Abstract:Phosphate-binding loop nucleoside triphosphate hydrolase (P-loop NTPase) are a class of conserved proteins widely present in organisms, characterized by a conserved P-loop motif that is responsible for binding and hydrolyzing ATP or GTP. The GPN family (GPN-loop GTPase family) is a newly discovered subfamily of P-loop NTPase with GTP-hydrolyzing activity, characterized by a conserved glycine-proline-asparagine (GPN) motif. In eukaryotes, the GPN family includes three members: Gpn1, Gpn2, and Gpn3. Among them, Gpn1 (also known as Npa3) in Saccharomyces cerevisiae, a classic model organism, has been extensively studied. Homologs of Gpn2 (Yor262W) and Gpn3 (Ybr016W) are also present in the yeast genome. It is noteworthy that the GPN-loop domain is highly conserved throughout evolution and is essential, particularly in the assembly and nuclear transport of RNA polymerase Ⅱ (RNAP Ⅱ). Focusing on the S. cerevisiae model, this article systematically reviews the structural characteristics of the GPN family proteins and their molecular mechanisms of influencing transcription by regulating the assembly of RNAP Ⅱ and then explores the role of this family in cancer development. This review provides a theoretical reference for the analysis of eukaryotic transcriptional regulatory networks and new ideas for the development of therapeutic targets for related diseases.

Abstract:Soil salinization is one of the serious problems in arid and semi-arid areas, inhibiting plant growth and reducing crop yields. Arbuscular mycorrhizal (AM) fungi as beneficial microorganisms in the soil help plants to relieve the harm caused by salt stress and improve plant salt tolerance. This paper reviews the harm of salt stress to plants, the effects of AM fungi on plant growth under salt stress, and the improving mechanisms of AM fungi for plant salt tolerance, and proposes the future research perspectives. This review is expected to provide new ideas for the enhancement of plant salt tolerance by microorganisms and the application of microorganisms in the improvement and utilization of salinized soil.

Abstract:Coal is one of the important energy substances in China. However, the exploitation of coal resources can cause serious eco-environmental problems in coal mining areas. With the improvement of environmental protection consciousness, the ecological restoration of coal mining areas has become a research hotspot in environmental ecology. Microorganisms, an important part of the soil environment, are of great significance to the biochemical cycling of carbon, nitrogen, phosphorus, sulfur and other elements and pollutant management in the deteriorated ecological areas due to coal mining. This paper introduces the roles and mechanisms of microorganisms in the ecological restoration of coal mining areas, especially the role of microorganisms in heavy metal pollution control and site substrate improvement. Through the practical application of microorganisms in ecological restoration of coal mining areas, we analyze the problems in the application of microorganisms in the restoration of deteriorate ecological areas due to coal mining and look forward to the future research directions.

Abstract:Background Saccharopolyspora is an important genus of rare actinomycetes. Strain BM8-3, isolated from sediments of the Mariana Trench, exhibits strong salt tolerance and antagonistic activity against pathogens. Currently, studies are limited regarding the functional genes, genomics, and metabolite biosynthesis of Saccharopolyspora from deep sea environments.Objective To analyze the genome sequence and functional genes and evaluate the secondary metabolite synthesis of Saccharopolyspora sp. BM8-3, thus laying a foundation for exploring the strategies of this strain for adapting to extreme environments and mining the gene resources involved in the synthesis of novel secondary metabolites.Methods The whole genome of strain BM8-3 was sequenced on the PacBio platform. Genome assembly was performed by SMRT Link 5.0.1, and gene prediction and functional annotation were conducted with cluster of orthologous groups of proteins (COG) and Kyoto encyclopedia of genes and genomes (KEGG).Results The genome of strain BM8-3 had a total length of 6 344 421 bp, with the G+C content of 72.5%. A total of 5 620 genes, 51 tRNA genes, and 12 rRNA genes were annotated. The annotation against COG and KEGG predicted 4 137 and 2 677 genes, respectively. The genome of strain BM8-3 contained rich genes related to osmotic pressure and high hydrostatic pressure adaptation, including genes involved in the synthesis and transport of compatible solutes such as trehalose (otsAB), glycine betaine (betAB), ectoine (ectABCD), and dimethylglycine/sarcosine (gsmt-sdmt). Additionally, the genome contained genes associated with sodium and potassium ion transport systems (e.g., multiple resistance and pH-related antiporter) and organic solute transport channels (e.g., MscL). Furthermore, the genes associated with cold adaptation were annotated, including cspA encoding cold shock protein, genes encoding the GroEL-GroES chaperonin system, and multiple genes involved in the metabolism of unsaturated fatty acids (e.g., desC and fabF). KEGG annotation revealed that strain BM8-3 possessed a complete taurine transport and metabolism pathway (TauABCD) as well as a thiamine salvage pathway. Additionally, 24 biosynthetic gene clusters (BGCs) for secondary metabolites were predicted in the genome, with more than 50% showing less than 60% similarity to known BGCs. This finding suggested the potential of strain BM8-3 to produce novel secondary metabolites.Conclusion We analyzed the whole-genome sequence of Saccharopolyspora sp. BM8-3, revealing its metabolic diversity and adaptation mechanisms in extreme environments. This study provides critical insights into the environmental adaptation strategies of deep-sea actinomycetes and their roles in sulfur cycling.

Abstract:Background In recent years, the pollution of antibiotics in natural water bodies has been aggravating. Because antibiotics can kill bacteria, they change the sedimentation and flocculating properties of sludge and affect the types of microorganisms living in the water, making it harder to remove pollutants.Objective Screening of a strain of denitrifying phosphorus-polymerizing bacteria for simultaneous removal of nitrogen and phosphorus, investigation of its growth under oxytetracycline stress and optimization of reaction parameters for the preparation of microbial agents.Methods In this study, we picked out, tested, and purified a strain P8 under oxytetracycline stress from a denitrifying and phosphorus- accumulating system which had been acclimated for a long time. Based on the 16S rRNA gene sequence and physicochemical properties identify this strain. Single factor tests and response surface methodology were employed to optimize the denitrifying and phosphorus-removing conditions of this strain and preparation of microbial agents by fermentation method.Results The results showed that this strain was identified Enterobacter chengduensis. And it could grow normally under oxytetracycline stress. The strain was able to remove 69.6% of total phosphorus and 96.7% of nitrate nitrogen under the conditions of 3.32 g/L sodium acetate as the carbon source, pH 7.0, an initial phosphorus level of 10 mg/L, and 30 ℃. The microbial agent carrier was composed of wheat bran and corn flour in an 8:2 mass ratio, with optimal efficacy observed at an inoculation volume of 20 mL.Conclusion We screened out a strain E. chengduensis P8 that can simultaneously remove nitrogen and phosphorus and good growth under oxytetracycline stress. This work provides new resources and scientific support for using microorganisms to remedy the environments polluted by oxytetracycline.

