Applied and Environmental Microbiology最新文献

Extracellular vesicles (EVs) released by Candida albicans are multi-antigenic compartments considered as promising prototypes for vaccine development. However, their stability, appropriate storage, and handling conditions are largely unexplored, which raises questions related to their biotechnological applicability. Here, we evaluated the physical and functional stability of C. albicans EVs under long-term storage. Furthermore, we conducted a comparative analysis of these properties in C. albicans EVs obtained through three commonly utilized isolation protocols documented in the literature. After identifying the most efficient isolation method for optimal yield, we devised a potential quality control for EV isolation based on protein and sterol ratios. Subsequently, we investigated the impact of drying EVs using vacuum centrifugation at room temperature or -4°C and the effect of freeze-thaw cycles on EV stability. Transmission electron microscopy (TEM) revealed that EVs maintained morphological stability after long-term (up to 4 years) storage at -80°C, as well as storage at room temperature, 4°C, and -20°C for 7 days with or without vacuum centrifugation, with a tendency of higher recovery when a lower temperature is used. Remarkably, all of the C. albicans EV suspensions maintained their biological properties as demonstrated by their ability to protect Galleria mellonella against C. albicans infection. However, the number of freeze-thaw cycles significantly impacted the protective effect of the EVs. Overall, our findings demonstrate that C. albicans EVs maintain notable morphological and biological stability under several conditions, enabling their efficient and reproducible utilization in research and potentially as therapeutic agents.

Importance: Extracellular vesicles (EVs) released by Candida albicans are promising vaccine prototypes due to their multi-antigenic nature. However, their storage and handling conditions are not well understood, raising concerns about their biotechnological use. This study evaluated the long-term physical and functional stability of C. albicans EVs. We compared three isolation methods to identify the most effective one and suggested a quality control measure based on protein and sterol ratios. We also examined the effects of vacuum drying and freeze-thaw cycles on EV stability. Our findings show that C. albicans EVs maintain their biological function after long-term storage at -80°C and under various conditions. Notably, their protective effect in an insect model was reduced through repeated freeze-thaw cycles. This research provides valuable insights for the efficient use of these vesicles in future studies.

Bisphenol F (BPF) is an emerging environmental pollutant widely present in surface water and wastewater systems. Microbial activity is crucial in driving its degradation, offering a potential avenue for mitigating its environmental impact. Although the degradation pathway for BPF has been identified in various bacteria, the biodegradation mechanisms remain unclear. In this study, we isolated a highly efficient BPF-degrading strain of Sphingobium yanoikuyae DN12, which could utilize BPF as the sole carbon and energy source for growth, from a river sediment in Anhui Province, China. Through ultra-performance liquid chromatography high-resolution mass spectrometry (UPLC-HRMS) analysis, we found that oxidation and hydrolysis are key steps for BPF biodegradation. Utilizing whole-genome sequencing, comparative transcriptomics analysis, and biochemical identification, a gene cluster bpf was identified to be involved in BPF degradation. BpfAB is a two-component oxidoreductase responsible for converting BPF to 4,4'-dihydroxybenzophenone (DHBP). BpfC is a Baeyer-Villiger monooxygenase responsible for converting DHBP to 4-hydroxyphenyl-4-hydroxybenzoate (HPHB). Isotope tracing demonstrated that the oxygen atom incorporated by BpfAB originates from water, whereas that incorporated by BpfC derives from molecular oxygen (O₂). BpfD is an α/β hydrolase responsible for converting HPHB to 4-hydroxybenzoate and 1,4-hydroquinone. Analysis of the taxonomic and habitat of 325 prokaryotic genomes revealed that BpfA-like homologs are predominantly found in the phylum Pseudomonadota, primarily inhabiting soil and aquatic environments. This study enhances our understanding of the biodegradation mechanism of BPF and provides guidance for the effective remediation of BPF-contaminated environments.IMPORTANCEBisphenol F (BPF) is a widely used alternative to bisphenol A and poses a growing threat to ecosystems and human health due to its environmental persistence and endocrine-disrupting effects. Although microbial degradation pathways for BPF have been reported, the key enzymes involved and their catalytic mechanisms remain unclear. This work reports the isolation of a Sphingobium strain capable of mineralizing BPF and the genetic basis for the catabolic pathway. Three enzymes-a two-component oxidoreductase, a Baeyer-Villiger monooxygenase, and an α/β hydrolase-were biochemically characterized and shown to catalyze the three critical steps in BPF degradation. These findings provide insights into the biochemical processes involved in the microbial degradation of BPF.

