Abstract: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.

Abstract:Electromagnetic radiation is a widespread physical phenomenon and exerts complex and profound effects on microorganisms. Understanding the state and function changes of microorganisms exposed to radiation is helpful to reveal the environmental response mechanisms of microorganisms and discover potential risk factors that threaten human health. By reviewing the relevant articles, we first discuss the damage of different types of electromagnetic radiation, including microwave, infrared, ultraviolet, X-rays, and γ-rays, to microorganisms. Furthermore, we elaborate on the molecular mechanisms by which electromagnetic radiation damages microorganisms from multi-omics. Finally, we reveal the potential relationship between the changes in the microbiome composition and the development of diseases in humans exposed to electromagnetic radiation.

Abstract:Escherichia coli, a facultative anaerobic, flagellated, Gram-negative rod bacterium commonly parasitic in the intestines of humans and animals, is one of the common zoonotic pathogens. E. coli is easy to form biofilms, which are special aggregates formed by bacterial cells attached to each other and encased with self-produced extracellular matrix. Biofilm formation is a major reason for the difficulty in curing bacterial infectious diseases in clinical practice. It not only helps bacteria evade the host defense system but also reduces or prevents drugs from working, thereby inducing biofilm-associated infections (BAIs). This review introduces the molecular mechanism of E. coli biofilm formation from the perspective of the gene regulatory system and related regulatory proteins and summarizes the strategies for the prevention and treatment of BAIs, providing references for finding appropriate drug targets and preventing BAIs.

Abstract:l-threonine is one of the eight essential amino acids that cannot be synthesized by the human body and must be taken from food. It is an important component of protein synthesis and is widely used in food, feed, medicine and other fields. At present, Escherichia coli can achieve a high threonine yield in fermentation, being the main bacterium used for industrial production of threonine. With the development of metabolic engineering, the modification of strains is no longer limited to mutagenesis, and the directed modification of strains greatly improves the production of l-threonine, facilitating the development of the l-threonine industry. This paper introduces the physicochemical properties and synthesis pathway of l-threonine and reviews the achievements in improving l-threonine production by metabolic engineering, aiming to enrich the knowledge about the modification of Escherichia coli for efficient synthesis of threonine.

Abstract:Microalgae are rich in lipids, proteins, and exopolysaccharides, serving as potential producers of high-value by-products. Compared with monoculture, microalgal co-culture offers advantages such as fast growth rates and strong resistance, increasing the microalgal biomass and lipid production. Biomass production by microalgae co-culture is influenced by environmental conditions, nutrient composition, and external stress, and the produced biomass can be utilized for biofuel production and food processing. This article introduces the types of microalgal co-culture systems and reviews the related studies on the production of high-value by-products. It summarizes the factors influencing biomass production in microalgal co-culture systems and highlights the potential of microalgal co-culture for resource utilization. Furthermore, this article discusses the prospects and challenges of microalgal co-culture.

Abstract:[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.

Abstract:[Objective] The protein SakA encoded by sakA is a member of the mitogen-activated protein kinase (MAPK) family in Aspergillus niger. However, little is known about the roles of SakA in A. niger. In this study, we constructed the A. niger strains with knockout of sakA to investigate the roles of this gene. [Methods] The Agrobacterium-mediated method was utilized to construct ΔsakA strains from A. niger RAF106 (the wild type, WT). The growth and spore production of ΔsakA and WT were observed on three different media. The sensitivity of ΔsakA and WT to different stress conditions was studied. The intracellular and extracellular levels of amylase, pectinase, and cellulase were compared betweenΔsakA and WT. Real-time quantitative polymerase chain reaction (qRT-PCR) was employed to determine the relative transcript levels of the genes associated with spore production, amylase, pectinase, cellulase, and hyperosmotic regulation. [Results] Three ΔsakA strains were successfully obtained and verified by PCR and qRT-PCR. The ΔsakA strains had slow growth, delayed spore production, and delayed conidiophore differentiation compared with WT. The ΔsakA strains showcased slower colony growth than WT under the stress conditions of 0.6 mol/L KCl, 0.8 mol/L NaCl, and 1.2 mol/L NaCl. Compared with WT, the knockout of sakA increased the extracellular amylase production by 20.68%–21.43% and decreased the intracellular amylase production by 19.18%–20.26%, decreased the extracellular pectinase production by 36.71%–38.30% and increased the intracellular pectinase production by 35.68%–36.53%, decreased the extracellular cellulase production by 28.04%–33.82% and increased the intracellular cellulase production by 15.28%–18.19%. Compared with WT, the knockout of sakA down-regulated the transcript levels of spore production-related genes (fluG,sfgA, flbA, flbB, flbD, laeA, brlA, abaA,vosA, stuA, and velB) by 8.53%–90.87%. Furthermore, it down-regulated the transcript levels of amylase-related genes (amyC, amyD, amyE, amyF, amyG, and amyH) and the transcription factor (amyR) by 8.87%–87.50%, the pectinase-related genes (aglB, lacA,pexB, pecA, pecC, pecB,endA, endC, and poly) by 23.23%–84.01%, the cellulase-related genes (xlnR, chbA, chbB, and eglB) by 3.75%–81.02%, and the hyperosmotic regulation-related genes (ena1, ena2,sho1, nik1, ypdl, pkA, and hAD) by 5.27%–94.36%. [Conclusion] The sakA gene of A. niger positively regulates spore production and is essential for spore production. The knockout of sakA affects the spore production of A. niger. Furthermore, SakA plays a crucial role in the synthesis and secretion of amylase, pectinase, and cellulase as well as osmotic stress response.

