[Objective] To investigate the acclimation mechanisms of straw-decomposing microbiomes in response to historically different climate conditions as characterized by extreme temperature distinction, we collected two native forest soil samples from the tropical (annual mean temperature: 25 ℃) and cold-temperate (annual mean temperature: −2 ℃) zones at a distance of 3 860 km. [Methods] Microcosm incubation was conducted at a low temperature (10 ℃), a high temperature (35 ℃), and alternated high and low temperatures (10 ℃/35 ℃). The two native forest soil samples were inoculated for targeted cultivation of straw-decomposing microbiomes. After 12 consecutive weeks of passage, 16S rRNA gene sequencing was carried out to analyze the microbial community composition. [Results] At 10 ℃, higher straw decomposition rate was observed in the forest soil from Changbai Mountain in the cold-temperate zone (15.5%) than that from the tropical zone. At 35 ℃, the decomposition rate in the soil from Sanya in the tropical zone (33.1%) was higher than that from Changbai Mountain The results of linear discriminant analysis effect size (LEfSe) showed that the dominant straw-decomposing genera included Duganella, Pedobacter, Janthinobacterium, and Serratia after 12 weeks of enrichment at 10 ℃ with the forest soil from Changbai Mountain. The dominant genera were Paenibacillus and Rhodanobacter after enrichment at 35 ℃, and Stenotrophomonas, Burkholderia, and Achromobacter after enrichment at 10 ℃/35 ℃. As for the forest soil from the tropical zone, the enriched dominant genera were Pseudomonas, Acinetobacter, and Flavobacterium at 10 ℃, Cupriavidus at 35 ℃, and Enterobacter and Cohnella at 10 ℃/35 ℃. [Conclusion] This study revealed the indicator microbial species for straw decomposition at different temperatures in native forest soils from geographically highly distinct regions with a 3 860 km distance. The results suggest that temperature could have likely played a pivotal role in shaping the microbiomes for straw decomposition. The findings provide a scientific basis for mining the straw-decomposing microbial resources in the cold zone in northeast China and the tropical zone in south China.

The spread of antibiotic resistance has aroused global concern. The development of technologies for detecting antibiotic resistance genes (ARGs) is essential for curbing the migration and spread of ARGs from the environment to plants/animals and human populations. This paper describes the development timeline of existing nucleic acid detection technologies and their first applications to the detection of ARGs and summarizes their detection principles, advantages and disadvantages, and development potential. Furthermore, this paper prospects that isothermal amplification combined with CRISPR/Cas might be the core technology for the development of in-situ rapid detection methods. By reviewing the development history of each technology, this paper aims to give insights into the development and applications of technologies for detecting ARGs and provide technical support for the research and control of antibiotic resistance transmission.

[Objective] To investigate the biological role played by the glucoside transporter Lmo0738 in the virulence of Listeria monocytogenes. [Methods] The lmo0738-deleted mutant (Δlmo0738) and complementation mutant (CΔlmo0738) were constructed by homologous recombination. The growth, hemolytic activity, cellular adhesion and invasion, and intracellular migration of the wild type strain and the mutants were assessed by the growth curves, sheep red blood cell hemolysis assay, infection of human epithelial cells (Caco-2), and infection of mouse fibroblastic cells (L929), respectively. Additionally, the mRNA and protein levels of the virulence factor listeriolysin O (LLO) were determined by real-time quantitative reverse transcription PCR (RT-qPCR) and Western blotting, respectively. [Results] The L. monocytogenes strain with the deletion of lmo0738 demonstrated weakened growth and diminished hemolytic activity. Notably, Δlmo0738 exhibited significantly reduced cell adhesion, invasion, and intracellular migration compared with the wild type strain. In addition, the mRNA and protein levels of LLO were significantly down-regulated in Δlmo0738. [Conclusion] This study provides the evidence that the absence of lmo0738 attenuates the virulence of L. monocytogenes, laying a crucial foundation to illustrate the mechanism of the phosphotransferase system (PTS) in regulating the sugar catabolism and the infection mechanism of major food-borne pathogens including L. monocytogenes.