Phage Felix O1 (FO1) is one of the most widely used bacteriophages for targeting Salmonella enterica and is applied in both research and food safety settings. Despite its broad host range, we observed significantly reduced efficiency of plating (EOP) on a subset of Salmonella enterica strains, including serovar Typhimurium 4/74 (S. 4/74). We found that O-antigen-mediated steric hindrance contributes to this resistance, which can be partially overcome by supplementation with the SP6 phage tailspike protein (TSP). However, full restoration of the EOP required deletion of the Gifsy-1 prophage, leading to the identification of a single prophage-encoded defense gene, gipd474 (Gifsy-encoded phage defense protein from strain S. 4/74). Heterologous expression of this gene in Escherichia coli increased resistance to multiple tailed phages. Bioinformatic analysis revealed that this gene is widely spread across the Enterobacteriaceae, suggesting a conserved role in phage resistance. Together, these findings uncover a dual barrier to FO1 infectivity in S. 4/74 and highlight gipd474 as a novel, prophage-encoded phage defense gene.IMPORTANCEPhage therapy and biocontrol are increasingly explored as alternatives to antibiotics, particularly against drug-resistant pathogens such as Salmonella. FO1 is a widely used lytic phage with broad activity across Salmonella serotypes. However, variability in phage infectivity limits its reliability against certain strains. Here, we report that S. 4/74 restricts FO1 through a combination of O-antigen-associated interference and a novel prophage-encoded resistance gene. This gene also impairs the infectivity of other tailed phages and is conserved across multiple Enterobacteriaceae genera. Our findings highlight the importance of prophage-encoded defense systems in shaping phage susceptibility and highlight the need to account for such elements when developing phage-based applications.

Plasmid DNA comprises various modules, including promoter and terminator regions, replication origins, and antibiotic resistance genes as selection markers. The 5'UTRs of the promoter and ribosome binding site (RBS) have been extensively studied, and multiple synthetic promoters and RBSs have been developed. In this study, we focused primarily on the components of plasmid vectors in Escherichia coli, excluding the 5'UTRs. Six replication origins and four selection markers were shuffled, resulting in the construction of 24 different plasmids. The gene encoding D-lactate dehydrogenase from Cupriavidus necator was cloned into each plasmid in this plasmid library, and the resulting 24 plasmids were evaluated based on D-phenyllactic acid production. The maximum concentration and yield of PheL from glucose were 5.45 g/L and 0.34 g/g, respectively. To the best of our knowledge, this yield represents the highest reported for PheL production. Furthermore, to examine the relationship between culture profiles and plasmid selection, clustering analysis was conducted, revealing the correlation between PheL production, and replication origins and selection markers. To demonstrate PheL production from biomass-derived resources, we also performed fermentation using mixed sugars of glucose and xylose with a metabolic pathway-compartmentalized Escherichia coli host. The gene cluster, including the Dahms pathway that converts xylose into acetyl-coenzyme A, was optimized, and the resulting transformant produced 3.5 g/L PheL with a glucose yield of 0.24 g/g.IMPORTANCEAs genetic and metabolic engineering has evolved, those technologies have enabled us to create synthetic microbes to produce chemicals of interest with high productivity. Although research on promoter regions and 5' UTRs in plasmid DNA for the effective expression of heterologous genes has been widely conducted, there are few reports about the effect of replication origins and antibiotic resistance markers on production. In this study, we focused on the combination of those two factors and the effect on culture profiles. Using D-phenyllactic acid as the model chemical, the production and selection of replication origins and antibiotic resistance were evaluated. Additionally, we applied the metabolic pathway compartmentalized technology to the transformant showing the highest production, and D-phenyllactic acid production from the mixed sugars with glucose and xylose was demonstrated. This study provides new insights of the heterologous gene expression in microbial biosynthesis.