Abstract:Background With the increasing depletion of peat resources, developing alternative substrates is of great significance for sustainable cultivation.Objective In the trials the amplicon sequencing is used to analyze the rhizosphere bacterial community structure and functional characteristics of strawberry plants grown on compound substrates containing varying proportions of food solid waste fermentation feedstock, aiming to provide a scientific basis for developing a novel strawberry cultivation substrate.Methods Five treatment groups (ASS1–ASS5) and one control group (ASS0) were designed with peat, perlite, and vermiculite as base materials, supplemented with food solid waste fermentation feedstock at 0%, 10%, 20%, 30%, 40%, and 50%, and the bacterial community structures and functions in different treatments were analyzed.Results Compared with the control (ASS0), treatment group showed increased electrical conductivity (EC), organic matter (OM), available nitrogen (AN), available phosphorus (AP), and available potassium (AK) in the rhizosphere substrates (P < 0.05). Among the treatment group, the strawberry plants in the ASS3 and ASS2 exhibited higher growth traits, yield per plant, single fruit weight, soluble sugar content, and vitamin C content. In the rhizosphere, the relative abundance of the main dominant phylum Proteobacteria showed an increasing trend, except in the ASS4 treatment, while the relative abundances of Actinomycetota and unidentified Bacteria decreased. Among the dominant genera, the relative abundances of Pseudomonas, Hirschia and Caulobacter exhibited an increasing trend. With increasing proportions of food solid waste fermentation feedstock, the α diversity of bacterial communities in the strawberry plant rhizosphere decreased. MetaStats analysis identified that Ketobacter, Salinicola, Alloprevotella, and Pseudomonas were enriched in the rhizophere of different treatments (P < 0.01). The Mantel test indicated that pH, EC, OM, and AK were key drivers of changes in the rhizosphere bacterial community structure. Spearman correlation analysis revealed functional divergence impacts of rhizosphere bacteria on strawberry plant agronomic traits, which were manifested as comprehensive growth promotion, photosynthesis-driven enhancement, architectural modulation, growth inhibition, yield improvement, and coordinated regulation of acid-sugar metabolism. Hirschia demonstrated growth-promoting, yield and fruit quality enhancing effects, making it a potential beneficial microbial resource for strawberry cultivation. Others, the uncharacterized bacterial genera, hold significant potential for future resource exploration. Tax4Fun-based functional prediction analysis revealed significant functional differences in the strawberry rhizosphere bacterial communities across different treatments. With increasing proportions of food solid waste fermentation feedstock, metabolic genes abundances were generally down regulated while unannotated functional genes showed up regulated abundance, concurrently elevating strawberry disease risk. ASS3 and ASS2 treatments demonstrated superior performance in key growth traits, yield and quality indicators. Specifically, substrate formulations incorporating 20%–30% food waste fermentation feedstock exhibit considerable application potential. For strawberry cultivation substrates amended with food waste fermentation feedstock should be formulated at appropriate ratios; Otherwise microbial activity and strawberry agronomic traits may be adversely affected.Conclusion Insights into the structure and function of the strawberry rhizosphere bacterial community provides a valuable reference for developing novel cultivation substrates.

Abstract:Background Dye-decolorizing peroxidases (DyPs) are members of the heme peroxidase family, and their catalytic activities depend on a non-covalently bound heme cofactor. DyPs are able to degrade a variety of toxic and harmful dyes, serving as promising biocatalysts. However, the low efficiency of heme binding to the apo-enzyme in the production of recombinant DyPs limits their catalytic activities.Objective To enhance the binding efficiency of heme with the apo-enzyme, we selected mutation sites based on structural information analysis and molecular docking to improve the enzyme activity.Methods Mutagenesis was performed on RhDyPB derived from Rhodococcus jostii RHA1 to obtain the high-activity mutant E156K, which bound more heme cofactors. The enzymatic properties and kinetic parameters of E156K were analyzed, and the binding capacity of RhDyPB and E156K to heme was evaluated.Results The heme binding capacity and specific activity of E156K were 14.14 nmol/mg and 19.5 U/mg, respectively, which were 57.5% and 56.0% higher than those of RhDyPB. This indicated higher heme binding capacity had a positive impact on the catalytic activity of this enzyme, while the optimal pH and temperature had no obvious difference between E156K and RhDyPB. Finally, the catalytic activity of E156K was further enhanced through fermentation condition optimization, 50.3% higher than that without optimization.Conclusion This study enhances the catalytic efficiency of RhDyPB by rational design based on the heme binding capacity, providing a basis for the molecular modification and industrial application of DyPs.

Abstract:Background Siderophores are small molecules that can specifically chelate trivalent iron ions, which not only maintain the balance of intra- and extracellular iron ions of bacteria but also inhibit pathogenic microbes through competitive and antibiotic effects.Objective The function of the siderophore gene in Bacillus velezensis B9601-Y2 (Y2) remains unclear. Therefore, we constructed a dhbA mutant of bacillibactin to study the effects of bacillibactin on the phenotype and biocontrol activity (against Exserohilum turcicum) of Y2. The results are expected to provide a new theoretical basis for regulating the biofilm formation and biocontrol activity of Y2.Methods The biosynthetic gene cluster gene member dhbA of bacillibactin was knocked out, and the phenotype, growth curve, swarming motility, and biofilm formation of the mutant ΔdhbA were observed and measured quantitatively. Chrome azurol S was used to measure the relative siderophore production of the wild type (WT) and ΔdhbA. Plate confrontation and in vitro inoculation methods were employed to explore the inhibitory effects of WT and ΔdhbA on the colony growth of E. turcicum.Results After the knockout of dhbA, B. velezensis B9601-Y2 showed whitening colonies, with the swarming motility, biofilm formation, and siderophore production decreasing by 51.9%, 19.5%, and 93.5%, respectively. In addition, the diameters of the inhibition zones of the cells and lipopeptide crude extract of ΔdhbA against E. turcicum decreased by 31.9% and 42.5%, respectively, compared with those of WT. Furthermore, the knockout of dhbA decreased the control efficacy on E. turcicum of detached maize leaves by 75%.Conclusion This study proved that dhbA was one of the indispensable genes for the biocontrol function of B. velezensis B9601-Y2, and the knockout of this gene negatively affected the normal growth, swarming motility, biofilm formation, siderophore synthesis, and biocontrol activity of Y2.

Abstract:Background Vibrio parahaemolyticus (Vp) is a major zoonotic pathogen that can cause foodborne gastroenteritis. The type Ⅲ secretion system (T3SS) is a complex nanomachine that delivers effectors into eukaryotic cells. V. parahaemolyticus possesses two distinct T3SSs: T3SS1 and T3SS2. VopD as a translocator of T3SS1 is involved in the delivery of effectors. However, the biological function of VopD remains unclear.Objective To investigate the role of VopD in the pathogenic mechanism of V. parahaemolyticus and the influence of VopD on the biological characteristics of the pathogen.Methods With POR-3 as the parental strain, a vopD-deleted mutant and a complementary strain were constructed by homologous recombination. The effects of vopD deletion on bacterial growth, biofilm formation, hemolytic activity, and cell adhesion were analyzed. The toxicity of vopD-deleted and complementary strains to HeLa cells was examined. Furthermore, we prepared polyclonal antibodies to detect the secretion of VopD via T3SS1.Results The deletion of vopD did not significantly affect the growth but significantly reduced the biofilm formation, hemolytic activity, and cell adhesion of V. parahaemolyticus. Compared with POR-3, the vopD-deleted mutant demonstrated significantly diminished toxic effects on HeLa cells 60, 90 and 120 min after infection, while the toxicity of the complementary strain was restored to the level of the wild type. VopD was successfully expressed via a prokaryotic expression vector, purified, and used to immunize mice for the preparation of polyclonal antibodies. Western blotting showed that the secreted VopD was not detected in the vopD-deleted strain or the T3SS1 function-deleted strain POR-2, while it was successfully detected in POR-3, RIMD 2210633, and the vopD complementary strain, which indicated that VopD was secreted via T3SS1.Conclusion vopD is involved in regulating the biofilm formation, hemolytic activity, and cell adhesion of V. parahaemolyticus and influences the cytotoxicity of this pathogen towards HeLa cells. The generated polyclonal antibodies effectively detect the secretion and expression of VopD.

