Abstract:[Background] Haloarchaea are able to survive in salt deposits for millions of years. Many haloarchaea produce extracellular subtilisin-like proteases (Halolysins). The Halolysins are folded in cells and transported across cytoplasmic or thylakoid membranes by Tat pathway. In addition, they mature automatically and most of them are produced at the late log phase and peak when the culture enters stationary phase. At the moment, the enzymatic properties, autocatalytic activation, and secretion mechanisms of Halolysins have been widely characterized. However, the physiological functions are rarely studied. Halolysin SptA is the major extracellular protease of Natrinema sp. J7-2. Previous studies showed that the growth phase-dependent production of SptA relies on the cooperative action of multiple cis-acting elements, allowing SptA to participate in the growth-phase transition of strain J7-2. SptA also contributes to the continued survival of strain J7-2 after the death phase. [Objective] To study the effect of SptA on long-term survival of strain J7-2. [Methods] strain J7-2 and ΔsptA1 mutant were cultured under nutrient-deficient, non-exogenous nutrient (liquid), and nutrient-rich (solid) conditions for a long time, respectively. The growth, survival, and SptA expression of the two strains were compared to further investigate the role of SptA. [Results] J7-2 strain produced more SptA under nutrient-deficient conditions, and number of viable J7-2 strain cells was significantly larger than that of viable ΔsptA1 cells after 33 days of growth under such conditions. strain J7-2 and ΔsptA1 underwent multiple events of cell division and cell death during long-term incubation in the absence of exogenous nutrients. The number of viable J7-2 strain cells was significantly larger than that of viable ΔsptA1 cells during the prolonged incubation (73-200 days). At the late stage of culture (160 days) on nutrient-rich solid plates, due to nutrient depletion, J7-2 strain benefited from SptA in terms of long-term survival by assimilating and utilizing the degradation products of proteins derived from dead cells.[Conclusion] The SptA-mediated cell death and degradation of dead cell-derived proteins enhance the long-term survival of J7-2 strain in response to nutrient starvation by helping J7-2 strain to scavenge dead cell-derived nutrients. This study provides new insight into the physiological role of Halolysins.

Abstract:[Backgroud] In recent years, with the increasing of land salinization in Gansu province, its impact on agricultural production and ecological environment has become more and more serious. [Objective] In this study, saline-alkali-tolerant strains were isolated and screened from the saline-alkali soils of Lanzhou new district of Lanzhou city and Heihe Ecological Reserve of Zhangye city, Gansu province. Then we identified the isolated strains, and evaluated and analyzed some of their functionality and growth-promoting properties, which provided microbial resources and theoretical basis for improving the saline-alkali land in Gansu province. [Methods] The isolated colonies were cultured in conditioned medium of different salinity and alkali concentrations, and those with high saline-alkali tolerance were screened out. The 16S rRNA gene sequencing was used to identify the types of the selected strains and test their various functional and growth-promoting effects in different media. [Results] Five highly saline-alkali-tolerant strains were obtained, which were Bacillus megaterium, Arthrobacter aurescens, Sinorhizobium fredii, Bacillus licheniformis and Acinetobacter sp.. All five strains had the ability to dissolve phosphorus and secrete indole-3-acetic acid (IAA),A. aurescens and B. licheniformis had the ability to dissolve potassium, and A. aurescens, S. fredii and Acinetobacter sp. showed nitrogen fixation ability. ACC deaminase activities secreted by B. licheniformis, B. megaterium and S. fredii reached 0.272, 0.217, and 0.159 U/mg, respectively, while A. aurescens and Acinetobacter sp. had almost no ACC deaminase activity.[Conclusion] The five saline-alkali-tolerant bacteria had different phosphorus-dissolving, potassium-solving and nitrogen-fixing functions and certain growth-promoting effects. This study provided species resources and theoretical basis for the later application of microorganisms to improvement of saline-alkali land.

Abstract:[Background] Yunnan boasts abundant Cordyceps species resources and the natural Cordyceps species community contains diverse endophytic fungi, which can be used for mining antimicrobial.[Objective] To investigate the diversity of entomogenous fungi and the endophytic fungi in Dashao village, Songming county, Yunnan province, and screen strains with antimicrobial activities. [Methods] Entomogenous fungi and the endophytic fungi were isolated from Cordyceps species samples with tissue separation method. They were identified and the diversity was analyzed based on morphological observation and sequencing of ITS, nrSSU,nrLSU, tef-1α, rpb1, and rpb2. The activities against 7 pathogenic bacteria and 5 plant pathogenic fungi were tested by plate confrontation assay.[Results] A total of 86 natural Cordyceps species samples were collected, which belonged to 7 species in 5 genera in 3 families:Cordyceps (1 species), Beauveria (2 species), and Samsoniella(2 species) in Cordycipitaceae, Metapochonia (1 species) in Clavicipitaceae, and Ophiocordyceps (1 species) in Ophiocordycipitaceae. Meanwhile, 26 strains of endophytic fungi in 9 genera of 9 families were isolated from the samples, among which Trichoderma(38%) and Fusarium (19%) dominated. In addition, 20 strains, which were selected from entomogenous fungi and endophytic fungi according to their origins and taxonomy, were tested for the antimicrobial activities. Results demonstrated that 11 strains showed activities against two or more pathogenic bacteria, and 13 strains against more than one plant pathogenic fungi. Trichoderma sp. Y3-1 and Fusarium sp. WZ3-1 demonstrated broad-spectrum antimicrobial activities. [Conclusion] The Dashao village in Songming county of Yunnan province enjoys rich resources of entomogenous fungi and endophytic fungi and the isolated fungi show activities against diverse pathogens. This study enriches the diversity of entomogenous fungi in Yunnan province, providing not only data support for the development and utilization of entomogenous fungi and the endophytic fungi in Yunnan province but also strains for mining active substances from them.

