Abstract:Inflammasomes are multi-protein complexes located in the cytosol and activate caspase-1. Subsequently, inflammasomes induce maturation and secretion of series of pro-inflammatory cytokines and pyroptosis. Inflammasome activation plays a critical role in host innate immune responses against infectious pathogens. Inflammasomes can protect host against most pathogens. However, the protection role of inflammasome seems sometimes less obvious, or it shows detrimental to the host and facilitates the pathogens. Pathogens evolved evasion strategies against inflammasomes under selective pressure, and could weaken or inactivate the functions of inflammasomes. In this review, we summarize the progress in research on the active role of inflammasomes in host immune response against pathogens and the inflammasome-evasion strategies of pathogens.

Abstract:Potassium ion (K⁺) is a ubiquitous monovalent cation necessary for all living cells. To maintain homeostatic intracellular K⁺ concentration, most prokaryotes possess several unique K⁺ uptake systems to transport K⁺. A newly found second messenger-cyclic diadenosine monophosphate (c-di-AMP), plays an important role in regulating K⁺ transport by binding to several K⁺ transport-related proteins, such as KdpD, KtrA and TrkA. When the intracellular c-di-AMP concentration is high, c-di-AMP can bind its receptor or effector proteins to inhibit transporter activity. In addition, riboswitch could also be targeted by c-di-AMP to control the transcription of downstream K⁺ transporter genes, including ktr, trk and kdp operon and kup gene. High intracellular c-di-AMP concentration depresses bacterial K⁺ uptake. Therefore, understanding the mechanism of K⁺ transport inhibition by c-di-AMP not only enriches the regulation mode of the K⁺ transport, but also sparkle new ideas for the control and applications of bacteria.

Abstract:Gliotoxin is a small molecular compound with a molecular weight of 326 Da, and its skeleton is a cyclic two-peptide synthesized by the non-ribosomal peptide synthetase GliP, catalyzing the condensation reaction of phenylalanine and serine. Gliotoxin belongs to the family of epidithiodiketopiperazines, is an important fungal secondary metabolite. Many studies have shown that gliotoxin has a variety of effects on plants and animals both in vivo and in vitro. Gliotoxin not only has the function of immune suppression, inducing host cell apoptosis, but also has potential application in biological control. The recent advances in biosynthesis process, mechanisms of gliotoxin on host cells and its potential application value are reviewed in this paper.

Abstract:Type 3 secretion system, as one of the secretion systems for Gram-negative bacterial, plays an important role in the pathopoiesia of Gram-negative bacteria. Bacteria can use T3SS as a canal that directly translocates effectors to host cell. Effectors in host cell manipulate a subset of signaling pathways of host cell to promote bacterial colonization in cells. The translocation of effectors is regulated by two factors, one is the signal sequences of effector, and the other is the regulation of T3SS-associated proteins. In this article, we review recent advances in studies on the constitute of T3SS and the mechanism of effectors translocation.

Abstract:[Objective] Proteomic analysis of Lactobacillus plantarum to salt stress could provide a theoretical basis for salt-tolerant mechanism involved in lactic acid bacteria.[Methods] Lactobacillus plantarum FS5-5 was originally isolated from traditional home-made fermented soybean paste from Northeast China. Growth was observed under 0%, 6.0%, 7.0% and 8.0% (W/V) NaCl, and protein expression was analyzed using iTRAQ technology.[Results] We collected bacteria at 5, 10, 12 and 12 h of the mid-logarithmic growth phase for proteomic analysis. Based on iTRAQ results, a total of 1271 differently expressed proteins were identified under different salt stress. They were involved in carbohydrate metabolism, amino acid metabolism, lipid metabolism, nucleotide metabolism, stress proteins, transporters, phosphotransferase system, ribosome, and so on.[Conclusion] The ability of L. plantarum FS5-5 to grow in high concentration of NaCl is connected with energy synthesis proteins, stress proteins and compatible solutes transporters.

Abstract:[Objective] We compared the difference of community structure of archaea between Qinghai Lake (the largest inland saltwater lake in China) and several other salt lakes in China.[Methods] Chaka Salt Lake in Qinghai Province, Huamachi Salt Lake and Gouchi Salt Lake in Shaanxi Province, and Yuncheng Salt Lake in Shanxi Province were randomly selected as the sample pool. From each lake five samples were taken and analyzed with the high-throughput sequencing technology for 16S rRNA gene.[Results] The dominant genera of Qinghai Lake were DHVEG-6_norank, Methanomicrobia_unclassified, Methanobacterium, Methanolobus, Candidatus_Methanomethylophilus, Miscellaneous_Euryarchaeotic_Group(MEG)_norank, AMOS1A-4113-D04_norank, Methanosarcina, Miscellaneous_Crenarchaeotic_Group_norank. Among them DHVEG-6_norank (70.46%) was absolutely dominant whereas it was hardly found in other salt lakes. On the contrary, the dominant genera in the other 4 salt lakes are Halonotius, Halorubrum, Natronomonas, Halobellus and Haloarcula. Degree of mineralization was the most influential factor that affected the structure of the archaea communities in Qinghai Lake and other salt lakes. The community structure of Qinghai Lake with a low degree of mineralization was significantly different from that of the other 4 salt lakes. The second factor is pH that affected the abundance of some species. However, no significant correlation between the altitude and the community structure was found.[Conclusion] The community structure and diversity of Qinghai Lake was significantly different from that of other 4 salt lakes and it was mainly influenced by the degree of mineralization.

