Western diets reduced in fiber promote dysbiosis and exacerbate colitis, with short-chain fatty acids (SCFAs) from fiber fermentation known to regulate glucagon-like peptide 1 (GLP-1) secretion. Whether GLP-1 dysregulation directly links diet-induced dysbiosis to colitis severity, and if this pathway can be therapeutically targeted independently of dietary fiber, remains unclear. Here, we show that dextran sulfate sodium (DSS)–induced colitis severity correlates with compensatory GLP-1 increases, while receptor blockade worsens damage, confirming GLP-1’s protective role during colitis. Fiber deficiency impaired L cell function and GLP-1 release, increasing colitis susceptibility. GLP-1 receptor agonist reversed these effects, restoring barrier integrity and accelerating recovery. We engineered a probiotic, releasing a microbial peptide, that locally elevates GLP-1, which normalized gut parameters in fiber-deprived mice and alleviated colitis via GLP-1–dependent mechanisms, including improved metabolism, antimicrobial defenses, and barrier restoration. Our findings mechanistically connect fiber deficiency to colitis through GLP-1 and demonstrate that probiotic-mediated GLP-1 modulation can bypass dietary fiber requirements to maintain gut homeostasis.

Untargeted tandem mass spectrometry (MS/MS)-based metabolomics enables broad characterization of small molecules in complex samples, yet the majority of spectra in a typical experiment remain unannotated, limiting biological interpretation. Reference data-driven (RDD) metabolomics addresses this gap by contextualizing spectra through comparison to curated, metadata-annotated reference datasets, allowing inference of spectrum origins without requiring exact structural identification. Here, we present an open-source RDD metabolomics platform comprising a user-friendly web application and a Python software package that perform RDD analyses directly from molecular networking outputs generated by GNPS. The tools support visualization and statistical analysis of RDD results, including interactive bar plots, heat maps, principal component analysis, and Sankey diagrams. We illustrate the approach using a hierarchical reference dataset of 3,500 food items to derive dietary patterns from stool metabolomics data of omnivore and vegan participants. The analysis reveals clear dietary group separation, demonstrating how RDD metabolomics can extract biologically meaningful patterns from otherwise unannotated spectra. Thus, the RDD metabolomics platform removes technical barriers for the metabolomics community to adopting reference data-driven analysis, with the functionality freely available at https://github.com/bittremieuxlab/gnps-rdd and https://gnps-rdd.streamlit.app/.

In Escherichia coli, RNase E, a central enzyme in RNA processing and mRNA degradation, contains a catalytic N-terminal domain, a membrane-targeting sequence (MTS), and a C-terminal domain (CTD). We investigated how MTS and CTD influence RNase E localization, diffusion, and function. Super-resolution microscopy revealed that ~93% of RNase E localizes to the inner membrane and exhibits slow diffusion similar to polysomes. Comparing the native amphipathic MTS with a transmembrane motif showed that the MTS confers slower diffusion and stronger membrane binding. The CTD further slows diffusion by increasing mass but unexpectedly weakens membrane association. RNase E mutants with partial cytoplasmic localization displayed enhanced co-transcriptional degradation of lacZ mRNA. These findings indicate that variations in the MTS and the presence of the CTD shape the spatiotemporal organization of RNA processing in bacterial cells, providing mechanistic insight into how RNase E domain architecture influences its cellular function.

The emergence of eukaryotes from a merger between an archaeon and a bacterial cell ~two billion years ago involved a profound change in cellular organization. While the order in which different features of the eukaryotic cell arose remains a matter of controversy, close archaeal relatives of eukaryotes have recently been identified that possess homologues of eukaryotic trafficking machinery and a complex cell architecture. The members of this phylum, the Asgard archaea (syn. Prometheoarchaeota) described so far, however, lack internal membrane-bound compartments, and therefore have shed little light on origins of the hallmark eukaryotic endomembrane system. Here we report the cell biological analysis of a member of the Heimdallarchaeia, Candidatus "Yibarchaeum umbracryptum" in enriched mixed microbial communities. Possessing a small genome encoding few homologues of eukaryotic membrane remodelling machinery, Ca. Y. umbracryptum cells in late-stage cultures resemble previously described Asgard archaea with extensive cellular protrusions. Surprisingly, during early stages of culture growth Ca. Y. umbracryptum cells have fewer protrusions but possess numerous intracellular vesicles, most of which have a luminal surface that morphologically resembles the outer coat of the plasma membrane. These data alter our view of eukaryogenesis by identifying a close archaeal relative of eukaryotes with a regulated endomembrane system.

