[Objective] To study the relieving effects and potential mechanisms of Lactiplantibacillus plantarum DY6 and the soybean meal fermented with this strain on constipation in mice and to develop non-toxic feed additives with probiotic potential. [Methods] Constipation was induced in male BALB/c mice by gavege of loperamide hydrochloride. The modeled mice underwent long-term feeding with the soybean meal fermented with DY6 or gavage with DY6 suspension. Linaclotide served as the positive control. We then systematically assessed the effects of fermented soybean meal on intestinal functions, immune factors, metabolic profiles, and gut microbiota in constipated mice. [Results] Compared with the model group, long-term intake of the soybean meal fermented with DY6 increased fecal moisture content and intestinal motility (P<0.05), ameliorated goblet cell reduction and lymphocyte aggregation in the colon tissue, lowered the serum levels of tumor necrosis factor-α, interleukin-6, and vasoactive intestinal peptide (P<0.05), and elevated the fecal levels of acetic acid and butyric acid (P<0.01). The fermented soybean meal modulated the gut microbiota structure. Specifically, the significantly enriched probiotics Odoribacter and Blautia were positively correlated with indicators of relieving constipation, while the significantly reduced pathogenic bacteria Helicobacter and Colidextribacter were negatively correlated with indicators of relieving constipation. [Conclusion] The soybean meal fermented with DY6 alleviates constipation by modulating the gut microbiota structure and metabolic profiles, restoring intestinal electrolyte homeostasis, and suppressing inflammation-associated signaling pathways. This intervention establishes novel applications and a theoretical framework for developing functional biological feed additives capable of both preventing and managing constipation in animals.

[Objective] To develop an efficient catalyst for organophosphorus pesticide degradation by immobilizing organophosphorus hydrolase (OPH) on the surface of Pichia pastorisvia the SpyCatcher/SpyTag (SpyC/SpyT) system, addressing the poor stability and low reusability of OPH in practical applications and providing a new method for the bioremediation of organophosphorus pesticide pollution. [Methods] The “bait protein” SpyCatcher (SpyC) was first displayed on the surface of P. pastoris, and the display efficiency was increased by increasing the copy number and optimizing the culture conditions. Then based on the specific interaction between SpyC and SpyT, OPH-SpyTag (OPH-SpyT) was efficiently displayed on the yeast surface. The thermal stability, pH stability, and reusability of immobilized OPH were evaluated, and the hydrolysis efficiency of immobilized OPH against methyl parathion, dimethoate, and chlorpyrifos was assessed. [Results] The display efficiency of SpyC on the P. pastoris surface reached over (97.0±0.4)%, with an optimized binding capacity of (21.4±0.7) mg green fluorescent protein for 1 g wet cells. OPH was successfully displayed on the cell surface via the SpyC/SpyT system. The immobilized OPH exhibited significantly enhanced thermal and pH stability, retaining more than 50% activity after five repeated uses. Under optimum conditions, the immobilized OPH showed the hydrolysis rates of (96.5±2.7)%, (79.5±2.3)%, and (82.6±2.8)% against 100 mg/L methyl parathion, dimethoate, and chlorpyrifos, respectively. This indicated that the method showed high hydrolysis efficiency for the organophosphorus pesticides. [Conclusion] The immobilization of OPH on P. pastoris surface via the SpyC/SpyT system effectively improves its stability and reusability, offering an efficient and environmentally friendly solution for the bioremediation of organophosphorus pesticide pollution. Meanwhile, this study provides a powerful tool and method for research in the field of P. pastoris surface display.

[Objective] To investigate the role of dppC2 in the survival of Yersinia pestis in macrophages. [Methods] The strain (201-ΔdppC2) with traceless knockout of dppC2 was constructed with a suicide plasmid via homologous recombination based on Y. pestis biovar Microtus strain 201. Phenotypes were compared between 201-ΔdppC2 and the wild type (201-WT) by the acid survival assay, hydrogen peroxide survival assay, macrophage intracellular survival assay, reactive oxygen species (ROS) detection, and cytotoxicity and mouse challenge assays. The gene expression was compared between 201-ΔdppC2 and 201-WT by transcriptomics analysis and RT-qPCR. [Results] Compared with 201-WT, 201-ΔdppC2 exhibited multiple phenotypic alterations, including significantly increases in intracellular survival rates in RAW264.7 and THP-1 cells and under acidic and hydrogen peroxide conditions, upregulation of the acid resistance gene hdeD and the catalase-related genes katA and katG, enhancement of catalase and peroxidase activities, and declines in intracellular ROS levels in 201-ΔdppC2 and RAW264.7 cells infected with the mutant. Furthermore, 201-ΔdppC2 showed reduced cytotoxicity to HeLa cells but no change in the virulence in mice. [Conclusion] The deletion of dppC2 has been demonstrated to enhance the fitness of Y. pestis to acidic and hydrogen peroxide environments, which promote the survival and replication of Y. pestis in macrophages.

