Abstract:[Background] Acetyl-CoA is an important intermediate for lycopene synthesis in Saccharomyces cerevisiae, and acetyl-CoA in cytosol is mainly derived from acetic acid catalyzed by acetyl-CoA synthase.[Objective] To improve cell growth and lycopene production by increasing the content of intracellular acetyl-CoA through adding acetate combined with regulation of acetic acid stress-responsive gene. [Methods] Lycopene recombinant yeast strains overexpressing acetyl-CoA synthase (acs2) were added with 10 g/L acetate during the fermentation process. The transcriptomic analysis was combined to excavate acetic acid stress-responsive genes for single and combined regulation. [Results] After adding acetate, the lycopene content of the recombinant Y02 strain increased by 19.14%, but cell growth was suppressed. The results of the transcriptional analysis indicated that the expression levels of adk2, fap7, hem13, elo3, pdc5, set5, pmt5, hst4, clb2, and swe1 were increased significantly. Therefore, a single-gene and dual-gene were overexpressed in the Y02 strain. It was found that the growth of Y02-set5-hst4 was significantly improved in the presence of acetate. At the same time, the intracellular acetyl-CoA concentration was increased by 78.21%, and the lycopene content and yield reached 12.62 mg/g-DCW and 108.67 mg/L, respectively, which were increased by 42.76% and 67.13%, respectively, as compared with the control strain Y02. In addition, the expression levels of key genes erg12, erg20, and hmg1 in the mevalonate pathway were increased by 1.70, 1.44, and 1.96 folds, respectively, as compared with the control strain. [Conclusion] On the basis of overexpression of acs2, the overexpression of set5 and hst4 can improve the tolerance of yeast to acetic acid stress and increase the synthesis level of acetyl-CoA and the metabolic flux of mevalonate pathway, thus promoting the synthesis of lycopene. The results of this study provide valuable references for the metabolic engineering of other isoprenoid products.

Abstract:[Background] Poly-γ-glutamic acid (γ-PGA), a homogeneous amino acid polymer produced by Bacillus, has application potential in a variety of fields. The cwlO in Bacillus can express a d,l-endopeptidase to influence γ-PGA production, the underlying mechanism of which remains to be clarified. [Objective] To explore the effect of cwlO deletion on γ-PGA production by using different precursors and decipher the underlying mechanism. [Methods] The recombinant strain with cwlO deleted was constructed on the basis of Bacillus licheniformis WX-02. The fermentation for γ-PGA production was conducted in 3-L fermenters with different precursors, and the performance was compared between the recombinant and the wild type. Moreover, the mechanism for the performance difference was explored by the analysis of transcriptional levels, cell morphology observation via inverted fluorescence microscopy, and the content measurement of peptidoglycan and its components. [Results] The cwlO-deleted recombinant showed an improved efficiency on l-glutamine assimilation. With l-glutamine and l-glutamic acid as the mixed precursor, the recombinant showed the γ-PGA production of 36.3 g/L, which increased by 48.8% compared with that of the wild type. The results of RT-qPCR indicated that the transcriptional levels of the key genes involved in γ-PGA synthesis and respiratory chain were up-regulated in the recombinant with l-glutamine as the precursor, compared with those of the wild type. The recombinant cells became shorter and rounder. The peptidoglycan content in the cell wall and the protein content in peptidoglycan of the recombinant were greatly lower than those of the wild type. [Conclusion] Deletion of cwlO in B. licheniformis decreased the peptidoglycan content in the cell wall and promoted l-glutamine utilization, which enhanced γ-PGA production. This study provides a new insight and research basis for further research on the role of cwlO in γ-PGA production.

Abstract:[Background] Han Yangling Museum has been the largest group of pottery figurines of the Han Dynasty so far found in China. A large number of nude figurines have been unearthed. Archaeologists believe that these nude pottery figurines were originally dressed and equipped with wooden arms, which are unique to the royal family and extremely precious. However, there is no scientific support for these conjectures. In addition, the microorganisms attached to the surface may corrode the pottery figurines. [Objective] To provide scientific evidence for the speculation that clothes and broken arms exist by comparing the structure of microbial community on the surface of the pottery figurines, and to provide targets for the prevention and control of the microbial corrosion of pottery figurines in the Han Dynasty. [Methods] The microorganisms on different parts of the pottery figurines were identified by high-throughput sequencing and pure culture method. [Results] The high-throughput sequencing results showed that the microbial diversity in female figurines was higher on the head. In male figurines, the microbial diversity was high on the upper body, moderate on the legs, and low on the head. The microbial taxa on the surface of the pottery figurines were clustered according to the types and different parts of the figurines. The dominant groups were Clostridia, Saccharopolyspora, Pseudonocardia, and Streptomycetaceae of Actinomycetes. The relative abundance of dominant groups varied in different parts, and the relative abundance of the functional microorganisms involved in the carbon cycle on broken arms was the highest. The culturable bacteria on the head of female figurines were more than those on the head of male figurines. The culturable microorganisms on the upper body and legs were more than those on the head in male figurines. The upper body and legs may have been covered by clothing, which provided the habitats with different nutrients for microorganisms. [Conclusion] There were significant differences in microbial diversity and composition between the head and the body of the pottery figurines in the Han Dynasty, which suggested that the nutrient composition varied in different parts of the pottery figurines. Such differences might be related to the covering of the pottery figurines with clothes or dye.

