Abstract:[Background] Xylanase, the core enzyme responsible for the degradation of xylan, can promote the digestion and hydrolysis of lignocellulose. Therefore, it has been widely used in animal breeding fields. The GH10 xylanase CbXyn10C derived from Caldicellulosiruptor bescii has good thermal stability at 80 ℃ with an optimum temperature of 85 ℃. With this property, it demonstrates a promising prospect for application in the feed industry. [Objective] To improve the thermal stability of CbXyn10C for meeting the technological requirements of feed granulation, especially aquatic feed processing, and decipher its heat resistance mechanism. [Methods] Based on the crystal structure of CbXyn10C, mutations were designed by introducing rigid amino acids and rearranging the hydrophobic network. After a single-point mutant with increased specific activity at 100 ℃ was obtained, the thermal stability of the enzyme was further improved by the stacking of beneficial mutation sites. Molecular dynamics (MD) simulation was employed to decipher the mechanism of thermal stability improvement. [Results] Four single-point mutants A45P, T69P, F309V, and A325P with improved stability were obtained, among which the mutant A45P showed the greatest stability improvement. The stacking of other three mutation sites on A45P gradually improved the thermal stability of the enzyme without compromising the enzyme activity. The obtained four-point mutant A45P/F309V/A325P/T69P showed the best heat tolerance, and its optimum temperature and melting temperature T_m were increased by 5 ℃ and 6.8 ℃, respectively, compared with those of the wild type. MD simulation showed that mutations at the four sites introduced new hydrogen bond forces and optimized the hydrophobic network, resulting in a more stable structure and conformation. [Conclusion] This study promotes the application of xylanase in the feed industry and provides theoretical support for molecular modification for improving enzyme stability based on its structure.

Abstract:[Background] CrgA is a key negative regulator of carotenoid synthesis in Blakeslea trispora (Bt). [Objective] To clone and determine the activity of the crgA promoter from B. trispora, laying a foundation for deciphering the regulatory mechanism of CrgA expression. [Methods] According to the genome sequence in the integrated microbial genomes (IMG), we cloned a sequence of 2 000 bp upstream of the translation initiation site of crgA, identified the cis-regulatory elements, predicted the transcription initiation region, and then Analysis of crgA transcription level of B. trispora under different light time by RT-qPCR. Furthermore, we constructed four recombinant expression vectors p1303-procrgAF, F1, F2, and F3 driven by truncated sequences of crgA promoters with different lengths, and integrated them into the genome of B. trispora via Agrobacterium tumefaciens. Under dark and light conditions, the fluorescence signals were observed and the β-d-glucuronidase (GUS) activity was determined. [Results] The crgA promoter contained not only the basic elements TATA-box and CAAT-box but also multiple elements related to light response. Both CaMV35S and the constructed four mutant promoters could drive the expression of downstream genes in B. trispora. The GUS activity indicated that procrgAF3 had the strongest activity, and the truncated promoter had stronger activity than CaMV35S. The btcrgA promoter sequence has a light-responsive element at 1 499−989 bp that activates downstream gene expression. [Conclusion] The transcription of crgA in B. trispora presented the phenomena of light activation and light adaptation. The core region of the crgA promoter was identified as procrgAF3, and the cis-acting elements on the btcrgA promoter that responded to light regulation were located in the region of 1 499−989 bp.

Abstract:[Background] Zabuye Lake located in the Qinghai-Xizang Plateau in China is characterized by high concentrations of CO₃²⁻, HCO₃⁻, and Na⁺. Due to the alpine climate, high altitude, and the location off the beaten track, few studies have been conducted on the microorganisms of Zabuye Lake. [Objective] To systematically explore the bacterial diversity and mine valuable strains in Zabuye Lake. [Methods] Illumina sequencing of the 16S rRNA gene V3-V4 region was carried out to analyze the bacterial community structure and diversity in Zabuye Lake. The pure culture method was used to isolate the culturable bacterial strains, and the taxonomic status, growth characteristics, and alkali-degrading capacity of the isolates were determined. Furthermore, the accumulation of ectoine, indole acetic acid (IAA), and extracellular polymeric substances (EPS) was determined for the isolates. [Results] Illumina sequencing yielded 583 genera of bacteria belonging to 86 orders, 44 classes, and 21 phyla. The dominant phyla were Proteobacteria (25.11%-67.60%), Bacteroidetes (4.84%-35.02%), and Firmicutes (1.24%-11.01%), and the dominant genera were Klebsiella (0.01%-9.53%), Halomonas (0.54%-8.75%), Gemmatimonas (2.39%-6.00%), and Nitriliruptor (1.27%-6.26%). A total of 38 strains of haloalkaliphilic bacteria were isolated, including 23 (60.53%) strains of Bacillus and 10 (26.32%) strains of Halomonas. All the strains showed salinity tolerance and alkali-degrading capacity (19.80%-29.68%). Most strains were able to accumulate ectoine, IAA, and EPS with the yields of 1.36-175.59 mg/L, 0.27-8.69 mg/L, and 0.02-0.22 g/g, respectively. [Conclusion] The bacterial community structure of Zabuye Lake is similar to that of other saline lakes, while there are a large number of undefined bacteria taxa. Most of the strains isolated from Zabuye Lake have salinity tolerance and alkali-degrading capacity. In addition, 4, 3, and 3 potential strains for the efficient production of ectoine, IAA, and EPS, respectively, were identified. This study lays a foundation for the development and utilization of microbial resources and saline environment improvement in Zabuye Lake.

