The cyclic oligonucleotide (CO)-based anti-phage signaling system (CBASS), an innate immune system widely distributed in bacteria, is composed of oligonucleotide cyclases cGAS/DncV-like nucleotidyltransferases (CD-NTases), CD-NTase-associated protein (Cap), and accessory proteins. When bacteria are infected by phages, CD-NTases generate COs to amplify signals. Subsequently, effectors are activated by COs, inducing cell death through multiple mechanisms such as damaging cell membranes, degrading DNA, and depleting essential metabolites. Accessory proteins are responsible for regulating the CBASS, ultimately inhibiting phage infection. This review introduces the composition and classification of CBASS and further discusses the process by which CD-NTases recognize and bind to phage RNA to activate the synthesis of the second messenger CO. Effectors encoded by Cap effector genes mediate cell killing by binding to COs, while accessory proteins encoded by Cap auxiliary genes are involved in regulating the activity of CBASS. In addition, the immune evasion of phages from CBASS is also discussed. This review helps to understand the detailed mechanisms and biological significance of the interactions between phages and their host bacteria from the perspective of CBASS.

The nucleotide-binding domain leucine-rich repeat and pyrin domain-containing receptor 3 (NLRP3) inflammasome, a crucial element of innate immunity, plays a pivotal role in immune responses and disease pathogenesis. Dysregulated activation of the NLRP3 inflammasome is strongly linked to the onset of various diseases. Recent studies have demonstrated that the Lactobacillaceae can exert anti-inflammatory effects by regulating the NLRP3 inflammasome activity. Therefore, this review outlines the anti-inflammatory mechanisms by which the Lactobacillaceae regulate the NLRP3 inflammasome activity both directly and indirectly. Additionally, we discuss the roles of specific strains, such as Lactiplantibacillus plantarum, Lacticaseibacillus casei, and Lacticaseibacillus rhamnosus, in intestinal inflammatory diseases, hepatic disorders, neurodegenerative diseases, and metabolic/immune-related conditions. This review aims to lay a foundation for an in-depth investigation of the precise mechanisms underlying the Lactobacillaceae-mediated regulation of the NLRP3 inflammasome and provides novel therapeutic strategies for inflammatory diseases.

Streptococcus pneumoniae is a common opportunistic pathogen that can cause various infectious diseases, including acute otitis media, bronchitis, sinusitis, community-acquired pneumonia, septicemia, and purulent meningitis. Autophagy, a lysosome-dependent intracellular degradation pathway, plays a dual regulatory role in both bacterial infection and host defense against pathogens. During S. pneumoniae infection, host cells can activate xenophagy to eliminate invading bacteria. However, this pathogen has evolved multiple evasion strategies, such as interfering with autophagosome maturation, escaping autophagic encapsulation, and even hijacking the autophagy pathway to promote intracellular survival and dissemination. Recent years have witnessed significant progress in understanding the molecular mechanisms underlying the dynamic interplay between S. pneumoniae and host autophagy systems during bacterial infection, yet a systematic review synthesizing these findings remains unavailable. This review focuses on the interaction network and key mechanisms of S. pneumoniae with host cell autophagy, aiming to provide theoretical foundations and research perspectives for developing novel targeted therapeutic strategies against S. pneumoniae infections.

Bacterial glycoproteins, glycolipids, and polysaccharides are collectively known as glycans, which can serve as pivotal pathogenic factors leading to infection. Bacterial protein glycosylation mainly includes N-glycosylation, O-glycosylation, S-glycosylation, and arginine glycosylation. Glycolipids and polysaccharides are also important glycoconjugates, mainly including lipopolysaccharides, lipoarabinomannan, rhamnolipids, peptidoglycan, teichoic acids, and capsular polysaccharides. Bacterial glycoconjugates can promote host-pathogen interactions, influencing bacterial virulence, drug resistance, and biofilm formation, thereby facilitating bacterial infection. In addition, bacterial glycoconjugates can exert dual effects by modulating the host immune system: on one hand, aiding bacteria in achieving immune evasion and causing host infection; on the other, activating host immunity to help eliminate bacteria and suppress infection. This article provides an overview of bacterial glycans regarding the types, structural characteristics, roles in bacterial adhesion and colonization, and regulation of host immune responses and summarizes the effects of bacterial glycans on infection, aiming to offer a distinct perspective from glycoimmunology and an alternative strategy for clinical prevention and treatment of bacterial infectious diseases.

The spread of antibiotic resistance has made bacterial infections a global public health crisis, posing serious challenges to conventional antibiotic therapy and creating an urgent need to develop novel antibacterial strategies. As viruses are capable of specifically lysing bacteria, phages represent a promising alternative therapeutic strategy due to their unique killing mechanisms and high host specificity. Nevertheless, they face limitations in monotherapy due to their narrow host ranges and the emergence of phage-resistant bacteria. In recent years, phage-antibiotic combination therapy has garnered significant attention. It demonstrates unique advantages in enhancing bactericidal effects, synergistically inhibiting dual-resistance mechanisms, broadening the host range, disrupting biofilms, and treating complex infections. This therapy not only overcomes the limitations of single phage therapy but also paves new avenues for treating multidrug-resistant bacterial infections. This review systematically summarizes the synergistic mechanisms, key influencing factors, current challenges, and optimization strategies of phage-antibiotic combination therapy, aiming to provide a theoretical foundation and practical guidance for further research and clinical translation in this field.

Pseudomonas aeruginosa (PA) is a major cause of hospital-acquired infections. It can survive in a variety of extreme environments, and is highly resistant to antibiotics. PA can attack immunocompromised populations to cause severe infections, being a major cause of mortality in clinical practice. Therefore, rapid diagnosis of PA is critical for infection control. Conventional detection techniques such as plate assays, immunoassays, and nucleic acid assays have excellent accuracy and sensitivity, while they are costly and time-consuming. In recent years, novel biosensing technologies have achieved ultrasensitive and precise detection of PA by integrating various biorecognition elements and signal enhancement strategies, providing an efficient, convenient, and cost-effective solution for the rapid identification, treatment, and control of PA. In this paper, we systematically review the principles, application progress, and challenges of magnetic separation biosensing technologies based on electrochemical, optical, CRISPR/Cas12a system, and magnetic nanoparticles, and provide future research directions, aiming to promote the innovation and clinical application of the technologies for rapid detection of PA.

