[Objective] Pseudoalteromonas, an endemic and dominant genus in the ocean, possesses multiple methyl-accepting chemotaxis proteins (MCPs), the functions of which remain unclear. [Methods] The chemotaxis ofPseudoalteromonas arabiensis N1230-9 isolated from surface seawater of the Pacific Ocean towards 23 carbon sources was analyzed by a plate-based assay for swimming motility. Two sCache domain-containing genes (woc28264 andwoc27036) encoding MCPs were deleted by homologous recombination. The chemotaxis of the two mutants to 10 carbon sources was then analyzed. [Results] Strain N1230-9 showed obvious chemotaxis to 10 carbon sources: trehalose, maltose, sucrose, N-acetylglucosamine, L-malic acid, sodium acetate, sodium propionate, sodium pyruvate, citric acid, and succinic acid. WOC28264 was a specific chemotactic receptor for L-malic acid and sucrose, while WOC27036 was a specific chemotactic receptor for citric acid and succinic acid. Both WOC28264 and WOC27036 were chemotactic receptors for N-acetylglucosamine and trehalose. [Conclusion] WOC28264 and WOC27036 have overlapping carbon source effectors.

[Objective] To investigate the community structure, diversity, and plant-growth-promoting functions of endophytic bacteria in the protocorms ofEpidendrum sp. and mine the core bacterial groups associated with the seed germination. [Methods] We collected five samples including three types of protocorms (germinated on MS1 medium, pine bark, and rotten oak leaves) and two types of substrates (pine bark and rotten oak leaves). High-throughput sequencing of the 16S rRNA gene was employed to compare the diversity and community composition of bacterial communities under different germination conditions. The conventional method was used to isolate the endophytic bacteria from the protocorms germinated on pine bark and rotten oak leaves (i.e., from symbiotic seed germination), and the growth-promoting potential of the isolates was evaluated. [Results] A total of 2 735 operational taxonomic units (OTUs) were obtained from the five samples, belonging to 876 genera, 453 families of 41 phyla, among whichProteobacteria andActinobacteria were the dominant phyla. The results of principal coordinates analysis showed that there were differences in the bacterial community structure between the protocorms ofEpidendrum sp. and the substrates, and the community structure of endophytic bacteria in the protocorms germinated on the MS1 medium was closest to that on pine bark. Functional prediction indicated that the endophytic bacteria in the protocorms germinated on rotten oak leaves had higher nitrogen-fixing ability than that in other types of protocorms. Nineteen isolates were obtained from protocorms geminated on pine bark and rotten oak leaves, belonging to 16 species of 12 genera.Tumebacillus flagellatus,Bradyrhizobium cenepequi, andWeizmannia ginsengihumi were the common species in the protocorms germinated on pine bark and rotten oak leaves.Pseudomonas koreensis andW.ginsengihumi had the potential of solubilizing phosphorus and producing indole-3-acetic acid (IAA) and siderophores. [Conclusion] TheEpidendrum sp. protocorms germinated in different environments harbor rich endophytic bacteria. The endophytic bacteria isolated from the protocorms from symbiotic seed germination had plant growth-promoting functions, such as fixing nitrogen, solubilizing phosphorus, and producing IAA and siderophores. This study provides a scientific basis for mining the microbial resources related to seed germination of orchids and studying the interactions between orchids and microorganisms.

[Background] Bacteria ofEnterobacteriaceae are the main pathogens of foodborne and clinical infections, posing a threat to human and animal health. The numerical identification method using API 20E as the "gold standard" is one of the main identification methods forEnterobacteriaceae. However, the existing numerical identification methods have problems such as laborious sample addition, low accuracy, and high prices. [Objective] To develop a semi-automatic, high-precision, and low-cost biochemical kit for identifying the bacteria ofEnterobacteriaceae based on the numerical identification method. [Methods] On the basis of the theoretical model and supporting software of the numerical identification system forEnterobacteriaceae established by our team, we designed and optimized 24 biochemical matrix trace formulations. Furthermore, a semi-automatic freeze-drying identification strip was developed. Using the commercialized numerical identification strip API 20E, MALDI-TOF MS, and 16S rRNA gene sequencing as the controls, we evaluated the performance of the developed biochemical kit that integrated semi-automatic biochemical identification strips and online analysis software. [Results] The biochemical spectra of 458 strains ofEnterobacteriaceae were obtained, with the overall identification accuracy of 98.5% at the genus level and 96.5% at the species level. The kit only needed twice sample addition to obtain the identification results, with the price only 4.46% that of API 20E. The shelf life of the product was 7 days, and the biochemical experiments had repeatability. [Conclusion] We develop a semi-automatic biochemical kit for identification ofEnterobacteriaceae based on the numerical identification method, which has simple operation, a low cost, and high accuracy. This study provides technical support for the identification ofEnterobacteriaceae and the development of biochemical products for numerical identification of other families and genera of bacteria.

