Objective To screen potential probiotics with antagonistic effects against Nocardia seriolae from the intestine of healthy largemouth bass (Micropterus salmoides). Methods Intestinal samples were collected from healthy largemouth bass, and strains were isolated and purified via serial dilution and spreading on TSA plates. The hole-punch method was adopted to select strains with antagonistic effects against N. seriolae as test strains for subsequent experiments. The species of the test strains were identified through morphological characterization, physiological and biochemical tests, 16S rRNA gene sequence alignment, and phylogenetic analysis. Additionally, the growth characteristics, adhesion ability, antibacterial activity of cell-free fermentation supernatants, and biosafety of the test strains were determined and analyzed. Results Among the 40 bacterial strains isolated from the intestinal samples of healthy largemouth bass, two strains (XXLC06 and XXLC08) with stable and significant antagonistic effects against N. seriolae were selected. XXLC06 was identified as Lysinibacillus macroides and XXLC08 as Lysinibacillus fusiformis. Both strains grew well at 25-37 ℃, salinities of 5‰-30‰, and pH 5.5-9.0, with survival rates higher than 53% at 0.3% and 0.5% bile salt concentrations. Auto-aggregation assays showed that the auto-aggregation rates of XXLC06 and XXLC08 after 8 h were 68.09% and 63.16%, respectively. In the three organic solvents, both XXLC06 and XXLC08 exhibited hydrophobicity rates exceeding 50%. After co-incubation with N. seriolae for 8 h, the co-aggregation rates of the two strains reached 54.62% and 52.44%, respectively. The diameters of the inhibition zones of cell-free fermentation supernatants of XXLC06 and XXLC08 against N. seriolae were (28.15±0.44) mm and (22.63±0.52) mm, respectively. Neither strain showed hemolytic activity, and both were sensitive to 23 tested antibacterial agents. Acute toxicity tests confirmed that they were non-pathogenic to largemouth bass. Conclusion Strains XXLC06 and XXLC08 exhibited favorable growth adaptability, adhesion capability, biosafety, and stable antagonistic activity in vitro . This study provides potential probiotic resources and experimental evidence for the control of N. seriolae.

Staphylococcus aureus, a common foodborne pathogen causing hospital-acquired infection, poses a grave threat to public health and safety, resulting in a substantial economic burden on the society. As a conditionally essential amino acid, arginine exhibits a dual role in the infection of S. aureus and the immune response of the host. On the one hand, arginine synthesis and catabolism are involved in pathogenic processes such as the biofilm formation and antibiotic resistance of S. aureus. On the other hand, arginine metabolites play an important role in anti-infective immunity, tissue repair, and wound healing through the modulation of macrophage polarization, immune modulation, metabolic reprogramming, and signaling. Recent studies suggest that arginine metabolism constitutes a regulatory hub for S. aureus-macrophage interactions, and its metabolic balance affects the progression and regression of infection and anti-infection. Consequently, targeting the arginine metabolic pathway to impede S. aureus infection by regulating host-pathogen metabolic interactions has emerged as a novel anti-S. aureus therapeutic strategy with significant translational medical relevance. In this review, we focus on the metabolic utilization of arginine to describe how S. aureus and macrophages exert their respective biological functions by competing for the utilization of arginine. In addition, we summarize the changes of arginine levels in macrophages during S. aureus infection to explore the feasible research directions and challenges of regulating arginine metabolism as a potential antimicrobial strategy in the future.

Objective To analyze the expression strategy, DNA-binding characteristics, and the role in heavy metal responses and transcriptional regulation of the UrcA-like membrane protein Chr1_2170 from Sphingobium xenophagum C1. Methods Chr1_2170 was expressed in Escherichia coli BL21(DE3) by codon optimization, dual-signal peptide guidance, and co-expression with homologous molecular chaperones. The interacting genes of Chr1_2170 were screened by constructing a functional promoter library of protein-bound genomic DNA fragments. The heavy metal response characteristics of Chr1_2170 were analyzed via the Chr1_2170-Luc reporter system. Results Chr1_2170 was successfully expressed in E. coli BL21(DE3). Six promoter regions specifically bound by Chr1_2170 were screened out and identified, with the conserved motif of 5′-AATXGCGXGTA-3′. Gene function annotation predicted that Chr1_2170 regulated multiple genes, including those encoding β-N-acetylglucosaminidase, two-component system ATP-binding protein, DNA topoisomerase IV subunit B, and serine hydrolase. Chr1_2170 showed dose-dependent responses to Cu²⁺ (1-80 μmol/L), Zn²⁺ (1-80 μmol/L), and Ba²⁺ (1-150 μmol/L). Conclusion Chr1_2170 functions not only as a heavy metal sensing element but also as a multifunctional transcriptional regulator. It regulates the expression of related genes by recognizing specific DNA sequences, playing a key role in environmental adaptation and stress responses of bacteria.

