Abstract:[Background] Phages are viruses obligately lysing bacteria. They have a significant impact on the industrial fermentation of Escherichia coli. It is necessary to seek highly effective control measures to reduce the damage caused by phages to production. [Objective] To isolate an E. coli phage strain from the contaminated fermentation broth and analyze its biological characteristics and genome sequence. [Methods] A phage strain was isolated from the contaminated liquid of an amino acid production enterprise, and its morphology and structure was observed by transmission electron microscopy. The host range, temperature tolerance, pH tolerance, optimal multiplicity of infection, and one-step growth curve were determined for this phage strain. Furthermore, whole genome sequencing was performed for this strain. Finally, a bacterial defense system was selected to control this strain. [Results] A virulent E. coli phage strain was isolated and named JNP1. It can form clear and translucent plaques. The phage consisted of an icosahedral head approximately 60 nm in diameter and a long slender tail about 170 nm in length. JNP1 can tolerate the conditions of 4-50 ℃ and pH 5.0-10.0. It showcased the optimal multiplicity of infection of 0.1, the latent period of 10 min, the lysis period of 10-70 min, and the burst size of 110 PFU/cell. The full-length genome of the phage was 40 865 bp, with the G+C content of 44% and a total of 65 open reading frames. The phylogenetic analysis showed that this strain was a novel E. coli phage. Based on the biological and genomic characteristics of JNP1, the control effect of DNA phosphorothioation on this phage was evaluated. [Conclusion] In this study, a phage strain JNP1 was successfully isolated from the fermentation environment of E. coli in production. An industrial strain with the DNA phosphorothioation system can defense against this phage. This achievement provides a solid theoretical basis for formulating prevention and control strategies against phage contamination during industrial production.

Abstract:[Background] Oxaloacetic acid (OAA), as an intermediate of the TCA cycle, is a crucial precursor for L-threonine biosynthesis. It not only participates in energy metabolism but also affects the synthesis and transformation processes of L-threonine. The concentration and metabolic rate of OAA directly influence the L-threonine production performance of the strain. Thus, optimizing the accumulation of OAA can effectively enhance the efficiency and yield of amino acid fermentation. Promoter engineering has been effectively employed in regulating gene expression and promoting the synthesis of metabolites. Particularly, the promoters responding to specific metabolites have demonstrated significant application potential in precise regulation. [Objective] To improve the production of L-threonine by constructing an OAA-responsive metabolic pathway in Escherichia coli WS022. [Methods] RNA was extracted under the conditions of 0, 0.5, and 2 g/L OAA, and transcriptome sequencing was conducted. An OAA-responsive promoter library was constructed by fluorescence characterization with eGFP. In this promoter library, 20 promoters were selected to regulate the expression of aspC and ppc related to OAA generation and consumption, respectively, on the basis of which the effects of different promoters on the growth and L-threonine synthesis of the strain were explored. [Results] The addition of 2 g/L OAA had a notable inhibitory effect on the growth of the strain in the early stage. The strain exposed to 0.5 g/L OAA showcased the same growth as the strain without OAA addition in the early stage, but had higher biomass in the later stage. Based on transcriptome data, 104 promoters were selected and characterized by eGFP fluorescence. Then, 20 promoters were selected to regulate the expression of aspC and ppc to construct the OAA-responsive metabolic pathway. The results indicated that the L-threonine production of 17 out the 20 strains with regulated expression of aspC increased compared with that of the control strain WN00. The L-threonine production of all the 20 strains with regulated expression of ppc increased compared with the control strain WN00. At the same time, the yields of strains WA16 and WA38 increased by 3.3 folds and 7.6 folds, respectively, compared with the control strain WN00. [Conclusion] The addition of OAA affected the growth of the strain. After regulation of the E. coli strain with different promoters, the production of most strains increased. Strain WA38 had the highest production, which was 7.7 folds higher than that of the control strain WN00, demonstrating the great potential of the OAA-responsive pathway in the biosynthesis of L-threonine.

Abstract:[Background] Cellulose is ubiquitous in nature and has great resource potential, and its efficient degradation is of great significance to alleviate the energy crisis and environmental pollution. [Objective] To screen cellulose-degrading bacteria and improve cellulase activity by optimizing enzyme production conditions. [Methods] Congo red staining was used for primary screening and DNS colorimetric method for secondary screening. The target strain was identified. The enzyme production conditions of the target strain were optimized by the single factor tests, Plackett-Burman design, path of steepest ascent method, and Box-Behnken design. [Results] A strain 26-2-2 with high cellulase activity was screened out and identified as Bacillus cereus. The optimal fermentation conditions of this strain were determined as follows: CMC-Na mass concentration of 31.2 g/L, (NH₄)₂SO₄ mass concentration of 13.6 g/L, KH₂PO₄ mass concentration of 4.0 g/L, rotation speed of 180 r/min, inoculation amount of 5.0%, fermentation time of 24 h, initial pH 7.0, and fermentation temperature of 35 ℃. Under these conditions, the cellulase activity of strain 26-2-2 reached (61.14±0.23) U/mL, which was 24.0% higher than that before optimization. [Conclusion] This study provides new strain resources for the bioconversion of cellulose and data reference for subsequent research on cellulose-degrading strains.

Abstract:[Background] Formate dehydrogenase (FDH, EC 1.2.1.2) is commonly used for NADH regeneration in the fermentation industry. Recent studies have shown that FDH can reversibly catalyze CO₂ reduction to formate, serving as a promising enzyme for microbial CO₂ fixation. However, poor thermal stability is the main factor limiting the application of FDH in in vitro CO₂ fixation. [Objective] This study aimed to enhance the thermal stability of a CO₂-reducing FDH from Thiobacillus sp. KNK65MA (TsFDH) via semi-rational engineering. [Methods] Nine different variants were designed via PROSS, which predicted stabilizing mutations based on sequence conservation and structural energy minimization. The enzymatic properties of the variants and the wild type were characterized, and structural mechanisms underlying stability improvement were analyzed through homology modeling. Finally, the engineered TsFDH variants were applied to the fixation of CO₂ in vivo by Komagataella phaffii GS115-4Δ. [Results] Compared with that of the wild type, the specific activities of TsFDH^A199G and TsFDH^P247K increased by 114.58% and 56.17%, respectively. The relative enzyme activities of TsFDH^A199G and TsFDH^P247K reached 19.32% and 39.47%, respectively, after incubation at 45 ℃ for 1 h, compared with that of the wild type. TsFDH^A199G and TsFDH^P247K showed the catalytic efficiency (k_cat/K_m) of 0.096 L/(mmol·s) and 0.042 L/(mmol·s) and the T_m increases of 0.3 ℃ and 0.7 ℃, respectively. These results indicated that the enzyme activity and thermal stability of TsFDH variants catalyzing CO₂ reduction were improved. Structural analysis showed that the hydrophobic interactions within TsFDH^A199G and TsFDHP^247K increased by two and four and the ionic interactions of TsFDH^P247K increased by one, compared with those of the wild type. Meanwhile, TsFDH^A199G optimized the microenvironment of the active center. It improves the affinity between the substrate and the enzyme, and thus increased the enzymatic reaction rate. Finally, TsFDH^A199G and TsFDHP^247K were transferred to K. phaffii GS115-4Δ for CO₂ fixation, increasing the biomass at the time point of 96 h by 118.04% and 106.11%, respectively. [Conclusion] TsFDH^A199G and TsFDH^P247K were successfully screened, which successfully improved the thermal stability of TsFDH. This study fills the gap of thermal stability modification of FDH for CO₂ reduction and provides two feasible enzymes for enzymatic fixation of CO₂.