Extracellular vesicles (EVs) from phytobacteria are emerging key ecological actors of plant-bacteria interactions. They can promote colonization of the host by delivering virulence factors and cell wall-degrading enzymes. Extracellular vesicles also modulate the plant immunity by transporting compounds that either induce or suppress plant defense responses. Furthermore, they mediate interspecies and inter-kingdom communication, influencing microbial community dynamics. This review highlights the involvement of bacterial EVs in many parts of the plant-bacteria dialog. We emphasize the need for studying EVs from phytobacteria to better understand plant-bacteria interactions and the possible use of bacterial EVs in a One Health context.

Toxin-antitoxin (TA) systems are widespread genetic modules in prokaryotes, implicated in diverse functions including stress adaptation, genome stability, and virulence. While extensively studied in pathogenic bacteria, their presence and roles in beneficial gut microbes like Bifidobacterium remain underexplored. This review consolidates current knowledge on type II TA systems within the Bifidobacterium genus, highlighting their diversity, genomic context, and potential functional roles. Genomic analyses reveal a predominance of MazEF and RelBE families, with other systems such as VapBC, YefM-YoeB, and PumAB also identified, albeit less frequently. Experimental validation is limited, with most studies focused on B. longum strains. Emerging evidence suggests that these systems may contribute to acid and osmotic stress responses and mobile genetic element maintenance. The species- and strain-specific distribution of TA loci suggests their potential utility as molecular markers for strain-level microbiome analysis. Given their multifaceted roles, further functional studies are warranted to elucidate the biological significance of TA systems in Bifidobacterium and their implications for gut health and probiotic efficacy.

The magnetotactic bacterium Magnetospirillum magneticum produces biogenic magnetic nanoparticles called magnetosomes, each comprising a magnetite core surrounded by a lipid bilayer. By expressing the human neurotrophin receptor TrkA, which is implicated in Alzheimer's disease, cancer, and other pathologies, on magnetosomes, we created a functional human transmembrane receptor platform that enables magnetic recovery and ligand screening. However, TrkA has limited activity within the native bacterial membrane, likely due to the absence of key eukaryotic lipids such as phosphatidylcholine (PC) and cholesterol (Chol). To overcome this limitation, we combined in vivo and in vitro membrane engineering to remodel the magnetosome envelope and closely mimic the human cell membrane. Specifically, PC was biosynthesized in vivo by co-expressing phosphatidylcholine synthase, and purified magnetosomes were subsequently enriched in vitro with Chol using methyl-β-cyclodextrin. This dual-lipid strategy significantly enhanced TrkA-ligand-binding and autophosphorylation. These findings show that orthogonal membrane engineering can precisely tailor the lipid composition of bacterial magnetosomes to recreate mammalian-like bilayers, rescuing the function of complex human receptors in a microbial environment. This approach expands the metabolic and synthetic biology toolkit for producing functional membrane proteins on scalable magnetic supports and establishes a cost-effective platform for the discovery of TrkA and other membrane receptors.IMPORTANCEMembrane receptors drive essential signaling in health and disease; however, they are difficult to study and screen because most platforms fail to reproduce human-like membranes at scale. Magnetosomes from the bacterium Magnetospirillum magneticum AMB-1 offer a simple alternative: lipid-bounded, magnetic nanoparticles that can be purified in one step. This work establishes human-like remodeling of magnetosome membranes by combining in vivo phosphatidylcholine synthesis with the first in vitro cholesterol loading of these particles. Displaying human tropomyosin receptor kinase A on the remodeled membranes preserved and enhanced the receptor function without complex purification or reconstitution. Because magnetosomes can be produced inexpensively and recovered magnetically, this approach enables practical, high-throughput assays for ligand discovery and inhibitor testing. The strategy is broadly applicable to other human membrane proteins, linking microbial biotechnology with human membrane biology to accelerate translational research.