Mobile genetic elements drive bacterial evolution, while exposing bacteria to the risk of invasion by “selfish genes”. In the arms race with mobile genetic elements, bacteria have evolved a range of immune systems that can protect hosts from invading nucleic acids. These immune systems are capable of preventing the invasion of mobile genetic elements, degrading invading nucleic acids, inhibiting the replication or transcription of invading nucleic acids, or inducing abortive infections to protect the population. Although much is known about the working mechanisms of these host immune systems, it remains unclear how bacteria orchestrate different defense strategies in response to different stages of nucleic acid invasion. Based on our research and different immune strategies of bacteria to limit mobile genetic elements in different spatiotemporal dimensions, this review summarizes and classifies the host immune systems. The elucidation of these multilayered immune mechanisms not only reveals the arms race between host and mobile gene elements in the evolutionary process but also underpins the development of new biotechnologies.

In multicellular organisms, cell death is perpetually in a dynamic process. Apoptosis as a pivotal form of regulated cell death, mainly encompasses two pathways: the intrinsic pathway and the extrinsic pathway. During the pathogen infection, host cells are capable of eliminating the infected cells through apoptosis. On the other hand, pathogens have evolved a multitude of strategies to regulate host cell apoptosis. These strategies involve the use of effector proteins to modulate cellular signaling pathways, the regulation of the expression of apoptosis-related genes, the control of key proteins within the apoptosis pathway, and the modulation of the activity of proteases in the Caspase family. This article provides a comprehensive review of the molecular mechanisms and strategies by which intracellular pathogens, such as viruses, bacteria, parasitic fungi, and parasites, regulate host cell apoptosis. The aim is to offer valuable references for further exploration of the intricate interaction mechanisms between pathogens and hosts.

There are a large number of non-rhizobia endophytes in addition to rhizobia in leguminous root nodules, which are of great significance for promoting plant growth and improving the bacterial abundance in root nodules. Some NREs can not only help rhizobia expand their host range but also participate in the leguminous plant-rhizobia symbiotic nodulation process. In this review, we systematically summarize the genetic diversity, classification, and functions of NREs, specifically describe the pathways of NREs entering rhizobia, the interaction mechanism between NREs and rhizobia, and the diversity of NREs in soil ecosystems, and discuss the application potential and future research directions of NREs. Furthermore, this article summarizes the current research progress in leguminous plant-rhizobia-NREs interactions and explores the methods of improving crop productivity and health through interactions mediated by nodule microecology, aiming to provide theoretical support for sustainable agricultural development.

With the development of human industrial activities, nitrogen (N) emissions and atmospheric N deposition have increased significantly. When atmospheric N deposition exceeds the critical load that plants can bear, it may exert a negative effect on plants and lead to a decrease in species abundance. As an important biotic factor affecting species abundance, mycorrhizae can affect plant diversity and community structure through nutrient supply and hyphal network mechanism. Objective To compare the critical load of N deposition of understory herbaceous plants with different mycorrhizal types and explore how mycorrhizal types affect the responses of understory herbaceous plants to N deposition. Methods According to the database of long-term N deposition critical load of forest herbaceous plants, and the published literature, critical load database of herbaceous plants under different mycorrhizal types of forest forests in response to N deposition was established. We identified three mycorrhizal types of forest dominant species, which included arbuscular mycorrhiza (AM), ectomycorrhiza (ECM), and AM+ECM. The effects of mycorrhizal types on the critical load of N deposition of understory herbaceous plants were investigated. Results The critical load of N deposition of understory herbaceous plants varied in forests of different mycorrhizal types (P<0.05). The critical load of N deposition was the highest [9.28 kg N/(ha·a)] in the forest of the AM+ECM type, the second [8.41 kg N/(ha·a)] in the forest of the ECM type, and the lowest [7.19 kg N/(ha·a)] in the forest of the AM type. In forests of different mycorrhizal types, the critical loads of understory herbaceous plants of different functional groups (gramineous and non-gramineous) in response to N precipitation were consistent with the responses of all understory herbaceous plants of different mycorrhizal types. N deposition caused changes in the abundance of understory herbaceous plants. The species abundance of understory herbaceous plants in the forest of the AM type showed an increasing trend, while that in the forest of the ECM type mainly decreased. Conclusion Mycorrhizal types affect the critical load of N deposition of understory herbaceous plants (P<0.05), which is related to the niche differentiation, N concentration of litter, and N acquisition strategies of different mycorrhizal types of plants. In addition, the abundance of understory herbaceous plants will also vary due to different mycorrhizal types.

Objective The outbreaks of infectious bovine rhinotracheitis (IBR) have been reported in multiple regions across China. To systematically characterize the molecular features and biological properties of the predominant strains of infectious bovine rhinotracheitis virus (IBRV) and provide etiological insights for evidence-based prevention and control against IBR. Methods We collected the bovine lung tissue for detection of common bovine respiratory viruses by PCR. Viral isolation was performed with MDBK cells, and then metagenomic sequencing was conducted to determine and analyze complete genome sequences of the viruses. Viruses were purified via the plaque assay and subcultured to the 9th generation (F9) for determination of the 50% tissue culture infectious dose (TCID₅₀), monitoring of one-step growth kinetics, and observation of viral particle morphology via electron microscopy. Two IBRV-seronegative healthy adults of cattle were intranasally inoculated with the F9 suspension (2.5 mL/nostril), while one additional head of cattle was housed in close contact. The clinical manifestations were monitored, including body temperature fluctuations and viral shedding dynamics. Results PCR detection revealed the presence of both IBRV and bovine coronavirus (BCoV) in the bovine lung tissue. After inoculation into MDBK cells, obvious cytopathic effects were observed at the third passage. Metagenomic sequencing confirmed the virus as IBRV, with a whole genome length of 134 678 bp. This isolate was designated as IBRV GSLT/04/2024. The TCID₅₀ of F9 was 10^5.5 TCID₅₀/mL, and no mutation was detected in the gB, gC, gD, or gE gene. Based on the gC gene and whole genome sequences, this strain was classified into the IBRV 1.2b subtype lineage. Viral shedding began on day 5 post-inoculation in the inoculated cattle and on day 10 in the contact cattle, lasting for approximately 10 days. The amount of viral shedding followed the order of nasal swabs>oral swabs>rectal swabs>ocular swabs. On day 30 post-inoculation, IBRV genes were only detected in the colon tissue. IBR-specific antibodies were detected on approximately day 7 in the inoculated cattle and on day 10 in the contact cattle. Conclusion We successfully isolated a novel IBRV subtype 1.2b strain with high infectivity in adult cattle. The findings provide epidemiological and etiological evidence to trace the recent surge in IBRV prevalence across China.

