Abstract:Protein synthesis is the cornerstone for cells to exert their biological activities. The proper activities and homeostasis of cells is crucial for organisms. Cells have evolved various regulatory mechanisms to deal with internal/external stimuli and adapt to environmental changes. Of note, ribosome-associated quality control is essential for cell homeostasis and activities. In addition to the translation mechanism, the studies about ribosomes focus more on the topics that ribosomes can monitor the abnormal protein synthesis and diminish the effects of protein synthesis errors on cell homeostasis. These hot topics involve antiviral activities, neurodegenerative diseases, and cancers. This review focuses on the molecular mechanisms of ribosome-associated quality control, which deal with protein degradation and disorders, and gives new insights into relationships between ribosome-associated quality control and the related diseases.

Abstract:Staphylococcus aureus, a common major zoonotic opportunistic pathogen, spreads widely in the community and the livestock and poultry environment, seriously threatening veterinary public health. The frequent occurrence of drug-resistant strains of S.aureus has posed a challenge to the antimicrobial strategies based on antibiotics. In response to the Chinese policy of reducing and substituting antibiotics, a large number of Chinese herbal medicines as the alternatives of antibiotics have been put into clinical research. This paper summarized the S.aureus infection situation, current status of drug resistance, and epidemic characteristics of drug-resistant strains in livestock and poultry, summarized the prevention and control strategies. Furthermore, this paper analyzed the mechanisms and targets of Chinese herbal medicines in the prevention and control of this pathogen, providing research contents for clinical research on the antibacterial strategies of Chinese herbal medicines.

Abstract:Conventional cancer therapies such as radiotherapy, chemotherapy, and surgery have obvious limitations in treating solid tumors. Bacteria-mediated tumor therapy has been proven effective in clinical studies and considered to be a promising strategy for tumor treatment. Salmonella, as a facultative anaerobic bacterium, can preferentially colonize tumors and activate the anti-tumor immune response of the host by remodeling the tumor microenvironment. Genetically engineered strains of Salmonella have good tumor targeting ability and controllability and are highly adaptable to different therapeutic requirements, thus serving as ideal carriers for drug delivery. Nevertheless, using Salmonella for tumor therapy faces two challenges: potential pathogenicity in the host and improvement of tumor-targeting ability. To develop safe and effective Salmonella-mediated therapies, researchers need to have deep insights into the interaction between Salmonella and the host immune system. We summarized the anti-tumor mechanisms of Salmonella, novel bacteria-mediated tumor therapies, and challenges and prospects of Salmonella-mediated tumor therapy.

Abstract:Recombinase polymerase amplification (RPA) can efficiently amplify the target sequence at constant temperatures and is praised for high sensitivity, short time consumption, and low equipment dependence. Therefore, RPA is a widely used isothermal amplification technology in recent years. However, non-specific amplification of RPA has limited its further development. The clustered regularly interspaced short palindromic repeats/associated protein 12a (CRISPR/Cas12a) is capable of specifically cleaving the double strands and has similar optimal temperature to the PRA temperature. Therefore, researchers have combined RPA with CRISPR/Cas12a to improve the detection specificity. Moreover, this method demonstrates high sensitivity, a wide application range, and simple operation. Particularly, the RPA-CRISPR/ Cas12a-based approach shows a promising application prospect in the detection of pathogenic microorganisms. This paper introduces RPA-CRISPR/Cas12a regarding the principles, product detection strategies, and application, providing a reference for the further development and application of RPA-CRISPR/Cas12a in the detection of pathogenic microorganisms.

Abstract:Nicotine is the most abundant alkaloid in tobacco and causes serious harm to both the environment and the human body. Microorganisms using nicotine as the energy source for growth can serve environmental remediation. Pseudomonas strains grow quickly and are highly adaptable to the environment. Moreover, they can grow with nicotine as the only carbon and nitrogen source, with excellent degradation performance and research potential. To elucidate the characteristics and mechanism of nicotine degradation by Pseudomonas, this paper introduced the strains and characteristics of Pseudomonas that can degrade nicotine, detailed the intermediates and pathways of nicotine degradation by Pseudomonas, and summarized the related genes. Finally, this paper prospected the ways to improve the ability of Pseudomonas in degrading nicotine, aiming to provide guidance for future research and applications of microbial degradation of nicotine.

Abstract:Human brucellosis is a zoonotic disease caused by the facultative intracellular pathogen Brucella. Humans often become infected through direct contact with infected animals or by eating animal products. Because of the unspecific clinical manifestations, brucellosis is easy to become chronic without timely diagnosis and treatment, which seriously affects the quality of life of the patients. Therefore, understanding the pathogenic characteristics of the pathogen, the mechanism of host-pathogen interaction, and the current treatment status can provide a new direction for the clinical diagnosis and treatment of brucellosis. This paper describes the infection mechanism of Brucella, the innate and adaptive response mechanisms of the host (human), and the current clinical characteristics and treatment strategies of brucellosis, aiming to provide reference for the prevention and treatment of human brucellosis.

Abstract:[Background] Acetic acid-producing bacteria can utilize one-carbon gas to produce high-value chemicals such as acetic acid by fermentation, which is an important way for carbon resource recycling and green biomanufacturing. In addition, most of the acetic acid-producing bacteria can utilize organic matter for metabolism. However, the two ways have different energy metabolism patterns. [Objective] To investigate the acid production characteristics of a typical acetic acid-producing bacterium, Clostridium aceticum, under heterotrophic/autotrophic conditions, and to reveal the mechanism of different culture conditions in affecting the metabolic patterns of this bacterium. [Methods] The acid production and metabolism of C.aceticum cultured with glucose and syngas (H₂ and CO₂ mixed in the ratio of 6:4), respectively, were investigated. The distribution of the products and the specific titer of acetic acid production were analyzed on the basis of the results of the experiments. The key enzyme activity indexes were employed to reveal the mechanism of the differences in the heterotrophic and autotrophic metabolism patterns. [Results] C.aceticum performed stepwise degradation in the presence of 10 g/L glucose. It firstly decomposed glucose to produce lactic acid, and subsequently produced 6.3 g/L ethanol, 13.2 g/L butyric acid, and 5.6 g/L acetic acid. However, only 1.96 g/L acetic acid was detected in the case of syngas as the substrate. The results of product distribution showed that the specific titer of acetic acid production and acid/alcohol ratio under autotrophic conditions reached up to 28.6 g/g and 17.57, respectively, which were much higher than those (8.23 g/g and 1.53) under heterotrophic conditions. The NADH and ATP production as well as pyruvate dehydrogenase activity were higher under heterotrophic conditions, while the acetate kinase activity showed an opposite trend. This result revealed that most of acetyl coenzyme A as an intermediate of pyruvate metabolism, flowed to cell synthesis and end products (ethanol and butyric acid) under heterotrophic conditions, while it flowed to acetate production under autotrophic conditions. [Conclusion] C.aceticum could achieve higher acetic acid production under autotrophic conditions, which provides a reference value for the utilization of syngas by microorganisms and the regulation of metabolic pathway of acetic acid-producing bacteria.