Abstract:[Background] The development and utilization of xylanase has been one of the hotspots in biological research. However, the industrial application and production of xylanase face problems, such as the high production cost of the enzyme and the non-reusability of the enzyme in the free state, which limit the application of xylanase in industrial production. [Objective] To construct a surface display strain of xylanase (XynT6) based on Escherichia coli BL21(DE3), thus improving the reuse rate and ultimately reducing the purification cost of xylanase. [Methods] The restriction-free cloning method was employed to fuse the outer membrane protein OMPA derived from Escherichia coli with XynT6 derived from Bacillus sonorensis. The fusion protein was transferred into BL21(DE3) to construct the surface display strain LOXynB. The activity and properties of this surface displayed XynT6 and the stability of the display system were explored by the 3,5-dinitrosalicylic acid (DNS) colorimetric method. The surface display of XynT6 was identified by SDS-PAGE, Western blotting, immunofluorescence, and flow cytometry. [Results] The E. coli strain LOXynB for the surface display of XynT6 was successfully constructed. The enzyme displayed on the surface of LOXynB showed the activity of 1 778 U/g and similar enzymatic properties compared with the free xylanase. The K_m and V_max of the displayed enzyme were (37.62±0.82) mmol and (0.49±0.01) mmol/min, respectively, which suggested that the displayed enzyme had enhanced substrate affinity and an increased maximal reaction rate compared with the free enzyme. Moreover, LOXynB maintained high enzyme activity after storage for 56 days and recycling for 16 times, which demonstrated that LOXynB possessed good storage and reuse stability. [Conclusion] We successfully displayed XynT6 on the surface of E. coli, providing a basis for the industrial production of xylanase.

Abstract:[Background] L-malic acid is widely used in biomedicine, food, and chemical industries. However, the wild-type Escherichia coli hardly accumulates malic acid. The T7 expression system has the ability to strictly control and induce expression at a high level, while its application is influenced by the presence of T7 RNA polymerase. [Objective] To construct a high-yield L-malic acid-producing chassis strain based on the T7 expression system. [Methods] Firstly, multi-gene knockouts in the malate consumption pathway of E. coli MG1655 were performed. Then, the gene expression cassette of T7 RNA polymerase was introduced into the chromosome of E. coli MG1655, and thus a high-yield L-malic acid-producing chassis strain MG1655W(DE3) was successfully constructed. [Results] The expression of the reporter gene in the chassis strain increased the fluorescence intensity to 114.0% compared with that of E. coli BL21(DE3). The L-malic acid accumulation of the recombinant strain was 12.9 times that of the original strain, and further overexpression of malate dehydrogenase via the T7 expression system increased the L-malic acid production to 26.7 times that of the original strain. [Conclusion] We successfully constructed a high-yield L-malic acid-producing chassis strain MG1655W(DE3) that could utilize the T7 expression system. This strain shows high application values in the production of metabolites such as malic acid and provides a new strategy for constructing excellent chassis strains for malic acid production.

Abstract:[Background] Developing and planting salt-tolerant plants has become an important strategy to improve the environment of saline-alkali land and ensure food security. Rhizosphere microorganisms, as the second genome of plants, can alleviate abiotic stress caused by the saline-alkali environment and promote plant growth. [Objective] To isolate microbial resources with saline-alkali tolerance and provide valuable strain resources for the development of bioagents and the comprehensive utilization of saline-alkali land. [Methods] Culturable bacteria were screened from rhizosphere soil samples of the salt-tolerant plant Suaeda salsa, which thrived in the coastal region of Weifang. The strains were identified by 16S rRNA gene sequence analysis. Subsequently, the salt tolerance and plant growth-promoting effects of these bacterial strains were assessed. To validate the plant growth-promoting effects of the strains, we monitored the seed germination and growth of the medicinal plant Isatis indigotica inoculated with these strains under saline-alkali stress (NaCl: Na₂SO₄: NaHCO₃: Na₂CO₃=1:9:9:1). [Results] A total of 306 strains of culturable bacteria were isolated and identified as 20 species, mainly belonging to Firmicutes and Proteobacteria, and the dominant genera were Bacillus and Peribacillus. Functional studies of the 20 different strains at the species level revealed varying degrees of indole-3-acetic acid (IAA) production. Additionally, 2, 5, and 6 strains demonstrated the abilities of solubilizing phosphorus, fixing nitrogen, and producing cellulase, respectively. The experiment on seed germination showed that Bacillus zhangzhouensis JP 176, Halomonas songnenensis JP 121, Pseudomonas yangonensis JP 207, and P. tianjinensis JP 208 had different degrees of plant growth-promoting effects under saline-alkali stress. Among them, H. songnenensis JP 121 and P. yangonensis JP 207 significantly enhanced the germination rate and bud lengths of I. indigotica seeds under saline-alkali stress, effectively reducing the effect of saline-alkali stress on the growth of this plant. [Conclusion] The rhizosphere soil of S. salsa contained a wealth of microbial resources. Isolating and screening plant growth-promoting rhizobacteria with saline-alkali tolerance is crucial for boosting the growth and reducing the saline-alkali stress of I. indigotica. These strains serve as candidates for the development of microbial inoculants, which can support the sustainable development of saline-alkali land and improve the value-added use of Chinese herbal medicine resources of saline-alkali land.

Abstract:[Background] Oxalis is a genus of plants with wide distribution and diverse species. Among them, Oxalis latifolia Kunth and O. corymbosa have become invasive weeds difficult to be controlled due to the strong environmental adaptability and dispersal ability. Plant endophytes play an essential role in plant growth and development, nutrient uptake, and stress resistance. As for plants of Oxalis, endophytes may also play a role that cannot be ignored in the physiological processes. [Objective] We explored the endophyte diversity in the bulbs of different Oxalis species and isolated and identified endophyte strains with plant growth-promoting effects, aiming to elucidate the potential influences of endophytes on the nutrient uptake and growth of Oxalis species. [Methods] The bulbs of O. corymbosa, common and Cornish forms of O. latifolia, and O. triangularis were collected and planted in pots with a diameter of 30 cm in the greenhouse of Yunnan Agricultural University. 30 plants were grown in each pot, and five replicates were set up. After six weeks of growth, the bulbs of 10 plants in each pot were cut off. After surface disinfection, high-throughput sequencing was conducted with the 16S rRNA gene V3-V4 and ITS1 universal primers to analyze the community structure of the endophytes of Oxalis. Furthermore, the endophytes of Oxalis with the functions of fixing nitrogen, solubilizing phosphorus, or solubilizing potassium were isolated and identified, and the functional intensity of these endophytes was quantitatively determined. [Results] The bacterial genus with the highest abundance among the endophytes of Oxalis was Steroidobacter (4.79%), followed by Allorhizobium_Neorhizobium_Pararhizobium_Rhizobium (4.45%), Niastella (3.87%), and Sphingomonas (3.64%). The fungal genera with higher abundance were Mortierella (4.05%), Wickerhamomyces (3.77%), Wallemia (3.32%), and Fusarium (2.83%). The results of alpha and beta diversity analyses showed significant differences in the community structure of endophytes in the bulbs of different Oxalis species. Specifically, in terms of bacteria, 13 genera belonging to 15 families, 11 orders, six classes of four phyla were enriched in the bulbs of different Oxalis species; in terms of fungi, 10 species belonging to 11 genera, 10 families, nine orders, six classes of one phylum showed significant differences in enrichment. A total of 10 endophyte strains capable of fixing nitrogen, solubilizing phosphorus, or solubilizing potassium were isolated from the bulbs of different Oxalis species, and their function intensities varied. Through the maximum likelihood method, they were preliminarily identified as members of Enterobacter, Pseudomonas, and Brucella. The microbial co-occurrence network constructed by high-throughput sequencing revealed that although these functional strains helped plants accumulate nutrients, they were not the key nodes of the network, and some microorganisms at the key nodes were not included in the NCBI database. [Conclusion] The endophytes in the bulbs of Oxalis have unique community structures and high diversity, and the endophyte community structures in the bulbs of different Oxalis species showcase extremely significant differences. The strains with nitrogen-fixing, phosphorus-solubilizing, or potassium-solubilizing function may play a role in promoting the growth and nutrient uptake of Oxalis species. The results of this study provide theoretical support for delving into the microecological mechanisms of the environmental adaptation and diffusion of Oxalis species.