Non-alcoholic fatty liver disease (NAFLD) is a common chronic liver disease around the world, and it has a complex disease progression. Recent studies have demonstrated that gut microbiota (GM) plays a crucial role in the pathogenesis of NAFLD. The theory of gut-liver axis provides a theoretical basis for understanding the relationship between GM and the liver. Dysbiosis of GM leads to immune dysfunction, inflammatory responses, damaged gut barrier, and insulin resistance, all of which promote the development and progression of NAFLD. Furthermore, GM can participate in the development and progression of NAFLD through endotoxemia and abnormal metabolism of short chain fatty acids, bile acids, and choline. How to mitigate NAFLD by modifying GM has become the focus of current research, and the measures include fecal microbiota transplantation, probiotics, prebiotics, Chinese herbal medicines, and lifestyle interventions. The review focuses on the impact of the pathological state of GM on NAFLD and discusses the research progress in GM-targeted therapy for NAFLD. It is expected to provide new strategies and targets for the prevention and treatment of NAFLD.

[Objective] To achieve efficient expression of the alkaline laccase PIE5 in Coprinopsis cinerea with molasses as a substitute for glucose as the carbon source. [Methods] We enhanced the laccase production in C. cinerea by either exogenous addition of the invertase GspInv or endogenous co-expression of GspInv to hydrolyze sucrose in molasses. The fermentation conditions were optimized based on the laccase activity. [Results] With 40 g/L molasses as the carbon source, strain CcPIE5-14 achieved the laccase activity of (11.9±1.2) U/mL, while sucrose remained unutilized in the fermentation liquid. Upon addition of exogenous GspInv, sucrose in the fermentation liquid was hydrolyzed into fructose and glucose, and strain CcPIE5-14 exhibited the peak laccase activity of (14.8±0.7) U/mL in the medium with 30 g/L molasses. Co-expression of GspInv in CcPIE5-14 generated the engineered strain CcPIE5-14-GspInv-12, which demonstrated the maximum laccase activity of (28.1±2.4) U/mL in the mKjalke medium. When using molasses as the carbon source for laccase production, strain CcPIE5-14-GspInv-12 achieved the peak laccase activity of (20.1±2.7) U/mL, which represented a 2.24-fold increase over that of the parental strain CcPIE5-14. Following fermentation condition optimization, strain CcPIE5-14-GspInv-12 attained the maximum laccase activity of (44.6±2.6) U/mL, which marked a 2.22-fold enhancement over the pre-optimization level. [Conclusion] The engineered strain CcPIE5-14-GspInv-12, co-expressing laccase and invertase, demonstrates efficient production of the alkaline laccase PIE5 in C. cinerea with cost-effective molasses as the carbon source.