Abstract:[Background] Photobacterium damselae subsp. damselae (PDD) can infect a variety of marine animals to cause diseases. Comparative genome analysis revealed that PDD strains harboring a large plasmid pPDD1608 exhibit strong pathogenicity. Furthermore, gene annotation analysis unveiled the presence of virulence genes and secretion system-related genes in this plasmid. However, there is a dearth of studies examining the functions of this plasmid. [Objective] In this study, a highly pathogenic PDD strain was selected and its plasmid-deleted mutant was generated through induced mutagenesis. The phenotypic characteristics and pathogenicity were compared between the wild type and the mutant to reveal the role of this plasmid in the phenotype and pathogenicity of PDD. [Methods] The plasmid-deleted strain ΔpPDD1608 was successfully constructed by the temperature variation-SDS method. Then, the phenotypes of PDD1608 and ΔpPDD1608 cultured on the TSB plates were observed by scanning electron microscopy. The physiological and biochemical characteristics were measured by the microbiochemical tests and compared between PDD1608 and ΔpPDD1608. The antibiotic susceptibility of PDD1608 and ΔpPDD1608 was determined by the K-B disk diffusion method. The phospholipase and hemolytic activities of the two strains were measured by the indicator media in vitro. The pathogenicity of PDD1608 and ΔpPDD1608 was assessed through the artificial challenge test by injection in Sebastes schlegeli. [Results] The large plasmid of PDD1608 was effectively eliminated by the temperature variation-SDS method, and a gene-specific PCR method was established based on the whole genome sequence of PDD1608. The experimental results revealed no discernible disparities in antibiotic susceptibility or physiological and biochemical characteristics between PDD1608 and ΔpPDD1608. However, ΔpPDD1608 showed reduced hemolytic and phospholipase activities. Furthermore, the artificial challenge test demonstrated that PDD1608 displayed higher pathogenicity towards S.schlegeli, resulting in the death of all the tested fish within 24 h, whereas ΔpPDD1608 exhibited lower pathogenicity towards S.schlegeli, causing a 24-h mortality rate of 56.6%. [Conclusion] The comparison on hemolytic and phospholipase activities and pathogenicity between ΔpPDD1608 and PDD1608 confirmed that the plasmid pPDD1608 played a crucial role in mediating the high pathogenicity of PDD1608.

Abstract:[Background] Bacillus velezensis can promote the growth of crops and inhibit the growth of a variety of pathogens. However, the knowledge is limited regarding the effects of B. velezensis on the rhizosphere microecology of crops. [Objective] The effects of B. velezensis HN-Q-8 on the microbial community structure and physical and chemical properties of potato rhizosphere soil were studied in order to provide a theoretical basis for the application of Bacillus in potato production. [Methods] Potato rhizosphere soil samples were inoculated with the fermentation liquid with bacterial cells (FLBS), fermentation liquid without bacterial cells (FL), and bacterial cell suspension (BCS) of strain HN-Q-8, respectively. The changes of microbial diversity and community structure of potato rhizosphere soil under different treatment conditions were determined by high-throughput sequencing. Furthermore, the changes in physicochemical properties of potato rhizosphere soil were determined. [Results] Strain HN-Q-8 had no significant effect on the microbial diversity but affected the microbial community structure in potato rhizosphere soil. Compared with the control, each treatment significantly increased the relative abundance of beneficial bacteria, such as Lysobacter, Bacillus, and Mortierella, in potato rhizosphere soil, which improved the microbial community structure of potato rhizosphere soil. In addition, strain HN-Q-8 enhanced the activities of urease, phosphatase, and catalase and significantly increased the content of available nitrogen, available phosphorus, and available potassium in potato rhizosphere soil. [Conclusion] B. velezensis HN-Q-8 regulates the microbial community structure and increases the relative abundance of beneficial microorganisms in potato rhizosphere soil. Furthermore, it enhances the enzyme activity and fertility of rhizosphere soil, shaping a favorable soil environment for potato growth and development of resistance against pathogen infection.

Abstract:[Background] Endophytes represent valuable resources for investigating microbial diversity and adaptive evolution, owing to their long-term co-evolutionary relationship with their hosts. Alhagi sparsifolia Shap. exhibits robust tolerance to stress conditions including drought and saline-alkali. However, the studies about the endophytes of A.sparsifolia remain limited. [Objective] To analyze the diversity of culturable endophytic bacteria and evaluate the stress tolerance and plant growth-promoting effects of the strains. [Methods] Endophytic bacteria were isolated from A.sparsifolia by the tissue homogenization and gradient dilution methods. The strains were identified by comparison of 16S rRNA gene sequences. Subsequently, we determined the stress tolerance, plant growth-promoting effects, and antimicrobial activities of the strains. [Results] A total of 14 culturable endophytic bacterial strains were isolated from A.sparsifolia, belonging to 9 genera (Bacillus, Brachybacterium, Microbacterium, Corynebacterium, Solibacillus, Glutamicibacter, Leucobacter, Brevibacterium, and Acinetobacter), among which Bacillus was dominant (33.33%). These strains exhibited varying levels of stress tolerance, plant growth-promoting effects, and antimicrobial activities. Notably, Bacillus subtilis L-1 demonstrated strong tolerance to 20% NaCl solution and 20% PEG6000 and inhibitory effects on four pathogenic bacterial species and three pathogenic fungal species. [Conclusion] Our study revealed the stress tolerance, plant growth-promoting effects, and antimicrobial activities of culturable endophytic bacterial resources in A.sparsifolia. The findings provide a scientific basis for further exploration and utilization of these valuable resources.

Abstract:[Background] As the intensification in aquaculture keeps increasing, nitrogen pollution and eutrophication in aquaculture water have become increasingly serious, which inhibit the activity of autotrophic ammonia-oxidizing bacteria. Therefore, it is of great significance to understand the distribution and functional characteristics of heterotrophic ammonia-oxidizing microorganisms such as Alcaligenes sp. in aquaculture systems to address nitrogen pollution. [Objective] We studied the distribution of Alcaligenes sp. in aquaculture areas, isolated the strain with efficient nitrogen conversion ability, and characterized the ammonia oxidation of the strain screened out, aiming to provide strain resources and theoretical guidance for the efficient nitrogen removal from aquaculture tail water. [Methods] The high-throughput sequencing of the 16S rRNA gene was employed to reveal the distribution of Alcaligenes sp. in the aquaculture areas in Guangdong. Alcaligenes sp. strain CHO6 with strong heterotrophic ammonia-oxidizing capacity was isolated from an aquaculture sediment sample by continuous enrichment culture. The single factor tests were carried out to explore the factors influencing the heterotrophic ammonia-oxidizing capacity of the strain CHO6. [Results] Alcaligenes sp. widely existed in the aquaculture areas in Guangdong. Alcaligenes sp. CHO6 isolated from an aquaculture sediment sample completely removed 10 mmol/L ammonia nitrogen within 48 h. The ammonia-oxidizing capacity of the strain CHO6 was influenced by a variety of factors including carbon source, temperature, pH, C/N ratio, dissolved oxygen, and initial ammonia nitrogen. [Conclusion] The prevalence of Alcaligenes sp. indicates strong adaptability to the aquaculture environment. Alcaligenes sp. CHO6 as a heterotrophic strain had strong ammonia-oxidizing capacity and resistance to high concentrations of ammonia nitrogen. This study provides strain resources for the removal of nitrogen in aquaculture environments, which is of great significance for the management of aquaculture tail water.

