[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.

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.

Streptomyces is a genus of diverse actinomycetes known for producing antibiotics, showcasing high value for research and development. However, the limited number of Streptomyces species included in commercial databases restricts the application of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) in the identification of Streptomyces. [Objective] To establish a MALDI-TOF MS database for the identification of Streptomyces based on the mining of whole genome sequencing data and test its accuracy. [Methods] By searching against a genome database, we selected all the genome-sequenced type strains of Streptomyces. Subsequently, the sequences of 12 ribosomal proteins were extracted from their genome sequences and theoretical MALDI mass/charge (m/z) values were calculated. Accordingly, a database was established based on the combination of 12 theoretically calculated m/z values, which can be used as the identification criteria for Streptomyces species. Finally, type strains were randomly collected for MALDI-TOF MS testing. To test the accuracy of the database construction method and comparison results, we matched the experimental m/z values with the theoretically calculated m/z values of the species in the database. [Results] We established a MALDI-TOF MS database including 615 Streptomyces species. The mass spectra of the tested strains were accurately matched with those of corresponding species in the database. In addition, we proposed a method for the identification of Streptomyces by database comparison. [Conclusion] The database established in this study laid a foundation for identifying Streptomyces based on MALDI-TOF MS.

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.

[Objective] To establish a triple qPCR detection method for Mycoplasma bovis, Pasteurella multocida (P.m) and Mannheimia haemolytica (M.h), and to conduct an epidemiological investigation of bovine respiratory disease complex (BRDC) in large-scale dairy farms with it. [Methods] Sensitive quality control samples were prepared using the purified strains isolated and identified in our laboratory. The conserved genes of the major prevalent strains in China were used as targets, including the uvrC of M. bovis, Kmt1 of P.m, and lktD of M.h, to establish a multiplex qPCR containing multiple pairs of primers and probes. By optimizing the parameters of the reaction system, a triple qPCR method was established and its sensitivity, repeatability and specificity were verified. The coincidence rate with the bacterial isolation and identification method was verified. A total of 1 252 clinical samples suspected of BRDC were tested using the established method, and the prevalence and spatiotemporal distribution characteristics of three pathogens were analyzed based on the test results. [Results] The R² values of the standard curves for the triple qPCR for detecting M. bovis, P.m, and M.h were 0.998 6, 0.994 6 and 0.998 6 respectively; the minimum detectable quantities were 2.50×10³, 1.26×10³ and 7.50×10² CFU/mL, respectively. The intra-batch and inter-batch coefficients of variation of the reaction system were both less than 2%, and only the specific amplification curves were observed for the three sensitive quality control samples established. The results of the test showed that the concordance rates of M. bovis, P.m, and M.h with bacterial isolation and identification methods were 100.00%, 98.40% and 97.60%, respectively. The epidemiological survey indicated that the total positive rate of bacterial pathogens in BRDC was 75.9% (95% confidence interval (CI), 73.4%-78.3%), among which the proportion of mixed infections was 48.8%. The incidence was higher in winter, and the positive rate in the northern region was significantly higher than that in the southern region (P<0.001). [Conclusion] The multiple qPCR method established in this study demonstrated excellent specificity, stability and repeatability, which provided a candidate solution for the detection of major pathogens of BRDC in China. The analysis of the co-infection characteristics and geographical-seasonal distribution patterns of BRDC bacterial pathogens offered meaningful references for the formulation of precise prevention and control strategies.