Abstract:[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.

Abstract:Potato scab caused by Streptomyces scabies has become a bottleneck threatening the sustainable development of the potato industry. Using biocontrol strains to combat bacterial infections is considered an ideal approach. [Objective] We screened the phosphorus-solubilizing antagonistic strains of S. scabies and investigated their effects on the prevention and control of potato scab, aiming to provide candidate strains for the development of composite functional bacterial agents. [Methods] The target strains were screened by plate confrontation and phosphorus-solubilizing tests. Their taxonomic status was determined by morphological observation, physiological and biochemical tests, and 16S rRNA gene sequencing. Pot and field experiments were carried out to measure the inhibitory effects of the target strains on S. scabies, and the relationship between phosphorus-solubilizing function and antagonistic effect was analyzed. [Results] Four antagonistic strains BN4-4, BN4-5, BN5-2, and YN17-2, were screened out. Among them, BN4-4 and BN5-2 showed strong ability to degrade inorganic phosphorus. BN4-4 and BN4-5 were identified as Bacillus atrophaeus, while BN5-2 and YN17-2 as Bacillus subtilis. The pot experiments showed that the relative control effects of the four strains were 71.35%, 38.70%, 62.18%, and 36.22%, respectively. In the field experiments, BN4-4 and BN4-5 showed the control effects of 69.07% and 56.20%, respectively. The four strains exerted inhibitory effects on four plant pathogens: Alternaria solani, Fusarium oxysporum, Rhizoctonia solani, and Verticillium dahliae. The four strains showed tolerance to pH 1.0–13.0, NaCl concentration of 1%–13%, and high temperatures (up to 80 ℃). Furthermore, they were insensitive to commonly used fungicides such as avermectin, zhongshengmycin, thiophanate-methyl, kasugamycin, and carbendazim. All the four stains exhibited the ability to metabolize and produce indole-3-acetic acid (IAA), which has been shown to promote the growth and increase the yield of plants. Notably, strains BN4-4 and BN5-2 capable of solubilizing phosphorus exhibited stronger inhibitory effects on S. scabies than the non-phosphorus-solubilizing strains. [Conclusion] B. atrophaeus BN4-4 with phosphorus-solubilizing function demonstrated promising preventive effects against S. scabies. This strain demonstrated broad-spectrum disease resistance, thermal stability, salinity tolerance, plant growth-promoting effect, and colonizing ability, serving as a potential functional strain for preventing and controlling potato scab and other soil-borne diseases in crops.