Abstract:[Background] Coal gangue dumps stockpile coal wastes with low calorific values and heavy metals, which are generated during coal mining, along with a large amount of acidic wastewater and deteriorated eco-environment nearby. [Objective] This paper aims to explore the structure and functional characteristics of microbial communities in coal gangue dumps. [Methods] The soil samples of the dump surface and gangue layers, as well as the sediment samples from wastewater leaching outlets and dump downstream river, of a typical gangue dump in Liuzhi special district, Guizhou Province were collected. Metagenomics was employed to reveal the microbial community structures and functional characteristics of the samples. [Results] The results showed that bacteria were more diverse and abundant than archaea. The dominant bacterial phyla were Proteobacteria and Actinobacteria, and the dominant genera were Leptospirillum and Sulfobacillus. The dominant archaeal phyla were Candidatus_ Thermoplasmatota and Crenarchaeota, and the dominant genera were Thermoplasma and Metallosphaera. The dominant genera of bacteria and archaea in different sampling sites varied from each other, and Fe-oxidizing bacteria (FOB) and sulfur-oxidizing bacteria (SOB) were more abundant in the gangue layer soil and wastewater leaching outlet sediment than in the other two sampling sites. The genes for carbon, nitrogen, and sulfur metabolism were abundant in coal gangue, with six carbon fixation pathways, six nitrogen metabolism pathways, and three sulfur metabolism pathways detected. The dominant genes for carbon fixation were ACAT and E2.2.1.1, and the dominant pathway was reductive tricarboxylate cycle. For nitrogen metabolism, the dominant genes were nirB, nasA and narG, and the dominant pathway was denitrification. For sulphur metabolism, the dominant genes were cysH and sir, and the dominant pathway was assimilated sulphate reduction. [Conclusion] The findings are expected to enhance the understanding of mine ecology and provide a theoretical basis for the ecological restoration and pollution treatment in mining areas.

Abstract:[Background] Candidate phyla radiation (CPR) and DPANN are unique groups that are significantly different from most known bacteria and archaea, and the knowledge about them is limited due to the late discovery. The two groups are known to be large and widespread, while their diversity and ecological roles in different habitats remain to be studied. [Objective] To analyze the diversity of CPR and DPANN in different groundwater samples and the influences of different methods on the detection and enrichment of CPR and DPANN. [Methods] Metagenomic sequencing was employed to determine the diversity of CPR and DPANN in four different groundwater samples around Hohhot after filtration through a 0.1 μm membrane. Metagenomic sequencing was compared with 16S rRNA gene amplicon sequencing regarding the detection of CPR and DPANN, and the effects of different filtration methods and membrane combinations on the enrichment of CPR and DPANN were compared. [Results] From the 4 groundwater samples, 33-64 phyla of CPR with the relative abundance of 0.17%-1.67% and 1-7 phyla of DPANN with the relative abundance of 0.000 93%-0.071% were detected. For CPR and DPANN, only Nanoarchaeota was detected by 16S rRNA gene V3-V4 amplicon sequencing. The combination of 1.2μm and 0.1 μm filters showed the best enrichment effect on CPR and DPANN, and the relative abundance increased to 13.33% and 0.58%, respectively, by timely replacement of filters. [Conclusion] There were abundant resources of CPR and DPANN with low relative abundance in groundwater, and the distribution of CPR and DPANN varied in different groundwater samples. The 16S rRNA gene V3-V4 amplicon sequencing missed the information of CPR and DPANN in groundwater. The enrichment effect on CPR and DPANN can be significantly improved by the combination of filters with different pore sizes and the timely replacement of filters. The findings underpin the further exploration of species, gene, and natural product resources and the strain culture of CPR and DPANN.

