Bacteroides thetaiotaomicron is a model Bacteroidota of the healthy human gut microbiota and a specialist in glycan utilization. Like other Bacteroides, B. theta has many highly regulated polysaccharide utilisation loci (PUL) that encode outer membrane (OM) TonB-dependent transporters (SusC), closely associated ''lid'' lipoproteins (SusD), and additional surface-exposed lipoproteins (SLPs) that bind and partially degrade specific glycans derived from host cells, diet, or other microbiota members. The canonical starch PUL products are thought to form a dynamic complex in the presence of starch. However, other PULs form stable complexes in the absence of substrate (recently named ''utilisomes''), with additional surface lipoproteins tightly associated with the core SusCD complex. In this study, we characterised the B. theta dextran utilisome, with a SusCDdex core and an associated glycoside hydrolase (GHdex) and surface glycan binding protein (SBGPdex). Via X-ray crystallography we solved high-resolution structures of SBGPdex in isolation and SusDdex and GHdex bound to dextran oligosaccharides. We used isothermal titration calorimetry (ITC) to quantify ligand binding of wild type and mutant SLPs. We further used single particle cryo-EM of the catalytically inactive dextran utilisome to visualize open and closed states of the complex. Three occupied dextran binding sites were observed across SusCdex, SusDdex and GHdex, with substrate observed in both open and closed states of SusDdex. 3D variability analysis showed a minority of particles in the process of SusDdex lid closure. Together our work defines commonalities and differences across utilisomes dedicated to the import of simple glycans.

Bacillus subtilis is a widely used microbial host for both fundamental research and the industrial production of enzymes and biopharmaceuticals. However, its four known signal peptide–dependent secretion pathways impose inherent limitations on the efficient extracellular expression of heterologous proteins. Here, we identify fructose 1,6-bisphosphate aldolase (FbaA) as a nonclassically secreted protein that achieves high-level expression and efficient extracellular export in B. subtilis. Structural and biochemical data indicate tetrameric FbaA as the predominant secreted form. Site-directed mutagenesis shows that the hydrophobic residue methionine 66 (M66) is critical for tetramer formation; substituting M66 disrupts oligomerization, abolishes secretion, and eliminates FbaA’s ability to mediate the export of heterologous proteins. Together, these results establish that FbaA oligomerization is essential for its nonclassical secretion and that FbaA functions as a modular export element capable of facilitating the secretion of fused heterologous proteins. This work provides mechanistic insight into oligomerization-dependent protein export and offers a promising strategy for engineering efficient secretion systems in B. subtilis.

Preozonation is widely used to enhance the effectiveness of coagulation and filtration in algae-laden water treatment, but cyanobacterial cell rupture and the subsequent release of intracellular organic matter and cyanotoxins can increase treatment burdens and pose health risks. In natural waters, cyanobacteria are often surrounded by symbiotic bacteria, whose influence on ozonation performance and underlying mechanisms remains unclear. Herein, we found that axenic filamentous cyanobacteria (Leptolyngbya sp.) exhibited strong resistance to ozonation (0.3 mg L–1, 20 min), whereas the presence of surface-associated bacteria markedly increased the cell rupture rate from 12 ± 6% to 76 ± 2%. Removal of loosely bound extracellular polymeric substances (LB-EPS) significantly reduced ozonation resistance in axenic cyanobacteria but unexpectedly enhanced that of xenic cultures. By integrating reactive oxygen species identification, extracellular metabolomics, and metabolic reconstruction, we demonstrate that surface-colonizing bacteria degrade the algal LB-EPS envelope, releasing metabolites that facilitate hydroxyl radical formation during ozonation, thereby intensifying cell rupture. Our results highlight surface-associated bacteria as a critical yet overlooked factor shaping cyanobacterial responses to preozonation, underscoring the need to re-evaluate ozone application strategies in bloom-impacted waters to minimize cell rupture and byproduct formation.

Protein translation is an error-prone process resulting in a random population of altered protein sequences in every cell. Here, we analyzed thousands of publicly available mass spectrometry datasets to detect amino acid misincorporations and quantify error rates in 14 model organisms. We find that overall error rates and the patterns of codon to amino acid error rates correlate across species. We estimate that on average 1-2% of protein molecules in a cell harbor a misincorporation, whereas this proportion can reach 10% for long proteins. Highly expressed and very long proteins have lower error rates, indicating evolutionary selection on codon usage to reduce the cost of translation errors. While both codon-anticodon mispairing and tRNA mischarging contribute to misincorporations, we estimate that ~70% of misincorporation events are due to mispairing. The more frequent an amino acid in the proteome, the more likely it is misincorporated (r = 0.53), likely because frequent amino acids are abundant in the cell, increasing the rate of mischarging, and have abundant tRNAs, leading to increased mispairing. Overall, we find that amino acid and codon usage explain error rates. The conserved patterns of amino acid misincorporations from bacteria to humans suggest universal mechanisms driving translational fidelity.

Colorectal cancer (CRC) is heavily influenced by gut microbiota and metabolites such as branched-chain amino acids (BCAAs), which provides essential growth materials for tumors and activates related cancer-promoting pathways. We engineered two E. coli Nissle 1917 strains (ECN)─ECN-Deg and ECN-Tra─to deplete BCAAs in the gut in previous work. In this work, using an AOM/DSS-induced CRC mouse model under the amino acid diet, we found that both strains significantly ameliorated CRC progression, improved survival, restored gut barrier function, and reduced systemic inflammation. Mechanistically, they lowered plasma BCAA levels, suppressed mTOR activation, and modulated retinol and drug metabolism pathways. Our results demonstrate that engineered probiotics targeting BCAAs catabolism can effectively inhibit colorectal tumorigenesis, offering a novel synthetic biology-based approach for cancer therapy.

Nonribosomal peptide synthetases (NRPS) represent a valuable yet underexplored resource for producing bioactive natural products. However, most NRPSs remain silenced potentially due to factors such as dysfunction of the initiation unit. The starter condensation (Cs) domain of the initiation unit catalyzes the lipoinitiation of nonribosomal peptides via the incorporation of an N-terminal fatty acyl chain. The concept of initiation unit engineering introduced herein encompasses the replacement of the native initiation unit of NRPSs with a foreign and well-characterized Cs domain-containing initiation unit to activate the NRPS and optimize its expression. This strategy was employed herein to successfully access three of the six previously silent NRPS pathways in Mycetohabitans rhizoxinica HKI 454, a bacterium of the class β-proteobacteria, resulting in the identification of three classes of lipopeptides. This strategy was then extended to access two NRPS pathways in Pseudomonas syringae (γ-proteobacteria) and obtain novel lipopeptides, thereby establishing a feasible complement to existing genome mining strategies for natural product discovery. Furthermore, change of the initiation regions of biosynthetic pathways of nonlipidated chitinimide (β-proteobacteria) and pseudotetraivprolide (γ-proteobacteria) with heterologous Cs-containing initiation units enabled the successful incorporation of fatty acyl chains into the N-terminus of both peptide backbones, launching a workable approach to create artificial lipopeptides. Overall, this study provides a practical strategy for the rational recovery of silent BGCs and introduction of fatty acyl chains into nonribosomal peptides, at least in Proteobacteria, thereby enriching genome mining and combinatorial biosynthesis approaches for accessing the underexplored biosynthetic potential of NRPSs from various bacteria.