Abstract:[Objective] To mine the differentially expressed genes (DEGs) of Kocuria rhizophila DC2201 exposed to clindamycin hydrochloride at 0.5 minimum inhibitory concentration (MIC) and reveal the response mechanism of Kocuria rhizophila DC2201 to clindamycin hydrochloride. [Methods] With the Kocuria rhizophila DC2201 cells cultured in LB liquid medium as the control, Illumina HiSeq platform was used for paired-end sequencing to determine the gene expression of Kocuria rhizophila DC2201 cells exposed to clindamycin hydrochloride at 0.5 MIC. Real-time fluorescence quantitative PCR was then conducted for validation. [Results] A total of 1 202 significantly DEGs were screened out from Kocuria rhizophila DC2201 under the stress of clindamycin hydrochloride, including 604 significantly up-regulated genes and 598 significantly down-regulated genes. After gene ontology (GO) annotation, 1 041 significantly DEGs were annotated into 35 GO terms of molecular function (MF), cell composition (CC), and biological process (BP). The Kyoto encyclopedia of genes and genomes (KEGG) enrichment analysis predicted 16 significantly DEGs related to DNA repair, 43 significantly DEGs related to ribosomal synthesis, 28 DEGs associated with ATP-binding cassette (ABC) transporters, 77 significantly DEGs associated with the pentose phosphate pathway, glycolysis, tricarboxylic acid (TCA) cycle, starch and sucrose, pyruvate, butyrate and other carbohydrate metabolisms, and 5 significantly DEGs related to peptidoglycan synthesis. [Conclusion] Kocuria rhizophila DC2201 exposed to clindamycin hydrochloride adopts a global response mechanism. It increases the efflux of clindamycin hydrochloride by up-regulating the gene expression of major facilitator superfamily (MFS) transporters in the multidrug resistance (MDR) family. By enhancing DNA repair and RNA metabolism pathways, the strain ensures the genomic stability and normal RNA function. In addition, it enhances the ribosome synthesis pathway to compensate for the protein synthesis barrier caused by the binding of clindamycin hydrochloride with the 50S ribosome. Furthermore, the strain reduces the absorption and transportation of carbohydrates to restrain the energy metabolisms pathways, thus slowing down the growth and reducing the energy demand. Correspondingly, the cell wall stability of Kocuria rhizophila DC2201 is also affected.

Abstract:[Objective] Ergothioneine, a rare natural amino acid, is a powerful antioxidant with important physiological functions in the body. It has been widely used in the fields of food, medicine, and cosmetics. However, extracting ergothioneine from mushrooms and chemical synthesis suffer from low yields and high costs. This study aims to use metabolic engineering approaches to improve the yield of ergothioneine in Actinoplanes sp. HS. [Methods] Firstly, we locked onto the genes potentially involved in ergothioneine synthesis in Actinoplanes sp. HS by bioinformatics analysis. Then, we identified the functions of these genes by heterologous expression in Escherichia coli BL21(DE3). Finally, the identified functional genes were combined and overexpressed in Actinoplanes sp. HS, and the yields of ergothioneine in the mutant strains were measured. By adding different concentrations of precursors to the fermentation medium, we investigated the impact of precursor concentration on the yield of ergothioneine. [Results] The enzymes encoded by BC03-04016, BC03-04015, BC03-04014, and BC03-04013 in Actinoplanes sp. HS could synthesize the ergothioneine precursor hercynine-cysteine-sulphoxide (HER-Cys-Sul). The enzymes encoded by BC03-04046 and BC03-04917 had the function of cleaving carbon-sulfur bonds, which could catalyze the formation of the final product ergothioneine from HER-Cys-Sul. The mutant strains YC313 and YC314 obtained through overexpression of genes in ergothioneine synthesis showed ergothioneine yields of 125 mg/L and 108 mg/L, respectively, which were 2.9 times and 2.5 times that of the wild-type strain Actinoplanes sp. HS. Additional supplementation of 0.35 g/L methionine in the fermentation medium increased the ergothioneine yield of YC313 by 24% compared with that in the original medium, and the addition of 10 g/L soybean meal resulted in a 19% increase in the ergothioneine yield. [Conclusion] In this study, we identified the genes and their functions in ergothioneine synthesis in Actinoplanes sp. HS and obtained two strains with high yields of ergothioneine through metabolic engineering. Furthermore, we investigated the impact of precursor supply on the yield of ergothioneine, which provided strategic support for the production of ergothioneine by Actinoplanes sp.

Abstract:[Objective] To study the inhibitory effect and mechanism of a novel antimicrobial peptide (BCE3) isolated from Penaeus vannamei processing waste fermented with Bacillus subtilis against Bacillus cereus. [Methods] The small peptide sequences in the fermentation broth were identified by ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS), and the potential antimicrobial peptides were screened by bioinformatics. The minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), and time-kill curve of BCE3 against B. cereus were determined by the microdilution method and plate colony counting method. Then, the alkaline phosphatase release assay, propidium iodide (PI) staining, nucleic acid and protein leakage assays, and flow cytometry were employed to examine the effects of BCE3 on the cell wall and cell membrane of B. cereus. The effect of BCE3 on bacterial DNA was explored by the gel retardation assay, fluorescence spectroscopy, and molecular docking. Finally, the antimicrobial effects of BCE3 in rice were evaluated by the colony counting method. [Results] The potential antimicrobial peptide BCE3 screened out showed the MIC of 62.5 μg/mL and MBC of 125.0 μg/mL against B. cereus. The time-kill curve revealed that BCE3 reduced the bacterial count by 86.0% within 3 h (62.5 μg/mL), outperforming nisin. BCE3 caused damage to the bacterial cell wall and membrane, leading to the leakage of cell contents. Moreover, it can bind with DNA to kill the bacteria. In addition, BCE3 (125.0 μg/mL) exerted a significant inhibitory effect on the growth of B. cereus in rice. [Conclusion] BCE3 inhibits B. cereus by altering the permeability of the cell membrane and binding to DNA, thus leading to bacterial death. These findings provide a theoretical basis for application of BCE3 in the control of B. cereus.