[Objective] To explore the protective effect of selenomethionine (Se-Met) on oxidative stress and intestinal barrier damage in mice infected with porcine deltacoronavirus (PDCoV) and the potential regulatory mechanism. [Methods] Forty female C57 mice were randomly grouped as follows: control, Se-Met (0.3 mg/kg Se), PDCoV, and Se-Met+PDCoV (0.3 mg/kg Se). After being fed with or without Se-Met for 23 days, the mice in the PDCoV group and the Se-Met+PDCoV group were administrated with 300 μL suspension of PDCoV HNZK-02-P5 strain (1×10⁶ TCID₅₀) by gavage, while those in the other two groups were administered with the same volume of Dulbecco’s Modified Eagle Medium (DMEM). All the mice were observed daily for clinical signs, food intake, and body weight changes until day 28. At five days post-inoculation (dpi), intestinal tissues were collected and PDCoV titers were determined. Hematoxylin staining and eosin staining were used to monitor pathological changes in intestinal tissues. Oxidative stress-related indicators such as malondialdehyde (MDA), superoxide dismutase (SOD), and glutathione peroxidase (GSH-PX) were investigated. The level of ROS in the jejunum tissue was measured via a 2′,7′-dichlorofluorescein diacetate (DCFH-DA) probe. Immunofluorescence was used to analyze the changes of small intestinal tight junction proteins (ZO-1 and Occludin). The mRNA levels of inflammatory cytokines (TNF-α, IL-1β, IL-6, and IL-10), intestinal tight junction proteins (ZO-1 and Occludin), and the Nrf2 signaling pathway-associated factors (Nrf2, HO-1, and NQO1) were determined by RT-qPCR. Western blotting was employed to assess the protein levels of factors related to the Nrf2 signaling pathway. [Results] The results of body weight, food intake, pathological examination, and viral RNA titers in different intestinal tissues revealed that Se-Met might increase the body weight, decrease viral titers in intestinal tissues, and attenuate PDCoV-induced structural damage of intestinal villi in PDCoV-infected mice. Se-Met attenuated PDCoV-induced inflammation by lowering the mRNA levels of major inflammatory cytokines, such as IL-1β, IL-6, and TNFα in the jejunum. Se-Met ameliorated PDCoV-induced intestinal mucosal barrier damage by up-regulating the mRNA levels of ZO-1 and Occludin in the jejunum. Se-Met ameliorated PDCoV-induced oxidative stress by decreasing the levels of ROS and MDA and increasing the levels of GSH-PX and SOD in the jejunum. Se-Met inhibited PDCoV-induced oxidative stress by activating the Nrf2 signaling pathway. [Conclusion] Se-Met may attenuate the intestinal injury in mice infected with PDCoV by activating the Nrf2 signaling pathway, which provides a theoretical basis for the prevention and treatment of PDCoV infection.

[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.

6′-sialyllactose (6′-SL) is an important component of porcine breast milk oligosaccharides, and sialic acid (SIA) is the monomer of sialylated breast milk oligosaccharides. Both can regulate the structure of human colonic microbiota. However, there are few reports regarding the effects of SIA and 6′-SL on the composition and fermentation characteristics of piglet colonic microbiota in vitro. [Objective] To investigate the effects of SIA and 6′-SL on the composition and fermentation characteristics of colonic microbiota in suckling piglets, aiming to provide a reference for using these substances to regulate gut health in piglets. [Methods] We used mixed colonic chyme from three suckling piglets as the microbial inoculum. The experiment was conducted with four groups: no carbon (NCB), lactose (LAC), SIA, and 6′-SL, each with four replicates. [Results] After 24 h of fermentation, the pH in the 6′-SL group was higher than that in the LAC group (P<0.05). After fermentation for 12 h and 24 h, the gas production in the SIA and 6′-SL groups was lower than that in the LAC group (P<0.05). At the time point of 24 h, the SIA and 6′-SL groups had higher acetic acid content and lower propionic acid and butyric acid content than the LAC group (P<0.05). Moreover, the SIA and 6′-SL groups had higher Chao1 and ACE indices than the LAC group (P<0.05). Beta diversity of the microbial community in the LAC, SIA, and 6′-SL groups changed (P<0.05). As fermentation progressed, compared with LAC, SIA increased the relative abundance of Bacillota, Anaerovibrio, and Faecalibacterium and decreased that of Pseudomonadota, Escherichia-Shigella, Mitsuokella, and Streptococcus (P<0.05); 6′-SL increased the relative abundance of Bacteroidetes, Prevotella, Bacteroides, and Roseburia and decreased that of Pseudomonadota, Bacillota, Megamonas, Escherichia-Shigella, Mitsuokella, and Prevotellaceae_NK3B31_group (P<0.05). [Conclusion] Both 6′-SL and SIA can effectively regulate the gut microbiota structure in piglets. Specifically, 6′-SL increases the relative abundance of Bacteroidetes, Prevotella, and Bacteroides, while SIA increases that of Bacillota and Anaerovibrio. Both substances promote colonic microbial fermentation in piglets to increase the production of short-chain fatty acids (SCFAs), especially acetic acid.

[Objective] To investigate the role of Listeria monocytogenes LPXTG motif-anchored protein Lmo0130 in infection causing diseases, the bacterial growth, infection in cell and host among the L. monocytogenes wild-type, lmo0130-deleted and lmo0130-complementary strains were compared. [Methods] The lmo0130-deleted strain Δlmo0130 and lmo0130-complementary strain CΔlmo0130 were constructed to investigate the effects of Lmo0130 on the abilities of bacterial growth, cell surface adhesion and invasion, intracellular proliferation, intercellular migration, survival of infected mice, and bacterial load in mouse organs, ultimately demonstrated the role of L. monocytogenes Lmo0130 in cell and host infection. [Results] LPXTG motif-anchored protein Lmo0130 contributed to cell surface adhesion and invasion, intracellular proliferation, specific colonization in the liver and spleen, and pathogenicity in mice. However, it had no effect on bacterial growth or intercellular migration. [Conclusion] Lmo0130 contributes to cell and host infection of L. monocytogenes finally.

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.