Abstract:[Background] Pentamycin is a polyene macrolide with significant inhibitory activities against Candida albicans, Trichomonas vaginalis, and several other vaginal pathogens and has been registered in Switzerland for treating vaginal candidiasis, trichomoniasis, and mixed infections. [Objective] To explore new biosynthetic gene clusters and production strains and improve the yield of pentamycin by genetic engineering, providing new ideas and a theoretical basis for further increasing the yield and promoting the industrial production and application of pentamycin. [Methods] BLASTp was used to search for the homologous proteins of known enzymes involved in the biosynthesis of pentamycin from bacterial genomes, and new gene clusters for synthesizing pentamycin and its analogues were identified. Then, multi-locus sequence analysis (MLSA) was performed to compare the genomic similarity among the strains possessing the biosynthetic gene clusters. The constitutive promoter kasOp* was used to overexpress the positive transcriptional regulatory gene ptnF for increasing pentamycin production. [Results] A total of 27 new strains containing the biosynthetic gene clusters of pentamycin/filipin were discovered from the bacterial genome. The results from medium screening, fermentation, and metabolite analysis indicated that Streptomyces misionensis can synthesize multiple compounds such as pentamycin and filipin III. The genetic engineering increased the yield of pentamycin by 4.34 folds (reaching 101.7 mg/L) compared with that of the original strain. The total production of pentamycin and filipin analogues by the engineered strain reached 146.1 mg/L, which was 3.28 times higher than that of the original strain. [Conclusion] This study systematically analyzed the biosynthetic gene clusters of pentamycin in bacterial genomes and the diversity of the strains carrying these gene clusters. The experimental data confirmed that S.misionensis can produce natural products including pentamycin and filipin. In addition, the production of pentamycin was improved by genetic engineering. This study provides new elements, pathways, strains, and theoretical foundations for constructing elite strains for the industrial production of pentamycin.

Abstract:[Background] How to effectively prevent and control plant diseases and achieve safe crop production without causing damage to the agro-environment is a key problem to be solved in the sustainable development of agriculture. [Objective] The strains and active metabolites of Streptomyces strains were screened out to provide the strain resources and approaches for the biocontrol of plant diseases, which is of great practical significance for the research on the potential application of strain S5-1 in the biocontrol of plant diseases. [Methods] The antimicrobial activity was examined by the plate confrontation method and the plate culture method with the fermentation broth of the strain. The strain was identified based on the morphological and physicochemical characteristics and 16S rRNA gene sequences. Furthermore, the single factor and orthogonal experiments were carried out to optimize the fermentation conditions, and the antimicrobial activity of the fermented crude extract was examined. [Results] The strain S5-1 had strong inhibitory activities on ten plant pathogens and was identified as Streptomyces toxytricini. The fermentations conditions were optimized as follows: TSB medium, initial pH 6.5, medium loading capacity of 100 mL/250 mL, inoculation amount of 10%, and fermentation at 28 ℃ for 7 d. The fermented crude extract inhibited the mycelial growth and spore germination of Phytophthora parasitica and Botrytis cinerea. Furthermore, it reduced the infection of the two pathogens in tobacco leaves and cherry tomatoes. The extract was stable at pH 6.0–7.0 and lost activity at the temperature above 37 ℃. In addition, the storage at ‒20 ℃ for 30 d and ultraviolet irradiation not exceeding 6 h caused slight changes in the extract activity. [Conclusion] The development and utilization of strain S5-1 contribute to the green prevention and control of plant diseases and provide excellent strain resources of actinomycetes for biocontrol. This study provide an experimental basis for the research and application of new antimicrobial agents.

Abstract:[Background] Colletotrichum graminicola, one of the main pathogens infecting maize, causes serious economic losses and directly threatens the global food security every year. Velvet proteins are fungal unique proteins with conserved functions, playing a role in regulating the growth and development of fungi. [Objective] To study the biological functions of a Velvet protein CgrVosA identified from C.graminicola. [Methods] The CgrvosA-knockout mutant and complementary strain were constructed by homologous recombination. The vegetative growth, stress response, conidial yield, germination, and pathogenicity of the strains were determined and compared to reveal the functions of CgrVosA. [Results] CgrvosA encoded a 425-residue protein with a typical Velvet protein domain. Compared with the wild type, ΔCgrvosA exhibited slow growth, high sensitivity to NaCl and H₂O₂, enlarged oval conidia, and reduced yield and germination rate of oval conidia. [Conclusion] The Velvet protein CgrVosA is involved in the regulation of vegetative growth, response to hyperosmotic and oxidative stress, and oval conidial production and germination of C.graminicola.

Abstract:[Background] Moesziomyces antarcticus has been extensively studied for its ability to produce excellent biosurfactants and lipases, while little is known about its expression elements for genetic manipulation. [Objective] To measure and compare the promoter activities of all the seven genes of the heat shock protein 70 superfamily in M.antarcticus, and screen out the strong promoters that can be used for regulating gene expression in M.antarcticus. [Methods] The gene information on seven members of the heat shock protein 70 family was obtained from the genome database of M.antarcticus JCM10317. Bioinformatics tools were used for evolutionary analysis of the heat shock protein 70 family, and key cis-acting elements in the heat shock protein 70 promoter (P_Hsp70) sequences were predicted. The recombinant expression vectors were constructed by fusing the promoters of the seven heat shock protein 70 genes with the gene encoding the enhanced green fluorescent protein. The positive transformants were measured for the fluorescence intensity and observed under a fluorescence microscope, on the basis of which the promoter activities of different members of the heat shock protein 70 family were compared. [Results] The seven heat shock protein 70 family members belonged to different subfamilies, and their promoters had different categories and number of cis-acting elements. Moreover, the average fluorescence intensities of transformants with P_Hsp701, P_Hsp702, P_Hsp703, P_Hsp704, P_Hsp705, P_Hsp706, and P_Hsp707 recombinant plasmids respectively were 12.8, 1.6, 2.9, 5.8, 4.6, 5.0, and 1.5 times of that in the control. The results are consistent with the fluorescence observation results under the microscope. [Conclusion] The results of bioinformatics analysis and enhanced green fluorescent protein expression revealed significant differences in the promoter activity among the heat shock protein 70 family members in M.antarcticus. P_Hsp701 showed the highest expression activity, serving as a strong promoter in M.antarcticus. P_Hsp704, P_Hsp705, and P_Hsp706 had lower activities than P_Hsp701 but could be used as alternative promoters for further research.

Abstract:[Background] Crops in high-altitude areas in southwestern China often suffer from low temperature stress. Tobacco as a thermophilic plant is particularly sensitive to low temperatures. [Objective] To isolate and screen out the strains capable of promoting tobacco seedling growth at low temperatures and study the effects of the strains on the growth and root exudates of tobacco seedlings. [Methods] We used the low temperature (10 ℃) culture method to isolate the psychrotolerant bacteria from the rhizosphere soil of tobacco seedlings planted in high-altitude areas and then employed the plate culture method to screen out the tobacco growth-promoting bacteria from the isolated bacteria. The plant growth-promoting strains were identified by 16S rRNA gene sequencing, and the growth-promoting characteristics were analyzed. Non-targeted metabolomics based on LC-MS/MS was employed to investigate the effects of the strains on the root exudates of tobacco seedlings. [Results] A psychrotolerant Bacillus sp. strain, named JZC10, was isolated. It significantly promoted tobacco seedling growth at 15 ℃ but had no significant growth-promoting effect at 25 ℃. JZC10 produced proteases and indoleacetic acid (IAA) and did not solubilize phosphorus or secrete siderophores or lipases. The strain showed stronger colonizing capacity at 15 ℃ than at 25 ℃, while it did not spread along the roots at neither temperature. At 15 ℃, the inoculation of JZC10 affected the composition of root exudates of tobacco seedlings. The content of 19 root exudates were significantly different between the JZC10 group and the control. One of the upregulated root exudates, lumichrome (5 nmol/L), significantly promoted the development of leaves and lateral roots of tobacco seedlings. [Conclusion] A psychrotolerant Bacillus strain that could promote tobacco seedling growth and root development at 15 ℃ was isolated in this study. Inoculating this strain changed the root exudate composition of tobacco seedlings. The results of this study provide a theoretical basis for screening plant growth-promoting bacteria and studying their interactions with plants.