Abstract:Bacillus paralicheniformis, a Gram-positive, facultative anaerobic, motile rod-shaped endospore-forming bacterium, can be used as a species of potential plant growth-promoting rhizobacteria (PGPR). In this study, B. paralicheniformis HMPM220325 was isolated from the fruit fermented milk. This strain can form biofilms at the gas-liquid interface during static cultivation. [Objective] To study the effects of different environmental factors on the biofilm biomass of B. paralicheniformis HMPM220325 and provide data support for the later development and application of HMPM220325 as a PGPR strain. [Methods] effects of different environmental factors and nutrients on the biofilm formation of B. paralicheniformis HMPM220325 were quantitatively detected by crystal violet staining, and the optimal conditions for the biofilm formation of the strain were optimized by orthogonal experiments. [Results] The optimal environmental conditions for the biofilm formation of B. paralicheniformis HMPM220325 were incubation at 50 ℃ and pH 9.0 for 36 h. The optimal medium was composed of maltose 15.0 g/L, urea 10.0 g/L, magnesium sulphate 20.0 mmol/L, disodium hydrogen phosphate 2.5 g/L, and bovine heart infusion 17.5 g/L. The optimized culture conditions increased the biofilm biomass by 58.28% compared with the original culture conditions. [Conclusion] This study explored the biofilm formation of B. paralicheniformis in a variety of environments and optimized the culture conditions for biofilm formation of this strain, providing an experimental basis for further development of PGPR.

Abstract:[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.

Abstract:[Objective] Poly(butylene adipate-co-butylene 2,5-furandicarboxylate) (PBAF) is a biodegradable furan-based copolyester plastic. In the chemical synthesis of PBAF, the randomness of oligomer polymerization leads to the formation of complex polymers, such as block copolymers, random copolymers, and alternating copolymers. In this study, dimethyl furan-2,5-dicarboxylate (FDME) and 1,4-butanediol (BDO) were used as substrates to synthesize bis-BDO ester by enzymatic reaction to provide the bioplastic precursor for controllable polymerization and avoid complex by-products. [Methods] RgPETase from Rhizobacter gummiphilus was heterogeneously expressed in Escherichia coli BL21(DE3). RgPETase exhibited acyltransferase activity for FDME and BDO. The reaction conditions including pH, temperature, content of BDO (as both substrate and solvent), and amount of enzyme for the synthesis of bis-BDO ester were optimized. [Results] The optimum reaction conditions of RgPETase were pH 8.0, BDO content of 30%, and reaction temperature within the range of 25–30 ℃. Under the optimum conditions (30 ℃ and enzyme concentration of 6 μmol/L), RgPETase can catalyze 10 mmol/L FDME to produce (2.96±0.01) mmol/L bis-BDO ester. [Conclusion] RgPETase exhibits high acyltransferase activity and catalyzes the generation of bis-BDO ester from FDME via acyl transfer reaction under mild conditions, which provides a green and sustainable approach for synthesizing the precursor of PBAF.

