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.

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.

Heat shock protein family A member 8 (HSPA8) is a widely distributed molecular chaperone in cells. HSPA8 is involved in multiple cellular physiological processes, mainly including the correct folding and transport of proteins, stress responses, presentation of antigenic proteins, mediation of autophagy, and immune regulation. It is also crucial for maintaining the homeostasis of the intracellular environment. In addition, HSPA8 plays a key role in multiple processes of virus infections. This paper reviewed the important roles and molecular mechanisms of HSPA8 in processes such as virus adhesion, formation of virus glycoprotein-receptor complexes, internalization, uncoating, genome replication, assembly, and regulation of host metabolism and immune responses, with the hope of laying a foundation for the subsequent development of drugs targeting HSPA8 for the treatment of virus infections.

Coronaviruses pose a serious threat to human and animal health. Their main protease (Mpro) plays a critical role in both the viral life cycle and host immune regulation, serving as a key target for the development of broad-spectrum anti-coronavirus drugs. The core function of Mpro lies in its specific cleavage of viral polyproteins pp1a and pp1ab to release functional non-structural proteins (NSPs), thereby driving the assembly of the viral replication/transcription complex. Additionally, Mpro can target and cleave key molecules in host immune signaling pathways, facilitating viral immune evasion. Given its dual roles and highly conserved catalytic center, the research on Mpro has become a major focus in the field. This review systematically outlines the structural features and functional diversity of Mpro, with an emphasis on its catalytic mechanism in the viral replication cycle and its role in mediating immune suppression. Furthermore, this article details the screening methods and design strategies for Mpro inhibitors, aiming to offer theoretical foundations and novel insights for the development of anti-coronavirus drugs targeting this critical protein.

Understanding the source, colonization rules, and dynamic evolution process of the gut microbiota is of significant importance for regulating host health, given its crucial role in nutrient metabolism, immune regulation, and intestinal barrier function. This article comprehensively reviews the composition and functions of the gut microbiota and explores the origins and transmission pathways, with a particular focus on the effects of maternal-infant transmission and paternal inheritance on the structure and functions of the gut microbiota. We chart microbial assembly across pivotal life stages, distill the driving factors involved in the community assembly process, and critically appraise the advances in metagenomic and source-tracking toolkits. The review provides an integrated framework for microbiome-targeted strategies aimed at reconstructing a health-oriented gut ecosystem.

Actinobacteria are Gram-positive bacteria of major ecological and biotechnological importance, responsible for organic matter turnover in nature and serving as a primary source of antibiotics and other bioactive natural products. Their complex physiological adaptation and life cycles are regulated by sophisticated signal transduction networks. This review examines the metabolism, signaling, and regulatory networks of nucleotide second messengers including cyclic diadenosine monophosphate (c-di-AMP), cyclic di-guanosine monophosphate (c-di-GMP), adenosine monophosphate (cAMP), and (p)ppGpp in the morphologically complex Streptomyces with rich secondary metabolites and pathogenic Mycobacterium with host adaptation. We discuss how these second messengers interact with other signaling systems, such as two-component systems, quorum sensing, and protein acylation, to integrate environmental and developmental cues. This coordination regulates the growth, development, secondary metabolite biosynthesis, and environmental adaptation of actinobacteria. By synthesizing current knowledge, this review provides reference for understanding the integrity and dynamics of the signal transduction system of actinobacteria, as well as their potential applications in the basic research of life sciences and in the fields of biotechnology and medicine.

The enzootic nasal tumor of sheep and goats is a progressive and contagious disease caused by enzootic nasal tumor virus. It is mainly characterized by tumor growth in the mucosal epithelial tissue of the ethmoid bone and nasal turbinates within the nasal cavity of sheep and goats. In the later stage of the disease, the significant enlargement of tumor volume can lead to upper respiratory tract obstruction, which subsequently causes the affected animals to die of asphyxiation. This disease markedly reduces the production performance of infected animals, causing economic losses to the livestock industry. Moreover, it poses a threat to precious local breeds and core breeding flocks, resulting in the loss of high-quality breeding sheep. Consequently, it has become one of the major diseases threatening the sheep and goat industry. Currently, there are no vaccines or specific treatments for this disease. This article reviews enzootic nasal tumor virus in terms of the etiology, epidemiology, clinical symptoms, main pathological changes, diagnosis, and prevention and control, providing references and ideas for the prevention and control of this disease.

