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.

Experimental approaches with a defined microbial community extend concepts from classical experimental evolution of single species to study the complexities of community level changes. Successfully assembling a microbial community, with traceable members, in a defined and manipulatable environment, would provide an effective opportunity to experimentally study general processes and principles in evolutionary ecology. The dynamics between species sorting and genotypic changes is a core, yet not well understood, topic across biological researc. The use of a synthetic microbial community inspired on natural communities reproduced in laboratory environments (for example, fermented foods), enables for experimental evolution of diverse communities. Appreciating the capabilities of communities to respond to changing environments and consequent selection pressures beyond genetic changes within single species must be understood in the face of rapid global environmental changes. Investigating the dynamics between intra- and interspecies processes is significant for understanding community responses to change in natural and managed systems across short and longer temporal scales

The gut microbiota contributes to cholesterol homeostasis by converting cholesterol into coprostanol, a non-absorbable sterol excreted in the feces. However, the enzymes mediating this process remain poorly defined. Here, we identify spiR, a steroid Δ5-4 isomerase/3-keto reductase from Eubacterium coprostanoligenes that catalyzes the initial oxidation of cholesterol to cholestenone, a requisite step in coprostanol production. We confirm that SpiR oxidizes both cholesterol and pregnenolone, and stereospecifically reduces 3-keto-steroids to 3β-hydroxylated forms. We show that SpiR preferentially binds to cholesterol over related steroids and functions as an NAD(H)-dependent homodimer. Through phylogenetic analysis, we show that spiR clusters with known Δ5-4 isomerases and is restricted to an uncultured clade within Acutalibacteraceae, where it frequently co-occurs with species encoding ismA, a gene previously implicated in cholesterol conversion. We analyze a multi-omic dataset from three human cohorts and find that spiR homologs were strongly enriched in individuals exhibiting cholesterol conversion. We also show that spiR homologs have a greater predictive power for cholesterol conversion than ismA homologs, establishing them as superior markers of microbial cholesterol metabolism. Our findings refine the enzymatic model of cholesterol metabolism in the gut and establish spiR as a critical biomarker and mechanistic driver for microbiome-mediated cholesterol reduction. Here, the authors identify SpiR, a gut bacterial enzyme that converts cholesterol, exclusively in a clade of uncultured gut bacteria, and show it is a superior predictor of microbial cholesterol metabolism in humans.

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.

Metagenomic binning is essential for reconstructing prokaryotic genomes from metagenomic samples. We benchmarked various binning tools using Critical Assessment of Metagenome Interpretation (CAMI)-simulated, custom-simulated, and real metagenomic datasets, primarily focusing on short-read sequencing data. Our analysis highlights critical factors influencing binning efficacy: (i) Sequencing depth and taxonomic complexity strongly impact binning performance, while CAMI-simulated benchmarking datasets exhibit substantially lower complexity than human gut and environmental metagenomes, (ii) Chimeric genome rates vary widely across tools, (iii) Multi-sample binning is most effective with about 20 samples, as using too few or too many samples can reduce its benefits, and (iv) Binning efficacy was lower for single-end sequencing samples due to reduced contig quality and assembly fragmentation. Neural network-based tools consistently outperformed others in genome recovery from both real samples and simulated samples with realistic taxonomic complexity, though at higher computational cost. By integrating and refining genome bins from the top three binning tools, we recovered >30% more high-quality genomes than previous methods. This study provides practical guidance for improving metagenomic binning to facilitate the reconstruction of prokaryotic genomes. Benchmarking metagenomic binning tools with simulated and real datasets reveals factors affecting genome recovery and provides practical guidelines for improving metagenome assembled genome reconstruction.