Abstract:[Objective] In this study, we explored the probiotic effect of Lactobacillus plantarum Lp3 on fatty rats.[Methods] In vitro Lp3 with high hypocholesterolemic effects was isolated from Yak yogurt in Qinghai Tibet Plateau. We established a Sprague Dawley rat model of hyperlipidemia to determine the effect of the strain on blood lipids in experimental animals, and to study the effects on the intestinal flora, fecal moisture, cholesterol levels and bile acids of fecal, and the changes of cholesterol and triglycerides levels in the liver of SD rats. Thirty rats were assigned to three groups and fed either a normal or a high-cholesterol diet. The LAB treated group received the high-cholesterol diet supplemented with Lactobacillus plantarum Lp3.[Results] Lactobacillus plantarum Lp3 had no obvious side effects in rats and had a good cholesterol-lowering effect in high fat model rats. Lactobacillus plantarum Lp3 could significantly reduce serum total cholesterol, triglycerides and LDL-cholesterol levels in rats fed a high fat diet compared with the control group (P<0.05), but no difference was observed in high-density lipoprotein cholesterol. Compared with other two groups of rats, number of E. coli in feces of high fat fed rats group (HC) had obvious increase, the number of Bifidobacterium and Lactobacillus decreased significantly. But in high fat and LAB fed rats group (HL), the number of Lactobacillus in the feces was slightly higher than that in control group. The number of E. coli and Bifidobacteria were almost same as that in the control group. The results indicated that Lp3 could maintain the balance of intestinal flora and increase the moisture content in feces about 6.44% compared with HC group. Cholesterol and triglyceride in the liver tissue of the high fat fed rats group (HC) were significantly higher than high fat and LAB fed rats HL group (P<0.05). Lp3 could reduce lipid deposition in liver tissue. From histology of liver steatosis, we could also draw out the above conclusion. Cholesterol and bile acid contents in feces of HL group were higher than the HC group.[Conclusion] Lactobacillus plantarum Lp3 has probiotic potentials.

Abstract:[Objective] We identified 16S rRNA genes in genomes of prokaryotes.[Methods] We constructed a 3-layer filtering model based on the three features of GC bases content of the gene sequences, 3-base periodicity and Markov chain to recognize the 16S rRNA genes from prokaryotic genomes.[Results] The specificity, sensitivity and Matthews correlation coefficients of the model were 99.58%, 91.60% and 91.49%, respectively.[Conclusion] The results showed that the 16S rRNA genes can be identified efficiently and accurately by using our model.

Abstract:[Objective] To uncover the diversity of extracellular protease-producing bacteria and to expand our knowledge on protease-producing bacteria in the sediments of North Yellow Sea, and to screen efficient protease production strains that might provide flora resources for mining marine protease-producing microorganisms.[Methods] Protease-producing bacteria were isolated by using casein gelatin plate from 5 sediment samples of the North Yellow Sea. The bacteria diversity was evaluated through phylogenetic analyses based on 16S rRNA genes. The protease diversity was evaluated through the inhibition tests for 39 strains with higher enzymatic activities by using 4 inhibitors:phenyl methyl sulfonyl fluoride (PMSF, serine protease inhibitor), 1,10-phenanthroline (O-P, metalloproteinase inhibitor), E-64 (cysteine protease inhibitor) and pepstatin A (aspartic protease inhibitor). Furthermore, degradation abilities for different protein substrates such as casein, gelatin and elastin were evaluated through observing hydrolytic zones.[Results] A total of 66 protease-producing strains were isolated from 5 sediment samples. These isolates were classified into 7 genera of 4 phyla including Bacteroidetes, Proteobacteria, Actinobacteria and Firmicutes, with Pseudoalteromonas (69.9%), Sulfitobacter (12.1%) and Salegentibacter (10.6%) as the dominant. The richness of the cultivable protease-producing bacteria reached 10⁴ cells/g in all sediment samples. The inhibition tests indicated that all the tested strains produced serine protease and/or metal protease, only a few strains produced cysteine protease or aspartic protease.[Conclusion] The cultivable protease-producing bacteria in the North Yellow Sea are diverse with Pseudoalteromonas, Sulfitobacter and Salegentibacterbacteria as the dominant groups and the extracellular proteases belong to serine proteases and/or metalloproteinases.