Abstract:[Background] With the progress in industrialization, microplastics causing "white pollution" have gradually become one of the main environmental pollutants. As an emerging microplastic adsorption method in recent years, microbial remediation of microplastics in water has important practical significance. [Objective] We modified CsgA to make the strain expressing the modified protein have the ability to efficiently remove microplastics and studied the influencing factors and mechanism of its adsorption of polystyrene (PS), aiming to provide reference for microbial remediation of microplastics. [Methods] After mutation of the negatively charged aspartic acid (D) on the R₂-R₄ structural unit of CsgA to positively charged arginine (R), the isoelectric point of the protein was increased. Accordingly, the protein surface had more positive charges, thereby binding with negatively charged microplastics to achieve the purpose of water restoration. The main factors (pH, initial concentration, temperature, etc.) affecting the PS adsorption capacity of the strain expressing modified CsgA were investigated, and the adsorption mechanism was discussed via isothermal adsorption models and kinetic analysis. [Results] Compared with CsgA, D104R/D127R increased the PS adsorption efficiency by 1.37 times from 40.88% to 97.08% and the adsorption capacity by 3.59 times from 1.27 g/g to 5.83 g/g. D104R/D127R demonstrated good PS adsorption ability within the range of pH 3.0 to pH 10.0. The adsorption of PS by D104R/D127R complied with the Freundlich isothermal adsorption model, which indicated multi-molecular layer adsorption. [Conclusion] D104R/D127R has a good ability to remove PS, and the research results on the influencing factors and mechanism of PS adsorption provide guidance for microbial remediation of microplastics pollution.

Abstract:[Background] Erwinia amylovora is the causative agent of fire blight, a bacterial disease that is destructive to pear and apple plants. At present, the control of fire blight mainly relies on antibiotics and copper-containing chemicals. Bacteriophages are considered to be promising biocontrol agents. [Objective] To isolate and characterize novel E. amylovora bacteriophages and contribute to the biocontrol of fire blight. [Methods] With E. amylovora strain Ea102 as the host, bacteriophages were isolated by the double-layer agar plate method from the soil of pear orchards suffering from fire blight. The bacteriophage particles were observed by transmission electron microscopy. PacBio sequencing was conducted to analyze the bacteriophage genome. Biological characteristics such as optimal multiplicity of infection, one-step growth curve, and thermal and pH stability were determined by the plaque counting method. The inhibitory effects of the bacteriophage against E. amylovora Ea102 on young fragrant pears and Pyrus betulaefolia seedlings were evaluated. [Results] A bacteriophage strain phB78 infecting E. amylovora was isolated. It formed clear plaques with translucent halo zones. phB78 was composed of an icosahedral head with a diameter of (108±3) nm and a long tail with a length of (210±6) nm. It had a circular genome of 261 824 bp (GenBank Accession No: PP942979) with the GC content of 49.3%. A total of 322 open reading frames were predicted, most of which had unknown functions. phB78 was closely related to the Alexandravirus bacteriophage pEa_SNUABM_30 (NC_061446.1), with 95.23% average nucleotide identity. phB78 kept stable lytic activity at pH 6-10 and temperatures below 50 ℃. With E. amylovora Ea102 as the host, phB78 showed the optimal multiplicity of infection of 0.1, the latent period of about 50 min, and the burst size of 20 PFU/cell. A host range analysis revealed that phB78 was able to infect 3 out of 4 E. amylovora strains and 7 out of 14 Pantoea agglomerans strains isolated from pear and apple plants. phB78 significantly reduced the pathogenicity of E. amylovora Ea102 on young fragrant pears and P. betulaefolia seedlings. [Conclusion] phB78 is a novel virulent bacteriophage of E. amylovora, demonstrating a great value for the biocontrol of pear fire blight.

Abstract:[Background] In recent years, Bacillus cereus has demonstrated an escalating invasive capacity and a broadening spectrum of hosts, with its biofilm formation being intricately linked to its invasive potential and virulence. [Objective] To characterize the biofilm-forming capability of B. cereus and elucidate the inhibitory mechanisms of lactic acid (LA) and peroxyacetic acid (PAA) on the biofilm formation. [Methods] The impact of LA and PAA on the biofilm formation of Bacillus cereus was comprehensively evaluated by analyzing biofilm inhibition growth curves, biofilm metabolic activity, extracellular matrix components (including polysaccharides and proteins), morphological characteristics, and the expression levels of biofilm-related genes. [Results] B. cereus could form biofilms. The minimum inhibitory concentration (MIC) for LA and PAA against B. cereus were 0.252% and 0.030%, respectively. The biofilm growth curves indicated that LA and PAA at 1/2 MIC and MIC levels significantly curtailed the biofilm formation of B. cereus. The CCK-8 assay demonstrated that LA and PAA at MIC levels reduced the biofilm metabolism activity of B. cereus by 61.02% and 69.27%, respectively. The sulfuric acid-phenol and Coomassie brilliant blue staining results revealed that MIC levels of LA and PAA diminished extracellular polysaccharides by 64.34% and 79.14% and extracellular proteins by 78.96% and 80.52%, respectively. Microscopic examination following crystal violet staining confirmed that even at 1/4 MIC levels, LA and PAA impeded the biofilm formation of B. cereus. Furthermore, real-time quantitative PCR indicated that the MIC levels of LA and PAA markedly downregulated the expression of biofilm-related genes (gapB, pgm, flgE, SigB, abrB, calY, sipW, and tasA) in B. cereus. [Conclusion] Collectively, our data suggest that LA and PAA serve as effective and safe anti-biofilm agents, capable of inhibiting the biofilm formation of B. cereus.