Abstract:[Background] In maize-soybean rotation system, the residual atrazine in maize field may influence the next crop soybean. [Objective] To screen an atrazine-degrading strain from the field soil in Anda City, Heilongjiang Province and explore the degradation characteristics. [Methods] A highly efficient strain was isolated and screened by enrichment culture method, and identified based on morphological observation, physiological and biochemical determination, and 16S rRNA sequencing. Different carbon sources, pH, temperatures, and atrazine concentration were set to explore the optimal fermentation and degradation conditions with the simple variable method. [Results] A highly efficient atrazine-degrading strain AD111 was screened out, which used atrazine as the sole nitrogen source in basic salt medium-glucose (BSM-G). It was identified as Achromobacter marplatensis. The optimal temperature, pH, and carbon source for atrazine degradation by AD111 were 35℃, pH 8.0, and sucrose. Within 24 h, it degraded 99.7% and 81.9% of atrazine at 50 mg/L and 300 mg/L, respectively. [Conclusion] AD111 has exceptional environmental adaptability and atrazine-degrading ability, which can remove the atrazine residue in alkaline land in Heilongjiang Province.

Abstract:[Background] Ochratoxin A (OTA) is one of the most potent carcinogens, threatening food safety and human health. In recent years, biodegradation of OTA has attracted the interest of scholars, but the currently available OTA-detoxifying enzymes are limited. Therefore, it is of great significance to explore efficient OTA-degrading and -detoxifying enzyme resources. [Objective] To screen efficient OTA-degrading strain, clone the degradation-related gene, and thus to provide gene and enzyme resources for eliminating OTA. [Methods] The selective medium with OTA as the sole carbon source was used to screen OTA-degrading strain from the sludge. The strain was identified based on the 16S rRNA gene sequence analysis. The degradation products of OTA by the strain were determined by high performance liquid chromatography (HPLC). The degradation-related gene was obtained by homologous sequence alignment, cloned into the expression vector pET-29a(+), and then expressed in Escherichia coli BL21(DE3). The expressed product was purified by Ni²⁺-NTA affinity chromatography, and its OTA degradation activity and enzymatic characteristics were determined. [Results] A strain with high OTA degradation efficiency was screened out, which could completely degrade 1 μg/mL OTA within 12 h. The strain was identified to belong to Niastella and named JX-6. OTA was transformed by JX-6 through the hydrolysis of the amide bond to generate non-toxic OTα. An OTA amidohydrolase was identified in JX-6 and named NcOTase. The sequence similarity between NcOTase and the reported OTA amidohydrolase was low (31%-53%). The purified NcOTase showed OTA hydrolysis activity with specific enzyme activity of 60.3 U/mg, which was significantly higher than that most of the characterized OTA-degrading enzymes. [Conclusion] NcOTase is a highly efficient OTA-detoxifying enzyme, which has good application prospects in the removal of OTA in food and feed.

Abstract:[Background] Studies have found that PrtV gene encodes a metalloprotease containing polycystic kidney disease (PKD) domains, which plays an important role in the pathogenic process of various bacteria. Vibrio mimicus is one of the important pathogenic bacteria that infects a variety of aquatic animals, but the role of PrtV gene in the pathogenicity of V. mimicus is not clear. [Objective] To explore the effect of PrtV gene on the pathogenicity-related biological characteristics of V. mimicus. [Methods] The natural transformation method was used to construct the PrtV gene-deletion strain (ΔPrtV) of V. mimicus, and the complement strain (ΔPrtV/pPrtV) was constructed by electrotransformation after gene combined with plasmid. The mutant's biochemical characteristics, biofilm formation, self-aggregation ability, extracellular product protease activity, pathogenicity, and cytotoxicity were analyzed.[Results] As compared with wild stains, the growth, biofilm formation, self-aggregation ability, and lecithinase activity of the ΔPrtV were hardly changed, but the physicochemical properties of decomposing urea, glycine, coumarinate, ornithine, and lysine were changed. The extracellular product protease activity and cytotoxicity of ΔPrtV were significantly decreased (P<0.05), and the pathogenicity to hybrid catfish was decreased by 10 times.[Conclusion] PrtV gene is related to various biological functions such as cytotoxicity and pathogenicity of V. mimicus. The results provide a basis for further analysis of the features of PrtV gene and its pathogenic mechanism in V. mimicus.

Abstract:[Background] Grains are easily contaminated by pathogenic fungi and toxigenic fungi during the growth, harvest, and storage stages, which results in serious losses. Practices and studies have proved that the pathogenic fungi of plants can be controlled effectively by Trichoderma species. [Objective] To identify and screen the strains for the biocontrol of Trichoderma on grains and develop biocontrol fungicides for ensuring grain security.[Methods] In this study, 35 strains of Trichoderma were isolated from grains. They were identified based on both multi-gene phylogenetic relationship and morphological characteristics. Plate confrontation experiments were conducted to select the strains with antagonistic effects on common deleterious fungi. [Results] Eight species of Trichoderma were identified, including Trichoderma afroharzianum, Trichoderma asperelloides, Trichoderma amoenum, Trichoderma paratroviride, Trichoderma obovatum, Trichoderma longibrachiatum, Trichoderma orientale, and Trichoderma atroviride. The antagonistic assay demonstrated that these eight Trichoderma species had antagonism on 10 deleterious fungal species from grains. The antifungal efficacy of T. afroharzianum and T. longibrachiatum was better than that of other species. The a91 strain of T. afroharzianum showed the best antifungal efficacy, with the inhibition rates more than 65% against 9 deleterious fungal species. [Conclusion] The Trichoderma strains screened out in this study have great application potential for biocontrol and strong inhibitory effect on common pathogenic fungi and toxigenic fungi isolated from grain crops.

