology and the development of artificial allosteric proteins is a key objective of synthetic biology and biotechnology. We show that machine-learning-engineered minimal ligand-binding domains act as efficient receptors in single-component allosteric switches, despite lacking global conformational change. Such colorimetric, luminescent and electrochemical biosensors of small molecules, peptides and proteins can be compiled into intramolecular YES and AND logic gates. Furthermore, we report fully synthetic allosteric switches composed of artificial receptor and reporter domains. Hydrogen/deuterium exchange mass spectrometry and 19F nuclear magnetic resonance analyses suggest that ligand binding reduces the conformation entropy of the system, increasing the catalytic activity of the reporter domain. The potential practical utility of this approach is demonstrated by engineering Escherichia coli cells with steroid-dependent antibiotic resistance and by developing bioelectronic devices capable of quantifying steroid hormones. Allosteric biosensors are constructed by combining reporter proteins with machine-learning-designed receptor domains.

The bacterial CRISPR–Cas9 nuclease has become a powerful genome manipulation tool for a wide range of organisms. However, it has yet to fully leverage the pervasive presence of DNA methylation in genomes. Here, to fill this gap, we report biochemical, structural and human genome-editing characterizations of a methylation-sensitive Cas9 (ThermoCas9). ThermoCas9 efficiently binds to and cleaves DNA upstream of its protospacer adjacent motif (PAM) 5′-NNNNCGA-3′ or 5′-NNNNCCA-3′ in vitro. Methylation of the fifth cytosine in either PAM sequence (5mCpG or 5mCpC), however, significantly inhibits ThermoCas9 activity. Cryo-electron microscopy structures of ThermoCas9 in pre-cleavage and post-cleavage states at 2.8 Å and 2.2 Å resolution, respectively, reveal the molecular basis for the stringent requirement of the unmethylated cytosine in PAM binding and provide guidance for further enzyme engineering. We demonstrate methylation-sensitive editing by ThermoCas9 in human cell lines with distinct DNA methylation landscapes. Moreover, we demonstrate that a catalytically enhanced ThermoCas9 efficiently targets luminal expression signature genes that are consistently hypomethylated in patients with breast cancer. Owing to its sensitivity to DNA methylation, ThermoCas9 can specifically target cells with disease-related hypomethylation, which adds another layer of precision to genome-editing technologies. ThermoCas9, a genome-editing enzyme that is sensitive to the DNA methylation status of the target locus, is characterized and shows promise for targeting hypomethylated DNA regions in cancer cells.

Fermentative Clostridium species associated with rice roots can contribute substantially to biological nitrogen fixation in anoxic paddy soils, yet whether their biological nitrogen fixation is regulated by the redox chemistry of rhizosphere remains unclear. Here we show that iron plaques on rice roots function as terminal electron acceptors that reprogram Clostridium fermentation and thereby enhance biological nitrogen fixation. In nitrogen-fixation microcosms, Clostridium sensu stricto I was selectively enriched under plaque-associated Fe(III)-reducing conditions, coinciding with elevated nitrogen fixation. Metabolomic profiling coupled with metabolic flux analysis revealed that Fe(III) reduction redirects a portion of carbon and electron flow from low-energy-yield solventogenesis toward high-energy-yield acidogenesis. This shift increases cellular ATP generation and expands the reductant pool, thereby benefiting the energetic and reductant demands of nitrogenase. Integrated transcriptomic and metagenomic analyses further identified NosR, a flavin mononucleotide-binding protein that is upregulated during Fe(III) reduction and may facilitate electron delivery to plaque-associated Fe(III). Our findings establish a mechanism in which iron plaque reduction optimizes fermentation for biological nitrogen fixation, providing fundamental insights into coupled Fe–N cycling in rice rhizospheres and suggesting potential strategies for sustainable nitrogen management in flooded agroecosystems.

Reactive oxygen species are essential for cellular signalling and redox homeostasis, but their accumulation causes cellular oxidative stress. In inflammatory bowel disease, oxidative stress is linked to chronic inflammation and alterations in the gut microbiota. We hypothesised that these alterations may result from the impact of reactive oxygen species on the interactions between bacteria and their viruses, bacteriophages. We followed the evolution of three E. coli strains and a virulent bacteriophage in a chemostat under continuous growth and studied the impact of oxidative stress on this community. We show that both the bacteriophage and its three hosts persisted in the system over 10 days, but the relative abundance of bacteriophages was decreased in the presence of reactive oxygen species. Oxidative stress also limited bacteriophage population diversity by favouring the selection of specialist bacteriophages with a narrower host range. Concomitantly, reactive oxygen species accelerated the evolution of bacterial resistance to bacteriophages and drove the fixation of genomic mutations in genes related to cell surface structures or located in mobile genetic elements. These results highlight that oxidative stress impacts the evolutionary dynamics between bacteria and bacteriophages with consequences for microbiota diversity and potential implications in the context of intestinal inflammation.