Abstract:[Background] Fusarium head blight (FHB) of wheat, caused by Fusarium graminearum, can result in severe yield losses or even complete crop failure. A crucial strategy employed by the pathogen to successfully colonize its host involves scavenging reactive oxygen species (ROS) generated during the oxidative burst in plants. This process can be facilitated by the glutathione peroxidase (Hyr1), which catalyzes the conversion of H₂O₂ into water. [Objective] To identify the glutathione peroxidase FgHyr1 in F. graminearum and elucidate its biological functions. [Methods] The FgHyr1 knockout mutant was generated by homologous recombination, and the corresponding complemented strain was subsequently constructed. To elucidate the function of FgHyr1, we compared the wild-type strain and the knockout mutant in aspects such as vegetative growth, asexual reproduction, stress responses, pathogenicity, and ROS-scavenging capacity. [Results] FgHyr1 encoded a protein composed of 171 amino acid residues, containing an active site and a signature motif of glutathione peroxidase. Compared with the wild-type strain, ΔFgHyr1 exhibited significantly reduced hyphal height and weakened aerial hyphal growth on the PDA medium, while its conidiation remained unaffected. The deletion of FgHyr1 decreased the sensitivity of F. graminearum to cell wall stress but enhanced the sensitivity to oxidative stress. Furthermore, ΔFgHyr1 showed significantly enhanced sensitivity to tebuconazole and prochloraz, which may be due to the significantly down-regulated expression levels of CYP51A/B/C. Pathogenicity assays revealed that ΔFgHyr1 had significantly reduced virulence to wheat spikes and corn silks, although the production of deoxynivalenol (DON) was not altered. Moreover, ΔFgHyr1 demonstrated significantly downregulated expression levels of ROS scavenging-associated genes and reduced the accumulation of ROS near the infection point of the host plant. [Conclusion] FgHyr1 plays a multifaceted role in F. graminearum, including the regulation of aerial hypha growth, positive regulation of oxidative stress responses, negative regulation of cell wall stress responses, and modulation of the sensitivity to azole fungicides. Moreover, FgHyr1 is essential for the host scavenging of ROS and is required for the full virulence of F. graminearum.

Abstract:[Background] Continuous cropping obstacles pose a critical challenge to sustainable development of the ginseng (Panax ginseng) industry, with benzoic acid (BA) as one of the key autotoxic substances contributing to this issue. [Objective] To screen the microbial strains capable of effectively degrading BA in the soil with continuous cropping of ginseng to alleviate the obstacles and support sustainable cultivation. [Methods] 60 rhizosphere soil samples were collected from the areas with continuous cropping of ginseng for the enrichment, domestication, and isolation of strains. HPLC was employed to assess the BA degradation efficiency of each strain. The taxonomic status of the selected strains was determined by morphological, physiological, biochemical, and 16S rRNA gene sequencing analyses. The degradation conditions of the target strain were optimized by single factor experiments and response surface methodology. [Results] A total of 213 bacterial strains were obtained, from which seven strains capable of efficiently degrading BA were screened out and identified. Among them, strain PJZ-1 was identified as Acinetobacter calcoaceticus (GenBank accession number: PQ772624.1). This strain demonstrated the degradation efficiency of 74.83%-83.11% for four phenolic acids including vanillic acid in addition to BA. PJZ-1 degraded 94.29% of 50 mg/L BA under the optimized conditions (pH 7.18, 33.83 ℃, and 2.02% inoculum). The fermentation broth supernatant of PJZ-1 exhibited the highest degradation rate (89.08%) for BA, while enzyme inhibitors reduced the degradation efficiency by 12.45%, which suggested extracellular component-mediated degradation of BA by strain PJZ-1. [Conclusion] A. calcoaceticus PJZ-1 is a novel microbial strain for mitigating BA accumulation in the soil with continuous cropping of ginseng. This study provides foundational insights for developing microbial agents to address soil autotoxicity and promote the sustainable development of the ginseng industry.

Abstract:[Background] Myxobacteria have attracted extensive attention due to their ability to produce structurally diverse secondary metabolites with significant potential in antifungal and antitumor applications. Potato late blight, caused by Phytophthora infestans, poses a severe threat to global potato production. Bayannur as a vital grain-producing area has diverse landform types. However, the resources of myxobacteria and their activity against potato late blight in this region have not been systematically analyzed. [Objective] To investigate the taxonomic status, distribution characteristics, and antagonistic activity against P. infestans of culturable myxobacteria in Bayannur, laying a foundation for the development of myxobacteria resources in the region and the biocontrol of potato late blight. [Methods] Forty-five soil samples were collected and their physicochemical properties were characterized. Myxobacteria were isolated from these samples by rabbit dung induction, Escherichia coli streaking, and filter paper induction methods. The isolated strains were identified by morphological observation and 16S rRNA gene sequence analysis. The antagonistic activity of the strains against P. infestans was determined through the plate confrontation method. [Results] The soil in Bayannur was arid and alkaline, with low content of organic matter and alkali-hydrolyzed nitrogen but rich available phosphorus and available potassium. A total of 249 strains of myxobacteria were isolated from this area, among which 147 strains were purified and identified as members of 5 genera: Myxococcus, Pyxidicoccus, Corallococcus, Archangium, and Cystobacter. Myxococcus was the dominant genus, accounting for 70.25%. Among the isolated strains, 83.33% had antagonistic activity against P. infestans. Myxococcus was the dominant genus against P. infestans, accounting for 66.29% of the total antagonistic strains. [Conclusion] Bayannur harbors rich resources of myxobacteria and most of the myxobacterial strains in this region have antagonistic activity against P. infestans. This study provides a theoretical basis and strain resources for the development of microbial resources and the biocontrol of potato late blight in this region.

Abstract:[Background] Tobacco black shank is a soil-borne oomycete disease caused by Phytophthora nicotiana, seriously impacting the yield and quality of tobacco in China. Selenium, a beneficial element for plant growth, exhibits broad-spectrum antifungal and antibacterial activities and plays a crucial role in enhancing plant disease resistance. [Objective] We investigated the inhibitory effects of various forms of selenium on P. nicotiana, aiming to identify new and effective materials for the control of tobacco black shank. [Methods] We assessed the effects of two selenium sources—sodium selenite and nano-selenium—with different selenium concentrations on the mycelial growth, cell membrane permeability, content of soluble sugars and soluble proteins, and PI staining of P. nicotiana. Furthermore, to explore the therapeutic effects of the two selenium forms on tobacco black shank, we conducted both detached leaf infection and tobacco plant infection experiments. [Results] Compared with the control group, both sodium selenite and nano-selenium inhibited the growth of P. nicotiana, achieving the highest inhibition rates of 44.28% and 51.54%, respectively, at a selenium concentration of 8 mg/L. As the selenium concentration increased, both the relative conductivity and malondialdehyde (MDA) content rose, while the content of soluble sugar and soluble protein in the mycelium decreased. Furthermore, the selenium treatments led to mycelial mortality, which showed a notable difference between the two selenium sources. Specifically, the mycelial damage inflicted by nano-selenium was considerably greater than that caused by sodium selenite. The results of detached leaf infection and tobacco plant infection experiments indicated that the two exogenous selenium treatments significantly inhibited the spread of pathogenic fungi and provided substantial protection to tobacco leaves infected with P. nicotiana. The two treatments effectively mitigated the seedling damage caused by tobacco black shank. Specifically, the spot lengths in the sodium selenite and nano-selenium treatment groups decreased by 52.65% and 65.53%, respectively, compared with that in the control group, which indicated that nano-selenium was more effective than sodium selenite. [Conclusion] Selenium demonstrates a significant ability to inhibit the growth of P. nicotiana, and nano-selenium exhibits greater potential as a novel material for the treatment of tobacco black shank.