Abstract:[Background] Root rot caused by Fusarium oxysporum is common in tobacco growing areas worldwide, severely threatening the quality and yield of tobacco. As chemical pesticides fail to effectively control the pathogen, biocontrol strains have attracted the interest of scholars. [Objective] To clarify the inhibitory effect of Bacillus velenzensis GDND-2 on the growth and development of F. oxysporium from tobacco. [Methods] The PDA containing 10% and 20% fermentation filtrate of GDND-2 was coated on glass slides, and then the conidia of F. oxysporum were inoculated. The effect of the fermentation filtrate on conidial germination, hyphal growth, sporulation, and pigment production of F. oxysporum was investigated, and the ultrastructure of F. oxysporum hyphae was observed by scanning and transmission electron microscopy. [Results] After the treatment with fermentation filtrate of GDND-2, the conidial germination was over 2 h late. The fermentation filtrate resulted in the malformation of germ tubes and early branching of hyphae. It inhibited the elongation of hyphae and thus they showed malformed spherical structure. The inhibition rates of hyphal growth by 10% and 20% fermentation filtrate were 53.41% and 61.58%, separately. The fermentation filtrate delayed the sporulation of the pathogen, significantly reduced the spore quantity, affected the conidial morphology, and promoted the pigment production by the pathogen. Under the action of 10% and 20% fermentation filtrate, the sporulation happened 20 h and 28 h late, respectively, and the inhibition rates of spore quantity were 52.11% and 78.85%, separately. The fermentation filtrate stimulated F. oxysporum to form microconidia. As for the ultrastructure of F. oxysporium, some hyphae swelled and malformed, with thinner cell wall, disappearance of cell membrane, cytoplasm exudation, and intracellular cavity structure. Some hyphae shriveled, twisted and thinned, and perforated in severe cases. Some hyphae cells were severely vesiculated and organelle distribution was uneven. In some cells, the number of liposomes increased obviously. [Conclusion] The fermentation filtrate of B. velenzensis GDND-2 inhibited the growth and development of F. oxysporum, including conidial germination, hyphal growth, and sporulation.

Abstract:[Background] Southern blight of Aconitum carmichaeli is a soil-borne bacterial disease caused by Sclerotium rolfsii, which seriously affects the production of the medicinal plant. [Objective] To screen a strain against the S. rolfsii in A. carmichaeli and use it for disease control. [Methods] The strains were isolated from Tenebrio molitor pupae by plate coating method and streak plate method. The strain with strong ability against S. rolfsii was identified by plate confrontation assay, and the taxonomic status was determined based on morphological observation, physiological and biochemical tests, and 16S rRNA sequencing. Fermentation conditions were optimized by single factor test. The inhibitory effect of suspension, volatile gas, and fermentation broth of the antagonistic strain on the mycelia of S. rolfsii was determined. [Results] An efficient antagonistic strain N2 was screened out and identified as Bacillus siamensis. The best medium formula (g/L) of strain N2 is tryptone 10.0, yeast extract 5.0 and sucrose 20.0. The optimum culture conditions were as follows:initial pH 6.0, temperature 36℃, rotating speed 240 r/min, liquid volume 30 mL, and inoculum 0.05%. The suspension, volatile gas, and fermentation broth of the strain all can effectively inhibit the growth of S. rolfsii mycelia. The results of extracellular enzyme assay showed that the strain could produce protease and cellulase but failed to synthesize siderophore. [Conclusion] Strain N2 has ideal inhibitory effect on S. rolfsii of A. carmichaeli, which has a good application potential and high research value in biological control.

Abstract:[Background] Panama disease of banana, an intractable disease in banana, restricts the development of banana industry. Therefore, it is of great significance to screen out strains with inhibitory activity against Fusarium oxysporum f. sp. cubense tropical race 4 (Foc4). [Objective] To isolate Actinomycetes from samples of banana plantation soil and preliminarily identify species, and subsequently determine the antagonistic activities of the isolated Actinomycetes against seven pathogens including Foc4, so as to acquire strains with high activity and biological control strategies to solve Panama disease of banana. [Methods] Soil samples of banana plantation soil in Guangxi were collected and pretreated by ultrasonic wave and other means. Actinomycetes were separated using a variety of specific media and identified based on their 16S rRNA gene sequences. Seven pathogenic bacteria were used as targets to screen out the bacteriostatic strains by the plate confrontation method, and the bacteriostatic rate against Foc4 was determined by the mycelial growth rate method. [Results] A total of 138 strains of Actinomycetes were isolated from the samples of banana plantation soil, all of which were Streptomyces. Among them, five strains numbered X1085, X1052, X2052, X3059, and X4046 were potential new species. Seventy-seven strains with antimicrobial activities were screened out, with the positive rate of 55.8%. Twenty strains exhibited inhibitory activity against Foc4, among which four strains manifested obvious antagonistic effects, and their inhibitory rates were greater than 80%. The inhibitory rate of the strain X4050 was 93.76%. [Conclusion] The species information of culturable Actinomycetes in banana plantation soil was preliminarily clarified, and some of them were unknown species. Results of activity analysis showed that more than half of Actinomycetes had antimicrobial activity, and some of them exhibited good antagonistic activity against Foc4. These results provide a theoretical basis for the research on the isolation, identification, and biological activity of culturable Actinomycetes in the banana plantation soil, and provide a new idea for biological control of Panama disease of banana.