Abstract:Background There are evidences that certain rhizospheric microorganisms can enhance crop growth under salt stress.Objective To isolate and select salt-tolerant strains from the rhizosphere of plants in the saline-alkaline soil of Lanzhou and Jingtai in Gansu Province and evaluate their plant growth-promoting characteristics.Methods Strains H3 and S10 were identified based on salt tolerance, physiological and biochemical characteristics, and 16S rRNA sequences. The plant growth-promoting effects of the two strains on wheat (Triticum aestivum) seedlings were evaluated under 150 mmol/L NaCl.Results Strains H3 and S10 exhibited the NaCl tolerance of 11% and 21%, respectively. They were capable of fixing nitrogen, solubilizing phosphorus and potassium, and producing siderophores and indole-3-acetic acid. Strain H3 was identified as Erwinia and S10 as Kushneria. Co-inoculation significantly enhanced the growth of wheat seedlings compared with inoculation of single strains under either normal or salt stress conditions, leading to increased accumulation of soluble substances and improved antioxidant enzyme activities. Moreover, co-inoculation was more effective in alleviating the adverse effects of salt stress on wheat seedlings.Conclusion Both co-inoculation and inoculation of single strains can significantly enhance the salt tolerance of wheat seedlings, and co-inoculation is more effective.

Abstract:Background Drechslera avenacea is the main pathogen of oat leaf spot, while the insufficient sporulation of this fungus has seriously affected the research progress in the interactions between oat and D. avenacea.Objective To screen the most suitable medium and culture conditions for the growth and sporulation of D. avenacea, and to provide a basis for the research and control of oat leaf spot caused by D. avenacea.Methods We determined the optimal medium, carbon source, and nitrogen source of D. avenacea, as well as the sporulation in the V8 medium under different temperatures, pH values, and ultraviolet (UV) irradiation time.Results The mycelial growth and sporulation of the three strains were different under different media, carbon and nitrogen sources, temperatures, pH values, and UV irradiation time. After 12 days of culture, the colony diameters of strains hsbzb and plpjg were more than 69 mm in the media with maltose as the carbon source and more than 77 mm in the media with peptone as the nitrogen source. Strain dttz had the largest colony diameter of 38.22 mm when maltose was used as the carbon source and 38.52 mm when peptone was used as the nitrogen source. After 4 weeks of growth in the V8 medium, the sporulation and colony diameters of the three strains showed consistent trends with the rise in temperature, and the optimal temperature was 25 ℃. An acidic environment (pH 4.5) was conducive to sporulation. UV irradiation promoted the sporulation of D. avenacea in a short period of time, and the optimal irradiation time was 8 h. Moreover, UV radiation induced the sporulation of strain dttz which did not produce spores.Conclusion This study clarified the optimal growth and spore production conditions of D. avenacea, providing key technical support for the efficient acquisition of pathogen spores, in-depth research on host-pathogen interactions, and the formulation of comprehensive prevention and control strategies for oat leaf spot disease.

Abstract:Background Dry rot is one of the diseases severely impacting pomegranate production, with increasing incidence and severity in Shaanxi Province.Objective To identify the pathogenic agents responsible for pomegranate dry rot in Shaanxi and evaluate their fungicide sensitivity.Methods The pathogenic agents were isolated from diseased pomegranate fruits by the spread plate method, and the target strains were identified by morphological characterization and multi-gene sequence analysis. The effects of temperature, humidity, light, medium, and nutrient on the growth of target strains were determined by the single-factor method. In addition, the fungicide sensitivity of each target strain was evaluated.Results Two pathogenic fungal strains ZGA1 and ZB2 were screened out and identified as Coniellus granati and Botryosphaeria dothidea, respectively. Pathogenicity tests confirmed that both strains could cause fruit lesions. The optimal growth conditions for strain ZGA1 were 25 ℃, 60% humidity, and potato dextrose agar, and those for strain ZB2 were 30 ℃, 60% humidity, and potato dextrose agar. The growth rates of both strains were positively correlated with light conditions. The results of the indoor toxicity test indicated that both strains displayed high sensitivity to difenoconazole and chlorothalonil. In contrast, they demonstrated strong resistance to carbendazim.Conclusion Both C. granati and B. dothidea are capable of causing pomegranate dry rot in Shaanxi Province. The findings offer valuable theoretical guidance for the effective prevention and management of this disease.

Abstract:Background Yaodu district, Linfen city, Shanxi province, is located in the core area of dryland agriculture in southern Shanxi. In recent years, the proportion of straw returned to the field here has been increasing, while the straw decomposition is slow due to the characteristics of brown soil which is clayey and compacted. Straw decomposition agents can improve the decomposition efficiency by enhancing microbial metabolism, while the effects of straw returning combined with a decomposition agent on soil microorganisms in the region are not well understood at present.Objective To clarify the effects of maize straw returning combined with a straw decomposition agent on the microbial community structure and function in brown soil.Methods We designed three treatments at the national agricultural microbial observation experimental site in Yaodu in the autumn of 2021. The three treatments were no straw returning (UC), direct straw returning (SR), and straw returning combined with a decomposition agent (SD). We employed metagenomic sequencing to analyze the microbial community structure and function in the 0-20 cm soil layer during the harvesting stage of wheat in 2024.Results Compared with SR, SD increased the wheat yield by 11.24% and the soil organic matter, available potassium, available phosphorus, and alkali-hydrolyzable nitrogen by 17.25%, 36.39%, 5.58%, and 9.87%, respectively, while reducing the disease index of wheat crown rot by 47.10%. Additionally, SD increased the relative abundance of Actinomycetota. At the genus level, SD elevated the relative abundance of Nocardioides and Chryseolinea. Characterization of carbon and nitrogen metabolism showed that the abundance of genes related to the citrate cycle pathway was higher in carbon fixation and that of genes involved in the assimilatory nitrate reduction pathway was higher in nitrogen metabolism. SD increased the abundance of functional genes involved in carbon fixation and nitrogen metabolism, which were closely related to Pseudomonadota, Actinomycetota, Acidobacteriota, Bacteroidota, and Nitrososphaerota.Conclusion SD enhanced soil organic matter and available nutrients and increased the abundance of soil microbial communities and functional genes, which provides a scientific basis for the popularization and application of decomposition agents.

