The emergence of superbugs with both high pathogenicity and high antimicrobial resistance poses a major challenge to public health. In Staphylococcus aureus, α-hemolysin (Hla) is a key virulence factor mediating infection and thus represents a crucial target for prevention and control. This review first summarized the structural characteristics of Hla in its monomeric crystalline form and heptameric pore assembly. It then systematically delineated the multi-layered and fine-tuned regulation of Hla expression, involving the hla gene locus, the Agr quorum-sensing system, the Sar protein family, the Staphylococcal accessory element regulator (SaeRS) two-component system, and other auxiliary regulators. The pathogenic mechanisms of Hla were further elaborated from the perspectives of both cellular effects and host receptors. Finally, this review summarized recent advances in prevention and treatment strategies targeting Hla, including vaccines, monoclonal antibodies, natural compounds, and nanoparticles, offering new insights and candidate approaches for the precise prevention of drug-resistant S. aureus infections.

The health risks posed by pathogenic microbial contamination in drinking water are becoming increasingly apparent. Following exposure to pathogens via water, the human body may experience reactions ranging from infection and illness to death. However, the current Standards for Drinking Water Quality (GB 5749—2022) primarily rely on indicator microorganisms to evaluate water quality, lacking precise targets for pathogen control and load levels, which makes it difficult to accurately assess the actual hazards posed by microorganisms in drinking water. Therefore, clarifying the relationship between the intake dose of pathogenic microorganisms in drinking water and the risk of human infection is of great significance for safeguarding public health. Quantitative microbial risk assessment (QMRA) can be employed to evaluate the health risks posed by pathogenic microorganisms in drinking water. It is currently combined with high-throughput molecular technologies such as real-time quantitative PCR, digital quantitative PCR, metagenomics, and metaviromics, overcoming the limitations of conventional culture-based methods in identifying pathogenic hazards and demonstrating significant advantages in pathogen hazard identification and multidimensional traceability. This paper reviews the research progress in the quantitative monitoring and risk assessment of pathogenic microorganisms in drinking water. On the basis of the distribution patterns and quantitative detection of pathogenic microorganisms, QMRA is employed to conduct risk prediction, screen high-risk pathogens, and identify critical control points, and thus a systematic approach for precise prevention and control and full-chain governance is ultimately established. This approach integrates multi-omics technologies, big data analysis, and health impact assessment, achieving a transition in water quality safety management from reactive response to proactive prevention and providing robust scientific support for ensuring drinking water safety and the health of vulnerable populations.

Driven by the continuous pressure of antibiotic selection, bacteria continue to evolve through multidrug resistance mechanisms, resulting in the increasing ineffectiveness of conventional antibiotic treatment and posing a severe challenge of no drug availability in clinical practice. Nano-delivery systems breathe new life into antimicrobial therapy with their designable physicochemical properties and versatile integration characteristics. Nano-delivery systems are not limited to simple drug carriers, but can be developed into a multi-functional intelligent platform integrating precise delivery, microenvironment perception, and immune reprogramming through precision design. This review systematically expounds the construction strategy of antimicrobial nano-delivery systems, sorts out the development context from drug carrier, synergistic vector, intelligent response vector to intelligent biomimetic vector. Furthermore, this review discusses the targeted design and antimicrobial component loading strategy in detail, including conventional antibiotic synergy and new antimicrobial co-delivery. The core thesis of this paper is that the application of nano-delivery systems in specific infection scenarios (e.g., intracellular bacterial infection and biofilm-associated infection) has gone far beyond simple delivery, and instead actively regulates host immune homeostasis, synergistically enhancing antibacterial effects by regulating innate immunity (e.g., macrophage polarization and neutrophil function) and activating adaptive immunity. Finally, this paper makes an outlook on the challenges and future development directions of this field, emphasizing that through cross-scale design and multi-mechanism synergy, nano-delivery systems are expected to provide a new generation of therapeutic strategies that are efficient, safe, and less likely to induce drug resistance for drug-resistant bacterial infections.

Lipid metabolism disorders have emerged as a global health challenge, and gut microbiota dysbiosis, along with the consequent metabolic endotoxemia, are key factors promoting this pathological process. This review summarizes the molecular mechanisms by which natural active components (NACs) improve lipid homeostasis through targeted modulation of gut microecology. At the microecological intervention level, NACs optimize the microbiota structure by increasing the abundance of functional bacteria such as Bifidobacterium and Akkermansia muciniphila, while inhibiting the proliferation of opportunistic pathogens like Desulfovibrio. At the “metabolite-host” interaction level, the lipid-lowering mechanisms mainly involve two aspects. The first is the targeted effects of metabolites, wherein short-chain fatty acids, secondary bile acids, and phenolic acid derivatives specifically modulate metabolic targets including 3-hydroxy-3-methylglutaryl-coenzyme A reductase and fatty acid synthase. The second is the host signaling responses, wherein the aforementioned metabolic mediators synergistically modulate key signaling nodes, such as adenosine 5′-monophosphate-activated protein kinase (AMPK)/sterol regulatory element-binding protein-1c (SREBP-1c) and peroxisome proliferator-activated receptors (PPARs), to inhibit de novo lipogenesis and promote fatty acid β-oxidation, while simultaneously inhibiting the Toll-like receptor 4 (TLR4)/nuclear factor-kappa B (NF-κB) pathway to alleviate inflammation-associated abnormal lipid deposition. The “component-microbiota-metabolite-host signaling” regulatory mechanism outlined herein provides a theoretical reference for elucidating the pharmacodynamic material basis of NACs and developing gut microecology-based personalized nutritional intervention strategies.

The skin microbiota refers to a complex ecosystem composed of diverse microorganisms that colonize the skin, which contributes to the maintenance of skin health through interactions with the host. Studies have demonstrated that an imbalanced skin microecology can cause various skin health problems through multiple pathways, such as the induction of DNA damage in skin cells and the promotion of pro-inflammatory factor secretion. In recent years, numerous studies have confirmed that probiotics play a significant role in improving skin health, with mechanisms including antioxidation, immunomodulation, and enhancement of skin barrier function. This review comprehensively summarized the composition of the skin microecology, its role in maintaining normal skin functions, and the research progress on the pathological mechanisms of skin problems induced by microecological imbalance. Based on this, this review systematically summarized the mechanisms and research progress of microecological therapy, particularly probiotics, in improving various skin health problems, especially skin cancer, skin inflammation and photoaging, aiming to provide a theoretical basis for the exploration of the mechanisms and application of probiotics in improving skin health.

Conventional cell culture relies on serum, which has the problems of unclear composition, high cost, and contamination risks. Microbial-derived additives, with clear sources, low costs, safe components, and obvious pro-proliferative effects, have become an ideal alternative to serum in cell culture. To provide a basis for the development of new sources for the culture media of cells in agricultural production, this review first summarizes the role of microbial-derived additives in the research and development of serum-free media, and then elaborates on the application status of microbial-derived additives in cellular agriculture. On this basis, it probes into the mechanisms through which microbial-derived additives provide precision nutrition and promote cell proliferation. This review aims to provide technical support for the research and development of serum-free culture media, the establishment of cellular precision nutrition, and the sustainable development of cellular agriculture.

The arms race between bacteria and phages has driven the evolution of diverse antiphage defense systems. In recent years, a series of defense mechanisms targeting the nucleotide pool have been discovered, revealing that nucleotides are not only fundamental molecules for metabolism but also serve as key signaling carriers, effector substrates, and toxic weapons in bacterial immunity. According to the interaction modes between defense mechanisms and the nucleotide pool, this review systematically summarizes seven representative antiphage systems and categorizes them into three major classes: (1) Systems mediated by nucleotide cyclization into signaling molecules [e.g., cyclic oligonucleotide-based antiphage signaling system (CBASS), pyrimidine cyclase system for antiphage resistance (Pycsar), and Panoptes], which utilize nucleotidyltransferases to synthesize cyclic oligonucleotide second messengers that activate downstream effector proteins, leading to abortive infection; (2) Systems mediated by nucleotide depletion [e.g., deoxycytidine triphosphate (dCTP) deaminase, deoxyguanosine triphosphatase (dGTPase), and Nezha], which specifically hydrolyze or modify dNTPs or NAD⁺, resulting in a nucleotide pool imbalance and an energy crisis that block phage replication; (3) Systems mediated by aberrant nucleotide conversion [e.g., restriction by an adenosine deaminase acting on RNA (RADAR) and type III CRISPR-Cas-associated deaminase (CAAD)], which convert canonical nucleotides into toxic analogs [e.g., inosine triphosphate (ITP) and deoxyinosine triphosphate (dITP)], poisoning the nucleotide pool and inducing abortive infection. This review focuses on the core components, protein structural features, assembly mechanisms, and antiphage regulatory strategies of each category of system and discusses the escape mechanisms evolved by phages against these systems. In summary, the regulation of nucleotide pool homeostasis has emerged as a core strategy in bacterial immunity. In-depth investigation of these systems not only helps elucidate the molecular basis of prokaryote-phage co-evolution but also provides important insights for developing novel antimicrobial strategies and tools in synthetic biology.