Objective To analyze the activity of ribose-5-phosphate isomerase B (RpiB) encoded by lmo0736 and explore its effect on the infection of Listeria monocytogenes (LM). Methods The recombinant protein Lmo0736 was obtained by prokaryotic expression and purification, and its catalytic activity for substrates was verified by the enzyme activity assay. The LM strain with lmo0736 knockout (LM Δlmo0736) and the complementary strain (LM CΔlmo0736) were constructed by bacterial homologous recombination. The growth curves of bacteria in vitro were plotted. The adhesion, invasion, and intercellular migration of bacteria were evaluated by in vitro cell infection models (Caco-2 intestinal epithelial cells and L929 fibroblasts). The ICR mouse infection model was used to measure the 7 d survival rate and 48 h organ load of each strain, and thus the pathogenicity of strains in mice was evaluated. Results Lmo0736 had typical RpiB activity and catalyzed the conversion of d-ribose-5-phosphate to d-ribulose-5-phosphate, with V_max=0.366 mmol/(L·min), K_m=4.489 mmol/L, k_cat=12.300 s^-1, and k_cat/K_m=2.740 L/(mmol·s). The growth rate of LM Δlmo0736 was not significantly different from that of the wild type EGD-e and LM CΔlmo0736in vitro, indicating that the deletion of lmo0736 did not affect the basic growth of bacteria. LM Δlmo0736 demonstrated significantly decreased adhesion and invasion in Caco-2 cells and intercellular migration in L929 cells and weakened colonization in mice, which indicated that lmo0736 regulated the pathogenicity of LM through a RpiB-dependent metabolic pathway. Conclusion This study reveals for the first time that the Lmo0736 of LM has typical RpiB activity. Although the functional loss of Lmo0736 does not directly affect the basic growth of the bacteria, it significantly attenuates the pathogenicity by weakening the adhesion, invasion, and intracellular migration in host cells and the colonization in vivo. The results accumulate experimental data for in-depth exploration of the biological functions of RpiB in LM. From the perspective of the association between metabolism and virulence, this study provides an experimental basis for delving into the infection mechanism of foodborne pathogens.

Objective To study the endophytic microbiota in different ecological niches of pepper (Capsicum annuum L.) seedlings at various stages following Ralstonia solanacearum infection, aiming to explore the endophyte migration dynamics triggered by R. solanacearum infection. Methods The copy number of R. solanacearum was quantified by quantitative real-time PCR (qPCR). Samples from roots, stems, and leaves of both infected and healthy pepper plants were collected at 1, 4, and 7 days post-inoculation (dpi). High-throughput amplicon sequencing was employed to analyze the endophytic bacterial and fungal communities. Dual-dimensional analyses integrating microbial copy number and community structures were conducted to elucidate the spatiotemporal dynamics of endophytic microbiota and pathogen proliferation characteristics under R. solanacearum invasion. Results The copy number of R. solanacearum continuously increased in roots and stems post-inoculation, peaking in leaves at 4 dpi and then significantly declining. The infection induced pronounced alterations in endophytic bacterial communities across all tissues, with temporal effects outweighing tissue-specific variations. Roots exhibited heightened sensitivity to pathogen invasion. At 7 dpi, the relative abundance of Bacillota in endophytic bacteria in the plants increased significantly. At the genus level, the relative abundance of Rhizobium, Pseudomonas, and endogenous fungi Fusarium and Aspergillus increased significantly compared with that in the control group. The beta diversity indices and structures of endophytic microbiota in all tissues underwent marked changes during infection. Pseudomonas emerged as a signature bacterial genus in roots, while Aspergillus dominated stems and leaves as a fungal indicator. The co-occurrence network analysis revealed greater complexity of the endophytic microbiota in infected plants, with an elevated proportion of negative correlation edges. Ascomycota served as pivotal network hubs, reflecting enhanced antagonistic interactions and inter-microbial associations under pathogen stress. Ten potential antagonistic microbial taxa were identified, including six bacterial taxa of Clostridia (Bacillota). Conclusion This study delineates the proliferation pattern of R. solanacearum in pepper seedlings and characterizes the structural and migration dynamics of endophytic microbiota following pathogen invasion.

Objective To breed Bifidobacterium adolescentis strains that can adapt to the gastrointestinal environment of felines and have strong intestinal colonization capabilities. Methods B. adolescentis was isolated from the feces of felines with long and regular life spans. The original strain QC-Y (with the life span extension rate reaching 33.85%) was selected through biomass assessments and mouse life span experiments and it was identified as B. adolescentis. After radiation-induced mutation, QC-Y-09 was screened out by the biomass assessment, gastrointestinal tolerance domestication, and evaluation. Results QC-Y-09 showed the biomass 55.667 times and the tolerance score 5.66 times that of QC-Y. Moreover, the tolerance of the strain to the feline gastrointestinal environment showed good genetic stability. The survival rates of the 10th generation of QC-Y-09 in the artificial gastric juice, intestinal juice, anaerobic, and micro-aerobic environments were 18.80%, 41.60%, 93.26%, and 48.39%, respectively, which were 62.67%, 108.53%, 97.92%, and 94.40% of those of the original generation. The intestinal colonization test showed that the colonization ability of QC-Y-09 in felines was significantly stronger than that of QC-Y and human-derived B. adolescentis. Seven days after the feeding of the microbial inoculum was stopped, the viable count of B. adolescentis in the feline feces of the QC-Y-09 group still reached 4.37 lg CFU/g, which was 1.74 lg CFU/g and 3.02 lg CFU/g, respectively, higher than those of the QC-Y group and the human-derived B. adolescentis group. In addition, QC-Y-09 had a good relieving effect on the feline food change stress, reducing the feline food change stress rate by 85.71%. The results of SNP analysis showed that QC-Y-09 was significantly different from QC-Y at the gene level, and the differentially expressed genes were mainly enriched in the ribosome structure and aminoacyl-tRNA biosynthesis pathway. Conclusion Feline-derived B. adolescentis QC-Y-09 bred in this study can effectively adapt to the gastrointestinal environment and colonize the intestines of felines. This study provides both theoretical and practical bases for the application of B. adolescentis in functional food for felines.