Abstract:[Background] Heterotrophic ammonia-oxidizing bacteria (HAOB) promote the regeneration of Italian ryegrass by increasing the content of leaf cytokinins, although the specific mechanism remains to be elucidated. [Objective] To elucidate how HAOB promote the regeneration of Italian ryegrass by regulating the synthesis of cytokinins via a dual-action mechanism (in the rhizosphere soil and outside the rhizosphere environment, respectively. [Methods] A strain (S2_8_1) of HAOB and potted Italian ryegrass were used in this study. The treatments were designed as follows: Regular watering (TA); Addition of blank medium for enrichment culture (TB); Addition of 3,4-dimethylpyrazole phosphate (DMPP) (TN1); Addition of DMPP and 20 mmol/L NO₃⁻-N (TN2); Addition of DMPP and 30 mmol/L NO₃⁻-N (TN3); Addition of DMPP and 40 mmol/L NO₃⁻-N (TN4); Inoculation with the strain S2_8_1 (TI). [Results] NO₃⁻-N of 30–40 mmol/L was most effective in increasing the content of leaf cytokinins and enhancing the transfer of these hormones from the roots to the leaves, thereby promoting leaf regeneration. When the nitration-induced increase in cytokinins in the rhizosphere soil reached the maximum, inoculation with strain S2_8_1 further boosted the synthesis of these hormones in the roots, leading to an increased concentration of cytokinins in the leaves. The HAOB strain S2_8_1 significantly elevated the level of cytokinins in leaves via a dual-action mechanism, thereby promoting the regeneration of Italian ryegrass. [Conclusion] This study unveils the dual-action mechanism by which HAOB strains in the rhizosphere enhance plant growth, offering a direct application value for improving forage yields and sustainable agriculture development.

Abstract:[Background] Tobacco bacterial wilt is a soil-borne disease caused by Ralstonia solanacearum, causing huge losses to tobacco production. [Objective] To investigate the inhibitory effect and control efficiency of two biocontrol strains of Beauveria bassiana HZXJ and Cordyceps javanica Bd01 on R.solanacearum. [Methods] The inhibitory effects of the two biocontrol strains and their fermentation broths on the growth of R.solanacearum were measured by plate confrontation test, agar diffusion method, and growth curve method. The effect of strain HZXJ and Bd01 1×10⁸ conidia/mL suspension on tobacco bacterial wilt was determined by indoor pot experiment and field experiment. [Results] The plate confrontation test showed that the inhibition rates of strain HZXJ and strain Bd01 on the fermentation broth at 10 days were 51.28% and 47.59%, respectively. The inhibition rates of the pasteurized fermentation broths of B.bassiana HZXJ and C.javanica Bd01 on R.solanacearum cultured at 10 days were 62.54% and 35.49%, respectively. The growth curves drawn by biocontrol bacteria and R.solanacearum OD₆₀₀ also showed that the aseptic fermentation supernatants of HZXJ and Bd01 both had inhibitory effects on R. solanacearum. The results the control efficiency of strain HZXJ and strain Bd01 on tobacco bacterial wilt after treatment with 1×10⁸ conidia/mL suspension for 15 days was 43.33% and 19.99%, respectively, and the control efficiency of 52% copper chloride sulfate soluble powder 800 times solution was 39.99%. The control effect of HZXJ and Bd01 on tobacco bacterial wilt at 30 days was 43.75% and 21.87% respectively, and the control effect of 52% copper chloride sulfate soluble powder 800 times solution was 46.90%. The results of field test showed that the control efficiency of strain HZXJ and Bd01 1×10⁸ conidia/mL suspension for 15 days was 80.00% and 35.00%, respectively, and the control efficiency of 52% copper chloride sulfate soluble powder 800 times solution was 40.00%. The control effect of strain HZXJ and Bd01 on tobacco bacterial wilt at 30 days was 52.63% and 63.16%, respectively, and the control effect of 52% copper chloride sulfate soluble powder 800 times solution was 53.98%. [Conclusion] B.bassiana HZXJ and C.javanica Bd01 showed significant inhibitory effects on R.solanacearum. Both strains had the potential of serving the biocontrol of tobacco bacterial wilt.

Abstract:[Background] There are numerous microorganisms in the rhizosphere soil, many of which can antagonize pathogens and thus protect plants from diseases. [Objective] To isolate and identify a biocontrol bacterial strain capable of promoting plant growth and explore the antifungal mechanism from genomics and bioinformatics. [Methods] The dilution-plate coating method was employed to isolate a bacterial strain with antagonistic effects against Colletotrichum orbiculare. The strain was identified based on 16S rRNA gene sequencing and genome sequence alignment. The effects of the strain suspension (5×10⁸ CFU/mL) on tomato seeds and seedlings were determined. The antifungal spectrum of the strain was determined by the plate confrontation test. Furthermore, the third-generation (PacBio, Nanopore) sequencing was combined with the second-generation sequencing to analyze the genomic information and gene function annotation of the strain. [Results] A strain of biocontrol bacteria isolated from the rhizosphere soil of Schefflera heptaphylla was named YN-2A. Based on the alignment results of 16S rRNA gene and nucleotide sequence database (NT), the strain YN-2A was identified as Bacillus velezensis. Strain YN-2A was an aerobic Gram-positive bacterial strain with the ability to produce biofilms. Strain YN-2A suspension (5×10⁸ CFU/mL) promoted tomato seed germination and seedling growth and increased the leaf chlorophyll fluorescence parameters. Specifically, the suspension increased the germination rate at the time points of 72, 96 and 120 h by 1.45 times, 1.42 times and 1.09 times, respectively, and it increased the plant height of tomato seedlings by 5.90 cm. Among the fluorescence parameters, photochemical quenching (qP) exhibited the most significant increase of 1.27 times in the strain treatment group compared with the control group. The results of the plate confrontation test showed that strain YN-2A can antagonize a variety of fungal pathogens. The results of whole genome sequencing showed that the whole genome length of strain YN-2A was 4 046 066 bp, with the G+C content of 46% and 4 090 coding genes. The whole genome sequencing data was submitted to NCBI to obtain the GenBank accession number CP139086. Most genes were annotated to the amino acid and carbohydrate metabolic pathways. Particularly, 905 genes were annotated to reduced virulence in the pathogen host interactions (PHI) database, and 69 genes were annotated to the loss of pathogenicity database. It is thus hypothesized that the antifungal effect of YN-2A is attributed to the disease-resistant and virulence-reducing genes. [Conclusion] A biocontrol strain B.velezensis YN-2A was isolated and identified. The strain promoted tomato seed germination and seedling growth and demonstrated a broad antifungal spectrum. The whole genome information of strain YN-2A was obtained by sequencing, which provided reference information for deciphering the antifungal mechanism of strain YN-2A.

