[Objective] We employed the wild-type strain of Listeria monocytogenes, the lipoprotein gene pplA- deleted strain, and the complementary strain to investigate the role of PplA in the infection of L. monocytogenes. [Methods] We compared the hemolytic capacity, cell adhesion and invasion, intracellular proliferation, cell-to-cell migration, mouse organ colonization, transcription levels of virulence factors in mouse organs, and transcription levels of quorum sensing-related genes among wild-type, pplA-deleted, and complementary strains to explore the role of PplA in the infection of L. monocytogenes. [Results] After the deletion of pplA, L. monocytogenes showed no significant change in intracellular proliferation or cell-to-cell migration. However, its hemolytic capacity, cell adhesion and invasion, mouse organ colonization, and transcription levels of virulence factors such as plcB, hly, and prfA in mouse organs were significantly reduced. Moreover, the transcription levels of quorum sensing-related genes agrA, agrB, agrC, and luxS were altered in the pplA-deleted strain. [Conclusion] Thelipoprotein PplA is involved in the virulence regulation and affects the pathogenicity of L. monocytogenes.

[Objective] Seed endophytes are key components of the plant microbiome, and their community structure and diversity are highly susceptible to environmental changes. Revealing the effects of fencing versus grazing management on the endophytic microbial communities of Caragana korshinskii seeds is essential for elucidating the dynamic interactions among plants, soil, and microorganisms. This study also provides a theoretical foundation for ecological restoration in arid and semi-arid grasslands. [Methods] The study was conducted in Otog Front Banner, Inner Mongolia, focusing on C. korshinskii under two management practices: fencing and grazing. The physicochemical properties of soil were analyzed by high-throughput sequencing of seed endophytic microbial communities to assess shifts in their structural composition and diversity under these contrasting regimes. [Results] Fencing management decreased soil pH while increasing moisture content, total carbon, total nitrogen, and total phosphorus of soil. Grazing management increased the abundance-based coverage estimator (ACE) index of seed endophytic fungi. Fencing increased the relative abundance of Pseudomonadota and decreased the relative abundance of Bacillota in seed endophytic bacteria, while promoting the relative abundance of Pantoea. In seed endophytic fungi community, Alternaria dominated under fencing management, while Trichoderma dominated under grazing management. Additionally, grazing management increased the number of modules and complexity in seed endophytic bacterial-fungal networks. [Conclusion] The contrasting management practices of fencing and grazing in desert stepps alters soil environment, thereby shaping the diversity and community structure of both bacterial and fungal endophytes in C. korshinskii seeds. This study reveals the impact of different management measures on plant-microbe interactions in desert steppes, providing theoretical support for related research.

[Objective] Transcription is the first step in gene expression, and the mRNA abundance to a certain extent determines the final protein expression abundance. Recent studies have found that different ribosome-binding sites (RBSs) located in the 5′ untranslated region (5′-UTR) can affect the mRNA abundance of the downstream gene. From the perspective of regulatory factors in the mRNA degradation process, the effect may be attributed to the binding strength between the Shine-Dalgarno (SD) sequence and the ribosome and the local secondary structure of the 5′-UTR. [Methods] We constructed a 5′-UTR mutant library with a size of 528. High-throughput sequencing was employed to efficiently collect the information on the mRNA abundance of downstream egfp corresponding to various 5′-UTR variants. The effectiveness was verified by RT-qPCR. [Results] The association between abundance of each mRNA mutant and its corresponding 5′-UTR sequence was analyzed. The results showed that the SD sequence with moderate to strong binding strength to the ribosome was most conducive to maintaining high mRNA abundance. Too high or low binding strength will lead to a reduction in the mRNA abundance. The completely conserved core SD sequence (GGAGG) was the key to ensuring high binding strength, and the decline in conservation would cause a significant decrease in the mRNA abundance. When the SD sequence was similar among different 5′-UTR variants, i.e.,the binding strength of the SD sequence to the ribosome was comparable, the local secondary structure of the 5′-UTR was instable and the abundance of corresponding mRNA was high. [Conclusion] This study delves into the regulatory effects of 5′-UTR sequence features 5′-UTR on the mRNA abundance and establishes a qualitative model of their interrelationships, providing a reference for the rational design of regulatory elements in metabolic engineering and gene circuits.