Pests cause up to 40% of global crops losses. Pesticide overuse drives resistance and poses notable risks to public health and the environment. Many hypocrealean fungi form symbiotic relationships with plants while antagonizing pests, making them valuable sources of biocontrol agents and biopesticides. However, little is known about their biosynthetic capabilities. Here we use phylogenomics, metabolomics and heterologous expression to catalog the biosynthetic repertoire of 82 plant-associated and insect-associated Hypocreales species. Annotation of 5,221 biosynthetic gene clusters reveals that ~80% of them encode unknown products. By linking biosynthetic gene clusters to molecules, we investigate the biosynthesis of several natural products, including pyridones, dethiosecoemestrin and efrapeptin. Additionally, by combining our metabologenomics workflow with synthetic biology, we characterize four nonribosomal peptide synthetase-like synthetases involved in the biosynthesis of hitherto unknown products. We believe that this work lays the groundwork for future efforts toward sustainable pest control in agriculture. Crop losses from pests threaten global food security. Here, the authors survey the biosynthetic repertoire of hypocrealean fungi, identifying over 5,200 biosynthetic gene clusters, most encoding unknown products, and linking several to bioactive molecules for sustainable pest control.

Postbiotics, defined as preparations of inanimate microorganisms and their components that confer health benefits, are emerging as stable and safe alternatives to probiotics. This review summarizes recent advances in the biochemical composition, mechanisms of action, and applications of postbiotics and paraprobiotics in human and animal nutrition. It also highlights innovations in omics technologies, artificial intelligence, and sustainable production strategies that are shaping next-generation microbiome-based functional foods and therapeutic interventions.

Cancer functional genomics using CRISPR base editors (BEs) holds great promise for molecular characterization and new target discovery. However, traditional BEs, using intact DNA deaminases as mutators, are often constrained by limited control and nonspecific toxicities. Here we developed a small-molecule-controllable system using split-engineered BEs (seBEs). By placing deaminase activity under small-molecule control, seBEs significantly reduced cellular toxicity and enabled robust and inducible in vivo functional genomics screens. High-density seBE genetic screens using ~11,000 single guide RNAs in vitro and ~3,700 single guide RNAs in vivo reveal known and previously unknown loss-of-function and dominant-negative mutations in cancer therapeutic targets. A deeper tiling seBE screen against Adar1, a key mediator in cancer immunotherapy, reveals critical residues within functional domains that show no phenotype in vitro but distinctively elicit non-cell-autonomous cancer dependencies in vivo. Overall, our seBE system offers a generalizable, controllable and highly efficient method to systematically identify key residues in cancer functional genomics. Inducible base editors enhance cancer genomic screens in vivo.

Microorganisms secrete extracellular vesicles (EVs) that transport bioactive molecules, including proteins and metabolites. While their functions are well studied in model microbes, their ecological contributions to natural ecosystems remain largely unexplored. Here we performed an integrative study investigating the role of environmental EVs in shaping microbial community assembly in the Xinglinwan Reservoir. By combining genome-scale metabolic models and multi-omics of field EVs, we found that EVs mediated metabolite exchanges mainly through carrying amino acids, disaccharides, carbohydrate-active enzymes (CAZymes) and signals. EVs can facilitate the growth of amino acid auxotrophic strains. Moreover, EVs act as an external reservoir of functional traits, potentially reinforcing stochastic assembly processes and conferring functional redundancy to the ecosystem. Collectively, our integrative data demonstrate that EV-mediated metabolic exchange is an auxiliary mechanism supplementing classical nutrient transport in aquatic environments. EVs emerge here as a significant, distinct vector in biogeochemical cycling, offering a critical layer for resolving complex natural microbial interactions. Microorganisms release extracellular vesicles, but their ecological roles in natural environments remain unclear. A year-long multi-omics study reveals that environmental extracellular vesicles mediate metabolite exchange central to carbon and nitrogen cycling while stabilizing microbial communities.

The root nodules formed by rhizobia and leguminous plants are specialized structures for nitrogen fixation. However, a large number of non-rhizobial endophytes (NREs) also coexist within the nodules, and their contribution to nitrogen fixation under abiotic stress conditions remains unclear. Here, using the wild leguminous shrub Sophora davidii as model system, we identified an important NRE (Bacillus siamensis BT-9-1) by analyzing keystone taxa within the bacterial cooccurrence network of root nodules. This strain could improve the survival of Mesorhizobium metallidurans YC-39 under saline-alkali stress. A mechanistic investigation revealed that the expression of ilvA, ilvH, and ilvD was downregulated, and the contents of (2S)-isopropylmalate and succinic acid decreased in M. metallidurans YC-39 under saline–alkali conditions, whereas B. siamensis BT-9-1 presented increased accumulation of these metabolites. These findings indicate that B. siamensis BT-9-1 cross-feeds M. metallidurans YC-39 with these metabolites, rescuing the compromised branched-chain amino acid synthesis pathway and the TCA cycle in saline–alkali environments. Eventually, coinoculation with B. siamensis BT-9-1 and M. metallidurans YC-39, along with (2S)-isopropylmalate and succinic acid supplementation, increased nitrogenase activity of the symbionts. Our study reveals a novel mechanism by which non-rhizobial endophyte Bacillus species enhances the growth and nitrogen fixation efficiency of M. metallidurans under saline-alkali stress through the delivery of key metabolites.