Abstract:[Background] Emmia lacerata, a white-rot fungus with global distribution, has a good inhibitory effect on the fungal pathogens of plants and can be developed as a biocontrol fungus. [Objective] To determine the antifungal ability and siderophore production ability of E. lacerata SR5 and explore its biocontrol potential. [Methods] The antifungal abilities of SR5 against 9 plant pathogens were determined by dual culture method and different concentrations of fermentation supernatants were used to determine the inhibitory effect of the metabolites. The chrome azurol S (CAS) method was employed to determine the siderophore production ability of SR5. [Results] SR5 inhibited 9 plant pathogens quickly in the way of overgrowth by competing for nutrients and survival space, with the inhibition rates of 23.7%–62.7%. It showed the strongest inhibition on Calonectria hongkongensis and Diaporthe sp., with the antagonism grade IV against Lasiodiplodia theobromae and grade III against the other 8 pathogenic fungi. SR5 demonstrated moderate capacity of producing secretory siderophore, with the highest siderophore unit (SU) of 44.1%. [Conclusion] SR5 plays the role of biocontrol by competing for nutrients and survival space and secreting siderophore, an extracellular anti-microbial metabolite. This study provides a scientific basis for the further development and utilization of E. lacerata in biocontrol.

Abstract:[Background] Fusarium solani, a ubiquitous pathogenic fungus, can cause a variety of soil borne diseases of plants and is one of the main pathogens causing root rot of Lycium barbarum. Potato glycoalkaloids (PGAs) have strong activity against F. solani and their source plants are widely cultivated with low cost. [Objective] To observe the effects of PGAs on the respiration and reactive oxygen species (ROS) metabolism of F. solani and decipher the possible antibacterial mechanism from the perspective of energy metabolism. [Methods] PGAs were extracted from potato buds by acetic acid and ammonia precipitation method, and F. solani was selected as the test pathogen. The inhibitory effect of PGAs on the mycelial growth of F. solani in PDA and PDB was investigated and the concentration for 50% of maximum effect (EC₅₀) was determined. The effect of PGAs on the respiration of F. solani was detected by an oxygen electrode. The effects of PGAs on the antioxidant enzyme system, ROS, and the metabolite malondialdehyde (MDA) of F. solani were studied in PDB. [Results] The PGA treatment decreased the mycelial respiration rate in a concentration and time-dependent manner and increased the content of intracellular hydrogen peroxide (H₂O₂) and superoxide anion (O₂⁻) (P<0.05). Furthermore, the treatment caused the imbalance of the peroxidase (POD), catalase (CAT), and superoxide dismutase (SOD) system, aggravated the membrane lipid peroxidation, and increased MDA content. Transmission electron microscopy results showed that the PGA treatment broke the mitochondrial membrane, caused the loss of mitochondrial contents, and destroyed the mitochondria of F. solani. [Conclusion] PGAs inhibit the respiration and reactive oxygen species metabolism, destroy mitochondria, the main organelle of the energy metabolism pathway, and finally inhibit the mycelial growth of F. solani.

Abstract:[Background] Fusarium solani is the main pathogen of Zanthoxylum bungeanum root rot in Longnan, affecting the production of Z. bungeanum. [Objective] To screen out a strain for the biocontrol of F. solani and provide new bioresources for the development of microbial agents for the control of Z. bungeanum root rot. [Methods] In this study, we employed the plate confrontation method and 16S rRNA gene sequencing to screen and identify the bacterial strain with strong antimicrobial activity from the rhizosphere soil of Z. bungeanum. Box-Benhnken design was employed to optimize the fermentation conditions of the strain. Liquid chromatography-mass spectrometry (LC-MS) was used to study the changes of metabolites in the organic solvent phase of the bacterial fermentation broth. [Results] The antagonistic strain screened out was identified as Bacillus velezensis W-1, which showed the inhibition rates of 89% against F. solani and 57%–90% against Gibberella fujikuroi, Fusarium graminearum, Fusarium syphilis, Fusarium proliferatum, and Colletotrichum fioriniae. The fermentation conditions of W-1 were optimized as pH 6.5, 30℃, inoculum amount of 5%, and 180 r/min. The OD₆₀₀value of the fermentation broth obtained under the optimal conditions reached 1.93, which was 2.6 times higher than that of the LB basal medium. The antimicrobial activity of the sterile fermentation broth remained stable in the range of pH 2.0−9.0 and gradually decreased after the broth was irradiated with ultraviolet light (UVC 200−280 nm, 100 μW/cm²) for 10 min. The antimicrobial activity significantly decreased when the temperature exceeded 80℃. Ethyl acetate was the best organic solvent for the extraction of antimicrobial ingredients. The organic phase of the extract contained rich antagonistic substances, such as lipopeptides (surfactants), phosphate oligopeptides (antibiotics), butirosin, polyketone compounds, lysobactin, cyclic dipeptides, and gramicidin. [Conclusion] B. velezensis W-1 can secrete a variety of antimicrobial substances and has strong inhibitory effects on multiple common pathogenic fungi of plants. Moreover, its fermentation broth can tolerate acid, alkali, and high temperature, demonstrating great potential for application in the biocontrol of Z. bungeanum root rot.