Abstract:Background Cotton verticillium wilt is a highly detrimental disease to the cotton industry, severely affecting cotton quality and the economic well-being of cotton farmers.Objective To isolate and screen strains with high-efficiency disease resistance and growth-promoting properties, so as to provide microbial resources for the biological control of cotton verticillium wilt.Methods The dilution-plating method and plate confrontation method were used to screen strains with antagonistic effects against cotton verticillium wilt. Morphological analysis, physiological and biochemical tests, and molecular biology were employed to identify the taxonomic status of the strains. The strains were assessed for enzyme production, nitrogen fixation, and phosphate solubilization capabilities. Cultivation conditions were optimized, and the practical application effects of the strains in antifungal and growth-promoting activities were evaluated through cotton pot experiments.Results A strain of Bacillus velezensis F41-14 with a high inhibitory activity against Verticillium dahliae was isolated and screened, showing an inhibition rate of 76.32% and an indole-3-acetic acid (IAA) production of 4.15 mg/L. Strain F41-14 exhibited the ability to produce cellulase, protease, and amylase and solubilize phosphate. The optimal cultivation conditions were 72 h of incubation, 3% inoculum size, 30 ℃, and initial pH of 7.0. Pot experiments demonstrated that the antagonistic strain colonized the root system of cotton, promoted cotton plant growth, reduced the disease index, and achieved a control efficacy of 78.62%.Conclusion B. velezensis F41-14 exhibits significant antifungal and growth-promoting potential, which makes it a viable biocontrol strain against cotton verticillium wilt, with substantial practical value.

Abstract:Background Gastrodia elata is a precious and widely used Chinese medicinal herb closely associated with fungi during the whole growth period. However, the fungal diversity during the seed formation of this herb remains unknown.Objective To explore the composition and diversity of endophytic and rhizospheric soil fungal communities at different seed formation stages of G. elata.Methods The pure culture method was adopted to isolate endophytic fungi from tissues and rhizosphere soil of G. elata at different seed formation stages. Fungal identification was performed by ITS sequencing, which was followed by diversity analysis.Results A total of 452 fungal strains were isolated, including 334 strains of endophytic fungi and 118 strains of rhizosphere soil fungi, belonging to 47 genera, 29 families, 18 orders, 9 classes of 3 phyla. Ascomycota dominated at all the stages. During the initial planting stage, endophytic fungi were primarily composed of Trichoderma, Aspergillus, Penicillium, and Chaetomium, while rhizospheric soil fungi were dominated by Trichoderma, Aspergillus, Penicillium, and Talaromyces. At the seedling emergence stage, both communities were predominantly characterized by Penicillium, Aspergillus, Talaromyces, and Trichoderma. At the bud formation stage, Ilyonectria emerged as an additional dominant genus in endophytic fungi, while rhizospheric soil fungi showed new dominance of Diaporthe and Sarocladium. During the flowering stage, Chaetomium regained dominance in endophytic fungi, while Penicillium became the predominant genus in rhizospheric soil fungi. At the fruiting stage, both endophytic and rhizospheric soil fungi were primarily dominated by Trichoderma, Penicillium, Aspergillus and Fusarium. The diversity, evenness, and richness of endophytic fungi peaked during the emergence stage, while those of rhizosphere soil fungi reached the highest levels at the fruiting stage.Conclusion We preliminarily explored the structures and diversity of culturable endophytic and rhizosphere soil fungal communities of G. elata during seed formation. The findings provide foundational research materials and a theoretical basis for exploring endophytic and rhizosphere soil fungal resources associated with G. elata.

Abstract:Background Exploring indigenous microorganisms for innovative research and development of green investment brands is an important way to boost agricultural development in plateau watersheds.Objective To excavate the indigenous plant growth-promoting rhizobacteria (PGPR) in the Erhai Lake Basin and evaluate their plant growth-promoting properties, thus providing strain resources for the development of microbial agents.Methods A total of 12 vegetable soil samples were collected from 3 major grain and vegetable planting towns in the Haixi area of the Erhai Lake Basin. The dilution coating method, functional culture medium screening, physiological and biochemical property tests, and 16S rRNA gene sequencing, and strain-seedling potting, and soil enzyme activity measurement were conducted to isolate and identify the strains and evaluate their plant growth-promoting properties.Results A total of 28 bacterial strains with plant growth-promoting functions such as nitrogen fixation, phosphorus solubilization, siderophore production, and indole-3-acetic acid (IAA) secretion were isolated from the soil of vegetable fields in the Erhai Lake Basin. Among them, six strains with better performance were identified, belonging to Bacillus, Alcaligenes, Providencia, and Myroides. The six strains could solubilize organic phosphorus, solubilize inorganic phosphorus, and produce IAA within the ranges of 41.16–158.63, 190.84–280.50, and 15.03–186.24 mg/L, respectively. In the pot experiments with seedlings of maize and asparagus lettuce, representative crops of grain-economy collaboration, inoculation with the isolated strains significantly promoted the growth and development of the aboveground and underground parts of the seedlings. Specifically, the inoculation averagely increased the plant height and total root length of maize seedlings by 31.80% and 58.96%, respectively. Meanwhile, it averagely increased the plant height and root fresh weight of asparagus lettuce seedlings by 41.47% and 83.87%, respectively. Moreover, the application of the isolated strains activated mineral elements in the soil to varying degrees and significantly improved the activities of some soil enzyme activities compared with the blank treatment.Conclusion In the Erhai Lake Basin with limited application of chemical fertilizer and weak research and development of local bio-organic fertilizer, the six strains of PGPR screened from vegetable fields in this study can be used as strain resources for the development of local indigenous microbial inoculants. The strains demonstrate great application potential in promoting the growth and development of maize and asparagus lettuce, improving the soil nutrient utilization efficiency and soil enzyme activity, and reducing exogenous nutrient input.

Abstract:Background Plant growth-promoting rhizobacteria (PGPR) are a type of beneficial microorganisms that effectively colonize plant roots, enhancing the growth and development of host plants while assisting in the defenses against pathogenic microorganisms through the production of complex signaling molecules.Objective To isolate and screen the bacterial strains with growth-promoting effects from the rhizosphere soil of healthy Pogostemon cablin. fields, thereby providing microbial resources to improve both the yield and quality of medicinal plants including P. cablin.Methods The culture-dependent method was employed to isolate and identify microorganisms in the rhizosphere soil of P. cablin. Pot experiments were conducted to screen the strains with plant growth-promoting effects, and the specific plant growth-promoting effects of the strains screened out were evaluated. Additionally, the growth-promoting effects of single strains and strain combinations on potted P. cablin seedlings were validated.Results Strains YFJ3LB2, YFJ3PDA8, and YFJ1LB8 significantly enhancing the yield, leaf dry weight, leaf number, and chlorophyll SPAD value of P. cablin were screened out. However, these strains did not have a significant effect on the content of patchouli alcohol and pogostone. All the three strains could produce indole-3-acetic acid (IAA) and NH₃ and form biofilms. Notably, strain YFJ3LB2 can solubilize phosphorus. The application of strain combinations did not exhibit superior growth promotion effects. Under the treatment with strain YFJ3LB2, the plant height, stem fresh weight, leaf fresh weight, and stem dry weight increased by 24.70%, 20.05%, 21.77%, and 21.05%, respectively. Under the treatment with strain YFJ3PDA8, the plant height and plant dry weight increased by 24.70% and 22.07%, respectively. Under the treatment with strain YFJ1LB8, the plant height, plant fresh weight, stem fresh weight, leaf fresh weight, and plant dry weight increased by 43.48%, 42.38%, 25.92%, 53.00%, and 35.17%, respectively. The treatments with strains YFJ3LB2 and YFJ3PDA8 showed no significant effect on the content of patchouli alcohol and pogostone. However, the treatment with strain YFJ1LB8 led to a decrease in content of pogostone. Strains YFJ3LB2, YFJ3PDA8, and YFJ1LB8 were identified as Pseudomonas knackmussii, Bacillus altitudinis, and Variovorax guangxiensis, respectively.Conclusion The three PGPR strains of P. cablin have the abilities of IAA production, NH₃ production, and biofilm formation. They can significantly increase the plant height, plant fresh weight, plant dry weight, and leaf development, while having no substantial effect on the levels of patchouli alcohol and pogostone. The strains serve as valuable microbial resources for the cultivation of P. cablin.