Antibiotics in water environments, one of the major global environmental problems, pose serious ecological and health risks, necessitating efficient removal strategies. Microalgae-bacteria systems have recently emerged as a promising approach for antibiotic removal due to their high efficiency, strong removal capacity, simple design, easy operation, and low costs. This review systematically summarizes recent advances in the removal of antibiotics by microalgae-bacteria systems. First, it introduces the current status of antibiotic pollution and its effects on the environment. Second, it elaborates on the interaction mechanisms between microalgae and bacteria, which include nutrient exchange, signal transduction, and gene transfer. Then, it analyzes the application of microalgae-bacteria systems in the degradation of antibiotics, involving the degradation effects and pathways for different types of antibiotics. Finally, this paper discusses the responses of bacteria and microalgae under antibiotic stress, including the changes in short-term and long-term physiological metabolic adaptation, and analyzes the key factors influencing the antibiotic removal performance of microalgae-bacteria systems.

Alcoholic liver disease (ALD) is one of the most common types of chronic liver diseases globally and an important cause of liver cirrhosis and liver cancer. Currently, the diagnosis and treatment of ALD face challenges such as delayed diagnosis, poor compliance with alcohol abstinence, and a lack of specific drugs. Therefore, it is of great significance to develop new intervention strategies. In recent years, the intervention effect of the medicinal herb Salvia miltiorrhiza (Sal) on ALD has received extensive attention. The crude extracts and active components of Sal can alleviate the pathological process of ALD through anti-inflammatory and lipid metabolism regulation, while the efficacy of single Sal treatment has limitations. Thus, combination medication has become an important strategy for ALD treatment. This article reviews the research progress in the combination strategies of Sal with natural products, antioxidants, liver-protecting drugs, and microecological regulators in the intervention of ALD. It is found that the combination medication with Sal can regulate the abundance of the gut microbiota and play a synergistic liver-protecting role through the mediation of gut microbiota. Relevant studies have confirmed that microecological regulators such as probiotics, prebiotics, postbiotics, and synbiotics can also ameliorate ALD by regulating the “gut-liver axis”, and they can be combined with Sal to intervene in ALD, demonstrating good clinical prospects. Developing microecological regulators targeting the gut microbiota homeostasis in ALD can not only provide new ideas for the development of bioactive preparations with high efficiency and low toxicity, but also have potential clinical value in improving the therapeutic efficacy and prognosis for ALD. It is an important research and development direction for new combination intervention strategies with Sal for ALD in the future.

Citrus is one of the most important economic crops in China at present. During the cultivation, it is threatened by multiple diseases. Citrus canker, as the most common disease in citrus cultivation, has caused great harm to the development of the citrus industry. The long-term use of chemical control agents has led to problems such as enhanced resistance of pathogenic bacteria, chemical residues on the surface of citrus fruits, and environmental pollution. Microbial control has attracted increasing attention due to its safety and pollution-free advantages. This review summarizes the sources and species of biocontrol microorganisms for citrus canker in the past decade, analyzes the biocontrol mechanisms of these microorganisms, evaluates the development and field control effects of biocontrol agents, and points out the problems existing in the microbial control of citrus canker, providing a reference for the research on biocontrol microorganisms for citrus canker.

[Objective] To improve the flavour quality of redried tobacco leaves and explore new microbial resources for fermentation. [Methods] A yeast strain PT-1 with pronounced fruit-aroma-producing ability was isolated and screened from commercially available Kyoho grapes. It was identified as Debaryomyces hansenii through rDNA-ITS sequence analysis. This strain was applied to the fermentation of redried tobacco leaves. By treating the tobacco leaves with a yeast suspension at an application rate equivalent to 4% of the tobacco leaf mass, we systematically evaluated the effects on the chemical composition, sensory quality, and microbial community structure of the tobacco leaves. [Results] After inoculation with D. hansenii, the total sugar and reducing sugar contents in tobacco leaves increased by 0.85% and 0.46%, respectively, while the total alkaloids and total nitrogen decreased by 0.07% and 0.05%, respectively. Sensory evaluation results indicated that the treatment group was superior to the control group in terms of aroma quality, sweetness, and aftertaste. High-throughput sequencing results showed that the bacterial diversity on the surface of tobacco leaves remained relatively stable after inoculation, while the fungal diversity significantly decreased. The microbial community structure was markedly simplified, with Debaryomyces becoming the overwhelmingly dominant genus (99.66%). Correlation analysis showed that D. hansenii was significantly positively correlated with total sugar, reducing sugar, aroma quality, and sweetness, and was negatively correlated with total nitrogen, total alkaloids, and strength, suggesting a potential association with improved tobacco leaf quality. [Conclusion] This study expands the pool of aroma-producing yeast resources and verifies the application potential of D. hansenii in tobacco leaf fermentation. The process is simple and low-cost, with a clear flavour-modulating effect. These findings provide a theoretical basis and a practical foundation for constructing an efficient and controllable functional microbial fermentation system.

[Objective] To elucidate the enzymatic-producing properties and environmental tolerance of culturable microorganisms from medium-high temperature Daqu, as well as their regulatory effects on the solid-state fermentation system of Baijiu, to investigate the differences of functional microorganism distribution across different Daqu grades, and to provide a theoretical basis for the development of fortified microbial starters and the optimization of brewing processes. [Methods] The isolation of functional microorganisms, along with the evaluation of enzyme-producing characteristics, growth properties, and environmental tolerance, was conducted on post-fermentation medium-high temperature Daqu of superior-grade and premium-grade used in the production of Nongjiangjianxiangxing Baijiu (strong-sauce aroma-type Baijiu). Moreover, solid-state simulated fermentation experiments were conducted to investigate the regulatory effects of four dominant strains on the physicochemical properties and volatile flavor compounds of the fermented grains. [Results] In this study, a total of 44 strains were isolated from Daqu of premium-grade and superior-grade used in the production of Nongjiangjianxiangxing Baijiu (29 from premium-grade Daqu and 15 from superior-grade Daqu), belonging to 5 phyla and 8 genera, with Bacillus being the dominant genus (accounting for 43.2%). A total of 28 amylase-producing strains (20 from premium-grade, 8 from superior-grade) and 4 cellulase-producing strains (3 from premium-grade, 1 from superior-grade) were primarily screened. Although the premium-grade Daqu contained a higher number of enzyme-producing strains, the strains from the superior-grade Daqu exhibited greater average enzyme production capacity. After secondary screening, strains T41, Y46, and T47 were identified as high-yield amylase-producing functional microorganisms, and strain Y22 as a high-yield cellulose-producing functional microorganism. The tolerance assays revealed that Y46 exhibited the strongest adaptability to high temperature, ethanol stress, and lactic acid stress, and Y22 exhibited the strongest acid tolerance whereas T41 showed the best performance under glucose stress. The results from the solid-state simulated fermentation indicated that all four tested strains contributed to maintaining fermentation ecosystem stability. The strains significantly enhanced starch degradation, achieving reduction rates (from 7.0% to 9.5%), with Y22 exhibiting the most pronounced degradation efficiency. The differential regulatory effects were observed on reducing sugar utilization and volatile flavor compound synthesis, particularly esters. Notably, T41 showed the most pronounced enhancement in flavor compound accumulation. [Conclusion] Post-fermentation medium-high temperature Daqu harbors abundant enzyme-producing functional microorganisms. Significant differences were observed in the quantity and enzyme-producing performance of functional strains across Daqu of different grades, with amylase-producing strains exhibiting a higher isolation rate than cellulase-producing strains. The four screened functional strains possess excellent enzymatic properties and environmental adaptability. In the simulated Baijiu fermentation system, these strains maintained acidity stability, enhanced starch utilization efficiency, and promoted the formation of flavor compounds. Among them, strains T41 and T47 exhibited superior overall fermentation performance and demonstrated considerable potential for practical application. These findings provide valuable microbial resources and a scientific basis for the development of functional microbial starters and the operation of Baijiu brewing processes.