Abstract:[Background] Hermetia illucens (HI) used for the treatment of organic wastes such as kitchen waste is rich in amino acids and nutrients, serving as a high-quality and economical source of protein. [Objective] To prepare peptone with H. illucens larvae (HIL) for bacterial culture and provide a new idea for the application of HI. [Methods] Peptone was prepared from HIL by enzymolysis. The optimal enzymolysis conditions were determined by single factor tests, and the biochemical and functional properties of HIL-derived peptone and commercial tryptone were compared. Escherichia coli ATCC25922 was cultured with two peptone products and the growth kinetics was compared. [Results] The optimal conditions for preparing HIL-derived peptone were enzymolysis with the enzyme complex (trypsin:alkaline protease=1:1) added at 1.3% (mass fraction ) and at pH 7.0 and 54 ℃ for 4 h, under which the hydrolysis degree of defatted HIL was (19.34±0.15)%. The functional and biochemical properties showed no significant differences between HIL-derived peptone and commercial tryptone (P>0.05). The X_max,and λ of E. coli ATCC 25922 grown in the medium supplemented with HIL-derived peptone were 6.44 and 2.45, respectively, and those of E. coli ATCC 25922 grown in the medium supplemented with commercial tryptone were 6.14 and 3.19, respectively. The X_max,and λ showed no significant differences between HIL-derived peptone and commercial tryptone (p>0.05).[Conclusion] HIL-derived peptone can replace commercial tryptone as a component of the medium for culturing E. coli.

Abstract:[Background] Colletotrichum siamense was the main pathogen of rubber anthracnose, which seriously restricted the yield of natural rubber. Homeobox transcription factors were widely found in plant-pathogenic fungi and involved in asexual reproduction, infection, and metabolism of fungi. [Objective] To determine the biological function of a homeobox transcription factor CsHtf1 identified in Colletotrichum siamense. [Methods] The gene-knockout mutant of Cshtf1 was obtained by homologous recombination, and its phenotypes such as vegetative growth, conidial production, and pathogenicity were analyzed. [Results] The Cshtf1 gene encoded 600 amino acids and contained a HOX domain. Compared with the wild type, the Cshtf1 knockout mutant showed no significant difference in vegetative growth and pathogenicity, whereas the conidial production of the mutant was significantly reduced, and the production of melanin increased. [Conclusion] CsHtf1 was involved in the regulation of conidiation and melanin production in Colletotrichum siamense.

Abstract:[Background] Processing tomatoes are one of the major crops for the “red” planting industry in Xinjiang. Due to the excessive application of chemical fertilizers in processing tomato production, bio-fertilizer has attracted increasing attention. [Objective] To explore the effect of a microbial mixture on the growth of processing tomatoes, stimulate the changes of functional microbial populations in the rhizosphere soil, and provide a theoretical and practical basis for the development and utilization of microbial agents in the future. [Methods] We applied the microbial mixture to the rhizosphere and then determined the agronomic traits of processing tomatoes during the growth period, so as to explore the effects of the microbial mixture on the growth of processing tomatoes. The 16S rRNA gene amplicon analysis was employed to explore the effects of the microbial mixture on the microbial community composition and structure in the rhizosphere soil. [Results] After the treatment with the microbial mixture, the fruit weight, fruit number per plant, and fruit sugar content increased, which improved the quality of fruits. Furthermore, the treatment altered the microbial population and community composition and structure. Specifically, the relative abundance of Actinobacteria, Acidobacteria, and Bacteroidetes increased. The correlation analysis indicated that the microbial mixture affected the growth of processing tomatoes. [Conclusion] The application of the water-dispersible microbial mixture composed of Bacillus tequilensis C-9 and Sphingomonas A1 in the rhizosphere of processing tomatoes affected the functional microbial community structure (increasing the relative abundance of some functional microorganisms) in the rhizosphere of processing tomatoes. It promoted the interaction between rhizosphere microorganisms and plants, thus improving the quality of processing tomato fruits.

Abstract:[Background] Strain LYEC03 is a major fermentation strain isolated from Fuzhuan tea in Jingyang county, Shaanxi province, China. [Objective] To reveal the genomic information of strain LYEC03 and the fermentation characteristics of the strain for loquat flower products, and clarify the fermentation mechanism of Eurotium cristatum at the molecular level. [Methods] Morphological and microscopic observation, ITS sequence identification, next-generation sequencing, and whole genome sequencing were employed to identify and analyze the genomic information of strain LYEC03. Furthermore, the strain was used to ferment loquat flowers and the effects of the strain on the main functional components and antioxidant properties of loquat flowers were studied.[Results] strain LYEC03 isolated from Fuzhuan tea in Jingyang, Shaanxi was identified as Eurotium cristatum. The genome of strain LYEC03 had a high coverage of the reference genome and contained abundant genes related to cellulase, protease, oxidase, and lipase. The genes associated with bacterial growth, energy metabolism regulation, fiber production, protease production, and oxidase production had undergone mutations, including hypothetical protein, carbohydrate kinase, cellulase family glycosylhydrolase, site-2 protease family protein, polyphenol oxidase, and glutathione peroxidase. The genome sequence of strain LYEC03 had a length of 30 623 602 bp and the GC content of 57.70%, encoding 13 033 genes, among which 55.74% were coding genes and involved in the carbohydrate metabolism, amino acid metabolism, xenobiotics biodegradation and metabolism, energy metabolism, lipid metabolism, and metabolism of terpenoids and polyketides. The growth of strain LYEC03 and the content of its main functional components presented consistent trends (first increasing and then slowly decreasing) with the three antioxidant indicators: total antioxidant activity, DPPH free radical scavenging rate, and hydroxyl free radical scavenging rate. [Conclusion] The fermentation characteristics of E. cristatum were related to the rich cellulase, protease, and oxidase genes in its genome. Probing into the genomic information and fermentation characteristics of E. cristatum will provide a basis for improving the fermentation performance and applying the strain in food, light industry, medicine and other fields in the future.