Abstract:[Background] At present, there is a lack of aerobic denitrification bacteria with significant nitrogen removal performance, high biological safety and alkali tolerance. Thus, it is difficult to treat high-alkaline industrial and aquaculture wastewater using biological methods. [Objective] The alkali-resistant and highly aerobic denitrification bacterium ZY-3 isolated from an aquaculture pond in Foshan city, Guangdong province was tested, in the hope of obtaining an efficient and safe aerobic denitrification bacterium which could be used in different pH environments for nitrogen removal. [Methods] The strain was identified by morphological observation, physiological and biochemical tests, and 16S rRNA gene sequence analysis. Antibiotic test and zebrafish challenge test were conducted to evaluate the environmental and biological safety of the strain. Three kinds of simulated wastewater containing different nitrogen levels were used to determine the nitrogen removal performance. [Results] ZY-3 was identified as Pseudomonas plecoglossicida, which was sensitive to a variety of clinically common antibiotics and had low aquatic toxicity. When this strain was cultivated in the three simulated wastewater with ammoniacal nitrogen, nitrite nitrogen or nitrate nitrogen as the sole nitrogen source at 28℃ and 180 r/min, the logarithmic phase appeared at 4−12 h. At 12 h, the removal rates of NH₄⁺-N, NO₃^--N and NO₂^--N reached 94.87%, 81.44% and 98.02%, respectively. The pH tolerance range of the strain was 6.0−10.0. [Conclusion] A safe and efficient aerobic denitrification bacterium P. plecoglossicida ZY-3 was obtained, with a wide range of pH adaptation and rapid removal ability against three nitrogen sources (NH₄⁺-N, NO₃^--N and NO₂^--N) under aerobic conditions.

Abstract:[Background] A survey of the species diversity of culturable fungi in tidal flats of China was carried out, aiming at enriching the fungal species resource in China. [Objective] To report two Talaromyces species producing ascospores new to China.[Methods] A polyphasic taxonomic method, which integrated morphological identification and the phylogeny based on sequences of β-tubulin gene (BenA) and rDNA ITS1-5.8S-ITS2, was employed. [Results] Three teleomorphic Talaromyces species were isolated and identified, namely, T. argentinensis (ZZ2-7-1h=CGMCC 3.16171), T. barcinensis (ZZ2-1-1=CGMCC 3.16172) and T. ucrainicus (JS11-5=CGMCC 3.16173), among which, the former two are new to China. T. argentinensis grows well at 25℃ and is restricted at 37℃, presenting floccose and funiculose colonies with white mycelia mingled with a light pink tint and producing sparse orange-yellow gymnothecia with echinulate ellipsoidal ascospores. T. barcinensis grows well at 25℃ but does not grow at 37℃, presenting velutinous colonies with white and yellow mycelia and bearing sparse, small gymnothecia in buff-yellow color with ellipsoidal, sparsely echinulate ascospores. [Conclusion] In review of the Talaromyces species reported from China hitherto, we confirmed that T. argentinensis and T. barcinensis are two new records of China.

Abstract:[Background] Ganoderma lingzhi polysaccharides (GLP) are the main active component of this mushroom. UDP-glucose 4-epimerase (UGE, EC 5.1.3.2), an essential enzyme in the generation of sugar donor in synthesis of GLP, catalyzes the interconversion of UDP-glucose and UDP-galactose and thus is closely related to the content of galactose residue in GLP.[Objective] Through heterogeneous expression of the UGE gene from G. lingzhi, this study aims to further explore the enzymatic properties of important enzymes in the generation of sugar donor in GLP synthesis and gain a clearer insight into the synthesis pathway of GLP. [Methods] With the cDNA of G. lingzhi CGMCC 5.26 as template, UGE gene was cloned and then expressed in Escherichia coli BL21(DE3). The yielded recombinant enzyme was then purified and the enzymatic properties and kinetics, substrate specificity, and conversion rate of the purified enzymes were investigated. [Results] The recombinant enzyme was 45 kDa. The optimum reaction pH was 6.0, and it was stable at pH 7.0−9.0. The optimum temperature was 30℃, and it was most stable at 40℃. Fe²⁺ and Mg²⁺ could activate UGE. With UDP-glucose as substrate, the enzyme had the K_m of 0.824 mmol/L, V_max of 769.230 μmol/(L· min), k_cat of 1.333 s⁻¹, and k_cat/K_m of 1.618 L/(mmol· s). It catalyzed D-glucose, galacturonic acid, and N-acetylglucosamine. The conversion rate was increased from 16.0% to 39.4% at the optimal pH, temperature, and ratio of substrate with the addition of metal ions.[Conclusion] UGE from G. lingzhi has similar enzymatic properties to plants-derived UGE, and the catalytic efficiency was higher than most of the UGEs from bacteria. This study enriches the enzymatic properties of important enzymes involved the sugar donor synthesis in GLP production, which helps enhance the understanding of GLP synthesis pathway.