Abstract:[Background] Fertilization is a common agronomic measure, while there are few studies about the effects of fertilization on the bacterial diversity and functions of potato roots. [Objective] To study the effects of non-fertilization and conventional fertilization on the community composition, structure, and function of bacteria in potato root samples collected from Zhangjiakou. [Methods] The 16S rRNA gene sequencing was employed to analyze the root bacteria of potato under fertilization and non-fertilization treatments. [Results] Fertilization affected the bacterial abundance of potato plants at the seedling stage and tuber formation stage. At the seedling stage, fertilization significantly reduced the abundance of Planctomycota, Cyanobacteria, and Firmicutes in the rhizosphere and significantly increased the abundance of Bdellovibrionota, Armatimonadota, Bacteroidota, and Patescibacteria on the root surface and the abundance of Chloroflexi and Planctomycetota in the roots, compared with non-fertilization treatment. At the tuber formation stage, fertilization significantly increased the abundance of Acidobacteriota, Firmicutes, Actinobacteriota, Bacteroidota, and Patescibacteria in the rhizosphere, Armatimonadota, Bacteroidota, and Bdellovibrionota on the root surface, and Armatimonadota and Hydrogenedentes in the roots. Meanwhile, it significantly decreased the abundance of Cyanobacteria in the rhizosphere, Gemmatimonadota, Cyanobacteria, and Myxococcota on the root surface, and Nitrospirota and RCP2-54 in the roots. In addition, fertilization affected the gene abundance of potato plants at the seedling stage and tuber formation stage. At the seedling stage, compared with non-fertilization treatment, fertilization significantly increased the abundance of genes involved in the biosynthesis of other secondary metabolites, glycan biosynthesis and metabolism, and replication and repair on the root surface and those for replication and repair in the roots. At the tuber formation stage, fertilization significantly increased the abundance of the genes associated with the metabolism of terpenoids and polyketides and lipid metabolism in the rhizosphere, replication and repair in the roots, and the metabolism of terpenoids and polyketides and lipid metabolism on the root surface. Meanwhile, it significantly reduced the abundance of genes involved in the energy metabolism, metabolism of cofactors and vitamins, metabolism of other amino acids in the rhizosphere and those participating in transcription, folding, sorting, degradation, and energy metabolism on the root surface. [Conclusion] Conventional fertilization could improve the root microbial structure and gene function abundance. That is, compared with the control group, fertilization increases the bacterial richness and diversity and the abundance of genes associated with the metabolism and energy conversion and utilization, thereby improving the potato yield.

Abstract:[Background] Colletotrichum graminicola (Ces.) G.W. Wilson is a major pathogenic fungus infecting plants, causing maize anthracnose. The APSES (Asm-1, Phd1, Sok2, Efg1 and StuA) transcription factor StuA, a fungal specific helix-loop-helix transcription factor, regulates the vegetative growth, conidial yield, germination, and pathogenicity of fungi. [Objective] To reveal the role of an APSES transcription factor CgrStuA identified in C.graminicola in the growth and development of C.graminicola. [Methods] The CgrstuA-deleted mutants were constructed by homologous recombination, and the obtained mutants were characterized in terms of the vegetative growth, conidial yield, germination, and pathogenicity. [Results] CgrstuA encoded a protein composed of 707 residues and containing a KilA-N domain. The CgrstuA-deleted mutants exhibited slow vegetative growth, decreased yields of two types of conidia, an extremely low germination rate of oval conidia, and significantly increased hyphopodia. However, there was no significant difference in the pathogenicity between the wild type and the mutants. [Conclusion] CgrStuA play important roles in the regulation of vegetative growth, conidial yield, germination, and hyphopodium formation of C.graminicola, while it did not affect the pathogenicity.

Abstract:[Background] The Brevibacterium antarcticum strain WN5, isolated from tobacco- cultivating soil in Guizhou, possesses ammonia transformation capabilities. However, there are few reports on its efficacy in promoting tobacco growth. [Objective] To investigate the growth-promoting effect of WN5 metabolites on tobacco seedlings. [Methods] We applied the WN5 culture to the tobacco seedlings with two leaves and then determined the agronomic traits, morphological traits and vigor of roots, nitrogen nutrients, and stress resistance of the tobacco seedlings. [Results] Four plant hormones and hormone precursors: 1-aminocyclopropane-1-carboxylic acid, trans zeatin, indole-3-acetic acid, and salicylic acid, were detected in the strain culture. The strain culture was then applied to the matrix of tobacco seedlings, which promoted the shoot growth, increasing the plant height, stem circumference, and fresh weight of the seedlings by 42.74%, 12.91%, and 5.00%, respectively. However, the maximum leaf area and leaf number increased without statistically significance, The chlorophyll content in tobacco seedlings increased by 32.86% compared to the control. Additionally, the strain culture enhanced root growth and vigor, increasing the total area, surface area, average diameter, average length, volume, and vigor of roots by 35.42%, 20.54%, 18.22%, 24.07%, 36.56%, and 29.01%, respectively. The growth-promoting effect of the strain culture was more significant on the roots than on the shoots, as the root-to-shoot ratio was increased by 41.67%. Moreover, the treatment with WN5 enhanced the nutrient absorption and stress resistance of tobacco seedlings. Specifically, the treatment increased the levels of nitrate reductase, catalase, superoxide dismutase, and soluble protein by 32.97%, 36.31%, 16.23%, and 18.50%, respectively. [Conclusion] B.antarcticum WN5 can promote the shoot and root growth, improve the root vigor, enhance the nitrogen absorption, and reduce the oxidative stress of tobacco seedlings by producing plant hormones. This finding suggests that WN5 has great potential for widespread application.

Abstract:[Background] It is of great significance to evaluate the fermentation characteristics and safety of Lactobacillus brevis D17, a high gamma-aminobutyric acid (GABA)-producing bacterium from fermented grains for the production of Chinese liquor. [Objective] To investigate the fermentation characteristics and evaluate the safety of L.brevis D17, laying a foundation for further assessment of probiotic properties. [Methods] The MRS medium and hydrolyzed oat milk were used to study the fermentation characteristics of strain D17. The whole genome sequence of D17 was compared with the virulence factor database (VFDB) and the comprehensive antibiotic resistance database (CARD) to assess the presence of virulence genes and antibiotic resistance genes. The antibiotic susceptibility profile of L.brevis D17 was established by antibiotic susceptibility tests. The toxicological test in mice was carried out to assess the in vivo safety of L.brevis D17. [Results] L.brevis D17 showcased good fermentation performance on oat milk, with the lactate production reaching 82.5% of that in the MRS medium. Moreover, it produced 480 mg/L GABA from the glutamate in oat milk. Additionally, L.brevis D17 improved the flavor profile of oat milk. L.brevis D17 did not carry virulence genes or antibiotic resistance genes. Despite inherent antibiotic resistance, D17 strain had no risk of resistance transfer. L.brevis D17 colonized the mouse intestine without causing any disease or death. Moreover, it exerted positive effects on serum indicators and gut microbiota in mice. [Conclusion] L.brevis D17 showcased strong potential for fermentation of plant-derived substrates and was confirmed to be safe, demonstrating significant probiotic properties.