As a globally prevalent malignancy with poor prognosis, liver cancer exhibits a well-established pathological association with the gut microbiota (GM). In recent years, increasing attention has been paid to the role of the GM in the initiation and pathological progression of liver cancer, which often evolves through stages of hepatitis, liver fibrosis, cirrhosis, and ultimately liver cancer. The GM influences the development of liver cancer through multiple mechanisms, including the regulation of the hepatic immune microenvironment by the GM and its metabolites, the mediation of epigenetic modifications and exosomal signaling pathways, and the synergy with other risk factors. Notably, patients with liver cancer commonly demonstrate reduced GM diversity and enriched pathogenic bacteria. These findings offer a new theoretical foundation and suggest potential therapeutic strategies such as probiotic supplementation, rational antibiotic use, fecal microbiota transplantation, combination therapies integrating GM modulation with conventional treatments, and integrated treatment regimens based on the above methods. This article reviews the pathogenesis of liver cancer mediated by GM dysbiosis and the research advances in GM-targeted interventions in recent years, providing reference for future studies on the pathogenesis and treatment of liver cancer.

Objective To investigate the mechanism by which the microbial fermentation product of the traditional Chinese medicine YA3D3 (YA3D3-MHF) improves cognitive function in the APP/PS1 transgenic mouse model of Alzheimer’s disease (AD) via the microbiota-gut-brain axis. Methods APP/PS1 mice were administered either the water extract of YA3D3 (YA3D3-HF) or YA3D3-MHF for 90 days. The gut microbiota structure was analyzed by 16S rRNA gene sequencing, and the fecal levels of short-chain fatty acids (SCFAs) were assessed by GC-MS. The neurotransmitter content in the brain tissue was measured via ELISA, and cognitive function was assessed via the Morris water maze. Network pharmacology and mass spectrometry were employed to identify active components and changes in chemical composition. Results Compared with the model group and the YA3D3-HF group, YA3D3-MHF significantly ameliorated cognitive impairment in mice. The Morris water maze test showed that the high-dose YA3D3-MHF (MH) group had the shortest escape latency and the highest number of platform crossings, approaching the performance of the normal control group. ELISA confirmed that the MH group had the highest levels of 5-hydroxytryptamine (5-HT), γ-aminobutyric acid (GABA), and glutamate (GLU) in the brain. The results of 16S rRNA gene sequencing revealed that the MH group exhibited the highest alpha diversity (Shannon index≈3.2) of gut microbiota and the highest abundance of beneficial bacteria, along with the lowest abundance of pro-inflammatory bacteria. GC-MS analysis indicated that the MH group had the highest levels of total SCFAs, acetate, and butyrate. MS demonstrated that YA3D3-MHF components exhibited reduced polarity and the emergence of new high-activity peaks. Conclusion YA3D3-MHF improves cognitive function in AD mice by modulating the gut microbiota-SCFAs-neurotransmitter axis, outperforming YA3D3-HF. This study provides experimental evidence for AD intervention targeting the gut-brain axis.

Objective To investigate the transcriptional regulation of quorum sensing (QS) regulators AphA, ToxR, and QsvR on the expression of the phosphodiesterase (GepA) gene gepA in Vibrio parahaemolyticus. Methods Total RNAs were extracted from the wild type (WT) and the mutant strains of aphA, toxR, and qsvR. Quantitative real-time PCR (qPCR) was carried out to calculate the transcriptional variation of gepA between WT and mutant strains. The regulatory DNA region of gepA was cloned into the upstream region of promoterless luxCDABEreporter gene in the pBBRlux plasmid. The recombinant plasmid was respectively transferred into the WT and mutant strains. Luminescence assay was used to test the regulatory effect of QS regulators on the expression of gepA. The primer extension assay was employed to detect the transcription start site and the promoter activity of gepA. The effects of QS regulators on gepA were evaluated based on the abundance of primer extension products. The regulatory DNA region of gepA was cloned into the upstream region of lacZ in the pHRP309 plasmid. The LacZ recombinant plasmid was transformed into EC100 λpir harboring pBAD33 or PBAD33-qsvR. Two-plasmid LacZ reporter assay was conducted to investigate the regulatory effects of QS regulators on the transcription of gepA in EC100 λpir. The regulatory DNA region of gepA was amplified by PCR, and the His recombinant proteins of QS regulators were purified. The electrophoretic mobility shift assay (EMSA) was performed to investigate whether QS regulators directly regulated the expression of gepA. Results At low cell density, the qPCR results showed that expression of gepA in ΔaphA and ΔtoxR were significantly lower than that in WT, indicating that AphA and ToxR activated the transcription of gepA. The luminescence assay showed that the transcriptional activity of the promoter region of gepA in ΔaphA and ΔtoxR was significantly lower than that in WT, further indicating that AphA and ToxR promoted the transcription of gepA. The primer extension assay detected that the transcription start site of gepA was located at the A nucleotide 30 bp upstream of the start codon ATG, and its transcriptional activity was activated by AphA. The EMSA result indicated that His-AphA and His-ToxR were unable to bind the promoter DNA region of gepA. At high cell density, both the qPCR and primer extension assay indicated that QsvR inhibited the transcription of gepA. The EMSA result demonstrated that His-QsvR directly bound to the promoter DNA region of gepA. Two-plasmid lacZ reporter assay demonstrated that QsvR inhibited the transcriptional activity of the promoter region of gepA in EC100 λpir. Conclusion AphA and ToxR indirectly activate while QsvR directly inhibits the transcription of gepA. Therefore, the transcription level of gepA is higher at low cell density and significantly decreases at high cell density.