Uropathogenic Escherichia coli (UPEC) is the leading cause of urinary tract infections (UTIs). It can adhere to and colonize uroepithelial cells, disseminate systemically, and induce severe sepsis and subsequent renal failure, posing a substantial threat to global public health. Emerging evidence indicates that lactylation, a key post-translational modification (PTM) in macrophages, plays a crucial role in the host defense against UPEC infection. Notably, the UPEC CFT073 strain harbors ldhA, which encodes lactate dehydrogenase (LDH), an enzyme critical for lactate biosynthesis. However, the mechanism by which LdhA (the ldhA-encoded LDH) regulates macrophage lactylation during UPEC infection remains elusive. Objective To elucidate how LdhA modulates macrophage lactylation and thereby impacts UPEC pathogenicity. Methods Online bioinformatics tools were used to predict the functional domains and transmembrane regions of LdhA. The recombinant protein rLdhA was generated via molecular cloning, and its LDH activity was measured by a commercial LDH activity assay kit. Western blotting was performed to assess the cellular entry of rLdhA into macrophages and its regulatory effect on macrophage lactylation. Enzyme-linked immunosorbent assay (ELISA) was employed to measure the secretion of inflammatory cytokines in macrophages treated with rLdhA. The drug resistance profile of UPEC CFT073 (wild-type and ldhA-knockout strains) was analyzed via an automated microbial identification system. To evaluate the role of LdhA in UPEC pathogenicity, we treated mice with the wild-type UPEC CFT073 (CFT073^wt), ldhA-deficient mutant (CFT073^ΔldhA ), or rLdhA (with or without pretreatment with an LDH inhibitor) through intraperitoneal injection or tail vein injection, and then observed and quantified pathogenic phenotypes. Results LdhA harbored a LDH domain and was secreted extracellularly. We successfully established an expression system for ldhA and achieved efficient expression and purification of rLdhA. Functional assays confirmed that rLdhA exhibited LDH activity and can enter macrophages via clathrin-mediated endocytosis, subsequently enhancing macrophage lactylation in a dose-dependent manner. Additionally, rLdhA significantly inhibited lipopolysaccharide (LPS)-induced inflammatory cytokine production in macrophages. Furthermore, LdhA was found to substantially modulate the drug resistance profile of UPEC CFT073. In vivo studies demonstrated that LdhA promoted the pathogenicity of UPEC in a mouse infection model. Conclusion Collectively, our findings demonstrate that LdhA enhances UPEC pathogenicity by upregulating macrophage lactylation and suppressing the production of proinflammatory cytokines. However, the underlying molecular mechanisms mediating this regulatory cascade remain to be fully elucidated and warrant further exploration. This study offers a new theoretical basis for deciphering the pathogenic mechanisms of UPEC infections.

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.

Objective To identify the key amino acid residues of the TetR family transcription factor BcPDR1 in Botrytis cinerea, thereby laying a foundation for elucidating the mechanism by which BcPDR1 regulates the growth, development, and pathogenicity of this pathogen. Methods The key amino acid sites of BcPDR1 were analyzed by bioinformatics methods, and four conserved regions (32-34 aa, 76-95 aa, 140-150 aa, and 189 aa) were selected for site-directed mutagenesis. On the basis of the knockout mutant ΔBcpdr1, the mutants BcPDR1-M1 (Δ32-34), BcPDR1-M2 (Δ76-95), BcPDR1-M3 (Δ140-150), and BcPDR1-M4 (mutation of Ile to Lys at 189 aa) were constructed. A comparative analysis of the phenotypic characteristics and pathogenicity was conducted on the four aforementioned mutants and the wild-type strain of B. cinerea, ΔBcpdr1, the complemented strain CE. Results The colony morphology, mycelial morphology, and growth rates of BcPDR1-M1, BcPDR1-M2, BcPDR1-M3, and BcPDR1-M4 were similar to those of ΔBcpdr1, but significantly different from those of BC22 and CE. These mutants could form lesions on tomato fruits and tobacco leaves, while their lesion areas were significantly smaller than those of BC22 and CE. Conclusion The regions 32-34, 76-95, 140-150, and the 189th amino acid are the regulatory sites for BcPDR1 to exert its functions.

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.