Photobacterium damselae subsp. damselae (PDD), a pathogenic bacterium widely found in seawater, can infect a variety of economic fish and cause huge economic losses to the global aquaculture industry. The flagellar gene flgK encodes the flagellar hook protein FlgK, which is essential for the normal formation of bacterial flagella. [Objective] To systematically analyze the influencing mechanism of flgK on the virulence of PDD. [Methods] The flgK-deleted mutant of PDD (ΔflgK-PDD) was constructed by homologous recombination mediated by a high-efficiency suicide plasmid, and the mutation was confirmed by gene sequencing. The biological characteristics, virulence gene expression, and pathogenicity were compared between ΔflgK-PDD and the wild-type strain (WT-PDD). [Results] There was no significant difference in the growth ability, hemolytic activity or phospholipase activity between ΔflgK-PDD and WT-PDD. However, the motility and biofilm formation of ΔflgK-PDD were significantly lower than those of WT-PDD. Transmission electron microscopy showed that ΔflgK-PDD failed to form a flagellar structure. The artificial infection experiments showed that the LD₅₀ of ΔflgK-PDD in Sebastes schlegelii was 557% that of WT-PDD, and the pathogenicity was significantly reduced. Real-time quantitative PCR results showed that compared with WT-PDD, ΔflgK-PDD demonstrated significantly down-regulated expression of the flagellar-related genes fliK and flgL, the type II secretion system (T2SS)-related genes gspC and gspD, and the virulence gene hlyA_pl. The expression levels of flagellar-related gene fliH, T2SS-related gene gspE, outer membrane-related genes ompP, lapB, and flhB were significantly up-regulated, and those of the remaining genes did not change significantly. [Conclusion] The mutation of flgK can lead to the failure of ΔflgK-PDD to form a complete flagellar structure and significantly change the relative expression levels of flagellar-related genes, thereby reducing the motility and colonization ability and ultimately weakening the pathogenicity of PDD.

[Objective] To clarify phyllosphere microbial responses to the invasion of areca palm velarivirus 1 (APV1), a virus causing yellow leaf disease of areca (Areca catechu), and provide a theoretical basis and technical support for the study of phyllosphere micro-ecology, exploration of excellent biocontrol resources, and green prevention and control of yellow leaf disease of areca. [Methods] We collected healthy leaves, mildly diseased leaves, and severely diseased leaves of areca. The phyllosphere microbial community structure and diversity were compared by high-throughput sequencing and bioinformatics methods. Furthermore, functional differences of phyllosphere microbial communities were analyzed. [Results] The dominant bacterial phyla in the phyllosphere of areca included Actinobacteriota, Proteobacteria, Acidobacteriota, Firmicutes, and Myxococcota, while the dominant fungal phyla were Ascomycota and Basidiomycota. As the disease became increasingly severe, bacterial richness initially increased then decreased while fungal richness initially decreased then increased. However, both bacterial diversity and fungal diversity showed a trend of first increasing and then decreasing. Firmicutes and Basidiomycota served as indicators of mildly diseased areca, with the relative abundance showing consistent trends with alpha diversity. The healthy plants and the diseased plants showed different phyllosphere microbial functions. Specifically, the environmental information processing function was significantly higher in severely diseased areca plants than in healthy ones. Additionally, the relative abundance of symbiotroph fungi in the phyllosphere were significantly higher in severely diseased areca plants than in healthy ones. [Conclusion] The yellow leaf disease significantly alters the phyllosphere microbial community structure and diversity of areca, with greater changes during the early disease stage. This suggests that areca may defend against APV1 infection by recruiting beneficial microorganisms, regulating cellular metabolism and biochemical reactions, and activating autoimmunity.