Abstract:[Objective] Ethyl carbamate is a common potential hazard in different fermented foods. Urea and ethanol are the major precursors of ethyl carbamate in rice wine. In this study, the accumulation of urea was reduced based on a high-throughput screening strategy, therefore decrease the accumulation of ethyl carbamate in rice wine.[Methods] An industrial strain Saccharomyces cerevisiae XZ-11 was used for the study. Lower urea accumulating strains were achieved by using Atmospheric and Room Temperature Plasma (ARTP) mutagenesis and high-throughput screening strategy. RT-qPCR analysis was used to detect the change of DUR1,2 and DUR3, two important urea metabolism and transport genes.[Results] A mutant strain 5-11C was obtained with the capacity of both efficiently urea using and genetic stability. The accumulation of urea was 50.6% lower than that of S. cerevisiae XZ-11. RT-qPCR results showed that the expression levels of DUR1,2 and DUR3 increased 3.3 and 2.2 folds, respectively.[Conclusion] High-throughput screening strategy can be applied to obtain mutants with reduced accumulation of ethyl carbamate precursor in rice wine.

Abstract:[Objective] We studied the regulation of crgA on carotenoid biosynthesis by Blakeslea trispora.[Methods] crgA was cloned from B. trispora and then disrupted by using split-marker strategy. Phenotypic characteristics, transcription of key enzyme genes and carotenoid accumulation between the wide-type and mutant strain were compared.[Results] In contrast to those of wild-type strain, the spore-forming ability of the mutant was weakened but transcription of key enzyme genes in the pathway of carotenoid biosynthesis increased and β-carotene production in the mycelia was improved by 31.2% after cultured for 120 h. After crgA was transformed into the mutant, the strain restored the phenotype as those of the wild-type strain.[Conclusion] crgA could regulate spore-forming and mycelia growth. Besides, the gene controlled carotenoid synthesis by regulating the transcription of key enzyme genes, which indicates that crgA is a negative regulator in Blakeslea trispora.

Abstract:[Objective] This study was aimed to isolate and identify Acetobacter strains from Drosophila melanogaster, and to study their roles in promoting the growth of hosts.[Methods] Selective culture method was used to isolate Acetobacter; Gram-staining and 16S rRNA gene blast was used to identify strains. Intestinal colonization test was used to verify the commensalism. Developmental time and growth rate were used to evaluate the effect of the bacterial isolates on hosts' development. Immunofluorescence staining was used to check the mitosis in gut. RT-PCR was used to assess the molecular biomarker of development and development-associated cellular signaling.[Results] The strain was identified as Acetobacter orientalis and it colonized the gut of Drosophila and fly medium, and significantly promoted the growth of the host. Furthermore, Acetobacter orientalis enhanced the number of mitotic cells in gut, and promoted the secretion of growth hormone of hosts by regulating the insulin signal pathway.[Conclusion] Acetobacter orientalis was one of commensal bacteria of Drosophila, and maintained the structure of intestine and promote the growth of hosts.

Abstract:[Objective] We purified and characterized L-sorbose dehydrogenase and L-sorbosone dehydrogenase from Ketogulonicigenium vulgare WSH-001.[Methods] L-sorbose dehydrogenase gene (sdh) and L-sorbosone dehydrogenase gene (sndh) from K. vulgare WSH-001 were amplified by PCR. The amplified fragments were inserted into pET-28a(+) to obtain expression plasmids, namely pET28a-sdh and pET28a-sndh. Escherichia coli BL21(DE3) harboring the above plasmids was used to express SDH and SNDH. Purified SDH and SNDH were obtained by using HisTrap^TM affinity chromatography and gel filtration chromatography.[Results] SDH and SNDH from K. vulgare WSH-001 were expressed in E. coli BL21(DE3) and purified. The molecular weight of SDH and SNDH were 64 kDa and 48 kDa on SDS-PAGE, respectively. Colorimetric assay showed that the enzyme activity of SDH and SNDH was 3.15 U/mg and 6.12 U/mg, respectively. The optimum temperature and pH of the purified SDH were 30℃ and 8.0, respectively, whereas the optimum temperature and pH of the purified SNDH were 35℃ and 8.0, respectively. The enzyme activity of SDH and SNDH was extremely low at pH 3.0, 4.0 and 5.0.[Conclusion] SDH and SNDH from K. vulgare WSH-001 were expressed in E. coli BL21(DE3) and characterized. The results could provide essential reference for the achievement of one-step fermentation of 2-KLG.