[Objective] To elucidate the signal sensing logic of the three-component EsrISR system, which is composed of the accessory protein EsrI, the histidine kinase EsrS, and the response regulator EsrR, in Corynebacterium glutamicum under hyperosmotic stress and its molecular mechanism for maintaining cell wall homeostasis. [Methods] The core stress-responsive gene cluster was identified through RNA-seq. A series of deletion mutants and overexpression strains were constructed through homologous recombination technology. The transcriptional fusion reporter system, field emission scanning electron microscopy/transmission electron microscopy (FESEM/TEM), electrophoretic mobility shift assay (EMSA), and real-time quantitative reverse transcription PCR, (RT-qPCR) were employed to systematically investigate the sensing threshold, stress response patterns, and global target gene networks of this system. [Results] RNA-seq showed that the esrISR gene cluster was significantly upregulated under 1.7 mol/L NaCl stress. Under the same stress conditions, the deletion of the regulator EsrR led to a significant decrease in biomass, with the maximum OD₆₀₀ dropping to 47.8% of that of the wild type. Moreover, severe damage and collapse of the cell wall were observed via electron microscopy. The construction and analysis of reporter plasmids showed that this system was driven by an internal promoter, peaking at 1.7 mol/L NaCl, with a maximum fluorescence intensity of 880 RFU/OD₆₀₀. The proposed regulatory model indicated that in the absence of external stress, EsrS mainly exerted phosphatase activity to repress the transcription of esrI, thereby maintaining the repressed state of the system (deletion of esrS led to an approximately 15.0-fold upregulation of esrI). Upon sensing hyperosmotic signals, EsrS changed its activity, promoting the phosphorylation of the regulator EsrR. Subsequently, the phosphorylated EsrR specifically bound to the core regulatory region, thereby activating the positive feedback regulatory loop. Transcriptomic analysis revealed significant alterations in the expression of key genes associated with cell wall homeostasis in the esrR deletion mutant (ΔesrR). RT-qPCR analysis confirmed that EsrR functioned as a crucial regulator of cell wall biosynthesis primarily by positively activating key pathway genes. Specifically, the peptidoglycan synthesis genes murB and CGL_RS01830 were downregulated by 95.0% in ΔesrR and upregulated by 2.0-6.5 fold in esrR-OE; the trehalose biosynthesis genes treS, treY, and treZ were downregulated by 75.0%-83.0% in ΔesrR and upregulated by 2.5-4.2 fold in esrR-OE; cmrA and gatC were significantly repressed in ΔesrR and upregulated by 3.5-folds in esrR-OE, thereby effectively maintaining the physical homeostasis of the cell wall. [Conclusion] This study provides new insights into the regulation of cell wall homeostasis by the three-component EsrISR system in Corynebacterium, providing critical genetic targets for the robustness engineering of industrial chassis strains.

[Objective] L-histidine is a high-value, imidazole-containing amino acid with broad applications in the pharmaceutical, food, and additive industries. The fermentative production of L-histidine remains constrained by the low carbon flux through the core biosynthetic route, limited precursor availability, and by-product accumulation. [Methods] With Escherichia coli as the chassis, L-histidine biosynthesis was systematically improved through rational metabolic engineering. [Results] The flux toward L-histidine was enhanced by elimination of transcriptional attenuation of the histidine operon, introduction of an efficient adenosine triphosphate (ATP) phosphoribosyltransferase mutant, and plasmid-based overexpression of key pathway genes. The pentose phosphate pathway was reconstructed to increase the precursor 5-phosphoribosyl pyrophosphate (PRPP) supply. In parallel, carbon loss was reduced by blocking formate- and acetate-forming pathways. Nitrogen sources were subsequently evaluated, and fermentation parameters, including temperature, pH, and dissolved oxygen, were systematically optimized. Under the optimized conditions, the L-histidine titer, yield, and productivity reached 34.84 g/L, 0.13 g/g, and 0.73 g/(L·h), respectively. In addition, the analysis of key gene expression and intracellular metabolites suggested that process optimization was conducive to enhancing the expression of the main synthetic pathway, improving energy and redox states, and increasing the ammonia donor supply. [Conclusion] The results establish an effective metabolic engineering strategy and a process basis for efficient microbial production of L-histidine.

[Objective] To obtain the strains with strong saline-alkali tolerance and plant growth-promoting traits from the severe saline-alkali soil in Xinjiang, thereby providing excellent strain resources for the development of microbial fertilizers and soil improvement agents. [Methods] Bacteria with strong saline-alkali tolerance were screened using modified LB medium. Subsequently, the strain was taxonomically identified based on morphology, physiological-biochemical tests and 16S rRNA sequencing, and its plant growth-promoting properties were characterized systematically. In addition, the strain was whole-genome sequenced via the Illumina NovaSeq X Plus platform. [Results] A strain with strong saline-alkali tolerance and multiple plant growth-promoting functions was isolated and designated as Bacillus zhangzhouensis A015 (CCTCC M20242724). It could tolerate 250 g/L NaCl and grow within a range of pH 2.0-12.0. Results of multifunctional growth-promoting tests revealed that strain A015 could produce ACC deaminase, and exhibited nitrogen fixation, potassium solubilization, organic phosphorus and inorganic phosphorus solubilization abilities. The nitrogenase activity of strain A015 was (185.36±4.16) ng/L. Its organic phosphorus solubilization index was 1.35±0.15, with lecithin conversion amount of (3.77±0.07) mg/L. The inorganic phosphorus solubilization index was 1.42±0.04, and the transformation amount of calcium phosphate reached up to (24.79±0.59) mg/L. Besides, the potassium solubilization index was 1.30±0.08, and the transformation capacity of feldspar powder was (5.16±0.18) mg/L. Wheat seed germination and growth promotion assays demonstrated that, compared with the control group (CK), inoculation with strain A015 significantly promoted the growth of wheat plumules and radicles on water agar media with 0-20 g/L NaCl. Specifically, under 5 g/L NaCl stress, remarkable growth promotion effects on plumule and radicle length were observed, with the growth increments of 43.86% and 20.14%, respectively. Notably, this strain could still effectively facilitate root growth under high salt stress of 15-20 g/L NaCl. The whole-genome sequence of A015 was deposited in NCBI, with the accession number JBSPZB000000000. Genome analysis revealed 25 saline-alkali tolerance-related genes, which were mainly involving mrp, mle, and nha gene clusters; meanwhile, 65 plant growth-promoting genes were identified, including nif, gdh, and kef gene clusters. [Conclusion] A strain of Bacillus A015 with strong salt-alkali tolerance and excellent plant growth-promoting ability was obtained from severe saline-alkali soil in Xinjiang. Genomic analysis further revealed genes linked to its salt-alkali tolerance and plant growth promotion capabilities. The findings provide theoretical basis and practical support for the development of saline-alkali soil microbial fertilizers in Xinjiang and the exploration of the strain’s stress tolerance mechanisms.

[Objective] To conduct site-directed mutagenesis on the Ca²⁺-binding sites, Leu²⁵³, and protein surface oxidation-sensitive Met residues of TSS, a collagenolytic protease derived from thermophiles, thereby screening out the variants with improved antioxidant activity, thermostability, and catalytic activity and revealing the intrinsic connection among them. [Methods] Site-directed mutagenesis was conducted on the Ca²⁺-binding sites (Ca1, Ca2, and Ca3) and Leu²⁵³ of TSS and the oxidation-sensitive Met residues on the protein surface. The derived variants were further evaluated for H₂O₂ tolerance, relative activity, and residual activity. Changes in antioxidant activity, catalytic activity, and thermostability after mutations were also studied. [Results] Mutations in amino acid residues at the three Ca²⁺-binding sites led to decreases in catalytic activity and/or thermostability of TSS. The Leu²⁵³ series variants constructed by simulating KP-43 did not enhance the antioxidant activity of TSS. Among the variants of oxidation-sensitive residues, M302L showed increases of 13% and 61% in the catalytic and antioxidant activities, respectively, while remaining unchanged thermostability. Ca²⁺ increased the catalytic activity and thermostability of TSS at high temperatures in a concentration-dependent manner. A high salinity environment with more than 2 mol/L NaCl prolonged the half-life of TSS at 75 ℃ and inhibited the maturation process of TSS proform. [Conclusion] The antioxidant and catalytic activities are positively correlated in the Leu²⁵³ and Met variants of TSS, which conforms to the “Trade-off” mechanism.