Microbial deodorization is an effective technology for treating odorous waste gases, in which microbial strains play a decisive role. [Objective] To screen the strains capable of degrading dimethyl disulfide (DMDS) and investigate their degradation efficiency and ability to produce surfactants under various conditions. [Methods] DMDS, a typical sulfur-containing odorous organic compound, was selected as the sole carbon source. A strain R1 capable of simultaneously producing biosurfactants and degrading DMDS was isolated from mangrove sludge. The strain was identified based on the physiological and biochemical characteristics analysis and 16S rRNA gene sequencing. The types of self-produced biosurfactants were determined using infrared spectroscopy and nuclear magnetic resonance spectroscopy analysis. [Results] Based on physiological and biochemical characteristics and 16S rRNA gene sequence, strain R1 was identified as Achromobacter sp. The strain was capable of degrading DMDS, with optimal degradation conditions of an initial DMDS concentration of 12.49 mg/L, a system temperature of 30 ℃, and an inoculum amount of 1.0 g/L, under which the DMDS degradation rate reached 70.74%. Emulsification experiments showed that strain R1 can use DMDS as a carbon source to produce biosurfactants, which were identified as glycolipids through nuclear magnetic resonance and infrared spectroscopy. [Conclusion] The main intermediate product in the biodegradation of DMDS is methyl mercaptan, and the transformation rate of sulfur to SO₄^2- is 65.99%. Strain R1 exhibits impressive performance in degrading DMDS and producing biosurfactants.

[Objective] To study the effects of the glycosyltransferase WekO involved in O-antigen synthesis on the biological characteristics of avian pathogenic Escherichia coli (APEC). [Methods] The mutant strain ΔwekO of APEC O₁ was constructed by Red homologous recombination, and the complementary strain CΔwekO was then constructed. The lipopolysaccharide (LPS) profile of each strain was identified by silver staining. Simultaneously, the growth rate and swimming motility were measured. The reactivity of each strain with rabbit anti-O₁ serum was determined by Western blotting. The ability of ΔwekO to form biofilms was measured by the crystal violet staining method. DF-1 cells were used to evaluate the adhesion and invasion of ΔwekOin vitro. Subsequently, chicks were selected as an animal model to evaluate the pathogenicity of ΔwekO. [Results] The mutant strain ΔwekO and the complementary strain CΔwekO were constructed. The LPS profile of ΔwekO was incomplete compared with that of the wild-type strain. The mutant lacked O-antigen bands and showed no reactivity to anti-O₁ serum. There was no significant difference in growth rate between different strains (P>0.05). However, the motility and biofilm formation capabilities of ΔwekO decreased (P<0.001). Additionally, ΔwekO demonstrated weakened adhesion to DF-1 cells (P<0.001) and demonstrated weakened invasion to DF-1 cells (P<0.01). Also, ΔwekO reduced pathogenicity to 7-day-old chicks (P<0.05). [Conclusion] The deletion of wekO results in impaired O-antigen synthesis, incomplete LPS profile, loss of flagellar and biofilm formation capabilities, and reduced pathogenicity of APEC. These findings are highly significant for improving the understanding of the role of glycosyltransferases involved in APEC O-antigen synthesis.

