[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 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.

[Objective] Episomal expression vectors typically have higher copy number to achieve strong gene expression than chromosomal expression vectors. Moreover, they are more convenient and flexible for DNA manipulation. However, the episomal plasmids suitable for the application inRhodosporidium toruloides remain to be determined, and the expression of heterologous genes or CRISPR/Cas9-based genome editing needs to be achieved by integration, which is a key reason for the slow progress in its genetic modification. Thus, this work aims to construct an episomal plasmid ofR.toruloides, which facilitates the expression of heterologous genes and promotes the gene editing in a time-saving manner. [Methods] First, the possible autonomously replicating sequences (ARSs) in the phenylalanine ammonia-lyase gene (PAL) ofR.toruloides were mined. Specifically,PAL and its upstream and downstream sequences were amplified in segments and constructed into a plasmid containing the β-isopropyl malate dehydrogenase gene (LEU2). The recombinant plasmids were then introduced intoLEU2-deficientR.toruloides by the electroporation method. An ARS was then identified according to transformation efficiency. Then, theBTS1 gene encoding geranylgeranyl pyrophosphate synthase was selected as the knockout target, and its gRNA was constructed into the episomal plasmid based on the identified ARS. The color change of the transformant was observed to verify whether the episomal plasmid was successfully applied to the CRISPR/Cas9 system ofR.toruloides. [Results] In this work, an ARS was identified, based on which an episomal plasmid was constructed and applied to CRISPR/Cas9 editing inR.toruloides. Finally, the episomal plasmid-based gene knockout ofR.toruloides was successfully achieved. [Conclusion] This work enriched the existing tool library and provided a research basis and technical support for the application ofR.toruloides in synthetic biology.

[Objective] To generateListeriamonocytogenes strains withlmo0880 deleted and complemented strains, so as to investigate the roles of Lmo0880 in bacterial infection in a host. [Methods] Thelmo0880-deleted strain was generated by homologous recombination, and the complementary strain was constructed by introducing an integrative plasmid carryinglmo0880 into thelmo0880-deleted strain. The growth, adhesion, invasion, and intracellular proliferation were compared between thelmo0880-deleted strain, complementary strain, and the wild type. [Results] The deletion oflmo0880 did not significantly impact bacterial growth or adhesion. However, it led to notable decreases in cell invasion, proliferation, and colonization in the liver and spleen, ultimately diminishing the pathogenicity in mice. [Conclusion] The LPXTG-anchored protein Lmo0880 plays a crucial role in bacterial invasion, proliferation, and colonization in a host. These findings provide a solid foundation for deeply understanding the pathogen-host interaction duringL.monocytogenes infection.

Ferric uptake regulator (Fur) is a key regulatory factor of iron metabolism and virulence inPseudomonas aeruginosa. Many research groups have failed to construct thefur-deleted mutant ofP.aeruginosa, sofur has always been considered to be an essential gene inP.aeruginosa, and the knowledge of its biological function is limited. [Objective] This study aims to construct afur-deleted mutant ofP.aeruginosa and analyze its phenotypes. [Methods] WithP.aeruginosa PAO1 as the parental strain, thefur-deleted mutant was constructed by homologous recombination. After that, we studied the effects offur on the growth, siderophore biosynthesis, resistance to oxygen stress, flagella formation, biofilm formation, and virulence ofP.aeruginosa. In addition, we explored the cause of the growth defect phenotype of thefur-deleted mutant by genetic analysis. [Results] Thefur-deleted mutant ofP.aeruginosa was successfully constructed. The deletion offur greatly limited the growth ofP.aeruginosa and reduced the growth adaptability ofP.aeruginosa to the iron-limited environment, while it did not affect the growth adaptability ofP.aeruginosa to the iron-rich environment. This growth defect phenotype of Δfur was caused by the slow cell growth and proliferation, rather than by cell death. Interestingly, heterologousfur could completely complement the growth defect phenotype of Δfur, suggesting that the Fur ofP.aeruginosa was not functionally unique. Although there was a functional relationship between Fur and the toxin-antitoxin system PacTA, the growth defect phenotype ofP.aeruginosa Δfur was not associated with PacT toxin. In addition to affecting the growth phenotype ofP.aeruginosa, the deletion offur also madeP.aeruginosa lose the inhibitory effect on siderophore biosynthesis and the ability to form flagella and have increased sensitivity to H₂O₂ and reduced virulence toGalleria mellonella larvae. Moreover, the deletion offur increased the intracellular cyclic diguanylate (c-di-GMP) level ofP.aeruginosa to induce the expression ofpelF andpslA, thereby promoting the biofilm formation ofP.aeruginosa. [Conclusion] fur is a non-essential gene that can be deleted and plays a crucial role in the normal growth, siderophore biosynthesis, resistance to oxygen stress, flagellum formation, biofilm formation, and virulence ofP.aeruginosa, which lays a foundation for the development of vaccines and agents againstP.aeruginosa.

[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.

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] 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] Antibiotics as emerging pollutants have aroused wide concern. In view of the shortage of effective tetracycline-degrading strains, this study aims to screen and identify the strains for tetracycline degradation, analyze degradation properties and type, pinpoint the localization of active substances for bio-degradation, and evaluate the physiological toxicity of degradation products. [Methods] Tetracycline was used as the sole carbon source to screen out the target strain from tetracycline-contaminated pig sludge. The strain was identified based on colony morphology, physiological and biochemical characteristics, scanning electron microscopy images, and the 16S rRNA gene sequence. Different carbon sources, pH, and removal kinetics were employed to characterize the degradation process of the strain. Different components of the strain were extracted to determine the degradation type of tetracycline by the strain. Furthermore, the intracellular and extracellular fluids of the strain were used to degrade tetracycline, so as to determine the location of the active substance for degradation. Finally, the toxicity of the degradation products was assessed. [Results] The strain MEH2305 was screened out and identified asEnterobacter hormaechei, which showed the best degradation performance at pH 7.0 and with tryptone as the carbon source. Strain MEH2305 showed a total tetracycline removal rate of 68% on the 7th day of culturevia abiotic degradation and bio-degradation, and the removal rates of oxytetracycline and doxycycline hydrochloride were 53% and 56%, respectively. The tetracycline removal efficiency by the intracellular and extracellular fluids of MEH2305 was 40.77% and 31.18%, respectively. Compared with tetracycline control without MEH2305, the tetracycline degradation products of MEH2305 had reduced physiological toxicity on Gram-negativeEscherichia coli K88 and Gram-positiveBacillus subtilis 168. [Conclusion] The strain MEH2305 can be used as an effective and safe tetracycline-degrading strain for the treatment of antibiotics in the environment.

[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.