Abstract:[Background] A preliminary study showed that vitamin C (VC) had a significant activation effect on the β-glucosidase (BGL) from Lactobacillus acidophilus GIM1.208. [Objective] To explore the effect of VC on BGL and the interaction between VC and BGL. [Methods] The 4-nitrophenol method, electrochemistry, Zeta potential, and nuclear magnetic resonance were employed to study the effect of VC on BGL activity and the interaction characteristics. [Results] VC exerted a significant effect on the catalytic activity of BGL, with K_m=1.167 mmol/L, and the V_max increased with the increase in VC concentration. BGL presented the highest activity when the VC concentration was 3.5%. At the VC concentration of 3.5% and a temperature below 30 ℃, the BGL activity was barely affected by temperature, and VC had a good stabilizing effect on BGL, which showed the relative activity above 90%. VC can bind to BGL, and the weak electrostatic interaction between VC and BGL facilitated the electroxidation of VC. We hypothesized that there was a hydrogen bond between the ethylene glycol branch chain in VC molecule and BGL. [Conclusion] VC can activate BGL. We studied the type and site of interaction between VC and BGL, aiming to provide a theoretical reference for the application of BGL and the efficient utilization of natural enzyme activators.

Abstract:[Background] Beak atrophy and dwarfism syndrome (BADS) caused by novel duck parvovirus (NDPV) infection leads to stunted growth and atrophy of the upper and lower beaks of ducklings. The outbreak of BADS has caused huge economic losses to the duck industry in China. [Objective] To construct NDPV virus-like particles (VLPs) in Escherichia coli, laying the foundation for the development of NDPV-related vaccines. [Methods] The full-length NDPV VP2 sequence was codon-optimized, synthesized, and then ligated into the pColdTF vector to obtain the pColdTF-NDPV-VP2 recombinant plasmid. After being identified by enzyme digestion and sequencing, the recombinant plasmid was transformed into E. coli BL21(DE3) for expression. SDS-PAGE was employed to analyze the solubility of the recombinant protein. The TF tag was removed by thrombin protease, and then recombinant protein was purified by Ni-NTA affinity chromatography. Western blotting was employed to examine the reactogenicity of purified VP2 protein and dynamic light scattering and transmission electron microscopy to observe the morphology of the recombinant protein and the formation of VLPs. [Results] The recombinant plasmid pColdTF-NDPV-VP2 was constructed successfully and expressed in E. coli mainly in the soluble form. The TF-VP2 fusion protein had a molecular weight of 115 kDa, and the molecular weight was 67 kDa after removal of the TF tag. Western blotting showed that the VP2 protein specifically bound to NDPV-positive duck serum. The VP2 VLPs with diameters of 20 nm to 25 nm could be observed by transmission electron microscopy. [Conclusion] We used the E. coli expression system to design NDPV VLPs, which can provide a basis for the development of subunit vaccines and biological-related products against BADS.