Pseudomonas plecoglossicida is the pathogen of visceral white spot disease in large yellow croaker (Larimichthys crocea). At present, the pathogenic mechanism of P. plecoglossicida has been partially understood, and some effective vaccines have been screened, whereas no commercial vaccine has been developed. [Objective] To deepen the understanding about the pathogenic mechanism and develop efficient attenuated live vaccines of P. plecoglossicida. [Methods] We targeted luxR and the RNA polymerase σ factor gene rpoE associated with quorum sensing to construct deletion mutants of P. plecoglossicida NB2011 by double homologous recombination. In addition, the strain ΔT6SS1ΔluxR with deletion of both the type VI secretion system 1 (T6SS1) gene and luxR was constructed. The biological characteristics and virulence of the mutants were analyzed. The relative percent of survival of fish after vaccination with the double deletion mutant was investigated. [Results] We successfully constructedΔluxR, ΔT6SS1ΔluxR, and ΔrpoE. Compared with the wild type, none of the three mutants showed significant changes in the growth rate, swarming ability or swimming ability, while ΔluxR and ΔT6SS1ΔluxR showed significant decreases in biofilm formation. The internalization, adsorption, and intracellular proliferation of the mutants in mouse macrophages were observed. All the three mutants could survive in J774A.1 macrophages and showed lower proliferation capacities than the wild type. Compared with the wild type, the two single mutants showed significantly reduced virulence to goldfish (Carassius auratus) after intraperitoneal injection at a concentration of 1.0×10⁷ cells/mL. The double deletion mutant ΔT6SS1ΔluxR showed no virulence when challenging goldfish at the same dose, with the LD₅₀>10⁸ cells/mL. In the immune protection experiment, the goldfish was vaccinated with ΔT6SS1ΔluxR, and the fish was artificially challenged with the wild type 28 days later. The relative percent of survival reached 78.60%, which indicated effective protection. [Conclusion] In this study, three gene deletion mutants were successfully constructed, and the virulence of the mutants was significantly decreased. Among them, ΔT6SS1ΔluxR is expected to be a candidate strain for the development of the attenuated live vaccine against P. plecoglossicida infection.

Yeast β-glucan has been widely used as a dietary supplement for its multiple biological activities, including immune stimulation. Previous studies in our laboratory have shown that yeast chs3Δ spores exposed in the glucan layer can activate immune effects more effectively than trophoblasts. However, chs3Δ spores are formed within the ascospores and are encapsulated by the ascospore cell wall, which greatly limits their direct application. [Objective] To explore whether the lysates of chs3Δ asci have the potential to be used as novel immune-stimulating dietary supplements. [Methods] We prepared chs3Δ asci lysates by ultrasonic freeze drying, enzymatic freeze drying, and enzymatic-assisted ultrasonic freeze drying and then explored their immune effects in depth. [Results] The chs3Δ asci lysate prepared by ultrasonic freeze drying induced the highest level of inflammatory cytokines compared with the vegetative cell lysate and chs3Δ spores, and the asci lysate achieved immunostimulatory responses through activation of Dectin-1 receptor. Further studies showed that the chs3Δ asci lysate also exhibited immunostimulatory activity and trained immunity-inducing ability in mice. [Conclusion] The chs3Δ asci lysate has great potential as a highly effective immunostimulatory dietary supplement.

[Objective] To systematically investigate the substrate promiscuity and catalytic performance of three UDP-glycosyltransferases: CsUGT75L12 (Camellia sinensis), CiUGT11 (Chrysanthemum indicum), and UGT73B1 (Arabidopsis thaliana). [Methods] The recombinant proteins of plant glycosyltransferases were heterologously expressed in Escherichia coli BL21(DE3) and purified for in vitro enzymatic assays. In vitro enzymatic reactions of the purified recombinant proteins were performed with six flavonoids including flavones (apigenin and acacetin) and flavanones (naringenin, eriodictyol, isosakuranetin, and hesperetin). The enzymatic products were characterized by HPLC and LC-MS and the conversion rates were calculated through comparative HPLC peak area analysis. [Results] CsUGT75L12, CiUGT11, and UGT73B1 exhibited broad substrate promiscuity towards the six tested flavonoids. The primary products were identified as flavonoid-7-O-glucosides. Notably, CiUGT11 and UGT73B1 demonstrated exceptional catalytic efficiency, achieving >96% conversion rates for hesperetin and naringenin. Leveraging this activity, we engineered CiUGT11 and UGT73B1 with high efficiency to produce hesperetin-7-O-glucoside and naringenin-7-O-glucoside through precursor feeding in E. coli. [Conclusion] The three glycosyltransferases display remarkable versatility in flavonoid recognition, with conserved preference for the C7-OH position. CiUGT11 and UGT73B1 show high catalytic efficiency for six flavonoids. These findings provide candidate gene elements for the efficient microbial production of flavonoid glycosides.