Abstract:[Background] Yersinia enterocolitica is a zoonotic bacterium with wide distribution in China and can cause a variety of intestinal symptoms and systemic diseases, posing a threat to human and animal health. [Objective] To understand the drug resistance phenotypes and genes, virulence genes, and multilocus sequence typing of Y.enterocolitica isolated from different raw meat sources in Liaoning Province. [Methods] A total of 26 strains of Y.enterocolitica were isolated from raw chicken, raw pork, and raw beef samples in Liaoning Province from 2018 to 2021. The drug sensitivity test and serotyping were carried out, and whole genome sequencing was conducted to reveal the genetic information of the strains. [Results] The 26 strains of Y.enterocolitica had the highest resistance to cefazolin. All the 26 strains were intermediately resistant to polymyxin E and sensitive to chloramphenicol and gentamicin. The virulence genes of the 26 strains were mainly ail⁻virF⁻yadA⁻ystA⁻ystB⁺, and there was a strain with the genotype of ail⁺virF⁻yadA⁻ystA⁺ystB⁻, which was from a pork sample. Most of the strains presented the serotype of O:5, accounting for 84.6%, and the strains with the serotype of O:8 and unserotyped accounted for 7.7%. All the 26 strains carried the same resistance genes of CRP, rosA, and rosB. Fourteen strains carried vat(F) and blaA, and 2 strains carried qnrD. A total of 21 sequence types (ST) were obtained by multilocus sequence typing, of which ST150, ST726, ST848, ST143, and ST820 each had two strains, and the remaining STs each had one strain. The STs of the same cluster had the same virulence genes and drug resistance genes. [Conclusion] The STs of Y.enterocolitica from different raw meat samples in Liaoning Province present scattered distribution and carry multiple drug resistance genes, posing a risk of drug resistance.

Abstract:[Background] As a common opportunistic pathogen, Enterococcus faecalis can cause severe infections and pose a serious threat to public safety as a carrier of high resistance/virulence factors. [Objective] To master the sequence type (ST) distribution, antibiotic resistance, and virulence of sheep-derived E.faecalis strains in Anhui Province, analyze the correlations of antibiotic resistance and virulence genes with ST, and evaluate the potential threat to the public health. [Methods] E.faecalis was isolated from 64 sheep anal swab samples collected from Anhui during 2021−2023. Multilocus sequence typing (MLST) was employed to classify and identify the isolates. Kirby Bauer (K-B) test was carried out to determine the sensitivity of E.faecalis isolates to 16 antibiotics. Furthermore, the distribution of 9 resistance genes and 14 virulence genes was tested. [Results] A total of 61 strains of E.faecalis were isolated and identified as 24 STs, of which 9 were newly discovered STs. Most of the strains showed resistance to netilmicin (72.13%), amikacin (75.41%), tetracycline (55.74%), and erythromycin (63.93%). A few strains were resistant to vancomycin (9.84%) and linezolid (11.48%). with And the multi-drug resistance (MDR) rate was 57.38%. The carrier rates of resistance genes ermB (77.05%), aac(6')/aph(2') (52.46%), and tem (55.74%) were high, while those of vanC and mefA were low at 8.20% and 1.64%, respectively. The carrying rates of virulence genes cpd (100%), efaA (98.36%), ebp (95.08%), and fsr (81.97%) were high, while it was the lowest for hyl (11.48%). The distribution of some resistance and virulence genes showed differences among different STs (P<0.05). [Conclusion] Enterococcus faecalis from sheep feces carry high resistance and high virulence genes, and the distribution of resistance and virulence is highly correlated with STs.

Abstract:[Background] Streptomyces anulatus 89-2-2, a moderately halophilic actinomycete strain, can produce secondary metabolites capable of inhibiting the tyrosinase activity in mushrooms and the melanin synthesis and tyrosinase activity in mouse melanoma B16 cells. Few studies report the genome sequence of S.anulatus 89-2-2, which limits the studies on the biosynthesis and regulation of tyrosinase inhibitors, melanin synthesis inhibitors and other secondary metabolites in the strain. [Objective] This study sequenced the genome of S.anulatus 89-2-2 and mined the genetic resources of secondary metabolites, aiming to lay a foundation for deciphering the mechanisms of tyrosinase inhibitor production and biosynthesis regulation in this strain. [Methods] Nanopore sequencing platform was used to uncover the genome sequence of S. anulatus 89-2-2. Bioinformatics tools were used for sequence assembly, gene prediction, functional annotation, phylogenetic analysis, synteny analysis, and prediction of the gene clusters for biosynthesis of secondary metabolites. The genomes of different Streptomyces spp. were compared, and the secondary metabolic potential and the evolutionary relationship of different Streptomyces spp. were studied. [Results] The genome of strain 89-2-2 was a single linear chromosome with a length of 8 117 999 bp and the G+C content of 71.52%. The sequence has been submitted to the GenBank of the NCBI, with the accession number CP137002. The genome of the strain contained 7 088 coding sequences. The annotation against COG, GO, KEGG, and NR predicted 5 300, 4 176, 2 513, and 7 013 genes, respectively. The antiSMASH predicted 34 gene clusters for the biosynthesis of secondary metabolites in the genome of 89-2-2, and these gene clusters were involved in the biosynthesis of a variety of natural products, such as terpenoids, non-ribosomal peptides, polyketides, and ribosomally synthesized and post-translationally modified peptides. Eleven clusters showed low similarities to the gene clusters for the biosynthesis of known compounds, which suggested that strain 89-2-2 had the potential to produce a variety of novel secondary metabolites. The genome of S.anulatus 89-2-2 presented high synteny with that of Streptomyces sp. AM2-1-1, which indicated their high homology. It showed large inversions compared with that of S.microflavus DSM40593, which suggested their low homology. Strain 89-2-2 had 51 specific gene families and 1 235 specific genes. The results indicated that Streptomyces spp. had strong ability to adapt to diverse environments, and partial fluctuations in the genomes occurred between species during the long-term adaptation. Further studies remain to be carried out to reveal the relationship between specific genes and adaptability of strain 89-2-2 to different environments. [Conclusion] This study analyzed the whole-genome sequence of S.anulatus 89-2-2, laying a foundation for further studies about the strain in terms of the association between the tyrosinase-inhibiting activity and the genome, the structures of gene clusters for the biosynthesis of secondary metabolites, and the secondary metabolic potential of Streptomyces spp. This study provides basic data for the discovery of novel compounds and new drugs.

Abstract:[Background] Akanthomyces lecanii is an important entomogenous fungus for biocontrol, and its mitochondrial genome has not been reported. [Objective] The mitochondrial genome of A. lecanii was systematically studied, which would help clarify the taxonomic status of this fungus and provide a molecular basis for the phylogenetic research and the resource protection and utilization of Cordycipitaceae. [Methods] The full-length mitochondrial genome of A.lecanii RCEF6920 was sequenced, and bioinformatics tools were used to probe into the composition, sequence repeats, phylogenetic relationship, and collinearity. Furthermore, the mitochondrial genome was compared between A.lecanii and other species of Cordycipitaceae. [Results] The mitochondrial genome of A.lecanii RCEF6920 was a circular DNA molecule of 24 577 bp and contained 43 genes, including 15 protein-coding genes, 2 rRNA genes, and 26 tRNA genes. Only one intron was detected in the mitochondrial genome, and it interrupted rnl and contained orf447 encoding ribosomal protein S3. The nucleotide composition of the mitochondrial genome was A+T biased (72.89%). The 26 tRNA genes could transfer all the 20 amino acids and presented a typical cloverleaf structure. AGA was the most frequently used codon of the protein-coding genes. Arginine and leucine were identified as the most common amino acids, while methionine and tryptophan were the least common amino acids in the protein-coding genes. The phylogenetic analysis based on the protein-coding genes revealed that Akanthomyces and Samsoniella were sister genera. Compared with other species of Cordycipitaceae, A.lecanii had a small mitochondrial genome. Except several species, other species of Cordycipitaceae had good mitochondrial genome collinearity. [Conclusion] We obtained the full-length mitochondrial genome sequence and related information of A.lecanii. The phylogenetic analysis supports the independent taxonomic status of A. lecanii, and the mitochondrial genomes of fungi belonging to Cordycipitaceae are conserved.