Abstract:[Background] Streptococcus suis serotype 2 (S. suis 2) causes severe meningitis in the host, posing a major threat to the pig industry and human public health. [Objective] The mouse model of meningitis induced by S. suis 2 infection was established, and its brain tissue was analyzed by transcriptomics to provide a theoretical basis for revealing the molecular mechanism of meningitis caused by S. suis 2 infection in mouse and discovering potential therapeutic targets.[Methods] S. suis 2 was used to infect mouse, and the histopathological changes in the mouse brain tissue were observed to verify the model of meningitis in mouse was successfully established. Furthermore, transcriptomic analysis was performed to identify the differentially expressed genes (DEGs) between the S. suis 2-infected and uninfected mouse. Moreover, gene ontology (GO) functional annotation, Kyoto encyclopedia of genes and genomes (KEGG) pathway enrichment analysis, and Venn analysis were performed for the DEGs. [Results] S. suis 2 successfully infected mouse and caused meningitis. The brain of the infected mouse showed infiltration of massive inflammatory cells and perivascular cuffing around the blood vessels. Moreover, the S. suis 2 strain was isolated from the tissues of the modeled mouse. The results proved that the mouse model of meningitis caused by S. suis 2 infection was successfully established. A total of 397 DEGs were identified, including 109 genes with down-regulated expression and 288 genes with up-regulated expression. GO functional annotation showed that the DEGs were mainly enriched in the cytoplasm, plasma membrane, signal transduction, regulation of transcription, apoptotic process, immune system process, response to bacteria, and protein binding. KEGG pathway enrichment analysis indicated that the DEGs were mainly enriched in multiple important signaling pathways regulating host cytokine-cytokine receptor interaction, immune response, apoptosis, phagocytosis, and metabolism. The Venn analysis showed that the brain-derived neurotrophic factor (bdnf) gene converged in meningitis-related genes set, deafness-related genes set, and DEGs set of S. suis 2 infection, with significantly down-regulated expression. The c4b, fas, icam1, cxcl1, csf3, lrg1, and nde1 genes converged in the meningitis-related genes set and the DEGs set of S. suis 2 infection, with significantly up-regulated expression. [Conclusion] A mouse model of meningitis induced by S. suis 2 infection was successfully established. The transcriptomics of the brain tissue of the model mouse revealed several key DEGs and signaling pathways, and the molecular signaling pathway network was built. The findings provide a new theoretical basis for revealing the molecular mechanism of S. suis 2 induced-meningitis and exploring potential therapeutic targets.

Abstract:[Background] Foot-and-mouth disease (FMD) caused by foot-and-mouth disease virus (FMDV) is one of the major diseases that infect cloven-hoofed animals such as cattle, sheep, and pigs. The structural protein VP1 of FMDV contains multiple sites that can cause host immune response, serving as a target for the research and development of subunit vaccines. As a safe production strain, Corynebacterium glutamicum is an elite cell factory for the production of pharmaceutical proteins. [Objective] To realize the heterologous expression of VP1 in C. glutamicum. [Methods] According to the gene sequence and function of VP1 and the codon preference of C. glutamicum, we designed and synthesized the VP1 gene and then ligated it with pXMJ19 to create the recombinant plasmid pXMJ19-VP1. C. glutamicum CGMCC 1.15647 was used to express VP1 protein. The protein expression elements [e.g., the promoter, 5′ untranslated region (5′UTR), and N-terminus] of VP1 protein and the culture conditions were optimized. SDS-PAGE and Western blotting were employed to determine the expression of VP1. Indirect enzyme-linked immunosorbent assay (ELISA) was employed to measure the immunocompetence of VP1 produced in this study. [Results] SDS-PAGE and Western blotting showed that VP1 was successfully expressed in C. glutamicum CGMCC 1.15647. Replacing P_tac promoter with the synthetic promoter P_H36 increased the protein yield. On this basis, the protein yield can be further increased by insertion of the 5'UTR sequence and mutation of the N-terminal amino acid of VP1. CspB signal peptide can be used to achieve secretory expression. Fermentation experiments showed that the optimum conditions of shake flask fermentation were 30 ℃ and 24 h. The results of ELISA suggested that VP1 could specifically bind to the positive serum. [Conclusion] The VP1 protein of FMDV was successfully expressed in C. glutamicum CGMCC 1.15647, and optimizing protein expression elements increased the protein yield, which laid a foundation for the development of immunodiagnostic reagents and safe and efficient subunit vaccines for FMD.

Abstract:[Background] Porcine deltacoronavirus (PDCoV), swine acute diarrhea syndrome coronavirus (SADS-CoV), and senecavirus A (SVA) are emerging pathogens which seriously endanger the development of the pig industry. The clinical symptoms of pigs infected with the three pathogens are difficult to be distinguished. Therefore, it is urgent to establish a multiplex reverse transcription-polymerase chain reaction (RT-PCR) method for the rapid diagnosis of suspected pigs and reduce economic losses. [Objective] To establish a triplex RT-PCR method for simultaneous detection of single or mixed infection of PDCoV, SADS-CoV, and SVA. [Methods] Three pairs of specific primers were designed according to the conserved regions of the N genes of PDCoV and SADS-CoV and the L/P1 genes of SVA registered in GenBank, and the optimal annealing temperature (T_m) was determined by the temperature gradient PCR method. The primer concentration was optimized by the array method. The recombinant plasmids PMD-PDCoV, PMD-SADS-CoV, and PMD-SVA were constructed as standards to determine the limits of detection (LODs). The specificity of the triplex RT-PCR method was determined with the nucleic acid samples of six common pig viruses including porcine transmissible gastroenteritis virus, porcine epidemic diarrhea virus, and porcine reproductive and respiratory syndrome virus. The repeatability of the established method was verified by inter-batch and intra-batch tests. Finally, we employed the triplex PCR method to detect the clinical samples and compared the results with those obtained with the reported detection methods, thus evaluating the clinical application performance of this method. [Results] The optimal T_m was 58.3 ℃, and the optimal primer concentrations were 0.50, 0.25, and 0.25 μmol/L, respectively. The established method had high sensitivity, with the LODs of 1, 1, and 10 copies/μL for PMD-PDCoV, PMD-SADS-CoV, and PMD-SVA, respectively. It had strong specificity, with specific bands only for PDCoV, SADS-CoV, and SVA and no bands for other viruses. Moreover, the method had good repeatability as the test results were consistent between and within batches. Finally, the positive rates of PDCoV, SADS-CoV, and SVA in the clinical samples detected by the established method were 4.4%, 0%, and 0.73%, respectively, which were consistent with the results obtained with the reported detection methods. [Conclusion] The triplex RT-PCR method established in this study is accurate and reliable for the simultaneous detection of PDCoV, SADS-CoV, and SVA in clinical samples.