Abstract:[Background] Caffeic acid (3,4-dihydroxycinnamic acid), a natural phenolic compound, has various biological activities and great medicinal value. Candida glycerinogenes, which harbors the caffeic acid precursor metabolic pathway, is a potential high caffeic acid-yielding chassis cell owing to the high acid tolerance, fast growth, and high fermenting rate. However, it has no episomal vectors, which limits the in-depth research on caffeic acid synthesis. [Objective] To explore whether it is feasible to construct episomal vectors with stronger expression of caffeic acid in C. glycerinogenes without natural episomal plasmids in a simpler way. [Methods] Autonomously replicating sequences (ARSs) were selected to establish the episomal plasmid for the synthesis of caffeic acid in C. glycerinogenes, and the plasmid was further optimized by modifying its ARS position, the marker gene promoter length, elements for gene expression, and using Kozak sequence. [Results] Of the five vectors constructed containing different ARSs, pTGAPU-CA-AOX1t-KLARS was able to self-replicate and express the genes for the synthesis of caffeic acid in C. glycerinogenes. After the vector was modified in different ways, such as ARS located upstream of the target gene expression element, URA5 promoter truncated by 250 bp, using Kozak sequence or URA5 terminator, the yield of caffeic acid was significantly higher. The maximum yield after the modification was 3.73 times higher than the initial yield, reaching 29.1 mg/L. [Conclusion] Caffeic acid was synthesized by episomal expression in C. glycerinogenes, with better results than that by integrated expression, which provides a new tool for future modification of the caffeic acid anabolic pathway with episomal vectors and a reference for the construction of episomal expression systems in other strains without episomal plasmids.

Abstract:[Background] Spermine plays an important role in plant defense response, animal anti-fatigue and anti-aging fight, and fungal growth and metabolism. However, there have been few reports on spermine function in the entomopathogenic fungi. [Objective] The study explored the molecular mechanism of spermine synthase (MrSPS) from Metarhizium robertsii during insect hemocoel colonization. [Methods] Insect bioassays of Galleria mellonella larvae were performed to examine the virulence of Mrsps deleted M. robertsii (ΔMrsps), and the growth characteristics of ΔMrsps in hemolymph were observed. Hemolymph of G. mellonella larvae injected with ΔMrsps and wild-type (WT) strains for 30 h were collected for transcriptome sequencing, and comparisons were made with genes from M. robertsii and G. mellonella separately. Real-time quantitative PCR was performed for verification of transcriptome sequencing. [Results] Compared to WT and complementation (ΔMrsps-cp) strains, ΔMrsps showed significantly decreased virulence, and the pathogenicity of ΔMrsps reduced markedly with decreasing of the injection concentration. After 36 h of infection, spores from both WT and ΔMrsps germinated normally and began to grow in a yeast-like state, while ΔMrsps biomass was less than that of WT after 60 h. A total of 3 202 genes were detected from M. robertsii, of which 1 769 were up-regulated and 922 were down-regulated in ΔMrsps. Lots of differentially expressed genes (DEGs) were involved in carbohydrate metabolism, and transport, catabolism, translation, and amino acid metabolism pathways. All identified 28 genes related to hemocoel colonization from DEGs were down-regulated in ΔMrsps. Real-time quantitative PCR showed that both collagen-like protein and Colonization of hemocoel 1 genes were decreased during the whole stage of hemocoel colonization in ΔMrsps compared to the conditions in WT and ΔMrsps-cp. A total of 13 249 genes were detected from G. mellonella, of which 4 026 genes had differential expression. KEGG analysis revealed most of DEGs were enriched in endocrine system and immune system. Furthermore, among the 22 genes involved in Toll and Imd signaling pathways, 18 were up-regulated in G. mellonella infected by ΔMrsps, which suggested ΔMrsps was more likely to cause the activation of the insect immune system than WT. [Conclusion] The molecular mechanism of MrSPS function in hemocoel colonization of M. robertsii was explored for the first time, which provided a theoretical basis for further revealing the mechanism of spermine function in fungi.

Abstract:[Background] Zinc (Zn) is an important trace element for the structure and regulatory system of bacterial cells. In the environment, bacteria exposed to high levels of Zn ions can be affected on their functions.[Objective] To study the effect of Zn on Bacillus amyloliquefaciens JK-JS8 biofilms and its antagonistic ability against Sphaeropsis sapinea, and to explore the possible mechanism between them, so as to provide theoretical basis for the application of biocontrol bacteria in different environmental conditions.[Methods] The formation of B. amyloliquefaciens JK-JS8 biofilms under different concentrations of Zn ions was observed, and the effect of non-lethal concentration of Zn on the antagonistic ability of B. amyloliquefaciens JK-JS8 against S. sapinea was explored by the plate confrontation method. Quantitative reverse transcription-polymerase chain reaction (RT-qPCR) was used to detect the expression of biofilm-related genes and the generation of antibacterial products after the treatment of ZnCl₂.[Results] The concentration of Zn ions at 300 μmol/L had no effect on the growth of B. amyloliquefaciens JK-JS8, but significantly inhibited the formation of its biofilms and the antagonism against pathogenic organisms. Under Zn stress, the expression levels of biofilm-related genes such as tasA, spo0A and bamD were markedly down-regulated compared with the conditions in control group. The production of bacillomycin D decreased by 39.1%, 58.8% and 61.0% at 24 h, 48 h and 72 h, respectively, which was determined by the liquid chromatography. [Conclusion] High concentration of Zn ions in the environment can affect the formation of B. amyloliquefaciens JK-JS8 biofilms and reduce its ability to antagonize the pathogenic organisms.

Abstract:[Background] Fusarium graminearum is one of the main fungal pathogens in wheat production.[Objective] To isolate and screen Streptomyces strains against F. graminearum and lay a theoretical foundation for the biological control. [Methods] Streptomyces strains were isolated with the dilution-plating method, and a plate confrontation experiment was conducted to screen out the strains with antagonistic effects on F. graminearum. The strain was identified based on morphological, physiological and biochemical characteristics, and sequence analysis of 16S rRNA gene. The mycelial growth rate method was used to analyze the fermentation conditions and the stability of the cell-free fermentation broth. The biocontrol effect and antifungal spectrum of the strain 21-6 were also evaluated.[Results] Strain 21-6, which was finally identified as Streptomyces stelliscabiei, showed inhibitory effect on F. graminearum, with the inhibition rate of 75.2%±2.1%. The optimized fermentation conditions for the antifungal activity were PDB medium, fermentation time of 5 days, and initial pH 7.0. The cell-free fermentation suppressed both the mycelial growth and conidial germination of F. graminearum. The antifungal activity of cell-free fermentation broth was free from high temperature, pepsin, trypsin, and proteinase K. However, the fermentation broth was resistant to acidic but sensitive to alkali environment. Moreover, the broth inhibited the infection of wheat coleoptiles by F. graminearum. Strain 21-6 harbors polyketide synthase pks-Ⅰ and pks-Ⅱ genes. It showed biological activities against five plant pathogenic fungi. [Conclusion] Strain 21-6 has a promising prospect in the biological control of F. graminearum.