Objective To study the protective effect of baicalin on mice infected by porcine extraintestinal pathogenic Escherichia coli (ExPEC) PCN033 strain and explore the underlying mechanism. Methods The mouse infection model and Western blotting were employed to determine the clinical features, survival rate, bacterial loads in different tissue samples, pathological changes, and expression levels of P65, IκBα, NLRP3, ASC, and Caspase-1 of mice in the infection group and the baicalin treatment group. Results After baicalin treatment, the mental state of mice in the baicalin treatment group was obviously better than that in the infection group. The survival rate of mice in the baicalin treatment group was higher than that in the infection group, and the colonization ability of PCN033 in the blood, brain, and lung of mice in the baicalin treatment group was lower than that in the infection group. Further studies showed that baicalin inhibited the PCN033 infection-induced activation of phosphorylation of P65 and IκBα and down-regulated the expression levels of NLRP3, ASC, and Caspase-1 in mice. Conclusion Baicalin alleviated the inflammatory response caused by porcine ExPEC infection by inhibiting NF-κB signaling pathway and blocking the activation of NLRP3 inflammasome, thereby reducing the clinical symptoms and tissue damage in mice. It is suggested that baicalin may be a potential drug to prevent and treat porcine ExPEC infection by regulating the inflammatory response.

Objective As a zoonotic pathogen, Proteus mirabilis poses a serious challenge to public health due to its multi-antibiotic resistance and the synergistic effect of virulence genes. To characterize the antibiotic resistance transmission of bacteria in the food chain in Zhejiang Province, this study systematically monitored the antibiotic resistance phenotypes and genes of isolates from cattle slaughterhouses and farmers’ markets, and analyzed the distribution differences of antibiotic resistance genes (ARGs), virulence genes (VGs), and mobile genetic elements (MGEs). Methods A total of 384 samples (feces, carcasses, environment, etc.) were collected from cattle slaughterhouses and farmers’ markets, and the strains were identified by 16S rRNA gene sequencing. Twenty ARGs and 10 VGs were detected by the K-B disc diffusion method and PCR, and the ARGs and VGs carried by P. mirabilis were analyzed. The ARG clusters were analyzed by sequencing of integron gene cassettes, and the co-occurrence network of ARGs, VGs, and MGEs was constructed. Subsequently, conjugative transfer experiments were carried out to explore the horizontal transmission potential of ARGs. Results A total of 101 strains of P. mirabilis were isolated, with the total isolation rate of 26.30%. The isolation rate of strains from slaughterhouses (33.85%) was significantly higher than that from farmers’ markets (18.75%). The resistance rates to ceftriaxone sodium, amoxicillin, and erythromycin were all over 90.00%. Among the ARGs, bla_TEM (89.09%), sul1 (77.71%), and tetA (63.29%) had the highest detection rates, and the distribution of ARGs in slaughterhouses was more complex. The VGs fliL (92.08%) and zapA (80.20%) were highly expressed in the isolates, which suggested potential pathogenicity. The detection rate of integrons in slaughterhouses was significantly higher than that in farmers’ markets, and PCR amplification results showed that there were a variety of ARGs, including aminoglycoside and trimethoprim resistance genes. Co-occurrence network analysis showed that ARGs, VGs, and MGEs had significantly positive correlations, and type I integron (intI1) was the hub gene. Conjugative transfer experiments confirmed that bla_TEM could be transmitted across species via horizontal transmission. Conclusion Compared with farmers’ markets, slaughterhouses are key nodes in the spread of antibiotic resistance due to the antibiotic exposure pressure, high organism density, and rich mobile components. The findings emphasize the importance of strengthening antibiotic management and monitoring the transmission chain of ARGs, providing a scientific basis for the prevention and control of antibiotic resistance under the framework of “One Health”.

Objective To elucidate and compare the diversity, structure, and functional characteristics of gut microbiota in sympatric fish species from a perspective of microbial ecology and explore the role of gut microbiota in feeding habit and ecological niche differentiation. Methods Foregut, midgut, and hindgut samples from Gymnocypris eckloni and Schizopygopsis pylzovi, along with their aquatic environmental samples, were collected from the upper Yellow River. The gut microbiota and potential functions were compared by 16S rRNA gene high-throughput sequencing and multiple bioinformatics approaches. Results Microbial alpha diversity followed the trend of aquatic environment>S. pylzovi>G. eckloni (P<0.05). In G. eckloni, alpha diversity was highest in the foregut, whereas in S. pylzovi, it decreased progressively from the foregut to the midgut and then to the hindgut. Beta diversity analysis based on clustering and CPCoA demonstrated that microbial communities derived from different gut segments of the same species were more similar to each other than those derived from the same gut segment of different species, indicating that the differences attributable to species were greater than those attributable to gut segments (P<0.001). Pseudomonadota accounted for over 50.00% of the gut microbiota in all sample types, while Bacillota comprised more than 25.00% in the guts of both fish species, which was significantly higher than that (3.80%) observed in the aquatic environment. Fusobacteriota and Cetobacterium were nearly absent in water and S. pylzovi but showed high abundance in the midgut and hindgut of G. eckloni. Enterococcus was specifically enriched in the hindgut of G. eckloni, while Lactococcus were predominantly found in the hindgut of S. pylzovi. PICRUS2 functional prediction revealed that gut microbiota of both species primarily enriched amino acid and carbohydrate metabolism pathways. The enriched metabolic pathways varied significantly across different gut segments of S. pylzovi, while significant differences in signaling molecules and interaction, cardiovascular diseases, and metabolism of terpenoids and polyketides were noted for the same gut segments between the two species. Conclusion There were significant differences in the microbiota composition and diversity in the gut between the two fish species and their aquatic environments, with distinct gut microbiota functions for each species. This study establishes a micrological foundation for research on the feeding habit and ecological niche differentiation of fish and provides theoretical support for exploitation of gut microbial resources and conservation and resource management of plateau fish species.