Abstract:[Background] Hematopoietic necrosis caused by cyprinid herpesvirus-2 (CyHV-2) has led to serious economic losses in aquaculture due to its spread in crucian carp, goldfish and other species. [Objective] To develop an on-site colloidal gold test strip for detecting and monitoring the level of IgM antibodies against CyHV-2 in crucian carp. [Methods] CyHV-2 was combined with colloidal gold as the gold-labeled antigen. Protein A was employed as the detection line, and rabbit-derived rORF66 polyclonal antibody as the quality control line. The optimal preparation and assembly conditions of colloidal gold test strips were analyzed, and the optimal pH and concentration of colloidal gold-labeled antigen, as well as the optimal marking concentrations of test line (T line) and quality control line (C line), were determined. [Results] The colloidal gold test strips prepared in this study did not cross-react with other common fish antibody-positive sera, such as grass carp reovirus antibody-positive serum, mandarin fish iridovirus antibody-positive serum, largemouth black bass iridovirus antibody-positive serum, and carp spring viremia virus antibody-positive serum. The test strips can detect serum antibodies at a minimum dilution of 1:100, and the density of the detection line can help roughly judge the antibody level. The test strips were used to detect 10 crucian carp serum samples and compared with the detection method of goldfish haematopoietic necrosis virus (GB/T 36194—2018). The Kappa value was 0.80, indicating a high coincidence rate between the two methods. [Conclusion] The test strips prepared in this study have high sensitivity, high specificity, simple operation, and low cost, demonstrating the potential for fish quarantine and evaluation of fish antibody levels against CyHV-2.

Abstract:[Background] Vibrio parahaemolyticus is one of the pathogens causing acute hepatopancreatic necrosis disease (AHPND) in cultured shrimp. PirAB, encoded by pirA and pirB located on the plasmid pVA, acts as the virulence factor of AHPND. However, the secretion pathway of PirABremains unclear. [Objective] To explore the effect of two T2SS homologous genes epsG2 and epsE2 of pVA on biological characteristics and PirAB expression and secretion of Vibrio parahaemolyticus. [Methods] The bioinformatics analysis revealed two type II secretion system (T2SS) homologous genes epsG2 and epsE2 on the pVA plasmid in V. parahaemolyticus VP220218. The mutants ΔepsG2 and ΔepsE2 were constructed by in-frame deletion. The growth, biofilm formation, swimming ability, and extracellular protease activity were compared between VP220218 and mutants. RT-qPCR and Western blotting were performed to compare the expression and secretion of PirAB between VP220218 and mutants.[Results] epsG2 and epsE2 suppressed the extracellular protease activity (P<0.001) and the growth during the logarithmic stage (P<0.05). In addition, epsG2 promoted the swimming (P<0.05) and inhibited the biofilm formation of VP220218 at the plateau stage (P<0.05); epsE2 restrained the swimming ability and promoted the biofilm formation of VP220218 at the middle and late logarithmic stage (P<0.05). The results of RT-qPCR showed that epsE2 had no effect on the expression of pirA or pirB; epsG2 inhibited the expression of pirA and pirB at the early and middle logarithmic stage, while it promoted the expression of pirA and pirBat the late logarithmic stage and plateau stage. The results of Western blotting showed that epsE2 promoted the synthesis and secretion of PirAB while epsG2 had no significant effect on the synthesis or secretion of PirAB. [Conclusion] The results indicated that epsG2 and epsE2 were involved in the physiological activities of V. parahaemolyticus VP220218. Moreover, epsE2 affected the synthesis and secretion of PirAB. The findings provided a reference for understanding the synthetic and secretory pathways of PirAB in AHPND-causing bacteria.