Cotton Verticillium wilt, caused by the soil-borne fungal pathogen Verticillium dahliae, is a devastating disease that severely impacts global cotton production. [Objective] To investigate the biocontrol potential and mechanism of endophytic Pseudomonas sp. NWSUAF303 against cotton Verticillium wilt and provide novel microbial resources for managing soil-borne diseases in cotton. [Methods] The strain was identified by phylogenetic analysis based on 16S rRNA gene sequences and phenotypic characterization. Its antifungal spectrum was evaluated via dual-culture and volatile organic compounds (VOCs) inhibition assays. VOCs were detected by headspace solid-phase microextraction coupled with gas chromatography-mass spectrometry (HS-SPME-GC-MS). Pot experiments were carried out to assess the disease control efficacy of the strain. RT-qPCR and enzymatic activity assays were employed to elucidate the resistance mechanism of the strain against cotton Verticillium wilt. [Results] Strain NWSUAF303 was identified as Pseudomonas alvandae, exhibiting plant growth-promoting properties including nitrogen fixation, phosphate solubilization, and indole-3-acetic acid (IAA) production. Its non-volatile metabolites inhibited six phytopathogenic fungi, whereas VOCs demonstrated broader antifungal spectrum against seven pathogens, showing the inhibition rates >95% against Sclerotinia sclerotiorum and of 89.27% against V. dahliae 592. The VOCs of this strain downregulated the expression of virulence genes (VdPR1, Vdpf, and VdGAL4) in V. dahliae (P<0.05). Three key antifungal VOCs were identified, including 2,3-butanedione, 2-nonanol, and 6-methyl-2-heptanol, with the inhibitory effect of 2,3-butanedione on V. dahliae being first reported. Pot experiments revealed the control efficacy of 54.40% against Verticillium wilt, which was comparable to that of carbendazim. Strain NWSUAF303 activated the salicylic acid/jasmonic acid (SA/JA) signaling pathway, upregulating the expression of defense-related genes GhPAL, Gh4CL, and GhCHI (P<0.01), while enhancing the activities of peroxidase (POD), polyphenol oxidase (PPO), and superoxide dismutase (SOD). [Conclusion] P. alvandae NWSUAF303 combats Verticillium wilt through dual mechanisms: producing novel antifungal VOCs and activating systemic resistance via SA/JA signaling and defense enzyme coordination. With broad-spectrum antifungal activity and plant growth-promoting properties, this strain represents a promising biocontrol agent for sustainable management of cotton Verticillium wilt.

UvrY is a key response regulator of the BarA/UvrY two-component system (TCS) and plays an important role in regulating bacterial virulence and environmental adaptability. [Objective] To investigate the regulatory role of UvrY in the biological characteristics and pathogenicity of Vibrio parahaemolyticus SH112. [Methods] The uvrY-deleted mutant (ΔuvrY) and its complementary strain (CΔuvrY) were constructed by homologous recombination. Phenotypes were systematically compared among the wild type, mutant, and complementary strains by growth curve plotting, motility (swimming and swarming) assays, biofilm formation assay, bacterial competition assay, HeLa cell adhesion and cytotoxicity assays, as well as a mouse infection model (analysis of bacterial loads in tissues and lethality). [Results] Compared with the wild type strain, ΔuvrY exhibited significant growth defects during the late exponential phase and weakened motility, with swimming and swarming reduced by 33% and 70%, respectively, while the biofilm formation of the mutant remained unaffected. Additionally, ΔuvrY showed weakened competitive inhibition against Escherichia coli, a 36.7% reduction in HeLa cell adhesion, and a 15.8% decrease in cytotoxicity. Mouse infection experiments further demonstrated that ΔuvrY had significantly reduced tissue colonization capacity and the attenuation of 75% in pathogenicity. [Conclusion] This study reveals that UvrY plays a crucial role in the pathogenicity of V. parahaemolyticus by regulating the growth, motility, competitive ability, and interaction with the host, giving insights into the regulatory network of the BarA/UvrY two-component system.

Cyanobacteria, commonly known as blue-green algae, are important primary producers in aquatic ecosystems and common dominant algae causing algal blooms in freshwater. Cyanophages, especially procyanophages, are important planktonic ecological factors that affect the evolution of blue-green algae and aquatic microbial communities. Yet studies on procyanophages in cyanobacteria remain scarce. So far, few studies have reported the procyanophages in Microcystis. [Objective] To investigate the prevalence of lysogeny in Microcystis and characterize the genomic features of procyanophages in Microcystis. [Methods] All the 354 genome sequences of Microcystis spp. in GenBank were downloaded. PHASTER was used to predict procyanophage regions in the Microcystis genomes. Resistance and virulence factors in intact procyanophages and questionable procyanophages were annotated via the virulence factors of bacterial pathogens (VFDB) and comprehensive antibiotic resistance database (CARD). Bioinformatics tools were used for gene annotation and phylogenetic analysis of the procyanophages in Microcystis. Microcystis flos-aquae FACHB-1344 and M. aeruginosa FACHB-1326 each was predicted to harbor an intact procyanophage by PHASTER. To evaluate the infection activities of these two procyanophages, mitomycin C induction assays and dot-ELISA were conducted. [Results] Among all the 354 Microcystis genomes, 98.3% were predicted to harbor intact procyanophages, questionable or imcomplete procyanophages. A total of 13 intact procyanophages, 5 questionable procyanophages and 725 incomplete procyanophages were predicted by PHASTER. The 13 intact procyanophages and 5 questionable procyanophages were named as WZ1-WZ13 and YS1-YS5, respectively. No antibiotic resistance or virulence gene was detected in them. The phylogenomic tree displayed distant evolutionary relationships between the 18 procyanophages and other known viruses. Bioinformatics analysis suggested that YS5 revealed a previously unknown novel genus. WZ2, WZ3, WZ4, WZ5, WZ6, WZ7, WZ9, WZ10, WZ11, WZ12 and YS3 together revealed a novel family. WZ1 and YS1 together revealed a novel family. YS2, YS4, WZ8 and WZ13 each revealed a novel family. The procyanophages in M. flos-aquae FACHB-1344 and FACHB-1326 were verified to be activated by mitomycin C. [Conclusion] Lysogeny widely exists in Microcystis spp. The novel procyanophages in Microcystis spp. unlock novel viral evolutionary lineages previously unknown. This study enriches the understanding about cyanobacterium-virus interactions and the diversity of aquatic viruses.