Abstract:[Objective] To study the morphological and physiological characteristics of hybrids compared with their parents and contribute to research on the mechanisms of speciation and evolution. [Methods] Sonneratia×hainanensis, a natural hybrid of the mangrove plants Sonneratia alba and S. ovata, usually presents hybrid weakness than its parents. In this study, Illumina high-throughput sequencing was employed to compare the rhizosphere microbiomes (including bacteria and fungi) between the hybrid and its parents, on the basis of which the reason for hybrid weakness was explored. [Results] The principal coordinate analysis (PCoA) revealed no significant difference in the rhizosphere bacterial or fungal community structure between the hybrid and its parents. However, the rhizosphere microbiome of the hybrid was different from that of the female parent S. alba with strong survival ability but similar to that of the male parent S. ovata. The rhizosphere bacteria belonged to 388 genera, 320 families of 76 phyla. The dominant phylum Pseudomonadota had the relative abundance above 41.00% in the rhizosphere of the three plant species, reaching 55.33% in the hybrid, which was higher than that in the parents. At the genus level, 18 common genera including Desulfococcus (3.23%) and Rhodoplanes (0.94%) in all the three mangrove plants showed the relative abundance of 15.77%. Among them, 8 salt-tolerant genera such as Mariprofundus showed decreased relative abundance in the hybrid, which may affect the salt tolerance. The rhizosphere fungi were dominated by Ascomycota and Basidiomycota with the relative abundance of 41.89% and 4.53%, respectively, which was significantly lower than that in the parents. Moreover, the predominant fungal genera were different in the three mangrove plants. Functional annotation of prokaryotic taxa (FAPROTAX) predicted that the mangrove prokaryotes were involved in sulfur metabolism and nitrogen metabolism. Although the hybrid had higher Shannon and Simpson indexes of rhizosphere bacteria than S. alba, some dominant taxa such as B-42 (unclassified Trueperaceae), Mariprofundus, and Sulfurimonas participating in the nitrogen cycle were not inherited by the hybrid. The soil total nitrogen (TN) and total phosphorus (TP) of the hybrid was significantly lower than that of S. alba. TN was significantly positively correlated with the relative abundance of Mariprofundus, B-42, Aspergillus, and Rhodotorula, which, however, demonstrated decreased relative abundance in the rhizosphere of the hybrid. [Conclusion] The results help to understand the mechanisms of hybrid weakness in Sonneratia×hainanensis.

Abstract:[Objective] To explore the effects of cell walls of Saccharomyces cerevisiae on the intestinal microbiota in finishing bulls by 16S rDNA and ITS sequencing. [Methods] A total of 40 simmental crossbred finishing bulls weighing about 550 kg were randomized into 4 groups, with 10 bulls in each group. The control group was fed with a basic diet, and 5, 10, and 15 g cell walls of S. cerevisiae were added to the diet of each bull per day in trial 1, 2, and 3 groups, respectively. The preliminary trial and trial lasted for 10 days and 94 days, respectively. Intestinal feces were collected 7 days before the end of the trial. [Results] 16S rDNA: (1) The Chao and ACE indices in the trial 3 group were higher than those in other groups (P<0.05); (2) Firmicutes and Bacteroidota were the dominant phyla, and Prevotella_9, Faecalibacterium, Succinivibrio,Bacteroides, and Bifidobacterium were the dominant genera; (3) The linear discriminant analysis effect size (LEfSe) revealed one differential species (LDA≥4.0, P<0.05) playing an important role in the trial 2 group. ITS: (1) There was no significant difference in the alpha or beta diversity among groups (P>0.05); (2) Ascomycota with the relative abundance above 50.00% was the dominant phylum. Penicillium, unidentified_Ascomycota_sp.,Aspergillus, Orpinomyces, and Eurotium were the dominant genera; (3) LEfSe revealed 8 differential species (LDA≥3.0, P<0.05), which included 3, 3, and 2 differential species playing an important role in the control, trial 2, and trial 3 groups, respectively. [Conclusion] Under conditions of this study, adding 10–15 g/d cell walls of S. cerevisiae in the basic diet increased the richness of intestinal microbiota and the relative abundance of beneficial bacteria Provetella_9, Tolypocladium, andTorulaspora, which were conducive to improve intestinal microecological environment of finishing bulls.

Abstract:[Objective] To explore the effects of compound lactic acid bacteria on the bacterial diversity and metabolites in the fermentation process of air-dried beef. [Methods] The lactic acid bacteria isolated in the early stage were inoculated into beef for fermentation with different strain combinations: C (Lactococcus garvieae TC-6:Pediococcus pentosaceus TMR-WJG, 1:1), D (L. garvieae TC-6:L. lactis TC-2, 1:1), G (P. pentosaceus TMR-WJG:L. lactis TC-2, 1:1), H (L. garvieae TC-6:P. pentosaceus TMR-WJG: L. lactis TC-2, 1:1:1), and K (without inoculation). High-throughput sequencing and non-targeted metabolomics were employed to study the bacterial community structure and metabolites, respectively, in air-dried beef inoculated with different combinations of lactic acid bacteria. [Results] A total of 304 species of bacteria belonging to 223 genera of 19 phyla were detected. Psychrobacter was predominant in the air-dried beef, with the average relative abundance of 52.21%. A total of 1 782 significantly differential metabolites were identified, including alkaloids, lipids, organic acids, and other metabolites related to flavor formation. The correlation analysis between microbiome and metabolome confirmed that the structures of bacterial communities differed significantly in the air-dried beef samples with different starters, which contributed to the differences of metabolites between groups, especially between groups C and K (P<0.05). Specifically, the inoculation lowered the levels of coumarin and corilagin and elevated the levels of camellianin A, octadecadienoate, and lippioside II. [Conclusion] The compound lactic acid bacteria selected in this study had a significant effect on the microbial community structure in air-dried beef. The quality formation of air-dried beef may be related to the microbial community structure and microbial metabolites. The compound lactic acid bacteria selected in this study demonstrated a potential application value.