Abstract:[Background] Cronobacter sakazakii is an emerging food-borne pathogen. The powdered infant formula (PIF) contaminated by this pathogen may cause necrotizing enterocolitis and meningitis in infants, threatening the life of premature or immunocompromised infants. Bacteriophages are well recognized for their antibacterial properties and may be novel biocontrol agents for C.sakazakii and other food-borne pathogenic bacteria. [Objective] To characterize a novel C. sakazakii bacteriophage isolated from sewage and evaluate its antibacterial effects in PIF. [Methods] The double-layer agar method was used to isolate phages, and then the pH stability, thermal stability, host range, one-step growth curve of the isolate were determined. The phage morphology was observed by transmission electron microscopy. Next-generation sequencing was performed for the genome of the phage, and the lysis efficiency was evaluated. [Results] A novel bacteriophage JC01 against C. sakazakii was isolated from sewage. The phage had an icosahedral head and a long non-contractile tail. The genome of JC01 was 61 736 bp in length, containing 76 the open reading frames (ORF) and no drug-resistance gene, virulence gene or tRNA. The phylogenetic analysis and genome comparison indicated that JC01 was a novel phage belonging to the genus Jacunavirus, the class Casjensviridae of the order Caudoviricetes, and its was named as Jacunavirus JC01. The phage was stable at −20 to 40 °C and pH 5.0−9.0 and capable of lysing C. sakazakii in the contaminated PIF. [Conclusion] JC01 appears to be a novel phage against C. sakazakii, and it might be an alternative biocontrol agent for C. sakazakii contamination in food processing and production environments.

Abstract:[Background] Bifidobacterium bifidum, an obligate intestinal bacterium that specifically metabolizes human milk oligosaccharides (HMOs) and mucin glycans of host gut epithelium, is essential for early life health and development. However, little is known about the genetic differences of this bacterium among different populations. [Objective] To investigate whether the B. bifidum strains from genetically similar populations with similar diets in a limited geographic area have population-specific regularities and to reveal the genetic differences of B. bifidum among different populations. [Methods] A total of 115 strains of B. bifidum were isolated and identified from school-age children of Uyghur and Kazak ethnic minorities in Yining, Xinjiang. Further, we performed multilocus sequence typing (MLST) for 53 representative strains screened out by rep-PCR to understand the genetic differences of B. bifidum between different ethnic groups. [Results] The 53 strains belonged to 37 sequence types (STs), showing high genetic diversity. Among them, 17 STs were identified for 26 isolates from the children of Uyghur ethnic minority, while 20 STs for 27 isolates from the children of Kazak ethnic minority. Only a few homologous genetic recombination events were detected between strains from both ethnic groups. goeBURST showed that B. bifidum isolates from the same population were more likely to be assigned to a specific phylogenetic clade or clonal complex than the isolates from the other population. [Conclusion] B. bifidum isolates from different ethnic groups showed high genetic diversity. The host (ethnic group) specificity of their genetic structure needs to be confirmed by larger sampling. Our findings provide a theoretical basis for further screening of elite probiotic strains tailored to localized population by in vivo and in vitro experiments.

Abstract:There are diverse geological landforms such as tundras, frozen soils, oceans, glaciers, lakes, and mountains in the Antarctic and Arctic regions. The low temperatures, dryness, strong radiation, and barren environments shape unique and diverse microbial communities, which are a treasure trove of microbial resources. This article introduces the functions and applications of low-temperature enzymes, polysaccharides, and metabolites (antimicrobial substances, surfactants, pigments, volatile organic compounds, etc.) of polar microorganisms, the diversity and collection of polar microbial resources and type strains, and the current status of polar microbial patent applications and related research institutions. Furthermore, this paper briefs the challenges of culturability, metagenomic analysis, and digital sequence data in polar microbial prospecting.

Abstract:Exploring the process of low temperature stimulating fruiting body formation (LSFF) can provide a reference for the industrialized low-carbon efficient cultivation of edible fungi and the breeding of varieties with wide temperature ranges. This study first performed principal component analysis to classify LSFF of edible fungi into the low-temperature type (11−19 °C) and the cold stress type (≤10°C). On this basis, the research progress in the two types of LSFF in edible fungi was reviewed. The LSFF in edible fungi involves the metabolic processes such as signal transduction, stress response, basic metabolism, cell differentiation, and cell structure changes. With the decrease in the stimulation temperature from the low-temperature type to cold stress type, the sugar metabolism may shift to lipid metabolism to provide energy for fruiting body formation.

Abstract:Seed coating is an efficient seed treatment technology, which combines exogenous materials with seeds to improve seed performance and further crop yield and quality. Plant beneficial microorganisms (PBM) refer to the microorganisms that can promote plant nutrient absorption, enhance its tolerance to biotic and abiotic stresses, promote plant growth or reduce the agricultural chemical input. Therefore, PBM can be used as a microbial seed coating agent. As an innovative technology, microbial seed coating can significantly improve crop yield and its economic benefits, and maintain sustainable development of the agricultural system, which was thought to be a promising alternative to conventional agricultural technology. The present paper reviewed the microbial seed coating technology and its application in crop production, and its limitation and inconsistence were also discussed.