Objective Senecavirus A (SVA) keeps posing a serious threat to the swine industry in China. This study aimed to characterize the biological properties and genetic evolutionary features of the latest circulating SVA strains. Methods In November 2024, vesicular lesion tissue samples were collected from pigs suspected of foot-and-mouth disease at a farm in eastern China. RT-PCR was performed, and positive samples were inoculated onto BHK-21 cells following standard virus isolation procedures. Indirect immunofluorescence assay was employed to preliminarily confirm viral isolation. The complete viral genome was amplified and sequenced, followed by genetic evolution analysis. Amino acid variations in the VP1 protein of the strain were identified by comparison with representative strains from key epidemic nodes. Viral replication characteristics were evaluated through plaque formation assay and one-step growth curve analysis. Viral particle morphology was observed via transmission electron microscopy (TEM). Results RT-PCR and virus isolation confirmed SVA as the causative agent of the disease, and the isolated strain was designated SDWF/11/2024. The viral genome of this strain was 7 292 bp in length, and its overall organization was highly consistent with that of previously reported SVA strains. Phylogenetic analysis revealed that SDWF/11/2024 belonged to the USA-like evolutionary clade, showing genetic divergence from Chinese strains circulating between 2015 and 2018, while exhibiting the closest relationship to a Chilean strain isolated in 2022. The isolate replicated efficiently in BHK-21 cells and induced typical cytopathic effects. Its replication kinetics was comparable to that of the early Chinese isolate HN/11/2017, although differences in plaque morphology were observed. TEM examination identified spherical viral particles with diameters of 25-35 nm, consistent with typical SVA virions. Conclusion This study successfully isolated and characterized the SVA strain SDWF/11/2024 circulating in China, 2024, and elucidated its molecular evolutionary features. The isolated SDWF/11/2024 provides a new reference strain for SVA surveillance in China and suggests that the virus may still persist at low levels in pig populations. These findings enhance our understanding about the genetic diversity and epidemic dynamics of SVA and support the improvement of molecular epidemiological monitoring and the development of prevention and control strategies.

Pasteurella multocida (Pm) and Mannheimia haemolytica (Mh) are major bacterial pathogens responsible for bovine respiratory diseases. However, the diversity of these two pathogens in transported calf populations remains poorly understood, which severely hinders the effective prevention and control of their infections. Objective To investigate the diversity of Pm and Mh in a group of fattening calves purchased from a calf trading market in Inner Mongolia and transported to a breeding farm in Hechuan, Chongqing. Methods After arrival, nasal swabs were collected from calves showing respiratory symptoms at four time points for bacterial isolation and culture. Suspected Pm and Mh colonies were selected based on colony morphology, hemolytic characteristics, and Wright-Giemsa staining results, followed by PCR identification and 16S rRNA gene sequencing for confirmation. Furthermore, the serotypes, biochemical and antibiotic resistance profiles, virulence genes, and resistance genes of the isolates were analyzed. Results A total of 23 strains of Pm serotype A, 10 strains of Mhserotype A6, and 1 strain of Mhserotype A2 were isolated from 68 nasal swabs collected at 4 different time points, and only 1 nasal swab harbored both Pm and Mh. Some isolates exhibited diversity in biochemical and antibiotic resistance profiles, which had no significant correlation with sampling time points. Antimicrobial susceptibility testing revealed that Pm and Mh isolates were resistant to most penicillins, aminoglycosides, and lincosamides but remained sensitive to cephalosporins and quinolones. Resistance gene detection showed that β-lactamase resistance (bla_TEM ) genes were detected in 73.91% of Pm isolates and 90.91% of Mh isolates, while sulfonamide resistance (sul2) genes were found in 69.57% of Pm isolates and 18.18% of Mh isolates. Only one Mh isolate carried aminoglycoside resistance genes (aadA25 and aadB). Discrepancies were observed between resistance phenotype and the presence of selected resistance genes. All Pm and Mh isolates were pathogenic. Virulence gene analysis confirmed that Pm isolates consistently carried tonB, hsf-1, nanB, oma87, and tbpA, while Mh isolates showed the detection rates of 100% for gapA and dnaN, 82% for lktA, plpB, and tbpB, and 0 for ptfA. Conclusion These findings suggest that calves purchased from trading markets and transported over long distances to new farms harbor Pm and Mh strains exhibiting diversity in biochemical characteristics and drug resistance, which pose challenges for effective infection control. This study provides critical insights for developing prevention and control strategies against Pm and Mh infections in transported calves.