Abstract:[Background] The abuse of antibiotics leads to increasingly severe drug resistance, which poses a growing threat to animal health and the environment. In response to this global public health threat, many countries have banned the use of antibiotics as food and feed additives. In this context, probiotics emerge as alternatives. [Objective] To comprehensively characterize bacteriocin-producing Enterococcus faecium F11.1G. [Methods] Tests were carried out to examine the thermal stability, acid tolerance, bile salt tolerance, antibiotic sensitivity, surface hydrophobicity, self-aggregation, and protease resistance of this strain. [Results] The fermentation supernatant of E.faecium F11.1G had strong thermal stability and maintained antibacterial activity at 110 ℃, with an optimal growth temperature of 37 ℃. The strain grew well and had the highest survival rate at pH 5.9. As the pH decreased, the growth was inhibited. However, at pH 1.8, the strain can still grow with a survival rate of 9%. The strain showed the survival rates of 140.0%, 126.6%, and 96.6% at the bile salt concentrations of 0.10%, 0.15%, and 0.20%, respectively. The strain was tolerant to 16 antibiotics: cefepime, amikacin, gentamicin, norfloxacin, erythromycin, roxithromycin, polymyxin B, lincomycin, streptomycin, neomycin, metronidazole, clindamycin, nystatin, trimethoprim, azithromycin, and kanamycin. It was moderately sensitive to ceftazidime, levofloxacin, and ciprofloxacin and highly sensitive to penicillin, cefazolin, cefuroxime, chloramphenicol, tetracycline, doxycycline, teicoplanin, clarithromycin and nitrofurantoin. The hydrophobicity of E.faecalis F11.1G to hexane, xylene, ethyl acetate, and dichloromethane was 84.29%, 25.75%, 31.26%, and 15.08%, respectively. The self-aggregation rates of E.faecalis F11.1G at the time points of 3, 6, 9, 12, 15, and 18 h were 51%, 54%, 76%, 80%, 83%, and 84%, respectively. In the presence of protease K, the inhibitory effects of the strain against Escherichia coli and Salmonella were significantly weakened, with the inhibition zone diameters of 13.05 mm and 11.28 mm and the inhibition rate decreases of 52% and 58%, respectively. In the presence of trypsin and pepsin, no inhibition zone was formed, which indicated that the strain almost completely lost the antibacterial effects. [Conclusion] E.faecium F11.1G demonstrates strong tolerance to high temperatures, acids, and bile salt, resistance to 16 antibiotics, high hydrophobicity to hexane, and high self-aggregation. However, it loses its antibacterial activity after treatment with trypsin and pepsin, which indicates that the antibacterial substance is a protein or peptide. The results provide basic data for the safe application of probiotics.

Abstract:[Background] Rimocidin is a tetraene macrolide with broad-spectrum antifungal activities, there being developed into a potential agricultural antibiotic. Until now, several Streptomyces producers (such as S.rimosus M527) have been discovered, while the low yield of rimocidin limits the application of these strains in the development of the agricultural antibiotic. [Objective] To systematically reveal the distribution and characteristic of the biosynthetic gene clusters (BGCs) for rimocidin (rim BGCs) in Streptomyces genomes, identify a new strain which can produce rimocidin, and improve its yield via genetic engineering and other strategies. [Methods] We employed BLASTp to search for the homologous proteins of the known synthetases of rimocidin in Streptomyces genomes to discover the strains with predicted rim BGCs and capable of producing rimocidin. The multi-locus sequence analysis (MLSA) was conducted to analyze the phylogenetic relationships among these strains and publicly available Streptomyces strains. A new rimocidin-producing strain was identified based on the metabolite analysis and the titer of rimocidin was increased by medium optimization and RimoR2 overexpression. [Results] A total of 36 Streptomyces strains carrying rim BGCs were predicted, in which S.albofaciens JCM 4342 was capable of producing rimocidin and its congener CE-108. The medium optimization led to a rimocidin titer of 172.5 mg/L. Moreover, the RimoR2 overexpression increased the rimocidin titer by 2.3 folds, which reached 397.1 mg/L. [Conclusion] This study systematically revealed the distribution of rim BGCs in Streptomyces. We confirmed that S.albofaciens JCM 4342 can produce rimocidin and its congener CE-108. Moreover, the titer of rimocidin was improved by genetic engineering. Our findings provide new catalytic elements, strains, and a theoretical basis for the design and construction of strains with high yields of rimocidin and the application and development of rimocidin.

Abstract:[Background] The microbial treatment of paper wastewater or the polluted environment is of great practical value. However, studies remain to be carried out regarding the physiological adaption mechanisms of microorganisms to the high temperatures of paper wastewater. [Objective] To investigate the physiological response of Serratia sp. AXJ-M, a thermophilic lignin-degrading bacterial strain preserved in our laboratory, to high temperature stress. [Methods] The bacterial cells were cultured at different temperatures, and the bacterial cell morphology, cell membrane fluidity and permeability, trehalose content, and activities of antioxidant enzymes were observed and measured. Illumina HiSeq 2000 was used for the de novo sequencing of AXJ-M, and then GO, COG, KEGG, and NR were employed to annotate the functional genes in the genome. [Results] Strain AXJ-M can survive in a thermal stress environment of 50 to 80 ℃, of which 50 ℃ is the optimal growth temperature. However, its growth was significantly hindered when the temperature was rises above 70 ℃. Strain AXJ-M exhibited marked morphological differences and augmented surface roughness at high temperature stress. Strain AXJ-M cultured at 80 ℃ experienced a decline in cell membrane fluidity, an upsurge in membrane permeability, and a rise in trehalose content. Moreover, the activities of catalase (CAT), superoxide dismutase (SOD), and peroxidase (POD) associated with oxidative stress increased by 50%–80%, 30%–47%, and 24%–31%, respectively, as the culture time was extended. By genomic analysis, strain AXJ-M contained the genes associated with the response to high temperature stress. [Conclusion] The culture at high temperatures can induce strain AXJ-M to develop heat tolerance and modify the cell morphology. Additionally, the increased trehalose content and antioxidant enzyme activities can protect the cells from external stress conditions. Moreover, a comprehensive analysis of the strain genome provides a basis for exploring the molecular response mechanism of thermophilic bacteria in high-temperature environments.