Abstract:[Background] Macrofungi are important food and drug resources, which can produce a variety of nutrients including essential amino acids. Glutamate, one of the important functional molecules of edible macrofungi, presents umami taste and is an important index to measure the nutritional value of edible fungi. [Objective] We evaluated the resources of macrofungal strains containing glutamate based on the conserved homologous genes of glutamate synthase in Agaricales, aiming to guide the exploitation of wild macrofungal resources and the manufacture of glutamate products. [Methods] We compared the glutamate synthase gene sequences from 25 strains and analyzed the conserved exon region. Two pairs of degenerate primers, GS Ag_les F1/R1 and GS Ag_les F2/R2, were designed for the conserved exon region of glutamate synthase and used for PCR screening of 65 strains belonging to 13 families of Agaricales. In addition, phenylisothiocyanate derivatization was combined with LC-MS and HPLC to quantify the glutamate produced by the 65 strains, and then the screening efficiency of this method was evaluated. [Results] The screening efficiency of primers designed in this study was 86.2%, and the screening efficiency of the primer pair GS Ag_les F1/R1 was significantly better than that of GS Ag_les F2/R2. The fermentation conditions of Agaricales for the production of glutamate were optimized as follows:LMM medium, 28℃, 200 r/min and 2-4 days. The results of PCR and HPLC showed that the primer screening method in this study had a true positive rate of 81.5%, and the application scope involved multiple families including Tricholomataceae,Physalacriaceae, Agaricaceae, Cortinariaceae, Bolbitiaceae, Lyophyllaceae, Pleurotaceae, Psathyrellaceae and Nidulariaceae of Agaricales. [Conclusion] The primers obtained in this study exhibit a good application prospect in the screening of glutamate-producing strains of Agaricales.

Abstract:[Background] The white mould disease caused by Fusarium oxysporum seriously affects the quality of muskmelon and causes economic losses.[Objective] To study the effect of Bacillus velezensis BG-2 on F. oxysporum and the storage quality of postharvest muskmelon.[Methods] The effects of storage temperatures (25℃ and 4℃) on fruit decay rate, weight loss rate, flesh firmness, titratable acid content, soluble solid content, and vitamin C content of muskmelon were studied with B. velezensis BG-2. At the same time, the activities of catalase (CAT), superoxide dismutase (SOD), and peroxidase (POD) were determined. Furthermore, the effects of B. velezensis BG-2 on F. oxysporum and the storage quality of postharvest muskmelon were explored. [Results] B. velezensis BG-2 suspension inhibited the growth of F. oxysporum. At the concentration of 1×10⁷ CFU/mL, B. velezensis BG-2 presented the inhibition zone diameter of (20.45±0.39) mm and the inhibition titer of 144.48 mm/mL. Meanwhile, B. velezensis BG-2 slowed down the declines in flesh hardness, vitamin C content, soluble solid content, and titratable acid content, and inhibited fruit weight loss and decay, which can maintain fruit quality and inhibit the weakening of defense enzyme activities.[Conclusion] B. velezensis BG-2 can significantly inhibit the growth of F. oxysporum, delay the post ripening of muskmelon, and maintain the fruit quality and defense enzyme activity of postharvest muskmelon, demonstrating good control effect on muskmelon rot.

Abstract:[Background] In industrial scale cultivation, Pleurotus eryngii features random fruiting, and the number of fruiting bodies is usually controlled by artificial bud thinning. However, there is no study available on the optimal number of fruiting bodies retained at the moment. [Objective] To explore the effect of different numbers of fruiting bodies retained by artificial bud thinning on the yield and quality of P. eryngii. [Methods] The substrate utilization in each treatment was monitored. The morphological indexes, yield, and texture characteristics of fruiting bodies after harvest were measured. [Results] The substrate utilization rate was the highest in the treatment with 3−4 fruiting bodies retained with little waste. The number of fruiting bodies retained had significant influence on the morphological indexes of fruiting bodies, weight of single fruiting body, and yield of single package. In the case of 1 fruiting body retained, the weight of single fruiting body was the highest, but the yield of single package was the lowest. When 4 fruiting bodies were retained, the yield of single package was high and the morphology of fruiting bodies was the same. As for the texture, when 4 fruiting bodies were preserved, the quality and taste of the fruiting body were the best. [Conclusion] In artificial bud thinning in industrial scale production of P. eryngii, preserving 3−4 fruiting bodies can help achieve high and quality at low cost.

