[Objective] To clarify the succession patterns of Lactobacillus and their regulatory effects on the ethyl acetate-to-ethyl lactate ratio during Xiaoqu Qingxiangxing Baijiu fermentation. [Methods] High-throughput sequencing was employed to analyze the species-level succession patterns of Lactobacillus in Xiaoqu and fermented grains (Jiupei). Modified culture media and enrichment methods were employed to isolate Lactobacillus from Jiupei. The dominant Lactobacillus strains were then evaluated for tolerance, subjected to single-carbon-source fermentation experiments, sorghum hydrolysate fermentation experiments, and lab-scale simulated solid-state fermentation with Lactobacillus. The physiological and biochemical characteristics of different Lactobacillus strains, their fermentation characteristics on single carbon sources, and their impact on the ethyl acetate-to-ethyl lactate ratio in the Baijiu were investigated. [Results] A total of 15 Lactobacillus strains were isolated from Jiupei, and high-throughput data analysis identified eight dominant species: Limosilactobacillus pontis, Lactobacillus helveticus, Lentilactobacillus buchneri, Lentilactobacillus hilgardii, Levilactobacillus brevis, Limosilactobacillus fermentum, Lactiplantibacillus plantarum, and Lactobacillus acetotolerans. Further tolerance tests and single-carbon-source fermentation characterization revealed that all the eight dominant strains could withstand actual fermentation conditions (ethanol/acetic acid/lactic acid). However, L. plantarum exhibited impaired maltose utilization, while L. acetotolerans showed defective d-galactose metabolism. L. helveticus and L. pontis displayed a single-product fermentation profile in single-carbon-source cultures, whereas L. brevis, L. buchneri, and L. hilgardii exhibited multi-product fermentation patterns in both single-carbon-source and sorghum hydrolysate media. In lab-scale simulated solid-state fermentation with Lactobacillus, compared with the control, supplementation with L. hilgardii increased the ethyl lactate and ethyl acetate content by 175% and 44%, respectively, reducing the ethyl acetate-to-ethyl lactate ratio to 0.357. Supplementation with L. buchneri increased the ethyl acetate content by 50% while decreasing the ethyl lactate by 71%, raising the ethyl acetate-to-ethyl lactate ratio to 4.496. [Conclusion] The dominant Lactobacillus strains in Xiaoqu Qingxiangxing Baijiu fermentation exhibit strong overall environmental tolerance, and their metabolic profiles vary in single-carbon-source fermentation. The supplementation with different dominant Lactobacillus strains differentially modulates ester formation. These findings provide a theoretical foundation for dynamically regulating key flavor compounds during Xiaoqu Qingxiangxing Baijiu fermentation.

[Objective] To investigate the effects of calcium ion-free (Ca²⁺-free) conditions on the gene expression, biofilm formation, and virulence of Vibrio parahaemolyticus. [Methods] Ethylene glycol-tetraacetic acid was used to chelate Ca²⁺ in culture media to create Ca²⁺-free conditions. Crystal violet staining was employed to evaluate the biofilm formation of V. parahaemolyticus. Swimming and swarming assays were performed to assess the motility. Additionally, the Kanagawa phenomenon test, HeLa cell adhesion assay, and cytotoxicity experiment were conducted to analyze the virulence phenotypes of V. parahaemolyticus. By comparing the expression profiles, we analyzed the effect of Ca²⁺-free conditions on the gene expression in V. parahaemolyticus. [Results] Ca²⁺-free conditions inhibited the growth and significantly reduced the biofilm formation, intracellular c-di-GMP levels, and motility of V. parahaemolyticus. Furthermore, Ca²⁺-free conditions suppressed the hemolytic activity and reduced the bacterial adhesion to HeLa cells, while enhancing the cytotoxicity of V. parahaemolyticus. Transcriptomic analysis revealed 359 differentially expressed genes (DEGs) under Ca²⁺-free conditions. These DEGs were mainly associated with biofilm formation, virulence factors, and regulators. Notably, the genes involved in lateral flagella and polar flagellum were downregulated, while most virulence genes were upregulated. The majority of putative regulator genes were downregulated. [Conclusion] Ca²⁺-free conditions significantly affect the biofilm formation, motility, virulence, and gene expression of V. parahaemolyticus.