Abstract:[Objective] We compared the microbial communities in the rhizosphere of plants with different genotypes and explored the relationship between microbial community and soil-borne disease occurrence, aiming to reveal the underlying mechanisms by which rhizosphere microorganisms assist plants in defending against pathogen invasion. [Methods] A pot experiment was conducted with the soil experiencing severe continuous cropping obstacles to compare the microbial communities in the rhizosphere of a susceptible watermelon variety ‘zaojia 8424’ and a resistant variety ‘xinong 8’ to Fusarium wilt. Furthermore, the relationship between microbial community and the occurrence of Fusarium wilt was explored. [Results] The resistant watermelon variety exhibited significantly lower disease index and pathogen (Fusarium oxysporum f. sp. niveum, FON) abundance than the susceptible watermelon variety. Although no significant difference was observed in the bacterial and fungal alpha diversity in the rhizosphere between resistant and susceptible varieties, the microbial beta diversity showcased significant difference between the two varieties. Moreover, both bacterial and fungal community composition was significantly correlated with pathogen abundance. Linear discriminant analysis effect size (LEfSe) further revealed that the resistant watermelon variety enriched more potential antagonistic or plant growth-promoting taxa represented by Actinobacteria and Rhizobiaceae in the rhizosphere. Interestingly, Fusarium was also enriched in the rhizosphere of the resistant variety, mainly composed of unclassified Fusarium and F. solani. Notably, the co-occurrence network of microorganisms in the rhizosphere of the resistant variety exhibited higher complexity and stability than that of the susceptible variety, with an increase of 18.18% in average degree and the nodes and edges dominated by Actinobacteria. [Conclusion] The watermelon varieties resistant and susceptible to Fusarium wilt demonstrate different microbial community composition in the rhizosphere. The enrichment of beneficial microbial taxa and interconnected co-occurrence network of the resistant variety contribute to plant defense against the pathogen invasion. This study disentangles the relationship between rhizosphere microbial community and soil-borne disease occurrence, providing important information and a theoretical basis for preventing and managing soil-borne diseases.

Abstract:[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.

Abstract:Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid notable for its involvement in the regulation of biological processes and the development of diseases. Sphingosine-1-phosphate phosphatase (S1PP) plays a role in regulating the intracellular metabolism of S1P, while the biological roles of S1PP in plant pathogenic fungi have not been reported. [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.

Abstract:[Objective] There are numerous non-rhizobia in addition to rhizobia in the root nodules of leguminous plants. Despite the extensive studies about the endophytic bacteria in other plant tissues, little attention has been paid to the species diversity of non-rhizobia in root nodules. Therefore, further research is needed to explore the significance and ecological roles of non-rhizobia. [Methods] The root nodules of wild Sophora davidii (Franch.) Skeels, a leguminous nitrogen-fixing shrub growing in the hilly-gully loess region of northern Shaanxi, were collected. The species diversity of rhizobia and non-rhizobia in the root nodules of S. davidii was systematically studied by the conventional culture method. The plant growth-promoting effects of the strains were evaluated and their growth-promoting effects on wheat seedlings were verified. [Results] A total of 320 strains of endophytic bacteria were isolated from the root nodules of S. davidii in six counties/districts in northern Shaanxi. The phylogenetic analysis based on the 16S rRNA gene sequences identified the strains into 55 genera, 35 families, 17 orders, 17 classes of 4 phyla. Pseudomonas (18.44%), Bacillus (17.81%), and Mesorhizobium (11.56%) were the dominant genera. The results of re-inoculation experiments showed that Mesorhizobium sp. and Ochrobactrum sp. HL-2 formed root nodules with the host plant. Furthermore, the plant growth-promoting characteristics of 192 strains isolated Baota District were studied. The results showed that 115, 20, 78, and 18 strains possessed the abilities of fixing nitrogen, solubilizing phosphorus, producing indole-3-acetic acid (IAA), and secreting siderophores, respectively. Four elite strains were selected and inoculated alone or in combination into the wheat seedlings in pots, and their growth-promoting effects on the agronomic traits of wheat seedlings were evaluated. The results indicated that the treatment HIJ increased the plant height and fresh weight of wheat seedlings by 49.65% and 140.00% respectively. The treatments HK and IK increased the root length and chlorophyll content by 45.84% and 25.48%, respectively. [Conclusion] There are diverse non-rhizobia in the root nodules of S. davidii. The results of this study have great scientific significance for exploring the role of such microbial resources in natural ecosystems and enriching the resource pool of endophytic bacteria. Additionally, the results provide a theoretical basis for the application of these strains in ecological restoration in the arid region of northern Shaanxi.

