[Objective] To isolate the pathogenic bacteria ofDrosophilasuzukii and study the mechanism of bacterial pathogenicity towards the host. [Methods] The pathogenic bacteria were isolated by the plate streaking method and identified based on the 16S rRNA gene sequences. The acidified ethanol method was employed to measure the ability of the isolate to cause browning. The pathogenicity of the isolate was examined by oral infection. Dihydroethidium (DHE) was used to determine the level of reactive oxygen species (ROS), and brilliant blue staining was used to examine the intestinal permeability. [Results] A strain was isolated from the brown culture medium ofD.suzukii and identified asGluconobacter oxydans. The strain reduced the survival rate ofD.suzukii to 49.41% on day 8 (P < 0.001). Furthermore, it weakened the tolerance ofD.suzukii to desiccation and starvation, reducing the survival rate to 58% and 50.9% at the time points of 24 h and 32 h, respectively (P < 0.001). Moreover, the flies treated withG.oxydans displayed impaired integrity of the intestine and had higher level of ROS in the guts than the control group (P < 0.001).G.oxydans robustly reduced the medium pH (pH 2.0), which compromised the survival rate ofD.suzukii and the growth ofLactobacillus plantarum, a probiotic ofD.suzukii. [Conclusion] G.oxydans was a potent pathogen capable of reducing the survival rate ofD.suzukii by lowering the medium pH and inhibiting the growth of probiotics, demonstrating the potential of serving the biocontrol ofD.suzukii.

Antibiotics are secondary metabolites produced by microorganisms during the stationary phase. They are widely used in the clinical treatment of bacterial infections because of their ability to kill bacteria or inhibit bacterial growth. In the long-term evolutionary process, bacteria have adopted several strategies to cope with the threats of antibiotics in the environment. In addition to the well-known antibiotic resistance, bacteria can develop tolerance and persistence to antibiotics, which seriously affects the clinical efficacy of antibiotics. Guanosine tetraphosphate (ppGpp) and guanosine pentaphosphate (pppGpp) (herein collectively referred to as ppGpp) are the alarmone signal molecules produced by bacteria in response to unfavorable environmental conditions such as nutritional starvation. ppGpp can regulate transcription globally and enable bacteria to survive in unfavorable conditions. An increasing number of studies have shown that ppGpp is closely related to antibiotic stress response. On this basis, this review summarizes the synthesis, hydrolysis, and mechanism of action of ppGpp in bacteria, with emphasis on the role of ppGpp in antibiotic stress response. This review aims to provide new ideas for the development of novel antibiotics.

African swine fever (ASF) caused by African swine fever virus (ASFV) is a severe infectious disease affecting both domestic pigs and wild boar. ASFV has a large genome, and the non-structural protein pD1133L is predicted to be one of the six helicases the genome encodes. We used the IP-MASS technology to screen the host proteins interacting with pD1133L and found that vimentin (VIM) is one of the host proteins that interacted with pD1133L. However, it remains unclear how the VIM affects ASFV replication. [Objective] To investigate the mutual regulation between ASFV and VIM and disclose the mechanism by which VIM enhances ASFV replication. [Methods] We employed the Co-IP assay to examine the interaction between pD1133L and VIM. Furthermore, we examined the effects of VIM on ASFV replication by designing and synthetizing VIM siRNAs and overexpressing VIM. Western blotting and quantitative real-time PCR (qPCR) were employed to determine the impact of ASFV on the protein and mRNA levels of VIM. Western blotting and indirect immunofluorescence assay (IFA) were used to explore the changes in the phosphorylation level and subcellular localization of VIM in macrophages infected with ASFV. The CCK-8 kit was used to determine the optimal concentration of KN-93, a VIM phosphorylation inhibitor, for treatment. The effects of KN-93 on the phosphorylation and subcellular localization of VIM and the replication of ASFV were examined by Western blotting and IFA. [Results] The overexpression of VIM promoted the replication of ASFV, while the knockdown of VIM inhibited ASFV replication. In addition, ASFV infection down-regulated both the protein and mRNA levels of VIM in a time-dependent manner. After ASFV infection, VIM was modified by phosphorylation and changed in subcellular localization, thereby promoting ASFV replication. [Conclusion] This study confirms the interaction between ASFV and the host protein VIM. After ASFV infection, pD1133L leads to the rearrangement of the subcellular localization of VIM towards paranuclear aggregation, which promotes ASFV replication.