Abstract:[Background] Salmonella, a group of Gram-negative intestinal bacilli, causes zoonotic diseases and threaten animal farming. Phage therapy, an emerging alternative to antibiotics, holds great promise for controlling bacterial infections. Understanding the molecular mechanisms of phage recognition of bacterial surfaces is crucial for the rational and effective use of phage therapy in preventing and treating bacterial infections. [Objective] To identify the receptors for phage recognition on Salmonella typhimurium and explore the biological characteristics of the strain with waaL knockout, giving insights into the mechanisms of phage receptor recognition. [Methods] Salmonella typhimurium SA55 and its specific phage φPK069 were used as model systems. The receptor type recognized by φPK069 on SA55 was identified by proteinase K treatment, sodium periodate oxidation, and lipopolysaccharide (LPS) adsorption inhibition assays. A waaL knockout mutant (ΔwaaL) and a complemented strain (C-waaL) were constructed by λ-red homologous recombination and plasmid recombination, respectively. The phage receptor was identified by comparing the adsorption rates and lytic activities of φPK069 on SA55 and ΔwaaL. The sensitivity of ΔwaaL to φPK069 was assessed by the spot assay, and the antibiotic susceptibility of ΔwaaL was evaluated based on the minimum inhibitory concentrations (MICs). The serum survival assay was employed to assess the survival of ΔwaaL in the serum. [Results] The receptor for phage φPK069 on Salmonella typhimurium SA55 was the O-antigen of LPS. Compared to SA55, ΔwaaL exhibited reduced susceptibility to 70% of the tested bacteriophages, an 8-fold increase in the MIC of azithromycin, a 4-fold increase in the MIC of gentamicin, and a survival rate of less than 50% in serum. [Conclusion] This study identified the O-antigen of Salmonella typhimurium SA55 as the specific receptor for bacteriophage φPK069. Deletion of the waaL gene led to resistance to the bacteriophage, enhanced resistance to azithromycin and gentamicin, and diminished survival in serum.

Abstract:[Background] Brucella multiplication in trophoblasts is the primary cause of abortions in animals. The proliferation of Brucella in host cells is mediated by expression of the type Ⅳ secretion system (T4SS), which is regulated by the acid environment of Brucella-containing vacuoles (BCVs). [Objective] Considering that hydroxychloroquine (HCQ) can deacidifying the acid environment of BCVs, we checked the effect of HCQ in suppresing Brucella-induced abortions of pregnant mice. [Methods] Brucella-infected pregnant mice were designed as the model to investigate the effect of HCQ in inhibiting abortions. Histopathological changes in the placenta were observed. The inflammation in the placenta was checked by ELISA. The proliferation of Brucella in trophoblasts were measured by the CFU assay. The intracellular T4SS expression of Brucella was quantified by RT-qPCR. [Results] HCQ inhibited the proliferation of Brucella in trophoblasts through suppressing T4SS expression, which mediated trophoblast necrosis. HCQ inhibited inflammation in the placenta and reduced the Brucella-induced abortion rate of pregnant mice. [Conclusion] HCQ suppresses Brucella-induced abortions of pregnant mice by targeting T4SS. This study provides data for further investigation of HCQ in treating or preventing Brucella-induced abortions of pregnant domestic animals.

Abstract:[Background] Since both the beneficial properties and pathogenicity of Bacillus pumilus are strain-specific, the in-depth biological characterization of B. pumilus is essential for evaluating its application value. [Objective] To study some biological characteristics of a strain of Bacillus pumilus isolated from the blood and parenchymal organs of Simmental cattle. [Methods] The blood and parenchymal organs of diseased and dead cattle were cultured in LB agar plates for the isolation of the target strain, which was then identified by 16S rRNA gene sequencing. The pathogenicity and some biological characteristics of the isolate were analyzed through mouse challenge tests, virulence gene detection, biofilm formation assessment, in vitro antimicrobial susceptibility tests, and acid and bile salt tolerance tests. [Results] A pathogenic strain was isolated and identified as B. pumilus. It was named NM01 (NM), with the GenBank accession number PQ136445.1. The strain carried 8 virulence genes (nheA, nheB, hblA, hblC, hblD, ces, cytK, and entFM) and had biofilm formation ability. It was sensitive to ciprofloxacin, doxycycline, streptomycin, penicillin, amoxicillin, and enrofloxacin, and moderately sensitive to oxacillin and chloramphenicol. The results of the mouse challenge test indicated that this strain caused pathological damage of varying degrees to the organs of mice. [Conclusion] The strain isolated in this experiment was pathogenic B. pumilus, which could cause the death of mice. This study provides data support for the clinical diagnosis and prevention of pathogenic B. pumilus infections.

Abstract:[Background] Ascomycin (FK520) is a macrolide synthesized by Streptomyces hygroscopicus, with various bioactivities. In addition, FK520 is the starting ingredient for chemical synthesis of the immunosuppressant pimecrolimus, demonstrating important application value. [Objective] To achieve large-scale production of FK520 by fermentation, we focused on the optimization and scaling-up of the fermentation processes in 1-L and 15-L fermenters with S. hygroscopicus ATCC 14891. [Methods] Strain ATCC 14891 was fermented in a 250-mL shake flask, which was further scaled-up to a 15-L fermenter. We appropriately reduced the initial carbon and nitrogen sources, removed soybean oil, and decreased manganese and magnesium salts in the 15-L fermenter to increase the FK520 titer. Further experiments in 1-L parallel bioreactors were conducted to reveal the optimal pH and ammonium ion concentration for fermentation. The exogenous precursor lysine was added during the process to further increase the FK520 titer in the 15-L fermenter. [Results] The FK520 titer reached 414 mg/L after fermentation in a 250-mL shake flask for 120 h, and the titer decreased by 30.92% to 286 mg/L when the fermentation was scaled-up to a 15-L fermenter. After appropriate reducing of the initial carbon and nitrogen sources, the titer increased by 56.99%. The removal of soybean oil and reduction of manganese and magnesium salts by 20% to improve dissolved oxygen increased the titer by 78.32% and 101.75%, respectively. Further experiments conducted in 1-L parallel bioreactors revealed that the optimal pH and ammonium ion concentration for fermentation were 5.5 and 5 mmol/L, respectively. Based on the optimal pH and ammonium ion concentration and the 20% reduction in manganese and magnesium salts, the exogenous precursor lysine was added during the process in the 15-L fermenter, under which the FK520 titer reached 717 mg/L, 1.5 times higher than that of the control fermenter. Moreover, the consumption of glycerol was significantly reduced, which was only 25.91% of that in the control fermenter. [Conclusion] The above fermentation control process laid a foundation for the subsequent process optimization and scaling-up of fermentation for FK520 production.