Isoquercetin is a flavonoid with antioxidant, anti-inflammatory, and immunomodulatory activities. However, the low content in plants poses a challenge to the large-scale production of isoquercetin by the extraction method.[Objective] α-L-rhamnosidase can specifically hydrolyze the terminal L-rhamnose residues of natural glycosides. In this study, we screened the strains capable of efficiently and specifically transforming rutin to produce isoquercetin with rutin as the sole carbon source and applied the α-L-rhamnosidase to the production of isoquercetin, aiming to provide new elements for the large-scale production of isoquercetin. [Methods] The selective culture medium with rutin as the sole carbon source was used to screen and identify the strains that can specifically hydrolyze rutin into isoquercetin. The transcriptome analysis was carried out to obtain highly efficient and specific α-L-rhamnosidase, the domain composition of which was determined by structural simulation. The enzymatic properties and substrate specificity of the α-L-rhamnosidase were studied. Furthermore, the hydrolysis effect of the enzyme heterologously expressed in Pichia pastoris in a 5 L fermenter was determined. [Results] AfRhase had five domains, including one α-domain (domain A) and four β-domains (domains N, E, F, and C). With rutin as the substrate, the recombinant enzyme AfRhase showcased the best performance at 55 ℃ and pH 4.5. AfRhase had a wide range of substrates including rutin, hesperidin, naringin, and epimedin C. In a 5 L fermenter for scaled-up production of isoquercetin, P. pastoris expressing AfRhase generated 61 g isoquercetin by hydrolyzing 120 g crude rutin (purity of 70%), with the molar conversion rate of 95.4% and production efficiency of 2.0 mmol/(L·h). [Conclusion] This study for the first time discovered a highly efficient and specific α-L-rhamnosidase from Aspergillus sp. XT-1 for the production of isoquercetin from rutin and heterologously expressed this enzyme in P. pastoris. The domain composition, enzymatic properties, substrate specificity, and hydrolysis efficiency in a 5 L fermenter of this enzyme were determined. In conclusion, this study broadened the function of a fungus-derived α-L-rhamnosidase for the transformation of rutin and laid a foundation for the industrial production of isoquercetin.

[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.

[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.

[Objective] To investigate the fungal diversity and community dynamics in the roots and root zone soil of the invasive species Spathoglottis plicata at different development stages and mine potential fungal resources. [Methods] The developmental period of S. plicata was divided into the protocorm, pre-seedling, middle-seedling, late-seedling, adult, and flowering stages. Fungal community composition and diversity in the roots and root zone soil were analyzed by QIIME 2 and other software. Fungal strains were isolated by the tissue culture method from the protocorms and seedlings of S. plicata, and the phylogenetic relationship of the strains was analyzed. [Results] The dominant fungi in the roots and root zone soil of S. plicata were Tulasnellaceae and Onygenaceae, respectively. The fungal community composition of S. plicata varied across different developmental stages. Tulasnellaceae was the most prevalent from the protocorm to middle-seedling stage, while Nectriaceae and Trichocomaceae became dominant during the late-seedling stage and adult stage. At the flowering stage, Ceratobasidiaceae presented high relative abundance. A total of 101 fungal strains were isolated from the protocorms and seedling roots of S. plicata, belonging to 13 families, in which strains of Tulasnellaceae accounted for 35.65%, being dominant. [Conclusion] The present study elucidated the fungal community composition and diversity dynamics in the roots and root zone soil of S. plicata at different development stages. The findings offer a theoretical foundation for comprehensively understanding of the intricate relationship between orchids and symbiotic fungi and facilitating the selection of optimal habitats for the conservation of rare and endangered orchid species.

[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.

[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.

Monocarboxylates such as lactate, pyruvate, and ketone bodies play an important role in the metabolic activities of organisms. As a monocarboxylate transporter, MpMch2 is mainly responsible for the transmembrane transport of monocarboxylates and the maintenance of glucose metabolism balance.[Objective] To analyze the functions of MpMch2 in Monascus purpureus. [Methods] The MpMch2 in M. purpureus Mp-21 was replaced with the hygromycin gene by homologous recombination to construct the deletion strain ∆MpMch2. The colony and cell morphology of Mp-21 and ∆MpMch2 on different media was observed, and the yield of monascus pigment, γ-aminobutyric acid, conidia and ascospores were determined. The expression levels of genes related to conidia and γ-aminobutyric acid were determined by RT-qPCR. [Results] There was no significant difference in the colony morphology between the wild type and ∆MpMch2 on different media. After knockout of MpMch2, the yields of conidia, ascospores, Monascus pigments, and γ-aminobutyric acid decreased, and the expression levels of related genes were down-regulated. [Conclusion] MpMch2 positively regulated the development of conidia and ascospores and the production of Monascus pigments and γ-aminobutyric acid.