[Objective] ALTA4H-deleted porcine kidney cell line (PK-15) was constructed by CRISPR/Cas9 to study the effect ofLTA4H on the replication of foot-and-mouth disease virus (FMDV), with a view to providing a theoretical basis for revealing the functions ofLTA4H and the mechanism of regulating virus replication. [Methods] Two small guide RNAs (sgRNAs) targeting porcineLTA4H were designed and integrated into the vector pX459-puro-MCS, respectively. The recombinant plasmid was transfected into PK-15 cells, which were then cultured in the medium supplemented with puromycin, and the monoclonal cells were selected by limiting dilution method. Western blotting and sequencing were performed to detectLTA4H knockout, andLTA4H-deleted cells were obtained. Furthermore, Western blotting, RT-qPCR, and virus titer assay were employed to examine FMDV replication and protein expression afterLTA4H knockout. [Results] Compared with the wild type cells, theLTA4H-deleted PK-15 cells exhibited inhibited FMDV replication. [Conclusion] This study successfully constructed a PK-15 cell line with LTA4H gene knockout, demonstrating that LTA4H can promote the replication of FMDV, and the results provided a theoretical basis for the subsequent research on the function ofLTA4H.

[Objective] To search for the strains with improved tolerance, probiotic properties, and safety, we isolatedLactobacillus plantarum HY21 from the intestinal contents ofApostichopus japonicus and evaluated the probiotic potential of the strain. [Methods] The shake flask fermentation, environmental factor simulation test, and antibiotic susceptibility test were carried out to study the growth characteristics and acid production of the strain, as well as the antioxidant capacity and aquatic pathogen-inhibiting effects of the fermentation broth. In addition,L.plantarum HY21 was characterized in terms of the tolerance to environmental factors, hydrophobicity, auto-agglutination and co-aggregation, antibiotic susceptibility, and intestinal adhesion ability. [Results] L.plantarum HY21 in shake flask culture with the initial pH 8.0 entered the logarithmic growth during the period of 2–10 h. The culture showed a strong acid-producing ability with the minimum pH 3.6 at the time point of 18 h. The fermentation broth of the strain showed an inhibition zone diameter of (13.96±0.30) mm againstVibrio alginolyticus and a 1, 1-diphenyl-2-trinitrophenylhydrazine (DPPH) free radical scavenging rate of 95.45%±1.56%.L.plantarum HY21 had a hydrophobicity rate of 60.42%±2.78%, an auto-agglutination rate of 22.69%±1.36% and a co-aggregation rate of 27.98%±1.45% withV.alginolyticus. Furthermore, the strain showed the adhesion number of (1.66±0.01)×10⁶ CFU/mL and (1.23±0.15)×10⁶ CFU/mL to fish body surface and intestinal mucus protein, respectively.L.plantarum HY21 was susceptible to 13 common antibiotics. The survival rate ofL.plantarum HY21 reached 99% and above in simulated gastrointestinal fluid (pH 3.0, pH 6.8) within 3 h, 68.11%±7.98% in 10% fish bile fluid within 3 h, and 97.58%±7.14% in 3% sea salt solutions within 16 h. [Conclusion] L.plantarum HY21 is a probiotic strain with strong colonization potential, antimicrobial activity, and antioxidant capacity, demonstrating a high survival rate and high safety in the applied environment. The findings provide a theoretical basis for the development of new probiotic strains as probiotic additives for aquaculture.