SnoaL family proteins belong to a class of the nuclear transport factor 2 (NTF2)-like superfamily, which are characterized by a distinctive α+β barrel fold structure. To date, proteins of this family have been demonstrated to catalyze a variety of reactions, such as aldol reaction, hydroxylation, decarboxylation, epoxide hydrolysis, and cycloaddition reaction, playing crucial roles in the biosynthesis of complex bioactive natural products. These proteins are widely distributed in various organisms including bacteria and fungi. However, research on proteins of this family is relatively limited at present. We reviewed the research progress on proteins of SnoaL family. The review not only provides beneficial guidance for the subsequent functional identification and mechanism research of related proteins but also helps to discover novel biocatalytic tools, laying a foundation for the research of bioactive molecule biosynthesis.

[Objective] Porcine circovirus type 3 (PCV3) is associated with the development of reproductive disorders in sows, piglet dermatitis, and myocarditis. However, due to the difficulty in stably passaging PCV3 in vitro, the animal infection model construction and specific pathogenicity of PCV3 remain to be investigated. This study aims to solve the above problems. [Methods] A PCV3 strain (PCV3-HK) was successfully isolated by inoculating PK-15 cells with lymph node samples from PCV3-positive weaned piglets. The strain was identified using PCR, indirect immunofluorescence assay, and transmission electron microscopy, and its complete genome was sequenced. The whole genome of the strain was sequenced. The pathogenicity of the strain was evaluated based on the clinical symptoms, body weight changes, virus excretion, and pathological changes of 21-day-old non-weaned piglets after inoculation with the 15th-passage viral cell culture at a dose of 10^6.4 TCID₅₀/mL. [Results] PCV3-HK had a genome length of 2 000 bp and belonged to PCV3c subtype. The viral particles were spherical, non-enveloped, about 15-20 nm in diameter, and capable of reacting specifically with the monoclonal antibody against PCV3 Cap protein. The results of infection in piglets showed that the PCV3-HK infected pigs developed obvious symptoms such as dermatitis, slow weight gain and even wasting, as well as anatomical changes such as lymph node edema and interstitial pneumonia. The viral nucleic acid was detected in oral swabs and anal swabs on day 7-21 post infection, and viremia was observed. The virus infected multiple organs, including the heart, lungs, kidneys, and lymph nodes. Notably, inflammatory cell infiltration and aggregation of necrotic cells were observed in the kidneys and lymph nodes. [Conclusion] We isolate a PCV3 strain with classical circovirus morphology and establish a piglet infection model, which provides key materials for pathogenic mechanism study and vaccine development regarding PCV3.

[Objective] To identify and develop a phage-derived lyase that can be heterologously expressed with high activity and stability and determine its optimal working conditions. [Methods] We employed the turbidity reduction assay to evaluate the bacteriolytic activity and identify the optimal parameters. [Results] Genome annotation and protein prediction of the Vibrio alginolyticus phage phiV208 showed that ORF30 encoded a lyase, named lysV208. This enzyme demonstrated soluble expression in Escherichia coli BL21(DE3), reaching a purified concentration of 204 μg/mL after 16 h induction with 0.25 mmol/L IPTG. Its bacteriolytic activity (turbidity reduction rate) increased from 24.2% to 68.0% in the presence of 0.5 mmol/L EDTA. Enzymatic characterization revealed that lysV208 exhibited the maximum bacteriolytic activity (75.6%) at 45 ℃ while maintaining high activity (52.8%-71.9%) within the temperature range of 25-37 ℃, which is typical for bacterial disease outbreaks in aquatic and terrestrial animals. The enzyme showed the maximum activity at pH 7.0 and retained substantial bacteriolytic activity (44.0%-63.2%) under alkalescence conditions (pH 7.0-9.0), demonstrating adaptability to marine and freshwater aquaculture environments. Divalent metal ions including Zn²⁺, Mg²⁺, Mn²⁺, and Fe²⁺ at 0.1-1.0 mmol/L moderately enhanced the bacteriolytic activity of lysV208, whereas those at 10.0 mmol/L reduced the activity (P<0.01). In addition, lysV208 displayed broad-spectrum lytic effects, showing the bacteriolytic activity of 59.7% against V. alginolyticus V039, 68.9% against Vibrio vulnificus H1, 65.8% against Vibrio parahaemolyticus GH32, and 38.0% and 65.6% against Vibrio harveyi TY13 and G1, respectively. [Conclusion] The recombinant lyase lysV208 demonstrates robust and stable in vitro bacteriolytic activity and a broader spectrum than its source phage. These findings highlight its potential for the control of bacterial infections and the development of phage-lyase synergistic agents.