Abstract:[Background] Lactic acid bacteria (LAB) are important microorganisms which affect the texture and flavor of fermentation foods such as sourdough and steamed bun. Amylolytic LAB have enormous application possibilities owing to their ability to colonize in flour. [Objective] To screen amylolytic LAB and study their starch utilization characteristics. [Methods] The medium with starch as carbon source was used to enrich target strains from Daqufor strong aromatic Chinese spirit, followed by testing the starch hydrolysis ablility. Then, expression level of amylase in the yielded strain and the enzyme activity were determined. [Results] Daqu which had been stored for 3‒6 months was optmal for screening the bactia. Amylolytic LAB can be quickly screened out by subculture with batter. The yielded strains were dominated by Lactobacillus plantarum and Lactobacillus paralimentarius. The starch utilization characteristics and amylase activity in the amylolytic L. paralimentarius LBM12001 were analyzed. The findings showed that it can hydrolyze starch at 10 g/L and well colonize in batter. The α-amylase and maltogenic amylase were extracellular enzymes. The specific enzyme activity of maltogenic amylase reached 1 240 U/mg at pH 3.5.[Conclusion] We developed a method to enrich about screen amylolytic LAB from Daqu for traditional Chinese spirit and a method for evaluating the starch hydrolysis ability, and screened out the extracellular maltogenic amylase-secreting strain which had great potential in the production of sourdough, steamed bun, and other fermentation foods.

Abstract:[Background] Streptococcus suis (SS) has a variety of serotypes, genotypes, and virulence factors. [Objective] To reveal the serotypes, virulence gene distribution, molecular typing characteristics and their associations of SS clinical isolates. [Methods] A total of 199 clinical isolates of SS were collected in this study. We employed PCR to detect the serotypes and virulence genes and carried out multilocus sequence typing (MLST). Further, we analyzed the prevalence and associations of serotypes, virulence genotypes and sequence types (STs). [Results] The 199 SS clinical isolates belonged to 16 serotypes (1, 2, 3, 4, 6, 7, 8, 9, 10, 12, 15, 16, 21, 24, 29, and 30). The serotypes 2, 4, and 3 were dominant, respectively involving 26.13% (52/199), 14.57% (29/199), and 12.06% (24/199) of the strains, and 21.61% (43/199) of the strains were untypable. We identified 72 STs, among which ST1, ST94, ST117, ST7, ST28, and ST87 were dominant, the strains of which accounted for 12.56% (25/199), 11.56% (23/199), 9.56% (19/199), 9.04% (18/199), 6.03% (12/199), and 3.01% (6/199), respectively. Twenty-four STs (ST1224-ST1227, ST1229-ST1235, ST1241−ST1242, and ST1300-ST1310) were newly discovered. In addition, these 72 STs involved 12 cloning complexes and 32 single STs. The detection rate of the virulence gene fbps in the 199 isolates was 96.98% (193/199). A total of 19 virulence genotypes were detected, among which epf‒/mrp‒/sly‒/gapdh+/fbps+/orf2+ were dominant (66 strains, 33.17%). [Conclusion] In recent years the serotypes 2, 4, and 3 were dominant among the clinical isolates of SS. ST had obvious genetic heterogeneity and high degree of intraspecific differentiation. The serotypes and STs showed certain crossover. The isolates in this study presented varied distribution of virulence genes and diverse virulence genotypes. In this study, the epidemiological characteristics of SS clinical isolates were investigated to provide scientific basis for diagnosis, treatment and prevention and control measures of SS disease.

Abstract:[Background] The increasing isolation rate of Streptococcus suis serotype 4 (SS4) causes serious harm to animal farming and public health, and there has been no effective vaccine for SS4 so far. [Objective] The purpose of this research was to screen out SS4 vaccine strains with strong pathogenicity and desirable antigenicity and genetic stability. [Methods] Seven SS4 isolates (code A1-A7) were selected as the tested strains. They were determined for the half lethal dose (LD₅₀), the IgG titer in serum of immunized mice and the immune protection rate by Reed-Muench method, enzyme-linked immunosorbent assay (ELISA) and challenge assay, respectively. Meanwhile, the histopathological changes of the mice organs were observed, and after continuous subculture, the 10th, 20th, 30th generations of the tested strains were subjected to pathogenicity and antigenicity tests. [Results] The LD₅₀ of A1-A7 for the mice were 2.19×10⁸, 1.76×10⁸, 1.83×10⁸, 1.01×10⁸, 4.05×10⁸, 1.19×10⁸, and 9.03×10⁷CFU, respectively. After 7 days of secondary immunization, the IgG titers of A1, A2, A3, A4, A6, and A7 immune group were 1:1 600, 1:1 600, 1:3 200, 1:6 400, 1:3 200 and 1:6 400, respectively, and the immune protection rates were 30%, 30%, 50%, 70%, 60% and 80%, respectively, with the pathological changes of A4 and A7 immune groups being milder than the other immune groups. After in vitro passing to the 30th generation, the LD₅₀ of A4 increased to 3.81×10⁸CFU, and the IgG titer and the immune protection rate decreased to 1:1 600 and 40%. By comparison the LD₅₀ of A7 elevated to 2.49×10⁸ CFU, and the IgG titer and the immune protection rate remained stable at 1:6 400 and 80%. Additionally, the pathological changes of A7 were milder compared with those of A4. [Conclusion] A7 (original number HBgu18-4) had strong pathogenicity and good antigenicity, with uniform and stable genetic traits. It could be used as a candidate strain for SS4 vaccine.