Obligate symbiotic bacteria in the human gut play a key role in maintaining microecological balance and are generally thought to be transmitted vertically across generations through breastfeeding. However, compared with that on Bifidobacterium, there is limited literature on the transmission and diffusion mechanisms of Bacteroides, a representative of obligate symbiotic microbiota in the human body, within populations. Additionally, the occurrence and cross-generational transmission of Bacteroides within families remain poorly understood. [Objective] We explored the vertical transmission and co-occurrence patterns of Bacteroides among family members, aiming to reveal the assembly mechanism of gut microbiota in the human body and provide a theoretical basis for the recommendation of microbiome-based interventions and the realization of personalized gut regulation. [Methods] A high-throughput sequencing dataset for Bacteroides-specific rpsD in fecal samples from four families with 50 members in 3-4 generations in Xinjiang, China was established. Then, the composition and diversity of Bacteroides communities among different families and member groups were measured at the species level and amplicon sequence variant (ASV) level by comparison and annotation. [Results] A total of 16 Bacteroides species and 3 704 ASVs were identified, of which 1 293 ASVs were common among the four families. The five species with the highest number of ASVs were B. fragilis (653), B. ovatus (619), B. uniformis (507), B. caccae (463), and B. finegoldii (314), which were also the five species with the highest relative abundance and prevalence. There were significant differences in community composition and abundance of Bacteroides among families, with B. fragilis, B. uniformis, and B. faecichinchillae being the most significant representatives. There was no significant difference in alpha or beta diversity among family members grouped according to gender and age (P>0.05). By contrast, the beta diversity analysis based on Bray-Curtis distance showed differences between families (P=0.001). According to the sharing rate of ASVs and Bray-Curtis distance, the Bacteroides strain similarity of mother-child and siblings was significantly higher than that of father-child, couple, and unrelated members. [Conclusion] The community structure and diversity of Bacteroides were characterized by family convergence, with significant differences among families. The similarity of Bacteroides strains was the highest within the mother-child and sibling groups among different social relationship groups, supporting transgenerational vertical transmission at the strain level. The conclusion remains to be validated by the combination of the strain isolation method and metagenomic sequencing.

[Objective] To investigate whether Fusobacterium nucleatum promotes the development of lung cancer and find out the underlying mechanisms. [Methods] The cell experiment proved that F. nucleatum can promote the proliferation and metastasis of lung cancer cells; Animal experiments showed that F. nucleatum can promote the development of lung cancer; 16S rRNA gene sequencing of alveolar lavage fluid and transcriptome sequencing of lung tissue were performed to analyze the effects of F. nucleatum on the lung flora of mice with lung cancer and the activation of relevant pathways in lung tissue. [Results] The cell experiment showed that F. nucleatum significantly promoted the proliferation and metastasis of A549 cells. The animal experiments confirmed that F. nucleatum infection in the lungs increased the number of nodules and enhanced the fluorescence intensity in the lung tissue. The 16S rRNA gene sequencing revealed the enrichment of Muribaculum and Simplicispira in the lung of infected mice. The transcriptome sequencing results showed that the mouse model of lung cancer activated the interleukin-17 signaling pathway, cytokine-cytokine receptor interactions, Staphylococcus aureus infection pathway, tumor necrosis factor signaling pathway, and other signaling pathways related to inflammation or immunity after being infected with F. nucleatum. [Conclusion] F. nucleatum infection in mice with lung cancer disrupts the microbiota and activates the immunity- and inflammation-related signaling pathways in the lung, thereby promoting the metastasis of lung cancer in mice.