Abstract:Background Sea cucumber cooking liquid (SCCL) represents a waste product difficult to be treated in the sea cucumber processing industry.Objective To investigate the potential application of SCCL in the cultivation of Cordyceps militaris by utilizing five different strains of this fungus.Methods We added different proportions of SCCL to the culture medium to analyze the effects of SCCL on the agronomic traits (including bioconversion rate and content of cordycepin and adenosine) of C. militaris. We then employed a NaCl solution with a concentration comparable to that of SCCL as the control to assess their effects on the agronomic traits of C. militaris and decipher the underlying mechanism.Results High- concentration SCCL and NaCl solutions exerted inhibitory effects on the growth of C. militaris, as evidenced by significant decreases in the bioconversion rates of the five strains under 0.3% NaCl treatment. However, notable differences in the tolerance of various strains to SCCL were observed, with NYC06 demonstrating strong tolerance, as indicated by the absence of a significant change in bioconversion rate compared with the control group when being exposed to a 60% SCCL solution. Both SCCL and NaCl solutions facilitated the synthesis of cordycepin in C. militaris, leading to a consistent variation trend in cordycepin content of the same strain. Notably, after treatment with both solutions, NYC06 showcased the highest increase in cordycepin content in the fruiting bodies and embryo among the five strains of C. militaris. The two solutions exhibited different impacts on the adenosine content of C. militaris. In particular, the SCCL solution demonstrated varied effects on adenosine content among different strains, whereas the NaCl solution had the potential to augment the adenosine level in C. militaris. This trend differed from the changes observed in the case of SCCL treatment.Conclusion We conclude that NaCl in SCCL is one of the factors influencing the fruiting body yield and cordycepin content of C. militaris. The findings lay a solid foundation for the subsequent industrial application of SCCL in C. militaris production to meet diverse demands.

Abstract:Background Porcine deltacoronavirus (PDCoV) is a porcine enteric coronavirus first reported in 2012, mainly causing gastrointestinal diseases characterized by diarrhea, vomiting, and dehydration in pigs. PDCoV infection is difficult to be distinguished from porcine epidemic diarrhea and porcine contagious gastroenteritis because of the similarity of their clinical symptoms. PDCoV has four structural proteins, and the spike (S) protein is located on the surface of PDCoV particles. The S protein consists of two subunits, S1 and S2, which are the main targets of virus-neutralizing antibodies.Objective To express the PDCoV S1 protein by the eukaryotic system and prepare polyclonal antibody against the purified PDCoV S1 protein.Methods The S1 gene of PDCoV CHN-JS-2018 strain was amplified by RT-PCR and ligated into the eukaryotic expression vector pCAGGS. Two eukaryotic expression plasmids named pCAGGS-PDCoV-S1-mFc and pCAGGS-PDCoV-S1-hFc were constructed and sequenced. Then the HEK293T cells were transfected with these two plasmids respectively, and the recombinant proteins were expressed, purified, and identified by indirect immunofluorescence assay (IFA) and Western blotting. The purified recombinant protein PDCoV-S1-mFc was used to immunize BALB/c mice, and polyclonal antibodies against it were prepared. The reactivity and titer of the polyclonal antisera were determined by IFA and indirect ELISA, respectively.Results The constructed eukaryotic expression plasmids pCAGGS-PDCoV-S1-mFc and pCAGGS-PDCoV-S1-hFc were expressed in HEK293T cells and the expressed proteins specifically bound to the positive serum of mice immunized with PDCoV. The titer of polyclonal antibodies against the recombinant PDCoV S1 protein reached more than 1:64 000, and the serum neutralization (SN) titer was 1:185, which indicated that the recombinant protein could be used in indirect IFA and Western blotting for detection of PDCoV.Conclusion The recombinant PDCoV S1 protein expressed by the eukaryotic expression system has strong immunogenicity and can induce mice to produce antibodies with a high titer. The prepared polyclonal antibody has good reactivity and neutralization activity. This study lays a foundation for the research on the diagnosis of PDCoV, preparation of monoclonal antibodies against PDCoV S1 protein, and development of subunit vaccines against PDCoV.

Abstract:Background Chlamydia psittaci is a zoonotic pathogen with diverse hosts, causing local or systemic diseases such as psittacosis. The oxidative stress of hosts is closely related to the Chlamydia infection process.Objective To explore the impact of C. psittaci infection on oxidative stress in HBE cells and the roles of miR-124-3p and nuclear factor I-B (NFIB) in this process.Methods HBE cells were infected with C. psittaci at a multiplicity of infection of 3. The levels of reactive oxygen species (ROS), superoxide dismutase (SOD), and malondialdehyde (MDA) and the expression levels of miR-124-3p and NFIB were measured to evaluate the impacts of C. psittaci infection on the oxidative stress and the expression levels of miR-124-3p and NFIB. After transfection with miR-124-3p inhibitor or miR-124-3p mimic or co-transfection with si-NFIB and miR-124-3p inhibitor, the levels of miR-124-3p and NFIB were measured to evaluate their correlation, and the levels of ROS, MDA, and SOD were determined to evaluate the impacts of miR-124-3p and NFIB on the oxidative stress induced by C. psittaci infection.Results The C. psittaci-infected HBE cells showed elevated levels of ROS and MDA, a weakened activity of SOD, an up-regulated expression level of miR-124-3p, and a down-regulated expression level of NFIB. After transfection with miR-124-3p mimic, the mRNA and protein levels of NFIB were down-regulated, and the oxidative stress caused by C. psittaci infection was enhanced, whereas miR-124-3p inhibitor produced opposite effects. In C. psittaci-infected HBE cells, the co-transfection with si-NFIB and miR-124-3p inhibitor elevated the levels of ROS and MDA and decreased the activity of SOD compared with transfection with only miR-124-3p inhibitor.Conclusion MiR-124-3p regulates oxidative stress in C. psittaci- infected HBE cells by targeting NFIB.

Abstract:Background The exacerbating antibiotic resistance of bacteria around the globe has rendered the development of novel antibiotics an urgent necessity. Deserts, with distinctive geographical conditions, harbor unique microbial resources, thus emerging as a promising reservoir for the discovery of new antibiotics.Objective To investigate the diversity of soil actinomycetes in the Gurbantunggut Desert, Xinjiang and the factors influencing their diversity, thus screening out new strains with potential antibacterial properties.Methods Actinomycetes were isolated by the dilution-plate coating method on six different media, and their taxonomic status was determined by 16S rRNA gene sequencing. The influences of soil factors on actinomycete diversity were explored, and the antimicrobial activities of fermentation products from 14 potential novel strains (with 16S rRNA gene sequence similarities < 98.7%) were tested.Results A total of 330 actinomycete strains were isolated, classified into 27 genera, 16 families belonging to 12 orders, with Streptomyces being the dominant genus. Soil pH, moisture content, total nitrogen, and concentrations of Ca²⁺ and Mg²⁺ were identified as the primary factors influencing the distribution of actinomycetes. The 14 potential novel strains showed inhibitory activities against at least one pathogen, with three strains exhibiting broad-spectrum antimicrobial activities.Conclusion The Gurbantunggut Desert harbors rich and novel resources of culturable actinomycetes, serving as a promising source for the discovery of new and efficient antimicrobial products.