Abstract:[Background] Mucosal microbiota from small intestine is an important part of gut microbiota. Many studies had shown that galacto-oligosaccharides (GOS) and manno-oligosaccharides (MOS) can regulate the gut microbiota from large intestine of pigs. However, there are few studies on its regulation of mucosal microbiota from small intestine. [Objective] This study aimed to explore the in vitro fermentation characteristics of GOS and MOS by mucosal microbiota from porcine jejunum and ileum. [Methods] The microbiota samples from the jejunum mucosa and ileum mucosa of growing pigs were used for the anaerobic fermentation of GOS and MOS. Total bacterial number, pH, ammonia nitrogen (NH₃-N), microbial crude protein (MCP), and organic acids were measured at 0, 6, 12, and 24 h during the fermentation. The bacteria were quantified by quantitative PCR after 24 h incubation. [Results] After 24 h incubation, the concentration of NH₃-N in the ileum mucosa group was lower than that in the jejunum mucosa group, while the concentration of MCP showed an opposite trend (P<0.05). After 6 h incubation, the pH changed slightly because of the small amount accumulation of organic acids. After 12 h incubation, lactate, acetate, butyrate, and total short-chain fatty acids in the MOS group were significantly higher than those in the GOS group (P<0.05), and only a small amount of propionic acid was produced in the ileum mucosa group. After 24 h incubation, the MOS group with the microbiota from ileum mucosa showed the highest yield of lactate and the lowest pH (P<0.05). The GOS group had higher propionate yield than the MOS group (P<0.05). The MOS group had higher yield of acetate than the GOS group, as well as that of butyrate and total SCFA in the jejunum mucosa group (P<0.05). After 24 h incubation, Firmicutes showed the close number to total bacteria, being the dominant phylum. Compared with MOS, GOS increased the number of Bacteroidetes, Streptococcus,Veillonella, and Faecalibacterium, as well as that of Clostridiumcluster IV in the jejunum mucosa group and Clostridium cluster XIVa in the ileum mucosa group (P<0.05). Compared with GOS, MOS increased the number of Escherichia coli and Lactobacillus, as well as that of Roseburia in the ileum mucosa group (P<0.05). [Conclusion] The mucosal microbiota from porcine small intestine had different fermentation patterns for GOS and MOS, as manifested by the varied production of organic acids and bacterial proliferation. GOS had an advantage of propionate production and increased the number of Bacteroidetes and Veillonella. MOS promoted the production of acetate, lactate in the ileum mucosa group and butyrate in the jejunum mucosa group, and increased the number of Escherichia coli and Lactobacillus.

Abstract:[Background] Colletotrichum siamense (C. siamense) is a major fungal pathogen causing rubber anthracnose, which has led to substantial economic loss to the world rubber industry. The fungi-specific Zn₂Cys₆ transcription factor regulates the growth and development of fungi.[Objective] A Zn₂Cys₆ transcription factor CsGcc1 was identified from C. siamense, which is homologous to Gcc1 in Magnaporthe oryzae, and its functions were studied.[Methods] The CsGCC1-knockout mutant was developed by homologous recombination, and the functions of CsGcc1 were clarified based on the vegetative growth, H₂O₂ sensitivity, conidial production and germination, cellophane assay and pathogenicity analysis. [Results] CsGcc1 encoded a protein of 646 amino acids and the protein had a GAL4 domain. CsGCC1 was highly expressed in the mycelia and conidia after 36 h culture. The CsGCC1-knockout mutant had slow vegetative growth and low tolerance to H₂O₂. The conidium yield, conidium germination rate, and appressorium formation rate of the mutant were lower than those of the wild type. Cellophane assay showed that deletion of CsGcc1 could weaken the penetration ability of conidia. The virulence of the mutant to rubber leaves was attenuated. [Conclusion] The Zn₂Cys₆ transcription factor CsGcc1 is involved in regulating vegetative growth, oxidative stress, conidium development, and pathogenicity inC. siamense.

Abstract:[Background] Microbial oxidation of highly toxic As³⁺ in the environment plays an important role in the biogeochemical cycle of arsenic, which has potential application value.[Objective] Bacillus sp. ZJS3 has been identified to be tolerant to As³⁺, among other heavy metals. This paper aims to clarify the morphological changes of ZJS3 in response to As³⁺stress and the genetic basis for arsenic response, which is expected to provide basic data for research on As³⁺-tolerant bacteria. [Methods] The whole genome of ZJS3 was sequenced by single-molecule real-time (SMRT) sequencing and Illumina, followed by functional annotation and bioinformatics analysis of the genes. Genes related to arsenic resistance and arsenic metabolism were analyzed by absolute quantitative PCR. [Results] The genome of ZJS3 was 5.82 Mb, which contained 1 chromosome and 3 plasmids, with GC content of 35.9%, 5 981 coding sequences (CDSs), 104 tRNA genes, 136 sRNA genes, 42 rRNA genes, 173 tandem repeats, 13 gene islands, 1 023 transporter-coding genes, 1 717 transmembrane protein-coding genes, and 160 two-component regulatory genes. A total of 97.66%, 69.30%, 78.52%, 65.49%, 67.65%, and 43.87% of the genes in the genome of ZJS3 were annotated in NR, Swiss-Prot, Pfam, COG, GO and KEGG, respectively. arsC expression was significantly higher in the arsenic treatment group than in the control group, while the level of arsB was significantly lower in the arsenic treatment group than in the control group. [Conclusion] Under As³⁺ stress, they block cell division, further influencing cell morphology. The presence of aqpZ, arsA, arsB and arsC in ZJS3 genome indicates that the strain has the ability of As³⁺ efflux and As⁵⁺ reduction, and the presence of phoUpstBACS suggests that the strain can absorb As⁵⁺. However, the expression ofarsB decreases in response to external As³⁺ stress.