Abstract:[Background] Our research team have discovered that Bacillus sp. DU-106 has good health benefits in previous studies. However, the conclusion is based on the studies of living bacteria and the studies of inactivated bacteria remain to be carried out. [Objective] To investigate the effect and mechanism of Bacillus sp. DU-106 lysate in the treatment of atopic dermatitis. [Methods] Pathological observation and immunohistochemistry were employed to study the therapeutic effect of Bacillus sp. DU-106 lysate on the mouse model of 2,4-dinitrofluorobenzene-induced atopic dermatitis. The expression levels of the proteins involved in the nuclear factor kappa-B (NF-κB) signaling pathway were determined to reveal the mechanism. [Results] Bacillus sp. DU-106 lysate alleviated the pathological changes, reduced the infiltration of mast cells, and lowered the levels of immunoglobulin E, tumor necrosis factor-α (TNF-α), and interferon-γ (P<0.05) in the model mice. Furthermore, Bacillus sp. DU-106 lysate down-regulated the expression levels and inhibited the phosphorylation of TNF-α, inhibitory kappa B kinase α (IKKα), and NF-κB in the NF-κB signaling pathway in the model mice. [Conclusion] Bacillus sp. DU-106 lysate can be used as a probiotic with therapeutic effects on atopic dermatitis.

Abstract:[Background] Aeromonas schubertii is ubiquitous in fresh water, seawater, and sediment. The pathogenic strains have been prevalent in cultured Channidae fish in China and infected other economic fishes, leading to death outbreak. [Objective] The pathogen was isolated and identified from the diseased Siniperca chuatsi. The pathogenicity and drug sensitivity of the isolate were determined, thereby providing references for the clinical treatment of this disease.[Methods] The spleen and kidney tissues of the diseased S. chuatsi were collected for polymerase chain reaction (PCR) or real-time PCR (RT-PCR) amplification of common viruses. The liver and ascites of the diseased S. chuatsi were pooled for the bacterial isolation and cultivation. The gyrB, 16S rRNA and virulence genes of the representative strain were amplified by PCR, and their physiological and biochemical characteristics were identified. The susceptibility test and the artificial infection test were carried out. [Results] The detection results of infection spleen and kidney necrosis virus, Siniperca chuatsi ranairidovirus and Siniperca chuatsi rhabdovirus in the diseased S. chuatsi were negative, but large amount bacteria were observed from the liver and ascites. The representative strain Gui210820 was confirmed as A. schubertii, and carried the virulence genes of hemolysin, aerolysin, elastase and lipase. The median lethal concentration (LD₅₀) of S. chuatsi infected by peritoneal injection was 3.16×10⁵ CFU/mL. The Gui210820 strain was resistant to 6 kinds of antibiotics such as tetracycline, kanamycin, and cotrimoxazole, and sensitive to 11 kinds of antibiotics, including doxycycline, enrofloxacin, neomycin, and florfenicol.[Conclusion] In this study, pathogenic A. schubertii has been isolated from the tissues of the diseased S. chuatsi. Aquatic approved drugs enrofloxacin, neomycin, and florfenicol can be used for the clinical treatment of this bacterial disease.

Abstract:[Background] Trueperella pyogenes is an opportunistic pathogen causing infections in humans and diverse animal species, particularly non-specific purulent infections in animals. [Objective] To characterize the suspected T. pyogenes strains from different hosts. [Methods] Strains were isolated from the subcutaneous abscess of forest musk deer and duck tarsal joint abscess and identified based on Gram staining and bacterial 16S rRNA gene analysis. The biological properties of the two strains were characterized through the analysis of the growth curves, drug sensitivity test, and detection of virulence genes. Then we analyzed the sequence and predicted the structure of the pyolysin gene plo. [Results] The strains isolated from forest musk deer and duck were named FTP-1 and DTP-1, respectively. According to the growth curves, the growth rates of the two were quite different. Drug sensitivity test showed that the two strains were resistant to β-lactams, sulfonamides, and rifamycins and sensitive to aminoglycosides and quinolones. Both strains carried plo, nanH, nanP, fimA, and fimE genes, and DTP-1 also harbored fimC gene. There were host-specific differences in the nucleotide sequence of plo gene between the two strains, but the amino acid sequences of encoded proteins were the same. The pyolysin (PLO) of T. pyogenes was highly similar to other members of the cholesterol-dependent cytolysin family. [Conclusion] One strain of T. pyogenes was respectively isolated from forest musk deer and duck in Yiliang of Yunnan Province, and the plo gene of two strains had close genetic relationship. The biological characteristics can serve as a reference for further research on and prevention and control of the pathogen.

Abstract:[Background] Streptococcus suis type 2 (SS2) may cause meningitis, arthritis, and sepsis in humans and pigs, which not only brings great economic damage to the pig industry but also seriously threatens public health safety. In the previous study, using the phage display library technique, this team found that the protein encoded by Orf207 may be involved in SS2-induced meningitis; however, its specific role in the pathogenesis of SS2 is still unclear. [Objective] To explore the effect of the Orf207 gene on SS2 pathogenicity. [Methods] In this study, the homologous recombination system mediated by the thermosensitive suicide plasmid was used to construct the SC19 Orf207 gene deletion strain ΔOrf207 and its complementing strain CΔOrf207, after which the differences in biological characteristics such as growth characteristics, colony morphology, tissue colonization capacity, virulence, cell adhesion and invasion, and anti-macrophage phagocytosis between the deletion strain and the wild strain were systematically compared. [Results] Compared to wild strains, the deletion strain had shorter chain length and slower growth rate. Moreover, Orf207 deficiency significantly increased the survival rate of mice and reduced the colonization of bacteria in the blood, heart, liver, spleen, lung, kidney and brain, alleviating pathological damage to lung tissues. It also remarkably reduced the adhesion and invasion ability of SS2 to Hale cells and its anti-macrophage phagocytosis. [Conclusion] The above results indicate that gene Orf207 can significantly reduce the pathogenicity of SS2 to the host. The findings of this study enrich the pathogenesis of SS2 and provide a new target for the development of SS2 vaccine.