Abstract:Background Sea cucumber is a marine organism with rich nutrients and a high medicinal value. The microbial community in the intestine of sea cucumber shows great potential in the synthesis of natural bioactive substances and the degradation of toxic and harmful compounds.Objective To explore the functional microorganisms in the intestine of sea cucumber, characterize their physiological and biochemical functions, and identify their metabolites, thus providing technical ideas for the development of new high-value natural products.Methods The functional strains in the intestine of sea cucumber were isolated by the plate dilution method and identified by morphological observation, physiological and biochemical tests, and 16S rRNA gene sequencing. The antibiotic resistance test and hemolysis test were carried out to examine the safety of the strains. Liquid chromatography-tandem mass spectrometry (LC-MS) was employed to identify the metabolites of the strains.Results A strain Lysinibacillus fusiformis UBIT-90 was isolated from the intestine of sea cucumber. Strain UBIT-90 was a Gram-positive bacillus sensitive to chloramphenicol, erythromycin, ceftriaxone, ampicillin, and penicillin. It presented no risk of antibiotic resistance and no hemolytic property, thus belonging to a potential safe strain. A total of 7 152 compounds were identified in the metabolites of strain UBIT-90 cultured in LB medium at 37 ℃ for 24 h, including 130 up-regulated compounds and 175 down-regulated compounds (P < 0.05) compared with those at 0 h. Among the up-regulated compounds, 26 natural drugs such as 1H-pyrazolo[3,4-d]pyrimidin-4-amine, methyl pyropheophorbide-a, homoharringtonine, and (–)-epigallocatechin and 8 functional food ingredients such as adenine, xanthine, and ethyl cellulose were identified. The down-regulated compounds included four harmful compounds: azaserine, trimethylcolchicinic acid, pyocyanin, and scilliroside.Conclusion L. fusiformis UBIT-90 is capable of synthesizing 26 natural drugs (such as anticancer and antioxidant homoharringtonine, (–)-epigallocatechin) and 8 functional food ingredients (such as adenine and xanthine, which promote growth and development and enhance immunity). Simultaneously, it can degrade 4 hazardous compounds including pyocyanin and scilliroside. This strain has significant application potential in the development of medicinal substances, production of functional food additives, and environmentally friendly biodegradation.

Abstract:Background In the context of the global population aging and the increasing burden on healthcare and elderly care, the research on aging and related diseases is of paramount importance. The relationship between intestinal flora and aging has garnered significant attention. As individuals age, the dynamic balance of intestinal flora is gradually disrupted, with a reduction in beneficial bacteria and an increase in pathogenic bacteria, which accelerates the aging process and increases the risk of inflammation and tumor development. Therefore, it is of great significance to investigate the dynamic changes of intestinal flora during the aging process, identify key probiotics that can delay aging, and explore their mechanisms of action.Objective To reveal the dynamic changes of intestinal flora during mouse aging, screen the taxa that can delay the aging process, and evaluate their anti-aging effects.Methods Fecal samples from young and aged mice were collected for metagenomic sequencing, and the potential beneficial bacterium Bacteroides thetaiotaomicron was identified by LEfSe. Aged mice were then administered with B. thetaiotaomicron at a dose of 2×10⁸ CFU per mouse via gavage. The anti-aging effects of B. thetaiotaomicron were evaluated based on the expression levels of senescence markers (CDKN1A and CDKN2A) in various organs of young mice, aged mice, and aged mice subjected to the bacterial intervention.Results The aged mice showed up-regulated expression of CDKN1A and CDKN2A in organs, altered fecal microbial richness, decreased abundance of Bacteroidota and Proteobacteria, and increased abundance of Firmicutes. The B. thetaiotaomicron intervention improved the exercise ability, eased anxiety, and down-regulated the expression of CDKN1A and CDKN2A in organs such as the heart, liver, spleen, lung, and kidney of mice.Conclusion This study reveals structural changes in the intestinal flora during mouse aging and demonstrates an association between B. thetaiotaomicron intervention and improvement in some aging-related indicators.

Abstract:Background Shewanella sp. is a Gram-negative conditional pathogen widely distributed in aquatic environments. It can infect economically important fish species such as crucian carp (Carassius auratus), causing diseases like ulcerative skin lesions and septicemia.Objective To investigate the growth characteristics, whole genome information, genome data-based evolutionary status, and distribution characteristics of virulence and antibiotic resistance genes of Shewanella sp. XWfb1, thereby providing a theoretical basis for elucidating its pathogenic mechanisms and guiding the prevention of antibiotic resistance risks.Methods We systematically evaluated the growth curve and environmental stress tolerance (including heat shock and salinity) of XWfb1. Illumina and PacBio platforms were used for the whole genome sequencing of XWfb1, followed by genome assembly via a hybrid strategy. Gene prediction, functional annotation, and PCR validation of key genes were then conducted. Phylogenetic and comparative genomic analyses were performed to clarify its evolutionary relationship.Results XWfb1 exhibited strong short-term heat shock tolerance and adaptability to moderate-high salinity environments (3% NaCl). The genome of this strain had a total length of 5 036 139 bp and the G+C content of 45.31%, containing 4 282 coding genes and 138 non-coding genes. Functional annotation against different databases identified 4 282 (NR), 1 476 (GO), 3 544 (COG), 2 508 (KEGG), and 2 660 (SWISS) genes. Phylogenetic analysis revealed that XWfb1 was most closely related to S. oncorhynchi Z-P2 [(average nucleotide identity (ANI, 97.39%), digital DNA-DNA hybridization (dDDH, 87.50%)]. Synteny analysis indicated local genomic rearrangements compared with closely related species. PCR validation confirmed high expression of key virulence genes (lon, luxS, dksA, hem, and fur) and detected antibiotic resistance genes (mcr-4 and Sul1).Conclusion Whole genome sequencing and comparative genomic analysis suggest that strain XWfb1 is a member of S. oncorhynchi. Only four genome sequences of S. oncorhynchi have been deposited in GenBank to date. This study enriches the aquatic microbial resource library, elucidates the virulence characteristics associated with motility and immune regulation, and predicts the potential risk of multidrug resistance of S. oncorhynchi. These findings provide a critical genetic foundation for advancing research on the molecular mechanisms of aquatic pathogens and developing precise prevention and control strategies.