[Objective] Glucanases serve as one of the main components in feed additives. This study identified and characterized a novel GH9 glucanase gene derived from rumen microbiota in herbivores, aiming to provide a reference for the research and development of feed enzymes. [Methods] We obtained theIDSGLUC9-25 gene from the rumen fluid cDNA of Hu sheep and heterologously expressed it inEscherichia coli. The recombinant protein was induced for expression by isopropyl β-d-thiogalactopyranoside, purified, and then subjected to functional characterization. [Results] IDSGLUC9-25 encoded a protein consisting of 527 amino acid residues, which included a CelD_N domain and a GH9 family catalytic domain. The recombinant rIDSGLUC9-25 protein exhibited a molecular weight of approximately 62.7 kDa and the highest enzymatic activity at 40 ℃ and pH 6.0. The enzyme displayed robust catalytic activity within the temperature range of 30–50 ℃. After preincubation at pH 4.0–8.0 for 1 h, rIDSGLUC9-25 retained the relative activity over 90%. The substrate spectrum analysis revealed that rIDSGLUC9-25 exhibited specific activities against barley β-glucan, moss lichenan, konjac gum, and xyloglucan, with the activities of (443.55±24.48), (65.56±5.98), (122.37±2.85), and (159.16±7.73) U/mg, respectively. The hydrolysis assay showed that rIDSGLUC9-25 primarily catalyzed the hydrolysis of β-glucan into cellotriose (representing 64.19%±1.19% of total reducing sugars) and cellotetraose (representing 26.24%±0.12% of total reducing sugars). Additionally, the enzyme predominantly generated cellotriose from the hydrolysis of lichenan (representing 78.46%±0.89% of total reducing sugars). [Conclusion] This study characterizes IDSGLUC9-25, an endo-β-1,4-glucanase (EC 3.2.1.4) derived fromTreponema sp. The enzyme exhibited robust activity in the conversion of polysaccharides into cellotriose and cellotetraose, establishing a foundation for the development of feed enzymes and functional oligosaccharides preparation.

[Objective] To study the inhibitory effect and mechanism of an antimicrobial peptide (A2M3) derived from alpha-2-macroglobulin identified in the human nasal cavity againstStaphylococcus aureus. [Methods] The mass spectrometry results of the human nasal liquid were analyzed, on the basis of which bioinformatic tools were used for the screening of potential antimicrobial peptides. The minimum inhibitory concentration (MIC) and time-kill curve of A2M3 againstS.aureus were determined by the microdilution method and plate colony counting method. Then, transmission electron microscopy, PI uptake assay, flow cytometry, and determination of nucleic acid protein leakage were employed to study the effects of A2M3 on the membrane integrity and permeability ofS.aureus. Finally, the effect of A2M3 on the genomic DNA ofS.aureus was investigated by the gel retardation assay and fluorescence spectroscopy. [Results] A2M3 showed an MIC of 125.0 µg/mL againstS.aureus and killed the bacteria completely within 3 h. A2M3 increased the cell membrane permeability to penetrate intoS.aureus cells, leading to leakage of nucleic acids and proteins as well as insertion into DNA base pairs to interfere with the gene function, resulting in the death of the cells. [Conclusion] The inhibitory mechanism of A2M3 againstS.aureus involves multiple targets. The antimicrobial peptide alters the permeability of the bacterial cell membrane and affects the gene function, thus exerting the inhibitory activity. The findings reveal that antimicrobial peptides could be screened and isolated from human body fluids for potential application.

[Objective] Nickel (Ni) is one of the heavy metal pollutants to which humans are widely exposed, and nickel exposure activates the cell wall integrity (CWI) signaling pathway, which lowers the level of intracellular histone acetylation. However, whether the CWI pathway is regulated by histone acetylation under nickel stress remains to be fully understood. [Methods] We used the histone-targeted mutant strain H4K5R (mimicking the deacetylated state) to study the regulation of the CWI pathway inSaccharomyces cerevisiae by H4K5 deacetylation under nickel stress, aiming to lay a foundation for unveiling the regulatory role of histone modifications in eukaryotes in response to heavy metal stress. [Results] Compared with the wildtype strain, H4K5R had strong nickel resistance, being able to grow in the presence of 5.0 mmol/L NiCl₂. The results of Western blotting and qRT-PCR showed that the CWI pathway of the wildtype strain BY4741 was activated under 5.0 mmol/L NiCl₂, with the expression ofMnn9 (encoding α-1,6-mannosyl transferase) andFks1 (encoding glucan synthase) being up-regulated by 3.13 folds and 1.49 folds, respectively. Moreover, the content of mannan and β-glucan were increased, which indicated that the wildtype strain activated the CWI pathway to increase the content of cell wall component. The activation of the CWI pathway in H4K5R was mild under the stress of 5.0 mmol/L NiCl₂. Although the expression ofMnn9 andFks1 was up-regulated, the changes in mannan content were not significant, and the increase in β-glucan content was less than that of the wildtype strain. [Conclusion] Under 5.0 mmol/L NiCl₂ stress, the deacetylation of H4K5 in the mutant strain regulated the CWI pathway, which affected the changes in cell wall components.