Electron transfer coupled to redox chemistry is at the heart of metabolism. The proteins responsible for moving electrons (protein electron carriers) must have emerged at the origin of life. The small iron–sulfur-binding bacterial ferredoxins were likely among these first proteins. Embedded within the ferredoxin sequence and structure is a symmetry that points to an ancient gene duplication event. Little is understood about the nature of ferredoxins prior to this duplication event or what environmental factors may have driven the selection for more complex forms. The deep-time molecular history of ferredoxins goes back billions of years and cannot be reconstructed by phylogenetic analyses based on amino acid sequences. Here, we use structure-guided protein design to model a fossil half-ferredoxin stage in the evolution of this fold, the semidoxins, and their symmetric full-length counterparts, the symdoxins. Semidoxin designs homodimerize, exhibiting structural, thermodynamic, and electrochemical behaviors in most cases identical to cognate symdoxins. However, the semi- and symdoxin fossil stages behave differently when incorporated into an in vivo electron transfer complementation assay. Both can support bacterial growth dependent on protein expression. Growth rates of bacteria expressing the semidoxins are much more sensitive to oxygen than those of bacteria expressing symdoxins. Motivated by the in vivo functionality of designed semidoxins, we identified putative naturally occurring semidoxins in extant anaerobic microorganisms. This is consistent with the observed in vivo oxygen sensitivity of the semidoxin designs. One natural semidoxin is shown to be folded and redox active. However, it exists as a mixture of monomers and dimers, suggesting a potential connection between semidoxins and even simpler single iron–sulfur cluster-binding peptides.

Surfactin, a lipopeptide antibiotic and quorum-sensing (QS) mediator from Bacillus subtilis, has dual functions in microbial ecology and plant disease suppression. This study engineered B. subtilis R31 to overproduce comK and phrC, key regulators of surfactin biosynthesis, increasing surfactin yield by 45% compared to the WT strain. While elevated surfactin enhanced antimicrobial potential, comK-mediated overproduction impaired biofilm formation and swarming motility, but rhizosphere colonization was mostly unaffected. 16S rRNA sequencing of banana rhizospheres showed that surfactin selectively shaped the microbial community by enriching beneficial Bacillus species. Mechanistic studies confirmed surfactin's dual role as an antimicrobial and an intercellular signalling molecule for coordinated development in Bacillus populations. These results reveal the molecular mechanisms of R31-mediated suppression of banana Fusarium wilt and offer a strategy for engineering synthetic microbial consortia by manipulating metabolic signalling pathways.

Miniature inverted-repeat transposable elements (MITEs) are nonautonomous mobile genetic elements (MGEs) that can be mobilized by transposases provided by the relevant autonomous MGEs. Composite transposon-like structures bounded by two MITE copies, designated MITE-COMPs, were previously shown to mobilize the intervening sequences. However, their association with antibiotic-resistance genes (ARGs) has not yet been systematically studied, and only a few MITE-COMP structures with ARGs have been reported to date. This study thus aimed to identify new MITE-COMP members containing ARGs in the genomic sequences of the clinically important bacterial family Enterobacteriaceae in public databases. MITE-COMPs were first searched for in the PLSDB database, focusing on small plasmids, using a self-against-self blastn approach. Then, newly and previously identified MITEs were searched for in the NCBI core nucleotide database to identify MITE-COMPs located on other replicons. Bioinformatics analyses identified multiple previously unreported MITE-COMP structures containing ARGs, which are bounded by directly or inversely oriented MITEs, including IS101, MITESen1, and a novel 244-bp MITE termed MITE244. All these MITEs seem to have originated from transposons of the Tn3 family. MITE244 contains a putative cointegrate resolution site related to Tn21, and MITE244 copies in MITE-COMPs were predominantly found in inverse orientation. The ARGs embedded in newly identified MITE-COMPs include blaKPC-2, qnrS1, tet(A), qnrD1, and floR. Notably, the blaKPC-2 carbapenemase gene was found in MITE-COMPs bounded by MITE244 and another MITE-COMP bounded by IS101. These findings highlight a potential role for MITEs in the spread of diverse ARGs in Enterobacteriaceae.