Objective The mesophilic salt-tolerant xylanase XynRBM26, a member of the GH10 family, holds significant application value in industrial fields such as animal feed. This study improved the thermostability of this enzyme by protein modification via rational design, aiming to lay a foundation for the industrial application of XynRBM26 preparations. Methods The bioinformatics software FoldX was used to conduct positionscan of the 3D structure predicted by AlphaFold 2.0 for XynRBM26. The mutants with free energy changes less than -0.5 kcal/mol were selected to construct an initial electronic library. According to the Pro effect and screening principles for potential mutants, an electronic library composed of Pro mutations was subsequently established. Finally, site-directed mutagenesis was employed to construct mutant genes, and positive mutants were screened after heterologous expression, purification, and experimental verification. Results After screening of the initial potential mutants, a small and precise mutant library consisting of 21 Pro effect mutants was constructed. All the mutants were experimentally validated, and positive single-point mutants D222P, V182P, D344P, and A352P with significantly increased T_m values were screened out. Through subsequent stacking screening, a three-point stacked Pro effect mutant with superior properties was obtained. The combination of this mutant with the experimentally screened positive mutant G115D produced the most stable mutant M4 (G115D-D222P-D344P-A352P). Compared with wild-type XynRBM26, M4 showed increases of 6.5 °C and 5 °C in T_m and optimal temperature, respectively. Moreover, M4 presented the half-life (t_1/2) at 55 °C 7.5-fold longer than the wild type, and its relative activity at the optimal temperature was 3.44 folds that of the wild type. Conclusion Screening thermostable mutants of the salt-tolerant xylanase XynRBM26 of the GH10 family based on the Pro effect and two different effect superimposing strategies is an effective approach.

Objective To investigate the effect of Lactobacillus reuteri CCTCC M 2016546 on chronic unpredictable mild stress (CUMS)-induced depression in rats and explore its potential mechanism. Methods Sixty male Sprague-Dawley (SD) rats were initially randomized into three groups: blank control (n=8), CUMS model (n=43), and prevention (n=9). The other groups except the blank control group received three CUMS stimuli daily. The prevention group was administrated with the L. reuteri CCTCC M 2016546 suspension (1×10⁹ CFU/d) via oral gavage prior to daily stress induction. After 4 weeks, depressive behaviors were assessed by sucrose preference, forced swimming, and open field tests. Successfully modeled rats (n=43) were re-randomized into five groups: model control (n=8), fluoxetine (2.1 mg/kg, n=8), combined therapy (2.1 mg/kg fluoxetine+1×10⁹ CFU/d CCTCC M 2016546, n=9), high-dose CCTCC M 2016546 (5×10⁹ CFU/d L. reuteri CCTCC M 2016546, n=9), and low-dose CCTCC M 2016546 (1×10⁹ CFU/d CCTCC M 2016546, n=9). Rats were administrated with corresponding agents daily for 4 weeks and then subjected to behavioral tests again. The levels of 5-hydroxytryptamine (5-HT) in the hippocampus and adrenocorticotropic hormone (ACTH) and cortisol (CORT) in the serum were quantified via ELISA. Western blotting was performed to determine the protein levels of brain-derived neurotrophic factor (BDNF), tyrosine kinase receptor B (TrkB), protein kinase B (Akt), phosphatidylinisitol 3-kinase (PI3K), extracellular signal-regulated kinase (ERK), and cAMP-response element binding protein (CREB) in the brain tissue. Results Compared with the model control group, all treatment modalities (fluoxetine, combined therapy, and CCTCC M 2016546) alleviated depressive behaviors: increasing sucrose preference (P<0.01, P<0.001), reducing immobility time in suspension (P<0.01, P<0.05), and enhancing horizontal locomotion distance/central zone exploration (P<0.01, P<0.05). Biochemical analyses revealed that treatments reversed the CUMS-induced alterations in 5-HT, CORT, and ACTH levels (P<0.01, P<0.05). Western blotting demonstrated upregulated protein levels of BDNF, PI3K, CREB, TrkB, Akt, and ERK in the fluoxetine and combined therapy groups (P<0.01, P<0.05). High-dose CCTCC M 2016546 elevated Akt, BDNF, CREB, and PI3K levels (P<0.01, P<0.05), while low-dose CCTCC M 2016546 raised Akt and BDNF levels (P<0.01). Prophylactic CCTCC M 2016546 administration primarily enhanced TrkB expression (P<0.05). Conclusion L. reuteri CCTCC M 2016546 ameliorates CUMS-induced depression in rats, potentially by modulating the hypothalamic-pituitary-adrenal axis hyperactivity and activating the PI3K/Akt/CREB/BDNF signaling pathway.