[Objective] To construct an Escherichia coli mutant strain that accumulates pyruvate by genetic modification guided by the genome-scale metabolic network model. [Methods] Using a genome-scale metabolic network model as a guide, we simulated pyruvate production of E. coli, screened key genes in metabolic pathways, and developed gene editing procedures accordingly. We knocked out the acetate kinase gene ackA, phosphate acetyltransferase gene pta, alcohol dehydrogenase adhE, glycogen synthase gene glgA, glycogen phosphorylase gene glgP, phosphoribosyl pyrophosphate (PRPP) synthase gene prs, ribose 1,5-bisphosphate phosphokinase gene phnN, and transporter encoding gene proP. Furthermore, we knocked in the transporter encoding gene ompC, flavonoid toxin gene fldA, and d-serine ammonia lyase gene dsdA. [Results] A shake flask process with the genetically edited mutant strain MG1655-6-2 under anaerobic conditions produced pyruvate at a titer of 10.46 g/L and a yield of 0.69 g/g. Metabolomic analysis revealed a significant increase in the pyruvate level in the fermentation broth, accompanied by notable decreases in the levels of certain related metabolic byproducts. Through 5 L fed-batch fermentation and an adaptive laboratory evolution, the strain finally achieved a pyruvate titer of 45.86 g/L. [Conclusion] This study illustrated the efficacy of a gene editing strategy predicted by a genome-scale metabolic network model in enhancing pyruvate accumulation in E. coli under anaerobic conditions and provided novel insights for microbial metabolic engineering.

[Objective] We isolated endophytes from the roots of Isatis indigotica Fort. and investigated the effects of endophytes on the growth, physiology, and medicinal quality of I. indigotica, aiming to provide a reference for microbial regulation of the cultivation and production of this plant. [Methods] The relevant culture media were selected to analyze the plant growth-promoting activities of the endophytes. Three endophyte strains (Bacillus sp. BC00, Bacillus sp. BV11, and Pseudomonas sp. PA28) were selected and applied singly to I. indigotica four times. The physiological indexes of the leaves were measured at different time points after the last treatment, and the growth and physiological indexes and the content of active ingredients of the roots and the leaves were measured at the harvesting stage. [Results] The photosynthetic pigment content and nutrient-metabolizing enzyme activities of I. indigotica leaves in each treatment group were higher than those in the control group at each time point. The three strains of endophytic bacteria significantly promoted the growth and biomass accumulation of I. indigotica. Strain BC00 had the best promoting effect on the dry weight of the above-ground part, which was increased by 74.16% compared with that of the control. Strain BV11 had the most significant promoting effect on the dry weight of the below-ground part, which was increased by 216.02% compared that of the control. The treatments with endophytic bacteria significantly affected the content of endogenous hormones and soluble substances. The three strains of endophytic bacteria had different effects on the active ingredients. Compared with the control, strain BC00 increased the content of indigo and epigoitrin by 7.83% and 5.64%, respectively, while strain BV11 slightly decreased the content of indigo and epigoitrin. The total active biomass of each index ingredient in the strain treatments was significantly higher than that of the control. [Conclusion] The endophytic bacterial strains BC00, BV11, and PA28 had significant promoting effects on the growth, physiological indexes, and active ingredients of I. indigotica, and strain BC00 demonstrated the best comprehensive promoting effect. The findings provide a basis for the later development of microbial fertilizers conducive to the growth of I. indigotica.