Abstract:[Background] Pseudomonas aeruginosa (PA) is a common opportunistic pathogen. The septic diseases caused by PA account for more than 50% of the deaths in captive forest musk deer populations. Phages have become popular substitutes of antibiotics due to the increased resistance of bacteria. [Objective] To isolate a bacteriophage using PA isolated from the lungs of dead forest musk deer as the host bacteria, and to conduct biological characterization, whole genome sequencing, and in vivo bacteriostasis test on it. [Methods] Double-layer agar culture method was used to isolated a phage against PA from the daily drinking water of forest musk deer in Sichuan Institute of Musk Deer Breeding. The same method was used to determine the host range, optimal multiplicity of infection (MOI), one-step growth, thermostability, and pH range of this phage. Phage morphology was observed by transmission electron microscopy (TEM). The whole genome of the phage was sequenced and analyzed, and in vivo bacteriostasis test was carried out in mice. [Results] A phage strain against PA was isolated and named as vB_PaeM_PAMD02. It produced a transparent plaque with clear edge and no halo ring and had a narrow host range. The phage strain had high thermostability and was stable in weak basic environment, with the optimal MOI of 0.1 and the incubation period of 40 min. The whole genome sequencing showed that the genome of this phage had a length of 66 264 bp and the GC content of 55.59%. The annotation results showed that the phage had 92 open reading frames and carried no virulence gene or resistance gene. The results indicated that the phage belonged to the Myoviridae family. Animal experiments showed that the phage effectively inhibited the pathogenicity of bacteria in mice. [Conclusion] The isolated phage vB_PaeM_PAMD02 has high lysis efficiency and good tolerance to adverse environments. It has great potential to be applied in clinical prevention and treatment of PA infection.

Abstract:[Background] Infections by antibiotic-resistant pathogens pose tremendous threats to human health, and there is an urgent need to develop alternative or adjuvant therapies for classic antibiotics. Phages as the natural predators of bacteria demonstrate promising prospects in treating bacterial infections. [Objective] To isolate virulent phages against carbapenem-resistant Acinetobacter baumannii (CRAB) and then treat CRAB-caused pulmonary infection with the isolated phages.[Methods] We isolated new phages from hospital sewage by using the CRAB clinical strain NAB11B as the host bacteria and then studied the biological and genomic characteristics of the isolated phages. Subsequently, we employed aerosol inhalation of a phage cocktail to treat a patient with CRAB pulmonary infection. Finally, we evaluated the efficacy and safety of phage therapy. [Results] We isolated a novel phage AB_SZL4, which could produce a transparent plaque with a halo in NAB11B lawn and inhibit the growth of NAB11B at low multiplicity of infection (MOI). AB_SZL4 showed the latent period of about 15 min and the burst size of 75 PFU/cell. AB_SZL4 remained stable at pH 3.0–11.0 and 70℃ and below. There was no detrimental gene in the genome of AB_SZL4. NAB11B was eliminated following phage therapy combined with antibiotics within a week, and no phage-related side effect was observed. [Conclusion] AB_SZL4 is a potent lytic phage with great potential for clinical application.

Abstract:[Background] Mycoplasma pneumoniae is an important pathogen causing respiratory tract infections in children and adolescents. It is easy to miss the optimal treatment period due to its nonspecific clinical manifestations for a long time.[Objective] To develop a method for rapidly detecting M. pneumoniae based on the multienzyme isothermal rapid amplification (MIRA) and nucleic acid test strip. [Methods] Primers and probes were designed using the community acquired respiratory distress syndrome (CARDS) toxin gene of M. pneumoniae as the target gene to optimize the temperature and time of the reaction system and evaluate the sensitivity. The specificity was analyzed by testing M. pneumoniae as well as the remaining 7 pathogens, and 35 clinical samples were validated. [Results] The MIRA nucleic acid test strip method completed the detection of M. pneumoniae within 15 minutes at 37℃, and the limit of detection was 10 copies/μL. Except for M. pneumoniae, the other 7 pathogens could not be amplified, showing good specificity. Using real-time fluorescent PCR detection as the standard, the diagnostic specificity, sensitivity, negative predictive value, and positive predictive value of MIRA nucleic acid test strip method for 35 clinical samples were 100.00%, 96.15%, 90.00%, and 100.00%, respectively. [Conclusion] In this study, the MIRA nucleic acid test strip method has been established for the detection of M. pneumoniae, which is fast, portable, sensitive, and specific, and is suitable for promotion at the grassroots level.