[Objective] To homologously overexpress an alkaline fungal laccase PIE5 (CcPIE5) in Coprinopsis cinerea FA2222 under the control of the Agaricus bisporus gpdII promoter. [Methods] The laccase activity reached (24.2±1.1) U/mL in the supernatant after 7 days of cultivation at 37 ℃ in the mKjalke medium. The purified CcPIE5 showcased the best performance at pH 8.0 and 60 ℃. [Results] Unlike other characterized fungal laccases, CcPIE5 was tolerant to high concentrations of NaCl. Particularly, both k_cat and K_m decreased when the concentration of NaCl was increased from 0 to 1.5 mol/L, which indicated that CcPIE5 demonstrated application potential in the dye decoloring of texile finishing. In dye decolorization, CcPIE5 efficiently degraded (92.9±2.3)% indigo carmine at pH 8.5 and 60 ℃, with syringic acid as the mediator. Isatin 5-sulfonic acid (ISA) was identified by LC-MS as the primary byproduct of indigo carmine degradation. [Conclusion] CcPIE5 is best-suited in decolorizing dyes under high temperatures and alkaline and salty conditions. It serves as a good candidate for specific applications in the environment and industry.

[Objective] To investigate the changes of pathogenicity and DNA methylation levels and patterns of Ralstonia solanacearum strains with different pathogenicity during consecutive subculture. [Methods] R. solanacearum strains with different pathogenicity were consecutively subcultured for 50 passages. The pathogenicity of different strains was determined by the attenuated index (AI) method and the pot experiments. Methylation sensitive amplification polymorphism (MSAP) analysis was performed to profile the DNA methylation levels of different strains. Moreover, the relative expression levels of genes related to methylases and demethylases were determined by real-time fluorescent quantitative PCR (qRT-PCR). [Results] After 50 passages, both of the virulent strain FJAT15304 and the intermediate strain FJAT445 evolved into avirulent strains, while the avirulent strain FJAT15249 remained to be avirulent. Compared with F₁ strains, FJAT15304.F₅₀ and FJAT445.F₅₀ showed the total methylation rates increasing by 7.82% and 38.22%, respectively. However, both of FJAT15249.F₁ and FJAT15249.F₅₀ had the total methylation rate of 33.33%. Full methylation was the main pattern in the virulent and intermediate strains, while hemi-methylation was the main pattern in all the avirulent strains. Compared with F₁ strains, strains FJAT15304.F₅₀ and FJAT445.F₅₀ showed up-regulated expression of three methylase-related genes dam, dcm, and ftsZ and down-regulated expression of demethylase-related gene alkB, which suggested that the change of DNA methylation might play a key role in the debilitation of pathogenicity. [Conclusion] The pathogenicity of R. solanacearum attenuates during the consecutive subculture, which might be related to the level of DNA methylation. The findings provide a scientific basis for the application of avirulent strains in the biocontrol of bacterial wilt.

[Objective] Porcine deltacoronavirus (PDCoV) is a major porcine enteric coronavirus, causing huge economic losses to the pig breeding industry worldwide. However, there is no commercial vaccine available for this virus. The spike (S) protein is a key factor inducing host immune response. In this study, the two sites 855 and 856 in the loop between the heptapeptide repeat-1 (HR1) and the central helix of PDCoV S protein were mutated to proline (E855P and V856P). Then, the recombinant S protein and mutated S protein (S2P) were expressed and purified by the ExpiCHO-S eukaryotic expression system, and their immunogenicity and immunoprotecive performance were evaluated for developing a PDCoV subunit vaccine with good immune effect. [Methods] The serum level of the specific antibody IgG in immunized mice was measured by indirect enzyme-linked immunosorbent assay. The serum neutralization test was carried out to determine the titer of neutralizing antibodies in the immunized mice. The proliferation of T lymphocytes in immunized mice was detected by flow cytometry. The secretion levels of interferon (IFN)-γ, IFN-α, interleukin (IL)-2, and IL-4 were determined. RT-qPCR was employed to measure the PDCoV load in the intestinal tissue of mice after challenge. Tissue sections were prepared to observe the intestinal lesions of mice. The distribution of PDCoV antigen in the intestinal tissue of mice was detected by immunohistochemistry. [Results] High levels of anti-PDCoV specific IgG antibodies were produced in mice after intramuscular injection of S and S2P subunit vaccines, and the serum of mice 42 days after immunization had a neutralizing effect on PDCoV. The 50% neutralizing protective titer of LLC-PK cells in the S2P group was significantly higher than that in the S group. In addition, the immunization with S and S2P significantly induced the proliferation of CD₄⁺ T lymphocytes in mice, which was higher in the S2P group than in the S group. The immunization with S2P induced the proliferation of CD₈⁺ T lymphocytes in mice, and the level of CD₈⁺ T lymphocytes showed no difference between the S group and the PBS group. The levels of IFN-γ, IFN-α, IL-2, and IL-4 in the S and S2P groups were significantly higher than those in the PBS group and had no difference between the S and S2P groups. The challenge assay results showed that the PBS group presented PDCoV, pathological damage, and a large number of PDCoV antigens in the intestinal tissue, while neither PDCoV nor intestinal tissue damage was detected in the S and S2P groups, which showed no significant difference between the two groups. [Conclusion] S2P induces higher level of the humoral immune response against PDCoV in mice than S. The vaccines prepared with both S2P and S have protective effects on mice. The findings lays a foundation for the follow-up study of PDCoV subunit vaccines.