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 investigate the effects of mannanoligosaccharides (MOS) on the in vitro fermentation characteristics and composition of intestinal microbiota in weaned piglets by using an in vitro microbial fermentation technique, with fructooligosaccharides (FOS) taken as the control. Methods Using microbial inocula derived from the jejunal and colonic chyme of piglets, with FOS and MOS as respective substrates, this study measured microbial gas production and fermentation broth pH at five time points (0, 6, 12, 24, and 48 h), and collected fermentation broth samples at each time point for subsequent microbial analysis. Results In the in vitro jejunal microbial fermentation system, both the fermentation broth pH and gas production in the MOS group were significantly higher than those in the FOS group (P<0.05). At 24 h of fermentation, compared with FOS, MOS significantly increased the concentrations of acetate, propionate, and total short-chain fatty acids (SCFAs) (P<0.01). The formate production in the MOS group was significantly lower than that in the FOS group (P<0.01). At 48 h of fermentation, the lactate concentration in the MOS group was significantly lower than that in the FOS group (P<0.01). In the in vitro colonic microbial fermentation system, gas production in the MOS group was significantly higher than that in the FOS group (P<0.05). At 48 h of fermentation, the production of formate, acetate, butyrate, SCFAs, and lactate in the MOS group was significantly higher than that in the FOS group (P<0.01). The 16S rRNA gene sequencing results of the jejunal fermentation broth revealed that at 48 h of fermentation, both the Shannon and Simpson indices in the MOS group were significantly higher than those in the FOS group (P<0.01). Furthermore, the microbiota composition exhibited disparity between the MOS and FOS groups. Moreover, the relative abundances of Bifidobacterium, Limosilactobacillus, and Megasphaera were significantly higher in the MOS group than in the FOS group (P<0.05). Conclusion Compared to FOS, MOS enabled the microbiota in the small intestine of piglets to significantly improve the microbial community structure, increase the abundance of beneficial bacteria such as Bifidobacterium, enhance gas production, and promote the generation of acetate and other SCFAs. These findings suggested that MOS held potential for modulating microecology in the small intestine of weaned piglets.

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.

Objective To screen indigenous rhizobia with high salt tolerance and plant growth-promoting traits from alfalfa nodules and clarify their phylogenetic status and functional potential, thereby providing strain resources and a theoretical basis for developing localized, efficient alfalfa symbiosis and further addressing the constraints of saline soil on alfalfa production in Inner Mongolia. Methods Indigenous alfalfa rhizobia were collected from six saline-alkali sites in Inner Mongolia via a trapping method. Following isolation and purification, the taxonomic status of the strains was determined by 16S rRNA gene sequencing. The plant growth-promoting functions were evaluated through nodulation tests, along with assays for nitrogen fixation, phosphorus solubilization, potassium solubilization, indole-3-acetic acid (IAA) production, and exopolysaccharide (EPS) content. Seed germination and pot experiments were carried out to evaluate the salt stress-alleviating effect of the target strain. Results A total of 250 strains were isolated, with Sinorhizobium meliloti being predominant (227 strains, 90.8%). Eight efficient nodulating strains (e.g., 1B1Y and 1B2Y) were screened out, all of which possessed nitrogen-fixing and potassium-solubilizing capabilities. Some strains (e.g., 2B3Y and 9B3Y) could solubilize organic phosphorus and produce siderophores. Strain 9B3Y secreted the highest amount of IAA (67.5 mg/L), while 16C1Y produced the highest EPS content (2.684 g/L). Under salt stress (0.4% NaCl), the aboveground fresh weight of alfalfa inoculated with strain 2B3Y increased by 29% compared with that of the saline control, and the strain significantly alleviated the inhibition on seed germination (notably increasing the root length). Conclusion The strains screened out, particularly strain 2B3Y, can effectively mitigate the inhibitory effects of salt stress on alfalfa through nitrogen fixation and the secretion of IAA and EPS. These strains show promise for application in alfalfa production and soil improvement in saline regions of Inner Mongolia.

Objective To investigate the effect of Kelch-like ECH-associated protein 1 (KEAP1) on the replication of herpes simplex virus type 1 (HSV-1) and thus provide theoretical support for anti-herpes simplex virus research. Methods The mRNA and protein levels of molecules in the KEAP1-NRF2 signaling pathway and viral molecules in ARPE-19 cells infected with HSV-1 were determined by qPCR and Western blotting, respectively. KEAP1-silenced and overexpressing ARPE-19 cell lines were constructed, and Western blotting was employed to assess the effects of KEAP1 silencing and overexpression on the nuclear factor erythroid 2-related factor 2 (NRF2) signaling pathway. The KEAP1-silenced and overexpressing cell lines were subsequently infected with HSV-1. Changes in viral mRNA expression were detected via qPCR, while immunofluorescence and Western blotting were used to evaluate alterations in viral protein expression. Additionally, a plaque formation assay was conducted to measure variations in viral titer. Western blotting was performed on KEAP1-silenced cell lines infected with HSV-1 to assess the expression levels of NRF2 signaling pathway and viral proteins at different time points. Results Silencing of KEAP1 activated the NRF2 signaling pathway and promoted HSV-1 replication, whereas KEAP1 overexpression downregulated the NRF2 signaling pathway and inhibited HSV-1 replication. These findings contradict previous studies suggesting that upregulation and activation of the NRF2 signaling pathway can suppress HSV-1 replication. Further investigation revealed that KEAP1 silencing-induced NRF2 upregulation was significantly inhibited following HSV-1 infection. Conclusion KEAP1 plays a crucial role in the host cell resistance to HSV-1 infection, and its interaction with NRF2 exerts complex biological functions in antiviral immune responses.