Abstract:[Objective] Trichoderma reesei is one of the major industrial fungi for cellulase production. A large amount of cellulases are secreted from the fungal cell through the protein secretion pathway. Therefore, knowledge of specific gene functions related to protein secretion would contribute to identification of key factors controlling cellulase production. In this study, the vacuolar protein sorting-associated gene VPS13 was deleted by gene knockout strategy in T. reesei and effects of VPS13 deletion on fungal growth and cellulase production were analyzed.[Methods] Double-joint PCR technique and homologous recombination method were used to delete VPS13 gene in T. reesei QP4 strain. Fungal cultivation, microscopic observation, sporulation detection, protein content and enzyme activity determination were applied to compare the growth characteristics, morphology, spore formation, and cellulase activity between the deletion strains and the parental strain.[Results] Two VPS13 deletion strains, ΔVPS13-1 and ΔVPS13-2, were obtained successfully. Compared with the parental strain, the mutants showed slower hyphae spreading, but their biomass increased significantly after the logarithmic growth phase. In addition, VPS13 deletion led to delay of sporulation. Cellulose plate cultivation revealed that VPS13 mutants produced clearer transparent halos around colonies than the parental strain, indicating that VPS13 deletion increased the ability to degrade cellulose. Furthermore, liquid fermentation showed that VPS13 deletion also improved protein yield and cellulase activity.[Conclusion] T. reesei VPS13 gene can be used as an important target to improve cellulase yield.

Abstract:[Objective] To explore the biosynthetic potential of endophytic fungi isolated from medicinal plants Paeonia lactiflora, we studied their population diversity and detected richness of their putative polyketide synthase (PKS) and nonribosomal peptide synthetase (NRPS) gene sequences.[Methods] Culture-dependent method was used to obtain endophytes from the surface-sterilized plant samples. Endophytes were identified based on morphological characteristics and ITS gene phylogenies. PKS and NRPS gene fragments were PCR amplified from genomic DNA with degenerate primers. The biosynthetic potential of endophytes was accessed by bioinformatic and phylogenetic analyses of PKS and NRPS gene sequences.[Results] A total of 105 endophytic isolates were isolated from P. lactiflora root tissues, yielding a total of 52 endophytic fungi through dereplication. The analysis results of endophtytic fungi ITS gene sequences showed that they affiliated to 15 genera of 13 families in seven orders, and Leptosphaeria, Ilyonectria and Fusarium were the predominant genera. Thirteen PKSs and eight NRPSs gene fragments were detected in a total of 52 endophytic fungi. Some fungi amino acid translated fragments were similar to sequences of PKS and NRPS involved in the biosynthesis of the antifungal compound in GenBank. Analysis of the detected endophyte gene fragments afforded consideration of the possible bioactivity of the natural products produced by endophytes from P. lactiflora.[Conclusion] This study demonstrated that the endophytic fungi from P. lactiflora roots had great significant diversity and potential to synthesize bioactive secondary metabolite, therefore, their natural products are worth of further research and development.

Abstract:[Objective] Pythium oligandrum is an environmental friendly mycoparasitic biocontrol fungus that can antagonize several plant pathogens and promote plant growth. We constructed Agrobacterium tumefaciens-mediated transformation of P. oligandrum.[Methods] Three A. tumefaciens strains:EH105, AGL-1 and LBA4404 were compared to find out the strain which showed the best genetic transformation efficiency within P. oligandrum. The factors of transformation system were screened and optimized to establish the genetic transformation system within P. oligandrum mediated by Agrobacterium tumefaciens.[Results] EHA105 strain showed the best genetic transform efficiency within P. oligandrum among three tested strains, followed by AGL-1 strain, and LBA4404 strain performed poorly. Then, the optimized genetic transformation system was as follows:A. tumefaciens EHA105 (OD₆₀₀=0.6) suspension was mixed with P. oligandrum (10⁶-10⁷ spores per milliliter) suspension in a proportion of 1-10:1, then incubated in dark conditions for 72 h between 25 and 26℃ in the presence of induction medium (pH 5.0) containing acetosyringone at 300 μmol/L. Afterwards, antibiotic was added in to eliminate A. tumefaciens before 24 hours reviving cultivation. In the end, the transformants were selected by medium containing 200 mg/L of Basta and 250 mg/L of cefotaxime sodium on PDA. In this work, egfp was successfully transformed into P. oligandrum with the efficiency of 130 transformants per 10⁶ spores.[Conclusion] We developed an Agrobacterium tumefaciens-mediated genetic transformation system for P. oligandrum for the first time to provide powerful tool for the study of P. oligandrum molecular breeding and biological control mechanisms.