[Objective] To investigate the role of WekM, the O-antigen glycosyltransferase of avian pathogenic Escherichia coli (APEC) O₁, in lipopolysaccharide biosynthesis and environmental adaptation. [Methods] The wekM-deleted strain ΔwekM of APEC O₁ was constructed by Red homologous recombination, and then the complementary strain CΔwekM was constructed. The impacts of wekM on bacterial growth and motility were examined. The lipopolysaccharide (LPS) profile and reactivity with rabbit anti-O₁ serum of each strain were identified by silver staining and Western blotting. Real-time fluorescence quantitative PCR was conducted to determine the transcriptional levels of flagellum-related genes, and ethidium bromide was used to measure the bacterial cell membrane permeability. Finally, the drug sensitivity test was carried out to identify the bacterial susceptibility to antibiotics such as ciprofloxacin. [Results] The constructed ΔwekM and CΔwekM were verified by PCR amplification and DNA sequencing. Compared with the wild type, ΔwekM showed incomplete LPS profile and absence of some O-antigen bands. Western blotting results showed that ΔwekM did not react with the anti-O₁ serum, suggesting that the loss of WekM impaired the LPS production. The deletion of wekM reduced the swimming motility and did not impact the bacterial growth rate compared with the wild type. The transcription levels of flagellum-related genes such as flgC were down-regulated in ΔwekM. The results implied that the reduced motility of ΔwekM was caused by the decrease in flagellar production. In addition, ΔwekM demonstrated increased cell membrane permeability compared with the wild type (P < 0.01), and ΔwekM improved bacterial sensitivity to 7 antibiotics including polymyxin. This result suggested that the adaptability of ΔwekM to the environment was inhibited due to the increased cell membrane permeability. [Conclusion] The deletion of wekM in APEC results in diminished swimming motility, increased antibiotic resistance, improved cell membrane permeability, and damaged LPS integrity. The findings lay a foundation for mining the role of wekM and enrich our understanding of the stress resistance mechanism of APEC.

[Objective] The plasmid interference system of CRISPR/LshCas13a was constructed in Escherichia coli MG1655-ΔrecA and Escherichia coli DH10B to analyze the escape phenomenon in RNA editing experiments by targeting the non-essential gene lacZ and the essential gene polA. [Methods] An inducible CRISPR/LshCas13a RNA editing system- associated plasmid was designed with LshCas13a from Leptotrichia shahii. MG1655-ΔrecA and DH10B were selected as the research objects. The Crisporo algorithm was employed to design the CRISPR RNA (crRNA) sequences targeting lacZ and polA, and the LshCas13a plasmid interference experiment was carried out to study the escape phenomena targeting lacZ and polA. The escape phenomenon of the LshCas13a system was evaluated based on the number and sequences of escaped colonies. PCR and Sanger sequencing were conducted to explore the escape events of the LshCas13a system. The escaped colonies carrying the LshCas13a system disrupted by the insertion sequence (IS) were selected, and OD₆₀₀ was measured to evaluate the growth recovery of the strains. [Results] The LshCas13a system was used to target lacZ and polA in MG1655-ΔrecA and DH10B. MG1655-ΔrecA escaped through point mutation of LshCas13a and IS-mediated transposition when lacZ was targeted. When polA was targeted, MG1655-ΔrecA and DH10B escaped by point mutation of LshCas13a, IS-mediated transposition, and mutation of the direct repeat (DR) sequence of crRNA. The mutation of LshCas13a promoted the recovery of strain growth. [Conclusion] The LshCas13a plasmid interference system successfully revealed the diversified escape phenomena during the RNA editing of E. coli, including IS-mediated the transposition of LshCas13a, point mutation of LshCas13a, and DR sequence mutation or recombination of crRNA. The results laid a foundation for optimization of the CRISPR/LshCas13a gene editing system.