Objective To compare the regulatory effects and underlying physiological mechanisms of Pseudomonas huaxiensis M11 and Bacillus megaterium M28 on the photosynthetic characteristics of maize subjected to low soil fertility stress. Methods A pot experiment was implemented with four treatments: normal soil control (CK), low nutrient treatment (LNT), and bacterial inoculation under LNT conditions (M11+LNT, M28+LNT). At the tasseling stage, measurements were taken for soil nutrients, plant growth indices, gas exchange parameters, chlorophyll fluorescence characteristics, and the fast chlorophyll a fluorescence induction kinetics (O-J-I-P chlorophyll a fluorescence transient, OJIP curve). Yield components were assessed at physiological maturity. Results Inoculation with M11 significantly increased the content of available phosphorus, available potassium, and organic matter, while reducing the electrical conductivity in soil. M28 significantly enhanced the total nitrogen content. Both bacterial treatments significantly promoted maize growth, increasing the plant height, leaf area, SPAD value, and biomass. Moreover, they highly significantly enhanced the net photosynthetic rate (P_n), stomatal conductance (G_s), transpiration rate (T_r), and water use efficiency (WUE), while reducing the intercellular CO₂ concentration (C_i). Chlorophyll fluorescence analysis revealed a decrease in minimal fluorescence (F_o) and increases in the maximum photochemical efficiency (F_v/F_m), actual photochemical quantum yield of PSII (Φ_PSII), apparent photosynthetic electron transport rate (ETR), photochemical quenching (qP), and the fraction of open PSII centers based on excitation energy (qL), with no significant change in non-photochemical quenching (NPQ). The OJIP curves indicated the absence of a K-step in inoculated plants, a decrease in fluorescence at the J-step, and increases at the I-step and P-step. The differential kinetic curves of relative variable fluorescence (ΔV_t analysis) confirmed that both strains synchronized the optimization of electron transport on both the donor and acceptor sides of photosystem II (PSII). The increased amplitude of the I-P phase suggested enhanced photosystem I (PSI) activity. Junction-intermediate-peak test (JIP-test) parameters demonstrated that inoculation significantly enhanced the performance index based on absorbed light energy (PI_ABS), the performance index on a cross-section basis (PI_CS), the probability that a trapped exciton moves an electron into the electron transport chain beyond Q_A (Ψ_o), the quantum yield for electron transport (φ_Eo), and the electron transport flux per reaction center (ET_o/RC). Conversely, dissipated energy flux per cross-sectional area (DI_o/RC) and quantum ratio for dissipated energy (φ_Do) decreased. Consequently, compared with the LNT group, the M11 and M28 treatments resulted in significant increases of 30.61% and 22.64% in maize fresh weight and 26.68% and 23.41% in dry weight, respectively. Conclusion P. huaxiensis M11 primarily enhances photosynthetic performance by increasing soil available phosphorus and potassium content, directly optimizing energy metabolism and stomatal movement, whereas B. megaterium M28 mainly acts by elevating soil total nitrogen content, focusing on stabilizing the structure of the photosynthetic apparatus. Together, they protect the integrity of photosynthetic apparatus and optimize the electron transport efficiency of photosystems, significantly improving the photosynthetic performance and yield of maize under low fertility stress. These findings provide a theoretical basis for the targeted application of microbial inoculants in sustainable agricultural production.

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.