Abstract:[Background] The isolation and identification of Cronobacter forms the theoretical basis for precise treatment in clinical medicine. However, the current biochemical identification methods can only identify them at the genus level. [Objective] To develop a simple and reliable method for species identification of Cronobacter, we established a numerical identification system and evaluated its identification performance. [Methods] The biochemical reaction formulations required for the identification system were concentrated and integrated into a set of plastic containers to prepare identification strips. The numerical identification system of Cronobacter was developed based on the numerical identification theory, and the identification accuracy was assessed with the standard strains and isolates of Cronobacter. The identification results based on the sequencing of fusA were taken as the reference and compared with those obtained with the numerical identification method. [Results] We established a biochemical reaction positive probability database for distinguishing the 9 species/subspecies of Cronobacter. Simultaneously, we defined the calculation methods and result evaluation criteria for the numerical method and developed a numerical identification system. Five standard strains of Cronobacter were correctly identified by the numerical identification system, and the results were evaluated as excellent. Among them, strain DSM 18707 was accurately identified as C.dublinensis subsp. lactaridi. The total identification agreement between the numerical identification method and the fusA sequencing method of the 84 Cronobacter isolates in the test set was 100%. The numerical identification method clearly identified other strains except C.sakazakii type IV (one strain) and C.dublinensis type III (one strain) with unacceptable identification results. [Conclusion] The numerical identification system developed in this study for Cronobacter. has simple operation, low cost, and high accuracy, which can provide an experimental basis for clinical diagnosis and food safety detection and demonstrates a promising prospect of application and popularization.

Abstract:[Background] Vibrio species pose a serious threat to human health worldwide. Vibrio parahaemolyticus, V.cholerae, V.mimicus and V.vulnificus capable of causing gastrointestinal infections and sepsis associated with consumption of raw or undercooked seafood are of particular concern. [Objective] To develop a real time fluorescence quantitative PCR method with improved efficiency and accuracy for the detection of V.parahemolyticus, V.cholerae, V.mimicus, and V.vulnificus. [Methods] The specific primers and probes were designed based on toxR of V.parahaemolyticus and V.mimicus, ompW of V.cholerae, and vvhA of V.vulnificus. A quadruplex real time fluorescence quantitative PCR method was established by optimizing the reaction system and conditions. [Results] The minimum detection limit of the real time fluorescence quantitative PCR method was 10 copies/µL, and the amplification efficiency was about 100%. In the specificity test, multiplex real time fluorescence quantitative PCR and conventional PCR were used to amplify the target bacteria genome, non-target bacteria genome and blank control strain genome, respectively. The results showed that only the target vibrio genome was amplified significantly, indicating that the method had good specificity. In the anti-interference experiment, high concentration Vibrio did not interfere with the detection of low concentration Vibrio. Three repeated experiments were performed for each concentration gradient, and the coefficient of variation of each group was less than 1.5%, indicating that the method was reproducible in this experiment. [Conclusion] The multiplex real time fluorescence quantitative PCR method was established, which could detect V. parahemolyticus, V. cholerae, V. mimicus, and V. vulnificus specifically and rapidly.

Abstract:[Background] Clostridium perfringens is the main pathogen causing sudden death syndrome in cattle, seriously endangering the cattle industry. Enzyme-linked immunosorbent assay (ELISA) is an important tool for diagnosis of this disease and antibody detection. [Objective] We expressed the recombinant α-β2-ε fusion protein and used this protein as the target antigen to establish an indirect ELISA method for antibody detection, aiming to provide technical support for the clinical detection of C. perfringens in yaks and epidemiological investigation. [Methods] The bioinformatic tools EditSeq and Protean and the Jameson-Wolf method were employed to analyze the antigenic indices of α, β2, and ε proteins of C. perfringens from cattle. The peptides with higher antigenic indices were captured, ligated, optimized for codons, and then cloned into the pET-32a(+) vector to construct the recombinant expression plasmid pET-32α-β2-ε, which was transformed into BL21(DE3) competent cells. The cells were cultured and induced for protein expression, and the expressed protein was purified and identified by SDS-PAGE and Western blotting. With the recombinant α-β2-ε fusion protein as the coating antigen, we optimized the reaction conditions by the checkerboard assay and single factor tests to establish an indirect ELISA method. The cut-off was determined by the receiver operating characteristic (ROC) curve, and the specificity, sensitivity, reproducibility, and clinical efficacy of the established method were evaluated. [Results] The peptides with high antigenic indices of α (aa 241–403), β2 (aa 161–355), and ε (aa 154–331) toxins were selected for fusion expression, and the fusion protein was determined as correctly expressed by SDS-PAGE and Western blotting. The optimal ELISA conditions for antibody detection were as follows: incubation with the coating antigen (5 μg/mL) at 4 ℃ overnight or 37 ℃ for 60 min; incubation with 5% skimmed milk at 37 ℃ for 1 h; incubation with the serum (dilution at 1:150) at 37 ℃ for 30 min; incubation with secondary antibody (1:2 000) at 37 ℃ for 30 min; color development for 10 min with protection from light. The cut-off of detection was 0.470 2. The established method demonstrated high specificity because of no cross-reactivity with positive sera of Mannheimia haemolytica, Salmonella, Escherichia coli, and Staphylococcus aureus. It showcased high sensitivity, with the maximum dilution of detectable serum being 1:3 200. The coefficients of variation within and between batches were less than 10%, which indicated high reproducibility of the established method. The detection for 277 yak serum samples from Ganzi Tibetan Autonomous Prefecture, Sichuan Province with the established method showed that the antibody positivity rate of C. perfringens was 89.89%, which was basically in agreement with the results obtained by the commercial ELISA kit for the detection of C. perfringens α-toxin. [Conclusion] We successfully constructed the recombinant α-β2-ε fusion protein and established an indirect ELISA method for the detection of C. perfringens.

Abstract:[Background] Gut microbiota is closely associated with hepatocellular carcinoma, impacting liver metabolism and immune responses. Ongoing attention is drawn to the study of gut microbiota-related mechanisms and treatment strategies. [Objective] Analyzing the trends, current status, and hotspots in domestic and international research on gut microbiota and hepatocellular carcinoma from 2003 to 2023, aiming to provide comprehensive and intuitive references for researchers in this field. [Methods] The articles about gut microbiota and hepatocellular carcinoma were retrieved from the Web of Science Core Collection (WoSCC) and China National Knowledge Infrastructure (CNKI). VOSviewer1.6.19 and CiteSpace6.1.R6 were used for bibliometric analysis. [Results] A total of 659 eligible English publications were retrieved from the WoSCC, and 70 Chinese publications meeting the criteria were screened out from the CNKI. The annual count and citation frequency of English publications showed an increasing trend, and the total number of Chinese publications also exhibited a rising trend. China was a leading country in terms of the publication number and international collaborations, while the United States exceled in citation frequency and international partnerships. The most influential research institution was the University of California San Diego, and the most impactful scholar/team identified was the Elinav Eran team. The International Journal of Molecular Sciences was the journal with the largest publication number. The co-occurrence analysis of keywords revealed that the research hotspots included the mechanisms of hepatocellular carcinoma occurrence, the relationship between gut microbiota and non-alcoholic fatty liver disease progression, and hepatocellular carcinoma treatment strategies targeting gut microbiota. The keyword burst analysis highlighted gut microbiome and immune checkpoint inhibitor as the current research frontiers in this field. [Conclusion] This study, from the perspective of bibliometrics, comprehensively analyzed the current status and development trends of the relationship between gut microbiota and hepatocellular carcinoma, and offered guidance for researchers in this field to delve into hotspots and frontiers and to choose suitable journals and collaborators.