Abstract:[Background] Abnormal expression of miR-107 can change the expression of the main proteins of Wnt/β-catenin signaling pathway in tumor cells, while miR-107 plays the same role in human cervical cancer cells (HeLa cells) infected with coxsackievirus B3 (CVB3) has not been reported. [Objective] To investigate whether miR-107 can affect the expression levels of glycogen synthase kinase-3β (GSK-3β), P-GSK-3β, and β-catenin in CVB3-infected HeLa cells. [Methods] The HeLa cells were cultured in vitro and infected with CVB3 for different time periods. The morphological changes of HeLa cells were observed by microscopy. The expression of miR-107 in HeLa cells was measured by real-time fluorescent quantitative PCR. The protein levels of GSK-3β, P-GSK-3β, β-catenin and viral capsid protein (VP1) in HeLa cells were determined by western blotting. [Results] After CVB3 infection for 6 h, the cytopathic effect was obvious. The expression level of miR-107 and the protein levels of GSK-3β, P-GSK-3β, and VP1 increased, while the protein level of β-catenin decreased, with the prolongation of CVB3 infection time (0−8 h). MiR-107 overexpression in the HeLa cells infected with CVB3 for 6 h increased the cell deaths, up-regulated the protein levels of VP1, GSK-3β, and P-GSK-3β (P<0.05), and down-regulated the protein level of β-catenin (P<0.001). The inhibition of miR-107 expression in the HeLa cells infected with CVB3 for 6 h reduced the cell deaths, down-regulated the protein levels of VP1, GSK-3β, and P-GSK-3β (P<0.05), and up-regulated the protein level of β-catenin (P<0.05). [Conclusion] Abnormal expression of miR-107 can affect the expression levels of proteins in the Wnt/β-catenin signaling pathway and VP1 in CVB3-infected HeLa cells.

Abstract:[Background] Microbial proteases have broad application prospects in industrial biotechnology. Among microbial proteases, alkaline proteases account for more than 50% of the world's total enzyme production. It is of great significance to explore new microbial resources that synthesize alkaline proteases. [Objective] Strains with high production of alkaline proteases were screened from the sea mud of Hainan offshore shellfish breeding base and their growth characteristics were explored. Moreover, the conditions for the enzyme production were optimized. Thereby, new protease-producing resources were obtained. [Methods] Casein medium was employed for screening the protease producers and the yielded strain was identified by morphological and phylogenetic analysis. The enzyme production conditions of the strain were optimized by response surface methodology. [Results] Strain F3 with high production of alkaline protease was screened out and identified as Serratia marcescens. The enzyme activity in the fermentation broth of the strain was up to (339.36±4.30) U/mL under the optimal conditions. [Conclusion] S. marcescens F3 has strong ability to produce alkaline protease.

Abstract:As the nitrogen cycle is cross-linked with the iron cycle in the nature, the metabolism and activity of anaerobic ammonia oxidation (anammox) bacteria involved in the nitrogen cycle are also closely related to the iron element. Being ubiquitous in the nature, iron minerals have been widely used for wastewater treatment, owing to the advantages of low operating cost, good stability, and low secondary pollution. The addition of a moderate amount of iron minerals not only can promote the enrichment of anammox bacteria and iron-reducing bacteria in the anammox system, and increase the abundance of functional genes and the activities of related enzymes, but also can regulate the sludge concentration, heme c content, extracellular polymeric substance (EPS) content, and granulation degree to improve the sludge properties and the anammox system stability. Meanwhile, iron minerals have the potential to promote the coupling of multiple nitrogen transformation pathways, such as the anammox, nitrate-dependent Fe(II) oxidation (NDFO), ferric ammonium oxidation (Feammox), dissimilatory nitrate reduction to ammonia (DNRA), and denitrification, which can improve the total nitrogen removal efficiency for the anammox system. Focusing on the good performance of iron minerals in promoting biological denitrification of wastewater and their varieties in the anammox system, the mechanism of iron minerals in enhancing the anammox system is systematically summarized in this study from nitrogen removal efficiency, sludge properties, microbial community characteristics, and enzymes activities. Moreover, the future research directions are prospected from the perspectives of the utilization of iron minerals and the uptake of the iron element by anammox bacteria, aiming to provide theoretical and technical guidance for the application of iron minerals in the anammox system.

Abstract:Salmonella are a major group of zoonotic foodborne pathogens. After infection, Salmonella can escape the clearance of host immune system by virtue of its unique immune escape mechanism and lurk in the host for over a year to establish persistent infection. The persistent infection of Salmonella is associated with the expression of Salmonella pathogenicity islands (SPIs), especially SPI-1 and SPI-2. SPI-1 effector proteins SipB and SipC affect bacterial invasion and induce autophagy or apoptosis in different ways. SPI-2 effector proteins SseI and SseL can assist the intracellular survival of Salmonella by regulating different signaling pathways, so as provide conditions for the persistent infection of Salmonella. This paper expounds the roles of different effector proteins such as SipB and SseL in the persistent infection of Salmonella and summarizes the effects of SPIs such as SPI-6, SPI-7, and SPI-19, hoping to provide new ideas for treating the persistent infection of Salmonella.

Abstract:Ganoderma lingzhi has long been used as medicine and the active components polysaccharides have extensive pharmacological activities. Liquid fermentation shows unique advantages in extracting G. lingzhi polysaccharides. In recent years, there has been an explosion of research on the liquid fermentation of G. lingzhi to yield polysaccharides, and this technology has attracted the interest of scholars. We summarized the research on the biosynthesis and biosynthesis regulation of G. lingzhi polysaccharides, the optimization of the medium for and process of liquid fermentation, among others, hoping to provide a reference and basis for liquid fermentation of G. lingzhi to yield intracellular and extracellular polysaccharides and its industrial application.

Abstract:Wide applications and improper treatments of organohalides in industry and agriculture lead to their ubiquitous presence in the underground environments, posing a great threat to ecosystem functions, drinking water safety and human health. Organohalide-respiring bacteria (OHRB) are crucial forin situ bioremediation of organohalide-contaminated environments. This review summarizes environmental factors (e.g., pH, temperature, salinity, electron donors and acceptors, oxygen) that may impact the growth, metabolisms and dechlorination activities of OHRB, and discusses the future research of OHRB, which is intended to provide theoretical and technical reference for the effective implementation of in situ bioremediation to clean up organohalides-contaminated sites.