Objective Tad pili are widely distributed in Gram-negative bacteria and are associated with the virulence of various pathogens. However, the studies about the Tad pili in Vibrio parahaemolyticus remain limited. This study aimed to elucidate the role of the Tad pilus secretin CpaC (VP2419) in the biological functions of V. parahaemolyticus. Methods The cpaC-deleted mutant (ΔcpaC) and complemented strain (CΔcpaC) were constructed from the wild-type (WT) strain (SH112) of V. parahaemolyticus. The strains were compared in terms of biofilm formation, competitiveness, swarming and swimming motility, cell adhesion, cytotoxicity, as well as virulence, tissue colonization, and pathology in mice. Results Regarding environmental adaptation, compared with the WT strain, ΔcpaC exhibited significantly decreased competitiveness, motility, and biofilm formation. In terms of pathogenicity, ΔcpaC demonstrated significantly reduced cell adhesion, cytotoxicity, as well as attenuated virulence, tissue colonization, and pathological damage in mice, compared with the WT strain. Conclusion As the Tad pilus secretin in V. parahaemolyticus, CpaC participates in multiple functions related to environmental adaptation and pathogenicity, including competitiveness, biofilm formation, motility, cell adhesion, cytotoxicity, and tissue colonization. These findings provide important insights for a deeper understanding of the biological functions of Tad pili.

Objective To perform biological characterization and genomic analysis for a multidrug-resistant Streptococcus strain isolated from the tonsils of a dead piglet, thus elucidating its phenotypic and genotypic characteristics. Methods The tonsillar tissue homogenate from the dead piglet was inoculated into the Streptococcus enrichment medium for enrichment culture. The enriched culture was then streaked onto Columbia blood agar medium for Streptococcus isolation. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), a biochemical identification system, and 16S rRNA gene sequencing were employed for strain identification. The minimum inhibitory concentrations (MICs) were determined by the broth microdilution method. Whole-genome sequencing was conducted via the Illumina platform. The ResFinder database was used to analyze the resistance genes, and mutations in the genes encoding penicillin-binding proteins (PBPs) were investigated through multiple sequence alignment. Results The phylogenetic analysis based on 16S rRNA gene sequences identified the isolate as Streptococcus orisratti. Antimicrobial susceptibility testing revealed that the strain was resistant to penicillin G, erythromycin, clindamycin, and compound sulfamethoxazole, and it exhibited reduced susceptibility to linezolid. Whole-genome analysis identified the presence of the macrolide and lincoamide resistance gene ermB and the oxazolidinone resistance gene optrA. Furthermore, multiple mutations were detected in the genes encoding PBPs. Conclusion This study reports the first isolate of S. orisratti harboring both ermB and optrA and exhibiting resistance to penicillin. It highlights the capacity of porcine-derived streptococcal strains to acquire multidrug resistance genes, underscoring the need for increased vigilance regarding the resistance traits and transmission risks of animal-derived streptococci.

Objective To determine the efficacy of Cucurbita pepo cv Dayangua (CPD) in alleviating hypoxia and explore the potential mechanisms involving the modulation of the gut microbiota and its metabolites. Methods Male Kunming mice were randomly assigned into two groups: a control group (normoxia ddH₂O, ND) and a CPD intervention group (normoxia CPD, CPD). The CPD group received a dose of 800 mg/(kg·d) of CPD, while the ND group received an equal volume of ddH₂O for 15 consecutive days. One hour after the final administration, mice from each group were placed in wide-mouth bottles, and the survival time was observed and recorded. Fecal samples collected prior to the last administration were subjected to 16S rRNA gene amplicon sequencing and targeted metabolomics analysis. Correlation analysis between gut microbiota and metabolites was subsequently performed. Results CPD intervention significantly prolonged the survival time of mice under hypoxic conditions compared to the ND group. CPD altered the structural composition of the gut microbiota in mice. Linear discriminant analysis effect size (LEfSe) revealed significantly different bacterial taxa between the ND group and the CPD group, with higher relative abundance of Bacillota, Lactobacillus, and Alistipes in the CPD group. Microbial genera, including Paraprevotella and Lactobacillus, showed a positive correlation with survival time. Targeted metabolomics identified 9 upregulated and 31 downregulated metabolites in the CPD group. Notably, metabolites such as palmitoleic acid, glyoxylic acid, hendecanoic acid, l-aspartic acid, O-succinylhomoserine, and allantoic acid were significantly enriched and positively correlated with the survival time of mice after CPD intervention. Kyoto encyclopedia of genes and genomes (KEGG) enrichment analysis of differential metabolites showed the highest enrichment in the tryptophan metabolism and glycine, serine, and threonine metabolism pathways. Conclusion CPD intervention significantly prolonged the survival time of hypoxic mice. CPD intervention enriched beneficial microorganisms, including Lactobacillus, and elevated the levels of beneficial metabolites such as choline and allantoic acid. These findings suggest that modulating the “gut microbiota-metabolite” axis may be one mechanism through which CPD enhances host hypoxia tolerance, providing a theoretical basis and potential targets for developing microecological intervention strategies against hypoxia-related diseases.

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.