Abstract:A growing number of studies have shown that intestinal microorganisms can influence the development of colorectal cancer. For example, enterotoxigenic Bacteroides fragilis and Fusobacterium nucleatum have been proved to be associated with advanced colorectal cancer and reduced patient survival. Changes of intestinal flora can lead to the disturbance of intestinal homeostasis, and the changes in the number and species of bacteria can lead to complex pathophysiological processes in the host that promote the development of colorectal cancer. Therefore, researchers need to investigate how intestinal microorganisms damage the intestinal barrier, mediate the substance metabolism, produce inflammatory cytokines, and activate signaling pathways, and how the gut microbial ecological dysfunction accelerate the disease process. Probing into the interactions between intestinal microorganisms and colorectal cancer can contribute to the early diagnosis, treatment, and prognosis improvement of colorectal cancer. We reviewed the research progress in the mechanisms underlying the interactions between intestinal microorganisms and colorectal cancer and the cutting-edge therapies of colorectal cancer.

Abstract:Spinal cord injury as a traumatic stress may trigger anxiety in patients. Studies have shown that intestinal dysbacteriosis is closely related to the occurrence of anxiety after spinal cord injury. This article expounds the mechanisms by which the alteration of gut microbiota affects the occurrence of anxiety after spinal cord injury from four aspects: serotonin system dysregulation, dopamine system dysregulation, brain-derived neurotrophic factor deficiency, and inflammatory response. This review aims to provide a theoretical basis for future in-depth research and drug development for the treatment of anxiety after spinal cord injury.

Abstract:Surface display technology, an emerging genetic engineering technology, can immobilize the target protein on the cell surface of filamentous fungi. Since filamentous fungi have strong abilities of protein secretion and post-translational processing, surface display technology has been developed for increasing filamentous fungi. We review the development and application of the surface display system of filamentous fungi and introduce the cell wall composition, anchored proteins, and genetic transformation methods of filamentous fungi which are closely related to the construction of the system.

Abstract:Diphenyl ether herbicides, a group of broad-spectrum herbicides with high efficiency and high selectivity, have been widely used for controlling annual and perennial broad-leaved weeds in soybean and peanut fields. The continuous use of these herbicides for years may lead to significant accumulation in the soil environments. In this paper, we briefed the basic structures of diphenyl ether herbicides and their impacts on organisms, summarized the microbial species capable of degrading diphenyl ether herbicides, degradation pathways, and the key enzymes and genes involved in the degradation of diphenyl ether herbicides, and analyzed the factors affecting the microbial degradation. Finally, the future research trends in microbial degradation of diphenyl ether herbicides were presented. This paper aims to provide references for further research on the biodegradation of diphenyl ether herbicides.

Abstract:The curriculum teaching of Microbiology Experiment of pharmaceutical engineering is designed according to the concept of outcome-based education (OBE) required by the professional certification of engineering education, with the aim of fostering students’ abilities of independent learning and practical innovation. The teaching projects of this course were set at three levels: introductory, advanced, and independent projects. The teaching process was student-centered, with the support of online teaching methods and competition mechanisms. The practical results showed that project-based teaching reform significantly improved both the teaching performance and the learning effectiveness. Students achieved significant progress in experiment operation and paper writing. Therefore, this teaching method is worth summarizing and promoting in the future.

Abstract:[Background] African swine fever (ASF), as a highly pathogenic infectious disease, has caused serious economic losses in China’s pig farming industry, so it is crucial to establish a fast and sensitive diagnostic method. [Objective] To develop a chemiluminescence immunoassay (CLIA) for the detection of the anti-pp62 antibody of African swine fever virus (ASFV). [Methods] The recombinant pp62 protein was used as the coating antigen, carboxylated magnetic beads as the solid-phase carrier, and alkaline phosphatase (AP)-conjugated rabbit anti-swine IgG as the secondary antibody. The reaction conditions were optimized, and the standard curve was established with the national reference product of ASFV as the traceable serum. Finally, a CLIA method for detecting the anti-pp62 antibody of ASFV was established. [Results] The established CLIA method was used to test 277 clinical samples, and the negative and positive thresholds were determined by the receiver operating characteristic (ROC) curve. The concentrations >140.02 U and <140.02 U were determined as antibody positive and negative, respectively. Furthermore, the established method was used to detect the serum antibodies to six different pathogens, which showed no cross-reactivity, the intra- and inter-batch coefficients of variation below 10%, and a compliance rate of 96.7% with the results obtained by commercial ASFV antibody detection kits. [Conclusion] The CLIA method established for the detection of ASFV has good specificity, sensitivity, and reproducibility, which can provide a reference for the early monitoring of ASF and the research and development of the relevant kits.