Abstract:[Background] L-cysteine is an important sulfur-containing amino acid widely used in the food, pharmaceutical, and chemical industries. Microbial synthesis of L-cysteine has attracted increasing attention in recent years. [Objective] To obtain a promising L-cysteine producer and identify new metabolic targets. [Methods] The atmospheric and room temperature plasma (ARTP) mutagenesis was employed to obtain a mutant library of the engineered Escherichia coli strain AR-0. Based on a biosensor capable of responding specifically to L-cysteine, a high-throughput screening platform was constructed to isolate positive mutants. Whole genome sequencing and comparative genomic analysis were performed to investigate the functions of key mutated genes. [Results] A positive mutant strain AR3-7 was isolated from a library of 4.5×10⁵ cells, achieving a 2.7-fold increase in L-cysteine production compared with the parental strain. Through whole-genome sequencing and subsequent functional analysis of key mutations, three nonsynonymous variants were identified in the genes crp, rpoS, and dauA, all of which are implicated in metabolic and transport processes. These mutations may be the reason for the increased yield of the mutant strains. The knockout of dauA reduced H₂S production and led to a 23% increase in L-cysteine production compared with the parental strain. [Conclusion] Using ARTP mutagenesis and high-throughput screening, we successfully screened out a strain producing L-cysteine at a high yield. It is the first to report the association of dauA with the synthesis of L-cysteine in E. coli, providing a research foundation for further selection and development of elite strains with high yields of L-cysteine.

Abstract:[Background] Drug resistance is a major clinical challenge impacting the prognosis of colorectal cancer. The resistance to oxaliplatin, a first-line chemotherapeutic agent for colorectal cancer, is a pressing issue in clinical settings. Fusobacterium nucleatum has been found to be closely related to chemotherapy resistance in colorectal cancer. Acidification of the tumor microenvironment is one of the key features of tumor metabolism. However, whether the acidic microenvironment regulates the adaptive evolution of microorganisms to affect the drug resistance of tumors remains to be studied. [Objective] To investigate the role of F. nucleatum evolved in the acidic tumor microenvironment in promoting oxaliplatin resistance of colorectal cancer cells and decipher the potential mechanism. [Methods] We cultured the original F. nucleatum strain ORI in an acidic environment for adaptive evolution and obtained an acid-adapted F. nucleatum strain LA57 at pH 5.7. The relative survival rates of colorectal cancer cells HCT-116 and HT-29 co-cultured with strain ORI or strain LA57 were examined. The original HCT-116 cells as well as the cells co-cultured with strain ORI or strain LA57 were subjected to transcriptome sequencing for identifying the key functions of relevant genes. An in vitro cell chemotherapy model was constructed by oxaliplatin treatment, and the cell proliferation and the mRNA and protein levels of relevant genes were determined after co-culture with strain ORI or strain LA57. Furthermore, a subcutaneous tumor model in nude mice was constructed with HCT-116 cells, with oxaliplatin being injected intraperitoneally and strain ORI or strain LA57 intratumorally injected at multipoints for interventions. The immunohistochemical assay was employed to examine the expression of tumor tissue-associated proteins, and quantitative immunohistochemical scoring was performed. [Results] strain LA57 could grow stably at pH 5.7, while strain ORI could not survive under this circumstance. The differentially expressed genes between the strain LA57 group and the strain ORI group were mainly enriched in necroptosis, among which RIPK1/RIPK3/MLKL demonstrated a significant difference in expression. The cell experiment results showed that the cell proliferation ability of the oxaliplatin and strain LA57 co-treatment group was significantly higher than that of the oxaliplatin and strain ORI co-treatment group. The RT-qPCR and Western blotting results showed that compared with strain ORI, strain LA57 demonstrated enhanced inhibitory effects on the oxaliplatin-induced up-regulation of the necroptotic molecules RIPK1/RIPK3/MLKL at both mRNA and protein levels. The results of in vivo experiments in mice showed the same trend. [Conclusion] Acid-adapted F. nucleatum promotes the resistance of colorectal cancer cells to oxaliplatin chemotherapy by inhibiting necroptosis.

Abstract:[Background] Skin microbiome is a critical component of the multi-dimensional barrier function of the skin and plays a pivotal role in the immune and regulatory systems. Skin fungi, an essential part of this microbiome, are crucial for maintaining microecological balance of the skin. However, the research on skin fungal communities remains limited. [Objective] To investigate the community diversity and composition of skin fungi at different sites of the human body, laying a theoretical foundation for deeply understanding the structures and functions of skin fungi and their intrinsic relationships with skin diseases. [Methods] High-throughput sequencing of the ITS was employed to analyze the fungal community diversity in 105 skin microbiome samples collected from the forehead, inner forearm, and scalp of 35 healthy adult volunteers in Guangzhou. Additionally, we conducted bioinformatics analysis to investigate the fungal community structures at different skin sites. [Results] A total of 6 885 414 clean reads and 1 866 operational taxonomic units were obtained from 105 samples, which were classified into 366 species belonging to 189 genera of 13 phyla. The alpha diversity analysis revealed that fungal diversity and richness were the highest on the inner forearm, moderate on the forehead, and the lowest on the scalp. The beta diversity analysis showed similar fungal communities between inner forearm and forehead, while the scalp exhibited a significant different fungal community from the other two sites. Although the skin fungi at all the three sites were dominated by Ascomycota and Basidiomycota at the phylum level and by Malassezia, Fusarium, and Aspergillus at the genus level, there were significant differences in the relative abundance of the main phyla, genera, and species between the scalp and the other two sites. Specifically, Malassezia at the genus level and Malassezia restricta and M. globosa at the species level served as key biomarkers distinguishing the scalp fungal community from the forehead and inner forearm fungal communities. [Conclusion] Significant differences in fungal diversity and community composition were observed among different regions of human skin, indicating that skin fungi exhibit distinct site-specific characteristics and dependencies.

Abstract:Emerging contaminants (ECs) present significant challenges for global environmental management, with river sediments serving as their primary accumulation site. Microorganisms, as the key drivers of substance degradation and transformation in sediments, directly influence the environmental behavior, fate, and ecological risks of ECs. Although many studies have focused on the degradation of free and readily bioavailable ECs via microbial respiratory metabolism, a large proportion of these ECs in natural river sediments exist bound to dissolved organic matter (DOM), and the mechanisms governing their microbial degradation and transformation remain unclear. Given the occurrence form, microorganisms may indirectly mediate the degradation and transformation of ECs by regulating DOM transformation. From this novel DOM perspective, we systematically explored four potential degradation and transformation mechanisms: (1) cometabolic degradation of ECs via microbial metabolism of DOM, (2) microbial-induced DOM-driven photodegradation of ECs, (3) cleavage of ECs by DOM via microbial-driven direct electron transfer, and (4) microbial-driven DOM-mediated humification transformation of ECs. Finally, we analyzed the research limitations of these mechanisms, and proposed future research directions based on the existing advances, aiming to provide new insights for the development of efficient bioremediation technologies for ECs in river sediments.

Abstract:With the advancement of high-throughput sequencing and bioinformatics, microbiome analysis has become increasingly mature, becoming a technical tool in forensic science. Microorganisms are characterized by high diversity, large abundance, strong environmental specificity, and regular succession of community structure. Consequently, the microbiome, as a novel type of biological evidence, holds potential for application in routine forensic casework and has emerged as a new focus in the research and development of technologies in forensic science. This paper reviews the recent research progress in the application of microbiome in forensic science, including regional traceability, body fluid identification, individual identification, and postmortem interval estimation, aiming to help readers better understand the current status of microbiome applications in forensic science.