[Objective] To explore the structural characteristics of microbial communities in the rhizosphere soil of Meconopsis baileyi along different altitude gradients. [Methods] This study employed high-throughput sequencing to investigate the microbial community composition and diversity in the rhizosphere soil of M. baileyi and conducted a correlation analysis on the microbial community characteristics with environmental factors. [Results] At different altitudinal gradients, there are significant differences in the common genera of bacteria and fungi in the rhizosphere soil of M. baileyi. The common genera at low altitudes are all greater than those at high altitudes, and show a downward trend with increasing altitude. Their diversity initially increased and then decreased with increasing elevation. Fungal species richness generally increased with elevation, whereas bacterial species richness also increased but declined sharply at 4 100 m. Pseudomonadota was the most dominant phylum among soil bacteria, and Ascomycota was the most dominant phylum among soil fungi. Their relative abundance generally showed a downward trend with the rise in altitude. Altitude exerted a more significant shaping effect on the fungal community structure. Although bacterial communities were also affected by altitude, the altitude specificity of taxonomic group distribution was weak, and diversity tended to be more stable.The co-occurrence networks of microorganisms at high altitude gradients were more compact, with core groups being more prominent, which indicated collaborative coexistence of microorganisms in extreme environments. In contrast, the networks at low altitude gradients were looser, with microorganisms surviving independently in suitable environments. Soil pH, electrical conductivity (EC), available phosphorus (AP), soil temperature (ST), and soil moisture content (SMC) were identified as the major drivers shaping microbial community structure across altitudinal gradients. [Conclusion] Altitude significantly affects the species composition and community distribution of rhizosphere microorganisms in M. baileyi. Diversity generally increased initially and then decreased with increasing elevation, whereas species richness showed an overall increasing trend; however, bacterial richness declined sharply at the highest elevation.The relative abundance of the dominant phyla of both communities generally decreases as altitude rises, and altitude has a more significant role in shaping the fungal community structure. In addition, the microbial co-occurrence networks are more compact at high altitudes but looser at low altitudes, with pH and EC acting as key environmental drivers of microbial community structure.

[Objective] To investigate the species composition, dominant taxa, and diversity characteristics of bacterial communities within Spodoptera frugiperda at different developmental stages, thereby providing a scientific basis for further exploring key microorganisms associated with S. frugiperda and developing green control technologies based on symbiotic bacteria. [Methods] High-throughput sequencing of the 16S rRNA gene V1-V9 regions of bacteria from S. frugiperda at different developmental stages was performed using the PacBio sequencing platform. The structure and diversity of bacterial communities across these stages were systematically analyzed. [Results] Bacterial communities within S. frugiperda at different developmental stages were annotated at multiple taxonomic levels, resulting in 35 phyla, 72 classes, 189 orders, 322 families, 678 genera, and 1 074 species. At the phylum level, Pseudomonadota and Bacillota were consistently dominant across all developmental stages. At the genus level, Enterococcus was the dominant genus in the 2^nd to 6^th instar larvae. During the pupal stage, Providencia became the dominant genus, with a relative abundance of 78.13%. Additionally, Serratia was the dominant genus in female adults, accounting for 71.00% relative abundance, whereas Enterobacter was the dominant genus in male adults, with a relative abundance of 39.18%. Diversity analysis revealed that the egg stage exhibited the highest Shannon diversity index and Chao1 richness index among all developmental stages, whereas the pupal stage displayed the lowest levels of both bacterial diversity and species richness. Functional prediction indicated that the abundance of functional categories in bacterial communities was generally consistent across different developmental stages, with metabolic pathways showing the highest abundance. [Conclusion] The composition and diversity of bacterial communities in S. frugiperda exhibited developmental stage specificity, with the highest bacterial diversity and richness observed in the egg stage and the lowest in the pupal stage. Dominant taxa varied dynamically across developmental stages. At the genus level, Enterococcus, Providencia, Serratia, and Enterobacter were the dominant taxa and may serve as key targets for green control strategies. This study clarifies the species composition and community differences of bacteria in S. frugiperda across developmental stages, providing fundamental data for further investigating the mechanisms underlying host-symbiont interactions and developing green control technologies.

Nymphaea candida C. Presl, a rare water lily species in China, is on the brink of extinction due to its extremely low natural germination rate. Obtaining robust seedlings suitable for artificial propagation has become a key for the conservation of this species. [Objective] To isolate and identify endophytic plant growth-promoting fungi from the roots of N. candida and evaluate their growth-promoting effects on seedlings. [Methods] Pot experiments were carried out with Chaetomium globosum isolated from N. candida to assess its effects on the growth of N. candida, soil physicochemical properties, rhizosphere microbial community structure, and leaf gene expression. [Results] The application of C. globosum significantly increased the leaf length, leaf width, number of leaves, and root length of N. candida by 46.74%, 96.08%, 64.71%, and 130.13%, respectively. Moreover, the application of C. globosum significantly increased soil organic matter, alkali-hydrolyzable nitrogen content, and sucrase activity. Rhizosphere microbial community analysis indicated that the application of C. globosum specifically enriched beneficial microorganisms, including norank-p-Cryptomycota, norank-o-Monhysterida, Surirella, and norank-p-Aphelidea. Leaf transcriptome analysis further revealed that C. globosum significantly upregulated the expression of the genes related to the steroid biosynthesis pathway. [Conclusion] C. globosum effectively promotes the growth of N. candida seedlings by improving the rhizosphere microenvironment, recruiting beneficial microorganisms, and regulating steroid biosynthesis in the leaves. Thus, the endophytic plant growth-promoting fungus C. globosum might be a reliable tool for the conservation of the endangered plant species N. candida.

Maize, an important food crop in China, directly affects national food security. In recent years, due to climate change and shifts in farming practices, maize stalk rot has become a major threat to maize production in China. [Objective] To screen and identify biocontrol bacterial strains against maize stalk rot, determine their optimal fermentation process, and develop effective bacterial agents, thereby providing theoretical support and a material basis for constructing a novel green prevention and control technology system. [Methods] Antagonistic bacterial strains against Fusarium graminearum were screened through a combination of dilution coating and plate confrontation methods. Candidate strains were identified through molecular biological techniques combined with physiological and biochemical characterization. Fermentation conditions were optimized via single-factor experiments, and seed coating agents were developed with the selected biocontrol bacteria. Indoor pot experiments and outdoor field plot trials were carried out for the biocontrol bacteria to systematically evaluate their maize growth-promoting effects and control efficacy against maize stalk rot. [Results] Two biocontrol strains, HS104 and HS107, with inhibition rates of approximately 70.00% against F. graminearum, were successfully screened out and identified as Bacillus velezensis. Both strains exhibited broad-spectrum antagonistic activity against major maize pathogens, including F. verticillioides, F. oxysporum, and Bipolaris maydis, with inhibition rates of around 60.00%. After optimization of fermentation conditions, indoor pot experiments showed that HS104 and HS107 not only significantly promoted maize growth but also achieved the control efficacy of 75.00% and 71.00% against maize stalk rot, respectively. The seed coating agents developed with the selected biocontrol bacteria performed well in field trials, significantly promoting maize growth and achieving the control efficacy of 63.93% and 77.33% against maize stalk rot, respectively. Moreover, they increased the yield of susceptible plants by 8.20% and 9.20%, respectively, compared with the control. [Conclusion] Strains HS104 and HS107 possess multiple excellent traits, including broad-spectrum antifungal activity, strong plant growth-promoting effects, and high disease control efficacy, demonstrating broad application prospects in the biocontrol of maize stalk rot and the enhancement of crop yields.