Sulfate-reducing bacteria (SRB) with unique reductive capabilities enable simultaneous sulfate reduction and heavy metal removal, demonstrating potential in heavy metal pollution remediation. Objective To isolate efficient SRB strains from marine sediments and investigate their reductive characteristics and application prospects in Cr(VI) contamination remediation. Methods We enriched and screened out an efficient SRB strain and systematically analyzed its sulfate reduction efficiency, Cr(VI) removal efficiency, and metabolic responses under environmental stressors (such as pH, sulfate concentration, and heavy metal concentration). This strain was then used to synthesize biological iron sulfide composite, the physicochemical characteristics of which were then investigated by scanning electron microscopy (SEM), X-ray diffraction (XRD), and Fourier-transform infrared spectroscopy (FTIR). Furthermore, the feasibility of applying the biological iron sulfide composite in the remediation of Cr(VI)-contaminated environments was explored. Results Strain S5 was identified as Desulfovibrio sp. with GenBank accession number OR140726. Its protein concentration and sulfate reduction followed an S-shaped curve. This strain exhibited a certain degree of acid tolerance, with the OD₆₀₀ and sulfate reduction rate reaching 0.16±0.01 and (83.71±1.49)% at pH 5.0, respectively. The strain exhibited sulfate reduction capability in the presence of 0.5-1.3 g/L sulfate, achieving a maximum reduction rate of (92.27±1.20)%. In the presence of 10-30 mg/L Cr(VI), strain S5 demonstrated efficient Cr(VI) removal. However, when the Cr(VI) concentration was higher than 30 mg/L, the Cr(VI) removal rate of this strain decreased significantly. The biological iron sulfide composite prepared based on strain S5 was porous, amorphous, and rich in functional groups such as C=O, N-H, and Fe-S, and its Cr(VI) removal rates were above 85% and did not differ significantly when exposed to Cr(VI) at high concentrations. Conclusion Desulfovibrio sp. S5 is a strain with high efficiency of sulfate reduction and Cr(VI) removal, and the biological iron sulfide composite prepared with it can overcome the limitation of higher Cr(VI) concentration and maintain high Cr(VI) removal rate, which has obvious advantages in the remediation of Cr(VI) pollution. The results of this study can provide a scientific basis for the application of SRB in the bioremediation of Cr(VI)-polluted environments.

Objective To identify the species and investigate the diversity of 120 Burkholderia cepacia complex (Bcc) strains isolated from industrial products and their production environments between 2022 and 2023. Additionally, the whole genome of a novel sequence type (ST) strain, Burkholderia aenigmatica ST2120, was analyzed to assess its virulence and pathogenicity. Methods Multilocus sequence typing (MLST) was employed to assign sequence types (STs) of Bcc strains. Multilocus sequence analysis (MLSA) was conducted for phylogenetic analysis and species identification of novel ST Bcc strains. Whole genome sequencing of ST2120 was performed on the Nanopore platform, followed by genome assembly, gene prediction, functional annotation, and prediction of biosynthetic gene clusters (BGCs) for secondary metabolites. Results Among the 120 Bcc strains, seven species (B. aenigmatica, B. cenocepacia, B. cepacia, B. contaminans, B. vietnamiensis, B. stabilis, and B. multivorans) and 38 STs were identified. Twenty-two novel alleles and 20 new STs were discovered. The novel ST strains were predominantly identified as B. aenigmatica and B. vietnamiensis. B. aenigmatica accounted for 55% of Bcc strains associated with industrial contamination, representing the most prevalent species within the industrial contamination-related Bcc. The genome (8 909 914 bp, G+C content: 65.73%) of B. aenigmatica ST2120 comprised 8 192 protein-coding genes, and the genome data were deposited in NCBI under the accession number CP184468-CP184476. Genomic analysis predicted siderophore-related BGCs for secondary metabolites (e.g., ornibactin C8 and chromobactin), five efflux pump-associated antibiotic resistance genes, and virulence genes linked to secretion systems, host adhesion/invasion, immune modulation, and quorum sensing. Conclusion B. aenigmatica has emerged as a predominant Bcc species in industrial contamination. The genome of B. aenigmatica ST2120 contains comprehensive virulence genes, indicating significant pathogenicity.

Objective To explore the functions of type XI histidine kinase genes in Botrytis cinerea, thereby establishing a foundation for further elucidation of the molecular mechanisms involving histidine kinase-related genes in the growth, development, and pathogenic processes of plant pathogenic fungi. Methods Through knockout vector construction, ATMT transformation, and PCR/qPCR validation, we obtained the mutant ΔBcHK91. The colony growth, sclerotium formation, conidial production and morphology, conidial germination rate, appressorium and infection cushion formation rates, pathogenicity, cell wall/membrane integrity assays and transcriptome profiling were conducted to evaluate the effect of BcHK91 knockout on B. cinerea strain B05.10, thereby elucidating the biological function of this gene. Results Two BcHK91 knockout mutants were successfully obtained and designated as ΔBcHK91-A and ΔBcHK91-B. The phenotypic analysis revealed that the knockout of BcHK91 reduced the conidial length, conidial production, conidial germination rate, appressorium formation rate, infection cushion formation rate, and pathogenicity. Moreover, the mutant formed no sclerotium and showed increased sensitivityto Congo red and SDS compared with the wild-type strain B05.10 and the ectopic insertion strain ET. To reveal the transcription mechanisms of BcHK91, we compared the transcriptomes of B05.10 and ΔBcHK91 by RNA-seq. A total of 1 533 differentially expressed genes (DEGs) were predicted in ΔBcHK91, including 1 017 genes with up-regulated expression and 516 genes with down-regulated expression. Gene ontology (GO) and clusters of orthologous groups (COG) annotation showed that the DEGs were mainly involved in cell process and metabolic process of biological processes, cellular anatomical entity and intracellular of cellular components, and catalytic activity, binding, and transporter activity of molecular functions. The Kyoto encyclopedia of genes and genomes (KEGG) pathway analysis showed that the DEGs were mainly involved in carbohydrate transport and metabolism, starch and sucrose metabolism, and mitogen-activated protein kinase (MAPK) cascade response and other physiological metabolic pathways related tothe phenotype and pathogenicity of ΔBcHK91. In silico analysis of the DEGs suggested that 17 DEGs were related to the growth and development, oxidative stress, cell wall biosynthesis, cell membrane integrity, sclerotial initials, and pathogenicity. The results of qPCR demonstrated that the expression levels of 13 genes matched the trends observed in RNA-seq data, confirming the high reliability of the transcriptome analysis. Conclusion In B. cinerea, BcHK91 plays critical roles in asexual development, environmental stress responses, and pathogenicity.