Abstract:The discovery and application of antibiotics have greatly contributed to the protection of human health and the development of animal husbandry. The abuse of antibiotics, however, has produced clinically resistant strains and superbugs, which have brought potential safety hazards to public health. Antimicrobial peptides kill bacteria rapidly and are not prone to drug resistance, thus being regarded as ideal antibiotic substitutes. Genetic engineering has brought opportunities for the industrial production of antimicrobial peptides. Microbial expression systems, such as Escherichia coli, Bacillus subtilis, Lactococcus lactis, Saccharomyces cerevisiae, and Pichia pastoris, have been developed for the preparation of antimicrobial peptides. However, the yields of recombinant antimicrobial peptides are not ideal due to the small molecular weights, easy degradation by proteases, and toxicity to host cells. This paper systematically discusses several strategies for yield enhancement, such as optimizing codon, increasing gene copy number, and developing co-expression strategies and tag fusion strategies, which, together with fermentation process optimization, can improve the yields of recombinant antimicrobial peptides.

Abstract:The remediation of heavy metal (HM)-contaminated soils is a significant global challenge in environmental protection. Phytoremediation technology has garnered considerable attention due to its eco-friendly nature, with root exudates serving as critical regulatory factors. They can optimize the rhizosphere microenvironment and modulate the bioavailability of HMs, thereby facilitating the phytoremediation process. However, the efficiency of phytoremediation is often constrained by HM concentrations and soil environmental conditions. In recent years, plant-beneficial bacteria (PBB) have been extensively studied as an auxiliary strategy. PBB not only enhance plant tolerance to HM stress but also improve phytoremediation efficiency by altering the chemical forms and mobility of HMs. Despite the initial progress achieved in collaborative remediation by plants and PBB, their interaction mechanisms and ecological functions remain insufficiently elucidated. This review focuses on the functional roles and synergistic interaction mechanisms of root exudates and PBB in the remediation of HM-contaminated soils and systematically evaluates the applicability of plant-microbe combined remediation technologies in typical polluted environments, such as farmlands and mining areas, aiming at providing theoretical support for the development of efficient and sustainable soil remediation strategies and exploring their potential in environmental remediation.

Abstract:Molnupiravir, a prodrug of the novel nucleoside analog N-hydroxycytidine, has broad-spectrum antiviral activity and can significantly inhibit the replication of pathogens such as influenza virus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and mpox virus. Both in vitro and in vivo studies have shown that N-hydroxycytidine inhibits viral proliferation by inducing lethal mutations in the viral RNA-dependent RNA polymerase during replication. However, the process by which molnupiravir is converted to N-hydroxycytidine in vivo to exert its antiviral effect may interfere with the normal life activities of host cells. This article systematically reviews the antiviral mechanism of molnupiravir converting to N-hydroxycytidine and its potential impact on cell activity, providing reference information for medical workers or researchers employing molnupiravir to treat viral infections.

Abstract:[Background] Microorganisms in natural environments often form biofilms, which enhance their resistance to environmental stresses. Extreme ecosystems, such as hydrothermal vents and caves, provide unique settings to investigate the structures, functions, and evolution of biofilms. [Objective] This study aims to reveal the research trends of biofilms in extreme environments regarding the subject fields involved, research progress in various countries, citations of relevant journals, and co-occurrence of key words in research hotspots. This is important for a comprehensive understanding of the status quo and direction of research on biofilms in extreme environments. [Methods] Web of Science Core Collection was searched for the articles related to biofilms in extreme environments that were published in recent 10 years. CiteSpace was used for statistical analysis. [Results] Since 2014, the number of publications in this field has been growing, and impactful journals and countries have played a role in advancing the development of this field. The co-occurrence of keywords indicated that biofilm formation was a research hotspot. Bacteria, Escherichia coli, sp. nov., and diversity etc. were the research focuses, and adaptation, antibiotic resistance, community, and ecology etc. represented potential research directions. Keyword clustering showed the latest clusters cluster#3 Enterococcus, cluster#7 nitrification, and cluster#12 toxic compounds, which indicated new research directions. The keywords bursts indicated that system, gene expression and DNA would continue to be of concern for a long time to come. [Conclusion] This article provides an overview of current research on biofilms in extreme environments and lays a foundation for identifying future directions and innovative approaches in studying biofilms.

Abstract:In order to address the disconnection between theory and practice in the teaching of Biochemistry under the emerging engineering education framework, a blended teaching model incorporating engineering practice was developed. The instructional program underwent a revision process, with the objective of incorporating observable indicators for the cultivation of engineering skill. The establishment of a cross-curricular knowledge system was undertaken with the objective of dismantling disciplinary barriers. The integration of virtual and real teaching scenarios has been designed to enhance engineering thinking. A three-dimensional practical platform encompassing scientific research, social applications, and biomimetic design was established. Concurrently, a dual-track assessment system incorporating engineering ethics and social responsibility education was implemented. The implementation outcomes demonstrated significant improvements in students' practical ability in engineering practice, with doubled disciplinary competition awards. The rate of qualification in corporate internships exhibited sustained annual increases, and the course satisfaction exceeded 95%. The present study lends further credence to the notion that the engineering practice-incorporated blended teaching model of Biochemistry is an efficacious means of cultivating bioengineering talents. In addition, it provides an implementable paradigm for engineering education accreditation and industry-education integration.

Abstract:Microbial Resources and Utilization is a degree course for professional degree graduate students majoring in resource utilization and plant protection. To address the common problems of graduate education in curriculum contents, teaching modes, and practical training, and to meet the learning needs of contemporary graduate students and their demand for access to high-quality teaching resources, the teaching team has carried out systematic explorations and practices in course system construction, teaching mode innovation, and assessment optimization. Through the construction of a modular course structure, ideological and political elements are integrated into the entire teaching process. Online academic conference videos and innovative use of WeChat official accounts have been employed to enrich teaching resources. A multi-dimensional assessment system incorporating process-based evaluation was also implemented. After three years of practice, these measures have significantly improved teaching quality, fostered students' research thinking, innovation awareness, and practical abilities, and gained widespread recognition. This course serves as a model for course construction in cultivating high-level application-oriented talents in the field of resource utilization and plant protection in the context of emerging agricultural education.

Abstract:The Animal Infectious Disease course is a core specialized course, mainly teaching the animal diseases caused by microorganisms. In the context of the integration of research, production, and education, how to enhance students' innovation consciousness is an important issue in vocational education. Taking the Animal Infectious Disease course as an example and considering the current epidemic situations of major animal diseases, we explored the factors of new quality productive forces and incorporate new technologies and new business forms from the workflow of typical positions and follow the requirements of integrating job, course, competition, and certification. The positions of clinical veterinary technician, laboratory tester, biosafety officer and the work processes of epidemiological diagnosis, clinical diagnosis, dissection, confirmed diagnosis, and control were considered in the reconstruction of the progressive course modules. Furthermore, ideological and political education was integrated into the teaching, and the learning situations and teaching objectives were clarified. The supervisors of both the school and other schools were invited to establish training bases in and out the campus. Blended teaching was implemented, and a three-dimensional classroom for disease prevention and control education was created. A vibrant classroom being task-oriented was created, implementing the "three-stage six-step" teaching strategy of exploration before class (clarifying tasks), practicing in class (initiating thinking, learning knowledge, practicing skills, and evaluating effectiveness), and expanding after class (expanding applications). The teaching framework internalized students' basic knowledge of microbiology, strengthened their ability to prevent and control diseases, and strengthened their belief in strengthening agriculture and promoting animal husbandry. This provides a theoretical foundation and practical experience for implementing the policy of integrating research, production, and education in micro courses and cultivating high-skilled talents with innovation consciousness and practical application ability.