Chromatin immunoprecipitation and coimmunoprecipitation assays are common approaches to characterize the genomic localization and protein interactors, respectively, for a protein of interest. However, these approaches require the use of specific antibodies, which often face sensitivity and specificity issues. On the basis of TurboID, we developed proximity-labeled affinity-purified mass spectrometry and sequencing (PLAMseq), which enables, in the same workflow, identification of the genomic loci and the interacting proteome of a protein of interest. Moreover, PLAMseq can also be applied to specifically map protein interactions and ubiquitin(-like)–modified proteins. We validated PLAMseq with two well-characterized proteins, RNA polymerase II, and CTCF, with excellent robustness and reproducibility. Next, we applied PLAMseq to characterize histone H1 SUMOylation, in which study has remained elusive due to the lack of specific reagents, and found that SETDB1 binds to SUMOylated histones H1.2 and H1.4 that also colocalize with H3K9me3 at repetitive regions of the genome.

Natural products are a major source of bioactive compounds, yet elucidating their biosynthetic pathways remains a major challenge due to complex genotype–phenotype relationships. Recent advances in computational approaches, particularly artificial intelligence (AI) and mechanistic modeling, are transforming this field. This highlight examines key databases that underpin computational studies, AI-driven methods for predicting biosynthetic pathways and enzyme–substrate interactions, and mechanistic simulations that provide energetic and structural insights. We also discuss current challenges and future opportunities for integrating these strategies to accelerate discovery, engineering, and application of natural products in drug discovery, biotechnology, and synthetic biology.

A synthetic cell is a membrane-bound vesicle that encapsulates cell-free transcription/translation (TXTL) systems. It represents a transformative platform for advancing bacteriophage therapy. Building on experimental work that demonstrates (i) modular genome assembly, (ii) high-yield phage TXTL systems, and (iii) smart hydrogel encapsulation, we explore how synthetic cells can address major limitations in phage therapy. The promising advances include point-of-care phage manufacturing, logic-responsive antimicrobial biomaterials, and new chassis to dissect the dynamics of phage-host interactions. We also propose a roadmap for the deployment of synthetic cells as programmable and evolvable tools in the context of laboratory research and translational clinical adoption. droplet

A sudden increase in temperature triggers Bacillus subtilis to activate expression of stress-specific heat shock proteins of the CtsR (class three stress gene repressor) regulon to withstand the adverse conditions. Key members of this regulon, such as ATPases, proteolytic subunits and their adaptors, which can assemble to the functional Clp protease system, perform crucial roles in maintaining cellular proteostasis, while their transcription is repressed by CtsR during vegetative growth. Upon heat shock, a conformational change in a thermo-sensing glycine-rich loop causes CtsR to detach from its DNA operators, enabling the transcriptional activation of the regulon. Novel data from a clpX-deficient strain demonstrated that in addition, the presence of the ATPase ClpX is essential for the CtsR dissociation from its DNA binding site. To further elucidate this role of ClpX, we constructed a conditional clpX strain, in which clpX induction is decoupled from its native transcriptional control. This conditional expression system mimicked a clpX-deficient phenotype under non-inducing conditions and restored the wild-type phenotype upon induction. Our results indicate that the full induction of the CtsR regulon, particularly clpE, requires both heat and the presence of ClpX, thereby extending the current model for the transcriptional activation of genes repressed by CtsR.

Bacterial extracellular vesicles (bEVs) produced by intestinal commensal bacteria mediate host-microbe interactions, but their proteomes have been explored in only a small number of species. We characterized bEVs from in vitro cultures of 12 species of Actinomycetota, Bacillota (Firmicutes), Bacteroidota, Fusobacteria, Pseudomonadota, and Verrucomicrobia. This is the first report of bEVs from Veillonella magna, an exceptional gram-negative Bacillota, and the gram-positives Peptostreptococcus russellii and Turicibacter sanguinis. The morphology and protein subcellular localization patterns of bEVs reflected the envelope structures of their parent bacteria. Notably, V. magna may be able to produce both outer membrane and cytoplasmic membrane vesicles. The proteome compositions were dictated by phylogeny, suggesting largely non-selective packaging of proteins. Annotation of protein functions indicated roles in nutrient metabolism and transport, and in host immune system modulation. Peptidoglycan modifying enzymes and the abundant bacteriophage proteins in Enterobacter cloacae and Limosilactobacillus reuteri bEVs may be involved in vesicle biogenesis. The functions of many abundant bEV proteins are unknown. The most abundant bEV proteins were largely species-specific, but we identified several conserved proteins that may be used as markers to distinguish commensal bEVs from host EVs. Comparison to previous in vitro and fecal metaproteomics data indicates that bEV proteome compositions are reproducible.