As an important grain production base and the main maize-producing area in China, the black soil region in northeast China can utilize multifunctional plant growth-promoting rhizobacteria (PGPR) to convert nitrogen and phosphorus into plant-available forms through nitrogen fixation and phosphate solubilization, thereby reducing chemical fertilizer input and promoting crop growth. [Objective] To systematically screen efficient multifunctional strains from the black soil in northeast China and evaluate their effects on soil fertility and maize growth under 20% nitrogen and phosphorus reduction through low-temperature (16 ℃) germination and pot experiments, thus providing excellent strain resources for developing microbial inoculants adapted to the black soil region in northeast China. [Methods] Functional strains were isolated from rice rhizosphere soil via selective media and identified by 16S rRNA gene sequence analysis. The plant growth-promoting traits of the obtained strains, including nitrogen fixation, phosphate solubilization, and indole-3-acetic acid (IAA) production, were quantitatively determined. Furthermore, maize seed germination and pot experiments were carried out to systematically evaluate the effects of these functional strains on seed germination under low-temperature conditions (16 ℃), as well as on soil physicochemical properties and seedling growth indicators during the seedling stage, on the basis of which the potential of functional strains in improving soil quality and promoting crop growth was comprehensively evaluated. [Results] Two PGPR strains, Niveispirillum sp. YM6 and Bacillus sp. A03, were screened from rhizosphere soil and identified by 16S rRNA gene sequence analysis. Functional characterization revealed that strain YM6 exhibited both nitrogen fixation and indole-3-acetic acid (IAA) production capabilities, while strain A03 displayed both phosphate solubilization and IAA production capabilities. Under low-temperature (16 ℃) conditions, inoculation with both YM6 and A03 achieved the best performance in promoting seed germination, with a germination rate of 49.00%, a germination index 51.44% higher than the control, and shortening of 0.18 d in the average germination time, demonstrating a synergistic growth-promoting effect superior to both single-strain treatments and ternary combinations. Pot experiments further demonstrated that compared with the reduced-fertilizer control (CK-R), the ternary combination YM6+A03+ZP (Penicillium bilaiae) significantly increased the maize plant height, total biomass, total chlorophyll content, and soil available phosphorus and ammonium nitrogen by 12.73%, 82.60%, 66.30%, 29.94%, and 30.03%, respectively. Notably, the binary combination YM6+A03 showed no significant difference from the full-fertilizer control (CK-F) in these indicators but increased them by 9.08%, 63.54%, 32.81%, 21.51%, and 15.31%, respectively, compared with CK-R, outperforming all single-strain treatments. These results indicated that YM6+A03+ZP exhibited a significant synergistic enhancement effect, capable of maintaining or even improving crop productivity under chemical fertilizer reduction. [Conclusion] Single-strain inoculation exhibited limited plant growth-promoting effects. The binary combination YM6+A03 under 20% reduced fertilization maintained crop productivity comparable to that of CK-F, whereas the ternary combination YM6+A03+ZP significantly outperformed CK-F in promoting maize growth while improving black soil nutrient status, holding promise for the development of region-specific microbial inoculants adapted to the black soil region in northeast China.

[Objective] To investigate the effects of planting patterns on the soil physicochemical properties and microbial community structures of Polygonatum cyrtonema Hua. [Methods] With the soil of wild P. cyrtonema as the control, the soil samples from P. cyrtonema grown under monocropping and rotation patterns were collected as the research subjects. The structural and functional characteristics of soil microbial communities under wild, monocropping, and rotation patterns were analyzed by Illumina MiSeq PE300 high-throughput sequencing. [Results] Compared with the control, monocropping and rotation significantly increased the biomass of P. cyrtonema and the polysaccharide content in the rhizomes. Compared with monocropping, rotation increased soil ammonium nitrogen (AN) by 37.96% and enhanced the activities of sucrase (SC), acid phosphatase (ACP), and urease (UE) by 63.15%, 58.72%, and 73.03%, respectively, and it exhibited neutral soil pH. The control and monocropping resulted in higher Shannon and Chao1 indices for both bacterial and fungal communities in the soil (P<0.05). Planting patterns significantly affected the structures of bacterial and fungal communities in the soil. Different planting patterns showed variations in the relative abundance of the dominant bacterial phyla Pseudomonadota, Actinomycetota, Acidobacteriota, and Chloroflexota as well as the dominant fungal phyla Ascomycota and Mortierellomycota (P<0.05). The relative abundance of Pseudomonadota, Actinobacteria, Ascomycota, and Mortierellomycota was the highest under rotation. Additionally, rotation increased the relative abundance of beneficial fungal genera, including Saitozyma, Podila, Mortierella, and Trichoderma. Correlation and redundancy analyses revealed that microbial community changes were correlated with soil pH as well as AN, UE, ACP, and SC activities (P<0.05). Functional prediction indicated that nitrogen fixation and ureolysis were the dominant bacterial functions under rotational cropping. FUNGuild analysis demonstrated that functions associated with ectomycorrhizal fungi and litter saprotrophs were significantly enriched in the rotation soil. [Conclusion] Monocropping and rotation change the structures and functions of bacterial and fungal communities in the soil of P. cyrtonema. Rotation enhances the activities of UE, ACP, and SC and increases the abundance of beneficial fungi in the soil and improves the polysaccharide content and biomass of P. cyrtonema, thus being conducive to seedling cultivation.

[Objective] To investigate the effects of partially substituting chemical fertilizer with bio-organic fertilizer on the structure and function of soil microbial communities in wheat fields, thus providing a theoretical basis and technical support for fertilizer reduction and efficiency improvement in wheat production. [Methods] This study compared three treatments—conventional chemical fertilizer (CF), partial substitution of chemical fertilizer with organic fertilizer (OF), and partial substitution of chemical fertilizer with bio-organic fertilizer (BOF)—with respect to soil physicochemical properties, microbial community structure and function, community assembly mechanisms at the wheat jointing stage, and plant growth traits at the harvest stage. [Results] Compared with CF, BOF increased the biomass by 33.33% (P<0.05). Compared with CF, BOF increased soil organic matter, available phosphorus, available potassium, sucrase activity, and urease activity by 29.82%, 35.13%, 5.32%, 14.93%, and 36.18%, respectively. Compared with OF, BOF showed the corresponding increases of 3.29%, 133.73%, 47.21%, 19.58%, and 14.78%, respectively. The soil quality index ranked as BOF>OF>CF. Microbial community analysis showed that BOF significantly increased the abundance of beneficial taxa such as RB41, Massilia, Mortierella, and Cystofilobasidium, while reducing the abundance of potentially pathogenic fungi such as Fusarium and Aspergillus and enhancing the complexity and connectivity of the molecular ecological network. Functional prediction indicated that compared with CF, OF and BOF increased potential carbon metabolic functions such as aerobic chemoheterotrophy and aromatic compound degradation. In addition, BOF enhanced the potential function of ureolysis while suppressing the potential function of nitrate reduction. [Conclusion] By comparing the comprehensive effects of different fertilization combinations, we found that BOF significantly improves soil nutrient status and enzyme activities at the wheat jointing stage and optimizes the microbial community structure and ecological network, thereby providing favorable soil fertility conditions for plant growth. This study lays a theoretical foundation for optimizing fertilization structure and reducing chemical fertilizer use while improving efficiency.

Cereibacter azotoformans has been widely applied in the water quality regulation and disease prevention of aquaculture owing to its efficient nitrogen and phosphorus removal capacity as well as its probiotic effects on aquatic animals. However, whether this bacterium possesses both capabilities of denitrification and polyphosphate accumulation (DPA) remains unclear. [Objective] To identify the DPA phenotype of C. azotoformans and to elucidate its underlying molecular regulation mechanism. [Methods] Using the C. azotoformans strain ATCC 17025 as a model, we first examined its metabolic characteristics under DPA-inducing conditions to determine the presence of a DPA phenotype. Subsequently, gene deletion, DNA affinity purification sequencing (DAP-seq), and physiological and biochemical analyses were employed to systematically investigate the regulatory role of the NtrX/Y two-component system in DPA of the model strain. [Results] The C. azotoformans strain exhibited a typical DPA phenotype: releasing phosphate during the anaerobic stage and excessively absorbing phosphate in synergy with denitrification during the anoxic stage. At the end of the experiment, the nitrate degradation rate reached 95%, and the phosphate removal rate exceeded 90%. The deletion of ntrY resulted in the loss of the DPA phenotype. Particularly, the environmental phosphate concentration continuously increased rather than decreased, and the peak synthesis of intracellular polyhydroxyalkanoates (PHAs) was significantly impaired compared with that of the wild type (0.11 mg/mL). DAP-seq analysis revealed that NtrX directly targeted key denitrification genes (nirK and norB/C) as well as the PHAs synthesis gene phaC, thereby coordinately regulating denitrification and polyphosphate metabolism. [Conclusion] This study demonstrates that the anoxygenic phototrophic bacterium C. azotoformans possesses excellent DPA capabilities, and it reveals a central regulatory role of the NtrX/Y two-component system in this process. These findings offer a new theoretical basis for the development of microbial resources for simultaneous nitrogen and phosphorus removal.