Myosmine, also known as 3-(3,4-dihydro-2H-pyrrol-5-yl) pyridine, is a tobacco alkaloid found not only in tobacco but also in various foods, fruits, and vegetables. It serves as one of the precursors for the formation of the carcinogenic tobacco-specific nitrosamine N′-nitrosonornicotine, posing a potential threat to human health. Objective To screen bacterial strains capable of degrading myosmine and preliminarily explore the pathways and mechanisms underlying myosmine degradation. Methods We used myosmine as the sole carbon source to enrich and isolate the myosmine-degrading bacterial strain from tobacco-growing soil. Taxonomic identification of this myosmine-degrading strain was achieved by a combination of morphological observation, physiological and biochemical testing, and molecular analysis. The myosmine degradation products by this strain were analyzed by HPLC and UHPLC-MS/MS. The degradation genes were predicted by BLAST comparison. Results A strain G-2 capable of degrading myosmine was successfully isolated. The strain was identified as a member of Shinella, designated Shinella sp. G-2. HPLC and UHPLC-MS/MS identified five degradation products. Genomic analysis showed that strain G-2 possessed a homologous gene cluster of a variant of the pyridine and pyrrolidine pathway (VPP) gene cluster. Conclusion In this study, a strain Shinella sp. G-2 with the ability to degrade myosmine was isolated. Strain G-2 might use enzymes in the VPP pathway to degrade myosmine through a metabolic pathway similar to the VPP pathway.

Objective The probiotic Escherichia coli Nissle 1917 (ECN) is engineered by synthetic biology to construct a tumor-targeting strain capable of colonizing the tumor tissue, converting glucose and metabolic waste ammonia in the tumor microenvironment into the photosensitizer precursor 5-aminolevulinic acid (5-ALA) and the immunomodulatory amino acid arginine, while synergizing with immune checkpoint inhibitors for enhanced antitumor efficacy. Methods The genes hemA^M, hemL, and argA were co-expressed in ECN, and thyA was knocked out via the λ-Red homologous recombination system to improve the tumor-targeting specificity. Shake-flask fermentation experiments, UV spectrophotometry, and HPLC were employed to quantify 5-ALA and arginine production. The antitumor effects of the engineered ECN were systematically evaluated by in vitro cellular assays and a murine colorectal cancer model. Results The engineered strain achieved 5-ALA and arginine yields of (173.00±11.46) mg/L and (1.70±0.09) g/L, which represented 8.2-fold and 20-fold increases, respectively, over that of wild-type ECN (P<0.000 1). The deletion of thyA enabled selective proliferation of the strain in tumor cells (HCT116 and CT26), with a two-fold increase in OD₆₀₀ compared with that in normal Vero cells (P<0.000 1), confirming enhanced tumor targeting. Both in vitro and in vivo experiments demonstrated sustained synthesis of 5-ALA and arginine in tumors. Compared with wild-type ECN, the engineered strain induced 2.7-fold and 1.9-fold increases in CD8⁺ and CD4⁺ T-cell infiltration (P<0.000 1), alongside 1.7-fold and 2.4-fold elevations in IL-6 and TNF-α secretion (P<0.000 1), respectively. The engineered strain combined with the anti-PD-L1 therapy achieved a tumor volume inhibition rate of 77.6% (P<0.000 1). Conclusion This study establishes a metabolically and immunologically dual-functional ECN platform that synergizes localized delivery of photodynamic therapy precursors, arginine-mediated immunometabolic reprogramming, and immune checkpoint blockade, providing a novel solution for the combined therapy against solid tumors. The engineered system offers a groundbreaking strategy for precise tumor microenvironment modulation, advancing the research on targeted cancer therapeutics.

Objective As biocatalysts, halohydrin dehalogenases can catalyze both cyclization and ring-opening reactions and are widely used in the synthesis of chiral epoxides and other compounds. The eco-friendly and efficient preparation of halohydrin dehalogenases is thus of great significance. In this study, we optimized the N-terminus of the coding sequence of the halohydrin dehalogenase HheC based on the mRNA secondary structure to achieve the efficient expression of this enzyme and then applied this enzyme in the synthesis of chiral epichlorohydrin. Methods The mRNA prediction tools was used to predict the secondary structure and thermodynamic properties of 5′mRNA. To reduce the stability of the mRNA secondary structure and increase folding free energy (ΔG), we designed the 5′mRNA sequence without changing the amino acid sequence. Furthermore, we characterized the expression efficiency and catalytic performance of this enzyme. Results The HheC mutant was obtained via the design of the 5′mRNA sequence, with the protein level increasing from 16.71% to 33.39% and the relative activity towards 1,3-dichloro-2-propanol increasing by three folds. Conclusion The optimization based on the secondary structure of 5′mRNA improves the expression level of HheC and enhances the synthesis efficiency of the target product, laying a foundation for constructing the route of enzymatic catalytic synthesis of chiral epichlorohydrin.

Objective To screen the biocontrol bacterial isolates with antagonistic activity against root rot pathogens of Panax notoginseng from the rhizosphere of Bletilla striata in a P. notoginseng-B. striata rotation system, identify the isolates, and evaluate their control effects, thereby providing a scientific basis for the application of P. notoginseng-B. striata rotation in alleviating continuous cropping obstacles and screening biocontrol strains from the rhizosphere of the plants for rotation rather than from that of target plants in the plant rotation system for controlling soil-borne diseases in the field. Methods The dilution plate method was employed to isolate culturable microorganisms from the rhizosphere soil of B. striata. The antagonistic activities of the isolates against root rot pathogens of P. notoginseng were evaluated via the dual culture assay. Molecular identification was performed based on the 16S rRNA gene sequence. In pot experiments, antagonistic bacteria were inoculated, and then the ability of antagonistic bacteria to control root rot was evaluated based on the root rot incidence of P. notoginseng. Results The rotation with B. striata significantly reduced the incidence and disease index of P. notoginseng root rot compared with continuous cropping. A total of 200 bacterial strains were isolated from the rhizosphere soil of B. striata, from which 25 strains exhibiting antagonistic activities against the root rot pathogens including Ilyonectria destructans RS6, Fusarium solani F3, and Fusarium oxysporum Z5 of P. notoginseng were screened out. The isolation efficiency of antagonistic bacteria was 12.5%. The 25 strains were identified as 12 species belonging to 5 genera, demonstrating rich diversity. There were 14 strains of Bacillus sp., including 4 strains of B. subtilis, 2 strains of B. velezensis, 6 strains of B. amyloliquefaciens, 1 strain of B. cereus, and 1 strain of B. toyonensis. There were 5 strains of Acinetobacter, including 2 strains of A. johnsonii, 2 strains of A. junii, and 1 strain of A. pittii. There were 4 strains of Pseudomonas, including 3 strains of P. putida and 1 strain of P. fulva. In addition, 1 strain of Enterobacter asburiae and 1 strain of Aeromonas caviae with antagonistic activities were isolated. Four strains (B. amyloliquefaciens BJ1, B. subtilis BJ7, B. amyloliquefaciens BJ8, and A. johnsonii YB10) antagonistic to all the three pathogens were applied to continuous cropping soil. They significantly reduced the root rot incidence and enhanced the fresh weight of P. notoginseng. Conclusion The rhizosphere soil of B. striata in rotation with P. notoginseng harbors diverse biocontrol strains against the root rot pathogens of P. notoginseng. This study lays a theoretical foundation for the rotation of P. notoginseng with B. striata to alleviate continuous cropping obstacles.