Objective To systematically isolate and purify the polysaccharide from the mycelium of Streptomyces rochei D74 (SRP), elucidate its fine structure, and evaluate the effect of the purified polysaccharide fraction on the growth of Salvia miltiorrhiza hairy roots and the biosynthesis of tanshinones, along with the underlying mechanism. Methods The crude polysaccharide was extracted using hot water, which was followed by ethanol precipitation and deproteinization via the Sevag method. Further purification was performed using DEAE-52 anion-exchange chromatography and Sephadex G-100 gel filtration chromatography. The physicochemical properties and structural features of the main active fraction, SRP-W-2, were systematically characterized by Fourier transform infrared spectroscopy (FTIR), high performance liquid chromatography-mass spectrometry (HPLC-MS), and nuclear magnetic resonance (NMR). The effects of SRP-W-2 on hairy root growth and the biosynthesis of tanshinones were assessed by measuring biomass, tanshinone content, and the expression levels of key biosynthetic genes. Results SRP-W-2 was obtained with a yield of 2.41%. It was primarily composed of glucose and galactose at a molar ratio of 12.53:1. Structural analysis revealed that the backbone of SRP-W-2 consisted of →4)-α-d-Glcp-(1→ and →4)-α-d-Galp-(1→ residues, with branching points at →4,6)-α-d-Glcp-(1→ and →4,6)-α-d-Galp-(1→. The side chain was identified as α-d-Glcp-(1→4)-α-d-Glcp-(1→. Bioactivity assays demonstrated that SRP-W-2 significantly enhanced both the biomass of S. miltiorrhiza hairy roots and the accumulation of tanshinones. After 15 d of treatment with 50 mg/L SRP-W-2, the dry weight of the hairy roots increased by 37.52%. Meanwhile, the content of cryptotanshinone (CT), dihydrotanshinone I (DT-I), tanshinone I (T-I), and tanshinone IIA (T-IIA) was increased by 19.0-fold, 6.4-fold, 2.8-fold, and 4.8-fold, respectively. Gene expression analysis further indicated that SRP-W-2 up-regulated key genes involved in the tanshinone biosynthetic pathway, including HMGR, DXS, DXR, and GGPPS. Conclusion The polysaccharide fraction SRP-W-2 from S. rochei D74 simultaneously promoted the growth of S. miltiorrhiza hairy roots and the biosynthesis of tanshinones, demonstrating its potential as an effective elicitor. This study provided a new strategy for the utilization and development of S. miltiorrhiza resources.