[Objective] To isolate and screen the algicidal strains from samples of a eutrophic water body and study their algicidal characteristics and mode against Microcystis aeruginosa, thus providing a theoretical basis for the biological control of cyanobacterial bloom. [Methods] Strain identification was performed through morphological observation, physiological and biochemical tests, 16S rRNA gene sequence analysis, whole-genome sequencing, and average nucleotide identity (ANI) analysis. The algicidal effects under different inoculation amounts, bacterial growth phases, and initial algal densities were investigated. The algicidal mode was clarified by comparing the algicidal efficiency of cell-free filtrate and cell resuspension of the strain. The morphology of algal cells was explored by means of scanning electron microscopy (SEM). In addition, changes in physiological characteristics of algal cells were investigated through three-dimensional excitation-emission matrix (3D-EEM) fluorescence spectroscopy. GC-MS was employed to identify the major algicidal substances after n-hexane extraction. [Results] A highly efficient algicidal strain L2 was obtained and identified as Bacillus velezensis (CGMCC No. 38496, GenBank accession No. PP693440). At an inoculation amount of 10%, the strain achieved the algal inhibition rate of 89.35% against M. aeruginosa after seven days. The algal inhibition rates of the cell-free filtrate and fermentation broth of L2 were 90.51% and 91.75% after seven days, respectively. While the algal inhibition rate of the cell suspension was only 15.10%. This phenomenon indicated that L2 indirectly inactivated M. aeruginosa by secreting extracellular active substances. The major algicidal substances secreted by strain L2 were identified as fatty amides and cyclic dipeptides. Algal cells exhibited shrinkage and surface roughening, with significantly decreased phycobiliprotein content during the algicidal process. In addition, the composition of extracellular organic matter shifted from protein-like substances to humic-like substances. [Conclusion] B. velezensis L2 exhibits high algicidal activity against M. aeruginosa. Algal cells are inactivated primarily through the indirect mode of extracellular substance secretion, which cause damage to the morphology of algae cells and inhibit the activity of the photosynthetic system. The strain L2 exhibits good potential for the biological control of cyanobacterial bloom.

[Objective] To elucidate the physiological and biochemical mechanisms of iron release from bacterioferritin (Bfr) in Agrobacterium fabrum, identify the physiological reductant that provides electrons for the iron release from Bfr in cells, and determine whether Bfr-associated ferredoxin (Bfd) is required to facilitate electron transfer from the reductant to Bfr, thereby exploring a new approach to inhibit the iron release from Bfr and further disrupt intracellular iron homeostasis as well as impair A. fabrum growth and proliferation. [Methods] Bioinformatics analysis was carried out to identify the gene encoding Bfd-like protein in the A. fabrum genome. Bfr and the predicted Bfd were heterologously expressed and the obtained proteins were purified. In vitro assays were conducted to determine whether the predicted Bfd could assist the physiological reductant in transferring electrons to Bfr. Subsequently, we knocked out the Bfd-encoding gene to assess the impacts of Bfd on the iron release of Bfr and A. fabrum growth at the cellular level. [Results] The A. fabrum gene ATU_RS13485 encoded a (2Fe-2S)-binding protein containing the conserved amino acid motif (…C-X-C…C-X₂-C…) existent in Bfd proteins, confirming it as the Bfd of A. fabrum. Purified recombinant Bfd was able to assist FADH₂ in delivering electrons to Bfr in vitro, triggering the release of Fe²⁺ from mineralized A. fabrum Bfr. Upon bfd knockout, intracellular Bfr iron release of A. fabrum was severely impaired, and A. fabrum growth was significantly affected. [Conclusion] ATU_RS13485 encodes the Bfd of A. fabrum. A. fabrum Bfr requires Bfd to utilize electrons from the physiological reductant FADH₂ for iron release. The absence of Bfd blocks iron release from Bfr and impairs A. fabrum growth, which suggests that designing inhibitors to disrupt the Bfr-Bfd interaction may offer a promising strategy for controlling crown gall disease caused by A. fabrum.

[Objective] As a key component of eukaryotic chromatin, histone variant H2A.Z plays an important role in gene expression regulation. This study explored the regulatory effects of H2A.Z on the biological characteristics and pathogenicity of Fusarium graminearum, aiming to decipher its epigenetic regulation mechanism in the pathogenicity of pathogenic fungi. [Methods] The genome of F. graminearum was excavated by bioinformatics methods, and the H2A.Z homologous genes were identified by multiple sequence alignment and phylogenetic analysis. The gene deletion mutants were constructed by homologous recombination combined with PEG-mediated protoplast transformation. The phenotypic differences in morphological development, pathogenicity, and environmental adaptability between the wild type and the mutants were systematically evaluated by morphological observation, pathogenicity determination, and abiotic stress response experiments. [Results] H2A.Z was encoded by FgHTZ1. Phenotypic analysis showed that the deletion of FgHTZ1 resulted in significant retardation of colony growth, abnormal mycelial polar growth and conidial morphogenesis, and significant attenuation in pathogenicity. In addition, the mutants showed significantly altered sensitivity to osmotic stress (NaCl and sorbitol), oxidative stress (H₂O₂), and cell wall damage (Congo red), exhibiting a complex phenotype of enhanced tolerance to cell wall disruptors but hypersensitivity to osmotic/oxidative stress. [Conclusion] FgHTZ1 is the coding gene of H2A.Z in F. graminearum. As a positive regulator, this gene not only regulates the vegetative growth, morphological development, and pathogenicity of F. graminearum but also participates in mediating osmotic stress and oxidative stress responses, playing a key role in maintaining cell wall integrity. This study provides an important theoretical basis for further understanding the molecular functions of histone variants in the pathogenic mechanisms of plant pathogenic fungi.

[Objectives] To investigate the effects of early fructo-oligosaccharide (FOS) intervention on the antioxidant capacity, colonic barrier function, and colonic microbiota and metabolites in suckling piglets. [Methods] A total of six litters (10 piglets per litter) of Duroc×Landrace×Yorkshire newborn piglets were selected. Within-litter grouping was adopted with equal average body weight per group: five piglets from each litter were assigned to the control group (CON), and the other five formed the experimental group (Fos). From the second day after birth, the Fos group received daily oral administration of a FOS solution at an effective dose of 3 g/d per piglet, while the control group received an equal volume of normal saline. The trial lasted for 21 days. [Results] Early FOS intervention reduced the total cholesterol level and increased the concentrations of urea nitrogen, total protein, and albumin in the serum samples of suckling piglets (P<0.05). Early FOS intervention enhanced the total antioxidant capacity and glutathione peroxidase activity (P<0.05), while reducing the malondialdehyde content (P<0.05) in the serum. In addition, it enhanced the catalase activity and decreased the malondialdehyde content in the colonic mucosa (P<0.05). Early FOS intervention decreased the diamine oxidase (DAO) level in the serum (P<0.05), increased the DAO and D-lactic acid levels in the colonic mucosa (P<0.05), and up-regulated the expression of colonic barrier function-related genes (Claudin-1, ZO-1, Occludin, MUC1, MUC2, and MUC4) (P<0.05). Early FOS intervention improved the alpha diversity indexes of colonic bacteria (P<0.05) and induced separation of microbiota in colonic chyme of 14- and 21-day-old piglets (P<0.05). It altered the composition of colonic chyme microbiota at both phylum and genus levels (P<0.05). Furthermore, early FOS intervention increased the concentrations of lactic acid and short-chain fatty acids in the colon of suckling piglets (P<0.05). [Conclusions] Early FOS intervention enhances the antioxidant capacity, upregulates the mRNA levels of colonic barrier function-related genes, modulates the colonic microbiota, and promotes the production of lactic acid and short-chain fatty acids in suckling piglets.