Abstract:[Background] Carbapenem-resistant Klebsiella pneumoniae strains have been on the rise with the extensive use and abuse of carbapenems. The production of carbapenemase is a common mechanism by which these strains resist killing by the carbapenems. The available detection methods for carbapenemase-producing K. pneumoniae are time- and labor-intensive, with poor specificity and sensitivity. [Objective] To develop an assay for the detection of K. pneumoniae with bla_KPC gene based on duplex chip digital PCR (duplex-cdPCR). [Methods] The specific primers and probes for the detection of the characteristic gene yhaI of K. pneumoniae and the carbapenem resistance gene bla_KPC were designed. The range of absolute quantification of nucleic acids, detection limit and optimal reaction conditions of the duplex-cdPCR for yhaI and bla_KPC were determined simultaneously. The specificity, sensitivity, and precision of the method were analyzed and the clinical strains were detected. [Results] Compared with duplex-qPCR, duplex-cdPCR had the sensitivity of about 1.5 orders of magnitude higher and the detection limit was 3.74 copies/μL (yhaI) and 1.93 copies/μL (bla_KPC), respectively. The optimized duplex-cdPCR showed the specificity consistent with that of duplex-qPCR. In this report, a total of 58 clinical strains were detected by the optimized duplex-cdPCR, of which 43 strains were detected as K. pneumoniae and 13 were K. pneumoniae harboring bla_KPC gene. This demonstrated 100% concordance with the results of mass spectrometry and drug resistance profile.[Conclusion] A duplex-cdPCR assay capable of simultaneously detecting the characteristic gene yhaI of K. pneumoniae and the carbapenem resistance gene bla_KPC in the same target was established. This assay appears to be highly specific, sensitive, and accurate. The duplex assay is suitable for nucleic acid detection and quantitative analysis of K. pneumoniae with bla_KPC gene. Moreover, it also provides a new technical reference for the detection of carbapenem-resistant pathogenic bacteria carrying other carbapenemase gene types.

Abstract:Pathogenic Escherichia coli infecting humans and domesticated animals can be classified into intestinal and extraintestinal pathogenic E. coli. Extraintestinal pathogenic E. coli (ExPEC) mainly colonizes tissues and organs outside the intestine and causes a wide range of extraintestinal infections, including uropathogenic E. coli (UPEC), newborn meningitis E. coli (NMEC), and avian pathogenic E. coli (APEC). Human ExPEC (including UPEC and NMEC) is the etiologic agent of urinary tract infections, pyelonephritis, and neonatal meningitis. APEC can lead to avian colibacillosis, causing huge economic losses in the poultry industry. In addition, mammary pathogenic E. coli and porcine ExPEC can bring forth cow mastitis, pig pneumonia, and acute sepsis. Studies have demonstrated that human and animal ExPEC strains have similarities in genomic structure, and they are essentially different from IPEC in pathogenic mechanism. ExPEC strains have a variety of similar virulence genes and resistance genes. The virulence genes and resistance genes in animal ExPEC can be transmitted to humans through edible animals, jeopardizing human health, which indicates that ExPEC strains from animals potentially serve as a reservoir of virulence genes and resistance genes for human ExPEC. ExPEC brings a tremendous burden on public health. Here, we review the hazards, virulence factors, pathogenic mechanism, and public health significance of ExPEC, hoping to enrich the knowledge about them.

Abstract:Lactococcus sp. and Lactobacillus sp. are lactic acid bacteria commonly used in industry and have long been used in food and beverage fermentation. In recent years, with the continuous improvement of molecular manipulation and genetic modification techniques, the basic research and application of Lactococcus and Lactobacillus have been promoted, and their important potential as functional strains as well as industrial microbial cell factories has been highlighted. In this paper, the research progress of genome editing technology of lactic acid bacteria commonly used in industry was reviewed, and the knockout knock-in based on integrative plasmid, fine modification and knockout knock-in based on genome recombination engineering, and gene editing of CRIPSR/Cas9 system based on clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated nuclease9 (Cas9) were emphatically introduced.

Abstract:Myxobacteria are predatory Gram-negative bacteria ubiquitous and often predominant in soil, ocean, and freshwater environments. According to the 16S rRNA gene sequences, myxobacteria were classified as an order Myxococcales affiliated to the class δ-Proteobacteria. The recent phylogenetic studies based on 120 conserved single-copy marker genes and the 16S rRNA gene sequences have upgraded Myxococcales to the phylum Myxococcota. This review briefly summarizes the characteristics of myxobacteria and the history of the phylogenetic research and then makes a perspective on the future studies and applications of myxobacterial resources.

Abstract:Irritable bowel syndrome (IBS) is the most common functional bowel disorder with the typical clinical symptoms such as abdominal pain, abdominal distension, and changes in bowel habits. Although the pathogenesis of IBS is complex and has not been fully understood, it has been proven to be related to the abnormal regulation of microbiota-gut-brain axis. The effects of derivative metabolites mediated by the microbiota, such as neurotransmitter, short-chain fatty acids, and bile acids metabolites, on the development of IBS symptoms (visceral sensitivity, abdominal pain, diarrhea and mental disorders) were systematically summarized. This study is expected to provide a new insight for the treatment of IBS with metabolites transforming bacteria as targets.

Abstract:Locust has been a major pest of China's agriculture, forestry, and animal husbandry since ancient times, causing huge losses to agriculture. Researchers at home and abroad have conducted in-depth research on this pest. With the advances in the microecology of insect gut, the gut microbiome of locust has become a research focus. Meanwhile, the rapid development of sequencing technology has facilitated the research in this field. This paper summarizes the research progress in the diversity, function, and research methods of locust gut microbiome and puts forward the future research prospects.

Abstract:Synonymous codons usage pattern as a bridge between nucleotides and amino acids, and their diversity mediates the ribosome scanning rate while expanding the genetic information storage of genes. With the application of new technologies, studies have demonstrated that specific codons and codon binding can modulate ribosome scanning rate and protein folding. Synonymous codon usage patterns affect the ribosome scanning rate in different ways and the stability of corresponding mRNAs. This paper briefly describes how the synonymous codon usage patterns regulate polypeptide chain extension in the process of ribosome scanning and translation of mRNA, which provides reference for the optimization of protein expression in bioengineering in the future.