Probiotic additives for feed play a crucial role in maintaining the health and improving the production performance of livestock and poultry. However, the application of most probiotics is limited by their sensitivity to environmental stresses (e.g., acid, bile salt, and temperature) in the animal intestinal tract, and microencapsulation serves as a key approach to enhance their stability. Objective This study constructed a metal-polyphenol-prebiotic (Fe-TA-GN) composite coating system for the probiotic strain Enterococcus faecium PL84 isolated by us and verified its protective effect on PL84, aiming to provide technical support for the industrial application of the strain. Methods Coating parameters (Fe³⁺-TA molar ratio and GN concentration) were optimized. Scanning electron microscopy (SEM) and the CCK-8 assay were employed to evaluate the effects of coating materials on the viability of mouse intestinal epithelial cells (IEC-6). The protective effect of the coating system was assessed through in vitro tolerance tests under acidic, bile salt, thermal conditions, as well as in simulated gastrointestinal fluids. Results E. faecium PL84 exhibited the highest cell viability during the logarithmic growth phase, being suitable for microencapsulation. When the molar ratio of Fe³⁺ to TA was 1:3, the PL84-Fe-TA composite particles showed the smallest nanoscale particle size and formed a dense metal-polyphenol network. At a GN concentration of 0.4 mg/mL, the Fe-TA-GN coating layer achieved the highest zeta potential and optimal structural stability. SEM revealed a uniform and continuous surface coating layer of PL84-Fe-TA-GN. In vitro tolerance assays demonstrated that the survival rate of PL84-Fe-TA-GN was higher than that of uncoated PL84 under conditions of pH 3.0 and 0.6% bile salt (P<0.01). After treatment at 60 ℃, the survival rate of the coated strain increased by 16.29% compared with that of uncoated PL84. Additionally, the survival rates of PL84-Fe-TA-GN in simulated gastric fluid and simulated intestinal fluid improved by 20.8% and 13.53%, respectively. The coating materials (Fe-TA, GN, and Fe-TA-GN) had no significant effect on the viability of PL84 (P>0.05). Conclusion When the molar ratio of Fe³⁺ to TA is 1:3 and the GN concentration is 0.4 mg/mL, the metal-polyphenol-prebiotic composite coating system is stable and can significantly enhance the environmental tolerance of E. faecium PL84. Moreover, the coating materials possess good biocompatibility, laying a solid technical foundation for the industrial application of E. faecium PL84.

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 evaluate the in vitro antifungal activity of quercetin against Trichosporon asahii and investigate its molecular mechanism of inducing fungal apoptosis. Methods According to the CLSI M27-A3 protocol, the inhibitory effects of quercetin on planktonic cells and biofilm formation of nine T. asahii strains were determined. On this basis, changes in intracellular reactive oxygen species (ROS) level, mitochondrial membrane potential (MMP), and cysteinyl aspartate-specific proteinase 3 (Caspase-3) activity were measured following quercetin intervention. Subsequently, transcriptome sequencing was utilized to verify and analyze the differentially expressed genes. Results The minimum inhibitory concentrations (MICs) of quercetin against T. asahii ranged from 8 to 32 μg/mL, and quercetin effectively inhibited biofilm formation. Cellular experiments indicated that quercetin triggered apoptosis by inducing ROS accumulation, reducing MMP, and activating Caspase-3. Transcriptomic data further confirmed the aforementioned mechanisms at the gene expression level. Conclusion Quercetin exerts antifungal activity against T. asahii primarily by inducing oxidative stress-mediated apoptosis.

Objective Mixotrophy that combines phototrophic autotrophy and phagotrophic heterotrophy is widespread among unicellular eukaryotic microalgae and plays a key ecological role in energy flow within food webs and in elemental biogeochemical cycles. However, identifying and characterizing mixotrophic microalgae in natural waters remains technically challenging. Improving current approaches to accurately reveal the diversity of mixotrophic microalgae is an urgent task in this field. Methods Fluorescently labeled prey surrogates and feeding experiments were employed to trace phagotrophic microalgae within plankton communities. Target organisms were captured at the single-cell level through fluorescence-activated cell sorting (FACS), followed by multiple-displacement amplification (MDA) and 18S rRNA gene sequencing for taxonomic identification. On the basis of this FACS-MDA workflow, we established a methodological framework for studying the functional groups of microalgae. Results Applying this approach to multiple freshwater and seawater samples from China, we identified twenty phagotrophic microalgal species belonging to six classes and twelve genera, as well as heterotrophic consumers representing one class and three genera, demonstrating the robustness and broad applicability of this method. Conclusion This study applies the combined FACS-MDA technology to the identification of functional groups of microalgae in natural water bodies. The established technology has broad application prospects in microbial ecology. It enables deeper insights into the functional diversity and in situ feeding activities of environmental microalgae.