[Objective] To investigate whether gut microbiota (GM) reshaped by exercise can ameliorate chronic unpredictable mild stress (CUMS)-induced depressive-like behaviors in mice via fecal microbiota transplantation (FMT), and to explore the underlying mechanisms. [Methods] Twenty SPF-grade male C57BL/6J mice (four weeks old) meeting the experimental criteria were randomly assigned to a control group (CON, n=5), a model group (CUMS, n=10), and an exercise group (SW, n=5). After four weeks of CUMS modeling, mice in the model group were further randomized into a PBS control group (CUMS+PBS, n=5) and an FMT group (CUMS+FMT, n=5). The latter received GM transplants from SW donors, and the experiment lasted for eight weeks. Depressive-like phenotypes were assessed by behavioral tests. GM composition was analyzed by 16S rRNA gene sequencing. Hippocampal and colonic structures were examined by histopathological staining. Hippocampal inflammatory cytokine expression was measured by quantitative real-time PCR (qPCR). Correlation analyses were performed between differential microbial taxa and environmental factors. [Results] Compared with the CON group, the model group exhibited typical depressive-like behaviors and disrupted GM structure, characterized by a decreased Bacteroidota/Bacillota ratio and enrichment of the pro-inflammatory genus Adlercreutzia. The SW group showed increased GM richness (P<0.05) and reversed behavioral abnormalities. After FMT intervention, depressive-like behaviors in recipient mice were markedly alleviated, indicated by an increased sucrose preference rate (P<0.000 1) and reduced immobility time in the forced swim test (P<0.01). Furthermore, hippocampal neuronal injury and colonic barrier disruption were effectively repaired. Microbiota analysis revealed that FMT restored GM diversity in recipients (P<0.01), shifted the microbiota structure toward that of the CON group, significantly reduced the relative abundance of Adlercreutzia, and increased the relative abundance of beneficial genera such as Candidatus_Saccharimonas and Alistipes. From a mechanism perspective, FMT suppressed the CUMS-activated nucleotide-binding and oligomerization domain (NOD)-like receptor signaling pathway and downregulated hippocampal mRNA levels of NOD-like receptor family pyrin domain containing 3 (NLRP3) and interleukin-18 (IL-18) (P<0.01). Correlation analysis further confirmed that the abundance of beneficial genera was positively correlated with positive behavioral indices and negatively correlated with pro-inflammatory cytokine expression. [Conclusion] Exercise can attenuate CUMS-induced depressive-like behaviors by reshaping the GM and inhibiting NOD-like receptor signaling pathway-mediated neuroinflammation, and this protective effect can be effectively transferred via FMT.

Tibetan pigs, a typical high-altitude indigenous breed in China, have developed unique physiological and metabolic characteristics through long-term natural selection under extreme plateau environmental conditions such as low oxygen and low temperature. However, stage-specific differences in their gut microbiota composition remain to be investigated. [Objective] To compare the rectal fecal microbiota composition and short-chain fatty acids (SCFAs) in Tibetan pigs at different growth stages from birth to the fattening stage. [Methods] Ten pregnant Tibetan sows with similar parity (2-3) and good body condition were selected and allowed to farrow synchronously. The offspring were reared from birth to 140 days old. A cross-sectional sampling design was adopted to collect rectal fecal samples from 10 pigs at 1, 7, 21, 70, and 140 days old for 16S rRNA gene sequencing and determination of SCFA concentrations. [Results] Microbial diversity in rectal fecal samples of Tibetan pigs gradually increased with age and stabilized during the later growth stage. Bacillota and Bacteroidota remained dominant throughout development. At the genus level, the gut microbiota of neonatal piglets was primarily colonized by Clostridium, Escherichia-Shigella, Streptococcus, and Fusobacterium. Notably, dominant genera in the gut during the suckling stage, such as Lactobacillus and Rikenellaceae RC9 gut group, remained abundant after weaning. Christensenellaceae R-7 group and Treponema showed low relative abundance before weaning but were significantly enriched after weaning and were among the dominant genera at 70 and 140 days old. PICRUSt2-based functional prediction analysis showed that the rectal fecal microbiota of Tibetan pigs at 21 days old was mainly enriched in pathways related to the citrate cycle and oxidative phosphorylation, whereas that at 70 and 140 days old was mainly enriched in amino sugar and nucleotide sugar metabolism, glycolysis/gluconeogenesis, and starch and sucrose metabolism. The concentrations of acetate, propionate, butyrate, and total SCFAs in rectal fecal samples at 70 and 140 days old were significantly higher than those at 21 days old. Correlation analysis revealed that Treponema and Rikenellaceae RC9 gut group had significantly positive correlations with acetate, propionate, butyrate, and total SCFA concentrations. [Conclusion] The gut microbiota composition differed markedly among Tibetan pigs at different growth stages. Beneficial bacteria such as Lactobacillus maintained relatively high abundance throughout the growth stages, while Christensenellaceae R-7 group remained abundant during the fattening stage. Moreover, the significant enrichment of complex carbohydrate-fermenting bacteria, represented by Treponema and Rikenellaceae RC9 gut group, during the growing-finishing stage suggests their key role in the hindgut fermentation of Tibetan pigs.

[Objective] To evaluate the inhibitory effects of polyunsaturated fatty acids (PUFAs) on oral microorganisms and to elucidate the response patterns of the oral microbiota to docosahexaenoic acid (DHA) treatment, as well as the genomic features associated with the sensitivity or tolerance. [Methods] The minimum inhibitory concentrations of four PUFAs were determined for five common species of oral bacteria. The effects of PUFAs at different concentrations on planktonic growth and biofilm formation were assessed in both single-species and mixed-species culture systems. Human intervention and metagenomic sequencing were employed to analyze the impact of DHA on oral microbiota composition and the abundance of specific bacterial taxa. The DHA sensitivity of Fusobacterium pseudoperiodonticum isolates from saliva was compared, followed by whole-genome analysis. [Results] The inhibitory effects of the four PUFAs varied among bacterial species. In single-species cultures, Streptococcus oralis, Streptococcus mutans, and Porphyromonas gingivalis were sensitive, whereas Fusobacterium nucleatum and Prevotella intermedia showed tolerance. In mixed-species cultures, the overall antibacterial effects were reduced. In the human intervention study, an 8-day mouth rinse with DHA did not significantly alter oral microbiota at the phylum level, while selectively modulating the oral microbiota at the genus and species levels, with increased relative abundance of Neisseria and Porphyromonas and decreased relative abundance of Prevotella. Dynamic monitoring indicated that DHA selectively influenced certain taxa over time, inducing a downward trend in F. nucleatum abundance. Strain-level analysis revealed differential DHA sensitivity among F. pseudoperiodonticum isolates, which was closely associated with variations in genes related to cell envelope and lipid metabolism (e.g., lapB, pilE, and ftsZ). [Conclusion] The inhibitory effects of PUFAs on oral pathogens exhibit species- and system-dependent heterogeneity. DHA selectively suppresses specific bacteria while maintaining the overall stability of the oral microbiota, and the strain-dependent variation in sensitivity may be associated with genomic characteristics. These findings provide new evidence for understanding the role of PUFAs, particularly DHA, in oral microbiota modulation and support their potential applications in this field.