Abstract:[Background] Riemerella anatipestifer (RA) causes enormous losses to the poultry industry due to its multiple serotypes and lack of effective cross protection. Therefore, establishing a rapid detection method to identify the serotypes of prevalent RA strains and preparing serotype-specific vaccines for immunization are of great significance for preventing RA infection. [Objective] To establish an ELISA method for detecting antibodies in the sera of ducks infected with RA serotype 2 strains or vaccinated with the serotype 2 vaccine. [Methods] The lipopolysaccharide (LPS) of RA serotype 2 strains was extracted by the hot phenol-water method and then used for coating the ELISA plate. By optimizing the antigen coating conditions, dilution ratio and incubation conditions of the serum sample and the HRP-conjugated anti-duck secondary antibodies, and substrate development time, we established an indirect ELISA method for detecting antibodies of RA serotype 2. [Results] The optimal reaction conditions were determined as follows: 5 μg/mL LPS as the coating antigen, PBS as the coating solution, 100 μL/well, coating ELISA plates at 4 ℃ for 12 h; 5% skimmed milk powder as the blocking solution, 200 μL/well, blocking at 37 ℃ for 2 h; serum sample dilution of 1:400 and incubation for 1 h; Rabbit Anti-Duck IgG (H & L)-HRP (secondary antibody) dilution of 1:10 000 and incubation for 1 h; color development time of 5 min. The highest P/N value was obtained under the optimal reaction conditions. By testing 80 serum samples from healthy non-immunized ducks, we established the positive judgment criterion as OD₄₅₀≥0.151 based on the mean (X)+3 times of standard deviation (SD). The specificity test of the established method showed positive results for the RA serotype 2-positive serum but negative results for the RA serotype 1, 6, 7, and 10-positive duck sera, avian pathogenic Escherichia coli-positive duck sera, avian Pasteurella-positive duck sera, avian Salmonella-positive duck sera, reovirus-positive duck sera, and duck hepatitis-positive duck sera. The sensitivity test showed that the limit of detection for positive duck sera was dilution factor > 50 000. The repeatability test showed that the inter- and intra-batch coefficients of variation of the method were less than 10%. The established ELISA method showed a 100% positive result for the serum samples from RA serotype 2-infected ducks on day seven post infection. It generated a 80% positive result for antibodies on day 14 and a 100% positive result on day 28 post vaccination with the inactivated vaccine against RA serotype 2. [Conclusion] The established method fills the gap in detection of RA serotype 2 antibodies and provides effective data support and a scientific basis for the epidemiological investigations and vaccine development of RA, showing both theoretical and practical values.

Abstract:[Background] Actinomycetes are an important reservoir for drug discovery, with Streptomyces being the primary source. However, the repeated discovery of Streptomyces strains is common, and rare actinomycetes have gained increasing attention. Nonomuraea is a rare actinomycete genus with significant potential for secondary metabolite production. However, targeted isolation of Nonomuraea strains is challenging. [Objective] By analyzing the antibiotic resistance genes of 263 known strains of Nonomuraea, we aim to identify suitable antibiotics and their concentrations as selective pressures to explore a targeted method for isolating Nonomuraea. This approach is designed to improve the isolation rate of Nonomuraea strains. [Methods] Genome mining was conducted to identify resistance genes in Nonomuraea. Both uncultured and cultured methods were employed to assess the differences in antibiotic resistance between Nonomuraea and Streptomyces. Suitable antibiotics and specific concentrations were selected as selective pressures to enhance the isolation of Nonomuraea strains. [Results] Genome mining revealed that Nonomuraea species commonly harbored genes conferring resistance to antibiotics such as aminoglycosides (e.g., gentamicin), β-lactams (e.g., ampicillin), macrolides (e.g., erythromycin), glycopeptides (e.g., vancomycin), and rifamycins (e.g., rifampin). The results from unculture experiments showed that the relative abundance of Nonomuraea under the selection pressure of gentamicin sulfate was significantly higher than that in the blank control and other antibiotic treatment groups. The culture experiment results demonstrated that Nonomuraea exhibited stronger resistance than Streptomyces to 4–16 μg/mL gentamicin sulfate and 2–4 μg/mL vancomycin. On the ISP 3 plates containing 2 μg/mL vancomycin, 124 strains were isolated, including 26 strains belonging to 12 species of Nonomuraea, with an isolation rate of 20.97%. On the ISP 3 plates containing 4 μg/mL gentamicin sulfate, 105 strains were isolated, including 44 strains belonging to 7 species of Nonomuraea, with a high isolation rate of 41.90%. [Conclusion] Leveraging genome mining, we identified high-frequency antibiotic resistance genes in Nonomuraea. By selecting the appropriate antibiotics and concentrations as selective pressures, we established a targeted isolation method for rare actinomycetes of Nonomuraea.

Abstract:[Background] Porcine rotavirus (PoRV) is one of the major pathogens causing porcine diarrhea, showcasing an increase in the infection rate recently. It is necessary to strengthen the clinical surveillance of PoRV. [Objective] To facilitate rapid detection and antibody screening in clinical practice, an indirect ELISA method targeting the VP6 protein of the prevalent PoRV strain was established. [Methods] The VP6 gene of the PoRV genotype G9P[23]I5 was cloned and expressed in a prokaryotic expression system, and the recombinant protein was purified. With the recombinant VP6 protein as the capture antigen, the checkerboard titration test was performed for PoRV-positive serum samples of pigs. The parameters of the indirect ELISA method were optimized, and the specificity, sensitivity, and repeatability of the method were examined. [Results] The parameters of the indirect ELISA method were optimized as follows: the VP6 antigen coating amount of 0.1 μg/well and coating overnight at 4 ℃; blocking with 5% skim milk at 37 ℃ for 2 h; positive serum dilution at 1:1 000 and incubation at 37 ℃ for 1 h; Goat pAb to Pig IgG H & L (HRP) at 1:15 000 and incubation at 37 ℃ for 1 h; color development with 3, 3', 5-5'-tetramethylbenidine (TMB) for 15 min. The established method showed the OD₄₅₀ cutoff value of 0.42 and the sensitivity of 1:8 000. It demonstrated great specificity, with the intra-batch and inter-batch coefficients of variation both less than 10%. A total of 130 clinical serum samples were detected by the established method and commercially available PoRV ELISA assay kit, which showed the result coincidence rate of 93.85%. The sensitivity of the indirect ELISA method established in this assay was higher than that of the commercially available PoRV ELISA assay kit. [Conclusion] The indirect ELISA method established in this study enables the rapid detection and large-scale screening of PoRV antibodies.