Objective To evaluate the effects of medium concentration, soil suspension dilution, and soil type on bacterial high-throughput cultivation outcomes, providing a reference for the exploration of bacterial resources in soda saline-alkali soils. Methods High-throughput cultivation and identification of bacteria from soda saline-alkali wildland and maize field soils were conducted. Three medium concentrations (1×TSB, 1/5×TSB, and 1/10×TSB), two soil suspension dilutions (optimal dilution and 2× optimal dilution), and two soil types (wildland and maize field) were set as experimental factors to analyze bacterial cultivation preferences under different treatments. Results The dominant bacterial phyla in both soils were Pseudomonadota, Actinomycetota, Acidobacteriota, Bacillota, and Chloroflexota. Among the top 10 dominant genera, only Bacillus and Rubrobacter were cultivable. A total of 2 256 positive cultures were obtained through high-throughput cultivation, with pure cultures accounting for 79.3%. A total of 153 amplicon sequence variants (ASVs) were identified, belonging to 52 genera of 4 phyla. On average, every 100 pure cultures yielded 6.8 ASVs or 2.3 genera. The 1/10×TSB medium resulted in the highest proportion of pure culture wells, while the 1×TSB medium showed the highest ASVs isolation efficiency. Cultivation with 2× optimal soil suspension dilution achieved higher pure culture ratios and isolation efficiency than the optimal dilution. The proportion of pure cultures, ASVs isolation efficiency, and genus isolation efficiency were all higher in wildland soil than in maize field soil, with more unique ASVs detected in wildland. The most frequently isolated genera via high-throughput cultivation were Pseudomonas, Hydrogenophaga, Bacillus, Paenibacillus, Acidovorax, and Arthrobacter, among which only Bacillus was a dominant genus in the soda saline-alkali soils. Conclusion High-throughput cultivation is an efficient method for obtaining numerous pure bacterial strains from soda saline-alkali soils within a short period. Natural wildland soil yielded more diverse cultivable bacteria than dryland soil. Moderately reducing medium concentration and soil suspension dilution improved pure culture isolation efficiency and diversity. However, most dominant soil taxa could not be cultivated via a single medium type, underscoring the need to diversify cultivation conditions to enhance the cultivability of dominant soil bacteria.

Objective To construct high-yield engineering strains of Halomonas campaniensis XH26 by introducing five recombinant plasmids (pHX01-pHX05), each carrying the P _tac promoter and combinations of the genes asd, lysC, ectA, ectB, and ectC. This metabolic engineering strategy was coupled with the response surface methodology (RSM) for optimization of the culture conditions, thereby enhancing ectoine accumulation. Methods The recombinant plasmids were conjugally transferred from Escherichia coli S17-1(λ-pir) into H. campaniensis XH26, with positive transconjugants selected via gentamicin (50 μg/mL). Recombinant strains were induced with 0.2 mmol/L IPTG, and ectoine accumulation was quantified by HPLC. Critical nutritional variables—NaCl, peptone, l-glutamate, and glucose—were optimized through one-factor-at-a-time experiments, Plackett-Burman design, and Box-Behnken design. Results Five recombinant strains (XH26/pHX01-XH26/pHX05) were successfully constructed. Culture in the MG medium revealed that strain XH26/pHX04 (overexpressing asd-lysC-ectA-ectB) achieved the highest ectoine titer of (1.32±0.04) g/L. Strains XH26/pHX05 and XH26/pHX03 achieved the ectoine titer of (1.19±0.07) g/L and (1.07±0.08) g/L, respectively, while XH26/pHX02 yielded a lower titer of (1.02±0.14) g/L. The medium composition optimized by RSM was composed of 116.08 g/L NaCl, 16.30 g/L peptone, 169.57 g/L l-glutamate, and 15.53 g/L glucose. Under these optimized conditions, the titer of ectoine produced by XH26/pHX04 increased to (1.81±0.02) g/L, representing a significant increase of 301.56% compared with that of the wild-type strain XH26. Conclusion This study demonstrates that using H. campaniensis as a chassis and overexpressing a key gene combination (asd, lysC, ectA, ectB) under a strong promoter, synergized with culture medium optimization via RSM, can significantly boost the ectoine yield of recombinant strains. The findings provide a robust technical framework for the subsequent industrial production of ectoine.