[Objective] Considering the important role of signal peptides in the secretory expression of heterologous proteins, we devised an automated high-throughput platform for the automatic screening of signal peptides, aiming to explore the effects of different signal peptides in Bacillus subtilis on the expression of heterologous proteins. [Methods] First, using the Escherichia coli-B. subtilis shuttle vectors pHP13 and pMA5 as the skeleton, we amplified the cell division B lethal gene (ccdB) and then ligated it to the middle of the promoter and the target gene to build the signal peptide screening vector. With the genomic DNA of B. subtilis 168 as the template, 173 signal peptides were amplified. An automated platform was established for the expression and screening of heterologous proteins in B. subtilis. Furthermore, the recombinant strains of heterologous proteins containing different signal peptides were constructed, and the effects of different signal peptides on the secretory expression of heterologous proteins were investigated. [Results] Five signal peptides (RpmG, AspB, CitH, LytF, and YkwD) showed strong abilities to induce the export of GFP from B. subtilis. Among them, RpmG had the strongest ability to induce the export of GFP, and the extracellular GFP fluorescence of the recombinant strain increased by 236% compared with that of the control strain. In addition, 41 signal peptides were not compatible with pullulanase (PulA), while the two signal peptides RpmG and AspB showed strong abilities to export PulA. The highest PulA activity of 116 U/mL was detected from the recombinant strain carrying the signal peptide RpmG, and the extracellular enzyme activity was 52 U/mL. The secretion rate of the PulA recombinant strain carrying the signal peptide AspB reached 74%, which was 68% higher than that of the control strain. [Conclusion] We developed an automated platform for high-throughput screening of the heterologous protein signal peptides in B. subtilis and obtained the signal peptides capable of improving the secretory expression of GFP and PulA. This automated platform allowed the parallel processing of a considerable number of samples, which simplified the repetitive manual laboratory work. This platform outperformed manual operation in terms of both time consumption and cost. The advantage of the automated high-throughput platform will be more significant with the increase in sample size. In summary, the established automatic high-throughput screening platform not only accelerates the screening process of signal peptides of heterologous proteins, but also provides new technical support for the modification and iteration of industrial strains of other value-added proteases.

[Objective] To explore the role of S1PP in the morphological differentiation, pathogenic process, and maintenance of sphingolipid balance of Magnaporthe oryzae. [Methods] We employed homologous recombination to delete the S1PP gene MoLCB3 from M. oryzae and characterized the obtained mutant ΔMolcb3 was by phenotypic analysis, gene complementation, and lipid metabolomics. Furthermore, we deleted the sphingosine kinase (SK) gene MoLcb4 from ΔMolcb3 to explore the relationship between MoLcb3 and MoLcb4. [Results] The deletion of MoLCB3 resulted in significant decreases in the mycelial growth rate and spore production and affected conidial malformation and initial appressorium formation. ΔMolcb3 completely lost the pathogenicity to barley. Moreover, the ΔMolcb3 mutant were significantly different from the wild type in responding to hyperosmic stress, cell wall integrity stress, high temperature stress, and fungal lipid synthesis inhibitors triadimefon and myriocin, suggesting that MoLcb3 was involved in these stress responses and lipid anabolism. Interestingly, the double mutant ΔMolcb3ΔMolcb4 basically compensated for all phenotypic defects of ΔMolcb3. In addition, lipid metabolomics showed that compared with the wild type, ΔMolcb3 presented significantly different levels of lipids, such as free fatty acids, ceramides, and phosphatidyl inositol. [Conclusion] MoLcb3 plays an important role in the mycelial growth, sporulation, spore germination, pathogenicity, stress responses, and lipid homeostasis. In addition, knockout of MoLCB4 can cushion the effects of MoLcb3 deletion. The results of this study provide new ideas for elucidating the sphingolipid metabolic pathway of M. oryzae and the development of inhibitors of fungal lipid biosynthesis.