Bacteriophages (phages), the dominant prokaryotic viruses that specifically target bacteria in the human gut microbiome, play a crucial role in maintaining intestinal balance, regulating bacterial populations, and preserving microbial diversity within the gut microbiota. While prophages can enhance bacterial virulence and antibiotic resistance, potentially posing health risks, they also provide beneficial functions, including enhancing host fitness, promoting immune modulation, and contributing to ecosystem resilience, which supports intestinal homeostasis. Human gut microbiota is essential for various physiological functions, including digestion, vitamin synthesis, immune modulation, and protection against pathogens. Dysbiosis, or microbial imbalance, is associated with various disorders such as inflammatory bowel disease, obesity, diabetes, and mental health disorders. Consequently, prophages are important considerations for developing therapies to prevent intestinal diseases. Recently, there has been significant interest in prophage induction in the gut due to its functional impacts on microbial dynamics, gut health, and disease modulation. Prophage induction can be regulated by diet, antibiotics, metabolites, gut health, lifestyle, and intestinal environments. However, compared with lytic phages, prophages remain underexplored, leaving gaps in our understanding of their functions within the gut. Therefore, further research is needed to fully elucidate the complex interactions between phages, prophages, and the gut microbiota, and their effects on health and disease. This knowledge could inform the development of phage-based therapies and improve therapeutic strategies for gut health.

Antibiotic 2,4-diacetylphloroglucinol (DAPG) is produced by many plant-associated beneficial bacteria of the Pseudomonas genus and plays an important role in plant disease control due to its broad antimicrobial activity against different pathogens. DAPG biosynthesis is activated by the conserved GacA/GacS two-component regulatory system. Here, we report that a ΔgacA mutant of Pseudomonas protegens Pf-5, lacking both DAPG production and pathogen inhibition in culture, controlled pea Aphanomyces root rot in a DAPG-dependent manner in the greenhouse. DAPG production of the ΔgacA mutant could be restored by exogenous phloroglucinol (PG), the first intermediate in DAPG biosynthesis, by culturing the ΔgacA mutant with either PG or PG-producing bacteria. We identified a new Gac-dependent promoter of phlD. Gene expression assays demonstrated that GacA is required to activate the phlD promoter. In vitro binding assays showed that the RNA-binding protein RsmE bound directly to the leader mRNA of phlA, another DAPG biosynthetic gene converting PG into DAPG, indicating that GacA regulates phlA expression post-transcriptionally. No detectable binding activity was observed between RsmE and the phlD leader mRNA. These results show that GacA regulates DAPG biosynthesis at multiple steps via different mechanisms and elucidate a novel layer of Gac-Rsm regulation in secondary metabolism. Targeted PG supplementation and/or partner microbe interactions may help to enhance the disease control efficacy and stability of the DAPG-producing bacteria.IMPORTANCEAntibiotic production is important for many beneficial bacteria to inhibit plant pathogens and control plant diseases. Understanding the molecular mechanism of how bacteria regulate antibiotic production can help improve the disease control effect. Previous studies have shown that the production of 2,4-diacetylphloroglucinol (DAPG) is activated by a global regulator GacA in strains of Pseudomonas spp. In this work, we found that two different regulatory mechanisms are used by GacA to regulate the DAPG production in Pseudomonas protegens Pf-5. Specifically, GacA regulates the expression of phlD and phlA, two DAPG core biosynthetic genes, at transcriptional and post-transcriptional levels, respectively. DAPG production of the Pf-5 mutant lacking GacA could be restored by amendment of phloroglucinol (PG), an intermediate of DAPG biosynthesis. The ΔgacA mutant protected pea plants in soil from root rot disease caused by an oomycete pathogen, Aphanomyces euteiches, that can be inhibited by DAPG. Results of this study advanced our understanding of the molecular mechanisms that regulate antibiotic production of plant beneficial bacteria and suggest that PG amendment may be used to improve the disease control stability and efficacy of DAPG-producing bacteria in the field.