Abstract:β-glucosidases have been widely used in food, medicine, bioenergy and other fields, and thus it is necessary to explore new and efficient β-glucosidases. [Objective] To realize the prokaryotic expression of a GH1 glucosidase derived from Devosia psychrophila and characterize the enzymatic properties of the expressed protein. [Methods] The gene encoding the β-glucosidase derived from D. psychrophila was synthesized, named bgl59, and then transformed into Escherichia coli BL21(DE3). After the gene expression was induced, and the obtained protein was purified and characterized for the enzymatic properties. [Results] Bgl59 had a molecular weight of 48.8 kDa, with the highest activity at 55 ℃ and pH 6.0. After treatment for 1 h within the range of pH 5.0–8.5, Bgl59 maintained the relative activity over 80%. Bgl59 had the highest hydrolysis ability for 4-nitrophenyl-β-d-glucopyranoside (pNPG) among the eight substrates tested, with the K_m of 3.090 mmol/L, V_max of 194 μmol/(min·mg), and k_cat of 159 s⁻¹. The presence of 1 mmol/L of Ca²⁺ and Co²⁺ had a significant activating effect on Bgl59, while the presence of 0.1% SDS resulted in a complete loss of enzyme activity. The presence of 0.10 mol/L glucose and 0.30 mol/L xylose increased the activity of Bgl59 by 74% and 91%, respectively. Moreover, the enzyme remained the relative activity above 50% even when being cultured with 1.25 mol/L glucose or 2.00 mol/L xylose. [Conclusion] Bgl59 exhibits outstanding enzymatic properties, robust pH stability, and tolerance to metal ions and chemical reagents. It is a rare glucose-activated β-glucosidase with exceptional tolerance to glucose, holding significant potential for future industrial production and application.

Abstract:[Objective] To study the physicochemical properties and active components of extracellular antibacterial substances from Bacillus subtilis subsp. spizizenii Bspi2104. [Methods] The Oxford cup method was employed to determine the antibacterial activities and physicochemical properties of B. subtilis subsp. spizizenii Bspi2104 with broad-spectrum antibacterial activity and B. subtilis subsp. spizizenii BspiL6 without antibacterial activity. Metabolomics was employed to detect the antibacterial components of the extracellular products of the strain Bspi2104. [Results] The treatment with trypsin, papain, protease K, pepsin, and lipase had no significant effect on the antibacterial activity of extracellular products. Extracellular products lost the antibacterial activity after treatment at 80 ℃ and 100 ℃ and presented decreased antibacterial activity at pH 9.0 and pH 11.0, especially at higher pH values. There was no significant difference in the antibacterial activity between the extracellular products treated with and without UV (P>0.05). The ammonium sulfate precipitates at 60%, 70%, and 80% saturation exerted antibacterial activities, which was the strongest at the saturation of 70%. The extracellular products of the two strains were extracted by hydrochloric acid precipitation, combined with methanol extraction, ethyl acetate extraction, and chloroform extraction, and all the extracts showed antibacterial activities. The ethyl acetate extract had the strongest antibacterial activity. LC-MS/MS was employed to analyze the composition of extracellular products of Bspi2104 and BspiL6 extracted with different methods. There were 35 common differential metabolites in the extracellular products of the two strains extracted with different methods. The differential metabolites belonged to 37 categories of compounds, including carboxylic acids and derivatives, fatty acids, organic oxygen-containing compounds, organic nitrogen-containing compounds, steroids and derivatives, pregnenolone lipids, phenols, alkaloids and derivatives, glycerol phosphates, isoflavonoids, and benzene and substituted derivatives. Some of these compounds, such as kurarinone, and surfactin B, had antibacterial activities. [Conclusion] The extracellular products of B. subtilis subsp. spizizenii Bspi2104 had good physicochemical stability and maintained high antibacterial activity after treatment with various proteases and lipases, and at −20 ℃ to 60 ℃, pH 1.0–11.0, and UV irradiation for 3 h. The ammonium sulfate precipitates and the extracts from hydrochloric acid precipitation combined with methanol extraction, ethyl acetate extraction, and chloroform extraction of the extracellular products of B. subtilis subsp. spizizenii Bspi2104 had antibacterial activities. Among them, the 70% ammonium sulfate precipitate and ethyl acetate extract had the best antibacterial effects. The extracellular products of the strain contained diverse categories of antibacterial compounds. The findings provide theoretical reference for the discovery and screening of antibacterial components of Bacillus, and the related metabolites have research prospects.