[Objective] Colony extraction and counting is essential in agriculture, food, and health industries. Currently, most of the available algorithms for automatic counting of colonies use colony culture dishes and has poor applicability to colony count plates. In addition, the current technologies have good performance in conventional segmentation of adherent objects, while their accuracy remains to be improved for the segmentation and counting of adherent colonies due to the unique morphological characteristics of colonies. [Methods] To solve such problems, we proposed a colony segmentation and counting algorithm based on target color base and gradient direction matching. Firstly, the color feature of the colony in the image was used as a base to convert the image into a base space to enhance the difference between the colony and the background. Secondly, the gradient magnitude feature of the colony image was used to filter the gradient direction, and then the matching was performed through the gradient direction, thereby segmenting the adherent colonies. Finally, non-maximum suppression was employed to screen and count the colonies. [Results] Through experiments, the counting accuracy of the algorithm in this study reaches 98.00%, demonstrating its capability to meet practical requirements. [Conclusion] In the context of targeted segmentation and counting of colonies, the algorithm studied in this paper not only exhibits high counting accuracy but also demonstrates good robustness. This algorithm had not only high counting accuracy but also good robustness, producing excellent results in the colony segmentation and counting of colony count plates from different manufacturers. However, it showed decreased counting accuracy in the detection and segmentation of large-area targets. Therefore, this algorithm is suitable for the detection and segmentation of small targets such as colonies.

[Objective] To explore the mechanism ofNeorhizobium petrolearium OS53 combined with alfalfa (Medicago sativa L.) in the remediation of petroleum-contaminated soil. [Methods] Illumina and Nanopore were employed to sequence the whole genome ofN.petrolearium OS53, and the complete genome map of the strain was constructed. Gene prediction and functional annotation were carried out to analyze the genes involved in nodulation and oil degradation. The abilities of strain OS53 to produce indole acetic acid (IAA), secrete siderophore, and solubilize phosphorus and potassium were tested. The activities of urease, dehydrogenase, polyphenol oxidase, and lipase in soil and the levels of chlorophyll, malondialdehyde, proline, soluble protein, soluble sugar, and superoxide dismutase in alfalfa were measured by kits. [Results] The genome of strain OS53 consisted of a circular chromosome of 5.56 Mb and two plasmids of 0.92 Mb and 0.38 Mb, respectively, with the G+C content of 60.2%. The genome encoded a total of 6 968 genes. The strain OS53 andN.petrolearium DSM 26482^T showed the 16S rRNA gene sequence similarity of 99.86%, and formed stable branches on the phylogenetic tree, indicating that strain OS53 andN.petrolearium were the same species. Therefore, OS53 was named asN.petrolearium OS53. The strain OS53 had the ability to produce IAA, and the related genes were identified in the genome. After 120 days of remediation of the soil with the initial oil content of (4 403.30±222.10) mg/kg, OS53 and alfalfa showed the remediation efficiency up to 57.53%, which was 44.26%, 41.69%, and 8.84% higher than that of no inoculation of strain OS53, inoculation of OS53 only, and planting alfalfa only, respectively. In the combined remediation system, alfalfa showed elevated the levels of chlorophyll, soluble protein, and soluble sugar and lowered levels of malondialdehyde, proline, and superoxide dismutase, and the soil showed increased activities of polyphenol oxidase, dehydrogenase, lipase, and urease. [Conclusion] The strain OS53 had the ability to produce IAA to promote the growth of alfalfa in the petroleum-contaminated soil, which increased the activity of enzymes involved in oil degradation in the soil. Finally, the combined system improved the remediation efficiency of the soil.