Abstract:Background Klebsiella pneumoniae is a common pathogen, whereas drugs are limited for its clinical prevention and treatment. Screening and developing new drugs is particularly important for the prevention and treatment of this pathogen. Allicin with multiple pharmacological functions has been studied regarding its antibacterial effects. The effect of allicin on the virulence of K. pneumoniae, however, has been rarely reported.Objective To study the effects of allicin on the growth and virulence factors of K. pneumoniae and decipher the underlying mechanism.Methods We inoculated K. pneumoniae in the liquid media containing different concentrations (25 μg/mL and 50 μg/mL) of allicin to plot the growth curve and determine the survival rate. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of allicin were determined by the microbroth dilution method. The morphology of bacteria was observed in the microscope, and the membrane potential and permeability were measured. The formation of biofilm, capsules, and fimbriae was detected. Furthermore, RT-qPCR was employed to determine the expression of virulence genes and quorum sensing (QS)-related genes. The binding of allicin to key proteins of QS was examined by molecular docking.Results Compared with the control group, the two allicin treatment groups showed no significant changes in the early growth rate of bacteria. The bacterial growth rate in the middle and late stages gradually decreased as the allicin concentration increased. The MIC and MBC of allicin against K. pneumoniae were 200 μg/mL and 800 μg/mL, respectively. Microscopic observation revealed that 50 μg/mL allicin resulted in damage to the membrane structure, changing the bacteria from short rods to spheres. The membrane potential and permeability of K. pneumoniae enhanced gradually with the increase in allicin concentration. Moreover, the capsule formation of K. pneumoniae was markedly diminished in the two allicin treatment groups. Allicin significantly inhibited the biofilm formation of K. pneumoniae, with no significant difference between the 25 μg/mL and 50 μg/mL groups. The formation of type Ⅰ fimbriae in the 50 μg/mL allicin group was significantly inhibited. The RT-qPCR results indicated that the genes associated with the formation of capsules (rmpA and magA), type Ⅰ fimbriae (fimD and fimK), biofilms (RcsB and bsmA) exhibited down-regulated expression. The expression of QS-related genes LuxS and LsrK was also down-regulated. Molecular docking results showed that allicin bound to and interacted with LuxS and LsrK.Conclusion Allicin affected the growth and virulence phenotypes including biofilms, capsules, and type Ⅰ fimbriae of K. pneumoniae by regulating QS. This study provides a theoretical basis for elucidating the inhibitory effect and mechanism of allicin against K. pneumoniae.

Abstract:Background Staphylococcus aureus is a common harmful bacterium that endangers human health. It is readily to attach both non-biological and biological surfaces to form biofilms during its growth. Consequently, S. aureus is prone to developing drug resistance in clinical practice, which poses a challenge to clinical treatment.Objective To decipher the mechanism of Turkish gall extract in inhibiting S. aureus ATCC 6538 for the development of plant-derived bacteriostatic agents.Methods The in vitro model of the S. aureus biofilm was established by Congo red plate and crystal violet staining. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of Turkish gall extract against S. aureus biofilms and the biofilm growth curve were determined by the 96-well plate micro-dilution method. The effects of Turkish gall extract on the cell membrane integrity, cell wall structure, and energy metabolism of viable bacteria in the biofilm were assessed based on the leakage of extracellular components, alkaline phosphatase (AKP) activity, adenosine triphosphate (ATP) content, and cell viability. Additionally, the impact of Turkish gall extract on bacterial biomacromolecules was investigated via DNA electrophoresis and quantification of bacterial DNA and protein concentrations. Concurrently, the performance of Turkish gall extract in eradicating mature biofilms was evaluated by scanning electron microscopy and transmission electron microscopy, on the basis of which the diverse inhibitory mechanisms of Turkish gall extract against S. aureus biofilms were deciphered.Results The MIC and MBC of Turkish gall extract against S. aureus were 56.25 μg/mL and 112.5 μg/mL, respectively. The Turkish gall extract inhibited the biofilm of S. aureus in a concentration-dependent manner. It destroyed the cell structure, causing obvious wrinkles and breakage of the bacteria and the leakage of intracellular components such as sugars and proteins, which diminished energy metabolism and overall cell viability. The extract inhibited the biofilm formation and destroyed mature biofilm components, hence inhibiting bacterial growth.Conclusion Turkish gall extract demonstrates the ability to inhibit the biofilm formation of S. aureus through multiple mechanisms. This study provides a theoretical basis for the further application of plant-derived natural products and clinical prevention and treatment of S. aureus biofilms.

Abstract:Background Uterine tissue damage is one of the common causes of infertility in animals, and gut microbiota is closely related to the occurrence and development of various diseases.Objective To study the interaction mechanism between gut microbiota and the uterus in mice.Methods We established mouse models of gut microbiota dysbiosis and fecal microbiota transplantation to investigate the relationship between gut microbiota dysbiosis and uterine tissue damage. Subsequently, we employed 16S rRNA gene sequencing to analyze the bacterial species positively associated with uterine tissue damage.Results Compared with normal mice, the mouse model of gut microbiota dysbiosis showed significantly upregulated expression levels of inflammatory mediators (TNF-α, IL-1β, and IL-10), while transplantation of normal mouse feces significantly downregulated the expression levels of TNF-α and IL-1β in the uterine tissue. Histological observation and hematoxylin-eosin (HE) staining results revealed that the uterine tissue damage in the mouse model of gut microbiota dysbiosis was significantly severer than that in normal mice and the mouse model of fecal microbiota transplantation. Compared with normal mice and the mouse model of gut microbiota transplantation, the mouse model of gut microbiota dysbiosis exhibited significantly elevated positive signals of neutrophils (Ly-6G) and macrophages (F4/80) and a declined positive signal level of tight junction protein Zonula occludens-1 (ZO-1). The 16S rRNA gene sequencing was performed on the feces of mice. At the family and species levels, it was found that the relative abundance of Prevotellaceae and Prevotellaceae_UCG-001 in the feces of mice with gut microbiota dysbiosis was significantly lower than that in normal mice and mice with intestinal flora transplantation. Prevotella exhibited a therapeutic effect on uterine tissue damage induced by gut microbiota dysbiosis, as evidenced by significant reductions in TNF-α and IL-1β expression levels and a significant increase in the IL-10 expression level in the uterine tissue after Prevotella treatment. Additionally, Prevotella treatment downregulated the expression levels of damage-related proteins HMGB1 and HABP2 in the uterine tissue. After treatment, the positive signals of neutrophils (Ly-6G) and macrophages (F4/80) in the uterine tissue were significantly downregulated, while the positive signal level of tight junction protein ZO-1 was significantly upregulated.Conclusion There is a close relationship between gut microbiota dysbiosis and uterine tissue damage in mice, with Prevotella playing a crucial regulatory role. Furthermore, Prevotella exhibits a therapeutic effect on endometritis induced by gut microbiota dysbiosis in mice. This study provides new insights and evidence for the prevention and treatment of diseases associated with uterine tissue damage.