Abstract:Staphylococcus aureus wall teichoic acids (WTAs) are the anionic glycopolymers containing phosphodiester-linked polyol units and contribute to the cell wall homeostasis, virulence, and pathogenicity of S. aureus after glycosylation. As the key targets of receptor binding sites and crucial antigenic epitopes in the host, S. aureus WTAs induce not only the secretion of pro-inflammatory cytokines in the innate immune system but also specific antibody response in the adaptive immune system. Furthermore, the WTAs promote S. aureus colonization by regulating the expression of virulence genes, thus demonstrating a great prospect as the targets in genetic engineering and phage therapy. We overview the biosynthesis of S. aureus WTAs, elaborate on the WTA-induced immunomodulation of host, and introduce the roles of WTAs in the invasion and colonization of S. aureus. Further, we summarize the mechanisms of the drug resistance of S. aureus WTAs and the research progress in the application of S. aureus WTAs as the targets in the development of novel therapies. This review aims to provide reference for deciphering the molecular mechanism of the pathogenicity of S. aureus WTAs and the corresponding host responses, as well as the development of preventative and therapeutic approaches against S. aureus-mediated diseases.

Abstract:Daqu, commonly used as a starter during the brewing of Chinese Baijiu, provides a variety of microbial strains and enzymes initiating the Baijiu fermentation, thus affecting the unique flavor and taste of Baijiu. In recent years, the microbial community structure of Daqu has become a research hotspot, and researchers have conducted extensive in-depth research on the microbial community structure, gene function, and functional microorganisms of Daqu. Therefore, the knowledge of the microbial composition, succession, and functions of Daqu has become increasingly richer. This review summarized the analytic methods of microbial community structure, the main microbial composition, functional microorganisms, and microbial traceability in the production of Daqu, aiming to provide a theoretical basis for investigating the microbial community structure of Daqu, optimizing the production process of Daqu, and improving the quality of Baijiu.

Abstract:Beauveria bassiana is a major entomogenous fungus characterized by strong pathogenicity, wide infection spectrum, safety, and free pollution. It can be prepared as a fungal insecticide to control pests in agriculture and forestry and used for the artificial production of the Chinese medicinal material Bombyx Batryticatus. Here we introduced the biological characteristics, infection characteristics, application status, and shortcomings of B. bassiana and put forward the development countermeasures, aiming to provide reference for further development and application of B. bassiana.

Abstract:Siderophores are small organic molecules produced by microorganisms under iron-limiting conditions which enhance the uptake of iron to microorganisms. Bacterial siderophores play an important role in antagonizing plant pathogens and promoting plant growth. In this paper, we summarized the mechanisms of bacterial siderophores against plant pathogenic fungi, such as nutrient competition, niche competition, eliciting inducible systemic resistance in plants, and disrupting the iron homeostasis of pathogens, and the plant growth-promoting effect of the siderophores, aiming at clarifying the potential of bacterial siderophores in the development of multifunctional microbial agents.

Abstract:The use of liquid fermentation technology can obtain functional raw materials from edible and medicinal fungi with stable quality, controllable products, and various active ingredients. This paper systematically summarized the research status and bottlenecks of liquid fermentation technology of edible and medicinal fungi, as well as the main components and efficacy of the liquid fermentation products of edible and medicinal fungi, and compared them with the cultivated fruiting bodies. Through the prospect of the breakthrough of liquid fermentation technology of edible and medicinal fungi, this paper is expected to provide references for the further application of liquid fermentation products of edible and medicinal fungi.

Abstract:Lignocellulosic biomass represents a crucial resource for sustainable development by replacing petroleum-based production systems. Lignin, a major component of plant cell walls, has been widely used in many industries. However, it poses a challenge to the utilization of lignocellulosic biomass due to the recalcitrance and complex structure. Therefore, decomposition or removal of lignin is the key to the utilization of other cell wall components. Lignin is naturally degraded by many different species of microorganisms, including fungi and bacteria, and the mechanisms underlying the degradation of lignin by microorganisms provide a host of possibilities to overcome the challenges of using harmful chemicals to degrade lignin biowaste in many industries. This review discussed the chemical constituents of lignin, the lignin-degrading microbial species, such as fungi and bacteria, and the mechanisms, and the lignin-degrading enzymes produced by a variety of microorganisms, especially the white-rot fungi, brown-rot fungi, and bacteria. Finally, the possible trends of research and applications of lignin biodegradation were prospected.

Abstract:The "post-course-competition-certificate" integration is a teaching reform model that integrates post, competition, and professional certificate into the course. Following this idea, we comprehensively implemented the teaching reform of Food Microbiological Examination Technology by setting the course based on post, improving the course by competition, and integrating course resources according to certificate. Specifically, considering the competence and responsibilities of posts needed by enterprises, we carried out modular and project-based teaching. Taking the skill competition and the innovation and entrepreneurship competition as carriers, we incorporated competition into teaching to trigger the endogenous motivation of students' growth and improve students' skills and comprehensive quality. According to the "1+X" certificate system, we connected the course standards with skill certificate standards and optimized the teaching resources. The nearly three years of teaching practice proved that the teaching reform of Food Microbiological Examination Technology based on "post-course-competition-certificate" integration can effectively improve students' vocational competitiveness, meet the needs of enterprises and society for talents, and provide reference for the reform of food-related courses in vocational education.