Abstract:[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.

Abstract:[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.

Abstract:[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_catand 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.

Abstract:[Objective] Microplastics (particle size<5 mm) with hydrophobic surface, strong adsorption capacity, and difficult degradation can be retained in the environment for a long time and easily colonized by microorganisms, which poses a potential risk to the ecosystem. To study the distribution characteristics of microorganisms on the surface of microplastics in the wetland of Poyang Lake in wet and dry seasons. [Methods] Samples of water, sediments, and microplastics in sediments were collected from the wetland during the wet and dry seasons. The bacterial diversity and community structure in the samples were analyzed by high-throughput sequencing of the 16S rRNA gene. [Results] The richness and diversity of bacteria in the environment were higher than those on the microplastic surface in wet and dry seasons. During the wet season, the bacterial community structure was similar between the water and sediment samples and had large differences between the environment and the microplastic surface. During the dry season, the bacterial community structure was different among different samples. At the phylum level, the bacteria in the environment were dominated by Proteobacteria, Bacteroidetes, and Actinobacteria, while the bacteria on the microplastic surface during the wet season mainly included Proteobacteria, Bacteroidetes, and Firmicutes. The dominant bacterial phyla on the microplastic surface were similar to those in the environment during the dry season. The relative abundance of Pseudomonas on the microplastic surface was higher than that in the environment. Most of the keystone bacterial species in the wet and dry seasons belonged to Proteobacteria, including Sphingomonas. [Conclusion] This study reveals the differences in the bacterial community structure in the environment and on the microplastic surface in the Poyang Lake wetland in wet and dry seasons. The findings can enrich the knowledge about microplastics in lake wetlands in China and provide a theoretical basis for the management of lake environments including the Poyang Lake wetland.

Abstract:[Objective] To use microorganisms to mobilize unavailable nutrients in soil for the utilization by leguminous crops and the sustainable and long-term utilization of soil resources. [Methods] Microbial culture and micro-plot experiments were carried out with Ceriporia lacerata HG2011, a new isolate of white-rot fungus, to investigate the fungal secretion, soil nitrogen (N) and phosphorus (P) mobilization, and influences on the nutrient uptake, growth, and yields of Vigna radiata and Vicia villosa. [Results] C. lacerata released cellulase, chitinase, β-l,3-glucanase, protease, phosphatase, and siderophore, and dissolved Ca₃(PO₄)₂ in pure culture. After being inoculated on the soil surface, this fungus formed colonies, with some mycelia extending into the soil, which decrease soil pH but increase the content of NH₄⁺-N, alkali-hydrolyzed N, water-soluble P and Olsen P, and the activities of protease and phosphatase. In general, C. lacerata inoculation improved soil N and P supplies, enhanced root activity, and promoted root growth, nodule formation and development, thus increasing the nutrient uptake, grain yield of V. radiata, and biomass of V. villosa. [Conclusion] C. lacerata dwelling in soil mobilized soil N and P to increase fertilizer use efficiency and promote crop growth. C. lacerata can be cultured with sawdust, straw, husk and other organic agricultural and forestry wastes, with low production costs. This study provides a new strategy for mobilizing soil nutrients, promoting the growth of leguminous crops such as V. radiata and V. villosa, and benefiting the conservation and sustainable use of cultivated lands.

Abstract:[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.

Abstract:[Objective] To develop a rapid nucleic acid detection method for Marburg virus based on clustered regularly interspaced short palindromic repeats/associated protein 13a (CRISPR/Cas13a). [Methods] According to the conserved region of Marburg virus nucleoprotein (NP) gene, specific primers for reverse transcription recombinase-aided amplification (RT-RAA) and CRISPR RNA (crRNA) were designed and synthesized. RT-RAA was employed to amplify the target sequence. The amplification products were detected by the CRISPR-Cas13a system, and the results were interpreted by easy-readout and sensitive enhanced (ERASE) lateral flow test strips. Finally, the national reference panel was used to evaluate the sensitivity and specificity of the new method. [Results] A set of high-efficiency RT-RAA primers and crRNA targeting Marburg virus NP gene was screened, on the basis of which a CRISPR-ERASE method for the detection of Marburg virus was developed. The target nucleic acid with a concentration of 1 copy/μL could be detected within 1 h, and there was no cross-reaction with other several pathogens. [Conclusion] In this study, a rapid, simple, highly sensitive, and specific nucleic acid detection method for Marburg virus was developed based on CRISPR/Cas13a.