Objective To isolate the Streptomyces hebeiensis strain JL9001 with significant biocontrol potential against tomato Fusarium wilt from Pseudostellaria heterophylla roots, elucidate the complete genome sequence and functional annotation of the strain, and extract genetic data pertaining to its secondary metabolites, thus offering a valuable microbial resource and a theoretical foundation for the biological management of tomato Fusarium wilt. Methods The colony morphology on various media was examined via the plate streaking technique. The antagonistic properties of strain JL9001 against Fusarium oxysporum were evaluated through the plate confrontation assay. The activities of metabolites (crude fermentation extract) against F. oxysporum were assessed via the microdilution method. The effectiveness of strain JL9001 in managing tomato Fusarium wilt was evaluated through root drenching with the fermentation broth. Whole genome sequencing of strain JL9001 was conducted, and the sequencing data were analyzed by appropriate software for species identification, gene prediction, functional annotation, and prediction of secondary metabolite biosynthesis gene clusters. Results Strain JL9001 demonstrated optimal spore production on the SIM medium. Antagonistic assays indicated that it inhibited the mycelial growth of F. oxysporum by 40.18%. Furthermore, the crude fermentation extract at a concentration of 1 000 μg/mL completely inhibited F. oxysporum. Pot trials revealed that irrigation with the fermentation broth of JL9001 resulted in a 51.61% reduction in tomato Fusarium wilt on day 13. The genome of strain JL9001 comprised 7 700 822 base pairs with the G+C content of 71.46%, encompassing 6 589 genes. Analysis predicted the presence of 27 biosynthetic gene clusters for secondary metabolites including terpenoids, polyketides, and siderophores, which may possess antimicrobial properties. Conclusion This study elucidates, through antagonistic and pot experiments, that strain JL9001 effectively mitigates the incidence of tomato Fusarium wilt. The analysis of the genomic composition and functional gene information of strain JL9001 provides a basis for exploring the antimicrobial mechanisms of natural products, examining secondary metabolite biosynthetic gene clusters, and assessing the potential of Streptomyces-derived secondary metabolites.

Biochar serves as an excellent carrier for non-symbiotic nitrogen (N)-fixing bacteria, enhancing their microbial activity and functions. However, the coupling mechanism between non-symbiotic N-fixing bacteria and biochar remains unclear. Objective To explore the effects of different biochar materials on the colonization pattern and N fixation efficiency of non-symbiotic N-fixing bacteria. Methods Non-symbiotic N-fixing bacteria were inoculated onto biochar samples derived from maize straw and wood chips, the particle sizes of which were >2.00 mm, 0.25-2.00 mm, and <0.25 mm. We compared the porosity and specific surface area of different biochar samples. Throughout the incubation period, the dynamic changes in nitrogenase activity and the number of N-fixing bacteria, pH, dissolved organic carbon (DOC), dissolved organic nitrogen (DON), microbial biomass carbon (MBC), and microbial biomass nitrogen (MBN) were monitored. Results N-fixing bacteria exhibited more uniform colonization and higher N fixation activity on straw biochar than on woody biochar, especially in the 0.25-2.00 mm group. The straw biochar with the particle size of 0.25-2.00 mm and inoculated with strains showed increases of 82.33%-160.55% and 231.46%-356.08% in the average MBC content and nitrogenase activity, respectively, compared with woody biochar. Moreover, significantly higher content of DOC and DON were maintained in all straw biochar groups, which provided a richer pool of available nutrients for microbial growth. The correlation heatmap indicated that pH significantly affected bacterial colonization and nitrogenase activity. Furthermore, nitrogenase activity showed strong positive correlations with DOC and MBC (P<0.001), which suggested that a carbon-rich environment was a key factor for the growth and N fixation of N-fixing bacteria. Conclusion Straw biochar with the particle size of 0.25-2.00 mm serves as an optimal carrier for non-symbiotic N-fixing bacteria. It provides a favorable microenvironment for the N fixation and some other functions of the bacteria.

Objective To investigate the differences and associations in endophytic microbial communities across four ecological niches of pepper varieties with varying pulp thickness and to delve into the microbial community disparities associated with different pepper pulp thickness. Methods We extracted DNA from the roots, stems, leaves, and fruits of pepper varieties with varying pulp thickness. The bacterial 16S rRNA gene and fungal ITS region of the endophytic microbial communities within these four niches were sequenced on the Illumina platform. Microbial taxa potentially associated with pulp thickness were identified and screened, followed by validation through pot experiments. Results Endophytic bacterial and fungal communities in the four ecological niches of pepper varieties with different pulp thicknesses all exhibited differences. Particularly, the bacterial community structure in the fruit displayed the most significant variations. Bar plots at the genus level and analyses of species disparities revealed that the genus Sphingomonas was significantly enriched in the pepper varieties with thick pulp and showed a positive correlation with pulp thickness. A total of 28 endophytic strains were isolated from pepper fruits. Among them, two strains belonged to the genus Sphingomonas, identified as S. aquatilis and S. yabuuchiae. Each of the two bacterial strains exhibited capabilities of both indole-3-acetic acid production and nitrogen fixation. Pot experiments demonstrated that inoculation with the two endophytic strains significantly promoted the fruit growth of pepper plants, increasing the pulp thickness by 75.44%. Conclusion The relative abundance of Sphingomonas in pepper fruits showed a significantly positive correlation with pulp thickness and Sphingomonas promoted fruit growth. This study is of great significance for revealing the role of endophytic microbial communities in the regulation of pepper fruit development and lays a theoretical foundation for improving pepper fruit quality.