Objective The mucosal microbiota of large yellow croaker (Larimichthys crocea) plays a vital role in host health and environmental adaptation. However, differences in the mucosal microbiota composition between wild and cultured populations of large yellow croaker, as well as their assembly mechanisms, remain unclear. This study compared the microbiota composition and characteristics in the skin, gill, and intestine of wild and cultured large yellow croaker, aiming to elucidate the impacts of aquaculture environments on host-microbiota interactions. Methods We employed 16S rRNA gene high-throughput sequencing to analyze the microbiota characteristics in the skin, gill, intestine, and surrounding seawater of wild and cultured large yellow croaker. Key bacterial strains were isolated and characterized. Results Significant divergence of mucosal microbiota was observed between cultured and wild populations. (1) Cultured fish exhibited homogenized mucosal microbiota across the skin, gill, and intestine, whereas wild fish maintained strong tissue-specific microbial signatures. (2) Cultured fish had more unique taxa in the skin (156 vs. 93) and gill (171 vs. 50) but fewer unique taxa in the intestine (118 vs. 253) than wild counterparts. (3) LEfSe revealed enrichment of potential opportunistic pathogens (e.g., Vibrio and Photobacterium) in the skin/gill of wild fish, while myxobacteria (e.g., Haliangium) were specifically enriched in the intestine of cultured fish. (4) Co-occurrence networks demonstrated predominantly cooperative interactions in both groups, yet wild fish microbiota displayed stronger competitive relationships. (5) Lysinibacillus sp. isolated from the mucosal tissue of large yellow croaker demonstrated cross-tissue colonization in cultured populations, exhibiting broad-spectrum antibiotic susceptibility and potential probiotic properties. Conclusion The aquaculture environment significantly reshaped the mucosal microbiota composition, diversity, and interaction networks in large yellow croaker, resulting in reduced microbiota stability and impaired metabolic processes, immune competence, and environmental adaptability of the host. These findings provide critical insights for refining aquaculture practices, developing probiotic resources, and mitigating microbiota dysbiosis in large yellow croaker.

Objective To screen out a strain with the ability to degrade penicillin G (PENG) and identify the key enzymes involved in PENG catabolism, providing strain and gene resources for the biological treatment of penicillin waste. Methods Bacterial strains capable of utilizing penicillin G potassium (PGK) as the sole carbon source were screened by enrichment culture. Key enzymes involved in the catabolism of PGK were identified by genome and transcriptome analyses, and their evolutionary origins were examined. The key enzymes were expressed and purified, and their kinetics were analyzed. The physiological roles of the key genes in bacterial growth on PGK were revealed by gene knockout and complementation. Results The obtained strain Delftia sp. PG-8 can degrade PGK and utilize it as the sole carbon source for growth. The strain showed the best performance in PENG degradation and growth at pH 7.0, 35 ℃, and 10.00 mmol/L PGK. PgkA catalyzed the rapid degradation of PGK, with K_m=(99.19±19.45) μmol/L and k_cat/K_m=(1.96±0.55)×10⁵ L/(mol·s). Compared with the functionally characterized β-lactamases, PgkA had a unique evolutionary origin. PgkB also had the ability to catalyze the transformation of PGK, while its substrate affinity was only 1/5 that of PgkA, in addition to the lower catalytic efficiency. The degradation and utilization of PGK for growth by strains PG-8-ΔpgkA and PG-8-ΔpgkB were significantly reduced, with PG-8-ΔpgkA showing a more pronounced decline. Although PG-8-ΔpgkAB, in which both pgkA and pgkB were knocked out, still degraded a certain amount of substrate, it was almost unable to use PGK as the sole carbon source for growth. Conclusion PG-8 is the first strain of Delftia capable of using PGK as the sole carbon source for growth. Both pgkA and pgkB play important physiological roles during PG-8 growth on PGK, with pgkA playing a dominant role.

Objective Bacteria of Pseudomonas are the main cause of food-borne and clinical infections. At present, mass spectrometry and 16S rRNA gene are widely used to identify Pseudomonas, while the facilities relied on are expensive and require cumbersome operation. Internationally, the numerical identification products require manual single-sample loading procedures, which are cumbersome. This study aims to develop a numerical identification kit for Pseudomonas with high accuracy and simple operation. Methods On the basis of the existing biochemical reaction data of Pseudomonas, we developed a numerical identification model via the branching diagram method and designed and optimized 11 biochemical matrix formulas for microquantization. We then used the kit to identify the standard strains and isolates of Pseudomonas and compared the results with those from mass spectrometry and PCR to evaluate the performance of the kit. Results A numerical identification kit was developed, and it was capable of identifying 10 species of Pseudomonas with just one sample addition. The accuracy rate of the kit in the identification for 5 standard strains and 135 isolates reached 97.04%, and the accuracy rate in actual samples was 97.74%. The biochemical test was stable and reproducible, and the identification cost (25 CNY/sample) of the kit was only 10% of that (240 CNY/sample) of the comparable product from bioMérieux (France). Conclusion The numerical identification kit developed in this study for Pseudomonas has simple operation, a low price, and high accuracy, which can be used in clinical diagnosis and food detection.