[Objective] To investigate the effects of Shouwu Pill (SWW) on physical performance, gut microbiota composition, and bile acid metabolism in the mouse model of doxorubicin (DOXO)-induced premature aging, and to preliminarily explore its potential mechanisms. [Methods] A mouse model of premature aging was established by intraperitoneal injection of DOXO. Mice were randomized into Control, DOXO, DOXO+Shouwu Pill low-dose (SWW-L), and DOXO+Shouwu Pill high-dose (SWW-H) groups. Grip strength test, hanging endurance test, and treadmill exercise test were performed to evaluate physical performance. Gut microbiota composition and diversity were analyzed by 16S rRNA gene sequencing, followed by PICRUSt functional prediction. Fecal bile acid profiling was performed via UHPLC-QQQ-MS. Spearman correlation analysis was further conducted to explore the association between differentially abundant gut microbial taxa and bile acid metabolism. [Results] Compared with the Control group, DOXO-treated mice exhibited increased systemic inflammatory cytokine levels and significantly decreased forelimb grip strength, hanging endurance, and maximal running speed, indicating successful establishment of the premature aging model. SWW treatment significantly reduced systemic inflammation and improved physical performance, with the DOXO+SWW-H group demonstrating greater efficacy than the DOXO+SWW-L group. The results of 16S rRNA gene sequencing revealed that DOXO induced gut microbiota dysbiosis characterized by altered microbial diversity and disrupted microbiota structure, including decreased abundance of Muribaculaceae and increased abundance of opportunistic pathogenic genera such as Desulfovibrio and Helicobacter. SWW treatment partially restored the microbiota structure by increasing the abundance of potentially beneficial bacteria, including Akkermansia, Lactobacillus, Bifidobacterium, and Roseburia. PICRUSt functional prediction indicated significant alterations in bile acid metabolism-related pathways following SWW intervention. Fecal bile acid profiling further demonstrated that DOXO treatment led to the accumulation of several conjugated bile acids, including T-α-MCA and T-β-MCA, accompanied by reduced levels of unconjugated bile acids. SWW intervention significantly lowered the levels of conjugated bile acids and partially restored the levels of unconjugated bile acids. Correlation analysis showed that several beneficial bacteria were positively correlated with unconjugated bile acids but negatively correlated with conjugated bile acids. [Conclusion] SWW ameliorates physical decline and gut microbiota dysbiosis in the mouse model of DOXO-induced premature aging. Its anti-aging effects may be associated with modulation of gut microbiota-related bile acid metabolism and restoration of bile acid homeostasis.

[Objective] To achieve efficient expression and comprehensive functional characterization of chitosanase (Csn, EC 3.2.1.132). [Methods] The chitosanase gene (CsnBh) was cloned from Bacillus halotolerans GXK615 and heterologously expressed in Escherichia coli BL21(DE3). [Results] Following purification via nickel-affinity chromatography, the recombinant CsnBh exhibited a specific activity of 400.47 U/mg, which represented 3.03-fold purification. Enzymatic characterization revealed that CsnBh demonstrated high specificity toward colloidal chitosan, with optimal activity observed at 40 ℃ and pH 6.0. The enzyme demonstrated remarkable thermal stability across a broad temperature range (30-70 ℃) and pH stability over pH 5.0-9.0. Among the metal ions tested, Mn²⁺ significantly enhanced enzymatic activity, whereas Cu²⁺, Fe²⁺, and Fe³⁺ completely inhibited the activity. Hydrolysis pattern analysis confirmed an endo-acting cleavage mechanism, with chitotriose to chitopentaose identified as the predominant oligosaccharide products. Further investigation showed that the chitooligosaccharide (COS) mixture generated by CsnBh-mediated hydrolysis exerted potent inhibitory effects against E. coli, Staphylococcus aureus, and Phytophthora nicotianae. Moreover, COS treatment significantly upregulated the activities of key plant defense-related enzymes, including superoxide dismutase, peroxidase, and phenylalanine ammonia-lyase, in tobacco leaves. [Conclusion] This study establishes both a robust theoretical foundation and a highly promising enzyme candidate for the efficient production of chitosanases and the green synthesis and application of bioactive COSs.

[Objective] Animal sera have the potential to induce predatory organ formation in nematode-trapping fungi, while the specific mechanism remains unclear. This study systematically compared the inductive effects of different animal sera on predatory organ formation in nematode-trapping fungi, determined the optimal serum type and concentration, and identified and validated key synergistic active metabolites, aiming to provide a basis for elucidating the chemical signaling mechanisms underlying predatory organ formation and for applications in agricultural biocontrol. [Methods] D. dactyloides capable of producing constricting rings was used as the model strain. Horse, pig, sheep, and bovine sera at different concentrations were applied in induction cultures to determine the optimal induction conditions. Constricting ring development was monitored over time. Ring inflation was assessed by stimulation with 55 ℃ hot water, and trapping and digestion activities against Caenorhabditis elegans were further evaluated. Untargeted metabolomic profiling was performed for horse, pig, and bovine sera with different inductive effects, followed by differential metabolite screening and clustering analysis. Lyso-platelet-activating factor C-16(lyso-PAF C-16), linoleic acid (LA), and glycochenodeoxycholic acid (GCDCA) were subsequently tested in synergistic induction assays. [Results] All the sera were able to induce constricting ring formation, although their induction efficiencies differed markedly. Horse serum showed the strongest induction activity at concentrations of 0.5%-1%. A small number of constricting rings appeared at 48 h, and ring formation peaked at 72-96 h. Rings formed at 48 h could not be stimulated to inflate, whereas those formed at 96 h exhibited efficient inflation and effective nematode trapping and digestion. Metabolomic analysis revealed clear separation among serum groups and identified several candidate metabolites enriched in horse serum. Functional validation showed that GCDCA significantly increased constricting ring formation when combined with 1% horse serum, producing the strongest effect at 0.04 μmol/L. [Conclusion] This study establishes a robust induction system for constricting ring formation in D. dactyloides, clarifies the optimal induction conditions and time, and identifies GCDCA as a key synergistic factor that promotes constricting ring formation.

[Objective] Mycoplasmoides pneumoniae is a major pathogen responsible for community-acquired pneumonia worldwide, and the aggravating antibiotic resistance underscores the urgent need to develop an effective vaccine. [Methods] According to the critical pathogenic mechanisms of M. pneumoniae, 13 key adhesins and the community-acquired respiratory distress syndrome (CARDS) toxin were selected as candidate antigens. Reverse vaccinology and immunoinformatics approaches were adopted to predict and screen cytotoxic T lymphocyte (CTL) epitopes, helper T lymphocyte (HTL) epitopes, and linear B-cell epitopes. The screened epitopes were then connected via specific linkers. Two adjuvants, human beta-defensin 2 (HBD-2) and 50S ribosomal protein L7/L12, were introduced at the N-terminus to assemble the complete vaccine sequence. Furthermore, the physicochemical and biological properties of the constructed vaccine sequence were comprehensively analyzed. [Results] A total of 15 CTL epitopes, 10 HTL epitopes, and 14 linear B-cell epitopes were obtained after screening. All the epitopes and adjuvants were ligated by specific linkers, and a final multi-epitope vaccine sequence consisting of 746 amino acid residues was successfully constructed. The computational analysis indicated that the vaccine construct exhibited favorable physicochemical properties, antigenicity, solubility, and a safety profile. Molecular docking results showed that this construct could bind to Toll-like receptor 2 (TLR2) and TLR4 stably, which was further confirmed with the results of molecular dynamics simulations. The immune simulation and epitope coverage analysis indicated that the vaccine could elicit potent humoral and cellular immune responses and provide potential protection for up to 99.74% of the global population. [Conclusion] Through systematic computational design and evaluation, this study develops a multi-epitope mRNA candidate vaccine with promising application prospects, laying a theoretical foundation for subsequent experimental validation and development.

[Objective] To address the current issue that pathogen detection methods rely on specialized personnel and precision instruments and are thus unsuitable for on-site rapid testing, this study established a portable and rapid detection method for Staphylococcus aureus (SA) based on the recombinase polymerase amplification (RPA)-CRISPR/dCas9 system and NanoLuc binary interaction technology (NanoBiT). [Methods] Firstly, targeting the highly conserved thermostable nuclease-encoding gene (nuc) of SA, RPA primers and single guide RNA (sgRNA) with high sensitivity were selected. Secondly, reaction parameters including the concentration ratio of sgRNA to dCas9 protein and the detection time were optimized, and detection signals were read by a self-developed portable photoelectric biosensor. Finally, with the optimized reaction system and conditions, this method was evaluated in terms of the sensitivity, specificity, adaptability under different environmental temperatures, and practical sample detection performance. [Results] The optimized method can complete the detection within 1 h. With the mean signal of the negative control plus three times the standard deviation (mean+3SD) as the threshold, the limit of detection (LOD) reached 10² copies/µL. The method was specifically sensitive only to the target bacterium SA, demonstrating high specificity, sensitivity, and environmental adaptability. [Conclusion] A highly sensitive, specific, portable, and low-cost detection method for SA was successfully established based on RPA-CRISPR/dCas9 and NanoBiT. This method is particularly suitable for on-site rapid testing in resource-limited settings and holds broad application prospects.