Abstract:Background Brucella, as a genus of facultative intracellular bacteria, can secrete effectors to modify multiple signaling pathways of host cells, thereby facilitating their intracellular proliferation. The identification of these secreted effectors is imperative for elucidating the pathogenic mechanism of Brucella. Conventional methods, such as fusion tagging and enzyme labeling, are frequently utilized to identify the effectors secreted by bacteria. However, these methods are often laborious and expensive.Objective To develop a simple and convenient plasmid system based on bimolecular fluorescence complementation for the identification of effectors secreted by Brucella.Methods On the basis of split-GFP, we first constructed the eukaryotic expression plasmid pMAX-sfGFP_1-10-IRES-NLS-tagBFP, which could express the sfGFP_1-10 fragment in the transfected cells, and the BFP protein with nuclear localization signal could indicate the transfected cells. Then, we constructed the plasmid pMCRt-mCherry-P31-4×GFP₁₁-Flag which could express 4×GFP₁₁ in Brucella. Finally, we employed the effectors BspA and BPE123 secreted by Brucella and the negative protein GST to verify the feasibility of the split-GFP system.Results HeLa cells transfected with pMAX-sfGFP_1-10-IRES-NLS-tagBFP were observed by fluorescence microscopy, which showed that tagBFP and sfGFP_1-10 were successfully expressed. Western blotting showed the fusion expression of BspA, BPE123, and GST with 4×GFP₁₁-Flag. The results of cell infection and fluorescence microscopy showed that split-GFP could successfully indicate the secretory activity of BspA and BPE123, while it did not show secretory activity of the negative protein GST.Conclusion The two-plasmid system constructed based on split-GFP in this study can successfully verify the secretory activity of Brucella effectors, serving as a novel tool for the identification of proteins secreted by Brucella.

Abstract:Background Rhamnolipids are widely used biosurfactants, while their production is limited by various factors, which make it difficult to meet the demands of large-scale production. Additionally, due to the high costs of raw materials and the complexity of the fermentation process, the production cost remains high.Objective Taking temperature as the core variable, we explored the influences of temperature on microbial growth and generation of metabolic products during the fermentation for rhamnolipid production. The aim is to optimize the fermentation conditions, reduce production costs, and increase the rhamnolipid yield, thus providing a theoretical basis for industrial production.Methods By constructing a GRU-attention-KAN neural network model, we predicted the fermentation conditions for rhamnolipid production at different temperatures.Results The GRU-attention-KAN model demonstrated good prediction performance across three experiments, with the best group achieving a root mean square error of 0.022 8, a mean absolute error of 0.119 2, an R² value of 0.825 9, and an explained variance of 0.920 6.Conclusion The model effectively captures the dynamic effects of temperature changes on the fermentation process, exhibiting high prediction accuracy and application potential, thus laying a foundation for subsequent optimization efforts.

Abstract:Background Phosphate-solubilizing microorganisms (PSMs) are integral to the cycling of phosphorus and mitigation of phosphorus deficiency in soil.Objective To reveal the historical background, current research focuses, and research perspectives by a thorough review of the existing literature related to PSMs, thereby laying a theoretical foundation for future studies.Methods We employed CiteSpace to analyze the publications associated with PSMs from the Core Collection of Web of Science (WOS).Results The research on PSMs has undergone three stages of preliminary exploration, stable exploration, and rapid development. During the stage of preliminary exploration, primary efforts were directed toward the development of specialized culture media and the establishment of standardized culture protocols for PSMs. In the stage of stable exploration, research priorities shifted to the screening of PSMs, molecular identification, and mechanism deciphering of phosphate solubilizing. During the stage of rapid development, the research focuses shifted from the single phosphate solubilizing function to multiple functions including promoting plant growth, remediating heavy metal pollution, and enhancing plant stress resistance. Furthermore, the focus has steadily progressed from laboratory research towards the field application of microbial fertilizer products. China had the largest proportion of publications in this field, which significantly improved our understanding of PSMs. However, the cooperation with other countries should be further strengthened.Conclusion Future research should adopt a holistic perspective encompassing the soil-microbiome-plant system, delving deeply into the interactions and interaction mechanisms among these three components. Additionally, emphasis should be placed on enhancing the ecological adaptability of PSMs, as well as designing and applying synthetic microbial communities specialized in phosphate solubilizing. This will provide theoretical and technical support for the advancement of green agriculture.

Abstract:Background Viruses are an important part of the natural environment and possess the characteristic of rapid mutation. They can play a crucial role in ecosystems and organisms by shaping the communities, altering the physiological characteristics, and accelerating the evolution of hosts. A virome is the collection of all viruses in a certain eco-environment. In recent years, the research on viromes has been growing exponentially.Objective To analyze literature on virimics to understand the research dynamics and hotspots in related fields.Methods We retrieved Viromics-related articles from the Web of Science (WOS) and used CiteSpace (6.2.R6) to conduct a visual analysis of the articles published in the past 13 years. Investigations were mainly carried out regarding the published papers on viromes, related disciplinary fields, publishing countries, research institutions, and authors. In addition, we identified the research hotspots in different periods in this field as well as the latest research trends.Results The research of Viromics will continue to be characterized by continuous innovations in research tools and contents. Further progress is expected to be made in this field, and future research hotspots will focus on the gut virome.Conclusion In the future, Viromics will remain a field with broad prospects, and relevant research in this field will help scientists understand viruses and their impacts on human health and the environment.

Abstract:In the era of digital intelligence, a staged flipped classroom teaching model is developed based on the construction of digital educational resources. Four stages are designed, including pre-class preview guidance, in-class knowledge internalization, open innovation practice, and post-class summary and expansion. According to the philosophy of student-centered teaching, integration of practice and innovation, and integration of digitalization and intelligence, this teaching model focuses on solving the problems of single teaching mode, poor student preview effect, lack of deep personalized guidance, and poor digital intelligence literacy of teachers in Microbiology Experiment. This teaching model builds a "bridge" for students' knowledge learning, ability cultivation, and quality improvement, empowering the improvement of teaching quality of Microbiology Experiment with digital technology.

Abstract:Microbiology is a compulsory basic course or a professional core course for life science majors in colleges and universities. According to the needs of cultivating top-notch talents in life sciences, the course teaching team, guided by the three standards of high-level, innovative and challenging put forward by the Ministry of Education, has carried out in-depth teaching reform in Microbiology and actively explored innovation in teaching models and methods. On the one hand, efforts have been made to strengthen the construction of high-quality teaching resources in Microbiology, promote the organic integration of online and offline teaching, and enhance students' independent learning abilities. On the other hand, special topic explanations, scientific and technological innovation, and competitions have been introduced to cultivate students' innovative thinking, practical innovation abilities, and teamwork spirit. Moreover, multiple measures have been taken simultaneously to improve the educational effectiveness of Microbiology, including integrating ideological and political education to fulfill the fundamental task of fostering virtue through education, opening up the course to promote the sharing of high-quality course resources, and conducting multi-dimensional integration to innovate the course evaluation system. Practice has shown that the teaching reform has effectively stimulated students' interest in learning, cultivated their independent learning abilities and innovative thinking, and strengthened their practical innovation abilities and teamwork spirit, significantly improving the educational effectiveness of Microbiology.

Abstract:Biocatalysis engineering, one of the core courses of the bioengineering major, combines theoretical education with practical application. Under the dual background of emerging engineering education and Double First-Class initiative, the teaching of Biocatalysis Engineering has two goals of improving students' engineering ability and scientist literacy. This course is designed to foster innovative talents with both patriotism and international vision by integrating professional abilities with industry mission, engineering ethics, and the spirit of scientists. To adapt to the emerging engineering education, the teaching team at the School of Biotechnology, Jiangnan University has carried out systematic reform and exploration in the teaching content, teaching methods, and evaluation methods of this course. Specifically, we incorporated the research frontiers and industrial cases into theoretical teaching and combined course assessment with brainstorming and thematic discussion. This round of teaching reform has improved students' engineering abilities and scientist literacy and enhanced their sense of professional identity.