Abstract:Entomopathogenic Beauveria bassiana shows a variety of morphological patterns such as aerial conidia, mycelia and single-cell form known as hyphal bodies. As a typical filamentous fungal species, B. bassiana plays important roles in conidial development and host-pathogen interactions. It is also widely applied for biological control of pests and thus is of great value in forest protection and agricultural production. Once related genes in the species are knocked out, the mutants show responses to oxidative stress associated with conidial development and virulence. This study summarizes the molecular genetics of B. bassiana involved in oxidative stress in recent years, which is expected to serve as a reference for the study of oxidative stress signaling pathway in filamentous fungi.

Abstract:During biofilm formation, the unique spatial structure formed upon active or passive biological processes among microbial populations is called spatial organization pattern. Microbial spatial organization patterns are ubiquitous in natural and artificial environments, such as the medical, industrial and ecological systems and processes. They are critical for the causes and consequences of microbial community structure, biodiversity maintenance, and ecological functions and thereby have received extensive attention. Nevertheless, mechanistic understanding of microbial spatial organization and ecological consequences remains elusive largely due to the extremely complex microbial communities and limited research methodologies. The paper aims to summarize the state-of-the-art in microbial spatial self-organization, the key determining environmental factors, and the impact on nutrient utilization, element cycling, evolution and diversity maintenance, and ecological functions of microbes.

Abstract:Antibiotic-resistant bacteria and antibiotic-resistance genes spread across species and habitats due to various human activities at the human-animal-environment interface. One Health that treats humans, animals and the environment as an organic whole is expected to become an effective strategy to mitigate such transmission. Due to human production activities, antibiotics and their metabolites are enriched in the environment, and then spread to people through animals or animal products, thus screening out resistant bacteria and causing the spread of resistance genes. This paper summarizes the main modes of transmission and the relationship between them and outlines Chinese and other countries՚ national plans to combat antibiotic resistance. We advocate that countries and regions use the concept and method of One Health to control the spread of antibiotic resistance. In addition, we should deal with the global challenge of antibiotic resistance through multisectoral collaboration among the medical and health sectors, food and drug administration, agriculture, forestry, fisheries, husbandry, education, and finance.

Abstract:Interspecific electron transport is of great significance to geobiochemical cycles and environmental pollution remediation since it can promote microbial co-metabolism. Interspecies electron transfer can be classified into direct interspecies electron transfer (DIET) and mediated interspecies electron transfer (MIET) according to the mode of electron transfer, in which the former has received extensive attention owing to its common occurrence and high efficiency. This study summarized the research progress in interspecies electron transfer, elaborated the involved pathways, compared the advantages and disadvantages between DIET and MIET, and suggested to mine more microorganisms with interspecies electron transfer function. This review aims to improve understanding of interspecies electron transfer and provide a reference for the future research in this field.

Abstract:A large number of studies have demonstrated that gut microbiota is closely related to the occurrence and development of a variety of diseases such as neurodegenerative diseases and metabolic diseases. The species and number of bacteria are affected by genetic factors, dietary habits, exercise and other factors. In metabolism-associated fatty liver disease (MAFLD), some metabolites of gut microbiota promote the progression of the disease by increasing hepatic steatosis and changing intestinal mucosal permeability. The relationship between the changes in the species and number of bacteria and the progression of disease has also been extensively studied. However, the casual relationship between them remains unclear. Exercise can increase the species and number of beneficial gut microbiota and alleviate the gut microbiota disorders caused by high-fat diet to mitigate MAFLD. On the other side, gut microbiota can regulate the exercise capacity of the body. However, the mechanism of how exercise alleviates MAFLD through gut microbiota remains to be studied. This paper elaborates the important roles of gut microbiota and exercise in MAFLD by reviewing the relationship between the three.

Abstract:Arbuscular mycorrhiza (AM) is a symbiont formed by the interaction and mutual recognition of soil-born AM fungi and the roots of most vascular plants in the long-term evolution. The development and function of AM, limited by environmental conditions, especially the soil nutrient level, drought, and salinity, depend on the precise "molecular dialogue" between AM fungi and host plants. Phytohormones are low-molecular-weight organics with low concentration and act as crucial signaling molecules in the regulation of AM symbiosis. There are mainly nine phytohormones participating in regulating AM development with different effects. Strigolactones (SLs) act at the first symbiotic recognition between AM fungi and host plants. At the early stage, abscisic acid (ABA) and brassinosteroid (BR) promote the fungal invasion, whereas salicylic acid (SA) and ethylene (ET) inhibit the fungal invasion. Auxin (Aux), ABA, and BR promote the subsequent arbuscule formation, whereas ET and gibberellin (GA) suppress the arbuscule formation. Jasmonic acid (JA) may have both positive and negative regulating effects on fungal invasion and arbuscule formation. However, the role of cytokinin (CTK) remains unclear in AM development. In addition, the signaling crosstalk among phytohormones normally determines AM development. This review summarized the characteristics of different phytohormones and their associated signaling crosstalk (synergistic or antagonistic) in regulating AM development, and the possible regulation mechanisms of different phytohormone signals involved in AM development under stress conditions. The profound research and systematical illustration of the physiological/molecular mechanisms of phytohormones in regulating the symbiotic relationships between AM fungi and host plants, will help the study on symbiology and the application of mycorrhizal technology.

Abstract:Environmental Engineering Microbiology is a compulsory course for environmental majors. To meet the talent cultivation needs in the new era, we focused on the four key issues in the teaching and reformed the teaching of Environmental Engineering Microbiology through the innovation in four aspects:teaching content, teaching method, evaluation method, and integration of ideology and politics. After several rounds of application and the evaluation of teaching quality by multiple parties, we concluded that the reform significantly improved the teaching quality of the course.