Bacterial Extracellular Vesicles from Chromobacterium subtsugae and Bacillus thuringiensis as Cell-Free Bioinsecticidal Nanocarriers Against the Soybean Pest Euschistus heros

Surfactin-like lipopeptides contribute to interference competition of Bacillus velezensis against the marine Flavobacteriaceae in phycosphere.

This study demonstrates that Bacillus velezensis SPE2, a low-abundance isolate from the phycosphere of dinoflagellate, exhibits a wide degree of antagonistic activity against multiple marine Flavobacteriaceae strains, a dominant taxonomic group across the phycosphere of diverse phytoplankton species.

A signal peptide-guided approach towards in situ functionalization of bacterial nanocellulose in Komagataeibacter rhaeticus.

BACKGROUND Bacterial nanocellulose (BC), produced by Komagataeibacter species, is an ideal scaffold for biological Engineered Living Materials (bioELMs) research. Current BC functionalization strategies often rely on secondary microbial hosts or post-production enzyme immobilization, limiting the scalability and modularity required for programmable bioELMs. Establishing a single-chassis system capable of simultaneous biopolymer synthesis and in situ functionalization remains a primary objective in bioELM research. This study addresses the need by benchmarking signal peptide-mediated protein translocation in K. rhaeticus iGEM, a model bacterium for BC-based bioELMs, enabling a synthetic biology framework for single-chassis based biomaterial functionalization. RESULTS Genome-wide analysis confirmed the presence of a complete Sec translocation machinery in K. rhaeticus. Through liquid chromatography-tandem mass spectrometry and SignalP 5.0 prediction, native signal peptides were identified and evaluated alongside previously characterized heterologous signal peptides using β-lactamase and mScarlet as cargo proteins. Protein translocation was found to depend on signal peptide identity, cargo type, and expression mode. Fluorescence imaging revealed cytoplasmic, polar, and peripheral localization patterns, confirming functional engagement with the native translocation machinery. A key limitation identified was the retention of recombinant proteins within the periplasm, restricting extracellular availability. Despite this, signal peptide-mediated translocation enabled the incorporation of enzymatic activity into BC during biosynthesis. A post-growth osmotic shock-release strategy increased measurable enzymatic activity by 30%, demonstrating a practical route to overcome this physiological bottleneck while maintaining the biomaterial production capacity. CONCLUSIONS This study benchmarks signal peptide-dependent protein translocation in K. rhaeticus and identifies periplasmic retention as a key constraint for extracellular protein release. By linking protein translocation to in situ BC functionalization, this work establishes a synthetic biology framework that supports the development of K. rhaeticus as a single-chassis platform towards the production of functionalized bioELMs.

Isolation and Proteomic Characterization of Outer Membrane Vesicles in Fusobacterium nucleatum.

Acinetobacter guillouiae, a lipolytic strain isolated from sludge capable of partially depolymerising polyethylene terephthalate: genomic, proteomic, and biochemical insights

Findings indicate that A. guillouiae I-MWF can mediate partial PET depolymerisation without assimilating the resulting monomers, while preferentially growing on lipid-like substrates, suggesting adaptation to hydrocarbon-rich environments.

Overcoming outer membrane barrier in gram-negative bacteria by PCNP-PVP-SE1 phage endolysin: Evidence from molecular docking, molecular dynamics and experimental analyses.

The increasing prevalence of antimicrobial resistance among Gram-negative pathogens necessitates the development of alternative antibacterial strategies. Bacteriophage-derived endolysins represent a promising class of antimicrobials; however, their limited ability to traverse the outer membrane of Gram-negative bacteria restricts their exogenous application. In this study, the native PVP-SE1 endolysin and a PCNP-fused Artilysin variant were expressed, purified, and comprehensively characterized using in vitro and in silico approaches. Antibacterial activity was evaluated against Escherichia coli and Salmonella Typhimurium using disk diffusion, minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), and time-kill assays, each performed in biological triplicate (n = 3). While the native endolysin required EDTA to exert antibacterial activity, the engineered variant was independently bactericidal, producing significantly larger inhibition zones and a significantly more rapid reduction in viable bacterial counts (p < 0.05). Structural modeling and molecular docking confirmed preservation of the catalytic core and substrate-binding residues following N-terminal PCNP fusion. Molecular dynamics simulations and MM-PBSA binding free-energy analyses indicated that the engineered complex achieved a more favorable binding free energy than the native form, driven primarily by stronger van der Waals and electrostatic interactions rather than hydrogen bonding. Collectively, these findings indicate that N-terminal PCNP fusion confers statistically supported bactericidal activity against E. coli and Salmonella Typhimurium with a markedly reduced dependence on EDTA, without compromising catalytic integrity, supporting further evaluation of PCNP-fused Artilysins as antimicrobial candidates.

Recent advances in lactic acid bacteria-derived extracellular vesicles.

Lactic acid bacteria (LAB) are globally recognized for their essential roles in food fermentation and impact on human health. Recent evidence identifies bacterial extracellular vesicles (EVs) as unique functional entities, serving as key mediators in microbe-host interactions. LAB-EV biogenesis is primarily driven by mechanisms that compromise cell-wall integrity, including prophage-encoded holin-endolysin systems and autolysin activity. EVs as nano-sized particles exert diverse beneficial effects by inhibiting pathogens, shaping microbiota composition, reinforcing the intestinal epithelial barrier, and modulating host immunity. Their ability to cross biological barriers and reach distal organs also implies systemic impacts. Consequently, LAB-EVs are emerging as a versatile platform for next-generation postbiotics and delivery systems, capable of protecting and transporting bioactive compounds. The interpretation of the effects conveyed by LAB-EVs requires critical considerations regarding mechanistic depth and cargo heterogeneity. A so-far overlooked factor is the influence of bacteriophage elements, which can influence both biogenesis pathways and host immune signatures. To successfully transition toward industrial and clinical applications, research must shift from observational studies to rigorous mechanistic evaluations and standardized production strategies, such as the use of prophage-cured strains or the development of artificial EV-mimics. This review synthesizes recent advances in LAB-EV biology and outlines the considerations necessary to harness these nano-couriers as valuable tools in modern biomedicine and food science.