[Objective] To investigate the molecular mechanism by which Listeria monocytogenes regulates the expression of mitochondrial calcium uptake 2 (MICU2) through the virulence factor listeriolysin O (LLO) and their interaction, thereby affecting mitochondrial calcium homeostasis and bacterial intracellular proliferation. [Methods] Western blotting was employed to analyze MICU2 expression in HeLa cells infected with L. monocytogenes EGD-e or Δhly. Gene silencing and eukaryotic overexpression approaches were used to examine how MICU2 regulated the intracellular proliferation of L. monocytogenes. AlphaFold3 was used to predict the interaction sites between LLO and MICU2, and co-immunoprecipitation (Co-IP) was performed to verify their interaction. Mitochondrial calcium fluorescence probe (Rhod-2 AM) was used to analyze the regulatory role of MICU2 in calcium homeostasis. [Results] EGD-e infection upregulated MICU2 expression at 4 h and 6 h post-infection (P<0.001), whereas Δhly showed no effect (P>0.05). The silencing of MICU2 enhanced bacterial proliferation (P<0.01) and elevated mitochondrial calcium levels (P<0.05), whereas overexpression of MICU2 reduced bacterial proliferation (P<0.01) and decreased mitochondrial calcium levels (P<0.05). AlphaFold3 predicted that alanine (Ala) at position 462 of LLO interacted with glutamate (Glu) at position 119 of MICU2 via a hydrogen bond, and Co-IP confirmed their interaction. [Conclusion] L. monocytogenes upregulates MICU2 expression via LLO, and MICU2 inhibits bacterial intracellular proliferation by reducing mitochondrial calcium levels. The interaction between LLO and MICU2 is a key molecular basis for this process. These findings provide insights into the pathogenic mechanism of L. monocytogenes.

Root-knot nematode, among the most destructive plant-parasitic nematodes, poses a severe threat to global agricultural production. The plant root microbiome is considered as the “second genome” of host plant and plays an indispensable role in plant growth, development, and stress response. Parasitism by root-knot nematode significantly disrupts the community structure and function of root-associated microbial communities in plants. The disturbance of host microbiome not only exacerbates plant pathological processes but also may induce cascade effects through tripartite interactions among microorganism, plant, and nematode. This review comprehensively elucidates the multifaceted impacts of root-knot nematode parasitism on the root micro-ecosystem of host plant, particularly focusing on the variation in the structural and functional characteristics of both the rhizosphere and endophytic microbiome, as well as their roles in the occurrence of nematode diseases and maintaining plant health. Investigating the interaction between pathogenic nematodes and plant microbiome on community level will not only advance our understanding the intricate network among plant, nematode, and microorganism, but also provide theoretical and practical insights for developing innovative strategies for controlling plant nematode diseases.

[Objective] To investigate the effects of fluoride exposure on the gut microecosystem of the model insect Bombyx mori and elucidate the toxic mechanisms of fluoride under different conditions on insect hosts. [Methods] We measured physiological enzyme activity changes in the gut tissue of B. mori following fluoride exposure and observed histopathological changes by using hematoxylin and eosin (HE) staining. We also analyzed the dynamic changes in gut microbiota by using 16S rRNA amplicon sequencing and examined the expression changes of immune-related genes in gut tissue by qPCR. [Results] Fluoride exposure significantly increased the malondialdehyde (MDA) level and decreased the reduced glutathione (GSH) level in the gut tissue. Enzyme activities of catalase (CAT), superoxide dismutase (SOD), acetylcholinesterase (AChE), carboxylesterase (CarE), alkaline phosphatase (AKP/ALP), and lactate dehydrogenase (LDH) were also markedly suppressed. Additionally, we observed epithelial cell rupture and separation from the basement membrane. The Toll immune pathway was inhibited, which resulted in significantly reduced expression of antimicrobial peptide genes, such as attacin, cecropin, lebocin, and lysozyme. Gut microbiota analysis revealed significant declines in the relative abundances of Glutamicibacter, Staphylococcus, Acinetobacter, and Methylobacterium. The gut microbiota exhibited notable structural heterogeneity with strengthened functions related to metabolic pathways and biosynthesis of secondary metabolites. [Conclusion] Fluoride exposure significantly weakened the host insect’s antioxidant capacity, basal metabolism, and immune function, damaged the integrity of gut tissue, and caused gut microbiota dysbiosis. This disruption of the gut microecosystem might be a major factor contributing to reduced insect species diversity and biomass.